Hepatitis E Virus: Identification and evaluation of the...
Transcript of Hepatitis E Virus: Identification and evaluation of the...
Hepatitis E Virus: Identification and evaluation of the potential for zoonotic
transmission in the pork food chain
Animal Health and Veterinary Laboratories Agency (AHVLA), Virology
Department, Addlestone, Surrey, United Kingdom & Faculty of Health and Medical
Science, Microbial Sciences Division, University of Surrey, Guildford, Surrey,
United Kingdom & Central Veterinary Institute, Wageningen University and
Research Centre (CVI), Department of Virology, Lelystad, The Netherlands
A thesis submitted in accordance with the requirements of the degree of Doctor of
Philosophy in Microbial Sciences
August 2012
©Alessandra Berto
( T AHVLAAnimal Health and
Æ Veterinary Laboratories j Agency
U N I V E R S I T Y O FSURREY W A G E N I N G E N
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STATEMENT OF ORIGINALITY
This thesis and the work to which it refers are the results of my own efforts. Any ideas, data, images or text resulting from the work of others (whether published or unpublished) are fully identified as such within the work and attributed to their originator in the text, bibliography or in footnotes. This thesis has not been submitted in whole or in part for any other academic degree or professional qualification. I agree that the University has the right to submit my work to the plagiarism detection service TurnitinUK for originality checks. Whether or not drafts have been so- assessed, the University reserves the right to require an electronic version of the final document (as submitted) for assessment as above.
Alessandra Berto (PhD Candidate)
Signature:............ Date:
Acknowledgments
No one walks alone on the journey of life. I would like to start to thank those that
joined me, walked beside me, and helped me along the way.
In fact a part of my big effort, the success of this project depends largely on the
encouragement and guidelines of many others.
First and foremost, I would like to thank to my supervisors, Dr. Malcolm Banks, Dr. Wim H.M. van der Poel, Dr. Francesca Martelli and Dr. Lisa Roberts for the valuable guidance and advice. They inspired me greatly to work in this project. Their willingness to motivate me contributed tremendously to my project and my scientific skills.
Besides, I would like to thank all my friends, mainly the VI5 PhD students at AHVLA, in particular Sosan Obulukola (Buki) that a part of feeding me with Nigerian food, she helped me to be stronger during these 3 years. Others two good friends are Victor Riitho my IT support and Sophie Morgan my English dictionary.
Sylvia Grierson not only helped me under the seientific aspect but she was/is my Scottish mentor.
An honorable mention goes to my parents and brother for their understandings and supports on me in completing this project.
Finally, I wish to express my love and gratitude to Ruben for his understanding & endless love through the duration of my studies.
Without the help of the particular that I mentioned above, I would have faced many difficulties while doing this.
Table of contentsABBREVIATION TABLE.............................................................................................. x
Abstract.............................................................................................................................. 1
CHAPTER 1 General Introduction..................................................................................3
.1 Hepatitis E virus aetiology......................................................................................... 4
.1.1 Hepatitis E in non-endemic regions........................................................................8
. 1.2 Hepatitis E in disease-endemic regions................................................................. 8
.2 Morphology and Genomic organization..................................................................10
.3 Viral proteins.............................................................................................................14
.3.1 Methyltransferase................................................................................................... 14
.3.2 Papain-like cysteine protease................................................................................ 15
.3.3 Helicase...................................................................................................................15
.3.4 RNA-dependent RNA polymerase (RdRp)......................................................... 15
.3.5 0RF2 and the major capsid protein......................................................................16
.3.6 ORF3 and its product.............................................................................................19
.4 The HEV replication cycle....................................................................................... 22
.4.1 Viral receptor and entry........................................................................................ 22
.4.2 Model of HEV replication.................................................................................... 22
.5 Potential targets for the development of antiviral drugs........................................ 25
.6 HEV inactivation studies......................................................................................... 26
.7 Taxonomy: Evolutionary History and Population Dynamics of Hepatitis E Virus
28
.8 Genotype classification.............................................................................................31
.9 Epidemiology of H EV ..............................................................................................34
.9.1 Epidemiology in humans...................................................................................... 34
.9.2 Epidemiology in pigs and other animals.............................................................. 43
.10 Pathogenesis, clinical signs and symptoms.......................................................... 44
.10.1 In humans............................................................................................................. 44
.10.2 In pigs....................................................................................................................48
.11 Diagnostic procedures.............................................................................................50
.11.1 ELISA...................................................................................................................50
.11.2 Conventional RT-PCR.........................................................................................51
. 11.3 Real time RT-PCR...............................................................................................51
IV
1.11.4 Negative strand detection.................................................................................... 52
1.11.5 Cell culture and new technology for in-vitro propagation of the virus............52
1.11.6 Microscopy.......................................................................................................... 56
1.11.6.1 Confocal microscopy....................................................................................... 56
1.11.6.2 Electron microscopy, transmission and scanning.......................................... 57
1.12 Vaccination............................................................................................................. 57
1.12.1 HEV vaccination modelling in pigs................................................................... 58
1.13 Aims of the VITAL PhD project...........................................................................61
CHAPTER 2 VITAL Ring Trial.................................................................................... 64
Introduction......................................................................................................................65
Materials and methods....................................................................................................66
2. 1 Virus concentration and nucleic acid extraction....................................................66
2.1.1 Sampling and virus concentration in pork liver tissue........................................ 66
2.1.2 Nucleic acid extraction from pork liver tissue.....................................................66
2.1.3 Sampling and virus concentration from soft fruit................................................67
2.1.4 Nucleic acid extraction from soft fruits............................................................... 68
2.2 Positive standards construction................................................................................ 69
2.3 Real time PCR protocols.......................................................................................... 70
2.3.1 Quantification of adenovirus by real-time PCR...................................................70
2.3.2 Detection and quantification of Murine Norovirus by real-time RT-PCR 71
2.3.3 The internal amplification controls (lACs)..........................................................71
2.4 Data interpretation:...................................................................................................77
Results..............................................................................................................................78
2.5 Detection of spiked Human Adenovirus in raspberries samples......................... 78
2.6 Detection of spiked Human Adenovirus in liver samples....................................81
2. 7 Discussion.................................................................................................................84
CHAPTER 3 Hepatitis E virus in the UK pork food chain......................................... 85
3.1 Introduction: VITAL Data gathering.......................................................................86
Materials and Methods.................................................................................................... 87
3.2 UK sampling scheme................................................................................................87
3.2.1 Sample collection................................................................................................... 87
3.2.1.1 Slaughterhouse.....................................................................................................87
3.2.1.2 Processing/cutting point:.................................................................................... 87
V
3.2.1.3 Point of sale:....................................................................................................... 88
3.3 Sample preparation and nucleic acid extraction:....................................................88
3.4 Real time PCR........................................................................................................... 89
3.4.1 HEV........................................................................................................................89
3.4.2 PAdV.......................................................................................................................90
3.4.3 MNoV.....................................................................................................................90
3.4.4 HAdV......................................................................................................................91
3.4.5 Internal assay controls........................................................................................... 91
3.4.6 Positive standards construction.............................................................................92
Results..............................................................................................................................96
3.5 HEV detection........................................................................................................... 96
3.5.1 PAdV detection......................................................................................................97
3.5.2 HAdV detection.....................................................................................................97
3.6 Discussion................................................................................................................. 99
CHAPTER 4 Replication of Hepatitis E virus in three-dimensional cell cultures system.......................................................................................................................103
4.1 Introduction............................................................................... ............................ 104
4.2 Use of the 3D Culture system to investigate the viability of HEV detected by RT-
PCR in UK pork sausage and French liver sausage (figatelli)..............................106
4.3 Propagation of HEV in cell cultures.................................................................107
4.3.1 Comparison of efficiency of the 3D and 2D cell culture for HEV replication
.........................................................................................................................................107
4.3.2 Inoculum preparation:.................................................................................... 108
4.3.3 Infection of the cells:........... 108
4.3.4 Comparison of 3D, 2D and 3D transferred to 2D cell cultures for HEV
replication.................................................................................................................109
4.3.5 RNA extraction from supernatant of 3D cell cultures, 2D cell cultures and 3D
cell transferred to 2D system infected with HEV..................................................110
4.3.6 Real Time RT-PCR.........................................................................................110
4.3.7 Positive strandard and copy number quantification..................................... 110
4.3.8 Definition of Ct values:.................................................................................. 112
4.4 Materials and Methods to investigate the viability of HEV in UK sausages and
figatelli samples................. 112
VI
4.4.1 Cell Preparation................................................................................................... 108
4.4.2 Inoculum preparation of figatelli sample and UK sausages:............................112
4.4.3 Cell inoculation....................................................... 113
4.4.4 Determination of infectivity of progeny virus...................................................113
4.4.5 RNA extraction and real time RT-PCR............................................................. 113
4.4.6 Electron microscopy............................................................................................113
Results............................................................................................................................ 115
4.5 Comparison of HEV replication in 3D cell and 2D cell culture systems........... 115
4.6 Evaluation of the infectivity of the viral progeny and comparison of HEV
replication in 3D and 2D culture systems and 3D cells transferred into 2D............. 115
4.7 Results of the use of the 3D cell culture system to investigate the viability of
HEV in UK sausages and French liver sausages (figatelli)....................................... 121
4.8 Discussion................................................................................................................125
4.8.1 HEV replication in the 3D cell culture system.................................................. 125
4.8.2 Discussion of the use of the 3D cell culture system to investigate the viability
of HEV in the UK sausages and French liver sausages (figatelli)............................. 129
CHAPTER 5 Inactivation studies................................................................................ 132
5.1 Heat inactivation..................................................................................................... 133
5.2 UV light and NaOCl HEV inactivation studies.................................................... 134
Materials and Methods.................................................................................................. 137
5.3 Cells preparation:.................................................................................................... 137
5.3.1 Heat inactivation experiment.............................................................................. 137
5.3.1.1 Inoculum preparation...................... 137
5.3.1.2 Infection of the 3D cells....................................................................................137
5.4. UV inactivation experiments................................................................................ 138
5.4.1 Preparation of the inoculum................................................................................ 138
5.4.2 HEV UV inactivation procedure......................................................................... 138
5.4.3 Inoculation of cultures and sample collection................................................... 139
5.4.4 Electron microscopye........................................................................................... 139
5.4.5 Sodium hypochlorite inactivation....................................................................... 140
5.4.5.1 Preparation.........................................................................................................140
5.4.5.2 Treatment...........................................................................................................140
5.4.6 RNA extraction and Real Time RT-PCR...................... 141
VII
Results............................................................................................................................ 142
5.5.1 Heat inactivation treatment................................................................................. 142
5.5.2 Homogenate of HEV positive liver exposed to UV light to test HEV
inactivation..................................................................................................................... 144
5.5.2.1 Homogenate of HEV positive liver treated for 30 min to UV light.............. 148
5.5.2.2 Electron microscopy result.............................................................................. 148
5.5.3 Inactivation of HEV positive supernatant with 5% of NaOCl........................152
5.6 Discussion............................................... 154
5.6.1 Homogenate of HEV positive liver heated at different temperatures.............. 154
5.6.2 Inactivation of HEV positive supernatant with UV light.................................. 156
5.6.3 Inactivation of HEV positive supernatant with 5% of NaOCl.......................... 159
CHAPTER 6 Prevalence and transmission of hepatitis E virus in domestic swine population in different European countries..................................................................162
6.1 Pig dynamics of transmission modeling study...................................................... 163
6.1.1 Introduction...........................................................................................................164
Materials and methods.................................................................................................. 167
6.2 Samplings.................................................................................................................167
6.3 RNA extraction and RT-PCR procedures..............................................................168
6.3.1 UK 2007 and 2008.............................................................................................. 168
6.3.2The Netherlands, Portugal, Italy, Spain and Czech Republic............................ 168
6.3.3 HEV transmission modelling.............................................................................. 169
Results.............................................................................................................................171
6.4 Discussion............................................................................................................. 175
CHAPTER 7 Overall discussion...................................................................................178
References...................................................................................................................... 187
Appendix........................................................................................................................200
Appendix A: Attempted construction of an interferon Knock-out cells line...........202
Appendix B: Multicenter collaborative trial evaluation of a method for detection of
human adenovirus in berry fruit................................................................................... 209
Appendix B.l: Transmission dynamics of hepatitis E virus in pigs: Estimation from
field data and effect of vaccination.............................................................................. 216
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Appendix B.2: Prevalence and transmission of hepatitis E virus in domestic swine
populations in different European countries............................................................... 223
Appendix C.l: VITAL SOP 001: Sampling and virus concentration from faeces..236
Appendix C.2: VITAL SOP 002, sampling and virus concentration from harvester's
hands.............................................................................................................................. 240
Appendix C.3 VITAL SOP 005, sampling and virus concentration from soft fruit
.........................................................................................................................................244
Appendix C.4: VITAL SOP 009, sampling and virus concentration from pork meat
and liver tissue.............................................................................................................. 250
Appendix C.5: VITAL SOP 010, nucleic acids extraction from faeces.................. 253
Appendix C.6: VITAL SOP Oil, nucleic acids extraction from pork meat and liver
tissue.............................................................................................................................. 257
Appendix C.7: VITAL SOP 012, nucleic acids extraction from soft fruit..............260
Appendix C.8: VITAL SOP: 013, nucleic acids extraction from harvester's hands..
.........................................................................................................................................263
Appendix C.9: VITAL SOP 014: general protocol for the quantification of
adenovirus by real time PCR....................................................................................... 266
Appendix C.IO: VITAL SOP 015, detection and quantification of porcine
adenovirus by real time PCR....................................................................................... 271
Appendix C .ll: VITAL SOP 020, detection and quantification of hepatitis E virus
by real time RT-PC R ...................................................................................................277
Appendix C.12: VITAL SOP 021 detection and quantification of murine norivirus
by real time RT-PCR....................................................................... 283
Appendix C.13: virus detection by RT-PCR: details on quality controls, virus
detection and quantification..........................................................................................289
Appendix C.14: VITAL SOP 023, protocol for the establishment of lAC
incorporation..................................................................................................................294
List of publication, training courses and conferences.................................................297
IX
ABBREVIATION TABLE
Abbreviation DefinitionATCC American Type Culture CollectionALT Alanine aminotransferaseCLD Chronic liver diseaseCSF Cerebrospinal fluidCt Cycle thresholdDpi Day post infectionELISA Enzyme-linked immunosorbent assayET-NANBH Enterically non-A non-B HepatitisODD or GAD Glycine Aspartate-AspartateHACCP Hazard Analysis and Critical Control Point
HAY Hepatitis A virusHCV Hepatitis C virusHEV Hepatitis E virusHPA Health Protection AgencyHAdV Human AdenoviruslAC Internal assay controlMC Monte Carlo modelMoNV Murine NorovirusNa2S203 Sodium thiosulphateNaOCl Sodium hypochloriteNsp Nonstructural proteinNTC No template controlOIE Organisation for animal healthORE Open reading framePaDV Porcine AdenovirusPBS Phosphate Buffered SalinePEG Polyethylene glicolPLC/PRF/5 Hepatocarcinoma cell lineRdRp RNA-dependent RNA polymeraseRT-PCR Reverse transcriptase- PCRRWV Rotating Wall VesselSIR Susceptible, infectious, recover (model)SOP Standard operating procedureSPC Sample process controlSTATl Signal transducer and activator of transcription 1UNG Uracil N-glycosylaseUSDA United States Department of Agriculture
X
UTR Untranslated regionUV light Ultraviolet lightVITAL Integrated Monitoring and Control of Food borne
Viruses in European Food Supply Chain
XI
PageFigures /Tables description number
Figure 1.1: Geographical distribution of human HEV disease pattern and human HEV isolates Page 5
Table 1.2: Differences in epidemiological and clinical featuresassociated with hepatitis E in disease-endemic and non-endemic region Page 10
Figure 1.3: Genome organization and proteins of HEV Page 11
Figure 1.4: role of the ORE 3 protein in HEV pathogenesis Page 19
Figure 1. 5: Proposed replication cycle of HEV Page 22
Figurel.6: Phylogenetic tree based on complete genomic sequences of selected human and swine hepatitis E virus Page 30
Figure 1.7: A phylogenetic tree based on the complete genomicsequences of 30 human, swine, and avian HEV strains Page 33
Table 1.8 Risk factors for asymptomatic hepatitis E virus infection in a random sample of Mornay population, Darfur, Sudan, September 2004 Page 36
Figure 1.9: Epidemic region, Kashmir, 1978 Page 37
Figure 1.10: Rotating Wall Vessel motor Page 52
Table 2.1: Graphic representation of pFBV2 containing the sequence ofthe synthetic DNA Page 70
Table 2.2: Adenovirus oligonucleotides Page 71
Table 2.3: Mumine norovirus oligonucleotides Page 71
Table 2.4: lAC constructions Page 72
Table 2.5: Results of analysis of raspberry artificially contaminated with HIGH titre of Human Adenovirus Page 75
Table 2.6: Results of analysis of raspberry artificially contaminated with LOW titre of Human Adenovirus Page 75
Table 2.7: Results of analysis of raspberry non artificially contaminatedwith Human Adenovirus Page 76
Table 2.8: Percentage of concordance for raspberry sample Page 76
XII
Table 2.9: Results of analysis of liver artificially contaminated with Page 78HIGH titre of Human Adenovirus
Table 2.10: Results of analysis of liver artificially contaminated with LOW titre of Human Adenovirus Page 78
Table 2.11: Results of analysis of liver non artificially contaminated with Human Adenovirus Page 79
Table 2.12: Percentage of concordance for liver samples between results provided at AHVLA and by the ring trial leader Page 79
Table 3.1: Source of surface swab samples Page 90
Figure 3.2 Graphic representation of pCR2. ITOPO-rSTD Page 91
Table 3.3: Number of samples PAdV, HEV and HAdV positive Page 94
Table 4.1: GTSF-2 complex medium Page 110
Table 4.2: Copy numbers of HEV genome detected in the 3D culture and2D cells Page 114
Figure 4.3: Comparison of Ct values in the 3 cell culture systems Page 115
Figure 4.4: copy number per ml detected in the 3D cells culture systemof the serial dilution Page 116
Table 4.5: HEV RNA detected by real time RT-PCR in supernatant ofHEV positive cells infected with French figatelli Page 118
Figure 4.6: supernatant of cells infected with UK sausages tested by realtime RT-PCR Page 119
Figure 4.7: HEV-like particle in HEV positive supernatant in figatelli sample Page 120
Figure 5.1: Treatment of HEV infected liver at 100° C leads to inactivation of the virus Page 139
Figure 5.2: Analysis of the Variation overtime the UV light inactivation experiment in the supernatant of the 3D cell cultures Page 142
Figure 5.3: HEV decay measured in the inoculum by real time RT-PCRafter the UV light treatment Page 143
Figure 5.4: analysis of CT values of supernatant of 3D cells infectedwith inoculum treated with UV light for 30 min. Page 146
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Figure 5.5: HEV-like particle Page 147
Figure 5.6: analysis of Ct values of HEV positive supernatant treatedwith NaOCl and untreated Page 149
Figure 6.1: HEV swine prevalence in six different EU countries. HEV prevalence plotted for six countries and 5 pig age groups Page 169
Table 6.2: Transmission rate parameter, average of infectious period and reproductive number. Page 170
XIV
Abstract
Hepatitis E is an acute hepatitis in humans, first recognised in 1980 and caused by
hepatitis E virus (HEV). The principal mode of spread of HEV is faecal-oral from
contaminated water supplies, almost exclusively in developing regions.
Accumulating evidence indicates that HEV transmission may be zoonotic in
developed regions from swine and perhaps other animal species serving as
reservoirs for the virus. The exact transmission routes are unclear, largely because
HEV is extremely difficult to propagate in vitro, but retail pig products have been
shown to contain HEV RNA.
This PhD project was part of the EU FP7 project VITAL (Integrated Monitoring
and Control of Food borne Viruses in European Food Supply Chains). The main
aim of this PhD project was to investigate the presence and residual infectivity of
HEV in the pork food chain. This helped to assess the potential importance of the
pig and its products in zoonotic transmission of HEV. A cell culture system for
HEV was further optimised for HEV detection in food samples.
A productive HEV infection was established in 3D cell culture (Alexander
hepatoma PLC/PRF/5) that was permissive for HEV replication. Furthermore, a
trial to compare the efficiency of 3D, 2D and 3D transferred to 2D cells culture
systems was performed indicating that replication in the 3D cell culture system was
the most efficient. In addition, these studies showed that cells grown in 3D and then
transferred to 2D for infection were able to support HEV replication. Further
refinements such as heat, UV light and sodium hypochlorite inactivation studies
were performed. These approaches should enable an assessment of the significance
of the pork food chain in transmission of HEV and facilitate the development of
control measures.
Within the VITAL project standard methods were developed to have common viral
detection and extraction methods between all laboratories, and ring trials were
organized between 15 EU laboratories to assess the efficacy of the Standard
Operating Procedures (SOPs) developed. Since all the laboratories involved were
able to detect the viruses with the common SOPs the ring trial was considered
successful and the second step of the project began, involving the screening by real
time RT-PCR for HEV throughout the pork food chain. One of 40 pig livers and 6
of 63 pork sausages were found to be HEV positive. Virus viability was tested using
the 3D cell culture system but no evidence of viral replication was detected. A
mathematical model suggested that the circulation of HEV in six European
countries is endemic. In addition, HEV prevalence in pig’s faeces was investigated
showing that pigs close to the slaughter age can still be HEV positive.
In conclusion, the work carried out in this PhD projected contributed to our
understanding of HEV replication in-vitro and provided useful information on the
prevalence of HEV in the pork food chain in the UK. In addition, progress was
made with possible inactivation methods and control strategies.
CHAPTER 1 General Introduction
1 Food-borne viruses
Foodborne viruses are a common and, probably, the most under-recognised cause of
outbreaks for example of gastroenteritis. Human infection can occur following
consumption of contaminated food, person-to-person body contact, or release of
aerosols. Food may be contaminated by infected food handlers or by contact with
water contaminated by treated or untreated sewage. Outbreaks of viral foodborne
illness have been associated with the consumption of shellfish that have been
harvested from sewage-polluted waters, for example. The greatest risk of foodborne
illness occurs with catering operations preparing ready to eat foods, although
foodborne spread is difficult to prove. The most common food borne vim ses are
Norovims and hepatitis A vims. Vimses require a host in order to multiply, and the
original source of all foodborne vimses is the human intestine. Usually, they cannot
grow in food. Contamination of food may occur either during preparation and
serving by infected food handlers or by contact with sewage or sewage-polluted
water.
Pathogenic vimses originate from two sources to contaminate the food chain:
humans and animals. To facilitate identification of whether contamination is of
human or zoonotic origin, monitoring the presence of human and animal vimses at
various points in the food supply chains is still necessary. Adenovimses infect both
humans and a wide variety of animal species, are shed in large numbers in the faeces
of infected individuals [4], and are capable of robust survival [5]. They have been
proposed as an index of viral contamination, and the specific detection of
adenovimses from human or animal origin should be a useful tool for tracing the
source of faecal viral contamination. Hundesa et al. (2006) stated that due to higher
prevalence in fecal and environmental samples of bovine adenoviruses, bovine
polyomaviruses are the best candidates for tracing a bovine source of viral
contamination [6]. As well as the index viruses, the presence of HEV in pork
production is necessary to be examined since that HEV is regarded as a model
zoonotic virus.
1.1 Hepatitis E virus aetiology
HEV is a hepatotropic virus and the causative agent of hepatitis E, an acute viral
hepatitis in humans. The infection may vary in severity from inapparent infection to
fulminant liver failure and death. Although considered an acute disease, chronic
infections have been observed in liver and kidney transplant and chronic liver
disease (CLD) patients. The mortality rate is between 1% and 4% [7], (higher than
hepatitis A virus - HAV, a Picomavirus) and in people with CLD and in pregnant
women it can reach 25-30%.
Hepatitis E is an important public health concern and a major contributor to
enterically transmitted hepatitis worldwide {Figure 1.1) [8]. Based on
seroprevalence data, an estimated one third of the world’s population has been
infected with HEV [7].
In endemic regions, hepatitis E occurs in epidemic forms meanwhile in developed
regions HEV occurs sporadically {Figure 1.1).
Hepatitis E is the second most important cause of acute clinical hepatitis in adults
throughout Asia, Africa and the Middle East where the infection is endemic. In
these countries, the infection mainly spreads through the contamination of water
supplies occasionally leading to large-scale outbreaks or epidemics. Hepatitis E is
rare in industrialized countries, where infection is historically mostly related to
travelling to endemic areas. However, more recently, significant numbers of
autochthonous cases have been documented in many developed countries [9].
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1.1.1 Hepatitis E in non-endemic regions
In developed regions, the transmission of HEV is most likely mainly via a zoonotic
route. Evidence of this is given by the many autochthonous (indigenously acquired)
eases worldwide where swine isolates show a very high RNA sequence homology
to human HEV isolates [10]. Additional evidence is the experimental transmission
of human isolates to pigs and of swine HEV to primates [10]. Hepatitis E
autochthonous transmission has been recorded in most developed countries and
regions including USA, Europe (including UK, France, the Netherlands, Austria,
Spain, Greece and Italy), and developed countries of Asia-Pacific (Japan, Taiwan,
Hong Kong, Australia) [11].
In the UK, the disease appears to be more common among residents of coastal and
estuarine areas [11]. Zoonotic transmission has been proposed [11] and is now
widely accepted; in some developed regions transmission appears to be seasonal
with peaks in spring and summer [11].
Patients with unexplained hepatitis are tested by serological tests and these are the
cases where the disease is most often recognized. Generally, the symptoms are
similar to those in endemic regions. In developed areas, the majority of the cases
have been in middle aged or elderly men, where often another disease already
coexisted [12] {Table 1.2).
1.1.2 Hepatitis E in disease-endemic regions
Although the majority of hepatitis E eases in resource limited countries are
sporadic, local epidemic outbreaks occur frequently. They are usually separated by
a few years and they can affect several thousand individuals [13, 14]. Usually the
8
majority of the outbreaks are due to consumption of drinking water contaminated by
human faeces and the longevity varies from a few weeks to over a year [14]. The
outbreaks frequently follow heavy rainfall and floods [15], conflict leading to
concentrations of displaced persons in refugee camps [15], or are associated with
disposal of human excreta into rivers [15]. Food-borne transmissions have been
described in resource-limited areas, but due to a relatively long incubation period
(up to 9 weeks), establishing a correlation between consumption of pork food and
occurrence of disease is difficult.
In India HEV is hyper-endemie, the majority of the cases reported are sporadic and
40% of sewage specimens obtained throughout all seasons are HEV positive [2, 16-
19]. Interfamilial spread is not common but multiple cases in one family have been
reported [2, 16-19]. It is suggested that this is due to shared infected water rather
than person-to-person transmission as the time interval between cases is too short.
Studies in endemic regions show high seroprevalence rates ranging from 15% to
60% [16, 20-24]. Notably, the age-specifie seroprevalence profiles for HEV are
found to differ from those reported for antibody to HAV even though, in endemic
countries, the transmission routes for these two viruses are similar [16]. HAV
seroprevalence rates reach more than 95% in children by the age of 10 years
whereas HEV infection is rarely detected in children [16].
The peak incidence in sporadic cases of hepatitis E in endemic regions occurs in
15-35-year-olds [2, 16-19]. Additionally, HEV infections are predominantly
reported in men with a male-to-female ratio ranging from 1/1 to 3/1 [25]. This sex
bias is, however, not seen in children presenting with hepatitis E [26]. The reason
why men more commonly develop hepatitis E infection is not understood but males
outnumbering females may be due to a greater risk of exposure to HEV infection
[26]. Morbidity rates during hepatitis E epidemics have ranged from 1% to 15%
[27]. Higher mortality rates and fulminant liver disease have been described among
pregnant women during hepatitis E outbreaks [27]. Furthermore, HEV infection
during pregnancy is not only associated with severe disease or higher mortality, but
also with an increased risk of prenatal mortality and low birth weight. In developing
regions neonatal vertical transmission rates have been estimated at 78.9% [28] but it
is yet unclear whether the high morbidity and mortality rates during pregnancy are
also seen in developed regions {Table 1.2). The exact cause for this predilection to
severe disease in pregnant women still needs to be better studied, including the
suspicion that it is due to hormonal or immunological factors [29].
1.2 Morphology and Genomic organization
HEV was designated in 2004 as the sole member of the genus Hepevirus in the
family Hepeviridae [30]. The HEV genome was first cloned from cDNA libraries
prepared from the bile of macaques experimentally inoculated with stool
suspensions from human patients [31]. A similar PCR was later used to clone the
genomes of multiple geographically distinct isolates of HEV [32-34].
HEV is a small, non-enveloped, single-stranded, positive-sense RNA virus. The
genome size is approximately 7.2 Kb [35, 36] {Figure 1.3). The genome of HEV is
capped at the 5' end and polyadenylated at the 3' end (Figure 1.3.A). It contains
short stretches of untranslated regions (UTR) at both ends {Figure 1.3.B, red box).
The HEV genome has three open reading frames (ORFs), shown in Figure 1.3B.
ORFl encodes the non structural polyprotein (nsp) that contains various functional
units: methyltransferase (MeT), papain-like cysteine protease (PCP), RNA helicase
1 0
(Hel) and RNA dependent RNA polymerase (RdRp) [3]. 0RF2 encodes the viral
capsid protein, the N-terminal signal sequence and glycosylation loci. ORF3
encodes a small regulatory phosphoprotein. Details of the 0RF3 protein are shown
in Figure 1.3. The roles of the 0RF3 protein in HEV pathogenesis are promotion of
cell survival, modulation of the acute phase response and immunosuppression [3].
1 1
Featm^ Eitdeinic regions NcMii-eiuleimc regions
Geographical locations Underdeveloped countries mostly in Asia and A&ica
Developed countries in Europe North America, parts of Asia, Australia,
Epidemiologicalpatterns
Large epidemics, small outbreaks and ^oradic cases
Only sporadic cases with occasional small clusters
Water-bornetransmission
Well known ,most common route Unknown, but has been detected and may be contributory
Zoonotic transmission Not reported Yes
Animal reservoir No Yes
Vims genotype Almost entirely genotypes 1 and 2, a few cases of genotype 4 in China
Genotype 3; occasional cases of genotype 4 in Taiwan
Age group Young men most commonly affected
Usually dderly
Chronic infection Not known Reported in tran^lant recipients receiving immunosuppressive dmgs
Severity Variable severity, including fulminant hepatic 6ilure
Severity and p oor outcome is related to coexistent disease conditions
Relationship with pregnancy
Particularly high rates of symptomatic disease and of more severe disease in pregnant women than in men and non-pregpant women
No data on pregnant women, but eady evidence indicates 1 ower mortality/morbidity in developed regions
Table 1.2 Differences in epidemiological and clinical features associated with hepatitis E in disease-endemic and non-endemic regions. The first column describes the HEV features, the second and third column describe the features in endemic and non endemic regions. Table adapted from Aggarwal et al, 2010 [1].
1 2
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1.3 Viral proteins
Open reading frame one (ORFl) is the largest (5079 nt) of the three ORFs and it
begins after the 5’ noncoding region (5'-NCR) of 27 to 35 nucleotides (nt). It
encodes a 1693 aminoacid polyprotein including viral non-structural proteins such
as methyltransferase, a papain-like cysteine protease, a helicase and an RNA-
dependent RNA Polymerase (RdRp) [37-41].
The region between the end of ORFl and start of ORF3/ORF2 appears to be
complex and contains regulatory elements [35] {see sections 1.3.5 and 1.3.6).
1.3.1 Methyltransferase
The Methyltransferase domain has been suggested by computer-assisted
assignments to encompass an amino terminal domain between 60 to 240
aminoacids. Downstream of the methyltransferase domain there is a Y domain of
200 aminoacids but at present no particular function is known. While the HEV
methyltransferase showed guanine-7-methyltransferase and guanyltransferase
activities [41, 42], the source of the RNA triphosphatase was not clear but it seems
that the RNA triphosphatases specifically cleave 5’-phosphate of the nascent
mRNAs, without attacking the P-phosphoryl group. The RNA triphosphatases from
RNA viruses are helicases or helicase-like proteins where the active site of the RNA
triphosphatase is shared or overlaps with the helicase/ NTPase catalytic site. This
suggested that the HEV helicase has RNA 5’-triphosphatase (RTPase) activity.
A recent report [43], suggested that when a purified recombinant HEV helicase
protein was incubated with either alpha-3 2p_ labelled RNA or gamma-3 2p_
labelled RNA, the HEV helicase had a gamma-phosphatase activity, which might
catalyze the first step in RNA cap formation. Two reports have shown the presence
14
of a 5’m 7G cap on the HEV genomic RNA. The HEV genomic RNA transcribed in
vitro from viral cDNA is infectious for primates only when it is capped [44]. A 5’
RNA ligase-mediated rapid amplification of cDNA ends (RACE) method designed
to select capped RNAs amplified the 5’ ends of the SAR-55 (genotype 1) and MEX-
14 (genotype 2) mRNA, confirming that the HEV genomic RNA is capped [44, 45].
1.3.2 Papain-like cysteine protease
A Papain-like protease domain follows the Y domain {Section 1.3.1) encompassing
440-610 aminoacids, and has been identified in other viruses such as alphavirus,
rubella virus and hepatitis C virus (HCV). It is postulated that this viral protease is
involved in either co- or post-translational viral polyprotein processing to yield
discrete non-structural protein products [42]. A conserved “X domain” of unknown
function flanks the papain-like protease domain, preceded by a proline-rich region
“P” that might constitute a flexible hinge between the X domain and the upstream
domains [37].
1.3.3 Helicase
The Helicase domain is similar to the typical Helicase superfamily and shows the
highest overall homology with the helicase of beet necrotic yellow vein virus
(>10%). It promotes unwinding of DNA, RNA or DNA duplexes required for
genome replication, recombination, repair and transcription [42].
1.3.4 RNA-dependent RNA polymerase (RdRp)
The RdRp domain, encompassing 1200-1700 aminoacids of the carboxy terminal
region of ORFl, shows a conserved amino acid motif recognised in all positive
strand RNA viruses as the canonical Glycine-Aspartate-Aspartate (GDD) motif. It
15
has been observed that mutations in this motif (GDD to GAD) generate replication-
deficient HEV viruses unable to replicate. The RdRp has a crucial role in binding to
the 3’UTR (untranslated region) of HEV RNA and directing the synthesis of the
complementary strand RNA [42]. Several linear B-cell epitopes have been
identified in the ORFl protein, and appear to be particularly concentrated in the
region of the RdRp [46].
1.3.5 ORF2 and the major capsid protein
Open reading frame 2 is about 1980 nt in length from nt 5147 to nt 7124,
downstream of ORFl. Translation of this region produces the HEV structural
polypeptide (pORF2) of 660/599 aminoacids [47] and this appears to be highly
conserved. The 5’ end of 0RF2 region presents an average of approximately 350-
450 nt most conserved among HEV isolates; recently it has been used for
classifying different subgenotypes of HEV [48].
In animal cells, the major capsid protein is expressed in a -74 KDa form (pORF2)
and a -88 KDa glycosylated form (gpORF2) that was immunoreactive with sera
from chimpanzees infected with HEV [49]. pORF2 is synthesized as an 82 KDa
precursor (ppORF2) that co-translationally translocates via the N-terminal signal
sequence to the endoplasmic reticulum (ER) membrane. The putative signal
peptides consist of three regions: an amino terminal region of 22 amino acids with
positively charged residues (Arg), a central hydrophobic core with 14 residues and a
third region containing a turn-inducing stretch of proline residues, followed by the
signal peptidase cleavage site. Processing of ppORF2 is by cleavage in the
endoplasmic reticulum into the mature polypeptide (pORF2), and then it is
glycosylated (gpORF2) at N-linked glycosylation sites “Asn-X-Ser/Thr” (N-X-S-T)
16
at residues 137, 310 (these appear to be the major sites of N-Glycan addition) and
561, attached as a core unit of oligosaccharides (Glc3Man9Glc-NAc2), while the
polypeptide chains are translocated across the ER membrane [50]. This process
occurs usually for the synthesis of envelope proteins. The glycosylation sites are
conserved in the 0RF2 sequences of all HEV isolates sequenced [32, 35, 51, 52].
Mutations in the pORF2 glycosylation sites prevented the formation of infectious
virus particles and resulted in low infectivity in macaques [53]. The 88 KDa
gpORF2 obtained is transported to the cell surface by a bulk flow mechanism in the
absence of any signal of retention in the endoplasmic reticulum. Final assembly
occurs at the cytoplasmic membrane with encapsidation of HEV positive-stranded
genomic RNA.
Expression of gpORF2 in mammalian cells (COS-1 and HepG2) showed that it is
expressed intracellularly, as well as on the cell surface, and has the potential to form
non-covalent homodimers [42, 49, 50, 54]. Recently, it has been suggested that
gpORF2 is an unstable form of the protein [55]. Although pORF2 is proposed to
take part in capsid assembly, the role of gpORF2 is not clear, being possibly
involved in apoptotic signalling [49].
ORF-2 has been expressed in vitro and characterized by heterologous expression
systems including Escherichia coli [56], mammalian cells using plasmids [49],
alphavirus vectors [55, 57], baculovirus expression systems [58], recombinant
vaccinia virus [59] and yeast [60]. The full length 0RF2 product expressed in insect
cells is insoluble, whereas the truncated products, mapping to aminoacids 112-660,
assemble into virus-like particles (VLP), indicating that cleavage and assembly of
the capsid protein occurs in the system [61-64]. The size of empty VLPs (23.7nm) is
17
smaller than the authentic native HEV virions (27nm) and similar virus particles
have not been found in the bile or stools from patients infected with hepatitis E or
from experimentally infected monkeys. Expressed VLPs were used as antigen for
enzyme-linked immunosorbent assay (ELISA) against antibodies to HEV,
appearing to be specific and sensitive enough to detect anti-HEV IgG as well as
IgM in human and experimentally infected monkey sera [65, 66]. Immunodominant
epitopes in ORF2 and 0RF3 have been included in commercially available
diagnostic ELIS As for HEV [67]. The 0RF2 epitopes are located at the extreme 3’
end of that reading frame [67]. The antibody response to pORF2 shows that it is
highly immunogenic and protective [7]. Currently, a single serotype has been
described, with extensive cross-reactivity among circulating human and swine and
chicken strains [47, 68].
To support the hypothesis that ORF2 is essential for the generation of infectious
virions, Parvez et al (2011) [69] constructed a recombinant baculovirus
(vBacORF2) that expressed the full-length 0RF2 capsid protein of a genotype (gt) 1
strain of HEV. Results showed that the baculovirus-expressed ORF2 protein was
able to transencapsidate the viral replicon and form a particle that could infect naïve
HepG2/C3A cells. Parvez et al (2011) [69] confirmed the results obtained by Xing
et al [70] that HEV virus-like particles formed in insect cells captured some of the
template 0RF2 RNA used to produce the particles. In conclusion it is strongly
considered that the 0RF2 protein transcomplements a replicon that is deficient in
capsid protein production and efficiently encapsidates the replicon viral RNA to
form stable HEV particles which are infectious for naïve hepatoma cells [69]. This
1 8
ex vivo RNA packaging-system could be further used to study many aspects of HEV
molecular biology [69].
1.3.6 ORF3 and its product
Open reading frame 3 (ORF3) partially overlaps with ORFl by 4 nt and shares most
of the remaining nucleotides of 0RF2 at the 5’ end [42]. 0RF3 encodes for a
123/122 amino acid immunogenic phosphoprotein of 13.5 KDa (pORF3) with yet, a
not fully defined function [35].
Recent studies using a HEV replicon with a deleted ORF3 in cell culture showed
normal RNA replication, suggesting that 0RF3 is not required for HEV replication,
virion assembly or infection of culture cells [71].
Yamada et al provided evidence that pORF3 is required for virion egress from
infected cells [72]. In addition, pORF3 is present on the surface of HEV particles
suggesting that the HEV particles released from infected cells are lipid-associated.
In its primary sequence, pORF3 contains two large hydrophobic domains in its N-
terminus that are rich in polycysteine [72]. Domain 1 may serve as a cytoskeleton
anchor at which pORF2 can assemble the viral nucleocapsid, although it was
reported that recombinant 0RF2 protein assembled into small but typical
icosahedrons in the total absence of ORF3 [73, 74] and also bound mitogen-
activated protein kinase phosphatase (MAPKP) [75]. Another smaller hydrophobic
domain (Domain 2) follows in the primary sequence, which has been shown to
homo-dimerize in a yeast cellular environment, and in human hepatoma cells it was
demonstrated to interact with another host protein endogenous hemopexin (Hpx), an
acute-phase plasma glycoprotein that plays important roles in inflammation. The
19
pORF3-Hpx interactions may have significant importance in viral pathogenesis
(Figure 1.4) [76].
Chandra et al [77] described studies that suggested that the 0RF3 blocks phospho-
STAT3 nuclear transport (Figure 1.4). A block in receptor mediated endocytosis
inhibits the nuclear transport of STAT3 [77]. It is known that STAT3 is involved in
the acute response and activation of acute phase proteins and it regulates the
transcription of a number of acute phase genes such as interleukin-6 (IL-6) [77].
The acute phase proteins (APPs) are expressed mainly by the liver and have a wide
range of activities that contribute to host defence. The main role of APPs is
neutralizing inflammatory agents and minimizing the extent of local tissue damage,
as well as participate in tissue repair and regeneration [77]. In conclusion, Chandra
at al. suggested that ORF3 could attenuate inflammatory responses and create an
environment for increased viral replication and survival mainly in the liver [77].
20
Receptor Tyrosine Kinase
Endocytosis
PSTAT3
(A)Promotion of cell survival m dm
I.KlWWlI
OIll-microglobulin
Numus
(B )Modulation of
acute phase response
ïicreasediil-microglobuBn secretion
(Cl knm unosuppreg ion
Figure 1.4 Role of the ORF3 protein in HEV pathogenesis. (A) Promotion o f cell survival. The ORF3 protein activates MAP kinase by binding and inactivating its cognate phosphatase (MKP). Additionally, it upregulates and promotes homooligomerization of the outer mitochondrial membrane porin, VDAC, and increases hexokinase levels, thus reducing mitochondrial depolarization and inhibiting intrinsic cell death. (B) Modulation o f the acute phase response. The ORF3 protein localizes to early and recycling endosomes, and inhibits the movement of activated growth factor receptors to late endosomes. This prolongs endomembrane growth factor signaling and contributes to cell survival. Through this mechanism, pORF3 also reduces the nuclear transport of pSTAT3, a critical transcription factor for the expression of acute phase response genes. (C) Immunosuppression. The ORF3 protein promotes the secretion of a 1-microglobulin, an immunosuppressive protein that could act in the immediate vicinity of the infected cell. Figure taken form Chandra et al 2008 [3].
21
1.4 The HEV replication cycle
1.4.1 Viral receptor and entry: The cell surface molecules that bind HEV or its
capsid proteins are not known yet. He et al (2008) described that a truncated peptide
of 0RF2 is involved in binding and entry of the following cell lines: HepG2, Huh-7,
PLC/PRF5 and A549 cells [78].
1.4.2 Model of HEV replication: The process by which HEV RNA enters the
target cells is still unknown (Figure 1.5:1-2). In the cytoplasm the genomic RNA is
translated into non-structural proteins (Figure 1.5: 3). The genome amplification
step involves replication of positive strand genomic RNA into negative strand RNA
intermediates (Figure 1.5: 4A). These are used as template for the synthesis of the
genomic positive strands (Figure 1.5: 4B). This is akin to alphaviruses and a region
homologous to alphavirus junction sequences is proposed to serve as the
subgenomic promoter. The subgenomic RNA can then be translated into the
structural protein(s) (Figure 1.5: 5). Based on in vitro expression and replicon
studies, some details have now begun to emerge. The genomic RNA is packaged
with the capsid protein to assemble new virions (Figure 1.5: 6). The mechanism by
which the virion is released from the cell has yet to be characterized [3].
It is unclear whether gut cells are infected following ingestion of the virus. It is
believed that the primary site of HEV replication is the liver, with hepatocytes being
the most likely cell type [79]. Results support infection and replication in non-
hepatic cell types such as A549 lung carcinoma cells and in Caco-2 colon
carcinoma cells. Although it is not efficient, viral replication has been demonstrated.
In pigs experimentally infected with swine HEV, positive-sense viral RNA was
detected in almost all tissues at some point during the infection, but negative-sense
22
RNA intermediates were detected primarily in the small intestine, lymph node,
colon and liver [79]. In a recent report, HEV RNA was detected in peripheral blood
mononuclear cells, but due to the lack of an efficient HEV in vitro cell culture
verifying the evidence of viral replication in this compartment in patients with HEV
infection was not possible [80].
23
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1.5 Potential targets for the development of antiviral drugs
Various steps in the HEV life cycle can be potential targets for the development of
antiviral drugs. The methyltransferase and guanyltransferase activities in the ORFl
protein {Section 1.3) are strictly virus-specific and thus good targets for antiviral
development [41]. The RNA helicase of HEV has been biochemically characterized
and it is essential for replication of the viral RNA genome [43], but it is not clear
how distinct it is from human helicases to be a potential drug target. The HEV
RdRp expressed in E. coli was shown to bind the 3’ end of the viral RNA genome
[81], but its biochemical activity has so far not been characterized. Since the RdRp
is unique to RNA viruses, it would again be a good drug target, and perhaps some
viral inhibitors can be explored against this target, for example the RdRp is used as
inhibitor of viral replication for HCV infections. Interference with HEV RNA
replication has been attempted using ribozymes and small interfering RNAs. Mono-
and di- hammerhead ribozymes designed against the 3‘ end of the HEV genomic
RNA were shown to inhibit expression from a reporter construct in HepG2 cells
[82]. In A549 cells infected with HEV, small interfering RNAs (siRNAs) against
the ORF2 region were also shown to offer protection [83]. While such approaches
are feasible in vitro, the delivery and targeting of such inhibitors in vivo would be
the real challenge. At least one study in immunocompromised transplant patients
with chronic HEV infection has also shown the efficacy of Ribavirin monotherapy
[84]. Again, the utility of this approach among the vast majority of HEV infections
that are acute remains questionable.
25
1.6 HEV inactivation studies
HEV has proven difficult to propagate in vitro [85], and despite some recent
improvements, there is no doubt that the failure to develop a repeatable and efficient
in-vitro propagation system for HEV has hindered attempts to understand the
environmental survival and other physical and pathobiological characteristics of
HEV. The determination of these qualities would potentially offer much valuable
information in understanding the epidemiology and control of HEV infections.
Feagins et al in 2008 [85] performed a HEV heat inactivation study in a pig animal
model. The objective of the study was to determine if traditional cooking methods
are effective in inactivating infectious HEV present in contaminated commercial pig
livers. The result obtained was that four of the five pigs inoculated with a pool of
two HEV-positive liver homogenates incubated at 56°C [86] for 1 h developed an
active HEV infection shedding virus in the faeces. The pigs inoculated with a
pooled homogenate of two HEV-positive livers stir-fried at 191°C [86] for 5
minutes and the group of pigs inoculated with a pooled homogenate of two HEV-
positive livers boiled in water for 5 minutes showed no evidence of infection as
there was no seroconversion, viraemia, or faecal virus shedding in any of the
inoculated pigs [87].
What is not clear is how effective the usual processing procedures for uncooked pig
products are in inactivating pathogens such as HEV. Moreover, the risk of HEV
infection via the consumption of HEV-contaminated pig tissues raises public health
concerns since it is not clear what cooking conditions will be effective in
inactivating the virus present in the contaminated pig tissues.
26
HEV can be found in the liver, blood, intestinal tract and skeletal muscle, all of
which are consumed in one form or another and often together, such as in sausages.
How safe are these products? The question is difficult to answer because HEV
grows poorly in cell culture, and testing HEV viability in vivo requires nonstandard
laboratory animals.
Other inactivation studies with HEV have not been performed thus far. Inactivation
studies with UV light were performed with other viruses such as HAV, calicivirus
or other enteric viruses, or with bacteria [88, 89]. Exposure to solar ultraviolet (UV)
radiation is a primary means of virus inactivation in the environment, and
germicidal (UVC) light is used to inactivate viruses in hospitals and other critical
public and military environments [90, 91]. Safety and security constraints have
hindered exposing highly virulent viruses to UV and gathering the data needed to
assess the risk of environments contaminated with high-consequence viruses [92].
UV sensitivity for some viruses has been extrapolated from data obtained with UVC
(254 nm) radiation by using a model based on the type, size and strandedness of the
nucleic acid genomes of the different virus families [93, 94]. Therefore, there was
little information to allow accurate modelling, confident extrapolation, and
prediction of the UV sensitivity of viruses deposited on contaminated surfaces,
conditions more likely to be relevant to public health or biodefence. One of the
goals of this study was to determine the inactivation kinetics produced by exposure
to UV light (UV, 254 nm radiation) of HEV since that is relevant to public health
(Section 5.2).
Other inactivation studies with disinfectants such as chlorine were not performed
until now with HEV. Sodium hypochlorite, a derivate of chlorine solution.
27
commonly known as bleach, is frequently used as a disinfectant or a bleaching
agent. US Government regulations (21 CFR Part 178) allow food processing
equipment and food contact surfaces to be sanitized with solutions containing
bleach, provided that the solutions do not exceed 200 parts per million (ppm)
available chlorine. A l-in-5 dilution of household bleach with water is effective
against many bacteria and viruses {Section 5.2).
1.7 Taxonomy: Evolutionary History and Population Dynamics of Hepatitis E
Virus
HEV segregates as four genotypes and the characterization is based on the genomic
sequence analysis of human and animal isolates [95, 96]. A genetically distinct
group has also been identified in avian samples, sharing 50% homology with
mammalian isolates [94].
Genotypes 1 and 2 appear to be anthroponotic whereas gts 3 and 4 are zoonotic
[97]. All four genotypes belong to a single serotype [30]. The recent discovery of
novel lineages of HEV in rabbits [98, 99], rats [100], and wild boar [101] has
expanded further the mammalian HEV diversity. It has been suggested that the
HEV sequences found in rabbits represent a novel genotype [102, 103]. However,
additional phylogenetic analysis indicated that rabbit HEV is closest to gt 3 [100,
104] and may have zoonotic potential. In addition, the discovery of a genetically
distinct avian HEV [105] indicates a very long evolutionary history for the HEV
group of viruses. Contrary to swine HEV (asymptomatic in pigs), avian HEV shows
hepatomegaly in poultry.
The first animal strain of HEV was detected in swine (swine HEV) in 1997 in the
USA [52]. Since then, swine HEV strains have been isolated from all over the world
2 8
and from several animal species (e.g. wild boar, mongoose and sika deer). In
developed regions the human and swine strains show a sympatric distribution [106].
Purdy et al [107] suggested that HEV can be segregated into two clades. One clade
is the enterically transmitted, epidemic form represented by gts 1 and 2, and the
other clade is the zoonotically transmitted, sporadic form exemplified by gts 3 and 4
[9, 97, 108].
Genotypes 1 and 2 have been identified only in humans, gts 3 and 4 have been
identified both in humans and in animals [42, 47, 52, 109]. Gt 1 HEV has been
identified from human cases in Asia and Africa [48] whilst gt 2 was firstly
identified in Mexico and subsequently in Africa. Gt 3 has been identified in humans
and animals in several developed countries, such as Europe, Japan, Australia and
New Zealand. Gt 4 has been identified in both animals and humans in China,
Taiwan, Japan and Vietnam and most recently in The Netherlands [110]. HEV
strains of gts 1 and 2 have less genomic variability than those of gt 3 and 4 [47].
This could be due to the differences in the transmission patterns between the
genotypes. In addition, the presence of an animal reservoir for gts 3 and 4 could
have caused an independent evolution of the virus in specific animal species [47].
That HEV has an animal origin [111] suggests that some ancestral HEV variants
could have subsequently developed the capacity to efficiently transmit to and
between humans. To prevent emergence of novel human diseases a better
understanding of epidemiological and evolutionary processes facilitating this
transition from enzootic to human-to-human transmission is necessary. The clear
division between HEV genotypes into two modes of transmission offers an
important opportunity for studying molecular evolutionary processes related to the
29
transition from one mode to another. Prudy et al [107] studied the evolutionary
history of HEV using several models estimating population dynamics, in terms of
time to the most recent common ancestor (TMRCA), and variation in selective
pressures acting on different HEV genotypes. Purdy et al [107] did not analyse
HEV gt 2 due to lack of available samples. ORF2 analysis suggests that the mean
time of emergence of the ancestor for modern HEV genotypes ranged from 536 to
1344 years ago. For gt 3, from 265 to 342 years ago; for gt 4, from 131 to 266 years
ago; and for gt 1, from 87 to 199 years ago. Thus, the anthroponotic gt 1 is the most
recent compared to the enzootic gts 3 and 4 [107].
Following Drummond et al [l\2 \, Purdy et al [107] decided to set up a model using
0RF2 sequences for gts 1, 3 and 4 to understand the genotype dynamics and to
study the demographic history of HEV genotypes. Gt 1 went through an increase in
population size between 25-35 years ago. Gt 3 population was stable since 1760,
but it had a dramatic shift in its size over the 20th century. The effective population
size of gt 4 remained constant until 20 years ago when it rapidly decreased over 10
years to the original level. [107].
Purdy et al [107] suggested that HEV has histories dating back tens of thousands to
millions of years but early members have been replaced by the modern variants
[107]. A more ancient TMRCA is suggested due to contacts between humans and
domesticated swine about 11.000 years ago [38] immediately after urbanization
started [39]. HEV gt 1 increased in the last 35 years. Gts 3 and 4 showed decreases
around 1990 [107] and this may be due to greater awareness of the HEV health
problem around the world and improved diagnostics rather than an actual expansion
of the HEV [107]. During the Second World War the increase of HEV cases was
30
probably related to the increasing population size rather than meat consumption
[111]. The country-specific HEV evolutionary history observed probably reflects
temporal variations in rates of transmission and/or exposure for HEV strains of the
same genotype circulating in different geographic regions [107].
1.8 Genotype classification
Extensive genomic diversity has been observed among HEV isolates, but a single
serotype is recognised [47, 113]. Genotype 1 was first identified and subjected to
sequencing in 1991 [35] from a sample that came from Myanmar (Burma strain)
showing more than 88% nucleotide identity with other gt 1 strains isolated in Asia
(China, India, Nepal and Pakistan) and Africa (Chad and Morocco) [47].
In 1992, a new strain which was completely different from the Burma strain was
sequenced from outbreaks in Mexico (1986) and classified as gt 2. Compared to gt
1, which is present in many geographic regions, gt 2 occurs in fewer countries [48].
Genotype 3 was identified in 1997 in the USA from an autochthonous infection in a
patient without history of travel abroad; it was sequenced and became the first strain
belonging to gt 3 [114]. Later on, gt 3 HEV was shown to be distributed in many
countries worldwide including Asia, Europe, Oceania, North and South America
[106, 115-117].
Currently, the four genotypes are classified into different subtypes, based on
approximately 300-450 nucleotides of sequence in the 5’ end of the ORF2 region
which are most conserved among all HEV isolates. The phylogenetic analysis
demonstrated that HEV can be divided into total 24 subtypes. Gt 1 was divided in 5
subtypes (la, lb, Ic, Id, le), gt 2 in two subtypes (2a, 2b), gts 3 segregate in 10
31
subtypes (3a, 3b, 3c, 3d, 3e, 3f, 3g, 3h, 3i, 3j) and gt 4 in 7 subtypes (4a, 4b, 4c, 4d,
4e, 4f and 4g) [48] (Figure 1.6) [118].
32
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1.9 Epidemiology of HEV
1.9.1 Epidemiology in humans
The epidemiology of HEV differs significantly in industrialized and non
industrialized countries. In resource-limited countries, the infection is endemic and
spreads mainly through contamination of water supplies.
Data from sero-surveys forced re-evaluation of the epidemiology of hepatitis E and
gave an indirect indication to vocationally acquired HEV infections in industrialized
countries [2].
In industrialized countries, Hepatitis E occurs sporadically and affects mainly
visitors returning from endemic areas. Some of the cases in industrialized countries
however, are non-travel-related and are considered as being autochthonous.
Autochthonous cases have been reported in N and S America, many European
countries and industrialized countries of the Asia-Pacific area, including Japan,
Taiwan, Hong Kong and Australia.
Zoonotic spread of the virus was first suspected when genomic sequences of HEV
isolates from two autochthonous cases in the USA were found to be closely related
to swine HEV [114].
In 2001 [119] HEV swine strains were identified in The Netherlands, showing close
genetic similarity to European human strains. In 2002 field isolates of swine HEV
were identified from different geographic areas [120] demonstrating nucleotide
identity between swine (88-100%) and human strains (89-98%). In 2004 in the
United Kingdom two UK swine HEV strains were identified with 100% amino acid
34
sequence identity over a partial sequence amplified by PCR, to one autochthonous
human case of HEV in the UK (Figure 1.7) [121].
In Spain, 2006, de Deus et al [122] identified swine affected by HEV with
nucleotide identity (85.7%-100%) between swine and human strains.
Recently, a hepatitis E outbreak on board in a UK cruise ship returning from an 80
night world cruise was investigated. The UK Health Protection Agency (HPA) was
informed of four cases of jaundice on board a cruise ship which departed from
Southampton on 7 January and returned on 28 March 2008. An epidemiological
investigation was launched by HP A to identify any additional cases of hepatitis E
and potential risk factor for infection. The investigation was a cohort study to
include all 2850 UK passengers who were on the cruise at any point. A total of 851
of the 2850 eligible passengers took part in the investigation. Finally, 33 (4%)
individuals were identified with recent acute HEV infection, although only 11 of
these were symptomatic cases. A common source outbreak was shellfish eaten on
board the cruise ship. The causative agent was identified as HEV gt 3 which was
closely related to the other gt 3 strains isolated in Europe [113, 123, 124].
The route of transmission has not been determined in most of these cases, although
zoonotic spread has been proposed [125]. To investigate the possible presence of
animal reservoirs, several animal species have been tested for anti-HEV antibodies.
HEV antibodies have been detected in different animal species, monkeys, pigs,
rodents, chickens, dogs, cats, cattle and sheep, both in resource-limited and
industrialized countries, suggesting that these animals could be infected by HEV
[113, 123, 124].
35
9996
92
95
92—E
f i
ri
AF503512 UKSW AY362357 UK Hu AF503511 UK Sw10Q----- AB073911 JAP Sw
L- AB093535 JAP Hu AF336292NLSW
A F 3 3 2 ^ 0 N L S w- AF336296 NL Sw— AF195063 SP Sw sew age API 95062 SP Hu AF336294 NL Sw
1 0 0cAF336295 NL Sw
AY032759NLSW- AF195061 SP Hu AY032758 NL Sw
AY032757 NL Sw
Figure 1.7 Phylogenetic tree. Human United Kingdom isolate (AY362357) is shown in bold and compared with closely related swine and human hepatitis E virus isolates. Bootstrap values greater than 70% are considered significant and are indicated. Figure taken from Banks et al, 2004, [121].
36
Water-borne (effectively faecal-oral) and food-borne transmissions, as well as
transfusion of infected blood products and vertical (maternal-foetal) transmission
[1], are now established routes of HEV transmission.
Aggarwal et al have reported an example of materno-fetal transmission of HEV
infection [126]. HEV-RNA or immunoglobulin (Ig) M anti-HEV antibodies have
been detected in seven of eight babies born to mothers with acute hepatitis E in the
third trimester of pregnancy [127].
Blood transfusion HEV infection has been described by Kriittgen et al in 2011
[128]. The study reported the youngest ever case of a five-month-old Caucasian girl
presenting with diarrhoea, emesis, and elevated ALT. Surprisingly, acute infection
with Hepatitis E virus (HEV) gt 3 was laboratory-confirmed by reverse transcriptase
polymerase chain reaction (RT-PCR) and sequencing [128]. In HEV endemic and
non-endemic areas, the presence of HEV viremia among healthy blood donors and
transmission of this infection to transfusion recipients has been documented [129].
Faecal-oral transmission of HEV occurs primarily through contaminated water in
endemic-regions where it is responsible for both sporadic and epidemic outbreaks
[130]. In epidemic form, the disease may involve tens of thousands of cases and is
the cause of considerable morbidity and mortality, posing a major public health
problem in endemic regions. In India alone, over 2.2 million cases of hepatitis E are
thought to occur annually. Hepatitis E in resource-limited countries has different
epidemiological and clinical features and investigation is patchy. Disruption of
water supplies in conflict zones has been shown to have caused major outbreaks of
hepatitis E amongst disrupted persons [131, 132]. During the conflict in Darfur,
Sudan, over 6 months in 2004, 2621 hepatitis E cases were recorded (incidence
37
3.3%), with a case-fatality rate of 1.7% (45 deaths, 19 of which involved were
pregnant women). Interestingly in this outbreak, as well as age, a risk factor for
infection was drinking chlorinated surface water (odds ratio, 2.49; 95% confidence
interval, 1.22-5.08) [132] {Tablel.8 [132]).
Although, supported by phylogenetic data, it is assumed the disease was around for
many years, hepatitis E was first recognised during an epidemic of hepatitis, which
occurred in Kashmir Valley in 1978. The epidemic involved an estimated 52,000
cases of icteric hepatitis with 1700 deaths (Figure 1.9) [1].
Based on these data, the possibility of another human hepatitis virus distinct from
post-transfusion non-A, non-B hepatitis was postulated. Balayan et al (1983) [130]
successfully transmitted the disease to himself by oral administration of pooled
stool extracts of 9 patients from a non-A, non-B hepatitis outbreak which had
occurred in a Soviet military camp located in Afghanistan. Over the years, hepatitis
E was identified as a major health problem in resource-limited countries with unsafe
water supplies and poor sanitary disposal.
38
Exposure
No. of individuals
Asymptomatic All HEV infection
in = 104) in = 491
Risk of asymptomatic
HEV infection, % RR (95% Cl)
Age group, years>45 17 5 29.4 Reference15-45 51 22 43.1 1.47 (0.48-4.47)0-14 36 22 61.1 2.08 (0.67-6.43)
SexFemale 73 35 47.9 ReferenceMale 31 14 45.2 0.94 (0.45-1.99)
Size of the family« 6 persons 65 29 44.6 Reference>6 persons 39 20 513 1.15 (0.57-2.30)
Presence of animals in the houseNo 54 23 42.6 ReferenceYes 50 26 52.0 1.22 (0.62-2.41)
Ever collected water from riverNever 76 35 46.1 ReferenceYes 28 14 50.0 1.09 (0.51-2.31)
No. of water reservoirs in house1 19 6 31.6 Reference2 37 16 432 1.37 (0.46^.07)>2 48 27 56.3 1.78 (0.63-5.00)
Source of drinking waterBorehole, unchlorinated 42 17 40.5 ReferenceSurface water, chlorinated 55 28 50.9 1.26 (0.61-2.59)Other 7 4 57.1 1.41 (0.37-5.45)
Use latrines.At least sometimes 81 37 45.7 ReferenceNever 23 12 52.2 1.14 (0.51-2.54)
Wash hands before eatingAt least sometimes 80 35 43.8 ReferenceNever 24 14 58.3 1.33 (0.62-2.88)
Wash hands after defecatingAt least sometimes 83 38 45.8 ReferenceNever 21 11 52.4 1.14 (0.50-2.61)
NOTE. RR, risk ratio.
Table 1.8 Risk factors for asymptomatic hepatitis E virus infection in a random
sample of Mornay population, Darfur, Sudan, September 2004. Figure taken
from Guthamann et al, 2006, [132].
39
MATERIAL REDACTED AT REQUEST OF UNIVERSITY
40
Food-borne transmission of HEV was first demonstrated in clusters of Japanese
patients that had eaten raw or undercooked meat of pig, wild boar or sika deer. The
genomic sequences of HEV identified from these patients were identical to those
recovered from the frozen leftover meat [133].
In addition, Colson et al [134] reported HEV evidence based on epidemiological
findings that 5 cases of autochthonous acute hepatitis E were linked to ingestion of
raw figatelli [134]. Figatelli are traditional sausages from Corsica, they are made
with pig liver and are commonly eaten uncooked, and they can be considered as a
possible source of HEV infection in France [134].
Legrand-Abravanel et al [135], studied 38 patients in south-western France with
HEV gt 3 infection. The patients were compared with matched control participants
in south-western France who had no evidence of HEV infection. According to the
results of a questionnaire, consumption of game meat, consumption of processed
pork and consumption of mussels were all statistically significantly more common
among case patients than among control participants. Eating undercooked pork and
pork products is quite common in Europe. Although the study by Legrand-
Abravanel et al [135] did not address the consumption of undercooked meat, other
studies have explored it’s association with hepatitis E. A case-control study by
Wichmann et al [136] in Germany, found that consumption of raw or undercooked
wild boar meat, and offal (liver, kidney, and intestine) was statistically significantly
associated with autochthonous HEV infection.
Other direct evidence of zoonotic transmission was recently reported by Kim et al
[137]. A sporadic case of acute hepatitis E was confirmed as gt 4 HEV in a 51 year
old Korean female. The case was reported as the first case of presumably zoonotic
41
transmission of HEV identified as gt 4 in a patient with acute hepatitis E after
ingestion of raw bile juice from a wild boar living on a mountain in South Korea.
Although drinking of raw bile juice is not a common practice in Korea, like other
parts of the world, some believe that bile juice could increase their energy or
stamina as a folk remedy.
Furthermore, it has been shown that commercial pig livers purchased from local
grocery stores as food in Japan, the United States (11%) and Europe [87, 138] are
contaminated by HEV and that some of the HEV-contaminated commercial pig
livers still contain infectious virus [87].
A study performed in an UK hospital tested 500 blood donors, 336 individuals over
the age of 60 years and 126 patients with chronic liver disease were tested for HEV
IgG. At the end of the study 40 cases of autochthonous hepatitis E (gt 3) were
identified [9]. These patients did not have a recent travel history and the major
probability was autochthonous hepatitis [9]. Autochthonous hepatitis E in developed
regions is frequently misdiagnosed as drug-induced liver injury, a common problem
that occurs with increased frequency in elderly people. The outcome can be poor in
those individuals with underlying chronic liver disease, with mortality approaching
70% [9].
Seroprevalence data from industrialised countries suggests that subelinical or
unrecognised infection is common. However, the real incidence of clinical
autochthonous hepatitis E in the UK is not known [139] but increased and improved
surveillance for hepatitis E has shown it may be more common than hepatitis A. [9,
140]. Data from France and Japan show similar trends [141, 142]. The literature
42
contains relatively few reports from the USA regarding autochthonous hepatitis E
[143]. It is known that the HEV human seroprevalence is around 21% in blood
donors, it is strongly possible that the majority of the patients with unexplained
hepatitis are “missed” since hepatitis E infection is often not considered a diagnostic
possibility in the USA [143].
1.9.2 Epidemiology in pigs and other animals
It is now accepted that autochthonous hepatitis E in developed regions has a largely
zoonotic source.
Evidence of this statement is described in many reports where HEV sequences
derived from pigs are closely related to HEV sequences from humans. Many
animals, for example domestic pigs, wild boar, deer, mongoose, trout and bivalves
are found to be HEV positive [144, 145]. In addition, HEV antibodies are detected
in domestic and feral animals. Gts 3 and 4 are the most commonly detected in this
wide range of animals. Data obtained from animal experiments suggest that
genotype 3 (zoonotic) is the most attenuated relative to genotype 1 and 2 (human to
human) where they cause more severe pathology [146-148]. Although genotype 3 is
considered by some to be the most attenuated for human beings, differences in
genotype virulence is still not well understood [149]. It is also suggested that gt 4 in
India differed relative to gt 4 subtypes found in China, Japan, and Taiwan. Data
show that Indian gt 4 is apparently not able to infect humans and it has been
suggested that this is probably due to the substitution of 26 amino acids, 16 in ORF-
1, 8 in ORF-2 and 2 in ORF-3 [150]. Autochthonous hepatitis E gt 3 was first
observed in the USA from comparing human sequences with pig sequences [52].
HEV seroprevalence in pig farms is high worldwide and it can be as high as 100%
43
in some pig herds [130]. Furthermore, in some studies it is demonstrated that
slaughterhouse workers, farmers, veterinarians and people that work in close contact
with pigs may be exposed to a greater risk of HEV infection. The evidence is based
on reports where this category of workers presents a higher HEV IgG
seroprevalence relative to non-pig workers [151]. More than 20% of pigs close to
the slaughter age are excreting HEV in faeces [152]. Watercourses may be HEV
contaminated due to run-off of pig faeces from outdoor pig units. In addition HEV
has been detected in slurry lagoons on pig farms, from urban sewage works, and
from pig slaughterhouses [153]. The risks of spreading untreated slurry on farmland
still need to be characterized but it should be remembered that rhesus monkeys have
been infected with HEV recovered from sewage and slurry [154].
1.10 Pathogenesis, clinical signs and symptoms
1.10.1 In humans
Studies on HEV have facilitated the understanding of elements of its replication,
host immune response, and liver pathology in HEV infected patients and primates
[39, 130]. It has been estimated that the infectivity titre of HEV for macaques is
10000-fold higher when inoculated intravenously compared with when it is ingested
[8]. Clinical signs of hepatitis E are dose-dependent in these animal models and
production of disease may require challenge doses 1000 times or more greater than
that required for infection [113].
After ingestion, the virus probably replicates in the intestinal tract (the primary site
of replication has not been identified yet) and reaehes the liver, presumably via the
portal vein [42]. It replicates in the cytoplasm of hepatocytes [155] and is released
into the bile and bloodstream, by mechanisms that are still poorly understood, and
44
excreted in the faeces [113]. The incubation period is 4-5 weeks based on an oral
infection study in human volunteers [130, 156]. Viral excretion in faeces begins
approximately 1 week prior to the onset of illness and typically persists for 2-4
weeks, in some cases RT-PCR has yielded positive results until 52 days after onset
[157]. The viremia can be detected in the first 2 weeks after the onset of illness
[156, 158, 159]. Viral excretion and viremia has been detected by RT-PCR also
prior to liver abnormalities, which normally appear with an elevation of
aminotransferase levels, and reach a peak by the end of the first week from the
clinical symptoms. Simultaneously the humoral immune responses appear. Anti-
HEV IgM or IgG levels are detected by enzyme immunoassay [160, 161]. Anti-
HEV IgM appears during clinical illness and then gradually disappears over a few
months (4-5 months). Some days later than IgM, anti-HEV IgG appears and persists
for few years [162, 163]. The persistence of HEV antibody in the sera is still
unclear. One study observed that 14 years after acute HEV infection anti-HEV
antibodies were still circulating in 47% of patients [164]. To diagnose acute HEV
infection, anti-HEV IgM is a useful tool, whereas IgG anti-HEV does not
necessarily indieate recent HEV infection [40].
Hepatitis E symptoms are typical of acute icteric viral hepatitis; the most common
recognizable symptom is an initial prodromal phase (preicteric phase) lasting a few
days, with a variable combination of flu-like symptoms, fever, mild chills,
abdominal pain, anorexia, nausea, aversion to smoking, vomiting, clay-coloured
stools, dark or tea coloured urine, diarrhoea, arthralgia, asthenia and a transient
macular skin rash [40]. These symptoms are followed in a few days by lightening of
the stool colour and jaundice appearance. Itching may also occur. With the onset of
45
jaundice, fever and other prodromal symptoms tend to diminish rapidly and then
disappear entirely. Laboratory test abnormalities include bilirubinuria, a variable
degree of rise in serum bilirubin (predominantly conjugated), marked elevation in
serum alanine aminotransferase (ALT), aspartate aminotransferase,
gammaglutamyltransferase activities and a mild rise activity in serum alkaline
phosphatase. The magnitude of transaminase rise does not always correlate well
with the severity of liver injury. The illness is usually self-limiting and typically
lasts 1-4 weeks [40]. Recent reports described evidence of chronie HEV infection
in transplant patients [165, 166]. A small number of patients with aeute HEV
infection have a prolonged elinical illness with marked eholestasis (cholestatic
hepatitis), including persistent jaundice and prominent itching. In these cases,
laboratories observed a rise in alkaline phosphatase and a persistent bilirubin rise
even after transaminase levels returned to normal [40]. The prognosis is good as
jaundice finally resolves spontaneously after 2-6 months. Within the past few years,
HEV has been demonstrated to be responsible for chronic hepatitis, which can
rapidly evolve to cirrhosis in immunocompromised patients [167-169]. However,
little data regarding HEV-related extrahepatie manifestations has been published,
although an association between neurologic manifestations (e.g., Guillain-Barré
syndrome, neuralgic amyotrophy, acute transverse myelitis) and acute HEV
infection has been suggested [170-174]. Previously, the association between
neurologic signs and symptoms and HEV infection has been based on detection of
anti-HEV immunoglobulin (Ig) M in serum. However, Rianthavorn et al [175]
reported a case of HEV gt 3-induced neurological amyotrophic in which HEV RNA
was detected in the serum of patients with neurologic signs and symptoms [176].
Recently, Kamar et al [176] detected HEV RNA in the cerebrospinal fluid (CSF) of
46
a kidney-transplant recipient with chronic HEV infection and neurological signs and
symptoms [177]. In addition, Kamar et al reported 7 chronic HEV gt 3 infections,
with development of neurological complications, from January 2004 until April
2009 [84] and the disappearance of the neurological symptoms were correlated with
a decreasing HEV titre.
Other infected individuals have a milder clinical course and develop only non
specific symptoms that resemble those of an acute viral febrile illness without
jaundice (anicterie hepatitis) [161]. Histological features of hepatitis E may differ
from other forms of aeute viral hepatitis. Nearly half of hepatitis E patients have a
cholestatic hepatitis, which is characterized by eanalicular bile stasis and gland-like
transformation of parenchymal cells. In these patients, degenerative changes in
hepatocytes are less marked [40, 178]. The Kupffer cells appear prominent. Portal
tracts are enlarged and contain an inflammatory infiltrate consisting of lymphocytes,
a few polymorphonuclear leucocytes and eosinophils. Polymorphonuclear cell
volume is particularly increased in the cholestatic type of lesion [40, 178]. In cases
with severe liver injury, a large proportion of the hepatocytes are affected, leading
to sub-massive or massive necrosis with collapse of liver parenchyma [40]. At the
beginning, HEV infection is entirely inapparent and asymptomatic. A small
percentage of patients have more severe symptoms with fulminant or subacute (or
late-onset) hepatic failure. The exact frequencies of asymptomatie infection and of
anicteric hepatitis are not known but a large proportion of individuals test positive
for anti-HEV IgG [40]. In resource-limited regions hepatitis E is common in young
adult and adults (15-40 years of age). Hepatitis E appears to cause more-severe
disease in pregnant women, particularly during the second and third trimesters [40].
47
HEV commonly causes intrauterine infeetion as well as substantial prenatal
morbidity and mortality [127], suggesting that the placenta may be the viral
replication site as Lassa fever [179, 180]. Death is usually due to encephalopathy,
haemorrhagic diathesis or renal failure. In a preliminary report [181] cynomolgus
monkeys infected intravenously with HEV developed acute tubular necrosis with
focal haemorrhages suggesting that HEV may replicate in monkey kidneys. In
pregnant monkeys, however, no increased mortality has been observed [182]. In
endemic countries such as India, the mortality rate of women with acute gt 1
hepatitis E in the third trimester of pregnancy is usually fairly high (26-64%) [183].
Why acute HEV infection in pregnant women causes severe liver dysfunetion is not
known.
Experimentally, HEV transmission has occurred from infected to uninfected in
contact pigs confirming that the virus is contagious [184].
Many reports described that [167, 176, 177] in immunosuppressed transplant
patients chronie HEV infection progress rapidly in cirrhosis [165]. Established
cirrhosis has been shown in two HIV-infected patients, in 2009, in UK and France
[185]. HEV and HIV coinfection still need to be better studied. [176] What it is
known to date is that that it seems that there was no difference in anti-HEV
seroprevalence between patients with HIV infection and control group [185].
1.10.2 In pigs
The mechanisms of HEV pathogenesis and replication are poorly understood due to
the lack of a practical animal model and an efficient in vitro cell culture system for
HEV. HEV might replicate in tissues and organs other than the liver [186].
48
Williams et al [72] confirmed clinical and pathological findings of HEV infection in
pigs previously reported by Halbur et al [149]. It is unclear how the virus reaches
the liver and extra-hepatic site(s), but it is presumably that HEV is transmitted by
the faecal-oral route. Primary the hepatocytes are the only known sites of HEV
replication [79]. It has been hypothesized that liver damage induced by HEV
infection may be due to the immune response to the invading virus and may not be a
direet eause of viral replication in hepatocytes [79, 187]. Several studies with
naturally infected pigs described HEV RNA detectable in different organs and
tissues, even after viremia was cleared [188]. For swine HEV-infected pigs, viral
RNA was detected in small intestines, colons, lymphnodes, and livers [79, 188].
Other extrahepatie tissues such as kidney, tonsil, and salivary gland had detectable
HEV RNA for only 1 or 2 weeks [79]. It appears that lymphonodes and the
intestinal tract are the main extra-hepatic sites of replication. The significance of
identifying extra-hepatic sites of HEV replication is unclear at this time.
Experimentally infected pigs do not present any clinical signs, histological analysis
shows signs of mild, focal liver necrosis but no fever or other signs (as for example
lack of appetite) are observed.
49
1.11 Diagnostic procedures
Enzyme-linked immunosorbent assays (ELISA), conventional reverse transcriptase
PCR (RT-PCR) and real time RT-PCR, cell culture, confoeal microseopy and
electron microscopy have been used for detection or confirmation of HEV infection.
These methods differ significantly in their sensitivity and specificity. The
eommonly used methods for HEV detection are described below in more detail.
1.11.1 ELISA
HEV recombinant proteins and synthetie peptides, corresponding to
immunodominant epitopes of the 0RF2 and ORF3 structural proteins of the virus,
have been sourced as the capturing antigen [113]. Subunits of ORF2 have been
expressed in different systems such as prokaryotic, insect, animal and plant cells in
order to obtain pure antigen for ELISA [189]. Recombinant antigens derived from
ORF2 generally have a superior sensitivity and specificity. In common with all
serological tests, ELISA can only be applied once antibody has developed, in most
cases at least 2 weeks after infection. However, serological tests are able to
discriminate between IgM and IgG, thus enabling distinction of the acute phase
from the convalescent phase of infection. HEV antibody prevalence has been
reported in several studies in industrialized countries [125, 189]. Commercially
available ELIS As have improved in recent years, but it is suspected some of the
earlier prevalence data reflected subelinical infections and serological cross
reactivity that may have contributed to this high seroprevalence in the non-endemic
areas [38, 113].
50
1.11.2 Conventional RT-PCR
Conventional RT-PCR assays are currently utilized in direct diagnosis of HEV. The
samples collected may be faeces, serum (from animal or human), cultures of
infected cells cultivated in 2D and 3D configurations, or post mortem tissue highly
positive as bile and liver [42, 190]. HEV is an RNA virus and the RNA needs to be
extracted before being subjected to the reverse-transcription reaction phase to
cDNA. This is a limiting step, because cDNA is easily degradable, if in the samples
the viral load is so low at initial state, may give rise at the end to false negativity.
Various sets of sense and antisense synthetic oligonucleotide primers may be used
for the detection of the HEV genome, differing based on conservative region targets
in the genome against the central or terminal part of ORFl or C terminal of ORF2
[42]. There are reports which indicate broad-spectrum degenerate primers, for
identifying positives samples from all genotypes. For example A l/S l and 3156/7
primers [191] are used to amplify the ORF2 region. Often the first product of PCR
amplification it is of insufficient quantity to be visualized by electrophoresis.
However, if the first product of PCR has been amplified by nested RT-PCR with the
internal primers A2S2 [192] and 3158/9 [191], respectively, the PCR product
became clearly visible on the eleetrophoresis gel through ethidium bromide
staining.
1.11.3 Real time RT-PCR
Real-time RT-PCR is becoming the most popular method for direct detection of
HEV in clinical samples. The technique enables both detection and confirmation of
specificity genotyping. In addition, real time RT-PCR is a sensitive tool in
epidemiological investigations since that this technique is fast and reliable. The full
51
viral genome of HEV was cloned in 1991 [35]. Since then several pairs of primers
have been designed to amplify various segments of the genome. The primers are
mainly designed to the conserved regions (heliease, polymerase and the terminal
fragment of ORF2) of the HEV genome [42]. The development of real time RT-
PCR, whereby the accumulation of the PCR amplicon can be deteeted in real-time,
has allowed for the quantification of HEV.
1.11.4 Negative strand detection
Since HEV is a positive strand RNA virus that putatively codes for a RNA-
dependent RNA-polymerase, HEV should replicate through a negative-strand RNA
intermediate [35]. Nanda et al [193] already showed HEV negative-strand RNA in
the liver tissue of infected rhesus monkeys, providing support for the putative
mechanism of HEV replication.
Varma et al [194] described viral HEV replication in transfected PLC/PRF/5 cells
and observed negative strand replication until 24h after the cells were transfected
with ORF2, with a maximum RNA negative strand peak after 8h post transfection
[194].
1.11.5 Cell culture and new technology for in-vitro propagation of the virus
Several cell lines for in vitro replication of HEV have been tested in the 2D
monolayer culture system [32, 195, 196]. These cell lines were hepatocytes from
non-human primates, human embryonic lung diploid cells (2BS), human carcinoma
alveolar basal epithelial cells (A549), hepatocarcinoma cells (PLC/PRF/5),
hepatocellular human carcinoma (HepG2) and primary hepatocytes from non
human primates. However, the majority of the cell lines did not support replication
52
of HEV or the virus growth was limited, i.e. low titre virus. The lack of an efficient
and reliable cell culture system and a practical animal model for HEV have
hindered studies on mechanisms of HEV replication, transmission, pathogenesis and
environmental survival.
In a recent study, Tanaka et al have tested 21 cell lines including PLC/PRF/5 cells
using a faecal suspension with high HEV load as inoculum [197]. A high load of
HEV was detected in the eulture supernatant of cultivated PLC/PRF/5 cells from
day 12 post inoculation. At AHVLA laboratory, several attempts were made to
reproduce Tanaka’s work using field swine HEV PCR positive faecal materials as
inoculum, but without success. Okamoto in 2011 described for the first time, a cell
culture system capable of secreting infectious HEV in high titres into culture media
[198]. The success with the original JE03-1760F strain has been extended to other
strains that can support the replication of HEV with an even higher efficiency, and
can be passaged through many generations [198]. Okamoto was able to infect
PLC/PRF/5 cells with both sera and faeces of patients and observe high HEV titre in
the cell culture system [198]. Furthermore Okamoto has engineered infectious HEV
cDNA clones, in addition he affirmed that this system, reinforced by reverse
genetics, will solve many mysteries and answer numerous questions surrounding the
epidemiology, viral absorption/entry, packaging and delivery of viral particles,
toward illuminating the life cycle of HEV. No other authors after Okamoto have
been able to reproduce those experiments [198].
Hence, an efficient in vitro propagation system for HEV is crucial for HEV research
in general, and to the VITAL project in particular. There are several reports in the
literature demonstrating the potential of a new 3D culture system Rotating Wall
53
Vessel (RWV) (Figure 1.10) [199], for the growth of fastidious viruses. The RWV
is a cylindrical bioreactor which is rotated on an axis parallel with the ground.
Subsequently, a solid body mass rotation of the culture medium is obtained, creating
a low-fluid-shear environment [200]. The RWV culture method has been shown to
be applicable for fluid shear stress-related studies in suspension. The cells are
maintained in suspension by the resolution of the centrifugal, gravitational and
Coriolis forces, so cells placed in the RWV bioreactor experience minimal
mechanical stresses and high mass transport (of nutrients, oxygen etc.) and are thus
able to assemble into tissue-like aggregates. This 3D culture system has been used
to grow fastidious Norovirus from faecal materials [201]. The system offers a
potential for in vitro cultivation of HEV. The RWV technology is used to simulate
the low shear environment inherent to microgravity [202].
54
B CFilling port Gas-pem cable membranî
\ \ A
/f Sampling ports
' f A '#
yDocking point
FRONT b a c k
Figure 1.10 Rotating Wall Vessel motor (RWV) or Rotary cell culture system.
A: The RCCS (RCCS-4DQ, Synthecon) is available as a one, two, four or eight
station rotator base. The system depicted consists of a four Station Rotator Base,
along with a power Supply with Tachometer. Each station is capable of rotation at
independent speeds, enabling four experimental conditions and/or experiments to be
run simultaneously. The system is supplied with four Rotary wall vessels (RWV).
(B): The cylindrical RWV is completely filled with culture medium, cells and micro
carrier beads through the filling port on the face of the vessel. All bubbles are
removed from the RWV through the sampling ports to reduce shear. The vessel is
attached to the rotator base by docking point and rotated on its axis that is parallel to
the ground creating a solid body rotation. Cell-beads aggregates in the RWV are
maintained in a gentle fluid orbit and do not collide with the walls or any others
parts of the vessel (i.e., suspension culture). As 3D tissues grow in size, the rotation
speed is adjusted to compensate for the increased settling rates of the larger
particles. The cells and/or tissue particles join to form larger tissue particles that
continue the differentiation process. Oxygen supply and Carbon dioxide removal
are achieved through a gas-permeable silicone rubber membrane that covers the
back of the RWV bioreactors. Schematic representation on how the system, works
(section c). Figure taken from Nickerson et al, 2001; [199].
55
1.11.6 Microscopy
Microscopy is the teehnical field of using microscopes to view samples or objects.
There are three well-known branches of microscopy, optical, electron and scanning
microscopy. Optical and electron microscopy involve the diffraction, reflection, or
refraction of electromagnetic radiation/ electron beam interacting with the subject of
study, and the subsequent collection of this scattered radiation in order to build up
an image. This process may be carried out by wide-field irradiation of the sample
(e.g. standard light microscopy and transmission electron microscopy) or by
scanning of a fine beam over the sample (e.g. confoeal laser scanning microscopy)
and scanning electron microscopy.
1.11.6.1 Confoeal microscopy
There has been a tremendous increase in the popularity of eonfocal microscopy in
recent years. The technique of laser scanning confoeal microscopy has become an
invaluable tool for a wide range of investigations in the biological and medical
sciences for imaging of optical section in living and fixed specimens ranging in
thickness up to 100 micrometers [203]. The basic key to the confoeal approach is
the use of spatial filtering techniques to eliminate out of focus light or glare in
specimens whose thickness exceeds the immediate plane of focus. Confoeal
Microscopy offers several advantages over conventional wide field optical
microscopy, including the ability to control depth of field, elimination or reduction
of background information away from the focal plane (that leads to image
degradation), and the capability to collect serial optical section. The choice of
fluorescent probes for confoeal microscopy must address the specific capabilities of
56
the instrument to excite and detect fluorescence emission in the wavelength regions
made available by the laser system and detectors.
1.11.6.2 Electron microscopy, transmission and scanning
The transmission electron microscopy (TEM) technique is specific, labour intensive
and expensive, but was a critical precursor for understanding the natural history of
HEV, being the tool used to detect the viral particle causing non-A non-B non C
hepatitis in 1975 [204]. The virus particle of 27-34 nm appeared non-enveloped,
was detected in stool samples collected during preicteric and early icterie phases
and to determine antibody titres in the sera [186]. In general, the TEM technique
does not serve as a diagnostic tool since it usually requires large amounts of antigen
and high antibody titre and further, virions are shed in degraded form in faeces [42].
The scanning electron microscope (SEM) produces very high-resolution of a sample
surface, revealing details about 1 to 5 nm in size. Due to the way these images are
created, SEM micrographs have a large depth of field yielding a characteristics
three-dimensional appearance useful for understanding the surface strueture sample
composition.
1.12 Vaccination
Due to lack of a reliable cell culture system for HEV, vaccine development has been
difficult. Two candidate vaccines have successfully completed phase 3 clinical trials
in humans. Baculovirus-expressed ORF-2 protein from a Pakistani strain of HEV
has been licensed by Smith Kline-Beecham [205]. In the Royal Nepalese Army, a
vaccination trial to prevent HEV clinical disease was conducted and it showed
95.5% efficacy (95% Cl). Also in China, another trial was conducted. The vaccine
57
was prepared with a recombinant protein from the HEV ORF-2 viral capsid
expressed in Escherichia coli (HEV 239) [83]. Vaccine efficacy after three doses
was 100% (95% Cl 72.1-100.0). It was considered that these two vaccines could
prevent HEV morbidity and mortality in pregnant women, patients with chronic
liver disease in endemie areas, patients with organ transplants and other
immunocompromised subjects who may contract HEV gt 3 in industrialized
countries.
As far as we know these two vaccines cover gt 1 but nothing is known about
prevention of gt 3 and it is quite unthinkable to set up a vaceination plan for the
entire worldwide population against HEV gt 3, mostly because in non-endemic
areas HEV is sporadic and incidence is generally still very low. The production of a
HEV vaccine for pigs would be more feasible and cheaper, but it is acknowledged
that in the absence of any disease in pigs, it might not be justified or practicable to
vaccinate pigs. However, in considering the options for control of autochthonous
acquired gt 3 and gt 4 HEV in humans, it is important to have some data on the
estimated impact and optional timing of HEV vaccination of pigs. This would be
useful feasibility data in case of changes in the incidence of human gt 3 infections in
developed regions or other events that may require the vaccination of pigs.
1.12.1 HEV vaccination modelling in pigs
Only a few studies regarding the dynamics of HEV transmission have been done but
no vaccination modelling in pigs has been performed to date. Bouwknegt et al 2008
[86] described HEV transmission among pigs from chains of one-to-one
transmission. The model describes HEV transmission in pigs and it can be used both
with animal contact exposure experiments and in the field. Each age group or
58
contact-exposure animal is subdivided into three distinct compartments that consist
of pigs that are susceptible (S), infectious (I) or recovered (R). The system
described by this SIR model is assumed to be in an endemic equilibrium. This
endemic equilibrium can only exist when the virus is suffieiently transmissible. The
transmissibility is expressed by the reproduction number (Rq) and it is the number
of infections by an infectious individual during its entire infectious period (in an
infinite susceptible population). The endemic equilibrium assumes that Rq > 1. This
SIR model means that the infected animals reach immunity after infection. The
latent period between infection and excretion of infectious virus, is observed to be 3
days in intravenously inoculated pigs [86].
The model is analysed by Monte Carlo (MC) sampling. This means that three
random numbers of infectious animals are drawn from the distribution depending on
the observed number of positive. Each Monte Carlo (MC) sample consists of three
numbers of animals that signify the numbers of infectious weaners, growers and
fatteners.
Bouwknegt et al 2008 [86] observed that Rq for contact-exposure was estimated to
be 8.8 (Cl 95%,) showing the potentia] of HEV to cause epidemics in populations of
pigs.
Casas et al reported a longitudinal survey study on swine HEV infeetion dynamics
conducted in different herds [206], but the dynamics of HEV transmission was
analysed using SPSS 15.1 software (SPSS Inc., Chicago, IL, USA) and not a
mathematical model such as the SIR model.
59
Only a couple of studies have applied mathematical model such as the SIR model in
field samples to better understand HEV dynamics of transmission [207]. This
mathematical model can better help in a theoretical way by mimicking the in vivo
system to understand of how HEV is circulating between pigs in the same farms and
between different age groups. Furthermore this model can also try to mimic how a
vaccination model can help to eradicate an endemic virus such as HEV and it can
help to understand at which age, during an early or later stage, it is more effective to
vaccinate the animals [86].
60
1.13 Aims of the VITAL PhD project
This PhD project was part of the European project VITAL (Integrated Monitoring
and Control of Foodbome Viruses in European Food Supply Chains). This project
included 15 laboratories in Europe and this PhD was developed to spend the first
year in the UK, one year in The Netherlands and the last year in the UK. The main
aims of the VITAL project were to:
i: Acquire data on virus contamination of food and environmental sources.
ii: Assess food borne viral risks for determining high-risk situations and efficacy of
interventions.
iii: Develop new measures to prevent virus contamination of food and the
environment.
iv: Develop and assess measures of reduction and control in case of virus
contamination.
The specific aims of this PhD project were to investigate HEV presence and
residual infectivity in the pork food chain in order to facilitate any future control
measures. During this PhD project samples across the UK pork food chain were
tested for HEV contamination. Furthermore, a cell culture system was optimised to
demonstrate the infectivity of the virus in the food samples tested and HEV
inactivation strategies were investigated. This will aid understanding of the
mechanisms of HEV replication, pathogenesis and environmental (including within
food matrices) survival. The knowledge derived from this study is going to be used
to develop codes of practice aimed at reducing or eliminating zoonotic transmission
of HEV via the food-borne route.
61
The 3 objectives of the PhD project were:
1) a) To evaluate a new 3D cell culture system to assess HEV infectivity. This was
set up to verify that the HEV virus content detected by PCR in pig products and
environmental samples is infectious.
b) To compare the efficiency of the 3D system to the conventional 2D cell culture
system. In addition, cells grown in the 3D system were transferred to a 2D system
and infected. This aimed to produce the best tool with which to examine large
numbers of samples being investigated for potential transmission routes.
c) The risk of HEV infection via the consumption or manipulation of HEV-
contaminated pig livers raises further public health concern since it is not clear
which conditions will be effective in inactivating the virus present in the
contaminated pig livers. Inactivation studies were performed to better understand
which is the best method to inactivate HEV in various matrices and environments.
The inactivation studies performed were heat, UV light and sodium hypochlorite
HEV inactivation. The heat inactivation was performed to better understand at
which temperature the virus is inactivated to produce guidelines for consumers,
particularly in relation to cooking conditions. The other two studies were set up to
provide information that could be incorporated in guidelines for pork chain workers.
2) To assess methods for HEV detection within the VITAL project, particularly
from sampling in the UK pork food chain. The first step of the VITAL project was
to optimise the Standard Operating Procedures (SOPs). It was requested that the
sample collection laboratories involved in the project tested the SOPs. This was to
be accomplished by means of blind ring trials. Samples were spiked with Human
62
Adenovirus (HAdV) and the results obtained were evaluated by the ring trial leader.
The second step was testing samples collected at the slaughterhouse (40 pig liver
sample and 40 pig faeces), processing point (40 pork muscles) and point of sale (63
pork sausages). The aim was to gain an insight into the frequency of HEV in the UK
pork foodchain.
3) HEV dynamics of transmission study: Since HEV is a zoonosis that is
widespread in the pig population in Europe, there might be an interest to produce a
pig vaccine to reduce the impact of HEV infection in the human population. Prior to
any vaccine development, modelling work is necessary to assess the impact of
vaccination in the reduction of HEV excretion by the pigs. I participated in the
collection of HEV prevalence data in European countries, and in the construction of
the dynamics of transmission model.
63
CHAPTER 2 VITAL Ring Trial
64
Introduction
This PhD project was part of the European project VITAL (Integrated Monitoring
and Control of Foodborne Viruses in European Food Supply Chains). This project
included 15 laboratories in Europe and one of the main aims was to assess methods
for the detection of Human Adenovirus (HAdV) and Norovirus in the soft fruit and
salad and detection of HEV in the pork products and shellfish. This PhD project was
focused on the pork foodchain and its initial phase was the validation of the
extraction and detection methods for two sample matrices (soft fruit and pork
products). These methods were developed as standard operating procedures (SOPs)
and assessed by means of a blind ring trial between all data gathering laboratories in
the VITAL consortium. Samples were spiked with HAdV, the target virus and with
Murine Norovirus (MNoV) the extraction control. All samples were tested by all the
laboratories involved in the ring trial and the results were sent to the ring trial leader
for evaluation.
Ring trial: In each data-gathering laboratory the first task was evaluating common
SOPs, developed for the project, to test the robustness of all methodologies from
virus extraction to detection methods (real time PCR).
65
Materials and methods
Liver tissue and raspberries were the matrices selected for the ring trial. HAdV and
MNoV, supplied from the Istituto Superiore della Sanita’, Rome (ISS), were used as
the target virus and extraction control virus (called sample process control SPC),
respectively. Each target virus suspension (HAdV) was tested blind and coded; the
concentrations were known only by the ring trial leader. Fifty pi of each target virus
suspension and 10 pi of SPC virus suspension were used to spike each sample. The
MNoV was used as control to monitor the success of the extraction process.
2.1 Virus concentration and nucleic acid extraction
2.1.1 Sampling and virus concentration in pork liver tissue
Two hundred and fifty mg of liver tissue, obtained from a local UK supermarket,
was cut from three different inner portions of a liver. Fifty pi of the coded sample
virus was spiked into the sample and incubated for 2 h. The liver was then
homogenized manually using surgical blades and mortar. The homogenized liver
tissue was transferred into 1 ml lysis buffer (containing 0.14 M D-mercaptoethanol).
Ten pi of the positive process control virus was added to the sample. The tubes were
centrifuged for 20 min at 10.000 x g. Eight hundred pi of the aqueous phase was
transferred to a new 2 ml microtube and the suspension used immediately for RNA
extraction (VITAL SOP 009, Appendix C.4).
2.1.2 Nucleic acid extraction from pork liver tissue
Trizol (Invitrogen) (0.75 ml) and 0.2 ml of chloroform were added to the
supernatant obtained in section 2.1.1. The samples were incubated for 5 min at room
temperature and the tubes centrifuged for 15 minutes at 12.000 x g. One ml from
66
the aqueous phase was transferred to a clean 2 ml microtube. An equal volume of
Phenol : Chloroform: Isoamyl alcohol (25:24:1) solution was added and the solution
was centrifuged for 15 minutes at 10.000 x g. Eight hundred pi from the upper
aqueous phase was transferred to a clean 2 ml tube. LiCl (0.1 ml, 5 M) solution was
added into the solution and mixed by vortexing. The tubes were incubated at -20°C
for at least 4 hours. The supernatant after centrifugation (10 minutes at 10.000 x g)
was removed and the pellet was washed with 70% ethanol, dried and resuspended in
50 pi of nuclease-free deionised-distilled-water. The extracted RNA was stored at -
80”C. (VITAL SOP Oil, Appendix C.6).
2.1.3 Sampling and virus concentration from soft fruit
Twenty five g of raspberries, obtained from a local UK supermarket, was weighed
and transferred to a sterile beaker and 50 pi of Adenovirus (yielding stock titres of
approximately 4x10^ plaque-forming units (PEU) ml“ ) was spiked into the sample
and incubated for 2 hours. Ten pi of the SPC and 40 ml of Tris Glycine 1% Beef
Extract (TGBE) Buffer Including 6500 U of pectinase (250 pi of Pectinex Ultra
SPL solutions) were added to the sample. The sample was agitated at room
temperature for 20 min by rocking at 60 rpm. The supernatant was decanted from
the beaker through a strainer into one 50 ml tube. The sample was centrifuged at
10.000 X g for 30 min at 4°C. The supernatant was decanted into a single clean
tube/bottle. The pH of the sample was adjusted to 7.2 with Hydrochloric acid (1 N).
5X electrolyte-polyethylene glycol / Sodium chloride (0.25 ml of solution) was
added to the sample and incubated with gentle rocking at 4°C for 60 min. The
solution was centrifuged at 10.000 x g for 30 min at 4°C and the supernatant
decanted and discarded. The pellet was resuspended in 500 pi of PBS. Five hundred
67
pi chloroform:butanol solution (1:1) was added to the solution and centrifuged at
10.000 X g for 15 min at 4°C. The aqueous phase was transferred to a clean tube and
stored at -20°C. (VITAL SOP 005, Appendix C.3).
2.1.4 Nucleic acid extraction from soft fruits
Nucleic acid extraction from the samples processed in step 2.1.3 was performed
according to the NucliSENSE lysis protocol (BioMérieux). Briefly 500 pi of the
concentrated solution obtained from the soft fruit {section 2.1.3) were transferred
into a clean centrifuge tube. Four and a half ml of NUCLISENSE lysis buffer were
added to the tube, and mixed by vortexing briefly. The samples were centrifuged for
2 min at 1.500 x g to ensure that the entire sample was brought down into the tube.
Fifty pi of well-mixed magnetic silica solution (BioMérieux) was added to the tube
and mixed by vortexing briefly. The supernatant was discarded after centrifuge for 2
min at 1.500 x g. Wash buffer 1 (400pl) was added and the pellet resuspended by
pipetting/vortexing. The suspension was transferred to a 1.5 ml screw-cap tube.
Another 2 washes (400 |il each time with washing buffer 2 and 3) were made, after
every wash the pellet attached to the silica beads was resuspened. The final step
consisted in adding 50 pi of elution buffer and transferring the tubes to a
thermoshaker for 5 min at 60°C at 1.400 rpm. The tubes were placed in a magnetic
rack to allow the silica to settle and the eluate was transferred to a clean tube. The
RNA was retained at 4°C for a maximum of 24 hrs or at -80°C for up to one week
(VITAL SOP 012, Appendix C.T).
68
2.2 Positive standards construction
Within the VITAL project synthetic multiple-target DNA oligonucleotides were
constructed for use as quantification standards for nucleic acid amplification assays
for Human Adenovirus, Porcine Adenovirus and Bovine Polyomavirus [208]. For the
DNA standard a synthetic DNA molecule was designed to contain target sequences
for real time PCR assays for BPyV [209], HAdV [210] and PAdV [211]. The
oligonucleotides were synthesised (Eurofins MWG Operon, Ebersberg, Germany)
and cloned into a pCR 2.1-TOPO plasmid (Invitrogen, Breda, The Netherlands)
{Figure 2.1) [208].
The DNA concentration was determined by UV spectrophotometry in a Nanodrop
ND-1000 spectrophotometer (ThermoScientific, Wilmington, NC, USA). The
measurement was performed in duplicate and concentration in grammes was
converted to molecule number using the following formula:
DNA molecules x pi *— [(g/pl)/(plasmid length in base pairs x 660)]
X 6.022 X 10“
The standards used for the quantification of the target viruses were designed by
Martinez-Martinez et al [208] and subsequently sent to all VITAL laboratories
involved in the VITAL ring trial.
69
2.3 Real time PCR protocols
2.3.1 Quantification of adenovirus by real-time PCR
This protocol was based on the SOP “General protocol for the quantification of
Adenovirus by Real Time PCR” {see SOP 14 VITAL, AppendixC.9). Briefly, this
assay was a duplex real time PCR using the primers and conditions described by
Hernroth et al (2002) [210], with the inclusion of an internal amplification control
(LAC) [212] to verify if PCR inhibitions occurred. The reaction contained
IxTaqMan Universal PCR Master Mix (Applied Biosystems). The primers were
used at a final concentration of 0.9 pM, Table 2.2. These primers targeted the
HAdV hexon gene. The reaction mix was prepared following the manufacturer
instructions (ABI PRISM HID 7000 SDA from Applied Biosystems) and consisted
of 0.225 pM adenovirus TaqMan probe (labelled with F AM), 50 nM lAC probe (0.1
pM, labelled with VIC), 100 copies of adenovirus lAC (Yorkshire Bioscience Ltd,
UK) and enzyme mix (12.5 pi). Ten p.1 of the diluted nucleic acid extract was added
to make a final reaction volume of 25 pi.
The total volume for one reaction after addition of target was 25 pi (15 pi mix plus
10 pi sample or standard). Ten pi of nuclease-free deionised-distilled-water was
added to the NTC samples (no template control). Two PCR replicates were
performed for each sample. In each PCR run, positive (Synthetic multiple-target
DNA oligonucleotides described in section 2.3) and negative (water) amplification
controls were included to exclude possible contaminations. Following activation of
the UNG (uracil Nglycosylase) (2 min, 50°C) and activation of the AmpliTaq Gold
for 10 min at 95°C, 45 cycles (15 sec at 95°C and 1 min at 60°C) were performed.
The data were analysed using the MX3000 software.
70
2.3.2 Detection and quantification of Murine Norovirus by real-time RT-PCR
This protocol was based on the methods described by da Silva et al, Svraka et al,
Loisy et al and Kageyama et al [201, 213-215]. The oligonucleodites used are
described in Table 2.3. The MNoV PCR was performed using RNA UltraSense™
One-Step Quantitative RT-PCR System (Invitrogen) and primers and probe were
designed by Baert et al in the ORFl/2 junction region: Fw-0RF1/0RF2 (5’- CAC
GCC ACC GAT CTG TTC TG-3’) (location 4972-4991), Rv-0RF1/0RF2 (5’-
GCG CTG CGC CAT CAC TC-3’) (location 5064-5080), MGB-ORF1/ORF2 (5’-
FAM-CGC TTT GGA ACA ATG-MBG-NFQ-3’) (location 5001-5015) [216].
Ten pi of RNA extracted from the samples was added into each reaction, including
the negative control (NTC) and 0.6 pi of lAC [213]. The total volume for one
reaction after addition of target was 20 pi (10 pi mix plus 10 pi sample or standard).
Ten pi of nuclease-free deionised-distilled-water was added in the NTC samples. The
Real Time RT-PCR was performed in a real-time PCR platform (MX 3000,
Stratagene): reverse transcription 50°C for 15 min, 2 min at 95°C followed by 40
cycles of 15 s at 95°C and 1 min at 60°C. The data were analysed using the MX3000
software.
2.3.3 The internal amplification controls (lACs)
Internal amplification controls (lACs) were constructed for incorporation into real
time nucleic acid amplification assays for Hepatitis E virus. Human Adenovirus
Murine Norovirus and Porcine Adenovirus. The addition of lAC into the assays was
to provide a robust PCR control that can be routinely applied in the analysis of foods
for viruses.
71
The lAC was a chimeric DNA molecule containing non-target sequences flanked by
target sequences complementary to the virus-specific primers [212]. This molecule
was then cloned into a plasmid {Figure 2.4) [212]. The plasmid or the RNA
transcript was the chimeric lAC which was co-amplified with the virus primers and
detected using a fluorescent probe complementaiy to the internal non-target
sequence [212]. When using a real-time PCR-based assay, the virus target
amplicons were detected with specific hydrolysis probes, labelled with one
fluorophore (e.g. FAM), and the LAC amplicons were detected with the specific
lAC probe, labelled with a different fluorophore (e.g. VIC). Each lAC was designed
by Diez.Valcarce [212] for the VITAL project as a DNA or RNA molecule
containing sequences from the prfA gene from Listeria monocytogenes (nucleotide
positions 2281-2348, AN AY512499) flanked by the sequences complementary to
the primers used in the specific assays [212]. The chimeric DNA molecules were
generated by PCR using as template 5 ng of L. monocytogenes strain CECT 935
DNA [212]. The PCR products were excised from a 2% Ix TBE agarose gel and
purified using QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany), then
cloned into the pCR 2.1-TOPO Vector (Invitrogen) in the case of lACs for the HEV
assays or into the pGEM-T Easy Vector (Promega, Madison, WI, USA) in the case
of lACs for the Human Adenovirus (HAdV), Porcine Adenovirus (PAdV) and
Murine Norovirus (MNoV) assays. lACP probe construction was also conducted by
Diez- Valcarce et al [212]. The probe was targeting portion of the target virus and
portion of the plasmid [212].
The lAC construction was performed by Diez-Valcarce et al [212], it was
subsequently sent to Yorkshire Bioscience for manufacturing and finally bought
from the VITAL members involved in the ring trial and data gathering.
72
73
00GCCCCTAGATCCTACCCTCAACGGAATTCTAGACAAAGATGGTGTGTATCCTGTTGAGTGTTGGTGTCCAGATCCAAGTAAC7r^C4r0C/lCm’ GCCGGGC4GG4CGK-CTCGGAG7ACCTGAGCCCaGGCCTGGrGC4G7TCGCCCGrGAACt3<3CCaCTACTGCAAOTTCCACATCCAGOTOCCiX:AAAAGTTCTTTGCÇÇTÇAAGAGCCTGCTOCTGCGGCCOC
pFBV2 4159 bp
Figure 2.1 Graphie representation of pFBV2 containing the sequence of the synthetic DNA. The length of the plasmid is 4,159 bp. The viral insert was flanked by Apal and Notl sites. The sequences of the qPCR assays are shown (BPyV—bold, HAdV-2—italics and PAdV— underlined. The sequences corresponding to the TOPO vector are in normal type. Figure taken from Martinez-Martinez et al [208].
74
Primers Sequences
Forward primer: AdP 5 ’- CWT ACA TGC ACA TCK CSG G-3’
Reverse primer: AdR 5 ’- CRC GGG CRA AYT GCA CCA G -3’
Adenovirus TaqMan Probe
5 ’- FAM- CCG GGC TCA GGT ACT CCG AGG CGT CCT-BHQ-3’
TaqMan probe: lACP 5 -VIC- CCA TAC ACA TAG GTC AGG -M GBNFQ- 3 ’
Table 2.2 Adenovirus oligonucleotides. The table describes primer sequences used
for the Adenovirus PCR detection method. Figure adapted from Diez- Valcarce et al
[212].
Primers Oligonucleodites
Forward primer FW -0RF1/0RF2 (5 ’- CAC GCC ACC GAT CTG TTC TG3’)
Reverse primer RV-0RF1/0RF2 (5 ’- GCG CTG CGC CAT CAC TC-3’)
Probe (Taqman MGB probe)
MGB-ORF1/ORF2 (5’-FAM- CGC TTT GGA ACA ATG -M G B N F Q -3 ’)
lACP lACP (5’-VIC- CCA TAC ACA TAG GTC AGG -M G B - NFQ- 3 ’
Table 2.3 Murine norovius oligonucleotides. The table describes primer
sequences used for the MNoV detection method. Figure adapted from Diez-
Valcarce et al [212].
Detection targetVims DNA/RNA
lAC targetL. monocytogenes DNA
Primer L.monoF # # # #
# * # #Primer L.monoR
Primer lACF # • • •
1st PCR
2nd PCR• • • •
Primer lACR
lACChimeric DNA
T7 RNA pol + DNase
DNA RNA
Duplex real-time PCR Duplex RT-real-time PCR
Figure 2.4 lAC constructions. PCR amplification of non-target DNA is performed using hybrid oligonucleotide primers. This produces a chimeric DNA molecule containing non-target sequences flanked by target sequences complementary to the virus-specific primers. This molecule is then cloned into a plasmid. If the lAC is for RNA virus detection, the plasmid should contain a T7 RNA polymerase promoter, and lAC RNA transcripts are subsequently produced by T7 RNA polymerase. The plasmid or the RNA transcript is the chimeric lAC which is co-amplified with the virus primers and detected using a fluorescent probe complementary to the internal non-target sequence. Figure taken from Diez- Valcarce et al [212].
76
2.4 Data interpretation: Results and data interpretation were described by
D’Agostino et al 2011 [217, 218]. Briefly, each participant sent to the trial leader
their data [217, 218]. When an assay showed a quantification cycle (Ct) value lower
or equal to 40 or 45 for Murine Norovirus or adenovirus, respectively,
independently of the corresponding lAC Ct value, the result was interpreted as
positive [217, 218]. When an assay showed a Ct value more than or equal to 40 or
45 for Murine Norovirus or Adenovirus, respectively, and the LAC Ct value lower
or equal to 40, the result was interpreted as negative [217, 218]. When an assay
showed both the target and its corresponding lAC Ct values > 40 or 45 respectively,
the reaction was considered to have failed. When a participant reported that at least
one of the HAdV replicates was positive, they were considered to have identified
the sample as being Adenovirus contaminated [217, 218]. When a participant
reported that both HAdV replicates were negative, but at least one replicate MNoV
assay was positive, they were considered to have identified the sample as being
Adenovirus uncontaminated [217, 218]. When a participant reported that both
replicate HAdV assays were negative and both replicate MNoV assays were
negative, they were considered to have reported that the analysis of that sample had
failed. Interpretation of the results followed the principles outlined by D’Agostino et
(2011) [219].
77
Results
2.5 Detection of spiked Human Adenovirus in raspberries samples
Nine batches of raspberry samples were spiked with an equivalent number of blind
coded samples, some of them known to contain human adenovirus (HAdV). Murine
Norovirus (MNoV) was used as internal extraction control. At the end of the ring
trial, the ring trial leader sent a feedback to each participant. The nine blind coded
samples were revealed to be divided into three groups: three positive with high viral
titre (5x lO" PFU) three positive with low viral titre (5x 10 PFU) and three
negative for HAdV. On three samples tested in duplicate for each category (high,
low level and blank) for HAdV all the samples tested by AHVLA showed the
expected Ct values (high HAdV contamination Ct values of 26, low HAdV
contamination 33, blank HAdV contamination no Ct values).
Table 2.5 shows the results from the analysis of raspberry samples artificially
contaminated with 5x 10 PFU of HAdV. The Ct values detected by real time PCR
for these samples had an average of 26 Ct. All samples were correctly reported as
contaminated with the target virus (HAdV) by real time PCR. Table 2.6 shows the
results, obtained by real time PCR, from the analysis of raspberry samples
artificially contaminated with 5x10^ PFU HAdV, in this case the Ct detected by real
time PCR were around 33 Ct. Table 2.7 shows the results from the analysis of the
non-artificially contaminated raspberry samples where no Ct values were detected
by real time PCR.
Sixteen out of 18 duplicates tested were positive for MNoV. Percentages of
concordance of the results provided at AHVLA by the ring trial leader are shown in
table 2.8.
78
Laboratoiy Sample A Sample B Sample C
HAdV MNoV HAd\^ MNoV HAdV MNoV
Rep. 1 Rep. 2 Rep. 1 Rep. 2 Int. Rq>. 1 Rqp. 2 Rep. 1 Rq>. 2 Int. Rep. 1 Rep. 2 Rep. 1 Rep. 2 In t
Table 2.5 Results of analysis of raspberry sample artificially contaminated with 5x10 PFU human adenovirus (HIGH). Twenty five g of raspberry was artificially contaminated with 50 p.1 of HAdV and with 10 jil of extraction control (MNoV). Samples A, B, C represent the samples run in duplicate of raspberries contaminated with HIGH level of HAdV (Human Adenovirus), and spiked with MNoV (Murine Norovirus). Rep. - replicate; + target signal present by real time RT-PCR, lAC signal present or absent by real time PCR; - target signal absent by real time PCR, LAC signal present; C-sample contaminated; Int -Interpretation. Figure adapted from D’Agostino et al, 2011 [217,218].
Laboratory Sample A Sample B Sample C
HAdV MNoV HAdV MNoV HAdV MNoV
Rep. 1 Rep. 2 Rep. 1 Rep. 2 Int. Rep. 1 Rep. 2 Rep. 1 Rep. 2 Int. Rep. 1 Rep. 2 Rep. 1 Rep. 2 Int.
Table 2.6 Results of analysis of raspberry sample artifîcially contaminated with 5x10 PFU human adenovirus (LOW). Twenty five g of raspberry was artificially contaminated with 50 jil of HAdV and with 10 |il of extraction control (MNoV). Samples A, B, C represent the samples run in duplicate of raspberries contaminated with LOW level of HAdV (human adenovirus), and spiked with MNoV (Murine Norovirus). Rep. - replicate; + target signal present by real time PCR, LAC signal present or absent by real time PCR; - target signal absent by real time PCR, LAC signal present; C- contaminated; Int - Interpretation. Figure adapted from D’Agostino erfl/,2011[217,218].
79
Laboratory’ Sample A Sample B Sample C
HAdV MNoV HAdV MNoV HAdV iViNoV
RqrJ Rqi. 2 R ep .! Rep. 2 Int. R ep .! Rep, 2 Rep. 1 Rqr. 2 bit. Rep. I Rep. 2 Rep. 1 Rep, 2 bit.
TIC nr nr.
Table 2.7 Results of analysis of the non-artifîcial contaminated raspberry sample.Twenty five g of raspberry was artificially contaminated with 50 |il of HAdV and with 10 jll of extraction control (MNoV). Samples A, B, C represent the samples run in duplicate of raspberries contaminated with no HAdV human adenovirus, and spiked with MNoV murine norovirus. Rep. - mean replicate PCR; + target signal present by real time PCR, LAC signal present or absent by real time PCR; - target signal absent, LAC signal present; UC - uncontaminated; Int - Interpretation. Figure adapted from D’Agostino et al, 2011 [217, 218].
HAdV MnoV
High level: 100% concordance 88.88% concordance
Low level: 100% concordance
Blank: 100% concordance
Table 2.8 Percentage of concordance for raspberry samples of the results provided at AHVLA by the ring trial leader. The first column describes that all 3 samples tested were reported as contaminated/uncontaminated with the High/ Low /Blank of HAdV. The second column represents the total MNoV concordance.
80
2.6 Detection of spiked Human Adenovirus in liver samples
Nine batches of liver samples were spiked with an equivalent number of blind
coded samples, some of them known to contain HAdV. MNoV was used as internal
extraction control. At the end of the ring trial, the ring trial leader sent a feedback to
each participant. The nine blind coded samples were revealed to be divided into
three groups: three positive with high viral titre (5x lO' PFU), three positive with
low viral titre (5x 10 PFU) and three negative for HAdV.
From the Collaborative Trial Table 2.9 shows the results from the analysis of liver
samples artificially contaminated with 5x 10 PFU of HAdV obtained by real time
PCR with an average of 29 Ct values. All samples were correctly reported as
contaminated but one was detected at a higher Ct than expected (40). Table 2.10
shows the results from the analysis of liver samples artificially contaminated with
5x10^ PFU HAdV, in these samples the average of Ct values detected was 34. All
samples were correctly reported as contaminated as judged by the ring trial leader.
Table 2.11 shows the results from the analysis of the non-artificially contaminated
liver samples and all samples were reported as negative where no Ct values were
detected by real time PCR in all samples.
Percentages of concordance of the results provided at AHVLA and those disclosed
by the ring trial leader are shown in Table 2.12. Of three samples tested in duplicate
for each category (high, low level and blank) for HAdV all but one sample gave the
expected Ct values. One replicate of sample contaminated with High HAdV level
gave a Ct over 40, and was considered by the ring trial leader as negative. Thirteen
out of 18 duplicates tested were positive for MNoV.
81
Laboratory Sample A Sample B Sample C
HAdV MNoV HAdV MNoV HAdV MNoV
Rep. 1 Rep. 2 Rep. 1 Rep. 2 In t Rep. I Rep. 2 Rep. 1 Rep. 2 Int. Rep. 1 Rep. 2 Rep. 1 Rep. 2 In t
Table 2.9 Results of analysis of liver artificially contaminated vrith 5x10^ PFU human adenovirus (HIGH). Two hundred and fifty mg of liver tissue was artificially contaminated with 50 jitl of HAdV and with 10 [il of MNoV (the extraction control).Samples A, B, C represent the samples run in duplicate of raspberries contaminated with HIGH level of HAdV (Human Adenovirus), and spiked with MNoV (Murine Norovirus). Rep. - replicate R; + target signal present by real time PCR, lAC signal present or absent; - target signal absent by real time PCR, LAC signal present; C - sample contaminated; Int - interpretation.
Laboratory Sample A Sample B Sançle C
HAdV MNoV HAdV MNoV HAdV MNoV
Rep. 1 Rep. 2 Rep. 1 Rep. 2 InL Rep. 1 Rep. 2 Rep. 1 Rep. 2 Int. Rep. 1 Rep. 2 Rep. 1 Rep. 2 In t
Table 2.10 Results of analysis of liver artificially contaminated with 5x10^ PFU human adenovirus (LOW). Two hundred and fifty mg of liver tissue was artificially contaminated with 50 |il of HAdV and with 10 jitl of MNoV (the extraction control). Samples A, B, C represent the samples run in duplicate of raspberries contaminated with LOW level of HAdV (Human Adenovirus), and spiked with MNoV (Murine Norovirus). Rep. - replicate; + target signal present by real time PCR, lAC signal present or absent; - target signal absent by real time PCR, LAC signal present; C - sample contaminated; Int - interpretation.
82
laboatarj* Sample A Sample B Sample C
RAdV MNoV HAdV MNoV HAdV MNoV
RepJ Rep. 2 Rep. I Rep. 2 bit. Rqi. 1 Rep. 2 Rep. 1 Rep. 2 bit. Rep, 1 Rep. 2 Rep. I Rep. 2 b it
Tir - no nr.
Table 2.11 Results of analysis of the non-artificial contaminated liver sample. Twohundred and fifty mg of liver tissue was artificially contaminated with 50 jil of HAdV and with 10 |Lil of MNoV (the extraction control).Samples A, B, C represent the samples mn in duplicate of raspberries contaminated with HIGH level of HAdV (Human Adenovirus), and spiked with MNoV (Murine Norovirus). Rep.- replicate; + target signal present by real time PCR, lAC signal present or absent; - target signal absent by real time PCR, lAC signal present, UC uncontaminated; Int - interpretation.
HAdV MNoV
High level: 83.33% concordance 72.22% concordance
Low level: 100% concordance
Blank: 100% concordance
Table 2.12 Percentage of concordance for liver samples of the results provided at AHVLA by the ring trial leader. The first column describes that all 3 samples tested were reported as contaminated/uncontaminated with the High/ Low /Blank of HAdV. Second column represents the total MNoV concordance.
83
2.7 Discussion
In general, the results obtained at AHVLA proved capability of detecting the target
virus (Human Adenovirus).
The method under trial proved capable of detecting Human Adenoviruses in berry
fruit at a level of at least 10 PFU per 25 g in artificially contaminated samples.
Concordance of 100% was obtained for detection of HAdV in raspberries, and
83.3% of concordance was obtained for detection of HAdV in pork liver, this is due
to one duplicate of the sample with high titre found to be negative. However, a
lower percentage of concordance (88.8%) for the raspberries (16 of 18 duplicates
tested were positive) and 72.2% for the pork liver (13 of 18 duplicates tested were
positive) for the process control virus (Murine Norovirus) (Tables 2.8 and 2.12)
indicated that the protocol was in need of some refinement. Template inhibition (too
much template in the reaction), pipetting error and some problems related to the
extraction methods of the pork liver SOP (such as presence of fat in the samples)
could have contributed to these differences. The SOPs of the EU VITAL project
were assessed with overall good results.
The ring trial assessed the efficacy of the SOPs developed during the first year of
the project and assessed the capability of the different data gathering laboratories in
their implementation, thereby providing a system for integrating the monitoring and
control of viruses in food supply chains.
84
CHAPTER 3
Hepatitis E virus in the UK pork food chain
85
3.1 Introduction: VITAL Data gathering
After optimisation of the SOPs during the VITAL Ring Trial, this project assessed
hepatitis E virus (HEV) contamination of the pork food chain from production to
point of sale.
Current systems for the monitoring and control of foodborne contaminations are
largely based on measuring contamination with bacterial and fungal pathogens, with
significantly lower emphasis on viral pathogens. As a consequence, the risks of viral
contamination of food at various points in production chains are largely unknown,
rendering construction of control measures and codes of practice very difficult.
HEV has been implicated in zoonotic foodborne acute hepatitis from contaminated
pig products [134] (see chapter 1). This study investigated the various stages of the
pork production foodchain from farm to retail outlet, to identify HEV contamination
levels. The knowledge derived from these studies will be used to develop codes of
practice aimed at reducing or eliminating transmission of HEV via the foodborne
route.
This chapter reports the findings obtained within the VITAL project in the pork
food chain in the United Kingdom.
86
Materials and Methods
3.2 UK sampling scheme
Samples were collected in a UK pig slaughterhouse (livers and individual faecal
samples), in a UK meat processing point (muscle samples), and in a UK
supermarket and a local butcher’s shop (sausages). In addition, surface swabs were
collected at the premises, in areas where viral contamination was considered more
likely. These included work surfaces (e.g. chopping boards, scales), utensils (e.g.
knives, points) and workers’ hands {Table 3.1). All samples collected were tested
for the presence of HEV (target virus). In addition they were tested for porcine-
adenovirus (PAdV) and HAdV, indicators of pig and human faecal contamination,
respectively. Nucleic acid extraction and real-time PCR were performed according
to standardised VITAL protocols. All samples were spiked with a control virus.
Murine Norovirus (MNoV) during nucleic acid extraction, to demonstrate the
extraction of amplifiable nucleic acid.
3.2.1 Sample collection:
3.2.1.1 Slaughterhouse: 40 carcasses were selected after slaughter. Ten carcasses
were randomly selected from each of 4 batches of pigs slaughtered on that day
(corresponding to 4 different farms). From each carcass the visceral pack was
removed during the slaughter process and 2 to 3 grams of liver and 8 to 10 grams of
faeces were collected. Ten surface swab samples were also collected at this point
{Table 3.1).
3.2.1.2 Processing/cutting point: 40 carcasses were selected. Ten carcasses were
randomly selected from each of 4 batches of pigs slaughtered (corresponding to 4
87
different farms, all slaughtered in the abattoir visited within the study). From each
carcass five grams of muscle were collected. Ten surface swab samples were also
collected at this point {Table 3.1).
3.2.1.3 Point of sale: 63 sausages were collected in 11 batches from 2 different
types of retail outlet (2 UK supermarkets and 1 butcher). Sausages were collected
on different days to ensure that they were from different batches of pigs. Eight
surface swab samples were collected at this point of the pork food chain {Table 3.1).
3.3 Sample preparation and nucleic acid extraction:
3.3.1 Faeces: Two hundred and fifty mg of soft faecal contents was suspended in
2.25 ml of gentamycin-containing PBS solution and centrifuged at 3.000g x 15 min.
Nucleic acid was extracted from 140 \i\ of the supernatant using the QIAamp® viral
RNA mini kit (QIAGEN), according to the manufacturer’s instructions. (VITAL
SOP 001, Appendix C.l, VITAL SOP 010, Appendix C.5).
3.3.2 Liver, meat, sausages: The samples were prepared according to the protocol
described by Bouwknegt et al, 2007 [151]. Briefly, two hundred and fifty mg of
pork meat or liver tissue taken from 3 different meat locations were disrupted in
lysis buffer and microcarrier beads (BlOspec products, cat. no. 110791 lOzx) using a
mechanical disruptor (3.000 rpm x 50 sec). Nucleic acid was extracted from the
supernatant using the RNeasy Midi kit (QIAGEN), according to the manufacturer’s
instructions. (VITAL SOP 009, Appendix C.4, VITAL SOP Oil, Appendix C.6).
3.3.3 Swabs: A sterile gauze square was swabbed five times in the operative’s hand
and transferred to a plastic bag containing 20 ml of gentamycin- PBS solution (see
recipe in Appendix C.2 and C.8). The gauze swab was squeezed to release the
88
contents of the swab, the contents were vortexed and the eluate was centrifuged at
3.000g X 5 min and stored at -20°C.
Nucleic acid was extracted using the NucliSENSminiMAGO kit (bioMérieux),
according to the manufacturer’s instructions. (VITAL SOP 002 and VITAL SOP
013 Appendix C.2 and C.8).
3.3.4 Extraction control: Each sample was spiked with 10 pi of a culture of MNoV
(titre: 4.7x10^ PEU) before the lysis step of the extraction. Detection of MNoV
RNA by PCR was used to demonstrate extraction of amplifiable nucleic acid.
3.4 Real time PCR: all real time PCR and real time RT-PCR were duplex PCRs
containing probe of the target virus and probe for the specific lAC.
3.4.1 HEV: PCR to detect HEV in the collected samples was performed using the
RNA Ultrasense™ One-Step Quantitative RT-PCR System (Invitrogen) and the
primers and probe.
Jothikumar’s primers [220] and probes were used and they were designed on a
multiple sequence alignment of HEV genome sequences in the ORF3 region
available in GenBank [220].
- JHEV-F (5’- GGT GGT TTC TGG GGT GAC -3’) (10 pM);
-JVHEV-R (5’- AGG GGT TGG TTG GAT GAA -3’) (10 pM);
- JHEV-P (Taqman probe) (5’-FAM- TGA TTC TCA GCC CTT CGC -BG Q l-3’)
(10 pM), [220].
89
Ten pi of RNA were added to a mix containing buffer RNA Ultrasense reaction mix
(5X), lAC probe (IpM), ROX reference dye (50x), RNA Ultrasense enzyme mix
and 0.6 pi of lAC to a total volume of 20 pi.
The real time RT-PCR reaction was carried out at 50°C for 15 min, 95°C for 2 min,
and 45 cycles at 95°C for 10 sec, 55°C for 20 sec and 72°C for 15 sec {VITAL SOP
020, Appendix C .l2).
3.4.2 PAdV: PAdV PCR was performed using TaqMan Universal PCR Master Mix
(Applied Biosystems). Hundesa et al (2009) [211] primers and probe were used:
PAdV-F (5’-AAC GGC CGC TAC TGC AAG-3’), PAdV-R (5’ AGC AGC AGG
CTC TTG AGG-3’), PAdV-P (5’- FAM-CAC ATC GAG GTG CCG C-BHQl-3’)
at a final concentration of 0.225 pM. Location of oligonucleotides refers to PAdV -3
hexon (GenBank accession number AJ237815).
Ten pi of RNA were added to a mix containing buffer reaction mix (2X) ,IAC-P and
0.5 pi of lAC (0.1 pM) to a total volume of 25 pi. The PCR reaction was performed
for 2 min at 50° C, 10 min at 95° C, and 45 cycles of 15 s at 95° C, 20 s at 55° C and
20 s at 60° C [221]. (VITAL SOP 015, Appendix C.IO).
3.4.3 MNoV: Real time RT-PCR was performed as described in section 2.3.2.
Briefly the MNoV PCR was performed using RNA UltraSense^^ One-Step
Quantitative RT-PCR System (Invitrogen) and primers and probe were designed by
Baert et al in the ORFl/2 junction region: Fw-ORFl/ORF2 (5’- CAC GCC ACC
GAT CTG TTC TG-3’) (location 4972-4991), Rv-0RF1/0RF2 (5’- GCG CTG
CGC CAT CAC TC-3’) (location 5064-5080), MGB-ORF1/ORF2 ( 5 -FAM-CGC
TTT GGA ACA ATG-MBG-NFQ-3’) (location 5001-5015) [216].
90
Ten pi of RNA were added to a mix containing buffer RNA Ultrasense reaction mix
(5X), lAC probe (IpM), ROX reference dye (50X), RNA Ultrasense enzyme mix
and 0.6 pi of lAC with a total volume mix of 20 pi. The RT-PCR reaction was
carried out at 50°C for 15 min, 95°C for 2 min, and 40 cycles at 95°C for 15 s and
60°C for 1 min (VITAL SOP 21, Appendix C .l3).
3.4.4 HAdV: Real time RT-PCR was performed as described in section 2.3.1 [210].
Briefly the HAdV PCR was performed using TaqMan Universal PCR Master Mix
(Applied Biosystems). Hernroth et al (2002) [210] primers and probe were used.
Primers have been selected from the conserved region of the first part of the
Adenovirus hexon gene. AdF (5’- CWT ACA TGC ACA TCK CSG G-3’). AdR
(5’- CRC GGG CRA AYT GCA CCA G-3’), AdPl (5’- FAM- CCG GGC TCA
GGT ACT CCG AGG CGT CCT-BHQ-3’) [210] at a final concentration of 0.225
pM. Ten pi of RNA was added to a mix containing buffer reaction mix (2X), lAC-P
(0.1 uM) and 0.6 pi of lAC to a total volume of 25 pi. The RT-PCR reaction was
performed at 2 min at 50°C, 10 min at 95°C, and 45 cycles of 15 s at 95°C and 1 min
at 60°C [210]. (VITAL SOP 015, Appendix C.9). Only swabs samples were tested
for HAdV.
3.4.5 Internal assay controls: All real time RT-PCRs and real time PCRs were
performed with an internal assay control (lAC). lAC construction was explained in
section 2.3.3. Briefly the lACs (lAC RNA or DNA depending on which virus was
going to be tested) were added in each reaction to test for inhibitors of PCR
amplification and to control for contamination of any of the real time RT- PCR
reagents [212].
91
The lAC was detected by a probe that targeted a different sequence to that of the
target virus probe, and was distinguished from the target probe by using a different
fluorescent label. A MGB TaqMan probe was used for each lACP assay, at a final
concentration of 0.1 pM {VITAL SOP 22 and 23, Appendix C.14 and C.75).
The constmction of lACs was performed by Diez-Valcarce et al [212] with PCR
amplification of non-target DNA using hybrid oligonucleotide primers containing
sequences from the prfA gene from Listeria monocytogenes (nucleotide positions
2281-2348, AN AY512499). This produces a chimeric DNA molecule containing
non-target sequences flanked by target sequences complementary to the virus-
specific primers [212]. The probes, labelled with one fluorophore (e.g. FAM), and
the lAC amplicons are detected with the specific lAC probe, labelled with a
different fluorophore (e.g. VIC) detected by real time RT-PCR with specific
hydrolysis [212].
The number of lAC copies was calculated by dividing the amount of lAC in each
stock solution by the weight of one lAC molecule [212].
3.4.6 Positive standards construction: Synthetic multiple-target RNA
oligonucleotides were constructed for use as quantification standards for nucleic acid
amplification assays for Human Norovirus genogroup I and II, Hepatitis E virus.
Murine Norovirus [208]. Briefly, a synthetic DNA molecule was designed to contain
target sequences for reverse transcription real-time PCR (RT-PCR) assays for HEV
[220], hNoV GI [214] and hNoV GII [222]. The oligonucleotide was synthesised
(Burofins MWG Operon, Ebersberg, Germany) and cloned into a pCR 2.1- TOPO
plasmid (Invitrogen, Breda, The Netherlands) [208], {Figure 3.2). The RNA
concentration was determined by UV spectrophotometry in a Nanodrop ND-1000
92
spectrophotometer (ThermoScientific, Wilmington, NC, USA). The measurement
was performed in duplicate and concentration in grammes was converted to molecule
number using the following formula:
RNA molecules x— [(g/|.il)/(transcript length in nucleotides x 340)]
X 6.022 X 1(P
The standards used for the quantification of the targets viruses were designed by
Martinez-Martinez et al [208] and subsequently sent to all VITAL data gathering
laboratories.
93
Slai^italioiBe Pl’ocesidng^ cutting po in t Point of sole
Bar under qp etator insp ecting livers B ench on v ^ c h meat is sold Chopping board
Floor under carcasses in dean area Box in which cuts collected Cold-room
Doorhandle
Hand 1 Doorhandle Hands
Hand 2
Hand 3
Hand 4
Hand 1
Hand 2
Hook
Kni fe us ed immediately after scrapin g Kni fe
Knife used on livers immediately after Point
Evisceration Saw
FI oor under v^iich livers are hung Scale
Boxes in which livers are collected prior to freezing and sale
Knives
Sausage maker
Sink
Sheer
Toilet
Table 3.1 Source of surface swab samples. The first column describes all the swabs samples collected at the slaughterhouse. The second column describes all the swabs samples collected at the processing point. The third and last column represents all the swabs collected at the point of sale.
94
OCGOCCOdTCOACGCCATCTTCATTCACAAAACTGOGAGCCAGATT<3CGATCOCCCTO:CACOTGCTCAGATCTOAGAATCTCATCCATCTOAACATfc-C7X4GMCGCCA7CA7CATTrACaKM7CQQQCAQQÂQA77QCQATCTCTaTCCA7AATCCGAG<n-CATOQMGCGCA7CCAGCQKQQOGTlGCllG<iMGWXMKG(i(i(iKXTGCGAAGGGCTGACAATCAACCCGGTCACCCCAGAAACCACCOCOGCCOCAATAAGGOCOAATTCTOCAOATATCCATCACACTOOCGOCCGCTCGAOa
GCOTGGGGCCC
pCR2.1TOPO-rSTD 4295 bp
Figure 3.2 Graphie representation of pCR2.1TOPO-rSTD containing the sequence of the synthetic rFBVl RNA. The length of the plasmid pCR2.1TOPO- rSTD is 4295 bp. The viral insert was flanked by Notl and Apal sites. The sequences of the RT-qPCR assays are shown (hNoV GII— within box, hNoV GI—italics, HEV—bold and MNV-1— underlined. The sequences corresponding to the TOPO vector are in normal type. Figure taken from Martinez-Martinez et al [208].
95
Results
UK pork products (livers, muscles and sausages) and faeces collected in the various
stages of the pork food chain (slaughterhouse, processing point and point of sale)
were tested for HEV to identify the possible HEV contamination levels.
The samples that tested positive for the different PCRs are listed in Table3.3. The
table describes the 3 points of the food chain where the pork samples were
collected.
3.5 HEV detection
HEV RNA was detected at all three sites of the pork food supply chain as evidenced
by real time RT-PCR. Table 3.3 shows the number of samples where HEV RNA
was detected. In the production point (slaughterhouse) we detected 5 HEV positive
faeces in a total of 40 samples collected (13%). One of the 40 livers (2.5 %) and 1
of 10 (10%) surface swabs, a hand swab of a worker along the chain, were HEV
positive.
In the processing plant none of the 40 pig muscle samples were HEV positive,
whilst 1 of 10 (10%) surface swabs from a metal point used to hook the carcasses
were HEV positive.
At the point of sale 6/63 (9.5 %) sausages and 2/8 (25%) surface samples (knife and
slicer swabs) were HEV positive. Five of the 6 positive sausages were in 1 of the 11
batches collected. All control results showed no evidence of cross contamination.
96
3.5.1 PAdV detection
The indicator of pig faecal contamination, PAdV, was detected at 2 of 3 sites.
Thirty-nine out of 40 (98%) faeces samples were PAdV positive in the production
point as were 6 of the 40 livers (15%) and 4 of 10 (25%) surface swabs (knife swab
immediately after evisceration, 2 hand swabs and floor swab from under which pigs
are hung). At the processing point PAdV was not detected in any of the pig muscle
samples (n=40) or swab samples (n=10) tested. At point of sale PAdV was not
detected in any of the sausages (n=63) tested but 1 of 8 swab samples (12.5 %) from
the door handle of the cold room was PAdV positive (Table 3. 3).
The highest number of PAdV positive swabs was observed in the production point
(4/ 10) whilst no PAdV was detected in any swab at the processing point (Table
3.5.2 HAdV detection
Swabs collected in the three points of the pork food chain were tested for HAdV,
but presence of virus was not detected in any of the swabs collected, as shown by
real time PCR. (Table 3.3).
97
Point in chain Sample type PAdVDNA + /n(% ) HEVRNA + /n(% )HAdV+/n
Production point (slaughterhouse)
Faeces 3 9 /4 0 (98) 5 /4 0 (12.5) -
Liver 6 /4 0 (15) 1 /40 (2 .5 ) -
Surface swab 4 /1 0 (40) 1 /1 0 (10) 0 /1 0
Processing point Muscle 0 /4 0 0 /4 0 -
Surface swab 0 /1 0 1 /1 0 (10) 0 /1 0
Point o f sale Sausage 0 /6 3 6 / 63 (9.5) -
Surface swab 1 /8 (1 3 ) 2 /8 (2 5 ) 0 / 8
Table 3.3 Number of samples PAdV, HEV and HAdV positive. The first column
represents the point of the chain: production point (sloughterhouse), processing
point and point of sale. The second column describes the sample type: faeces, liver,
surface swabs of the slaughterhouse. In addition it describes muscle and surface
swabs of the processing point and sausages and surface swabs of the point of sale.
The third columns describes the number and percentage of sample tested PAdV +
(positive)/-(negative) as assessed by real time RT-PCR. The fourth column
represents the number and percentage of samples tested HEV + (positive)/-
(negative). The last column describes the number and percentage of samples tested
HAdV +(positive)/-(negative).
98
3.6 Discussion
The presence of HEV and/or faecal contamination was investigated at three points
in the pork food supply chain in the UK, in the slaughterhouse, in the processing
plant and at the point of retail sale. Samples of pig liver and faeces were collected at
slaughter, samples of pig muscle (meat) during processing, and pork sausages at the
point of sale. In addition, swab samples were collected from various surfaces
considered likely sources of HEV and/or faecal contamination. All samples were
tested by real time RT-PCR for HEV and real time PCR for PAdV, and for HAdV
(swab samples only).
HEV has a high seroprevalence in the UK pig herds [121]. In this study HEV was
detected in the faeces of 12.5% of pigs at slaughter-weight. In a previous study
conducted in the UK [223] a similar percentage (13%) [121] of faeces collected at
slaughter weight was positive for HEV. The presence of HEV in pig liver at
slaughter has not been investigated in the UK prior to this study, but at 12.5%
indicates that a high percentage of HEV faeces-positive slaughter pigs may have
HEV present in the liver.
The failure to detect HEV in pig meat in the cutting (processing) plant compared to
the detection in 9.5% of pork sausages at the point of sale is interesting. Liver is not
permitted as a constituent of pork sausages in the EU (Commission Directive
2001/101/EC), but it is possible that the samples of muscle tissue scanned for HEV
at the processing point were not as representative as those for sausage meat, where
mixing and mincing of meat occurs prior to sausage production. The sausages were
collected on different days to ensure they originated from different batches of pigs.
99
The choice of sausages as the type of point of sale pork product investigated for
HEV was made because this product is consumed widely across the UK, unlike pig
liver for instance, and a 9.5% HEV detection rate in pork sausages at point of sale
could be a cause for concern.
In terms of viral transmission potential, the surface swabs provided evidence that
both PAdV and HEV contamination does occur in the slaughterhouse and
interestingly at the point of sale. In the processing point HEV was detected in just 1
surface swab. The 98% positive rate recorded for pig faeces with the PAdV indicator
provides validation of this approach for detection of faecal contamination of porcine
origin. The detection of PAdV on a door handle swab is interesting. This may have
been the result of transfer from a contaminated pig carcass, but the in-test controls
and method of sampling exclude this contamination as a source of the HEV in the
sausage meat.
No evidence of human faecal contamination was detected in any sample at any
point in the chain, indicating that personal hygiene standards were high, and that the
HEV detected was unlikely to have come from human contamination of the
samples.
In industrialized regions, although the incidence of clinical hepatitis E in humans is
low, the seroprevalence is relatively high, indicating a high proportion of subclinical
disease and/or underdiagnosis. Whilst it is likely that a small proportion of this
exposure to HEV results from travel to or migration from, endemic regions [117,
142], this still leaves a substantial level of exposure to HEV that appears to have an
indigenous source.
1 0 0
Pork food products have been shown to contain HEV in several industrialized
regions, including the UK and recently a cluster of cases in Southern France
associated with the consumption of raw figatelli, a pig liver sausage mainly eaten
raw [134]. However, these pork foodborne reports have to date involved pig liver,
and although in other studies pig muscle tissue was shown to carry HEV [224], this
current study shows that in the UK, a proportion of a point of sale pork product with
a high volume, nationwide consumption (>193,000 tonnes of pork sausages
consumed in GB in the year to February 2012, BPEX, UK), may be contaminated
with HEV.
In efforts to determine the transmission routes of autochthonous hepatitis E, this
data does indicate that the potential for exposure to HEV via consumption of
undercooked pork sausages does exist in the UK.
It has to be remembered that the numbers of samples tested for viral contamination
were relatively small in this study, so these results should be taken as indicators
only, and for greater confidence in the results, a greater number of samples would
have to be tested.
A corollary question to ask from these observations is in relation to the viability of
the HEV detected in the pork sausages. Feagins et al [85, 138] have modelled the
survival of HEV under various times and temperatures of cooking [85] observing
that HEV is not completely inactivated when heated at 56 ° C for 1 hour. So from
this evidence, adequate cooking of pork sausage should at least remove the threat of
infection. Whilst the findings reported here do not provide any indications regarding
the viability of the detected HEV, viability of HEV in the positive samples from this
1 0 1
study was determined using a 3D cell culture system which we have shown is more
sensitive than monolayer culture for in-vitro propagation of HEV {Chapter 4).
1 0 2
CHAPTER 4
Replication of Hepatitis E virus in three-
dimensional cell cultures system
103
4.1 Introduction
In addition to the data gathering on the presence of HEV in the food chain, this
project also aimed to develop a 3D cell culture system able to support the
replication of HEV and investigate if HEV detected by real time RT-PCR in pork
products corresponds to the presence of viable virus.
To date attempts to confirm the routes of transmission in epidemiological
investigations of cases of autochthonous hepatitis E in developed regions have
failed [113] but it is suggested that there may be several routes of zoonotic
transmission, contributing to exposure to HEV and disease in humans [125]
{Chapter 1).
A major impediment to the investigation of potential HEV routes of transmission
from pigs to humans is the limited knowledge relating to the survival of the virus in
pig tissues and faeces and in the environment. To a large extent this is due to the
difficulty in propagating HEV in-vitro. A method using hepatocellular carcinoma
HepG2/C3A has been reported by Emerson et al [225]. However, the infection of
HepG2/C3A with HEV was not able to be repeated at AHVLA (data provided by
Malcolm Banks). Tanaka et al [197] reported that PLC/PRF5 cells were able to
support replication of HEV. Moreover, Tanaka et al [197] reported that the virus
progeny was infectious, as demonstrated by passage in the PLC/PRF/5 cells [197].
Infection of PLC/PRF/5 using as inoculum swine faeces, instead of human faeces,
was attempted at AHVLA without success. There are several reports in the literature
demonstrating the potential of a 3D culture system utilising a Rotating Wall Vessel
(RWV), for the growth of fastidious viruses [201, 226-228]. This RWV low-shear,
suspension culture system was introduced as a novel method to cultivate cell lines
104
able to support bacterial replication in varying shear conditions [200]. The RWV is
a cylindrical bioreactor that is rotated on an axis parallel with the ground.
Subsequently, a solid body mass rotation of the culture medium is obtained, creating
a low-fluid-shear environment {Figure 1.10, chapter 1) [200, 229, 230]. The cells
are maintained in suspension by the resolution of the centrifugal, gravitational and
Coriolis effects, so cells placed in the RWV bioreactor experience minimal
mechanical stresses and high mass transport (of nutrients, oxygen etc). It has been
shown that several 3D lines changed molecular mechanisms in the transduction of
mechanical culture conditions into cellular effects [231]. Possible changes of the 3D
cells could be in cell cycle and cell death pathways or upstream regulation of
secondary messengers [231]. The cells are attached to porous, collagen-coated
microcarrier beads and this allow the cells to assemble into tissue-like aggregates
with a functionality similar to tissues in the human body [231]. The system offers a
potential for in vitro cultivation of HEV, therefore, we investigated the use of 3D
cultures as a means of improving the efficiency of HEV propagation.
Since that literature reported that the 3D cell culture system is an efficient and
reliable cell culture system able to support the propagation of viruses, during my
PhD project I aimed to:
1) Evaluate a new 3D culture system to assess HEV infectivity. Homogenate of
HEV positive pig liver obtained from an animal experiment was used as inoculum
to evaluate the 3D cell culture system. This was needed to verify if the HEV
detected by PCR in pig and environmental samples was infectious.
2) Compare the efficiency of the 3D system to the conventional 2D cell culture
system (PLC/PRF/5 cells grew in monolayer). In addition, cells grown in the 3D
105
system were transferred to a 2D system and infected. Since that the 3D cell culture is
difficult for a number of reasons (i.e limited number of samples for each experiment)
the testing of 3D transferred to 2D was an attempt to exploit these cell
receptor/differentiation advantages in a format i.e. microplate, that would allow for
larger numbers of samples to be tested.
4.2 Use of the 3D Culture system to investigate the viability of HEV detected by
RT-PCR in UK pork sausage and French liver sausage (figatelli)
The detection of HEV RNA by real time RT-PCR in six of 63 pork sausages
collected at UK retail outlets {Section 3.5.1) needed further investigation to clarify
the risks of foodborne transmission of HEV. The concern was: is the virus viable or
is it present but inactivated?
This section describes the work undertaken to use the 3D culture system as a means
of determining the infectivity of the HEV real time RT-PCR positive UK sausages.
Pork liver sausages, known as figatelli, which are often eaten raw after cold
smoking, have been linked to cases of clinical hepatitis E in France. A collaboration
was made with the French ANSES Institute in Paris. A contact was made with Dr
Nicole Pavio of ANSES, with the suggestion that by using the 3D system, the
viability of HEV detected in the figatelli could be confirmed. The figatelli saiisages
were then sent to AHVLA for further investigations.
The main aim of this section was testing via the 3D cell culture system if the UK
sausages collected during the VITAL data gathering {chapter 3) and French figatelli
contains viable virus, for this reason the UK and French sausages were used as
inoculum to infect 3D cell cultures and evaluate the infectivity of those samples.
106
Materials and Methods
4.3 Propagation of HEV in cell cultures: The Alexander hepatocarcinoma cell line
(PLC/PRF/5) from the American Type Culture Collection (ATCC 8024) was used
in the experiments. The cells were initially grown as 2D monolayers inside
conventional cell culture flasks (BD Bioscience, USA) in the complete growth
medium GTSF-2 [228] {Table 4.1) in preparation for seeding into the Rotating Wall
Vessel (RWV, Synthecon, Inc, Houston TX, USA), at 3TC in a 5% CO2
environment. Cells were trypsinised at 95% confluence and resuspended in fresh
medium at a density of 2x10^ cells/ml, the cell density required before being
transferred in the vessel. PLC/PRF/5 cells were introduced into a RWV cell culture
vessel with 10 mg/ml of porous Cytodex-3 microcarrier beads (collagen type-I-
coated porous microspheres, average size 175 \xm in diameter - Cat number C0646,
Sigma). Cells were cultured in the RWV in GTSF-2 at 37°C and 5% C02, with a
rotation speed appropriate to maintain the cell aggregates in suspension during the
entire culture duration (approximately 17-25 rotations/min initially with subsequent
increase to 27-35 rotations/min after the infection) [232]. The cells were grown for
at least 28 days before being infected to allow differentiation as described by
Navran [232]. For the 2D system experiments the cells were seeded in 48-well
plates, each well containing 2x10" cells.
4.3.1 Comparison of efficiency of the 3D and 2D cell culture for HEV
replication: The first experiment aimed to compare the efficiency of the 3D and 2D
cell culture systems when infected with the same HEV PCR positive inoculum.
Details of the protocol used are listed below.
107
4.3.2 Inoculum preparation: The positive HEV pig liver sample obtained from an
animal experiment was provided by Central Veterinary Institute, Wageningen
University and Research Centre [86]. A sample of the liver (0.3g) was homogenized
manually using a pestle and mortar in 2.7 ml of GTSF-2 media. The homogenate
was centrifuged at 8.000 x g for 3 minutes and the supernatant was filtered through
a sterile spin-X centrifuge tube filter (0.22pm; Costar) at 10.000 x g at 4°C for 15-
25 min. Two and half ml of inoculum was used to inoculate the cells.
4.3.3 Infection of the cells:
3D: the medium was removed from the vessels and 2.5 ml of viral inoculum was
added to the cells in the vessel. One vessel was inoculated with the virus and one
was used as a negative control (2.5 ml GTSF-2 non-infected media). Cells were
incubated for two hours at 35.5°C and inserted into the Rotating Wall Vessel. After
two hours the vessel was filled with 47.5 ml of fresh medium. Subsamples of
medium (140 pi) were collected in duplicate and added to 560 pi of lysis buffer
(Viral RNA, Qiagen), and stored at -20°C (0 days post infection, dpi). Samples were
collected as described above at the following dpi: 3, 6, 9, 12, 15, 18, 24, 27, 30, 32,
36, 39, 42,46,49, 58, 62, 67, 70, 85, 107, 126, 134, 155 and 175.
2D: the medium was removed from the cells and 200 pi of virus inoculum was
added to each well of a 48 well plate. One column of the plate was used as negative
control (200 pi of GTSF-2 non-infected media). The plate was incubated at 35.5°C
for two hours and each well was replenished with fresh medium (300 pi) without
removing the inoculum. A subsample (140 pi) of medium was collected in duplicate
and added to 560 pi of Lysis buffer and stored at -20°C (0 dpi). Fresh medium (280
1 0 8
pi) was added to replace the medium that was removed. Samples were collected
twice a week for 27 days.
4.3.4 Comparison of 3D, 2D and 3D transferred to 2D cell cultures for HEV
replication: In a further experiment, the 3D cells from a 3D vessel were transferred
to a plate to be infected simultaneously with the 3D cell culture and the
conventional 2D cell culture.
3D cells transferred to 2D: Each well of the plate contained 50 pi of cells and media
from one vessel (33 days of differentiation in the 3D system) plus 450 pi of GTSF-2
media. The plate was left in the incubator for 6 hours at 37°C to allow cell adhesion.
The preparation of the 3D and 2D cell culture was performed as described in section
4..?..?.
The inoculum was the supernatant (real time RT-PCR positive for HEV) of the cells
of the first experiment at 58 dpi {section 4.3.1).
The virus was used neat and diluted from 10' to 10' in the 2D and 3D transferred
to 2D systems. In the 3D system only four vessels were available and they were
infected with the virus undiluted, diluted 10 times (10'^), diluted 100 times (10'^)
and a non-infected control.
The infection of the three systems for HEV replication was performed following the
protocol described in section 4.3.3. The experiment was carried out for 40 days and
subsamples (140 pi) were collected at the following dpi: 0, 5, 8, 12, 15, 19, 22, 26,
29, 33, 36 and 40, for both 2D cells and 3D cells transferred to 2D, while for the 3D
cells the experiment lasted 96 days and samples were collected once a week.
109
4.3.5 RNA extraction from supernatant of 3D cell cultures, 2D cell cultures and
3D cell transferred to 2D system infected with HEV: Nucleic acid extraction was
performed according the Qiagen viral RNA kit (Qiagen) protocol. A subsample
(140 pi) of medium was collected in duplicate, added to 560 pi of Lysis buffer and
stored at -20°C (0 dpi).
4.3.6 Real Time RT-PCR: The real time RT-PCR reaction was set up according to
the protocol of Jothikumar et al 2006 [220] using the Superscript III Platinum one-
step quantitative RT-PCR kit (Invitrogen). The RT-PCR reaction was set up and
performed according to the manufacturer’s instructions. Jothikumar’s primers and
probes were used and they were designed on a multiple sequence alignment of HEV
genome sequences in the ORF3 region available in GenBank [220].
-JHEV-F (5’- GGT GGT TTC TGG GGT GAC -3’)
-JVHEV-R (5’- AGG GGT TGG TTG GAT GAA -3’)
- JHEV-P (Taqman probe) (5’-FAM- TGA TTC TCA GCC CTT CGC -BG Q l-3’).
The 20 pi reaction contained 10 pi of 2x RT-PCR kit Master Mix (Qiagen), 0.2pl of
enzyme, 2pl of RNA, and primers and probe at concentrations of 250 and 100 nM,
respectively. The real time RT-PCR reaction was carried out at 50°C for 15 min,
95°C for 2 min, and 45 cycles at 95°C for 10 sec, 55°C for 20 sec and 72°C for 15
sec.
Negative (water) and positive (synthetic RNA constructed by Martinez-Martinez et
al [208]) controls were included in each run.
4.3.7 Positive standard and copy number quantification: The standard used for
the quantification of the HEV nucleic was constructed by Martinez-Martinez et al
1 1 0
[208]. The plasmid construction was described in section 3.4.6. Briefly the
construction of a plasmid for transcription of synthetic RNA was performed. A
synthetic DNA molecule was designed to contain target sequences for real-time RT
PCR assays for HEV [220], hNoV GI [214] and hNoV GII [222] [208]. The
oligonucleotide was synthesised (Eurofins MWG Operon, Ebersberg, Germany) and
cloned into a pCR 2.1- TOPO plasmid (Invitrogen, Breda, The Netherlands).
The RNA concentration was determined by UV spectrophotometry in a Nanodrop
ND-1000 spectrophotometer (ThermoScientific, Wilmington, NC, USA). The
measurement was performed in duplicate and concentration in grammes was
converted to molecule number using the following formula:
RNA molecules x |il ^= [(g/|il)/(transcript lengtii in nucleotides x 340)]
>< 6 .02:2 >( 1()23
The 20 gl reaction contained 10 gl of 2x RT-PCR kit Master Mix (Qiagen), 0.2ml of
enzyme, 2gl of standard, and primers and probe at concentrations of 250 and 100
nM, respectively. The real time RT-PCR reaction was carried out at 50°C for 15 min,
95°C for 2 min, and 45 cycles at 95°C for 10 sec, 55°C for 20 sec and 12°C for 15
sec.
This synthetic plasmid, as previously mentioned, was designed by Martinez-
Martinez et al [208] as part of the VITAL project.
The copy number of the samples was extrapolated from a standard curve produced
from logio titrations of cloned amplicon. Copy number (HEV RNA copies per ml
sample) was calculated as follows:
1 1 1
copy number per 2 gl template RNA
X 30 (per 60 gl extraction elute = 140 gl sample, HEV positive supernatant)
X 7.14 (1000 gl/140 gl)
4.3.8 Definition of Ct values: Cycle threshold (Ct) is a measure of the number of
PCR cycles (in Real-time RT-PCRs) needed to observe a fluorescent signal. Our Ct
+/- cut off value was fixed at 40 to avoid false positive and non-specific signal; this
means that only sample with Ct < or equal to 40 were considered positive. The Ct
values were determined fixing the threshold just above the non-specific background
fluorescence.
4.4 Materials and Methods to investigate the viability of HEV in UK sausages
and figatelli samples
4.4.1 Cell Preparation: cell culture preparation was performed as described in
section 4.3.
4.4.2 Inoculum preparation of figatelli sample and UK sausages: After one year
from the first HEV RNA detection only three of the six sausages (section 3.1) were
still HEV RNA positive by real time RT-PCR, possibly due the degradation of the
HEV RNA after prolonged storage. The HEV real time RT-PCR-positive figatelli
samples were obtained from a French processing point (Dr Nicole Pavio, ANSES,
France). Two and half g of each of the four figatelli samples (four different subtypes
of genotype 3) and the three UK sausages were homogenized manually using a
pestle and mortar in 5ml of GTSF-2 media. The homogenate was centrifuged at
1 1 2
8.000 X g for 3 minutes and the supernatant was filtered through 1.2 gm, 0.45 \im
and 0.2 gm filters to reduce the risk of bacterial contamination.
4.4.3 Cell inoculation: cell inoculation was set up as described in section 4.3.3.
4.4.4 Determination of infectivity of progeny virus: To evaluate the infectivity of
progeny virus from the primary inoculations, HEV real time RT-PCR positive
supernatant from dpi 16, of one sample named as figatelli 84, was used to infect
fresh 3D PLC/PRF5 cultures. Two and half ml of HEV positive supernatant was
used as inoculum to infect the 3D cell cultures. The cell inoculation was performed
as described in sections 4.3.3.
4.4.5 RNA extraction and real time RT-PCR: HEV RNA extraction and real time
RT-PCR was performed as described in section 4.3.5 and 4.3.6.
4.4.6 Electron microscopy: In order to provide further confirmation of the validity
of the real time RT-PCR results, a sample was sent to Reimar Johne and Jhone
Reetz at the Bundesinstitut fur Risikobewertung (BfR) in Germany and submitted to
electron microscopy examination. Supernatant of the cell cultures collected at 33
dpi was exposed to polioformcarbon-coated, 400-mesh copper grids (Plano GmbH,
Wetzlar, Germany) for 10 min, fixed with 2,5% aqueous glutaraldehyde (Electron
Microscopy Science Company , Germany) solution for 1 min and stained with 2%
aqueous uranyl acetate solution (Electron Microscopy Science Company,
Germany) for 1 min. The specimens were examined by transmission electron
microscopy using a JEM-1010 (JEOL, Tokyo, Japan) at 80 kV accelerated voltage.
113
ComponentConcentration or volume ^
Source/order number of designation
MEM -0 1 supplemented with 2.25 g/liter of L-Gln 400 ml (40%) Sigma
L-15 600 ml (60%) GIBCO
NaHC03 1.35gperL Sigma/S-5761
HEPES 3.0g Research Organic s/6003H-2
Folic Acid 67 /ig/ml lOOul SIGMA/F-8758
0.5% Nicotinic Acid 0.66 ul Sigma/N-4126
Bactopeptone 0.6g Difco/0118-01
I-inositol 0.024g Sigma/I-5125
Fructose 0.13g Sigma/F-3510
Galactose 0.25g Sigma/ G-5388
D-Glucose 0.33g Sigma/G-5250
200mML-Gln [2Mm] 18.3ml Sigma/G-5763
Gentamycin 1ml Gibco/600-5750AD
Fungizone 1ml Sigma A 2942
Ins ulin - Trans ferrin- S o dium-S e lenite (ITS S) 5ml Sigma/1-1884
Fetal bovine serum (FBS)
6% during differentation 2% after infection Autogenbioclear
Table 4.1 GTSF-2 complex medium with relative supplements \
Concentrations are provided for the preparation of approximate by 1 L volume of
medium.
114
Results
4.5 Comparison of HEV replication in 3D cell and 2D cell culture systems
This experiment was set up in order to assess the efficiency of HEV replication in
the 3D cells culture system and to compare it to the conventional 2D cell culture
system.
After the inoculation with homogenate of HEV positive liver obtained from an
animal experiment, in the 3D culture system, HEV nucleic acid was detected in the
supernatant of the infected cells at all collection points (Figure 4.2 A). In contrast,
no HEV nucleic acid was detected at any collection points in the 2D culture system
(Figure 4.2 E), for this reason the 2D experiment was terminated at 27 dpi. In the
3D culture system, the virus copy number showed a significant increase between 24
to 39 dpi, peaking at 1.5 xlO^ viral RNA copies/ml of supernatant, followed by a
second increase between 100 dpi to 155 dpi, peaking at 2.0 xlO^ viral RNA
copies/ml of supernatant (Figure 4.2 A). HEV nucleic acid was still detectable at
175 dpi. In both culture systems, the non-infected control cells remained HEV
negative throughout the experiment. The real time RT-PCR controls (synthetic
plasmid and water) performed as expected, with the positive control being positive
while no Ct values were detected for the water samples (real time RT-PCR negative
control).
4.6 Evaluation of the infectivity of the viral progeny and comparison of HEV
replication in 3D and 2D culture systems and 3D cells transferred into 2D
During the secondary infection, where the inoculum was the supernatant collected at
58 dpi from a vessel of the previous experiment (section 4.5), viral RNA was
detected by real time RT-PCR at all dpi in the 3D cell culture system (Figure 4.3 A),
115
and at all dilutions of inoculum {Figure 4.3 A, shows the Ct values and Figure 4.4 A
shows the copy number/ml). In the supernatant of the cells infected with undiluted
inoculum the viral RNA copies/ml was low and constant throughout all the
experiment. In this experiment no trend was observed, the expectation was that in
the non diluted inoculum the copy number detected by real time RT-PCR would be
the highest followed by the sample infected with inoculum diluted 1 in 10 (10'^) and
the lowest copy number should have been detected in the sample infected with
inoculum diluted 1 in 100 (10'^).
In the supernatant of 3D cells infected with inoculum diluted one in ten, the number
of viral RNA copies/ml increased sharply between 61 and 82 dpi, peaking at 3.5
xlO \ After 82 dpi the number of genome copies in this sample remained constant
{Figure 4.3 and Figure 4.4 A). The number of copies in the supernatant of the
inoculum diluted one in 100 (10'^) was higher compared to the other 2 inocula
during almost all the follow up of the experiment. Several other peaks were
observed throughout the incubation period suggesting HEV replication.
In the 3D cells transferred to 2D system the trend was similar for the undiluted and
10' dilution of inoculum and Ct values indicative of a positive signal were detected
at all dpi. In the supernatant of the 3D cells transferred to 2D infected with
inoculum diluted 100 times, Ct values were detected at all dpi but 12 dpi. However,
all the other dilutions (from 10" to 10' dilutions) were considered negative as Ct
values were above 40 for almost all dpi (Figure 4.3 C).
In the 2D system, the Ct values (Ct values range 25-35) for undiluted and 10'
dilution of inoculum remained almost unchanged, throughout the experiment whilst
116
the values for the 10' were negative at 26 dpi. All the other dilutions were equal to
or above 40 and for this reason considered as being negative (Figure 4.3 B).
117
2 50E+08
150E+08
OL 1 OOE+08
OOE+07
0 O O E + 0 0 I # I # I # i 0 1 # 1 — I— I— — r— I— r —i— i— i— i— i— i— i— r
3D cells
2 0 cells
days post infection
ICXG COPY number/ml 0 3 6 9 12 15 18 24 273D cells 0 0000 00033 0 0046 00033 0 0240 0 0480 36000 822% 11312D cells 000 000 000 000 0 00 000 0.00 000 000
Figure 4.2 Cq)v niiinbeis of HEV genome ml detected by real time RT-PCR in
the 3D culture system. 3D and ZD PLC/PRF/5 cell cultures were infected with
homogenate of HEV positive liver and supernatant was tested by real time RT-PCR.
A ------ represents the copy number/ml of supernatant of the 3D PLC/PRF/5,
represents the copy number/ml of the supernatant of 2D cells. Both samples
were infected with homogenate of HEV pork liver obtained from an experimentally
infected animal.
B Copy numbers per ml detected in the supernatant of 3D cells and 2D cells.
The table shows 10 viral copy numbers per ml detected by real time RT-PCR. It
details the viral copy number displayed in Figure 4.2 A to better describe that the
copy number/ml observed in the first 27 days were not zero.
118
20
2 5 -
3D cells» 3 0 - u n d i l u t e d
10''-!10''-2Ic 3 5 -
4 0 -
4 57 14 21 27 34 4 0 4 7 5 4 61 68 75 82 8 9 960
days p o s t in fe c tio n
T ’ W ' W A \ ,
3D cells transferred to 2D
■ u n d i l u t e d
- 1 0 ''- 1
■ 1 0 ''-2
■ 1 0 ''-3■ 10 ''-4
■ 10 ''-5
- 10''-6
12 15 19 22days p o s t in fe c tio n
2 0 -r -
2 5 -
3 0 -
Ô 3 5 -
4 0 -
3 60 5 8 12 1 5 19 22 26 2 9 33 40
2D cells
- u n d i l u t e d
- 1 0 » -1
. 10''-2 - 10 ''-3 . 10 ''-4 . 1 0 ''-5 . 1 0 ''-6
days p o s t in fe c tio n
Figure 4.3 Comparison of Ct values detected in the supernatant of the 3 different systems infected with different dilutions of inoculum. The 3 different cell cultures were infected with HEV positive supernatant diluted serial times obtained in the previous experiment. The supernatant collected at different days post infection (X axis) was tested by real time RT-PCR. The graphs represent the Ct values during the course of the experiment. A Ct values in the 3 cell cultures system black dashed line represent the cut off, B Ct values in the 3D cells transferred into 2D, black dashed line represents the cut o ff; C Ct values in the 2D cells cultures, black dashed line represent the cut off.
119
I 2.50E-K)7 - SI 2.00E+07 -
I 1.50E+07 -
l.OOE+O'
OOOE+OO
0 7 14 21 34 40 47 54 61 68 75 82 89 96
■ undiluted
10-1 • 10-2
c b \- s p o s t i n f e c t i o n
10^6 copy number/ml 0 dpi 7 14 21 27 34 40 47 54 61 68 75 82 89 96 dpi
Undiluted 0.34 0.99 0.19 0.42 0.45 0.39 0.27 0.18 0.20 0 26 0.79 1.55 1.83 1.64 0.47
0 38 0.11 1.04 1.47 083 0.14 0 06 0.00 0.55 0 99 19.97 36.21 0.29 0.92 0.01
iO'^-2 8.21 1,38 11.36 17.18 8.29 0.48 12.23 22.28 28.69 31.03 13.11 1.08 9.77 12 64 3.42
Figiire 4.4 copy num bers m l detected in th e supernntnnt o f the 3D cell cu ltures infected w iüi m i diluted m oculm n progeny, inoculm n diluted 10' or inocu lum diluted ICr .
A Co])y num bers/m l of H E V genom e detected b y R T-PC R in the 3D culture system (W hile figure 4.3 A describes the Ct values figure 4.4 A describes the copy num ber/m l observed in th e 3D cell culture during the course o f th e experim ent).
The 3D cells w ere infected w ith HEV positive supernatant undiluted, diluted 10
tim es (10'^) and diluted 100 tim es (10"^). The supernatant w as tested by real tim e
R T -P C R . represents supernatant of PLC/PRP/5 infected w ith hom ogenate o fH EV pork liver obtained from the supernatant o f the first experim ent (inoculum
undiluted). ------ represents supernatant o f cells infected w ith inoculum diluted 10
tim es and describes supernatant o f cells infected w ith inoculum diluted 100
times.
B Co%)y m im bers per m l detected iir the serial d ilution exi>erimeiit. T he table
shows 10^ viral copy num bers per m l detected by real tim e RT-PCR. I t details the
viral copy num ber displayed in F igure 4 .4 A.
1 2 0
4.7 Results of the use of the 3D cell culture system to investigate the viability of
HEV in UK sausages and French liver sausages (figatelli)
Homogenates of 3 UK sausages and 4 French figatelli were used as inoculum to
infect PLC/PRF/5 cells in the 3D cells culture system.
HEV RNA was detected by real time RT-PCR only in the supernatant of the 3D
cells infected with 1 of 3 French figatelli samples (figatelli 84). HEV RNA was
detected at all dpi in the cells inoculated with the figatelli homogenate (Figure 4.5
A). At 0 dpi the viral RNA copies were 6 . 4 x 1 /ml, the HEV viral RNA copy
number fell to 3.35x10^ /ml on 5 dpi, and then began to increase on dpi 26 to a peak
of 1.75x10^ /ml, at dpi 49. At the last sampling point on dpi 55, the copy number
was 8.9x10"^/ml. No further collections were performed due to mould contamination
in the vessels.
The cells infected with progeny virus from the original figatelli homogenate
cultures had detectable HEV RNA on all dpi tested. The copy numbers remained
fairly constant from just after inoculation (0 dpi) to the final reading at dpi 35, and
varied from 4.14x10^ to 1.71x10^ copies per ml suggesting viral replication (a slight
increase of HEV RNA copy numbers/ml was observed).
HEV RNA was detected in the supernatant of 3D cells infected with the UK
sausages until five dpi only in two out of three sausages used as inoculum to infect
the 3D cells (Figure 4.6).
4.7.1 HEV viral particles observed by electron microscopy: In the EM picture
(Figure 4.7) four HEV viral particles were detected by EM. The sample tested by
EM was supernatant of figatelli 84 collected at 33 dpi.
1 2 1
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UK 46 UK47
■ figatelli 87
• figatelli 100
■figatelli 116
days post infection
Figure 4.6 Supernatant of cells infected with UK sausages and French sausages tested hy real time RT PCR. Ct values detected by real time RT-PCR in the supernatant of PLC/PRF/5 cells infected with UK and French sausages (samples UK sausages 44, 46, 47, and French sausages 87, 100, 116)
123
'4'5
'4 ^
(
i-
. A~C
Figure 4.7 HEV-like particles in HEV positive supernatant obtained from the
3D cell culture system infected with homogenate of Hgatelli 84. Four HEV like
particles were observed by electron microscopy in the supernatant of 3D cells
infected with homogenate of HEV positive figatelli and collected at 33 dpi. The
arrows show four different HEV-like viral particles.
1 2 4
4.8 Discussion
4.8.1 HEV replication in the 3D cell culture system
The aims of this work were to investigate an in vitro 3D culture system to facilitate
studies into the viability of HEV detected by real time RT-PCR in pig products, and
to compare the system with the conventional 2D system.
In a study by Tanaka et al [III, 197] the potential of in vitro replication of HEV in
2D cultures of 21 cell lines, including PLC/PRF/5 (human hepatocarcinoma cell
line) [233] was investigated. The PLC/PRF/5 cell line was able to support in vitro
replication of HEV, yielding a high titre of HEV from 14 dpi to the end of the
observation period at 88 dpi [197]. However, the methodology described by Tanaka
et al [111] proved difficult to reproduce in our laboratory, prompting the
investigation of 3D culture system for more efficient virus propagation as described
by Straub [231].
The real-time RT-PCR results obtained in the 3D cultures, inoculated with
homogenised liver samples from an experimentally infected pig, showed detectable
HEV RNA at all dpi. In contrast, in the 2D system infected in parallel with the same
sample, HEV RNA was not detectable at any dpi.
In the primary inoculation there was evidence of virus replication by the
maintenance of the HEV RNA copy number close to the 0 dpi titre up to 24 dpi,
followed by a burst of replication peaking at 36 dpi with a decline back to the level
observed at 0 dpi at 42 dpi. This decline may have been due to synchronized
infection of uninfected cells and subsequent internalization of virus. Thereafter the
copy number gradually increased to reach a peak at 136 dpi (2 x 10 viral RNA
125
copies number per ml) followed by a gradual decline back to below the level
detected at 0 dpi by the end of the experiment at 175 dpi, when probably cell and
virus damage caused the rate of viral RNA production to be exceeded by that of
degradation.
By setting up a secondary inoculation {Figure 4.3 A), using progeny virus from the
first replication round, the viability of the virus detected by real time RT-PCR was
demonstrated. This data illustrates that in our hands, the 3D system was more
efficient in terms of demonstration of infectivity compared to the 2D system, since
the virus was able to replicate up to five months in the 3D cell culture system with
higher copy number/ml detectable by real time RT-PCR. Other studies have also
demonstrated that the 3D cell culture system is a useful tool in the cultivation of
fastidious bacterial and viral pathogens [199, 231, 234].
In the secondary passage titration experiment, the efficiency of propagation
appeared to be indirectly proportional to the concentration of the inoculum. Walker
et al [235] described that, depending on the cell line and the concentration of the
cells, a lower multiplicity of infection (MOI) can ultimately result in a higher peak
titre during the incubation period, and this phenomenon was also demonstrated by
others using suspension cultures [235]. The inoculum with the highest dilution
showed a phasic pattern of viral RNA copies number/ml not dissimilar to that of the
primary inoculation, whilst the intermediate dilution maintained the RNA copy
numbers/ml until a late single peak between 61 and 82 dpi. The undiluted inoculum
maintained the HEV copy at or around that of the TO level for the duration of the
experiment. Higher MOI represented by high copy number in real time RT-PCR
could be attributable to the same phenomenon explained by Walker et al [235].
126
The inverse relationship between inoculum concentration and efficiency of
replication, as measured by HEV copy number in the cultures, may indicate the
presence of a high proportion of non-viable HEV particles in the inoculum. These
could have a direct interfering effect by physical competition for receptor sites
[236], or an indirect effect by induction of the interferon response [236]. Dilution of
the inoculum would have the effect of reducing this interference. Since the cell line,
PLC/PRF5 appears not to produce IFN as measured by CAT ELISA (see Appendix
A), then the former interpretation is more likely to be correct.
In the serial dilution experiment, a small titration range was introduced to give some
impression of the relative sensitivity of 3D, 3D transferred to 2D, and 2D alone. The
virus was detectable by real time RT-PCR in all three systems until the end of the
experiment {Figure 4.3). In both the 2D and the 3D transferred to 2D systems the
virus was detectable at several dilutions at most dpi {Figure 4.3). The Ct values
among the dilutions were very similar and did not follow a regular trend (the
expected reduction would be three Ct of difference between each ten fold dilution).
The global examination of the data indicated a similar sensitivity of the 3D
transferred to 2D compared to the conventional 2D system in detecting HEV RNA.
A possible explanation for the inconsistency in detection of HEV RNA observed
with the 3D transferred to 2D system could be that not all the cells adhered to the
2D wells when transferred. Consequently, every time that the supernatant was
collected an undefined amount of cells was also removed. This would cause a
gradual reduction of the cells in the wells which consequently might have limited
the availability of cells for virus replication.
127
An end point of the serial dilutions was not achieved but it was observed that with
the increase of dilution, the Ct values progressively became higher in all systems
except the 3D system. Since there was no true trend in the Ct values detected, the
results obtained in the serial dilutions experiment were insufficiently consistent to
draw any measurable conclusion in relation to the relative sensitivity of the 3D cells
transferred to 2D and 2D system.
Regarding the results obtained from the serial dilutions of the virus in the 3D
system, no conclusion can be made since the virus was able to infect the cells in all
the dilutions. No trend was observed between the different dilutions, in terms of
higher virus concentration higher copy number. In fact, higher HEV RNA copy
number was detected in the cells infected with the inoculum diluted 100 times (10'
). This may be because the cells better tolerated a lower concentration of virus
allowing more efficient replication.
In conclusion, we demonstrated that the PLC/PRF/5 cells grown in the 3D culture
system offers an efficient tool for HEV propagation. The same cell type grown in
monolayer did not show significant evidence of supporting HEV replication. The
described system, including the diagnostic procedures, is useful tools to investigate
the biology of HEV virus and the viability of HEV in pork samples.
Research to optimise the described cell culture systems for the assessment of the
infectivity of the HEV in food samples should be planned. This may contribute
towards understanding the mechanisms of HEV replication, pathogenesis and
environmental (including within food matrices) survival.
128
4.8.2 Discussion of the use of the 3D cell culture system to investigate the
viability of HEV in the UK sausages and French liver sausages (figatelli)
Figatelli sample 84 was shown to contain viable HEV that was able to replicate in
the 3D cell culture system. There was an increase in the RNA copy numbers
between 29 and 44 dpi. The relatively low copy number in the inoculum used did
not affect the onset of viral replication in the 3D cell culture system {Section 4.8.1).
It is possible that the virus needs a specific threshold for optimal, sustained,
productive replication of HEV, and a low copy number in the inoculum would
influence the time taken for this threshold to be reached [197]. The observation that
at least one of the figatelli samples contained viable HEV provides a very good
corroboration of the reports from France implicating consumption of these products
as a cause of hepatitis E [134].
Regarding the culture of progeny HEV, RNA was detected at all dpi, but no
significant increase in copy number was observed at the time of last sampling (dpi
34). This result may indicate that to observe higher viral titre the virus probably
needs more time. Tanaka et al [197] observed that HEV appears to require a high
titre (between 10 and 10 ) to be able to infect 2D PLC/PRF/5 and HEV RNA was
first detectable in the progeny at 36 days by real time RT-PCR [197]. In this
experiment the viral copies number/ml was relatively low in comparison with the
Tanaka’s experiment and probably for this reason the figatelli progeny could not
replicate rapidly in the 3D system, giving a constant low copy number throughout
the experiment (34 dpi). Unfortunately, the experiment had to be terminated due to
mould contamination in the vessel. Due to this contamination we could not
determine if the HEV copy number would have increased in the same way as that of
129
figatelli 84, where the copy number began to increase around 36 dpi. Unfortunately
due to time limit and the economical restraints of the project the experiment could
not be repeated.
To provide further confirmation of the HEV real time RT-PCR results figatelli 84,
samples of culture supernatant from dpi 33 were examined by EM. Several entire
viral particles were observed in the sample showing that cell-free virus was present
in the supernatant after replication and release from cells.
Three other figatelli samples (87, 100 and 116) and the 3 UK HEV real time RT-
PCR positive sausages were tested using the 3D cell culture system, but other than 0
and 5 dpi for the UK sausages and 8 dpi for the 2 figatelli samples number 87 and
116, HEV RNA was not detected at any other time point. It may be that viral titre in
the inoculum was not high enough to obtain viral replication in the cells, as
previously reported by Tanaka et al [197] or because the virus contained in the
figatelli and UK sausage samples was not viable. A consideration that should be
taken into account is that in the two figatelli samples HEV RNA was detected until
8 dpi and for the UK sausages HEV RNA was detected until 5 dpi, these results
could be due to the fact that no viable virus in the UK sausages (due to bad
conservation of the samples) and that for the other two figatelli sample the viral titre
was not enough to support an in vitro infection. This would require further testing
using greater numbers of field samples such as sausages.
In conclusion, these results showed a significant finding outside the normal range of
experimental error. It is possible that in different homogenates or supernatants there
will be variable proportions of intact, viable virus, defective interfering particles,
free viral genomic RNA and degraded but still PCR reactive RNA. The differing
130
proportions will be manifested by a different relationship between apparent copy
number and kinetics of replication in-vitro. In theory, if all the RNA detected in the
real time RT-PCR is inactivated/degraded but still PCR reactive there should be a
decreasing in detection HEV RNA by real time RT-PCR.
131
CHAPTER 5
Inactivation studies
132
Introduction: After having evaluated the new 3D cell culture system the next step
was to carry out inactivation studies to better understand how and if HEV can be
inactivated.
In addition, to harmonise the VITAL project, three post-graduate students were
focused on inactivation studies of three different viruses: Norovirus, HEV, and
Adenovirus. In my case, the survival of HEV in pork products under various
inactivation conditions was investigated.
This chapter is subdivided into the following sections 1) heat inactivation 2) UV
light and NaOCl inactivation.
5.1 Heat inactivation
The risk of HEV infection via the consumption of HEV-contaminated pig livers
raises public health concerns, since it is not clear whether cooking conditions will
be effective in inactivating the virus. Feagins et al (2008) [85, 87] performed a HEV
heat inactivation study in an animal model. The objective of this study was to
determine if traditional cooking methods are effective in inactivating infectious
HEV present in contaminated commercial pig livers. Four of the five pigs
inoculated with a pool of two HEV-positive liver homogenates incubated at 56°C
for 1 h developed an active HEV infection. The pigs inoculated with a homogenate
of two HEV-positive livers stir-fried at 19UC for 5 min and the group of pigs
inoculated with a homogenate of two HEV-positive livers boiled in water for 5 min
showed no evidence of infection since there was no seroconversion, viremia, or
faecal virus shedding in any of the inoculated pigs.
133
HEV can be found in the liver, blood, and intestinal tract, which are all consumed in
one form or another and often together, such as in sausages. How safe are these
products? The question is difficult to answer because until recently it was difficult
to propagate HEV in cell cultures and testing HEV viability in vivo requires the use
of experimental animals, usually primates or pigs.
The in vitro 3D cell culture system described in the previous chapter was used to
propagate HEV for a heat inactivation experiment based on Feagins’s work but
replacing the use of pigs with 3D cell culture system.
5.2 UV light and NaOCl HEV inactivation studies
After having optimised the in vitro 3D cell culture system at AHVLA, as part of the
PhD project, I moved for one year to the Central veterinary Institute (CVI, The
Netherlands) transferring the 3D technology to continue the HEV in vitro studies
and subsequently perform virus inactivation experiments.
The following inactivation strategies were selected in this project: UV light
inactivation; NaOCl inactivation.
1) UV inactivation was investigated to clarify whether it could be a useful tool to
inactivate HEV on tools such as knives used to process the pork meat, on surfaces
and equipment such as found in farms, slaughterhouses, processing plants and
points of sale.
In this study the effect of UV light on HEV was evaluated. A homogenate of HEV
positive liver was exposed for 20, 30 and 50 minutes to UV light and the inoculum
was used to infect 3D cell cultures. This experiment was set up because exposure to
solar ultraviolet (UV) radiations is a primary means of virus inactivation in the
134
environment, and germicidal (UVC) light is used to inactivate viruses in hospitals
and other critical public and military environments [90, 91]. Safety and security
constraints have hindered exposing highly virulent viruses to UV and gathering the
data needed to assess the risk of environments contaminated with viruses that can
cause high consequence in humans [92]. UV sensitivity of some viruses has been
extrapolated from data obtained with UVC (254 nm) radiation by using a model
based on the type, size and strandedness of the nucleic acid genomes of the different
virus families [93, 94]. These predictions were based on viruses suspended in liquid
solutions, instead of a dry state. Therefore, there was little information to allow
accurate modelling, confident extrapolation, and prediction of the UV sensitivity of
viruses deposited on contaminated surfaces, conditions more likely to be relevant to
public health.
2) HEV inactivation by sodium hypochlorite (NaOCl) was also performed. Sodium
hypochlorite solution, commonly known as bleach, is frequently used as a
disinfectant. This disinfectant is one of the most common used in farms, in high
containment level laboratories, in water and or surfaces to kill bacteria and viruses.
US Government regulations (21 CFR Part 178) and the CDC: Guideline for
Disinfection and Sterilization in Healthcare Facilities (2008), allow food processing
equipment and food contact surfaces to be sanitized with solutions containing
bleach, provided that the solution is allowed to drain adequately before contact with
food, and that the solutions do not exceed 200 parts per million (ppm) available
chlorine. Furthermore Zand et al (2012) [237] observed that different concentrations
of NaOCl from 0.5% to 5.25% were able to inactivate E. Faecali growth [237].
135
Only a few studies have been deseribed with sodium bypocblorite inactivation of
viruses. Sabbab et al in 2010 [238] described that 5 minutes with peracetic acid or
with chlorine dioxide are sufficient to reduce the level of bacteria in environmental
surfaces as indicated in the disinfectant criteria standard guideline submitted by U.S
Protection agency (EPA) Guidance manual showing that this disinfectant is a good
tool to inactivate pathogens. Furthermore, this statement was also confirmed by
Tburston-Enriquez et al in 2003 demonstrating that viruses like FCV, adenovirus
and poliovirus type 1 are inactivated by chlorine [239].
Since NaOCl appears to be commonly used in the field we decided to set up an
inactivation study with NaOCl. HEV positive supernatant was treated with NaOCl
to a final concentration of 5% and the effect of the NaOCl was neutralised after 5
minutes with 10% of sodium tbiosulpbate (NazSiOg). This approach for neutralising
the cytotoxic effects of NaOCl was adopted from Sabbab et al, Benarde et al and
Tburston-Enriquez et al [238, 240, 241] who performed studies to verify if bacteria
and viruses were killed by the disinfectant.
136
Materials and Methods
5.3 Cells preparation; The cells were propagated in the 3D cell culture system as
deseribed in section 4.3.
5.3.1 Heat inactivation experiment
5.3.1.1 Inoculum preparation: The positive HEV sample was provided by the
Central Veterinary Institute, Wageningen University and Research Centre - CVI.
The sample was a liver tissue from an experimentally HEV infected pig [86]. The
liver tissue (Ig) was homogenized with a mechanical disruptor in 1 ml of GSTF-2
media and subsequently 8 ml of GTSF-2 media was added. The bomogenate was
centrifuged at 8.000 x g for 3 minutes and the supernatant was filtered through a
sterile spin-X centrifuge tube filter (0.22pm; Costar) at 10.000 x g at 4°C for 15-25
min. [87].
The human bepatocareinoma cell line was infected with inoculum untreated, heated
at 56‘ C for Ibour or heated at 100°C for 15 minutes. In addition one vessel was
used as non infected control.
5.3.1.2 Infection of the 3D cells: The medium was removed from the vessels and
2.5 ml of inoculum was added. The vessels were incubated for 2 hours at 35.5°C,
and gently agitated every 20 minutes. After two hours, 47.5 ml of fresh medium was
added to each vessel (the inoculum was not removed).
The whole experiment lasted 69 days. The collection of the sample was performed
on day: 0, 7, 13, 22, 33, 40, 48, 55, 62 and 69. On each collection day the following
aliquots were collected: 140 jil in duplicate for each vessel added to Lysis buffer
137
(Qiagen Viral RNA kit, Qiagen), to be stored at -20°C before RNA extraction. Fresb
medium (47.5 ml) was added to eaeb vessel to restore tbe full volume (50ml).
5.4. UV inactivation experiments
5.4.1 Preparation of the inoculum: Tbe preparation of inoculum was performed as
described in section 5.3.1.1.
5.4.2 HEV UV inactivation procedure: A 30 W UV lamp, 91 cm long (TUV
30WAT, 254nm, UVC, Philips) was warmed up for ca 20 min before starting tbe
experiments and tbe UV lamp was previously used for 30 hours (an UV light lamp
can be used for ca. 8000 hours). This represented tbe range of time recommended to
ensure that tbe light was 100% efficient. Tbe lamp was positioned above tbe sample
Petri dish to allow a distance from tbe UV source to tbe sample surface of 20 cm,
with tbe agitation set at 100 rpm.
Seven and half ml of liver bomogenate, prepared as previously described {Section
5.4.1) was exposed for 20, 30 and 50 minutes respectively under UV light. Tbe UV
irradiation dose that tbe inoculum received was: Dose UV light for 20 min= 99.6m
(W*s)/cm^; 30 min= 149.4m (W*s)/em^; or 50 min= 256.6m (W*s)/cm^. These data
were obtained from Philips website (bttp://www.pbilips.co.uk/) and they were
calculated as if tbe sample was Im from tbe centre of tbe lamp. Tbe Intensity was
83uW/cm^. Tbe depth of tbe inoculum in tbe Petri dish was 4mm. Tbe temperature
of tbe inoculum exposed under UV light was tested and it did not change during tbe
UV light treatment (ca 18°C).
A second experiment was performed as above but decreasing tbe depth of tbe
inoculum (from 4 mm to <1 mm) whilst exposed to tbe UV light for 30 minutes.
138
The duration of the first experiment where the inoculum was exposed under UV
light for different length of time was 60 days, whilst the second experiment where
the inoculum was exposed under UV light for 30 min and the depth of the inoculum
in the Petri dish was less than 4mm was terminated after 36 days due to
mycoplasma contamination. Each experiment was run with a positive control
(bomogenate of HEV positive liver and a non infected control).
5.4.3 Inoculation of cultures and sample collection: tbe infection was performed
as already deseribed in section 4.3.3. Briefly tbe medium was removed from tbe
vessels and 2.5 ml of infected supernatant (bomogenate of liver) (previously UV
inactivated) was added to tbe cells. Tbe vessels were incubated for 2 hours at
35.5°C, and inserted in tbe Rotating Wall Vessel (RWV). After two hours 47.5 ml
of fresb medium was added to each vessel (tbe inoculum was not removed). On
each collection day (tbe samples were collected once a week for two months) tbe
following aliquots were collected: 140 pi in duplicate for eaeb vessel was added to
Lysis buffer, to be stored at -20°C before extraction and an aliquot of media (20 ml
ca. from tbe infected vessels) was collected and stored at -80°C. After each
collection tbe vessels’ volumes were restored to 50 ml by addition of fresb GTSF-2
medium {Table 4.1).
5.4.4 Electron microscopy: Tbe electron microscopy procedure was performed as
described in section 4.4.6. Briefly, R. Jobne and J. Reetz at BfR in Germany
performed tbe EM examination, to provide more evidence of HEV replication.
Supernatants of tbe cell cultures that received tbe inoculum treated for 20 min under
UV light and collected at 21 dpi were applied to polioformcarbon-eoated, 400-mesb
copper grids (Plano GmbH, Wetzlar, Germany) for 10 min, fixed with 2,5%
139
aqueous glutaraldehyde (Electron Microscopy Sciences Company, Germany)
solution for 1 min and stained with 2% aqueous uranyl acetate solution (Electron
Microscopy Sciences Company, Germany) for 1 min. The specimens were
examined by transmission electron microscopy using a JEM-1010 (JEOL, Tokyo,
Japan) at 80 kV accelerated voltage.
5.4.5 Sodium hypochlorite inactivation
5.4.5.1 Preparation: In this experiment HEV positive supernatant was used as a
surrogate to better simulate environmental surface disinfection in premises where
pork and pork products are bandied. Five ml of a HEV positive supernatant
collected at 13 dpi exposed under UV light for 20 min in tbe previous UV
inactivation experiment and shown to be viable, was chosen to be treated with 5%
Sodium hypochlorite for 5 minutes. Tbe Sodium bypocblorite was neutralized with
10% of sodium tbiosulpbate [238, 240, 241].
Four vessels were used for this experiment. One vessel was used as positive control
(positive supernatant). Tbe second vessel was infected with 2.5 ml of HEV positive
supernatant treated with 5% NaOCl for 5 min. Tbe third vessel was tbe HEV
negative supernatant treated with 5% of NaOCl. Tbe last vessel was tbe non
infected control.
5.4.5.2 Treatment: Before tbe inoculum was added to tbe cells, to remove possible
bacteria contaminant, tbe inoculum (HEV positive supernatant treated or non
treated with NaOCl) was filtered with 0.45 pim filter. Infection was performed as
described in section 5.4.3. Briefly, 2.5ml of HEV positive supernatant (previously
tested by real time RT-PCR) was collected from tbe total 5 ml previously exposed
to 5% NaOCl for 5 minutes and used as inoculum to infect tbe 3D cell cultures.
140
Sample collection was performed once a week as described in section 5.4.3 for 36
days before termination due to mycoplasma contamination.
5.4.6 RNA extraction and Real Time RT-PCR: RNA extraction and PCR of tbe
supernatant collected from tbe vessels was performed as described in section 4.3.5
and 4.3.6. Briefly nucleic acid extraction from 140|il of eaeb sample was performed
using tbe Qiagen viral RNA kit (Qiagen) following tbe protocol deseribed by tbe
manufacturer’s guidelines.
Real time RT-PCR testing was performed according to tbe protocol described by
Jotbikumar et al (2006) [220] using tbe Superscript III Platinum one-step
quantitative RT-PCR kit (Invitrogen). Tbe real time RT-PCR reaction was set up
and performed according to tbe manufacturer’s instructions as described in section
141
Results
5.5.1 Heat inactivation treatment
Three aliquots of bomogenate of HEV positive liver were non treated, heated for lb
at 56°C or heated for 15 min at 100°C and subsequently used as inoculum to infect
tbe 3D cell culture system to better understand tbe optimal temperature to inactivate
tbe virus.
HEV RNA was detected at all dpi except for 22 dpi in tbe cells infected with tbe
untreated inoculum {Figure 5.1). At 33 dpi tbe Ct values decreased and remained
almost constant until tbe end of tbe experiment (69 dpi). HEV RNA was also
detected in tbe 3D system infected with tbe inoculum heated at 56^C for one hour, at
0, 7, dpi with Ct values ranging between 43 and 40 and from 48 dpi until 62 dpi (Ct
values between 35 and 40) {Figure 5.1).
No viral RNA was detected at any dpi in tbe 3D cells infected with tbe inoculum
that was heated at 100°C for 15 minutes {Figure 5.1). In this experiment we set tbe
cut-off at 40 Ct to exclude non specific signal meaning that all samples detected
above 40 Ct were considered negative.
142
•3 • -
23 -<ü
>o
3 12
■2D unt'=3t5d ■ 2D % : C ■2D 133= D
days post infection
Figure 5.1 Tientment of HEV infected liver nt 100 "C lends to irrnctiv atiair of
the vims. 3D PLC/PRF/5 were infected with bomogenate of HEV positive liver and
the inoculum previous the infection was untreated, heated for Ih at 56°C and heated
for 15 min at 100 °C. Supernatant of the 3D cells was tested by real time RT-PCR.
— ♦ supernatant tested by real time RT-PCR of the cells infected with non heated
bomogenate of HEV positive liver. — supernatant of cells infected with
bomogenate of HEV positive liver heated for Ih at 56°C . — supernatant of 3D
cells infected with bomogenate of HEV positive liver heated for 15 min at 100°C.
■ represents +/- cut off at 40 Ct. Samples above this line are considered
positive for HEV.
143
5.5.2 Homogenate of HEV positive liver exposed to UV light to test HEV
inactivation
A homogenate of a HEV positive liver previously shown to contain viable virus was
exposed to UV light for 20, 30 and 50 min and aliquots of 2.5 ml were used to infect
3D cell cultures and HEV infectivity was evaluated by real time RT-PCR. The
experiment was repeated, decreasing the depth of the inoculum in the Petri dish
during the 30 min of UV light exposure.
The real time RT-PCR analysis showed that Ct values were detected at almost all
dpi in the supernatant of 3D cells infected with inoculum exposed to UV at different
times.
In the 3D cells infected with non-treated inoculum, HEV RNA was detected at all
except two dpi, 7 and 35 dpi. Ct values increased significantly at 13 dpi, indicating
lower viral titre (Ct values during the experiment ranged from 25 to 40). From 13 to
28 dpi, there was a difference of 8 Ct values (Ct values were between 30 and 38).
HEV RNA was detected in the 3D system infected with inoculum treated with UV
light for 20, 30 and 50 minutes at 0 ,7 , 13, 21, 28 and 42 dpi {Figure 5.2). At 35 dpi
no Ct values were detected in the supernatant of all samples by real time RT-PCR in
all the different treatments, suggesting that possibly the virus was replicating inside
the cells or due to a problem with the RNA extraction on that particular dpi.
Figure 5.3 describes the decay of HEV in terms of Ct values observed by real time
RT-PCR immediately after the UV light treatment. The non UV light treated (NT)
inoculum showed higher Ct values in comparison with the Ct values observed in
vessels receiving inoculum exposed to the UV light treatment during the 20, 30 and
50 min, indicating no inactivation. It should be noted that the UV dose calculation
144
provided by the UV light producer was made considering the UV lamp Im distant
from the sample while in this case the samples were 20 cm distant from the UV
lamp. Although the UV dose calculations are approximate, figure 5.3 shows there
was a partial increase in Ct in parallel with increase of the UV dose.
145
tu
13>Ô
21G 7 1 2 28 31 42 ec
“ ♦ “ untreated
UV
UV
- # “ 50 UV
days post infection
Figure 5.2 Analysis of the variation of Ct values overtime iir the UV light irractivatioir experiment irr tire supernatarrt of tire 31) cell cultures. 3D cells were infected with homogenate of HEV positive liver not treated, treated for 20 min under UV light, treated for 30 mm under UV light and treated for 50 min under UV light Supernatant o f the 3D cells cultures was tested by real time RT-PCR.— represents the supernatant of cells infected with the homogenate of liver not treated under UV light, represents the supernatant of cells mfected with homogenate of liver treated for 20 minutes under UV light. — represents the supernatant of cells infected with homogenate of liver treated under UV light for 30 min. — represents the supernatant of cells mfected with homogenate of liver treated for 50 mmutes under UV light
IS the cut off at 40 Ct values.
146
0.045i
0.040“
u
0.035“
0.030
r300
-200 0
“100
20 30 50
Treatment time (min)
Figure 5.3 HEV decay measured in the inoculum by real time RT-PCR after the UV light treatment. The graph describes the variation of the Ct values observed in association with the UV light dose that the inoculum (homogenate of HEV positive liver not exposed and exposed under UV light for 20, 30 and 50) received previous the 3D cell cultures infection. The UV dose was calculated considering the light at 1 m of distance from the centre of the lamp. The UV dose showed in this graph is an approximation of the UV dose during the time of the experiment. Black columns represent the increasing of UV light dose during the time. W hite column represent the Ct values detected after the UV light treatment.
1 4 7
5.5.2.1 Homogenate of HEV positive liver treated for 30 min to UV light
An homogenate of HEV positive liver was exposed to UV light for 30 min and 2.5
ml of the inoculum was used to infect 3D cell cultures; the HEV infectivity was
evaluated by real time RT-PCR. This UV light experiment was repeated, decreasing
the depth of the inoculum in the Petri dish.
HEV RNA was detected by real time RT-PCR during the entire experiment in the
supernatant of cells infected with an untreated homogenate of HEV positive liver
{Figure 5.4). Ct values increased from 0 dpi to 14 dpi from 20 to 30 Ct, at 18 dpi
there was a modest decrease in Ct and then an increase again to 26. From 29 dpi
until 36 dpi Ct values remained stable around 30, suggesting a stable replication.
The supernatant of 3D cells infected with the homogenate of HEV positive liver
where the inoculum prior to infection was exposed for 30 minutes to UV light, was
HEV positive by real time RT-PCR at all dpi but 3 dpi (10, 18, 29). The Ct values
were slightly higher (around 5 Ct higher) compared to the non treated inoculum
suggesting that viral particles may have been partially inactivated by the UV light.
From 0 until 10 dpi, Ct values increased gradually then decreased at 14 dpi,
increased again at 18 dpi and decreased at 26 dpi. At 29 dpi no RNA was detected
by real time RT-PCR but lower Ct values were detected at 33 dpi, followed by a
modest decrease of Ct at 36 dpi, suggesting that the virus was replicating.
5.5.2.2 Electron microscopy result
Following negative staining with uranyl acetate HEV-like particles were detected in
the supernatant of cells infected with homogenate of HEV positive liver that had
148
been exposed under UV light for 20 min and collected at 21 dpi. However, HEV
viral particles were very sparse and only as single particles {Figure 5.5).
149
20 -
25 -
>
35 -
40 -
0 3 7 10 14 18 22 26 29 33 36
■ liver
•liver 30'UV
days post infection
Figiire 5.4 Ann lysis of the Ct values observed in the supernatant of 3D cells infected with inoculiun treated with XJ\' light for 30 min 3D cells were infected with homogenate of HEV positive liver not exposed under UV light and exposed under UV light for 30 min. The supernatant collected at different days post infection (X axis) was tested by real time RT -PCR. The inocula were untreated homogenate of liver (— ) or homogenate of liver exposed for 30 minutes under UV light (— ).The IS the cut off at 40 Ct.
1 5 0
3 ^ j*
M.: ' 'S<'
Figure 5.5 HEV-like particles. The figure shows two HEV-like particles (arrow)
obtained by negative staining with uranyl acetate. The two particles were detected
in the HEV positive supernatant of 3D cell culture collected at 21 days post
infection and infected with the inoculum exposed for 20 min under UV light and
tested by electron microscopy.
151
5.5.3 Inactivation of HEV positive supernatant with 5% of NaOCl
Figure 5.6 describes the results obtained in the NaOCl inactivation study. The
supernatant (the inoculum was supernatant of 3D cells infected with HEV positive
supernatant, exposed for 20 minutes under UV and collected at 13 days post
infection) of cells not treated with NaOCl was positive at all time points except for
supernatant collected at 26 and 36 dpi. Ct values after a peak at 3 dpi with a Ct of 20
ranged between 32 and 40 during the course of the experiment. At 3 dpi, Ct values
decreased then increased slowly until 26 dpi and then HEV RNA was detected
again at 29 and 33 dpi, suggesting viral replication. Supernatant of the 3D cells
infected with inoculum treated with 5% NaOCl was positive by real time RT-PCR
at 0, 3 and 7 dpi.
In all experiments, to exclude a non specific signal, a cut off of 40 Ct was selected.
152
2 0
25 -
30 -
o35 -
40 -
450 3 7 10 14 18 22 26 29 33 36
• sup progeny •NaOCL
day s p o s t in fe c t io n
Figure 5.6 Analysis of the Ct va hies of HEV positive supernatant treated with NaOCl and untreated. PLC/PRF/5 cells were infected with HEV positive supernatant obtained form the UV light experiment. The cells received inoculum not treated with NaOCl and treated with 5% of NaOCl for 5 min. — represents the Ct values detected by real time RT-PCR of 3D cells infected with HEV positive supernatant exposed for 20 min under UV light and collected at 13 dpi but nottreated with NaOCl. represent the Ct values detected by real time RT-PCR of3D cells infected with HEV positive supematant exposed for 20 min under UV lightand collected at 13 dpi then treated for 5 minutes with 5% of NaOCl. is the cutoff at 40 Ct.
153
5.6 Discussion
5.6.1 Homogenate of HEV positive liver heated at different temperatures
A homogenate of pig liver known to contain infectious HEV was subjected to
heating, simulating some normal cooking conditions, and was applied to 3D cell
cultures to determine the effect of the virus inactivation as measured by HEV RNA
copy numbers in cell supernatants.
Differences in the Ct values were observed between the supernatant of the cells
infected with non- heated liver and supernatant of cells infected with HEV positive
liver heated at 56°C for one hour. As we can see in figure 5.1 the Ct values were
lower (ranging between 40 and 29) in the sample infected with the homogenate of
non-heated liver compared to the supernatant of cells that received as inoculum the
homogenate of liver heated at 56°C for one hour. The Ct values in the supematant of
cells infected with HEV positive liver heated at 56°C for one hour were higher,
probably reflecting partial virus inactivation. Full HEV inactivation was observed in
the inoculum heated at 100°C since no HEV RNA was detected by real time RT-
PCR at any point of the experiment. The results are similar to those of Feagins et al
[85, 87] where the pigs infected with HEV positive liver heated at 56°C were
shedding virus in the faeces, showing that the treatment was not sufficient to
inactivate HEV. Furthermore, the similarity of the results of in vivo and in vitro
experiments of this study underline the potential of the 3D cell culture system in
replacing the traditional in vivo infectivity studies.
HEV transmission in industrialized regions is not fully understood. It has been
suggested and is now widely accepted that HEV transmission is zoonotic [138,
154
242]. Tel et al [133] reported direct evidence of zoonotic HEV transmission via the
consumption of grilled or undercooked commercial pig liver purchased from local
grocery stores in Japan [133]. The majority of the patients in that study had a history
of consuming undercooked pig livers prior to the onset of the disease, indicating
that consumption of pig livers is a risk factor for hepatitis E [133]. Eleven percent of
livers purchased from local grocery stores in the United States, 6% in The
Netherlands [243] and 9.5% in the United Kingdom were found to be contaminated
by HEV (Chapters, section 3.5.1).
HEV inactivation and environmental resistance is not a well-covered topic and little
information is available. As an orally transmitted virus, HEV is most likely resistant
to inactivation by the acidic conditions of the stomach. The ability of HEV to
survive harsh or extreme environmental conditions can be attributed at least in part
to its non-enveloped viral structure [85, 87].
In Europe most pork meat is cooked prior to consumption, but there are some
exceptions where pork meat is eaten raw, as for example liver sausages in France.
The United States Department of Agriculture (USDA) and the United States
National Pork Board (NPB) recommend a cooking method for fresh pork that will
result in a minimum internal cooking temperature of 71 °C (http://
www.fsis.usda.gov/is_it_done_yet/, accessed on March 15, 2007). A time
stipulation is suggested based on the level of heat but many of the recipes do not
specify a minimum cooking temperature. Stir-frying and boiling are the two most
widely used and accepted methods for cooking pig livers for consumption. Feagins
at al evaluated that stir-frying and boiling of HEV-contaminated pig livers can
effectively inactivate the virus by using a swine bioassay to determine the virus
155
infectivity [85, 87]. By using an in vitro system, Emerson et al [225] reported that
HEV is approximately 50% inactivated when heated at 56°C for 1 h. In this study
we demonstrated that incubation of homogenate of contaminated pig livers at 56°C
for 1 h (temperature that produced an internal cooking temperature slightly below
the recommended 71°C without burning the tissue) did not fully inactivate the virus,
as HEV RNA was detected during the course of the experiment. Our results support
the in vitro results of Emerson et al [225] and the in vivo results of Feagins et al
[87] confirming that adequate cooking of HEV-contaminated commercial pig livers
will inactivate HEV in the tissue, thereby decreasing the risk of food-borne HEV
transmission. Importantly these results confirm that partial inactivation of HEV
(heat at 56°C for 1 h) may allow the virus to initiate an active infection in vitro
while the treatment of the liver at 100°C appears to be efficient to inactivate the
virus completely.
5.6.2 Inactivation of HEV positive supernatant with UV light
UV light inactivation studies are mostly performed with bacteria such as
Sphingopyxisalaskensisa marine bacteria. Salmonella and E. Coli [244, 245]. Only a
few UV light inactivation studies have been performed with viruses such as
Hepatitis A virus (HAV), Feline Calicivirus (FCV) and two Picornaviruses [246,
247] but never with HEV. This study was performed to find out if UV light would
inactivate HEV under the conditions described. From the results obtained in this
study the UV light applied was insufficient to completely inactivate the virus and
the same results were also obtained in other studies. In fact it has been observed a
decrease of 2 log in samples (lettuce, strawberry and onion) artificially
contaminated with HAV and Feline Calicivirus [246, 247]. Using relative Ct as a
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crude measure of viral copy number, the amount of viral RNA detected did not vary
significantly between cells infected with UV light treated and non UV light treated
inoculum. HEV RNA was detectable in the 3D system by real time RT-PCR in the
cells that received the UV light treated inoculum. The data showed that the Ct
values for the 3D cells were not significantly different for all the different UV light
treatments. This could be due to the fact that the UV light treatment is not effective
in inactivating HEV or that the inactivation was partial and remaining viable
particles were able to infect the cells.
When the experiment was repeated, reducing the depth of the inoculum during
exposure, viral RNA was detected throughout the experiment, suggesting that viable
virus was present and inactivation had been incomplete although an increase of
almost 7 Ct values was observed during all the experiment in the supernatant of the
cells infected with inoclulm exposed for 30 min under UV light suggesting partial
inactivation of HEV. The depth of the inoculum in the Petri dish was reduced
because in the first experiment the depth of the inoculum in the Petri dish was 4 mm
and the literature advises to have less then 3mm of depth during the UV inactivation
[246, 247]. Also in this second experiment, where the inoculum was previously
exposed for 30 min under UV light, we detected RNA by real time RT-PCR at
almost all dpi, confirming that under the conditions employed, UV light did not
inactivate all the viral particles allowing some HEV replication in the cells.
The temperature of the inoculum exposed under UV light was tested and remained
constant during the inactivation treatment, avoiding any chance that the temperature
was affecting the experiment.
157
A possible explanation of this UV inactivation result can be that the RT-PCR and
3D sampling system is insufficiently sensitive to detect small variations in viral
particles once the cells are infected with the virus under different treatments, in
other words the 3D cells are able to pick up infectious virus also when it is present
in small quantities. As we already observed similar results were obtained in another
experiment where serial dilutions of the inoculum were performed {Chapter 4). In
the HEV serial dilution experiment, as obtained in the UV inactivation study, no
dose-related trend was observed after the cells were infected with HEV treated with
UV light or with serially diluted inoculum {Chapter 4).
Fino et al in 2008 [248] showed that HAV and other viruses are partially inactivated
in lettuce and that bacteria as for example E. Coli is inactivated by 99% with the
same treatment. Our results confirm partially those of Fino et al [248] where viruses
were partially inactivated by UV light.
In this study, we have shown that HEV, albeit partially inactivated by UV light
(higher Ct values at 0 dpi compared to the non-treated inoculum), is able during the
course of the incubation to replicate reaching the same Ct values or higher than the
inoculum without UV light treatment.
Electron microscopy was performed on HEV positive supernatant exposed to UV
light for 20 min and collected at 21 dpi. Only a few viral particles were detected,
probably because the viral replication was inhibited by mycoplasma contamination
in the 3D cell culture system and since the cells were supporting double replication
from the bacteria and from the virus [249]. Clearly, the demonstration of HEV
particles by EM is not easy since this is only the third electron micrograph since
1983 to show a hepatitis E virion [130, 250]. These results show that hepatitis E
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virus is replicating in the 3D cells, since intact viral HEV particles have been
detected in the supernatant of the 3D cell culture system after several days from the
infection. In addition it is highly unlikely that the particles detected were from the
residual inoculum because at every collection point almost half of the media (23 ml)
content in the vessel was refreshed once a week. In conclusion, UV light under the
conditions employed appeared to be insufficient to fully inactivate HEV.
5.6.3 Inactivation of HEV positive supernatant with 5% of NaOCl
In this experiment a sample of HEV positive supernatant (HEV infectious progeny
virus obtained from a previous experiment) was treated with NaOCl at the final
concentration of 5% and the effect of the chemical was neutralised after 5 minutes
with 10% of sodium thiosulphate (Na2S203).
HEV RNA was detected only until 7 days post infection and Ct values detected by
real time RT-PCR were increasing each successive day post infection, indicating
that the virus was possibly inactivated by the NaOCl and the high Ct values detected
were residuum of inoculum.
The lack of detection of HEV RNA by real time RT-PCR in further collection
points could be also due to the fact that live virus was still present but the cells were
damaged by the chemical and could not support viral replication. To minimize the
cell damage 10% of sodium thiosulphate was used [237] to neutralize the effect of
the NaOCl. Despite this, visual confirmation of partial cell damage was observed
also in the negative control vessel (cells in contact with non-infected media treated
with 5% NaOCl and 10% Na2S203). The cytotoxic effect of the NaOCl and
thiosulphate should have been tested in this particular cell-system scenario before
the experiment in the cells or the virus removed from the inactivation mixture by
159
pelleting and washing in PBS, despite the literature describing the neutralisation of
the chemical (NaOCl) with 10% of sodium thiosulphate [237].
Alternatively, the inactivation might have worked, the treated inoculum was not
cytotoxic and the RNA detected at 7 dpi was just residual inoculum as is shown in
section 5.5.3 {Figure 5.6). Although definitive conclusions cannot be made, the
consideration that NaOCl was efficient enough to inactivate the virus should be
taken in consideration.
Guthmann et al [132] in 2006 reported a large outbreak of hepatitis E in the region of
Darfur of Sudan in 2004. In 6 months, 2621 cases of hepatitis were recorded where
contaminated water seemed to be the cause of this outbreak. Although the water
before being distributed was chlorinated with standard level (0.3/0.6 mg/L) of
chlorine, the drinking of chlorinated water was assessed as a risk factor for
contracting hepatitis E [132]. So it appeared that during that outbreak the water
disinfection was not effective to inactivate HEV. This highlights a need and it would
be useful to set up another disinfectant study to better prove which chlorine or other
chlorine derivate dose/time is effectively able to eliminate the virus from surfaces
and HEV contaminated water [132].
In conclusion, we determined that probably the NaOCl could be a good tool to
disinfect surfaces been in contact with pork products since that after the first week
no HEV RNA was detected in the HEV positive supematant derived from 20 min
UV light experiment and collected at 13 dpi treated for 5 minutes with 5% of
NaOCl [238, 240, 241]. Furthermore, we also showed that the HEV progeny vims
was able to infect other 3D cells providing once again that progeny is infectious and
is able to infect 3D cells {Figure 4.4, chapter 4) until 33 days post infection.
160
HEV can be identified at different points of the pork food chain and zoonotic
transmission through consumption of contaminated pork meat has been
demonstrated. There is still the need to understand which chemical and physical
conditions can be utilized to inactivate viable HEV particles that could be present
along the chain and in the final products.
In these studies we exposed viable HEV to heat, UV light and NaOCl. The
effectiveness of these selected inactivation strategies was evaluated in a 3D in vitro
system, previously shown to be able to support HEV replication.
Between the 3 methods used, the only one that gave indications of a consistent
successful inactivation was the heat treatment. These data confirm results obtained
by other authors in previous in vitro and in vivo models. Thoroughly cooking pork
meat is an effective means of inactivating HEV, and should therefore be
recommended.
161
CHAPTER 6Prevalence and transmission of hepatitis E virus
in domestic swine population in different European countries
1 6 2
The final goal of this PhD project was estimating HEV prevalence in 6 different
European countries and applied a mathematical model (SIR, described below)
developed by Backer et al [251] to determine the HEV dynamics of transmission.
Below is a brief explanation about the mathematical model that Backer et al [251]
applied to study HEV dynamics of transmission follow by the description of HEV
prevalence in 6 European countries.
6.1 Pig dynamics of transmission modeling study
Field studies, both cross-sectional [192, 252] and longitudinal [206, 253] have
shown a peak prevalence of HEV RNA in grower pigs, and a non-zero prevalence
in finishing pigs at slaughter age. A mathematical model determined the prevalence
pattern by how fast a susceptible animal can be infected (expressed by the
transmission rate parameter) and how long an infectious animal excretes virus
(expressed by the average infectious period). The product of these two parameters is
the reproduction number Rq that represents the number of infections one infectious
animal can cause in a fully susceptible population. However, the proportion of
infectious animals at slaughter age depends on all transmission parameters and these
have been determined in an experimental setting only [86]. Backer et al [251]
estimated all parameters that determine the transmission dynamics of HEV between
pigs, from field data confirming that HEV in pigs is endemic.
Briefly, each age group is subdivided in three distinct compartments that consist of
pigs that are susceptible (S), infectious (I) or recovered (R) [254]. The SIR model
assumed an endemic equilibrium. The virus is assumed not to be introduced by
infected weaners or other external sources but the disease can sustain itself in the
163
regenerating pig population. This endemic equilibrium can only exist when the virus
is sufficiently transmissible. The transmissibility is expressed by the reproduction
number Rq that represents the number of secondary infections caused by one
infectious animal during its entire infectious period in a fully susceptible and
infinite population [254]. When this number is smaller than one, the outbreak
cannot sustain itself and will die out. Therefore, the endemic equilibrium
assumption also contains the assumption that Ro > 1. This SIR model choice means
that the latent period was ignored and the infected animals reach immunity after
infection.
The same model described above was used to study HEV circulation in 6 different
EU countries (Following section).
6.1.1 Introduction
In 2008, Di Bartolo et al [192] investigated the prevalence of swine HEV in 274
pigs from six different swine farms of Northern Italy. Viral RNA was tested in
faeces and HEV RNA was detected in 42% of the samples. All farms tested positive
for HEV, with a prevalence ranging between 12.8% and 72.5%. All age groups
tested HEV-positive, although infection was more prevalent in weaners than in the
fatteners (42.2% vs. 27.0%).
Fernandez-Barredo et al [252] in 2006, tested 146 faecal samples of pigs from 21
farms. HEV RNA was detected in faecal samples from 34 pigs (23.29%). Pigs in the
first month of feeding (60%) and weaners presented the higher HEV prevalence
(41.7%).
164
De Deus et al [253] conducted a prospective study, where 19 sows and 45 piglets
were tested for antibodies to HEV. HEV IgG and IgM antibody was detected in
76.9% and 15.4% of sows, respectively. HEV RNA was detected in serum at all
ages analysed with the highest prevalence at 15 weeks of age. HEV was detected in
faeces and lymph nodes for the first time at 9 weeks of age and peaked at 12 and 15
weeks of age [253]. This peak coincided with the occurrence of mild to moderate
focal hepatitis as well as with HEV detection in bile, liver, mesenteric lymph nodes
and faeces, and with highest IgG and IgM at 15 weeks [253].
Few HEV transmission dynamics studies have been performed so far in pigs. The
common aim of those studies was evaluating the Rq that represents the number of
infections that one infectious animal can cause in a fully susceptible population.
Backer et al [251] estimated transmission parameters to explain the prevalence
pattern between pigs of different age groups. Briefly, the model describes how soon
after exposure a susceptible animal can be infected (expressed by the transmission
rate parameter) and how long an infectious animal excretes virus (expressed by the
average infectious period). The product of these two parameters is the reproductive
number Rq that represents the number of infections once that one infectious animal
can cause in a fully susceptible population.
Satou et al [207], using serology, tried to clarify the mechanisms of transmission
within farms in order to facilitate an understanding of the age-specific patterns of
infection, especially just prior to slaughter, estimating that more than 95% of pigs
are infected before the age of 150 days at which pigs are ready to be slaughtered.
The objective of this study was to evaluate HEV prevalence and HEV transmission
rates in different pig age groups in different countries. For this work, results from
165
pig samples obtained from farms in United Kingdom, Portugal, The Netherlands,
Italy, Spain and Czech Republic were used. For comparison of HEV transmission
rates and HEV infectious periods the model developed by Backer et al was used
[251].
166
Materials and methods
6.2 Samplings
The UK data sets (UK2007 and UK2008) consisted of 10 herds sampled by age
class: weaners (6-9 weeks of age), growers (10-12 weeks of age), fatteners (13-26
weeks of age) and sows. Pig faecal samples were collected from 10 different pig
farms in 2007 and 10 pig farms in 2008. Five faecal samples were obtained from
each age group.
In the Portugal data set, each herd was tested at entering (weaning age of 3 weeks),
growing (7 weeks) and at departure (slaughtering age of 21 weeks). A total of 200
pig faeces samples were collected from 5 commercial pig farms (40 samples per
farm) between December 2010 and February 2011. From each farm a total of 10
stool samples were obtained from each age group.
The data sets of Italy and The Netherlands comprised of test results of one fattening
group (21 weeks) of one single farm for The Netherlands (60 samples tested) and 3
farms for Italy (100 pigs faeces tested, age of the pigs 150 days), whereas the data
set obtained from Spain comprised of one group of sows in one single farm, and 23
boars in 5 different farms where faeces were tested for HEV RNA.
Ten pig farms were selected in Czech Republic, faecal samples from 200 pigs of
different age groups, weaners, growers, fatteners, sows and boars were tested for
HEV.
In all farms, samples of a minimum of 1 g of faeces were collected aseptically in a
sterile plastic container and maintained at 4°C (max. 24 h) or frozen at -20°C until
processing.
167
6.3 RNA extraction and RT-PCR procedures
6.3.1 UK 2007 and 2008
RNA extraction and PCR was performed as described by McCreary et al 2008
[223]. Briefly, 0.2 g of faeces was suspended in 1.8 ml phosphate-buffered saline,
140 pi of the supernatant was used to extract RNA, using the QIAamp Viral RNA
mini kit (Qiagen) according to the manufacturer’s instructions. The first round of
the PCR used 2 pi of RNA. The reaction conditions were 96°C for five minutes,
then 35 cycles of 96°C for five seconds, 55°C for five seconds and 75°C for 30
seconds, followed by 72°C for one minute. A second round was carried out with a
nested PCR, using a fast cycling PCR kit (Qiagen). The primers targeted the ORF-2
region; 3158N (forward): 5’ GTT(A)ATGCTT(C)TGCATA(T)CATGGCT-3’ and
3159N (reverse): 5 -AGCCGACGAAATCAATTCTCTC-3’ (Huang et al 2002).
The products of the amplification process were separated by gel electrophoresis, and
visualised with UV light [223].
6.3.2 The Netherlands, Portugal, Italy, Spain and Czech Republic
Two hundred and fifty mg of soft faecal contents was suspended in 2.25 ml of
gentamycin-containing PBS solution and centrifuged at 3.000g for 15 min. Nucleic
acid was extracted from 140 pi of the supernatant using the QIAamp® viral RNA
mini kit (QIAGEN), according to manufacturer’s instructions.
The real time RT-PCR was performed using RNA Ultrasense"^^ One-Step
Quantitative RT-PCR System (Invitrogen) and primers and probe: JHEV-F (5’-
GGT GGT TTC TGG GGT GAC -3’); JVHEV-R (5’- AGG GGT TGG TTG GAT
GAA -3’); JHEV-P (Taqman probe) ( 5 -FAM- TGA TTC TCA GCC CTT CGC -
BHQl-3’). Ten pi of RNA were added to a mix containing buffer RNA Ultrasense
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(Invitrogen) reaction mix (5X), ROX reference dye (50X) and RNA Ultrasense
enzyme mix.
The real time RT-PCR was carried out at 50°C for 15 min, 95°C for 2 min, and 45
cycles at 95°C for 10 sec, 55°C for 20 sec and 12°C for 15 sec.
6.3.3 HEV transmission modelling
The model to describe HEV transmission in a pig herd with the same structure has
been described by Backer et al [251]. Each age group was subdivided into three
distinct compartments consisting of pigs which are susceptible (S), infectious (I) or
recovered (R) [21]. For the analyses, it was assumed that each susceptible animal
can be infected by an infectious animal in its own group or any other group with the
same probability.
These dynamics are characterized by the average infectious period p and the
transmission rate parameter P that signifies the number of infections one infectious
animal can cause per time unit. The product of these two parameters is the
reproductive number Rq = p*p that expresses the number of infections one
infectious animal can cause during its entire infectious period in a fully susceptible
population. When the reproduction number is larger than one unity, Rq > 1, an
outbreak can grow exponentially. Otherwise, when Ro < 1 the outbreak will die out.
Our model assumes HEV transmission to be in endemic equilibrium, i.e. the disease
can sustain itself in the regenerating pig population. For this reason, we have
omitted the herds with few positive or only negative results, as endemic equilibrium
could not be justified.
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The UK data sets (UK2007 and UK2008) consisted of herds subdivided into three
groups: weaners (6-9 weeks of age), growers (10-12 weeks of age) and fatteners
(13-26 weeks of age). Animals entering the weaning group are assumed to be
uninfected. In the Portugal data set, the herds were assumed to consist of one group
that was tested at entering (weaning age of 3 weeks) and at departure (slaughtering
age of 21 weeks). The test results of the growers (age of 7 weeks) are used as proxy
for the infection pressure in the entire herd. The data sets of Italy and The
Netherlands comprise of test results of just one fattening group. For this reason, we
cannot estimate the transmission rate parameter and the average infectious period
separately, but only their product, the reproduction number. For both data sets the
total residence time is assumed to be 20 weeks from weaning to slaughtering age.
The data set of Spain and the Czech Republic did not include a significant number
of positive samples. For this reason, we cannot estimate the reproduction number.
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Results
HEV prevalence in different age groups in the UK (2007, 10 farms and 2008, 10
farms), in Portugal (2011, 5 farms), Italy (2010, 3 farms). The Netherlands (2011, 1
farm), Czech Republic (2010, 10 farms), Spain (one farm between 2010 and 2011)
are depicted in Figure 6.1. Briefly, the prevalence of weaners, growers, fatteners
and sows in UK 2007 was 26%, 44%, 10% and 6% respectively. The prevalence of
prevalence of weaners, growers, fatteners and sows in UK 2008 was 8%, 22%, 8.8%
and 2%. The prevalence of weaners, growers, fatteners and sows in Portugal was
30%, 20%, 30% and 4% respectively. The prevalence of fatteners in Italy was 23%.
The prevalence of fatteners in The Netherlands was 73%, meaning that 44 out of 60
pigs were shedding virus in the faeces on the day of the sample collection. The data
set is similar between the age groups and the prevalence is in line with other studies.
The prevalence in The Netherlands was relatively higher in the fattening groups
compared to the other European fattening groups. One hundred and forty-four faecal
samples from sows collected in Spain and tested by real time RT-PCR were found
to be HEV negative, while 4.3% of the boars (1 positive out of 23) was positive. In
none of the weaners and fatteners tested in the Czech Republic, HEV RNA was
detected. Only one grower out of 32 (3.1%), 5 sows out of 103 (5%) and 1 boar
(3.5%) out of 28 tested HEV positive by real time RT-PCR.
Table 6.2 shows the transmission rate parameter p, average infectious period p and
reproductive number Rq of UK 2007 and 2008 and Portugal and the reproductive
number R q for Italy and The Netherlands. The data set from Spain and Czech
Republic could not be used in this study since all or almost all animal tested were
HEV negative and we could not apply the model to those data. Briefly the
transmission rate parameter p, that means how often a pig gets infected with HEV is
171
one pig every 9 days for UK 2007, one pig every 11 days for UK 2008 and one pig
every 27 for Portugal. The average of the infectious period p that means how long
an animal stays infected with HEV is 43 day for both UK 2007 and 2008, and 101
days for Portugal. The reproductive number for all countries where the model has
been applied was greater than one, indicating that HEV is endemic.
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mean prevalence
4>OC0>(V>2a.
100% T
20% -
weaners
□ UK 2007
m UK2008
■ Portugal
□ Spain
■ The Netherlands
□ Italy
■ C zech Republic
growers fatteners
age group
sows boar
Figure 6.1 HEV swine prevalence in six different EU countries. HEV RNA
prevalence plotted for six countries and 5 pig age groups. The X axis represents the
age groups weaners (UK 2007, UK2008 and Portugal), growers (UK 2007, UK
2008, Portugal and CZ), fatteners (UK 2007, UK 2008, Portugal, The Netherlands
and Italy), sows (UK 2007, UK 2008, Portugal and CZ) and boar (Spain and CZ).
The Y represents the HEV prevalence in percentage observed in the different age
groups and in the different EU countries. Error bars denote the standard error of the
mean.
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Dataset transmission rate parameter p average infections reproductive number(dayh period ) i (days)
UK 2007 0.11 (0.070-0.17) 43 (3 3 -5 9 ) 4.7 (3 .6 -6 .4 )(10 herds)
UK 2008 0.071 (0.041 -0 .13) 43 (2 9 -7 3 ) 3.1 (2 .5 -4 .1 )(8 herds)
Portugal 0.037 (0.0035-0.16) 101 (70 -403) 3.7 (1 .2 - 14)(6 herds)
Italy - - 2.0 (1 .4 -3 .6 )(3 herds)
Netherlands - - 8.4 (5 .3 -15 )(1 herd)
Spain - - -
Czech Republic - - -
Table 6.2 Transmission rate parameter, average of infectious period and
reproductive number. The first column describes the dataset (UK 2007, UK 2008,
Portugal, Italy, The Netherlands, Spain and Czech Republic). The second column
describes the estimated transmission rate parameter p. The third column shows the
average infectious period \x and the fourth column describes the reproductive
number Rq of each country. Median maximum likelihood estimates and 5% - 95%
credible interval between brackets.
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6.4 Discussion
The HEV transmission dynamics in commercial pig farms in six different European
countries (UK, Portugal, Italy, The Netherlands, Spain and Czech Republic) was
studied.
The data collected show the HEV RNA prevalence in weaners ranging from 8% to
30%. The average HEV prevalence in growers was between 3% and 44%. The
fatteners prevalence ranged between 8% and 73%. Sow prevalence was similar in
all countries ranging between 2% and 6%. Boar faeces were tested for HEV only in
Spain and Czech Republic, and the prevalence was 4.3% and 3.5% respectively.
The prevalence detected in these 6 European countries shows that HEV is actively
circulating.
Overall, Figure 6.2 describes HEV RNA prevalence comparing Czech Republic,
Italy, Portugal, Spain, The Netherlands and UK 2007, 2008. The data set is similar
between the age groups and the prevalence is of the same order as with other studies
[223, 252]. The prevalence in the Dutch fattening group was relatively higher
compared to other European fattening groups [255] possibly due to an outbreak
during the sampling collection.
Our data are similar to previously published Italian [255] and Spanish [252] data,
confirming that HEV circulation during time is constant in terms of HEV
prevalence detected in faeces and HEV is circulating in all farms in all age groups,
from weaners to fatteners and that pigs close to the slaughter age can still be
infected with HEV.
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The collected data sets were analyzed using a recently developed model to estimate
the transmission dynamics of HEV in the different countries.
Satou et al in 2007 [207] using serology, studied HEV transmission in 6 different
Japanese provinces and found the reproductive number in the order of 4.02 - 5.17,
which agrees with our estimated reproductive numbers ranging from 2.0 to 8.4. The
study by Satou et al [207] was the first report on HEV transmission estimated from
field data. Bouwknegt et al in 2008 performed the first HEV transmission dynamics
study in an animal experiment [86]. In this study, the Rq was found to be 8.8 and 32
in two separate experiments, much higher than 1.0, indicating that swine could be
assumed to be a true reservoir of HEV. The Rq values calculated by us are lower
than the Rq values calculated by Bouwknegt et al [86]. This is because the
infectious periods are comparable, but the transmission rate parameters for the
experimental and field situation are different.
The average infectious period p in UK 2007 data was for instance estimated to be
43 (33 - 59) days, whereas Bouwknegt et al [86] estimated average infectious
periods of 49 (17-141) days and 13 (11 - 17) days.
The transmission rate parameter in our study was 0.11 (0.070 - 0.17) day'^ for UK
2007, meaning that one infectious animal infects another animal every 9 days. The
transmission rate parameters were 0.071 (0.041-0.13) day'^ for UK 2008 and 0.037
(0.0035-0.16) day'^ for Portugal 2011. In the animal experiments, Bouwknegt et al
[86] estimated a higher rate of transmission , i.e. 0.66 (95% Cl: 0.32-1.35) day '\
The difference can be explained by the fact that transmission experiment encounter
animals that are in the early and possibly more infectious stages of virus shedding
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since they have been infected intravenously while in other hand the animals in the
commercial farms are infected due to faecally-orally transmission.
The transmission rate parameters for the other EU countries could not be estimated
because either only one age group was tested or the majority of the animals were
negative and the model was not applicable.
This study gave a genuine contribution to better understand HEV prevalence in six
different European countries by a mathematical model.
In conclusion, HEV is widely circulating in many pig farms in Europe and can be
present in fattening pigs, where usually this age group is the one arriving to the
table. In industrialized regions, although the incidence of clinical hepatitis E in
humans is low, the seroprevalence is relatively high [86], indicating a high
proportion of subclinical disease and/or underdiagnosis [124]. It is likely that a
small proportion of this exposure to HEV results from travel to endemic regions, or
migration from endemic regions [117], this still leaves a substantial level of
exposure to HEV that appears to have an indigenous source and might be related to
the presence of endemic HEV infections in the pig population.
HEV positive fatteners were found in all European countries where the fattening
group samples were collected. This may pose an important risk for public health
especially in those countries where pork products are eaten undercooked or raw.
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CHAPTER 7 Overall discussion
178
This PhD project was funded by the EU FP7 project VITAL (Integrated monitoring
and control of foodborne virus in European food supply chains).
The EU FP7 project VITAL aimed to develop a system for monitoring viral
contamination of foodstuff intended for human consumption, by examination of
selected food chains from production through processing to point of sale.
The main areas investigated during this PhD were:
• Standardization of methods for detection of viruses in different foodstuff
(for example: soft fruit, fresh vegetables and pork products) via the VITAL ring
trial.
• Investigation of HEV prevalence in the pork food supply chain in the UK
(slaughterhouse, processing plant and points of sale).
• Development of a cell culture system for HEV.
• Investigation of resistance of HEV to different inactivation strategies.
• Investigation of HEV prevalence and transmission dynamics in pig farms in
Europe.
As part of the VITAL project, standard methods were developed to facilitate
harmonization of testing between the partner laboratories. In the first instance, this
harmonization took the form of a Ring Trial where a panel of samples of soft fruit
and pork products were tested blind by each data gathering laboratory. The aim of
the ring trial was to assess the efficacy of the SOPs developed during the first year
of the project, and to assess the capability of the different data gathering laboratories
in their implementation. Developing and validating SOPs for detection of viruses in
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foodstuff was needed considering the complexity of these matrices and the number
of participating laboratories. Furthermore, viruses present in food matrices do not
replicate in situ, and can therefore be present in small numbers, close to the limit of
detection of the technique used but still potentially infectious. The nucleic acid
extraction process is for this reason normally preceded by a concentration step and
by a lysis step in the case of intracellular viruses. Particular attention had to be paid
in reducing the concentration of inhibitors in the viral suspensions and extracts,
such as not to compromise the PCR reactions. Real Time RT-PCR was selected as
the best detection method for it's sensitivity in detecting viruses and the potential
use for quantification.
Data on the presence of HEV in abattoirs and points of sale have been published
previously [121] but a systematic investigation of the pork food chain was needed to
assess where a risk of HEV contamination can occur. The results obtained in this
project confirm the presence of HEV at slaughter, and underline the presence of
HEV in fresh pork products at point of sale. Detection by real time RT-PCR showed
the presence of HEV nucleic acid but gives no information of the virus viability,
and therefore the infection transmission risk of PCR-positive food and
environmental samples. The virus detected at point of sale was not able to cause
active infection in cell cultures, most likely because it was inactivated during the
meat preparation process or because the RNA detected by real time RT-PCR was
not enough to infect the cells. The failure of the infection of the 3D cells culture
could be also due to a prolonged -20°C storage or multiple freeze/thaw of the UK
sausages. The sample size was very small however, and it would be valuable to
follow up these results with a more focussed study involving a greater number of
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samples, with the power to generate significant results in order to inform evidence-
based risk assessments and codes of practice for the food industry.
Detection by real time RT-PCR shows the presence of nucleic acid and gives no
information of the virus viability, and therefore the risk, of PCR-positive food and
environmental samples. The lack of a reliable HEV cell culture system for viral in
vitro culture inhibits studies into the replication and environmental survival
properties of HEV and into vaccine research. As HEV has proved difficult to
propagate in conventional cell monolayer systems, we investigated the 3D cell
culture [200, 231] for more efficient virus propagation. The results obtained with
HEV-inoculated 3D cultures have showed detectable HEV RNA in real time RT-
PCR at all dpi in the first 3D cell culture infection, although a big variation in copy
number was detected during the data analysis. The wide copy number variation could
be due to virus internalisation in the cells while it is replicating. In contrast, in the 2D
cell culture system HEV RNA was not detectable at any dpi. These data illustrate
that the 3D system is more efficient when compared to the conventional 2D system.
Other studies have also demonstrated that the 3D cell-culture system is a useful tool
in the propagation of fastidious viral pathogens such as Norovirus [256]. Although it
proved very useful during the course of this project, the 3D cell culture system could
still benefit from further optimisation and standardisation such as be able to run the
experiments in duplicate to have a better and more efficient overview of the results
obtained. For example, further studies could examine the reasons why there is a big
variation in Ct values during the experiment.
The observation of HEV replication in PLC/PRF/5 cells in this system indicates that
the 3D system may potentially be used as a tool to investigate elements of the
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pathobiology of HEV, which may, in turn, facilitate vaccine research, monitoring of
HEV contamination and survival through processing to point of sale, and survival in
other environmental samples and viricidal agents. Once developed, the 3D cell
culture HEV infection system was used to investigate the infectivity of selected
foodstuff that tested positive by RT-PCR (three UK sausages and four smoked
French sausages-figatelli). Only one of the French sausages used as inoculum to
infect the 3D cells culture system showed HEV replication in the 3D cell culture
system, suggesting the presence of viable virus in the original sample and providing
further corroboration of the evidence implicating consumption of these sausages with
outbreaks of clinical hepatitis E in France. Furthermore, to better confirm that the
HEV positive supernatant of cells infected with homogenate of HEV positive
figatelli contained viable virus, the supernatant was tested by EM and a rare image of
several HEV-like particles was obtained from the supernatant of the infected culture.
The presence of HEV along the pork food chain is a cause for concern, and
inactivation strategies have been explored to reduce the contact of the consumer
with viable virus. We investigated inactivation strategies that could either be applied
during the production and processing phase of the pork meat, or during the
preparation of foodstuff in the kitchen. Ultraviolet light inactivation (that can be
applied in processing plants for disinfection) did not appear to be sufficiently
effective in inactivating HEV under the conditions applied. The use of NaOCl
caused a complete inactivation effect, but this could have been due to the toxic
effect of this chemical on cell culture systems. The lesson learned from this is to be
cautious when directly adopting published work without some initial pilot trial. Heat
inactivation at 100°C caused viral inactivation, whilst viable virus was still
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detectable after exposure of pig tissue at 56°C for an hour. These data stress the
importance of thoroughly cooking pork meat and other pig products prior to
consumption.
Data on HEV prevalence in pigs of different age classes were collected across
Europe, to study transmission dynamics and develop a model that could help the
understanding HEV transmission dynamics in the pig population. HEV was
confirmed to be endemic in pig farms across Europe. A mathematical model (SIR)
was applied by Backer et al for studying HEV transmission dynamics in the field
[251]. The results of this model suggested that the circulation of HEV is endemic in
pig farms in all age groups (weaners, growers, fatteners).
It is now generally accepted that HEV gt 3 is zoonotic and strict safety measures
should be taken to prevent the increasing of number of people detected with HEV.
Until now, the only preventative advice can be found on the website of the America
Ministry of Agriculture and DEFRA. The two websites suggest that pork foodstuff
should be safe to eat within an internal temperature of 71°C [87]. Both Defra and
the UK Food Standards Agency have been informed of the data relating to the
presence of HEV in the UK pork chain and HEV inactivation and guidelines will be
written and available for the public. For example providing cooking information and
conditions in all pork foodstuff products could be a way to control HEV infection in
humans.
In conclusion, the work carried out in this project helped in progressing the
knowledge on HEV epidemiology and pathogenesis, with particular attention to the
public health implications related to the consumption of pork meat [197].
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During the course of this PhD a broad range of biological disciplines were
employed, including, classical and molecular virology, epidemiology, HEV
transmission dynamics, advanced cell culture techniques, experimental design and
data interpretation. With this PhD a better figure regarding HEV has been generate
and it will hopefully help to improve future studies on this virus.
Future plans
Without doubt more studies are still necessary to better understand hepatitis E Virus
in all its characteristics. I would mainly like to focus on 3 aspects:
1) Hepatitis E virus monitoring in the pork production chain in a larger scale: A
bigger UK study investigating the presence of HEV in pork food stuff is necessary to
provide more confidence in the data on the prevalence of HEV in pork products in
the UK. Furthermore, HEV investigation in pork food stuff should be planned also in
resource limited regions to evaluate and confront which genotype is circulating in the
humans and in the pig population.
Furthermore, thinking of what are the major unknown areas, principally on the
veterinary side, but with links through to HEV in humans it is pretty well accepted
now that the only credible source for the autochthonous, clinical hepatitis E
infections in developed regions is the pig. Despite our evidence of foodborne virus, a
significant number of the clinical cases of hepatitis E in the UK and other developed
regions appear not to have this risk factor, according to retrospective questionnaires,
indicating that other (i.e. other than direct foodborne) transmission routes from the
pig to people may be contributing to the clinical (and possibly subclinical) cases. In
this context the presence and survival (i.e. viability) of HEV in all sorts of
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environmental samples could shed light on some of the possible alternative
transmission routes. These could include soil and water samples close to pig farms or
sewage outfalls, slurry lagoons, vegetables and fruits at various points, including the
water used to irrigate them (in fact one task of the VITAL project was the detection
of HEV on fruit and vegetables) and shellfish samples. Analysis of these samples by
the real-time RT-PCR and the 3D culture system could provide quantitative and
qualitative information on the potential risk pathways, enabling an appropriate
response to reduce or eliminate the HEV contamination. In addition, (as I already
mentioned in chapter 5 but it would be good to re-emphasise at this point) this work
could be supported by an extended examination by means of the 3D culture system,
of HEV inactivating agents to improve our ability to eliminate HEV contamination at
appropriate or practicable points in the transmission cycle. It should be remembered
though that achievement of these objectives would be enhanced by further
refinement of the 3D cell culture system to improve sample throughput numbers and
robustness.
2) In vitro studies
a) The 3D cell culture system, with a little more refinement, should be employed to
undertake cell infection and replication characteristics, to understand how this virus
enters the cells and which mechanism is used to replicate in and exit the cells.
b) Since that the 3D cell culture system is an expensive technique and it allows the
testing of maximum 8 samples for each experiment and it is time consuming (i.e. 28
days are required to allow the cells to differentiate in the 3D configuration before
infection). It is still necessary to study different cell lines (i.e. stem cells) that allow
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HEV replication with the same efficiency but reducing the costing, the time and
more important to have as many sample is possible in each experiment.
3) Diagnostic tools: Real-time RT-PCR, conventional RT-PCR, and ELISA, are the
only practicable and reliable techniques able to detect HEV and HEV antibodies
respectively. In developing countries, there is a need for reliable techniques able to
detect HEV (RNA) faster and without the need of trained personnel and specialized
laboratories.
a) Evaluate the use of isothermal nucleic acid amplification techniques, especially
LAMP (loop mediated isothermal amplification). The main characteristics of this
techniques include high sensitivity and specificity, rapid testing, constant
temperature operation, easy to perform and interpret and the possibility of combining
it with portable detection devices. This technique is used with great success for the
detection of other RNA viruses and it could represent a great advantage for point of
care screening of HEV in both specialized and non-specialized diagnostic labs,
hospitals and pork production points.
b) The PCRs currently available are genotype specific or in the case of Jothikumar’s
real time PCR based on recognising the 4 genotypes but without distinction, so
sample sequencing is necessary to distinguish which genotype the possible positive
sample belongs. The need of a multiplex RT-PCR able to detect and discriminate all
four major genotypes it would be beneficial.
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References1. Aggarwal R, Naik S: Epidemiology of hepatitis E: current status. Journal of
gastroenterology and hepatology 2009, 24(9): 1484-1493.2. Khuroo MS: Seroepidemiology of a second epidemic of hepatitis E in a population that
had recorded first epidemic 30 years before and has heen under surveillance since then. Hepatol Int 2010, 4(2):494-499.
3. Chandra V, Taneja S, Kalia M, Jameel S: Molecular hiology and pathogenesis of hepatitis E virus. Journal of biosciences 2008, 33(4):451-464.
4. Granoff DM: Correlates of clinical performance of acellular pertussis vaccines: implications for development of DTaP combination vaccines. Biologicals 1999, 27(2):87- 8&
5. Rzezutka A, D A gostino M, Cook N: An ultracentrifugation-hased approach to the detection of hepatitis A virus in soft fruits. Int J Food Microbiol 2 0 0 6 ,108(3):315-320.
6. Hundesa A, Maluquer de Motes C, Bofill-M as S, Albinana-Gimenez N, Girones R: Identifîcation of human and animal adenoviruses and polyoma viruses for determination of sources of fecal contamination in the environment. Appl Environ Microbiol 2006, 72(12):7886-7893.
7. Purcell RH, Emerson SU: Hepatitis E: an emerging awareness of an old disease. J Hepatol 2008, 48(3):494-503.
8. Meng XJ: Swine hepatitis E virus: cross-species infection and risk inxenotransplantation. Curr Top Microbiol Immunol 2003, 278:185-216.
9. Dalton HR, Bendall R, Ijaz S, Banks M: Hepatitis E: an emerging infection in developed countries. Lancet Infect Dis 2008, 8(11):698-709.
10. Erker JC, Desai SM, Schlauder GG, Dawson GJ, Mushahwar IK: A hepatitis E virus variant from the United States: molecular characterization and transmission in cynomolgus macaques. J Gen Virol 1999, 80 ( Ft 3):681-690.
11. Ijaz S, Arnold E, Banks M, Bendall RP, Cramp ME, Cunningham R, Dalton HR, Harrison TJ, Hill SF, Macfarlane L et al: Non-travel-associated hepatitis E in England and Wales: demographic, clinical, and molecular epidemiological characteristics. J Infect Dis 2005, 192(7):1166-1172.
12. Mansuy JM, Peron JM, Abravanel F, Poirson H, Dubois M, Miedouge M, Vischi F, Alric L, Vinel IP, Izopet J: Hepatitis E in the south west of France in individuals who have never visited an endemic area. J Med Virol 2004, 74(3):419-424.
13. Zhuang H: [Advances in the research on non-A, non-B hepatitis in China]. Zhonghua Liu Xing Bing Xue Za Zhi 1991,12(6):377-379.
14. Naik SR, Aggarwal R, Salunke PN, Mehrotra NN: A large waterborne viral hepatitis E epidemic in Kanpur, India. Bull World Health Organ 1992, 70(5):597-604.
15. Corwin AL, Tien NT, Bounlu K, Winarno J, Putri MP, Laras K, Larasati RP, Sukri N, Endy T, Sulaiman HA et al: The unique riverine ecology of hepatitis E virus transmission in South-East Asia. Trans R Soc Trop Med Hyg 1999, 93(3):255-260.
16. Arankalle VA, Tsarev SA, Chadha MS, Ailing DW, Emerson SU, Banerjee K, Purcell RH: Age-specific prevalence of antibodies to hepatitis A and E viruses in Pune, India, 1982 and 1992. J Infect Dis 1 9 9 5 ,171(2):447-450.
17. Khuroo MS: Chronic liver disease after non-A, non-B hepatitis. Lancet 1980, 2(8199):860-861.
18. Wong KH, Liu YM, Ng PS, Young BW, Lee SS: Epidemiology of hepatitis A and hepatitis E infection and their determinants in adult Chinese community in Hong Kong. J Med Virol 2004, 72(4):538-544.
19. Wong DC, Purcell RH, Sreenivasan MA, Prasad SR, Pavri KM: Epidemic and endemic hepatitis in India: evidence for a non-A, non-B hepatitis virus aetiology. Lancet 1980, 2(8200):876-879.
20. Arankalle VA, Chadha MS, Mehendale SM, Tungatkar SP: Epidemic hepatitis E: serological evidence for lack of intrafamilial spread. Indian J Gastroenterol 2000, 19(l):24-28.
21. Assis SB, Souto FJ, Fontes CJ, Caspar AM: [Prevalence of hepatitis A and E virus infection in school children of an Amazonian municipality in Mato Grosso State]. RevSoc Bras Med Trop 2002, 35(2):155-158.
187
22. Ataei B, Nokhodian Z, Javadi AA, Kassaian N, Shoaei P, Farajzadegan Z, Adibi P: Hepatitis E virus in Isfahan Province: a population-hased study. Int J Infect Dis 2 0 0 9 ,13(1):67-71.
23. Clayson ET, Shrestha MP, Vaughn DW, Snitbhan R, Shrestha KB, Longer CF, Innis BL: Rates of hepatitis E virus infection and disease among adolescents and adults in Kathmandu, Nepal. J/n /ecr Dw 1 9 9 7 ,176(3):763-766.
24. Tran HT, Ushijima H, Quang VX, Phuong N, Li TC, Hayashi S, Xuan Lien T, Sata T, Abe K: Prevalence of hepatitis virus types B through E and genotypic distribution of HBV and HCV in Ho Chi Minh City, Vietnam. Hepatol Res 2003, 26(4):275-280.
25. Margolis H AM, Hadler SC. Viral hepatitis. In Evans AS, Kaslow RA, eds. Viral infections of humans: epidemiology and control, 4th edn. New York: Plenum Medical Books Co, 1997: 363-418.
26. Balayan MS: Epidemiology of hepatitis E virus infection. J Viral Hepat 1997, 4(3):155-165.
27. Khuroo MS, Teli MR, Skidmore S, Sofi MA, Khuroo MI: Incidence and severity of viral hepatitis in pregnancy. Am J Med 1981, 70(2):252-255.
28. Khuroo MS, Kamili S, Khuroo MS: Clinical course and duration of viremia in vertically transmitted hepatitis E virus (HEV) infection in babies horn to HEV-infected mothers. Journal of viral hepatitis 2009,16(7):519-523.
29. Kar P, Jilani N, Husain SA, Pasha ST, Anand R, Rai A, Das BC: Does hepatitis E viral load and genotypes influence the final outcome of acute liver failure during pregnancy? Am J Gastroenterol 2 0 0 8 ,103(10):2495-2501.
30. Emerson SU, Nguyen H, Graff J, Stephany DA, Brockington A, Purcell RH: In vitro replication of hepatitis E virus (HEV) genomes and of an HEV replicon expressing green fluorescent protein. J Virol 2004, 78(9):4838-4846.
31. Reyes GR, Yarbough PO, Tam AW, Purdy MA, Huang CC, Kim JS, Bradley DW, Fry KE: Hepatitis E virus (HEV): the novel agent responsible for enterically transmitted non-A, non-B hepatitis. Gastroenterol Jpn 1991, 26 Suppl 3:142-147.
32. Huang RT, Li DR, Wei J, Huang XR, Yuan XT, Tian X: Isolation and identification of hepatitis E virus in Xinjiang, China. J Gen Virol 1992, 73 ( Pt 5): 1143-1148.
33. Panda SK, Ansari IH, Durgapal H, Agrawal S, Jameel S: The in vitro-synthesized RNA from a cDNA clone of hepatitis E virus is infectious. J Virol 2000, 74(5):2430-2437.
34. Purcell RH, Emerson SU: Animal models of hepatitis A and E. ILAR J 2001, 42(2): 161-177.
35. Tam AW, Smith MM, Guerra ME, Huang CC, Bradley DW, Fry KE, Reyes GR: Hepatitis E virus (HEV): molecular cloning and sequencing of the full-length viral genome. Virology 1991,185(1):120-131.
36. C M. Fauquet M, M.A., Maniloff, J., Desselberger, U., Ball, : Taxonomy London; 2005.37. Koonin EV, Senkevich TG: Evolution of thymidine and thymidylate kinases: the
possibility of independent capture of TK genes by different groups of viruses. Virus Genes 1992, 6(2): 187-196.
38. Krawczynski K, Aggarwal R, Kamili S: Hepatitis E. Infect Dis Clin North Am 2000, 14(3):669-687.
39. Krawczynski K, Mast EE, Purdy MA: Hepatitis E: an overview. Minerva Gastroenterol Dietol 1999, 45(2): 119-130; discussion 130-115.
40. Aggarwal R, Krawczynski K: Hepatitis E: an overview and recent advances in clinical and laboratory research. J Gastroenterol Hepatol 2 0 0 0 ,15(l):9-20.
41. Magden J, Takeda N, Li T, Auvinen P, Ahola T, Miyamura T, Merits A, Kaariainen L: Virus-specific mRNA capping enzyme encoded by hepatitis E virus. J Virol 2001, 75(14):6249-6255.
42. Panda SK, Thakral D, Rehman S: Hepatitis E virus. Reviews in medical virology 2007, 17(3):151-180.
43. Karpe YA, Lole KS: RNA 5'-triphosphatase activity of the hepatitis E virus helicase domain. J Virol 2010, 84(18):9637-9641.
44. Zhang M, Purcell RH, Emerson SU: Identification of the 5' terminal sequence of the SAR- 55 and MEX-14 strains of hepatitis E virus and confirmation that the genome is capped. JMed Virol 2001, 65(2):293-295.
45. Zhang M, Emerson SU, Nguyen H, Engle RE, Govindarajan S, Gerin JL, Purcell RH: Immunogenicity and protective efficacy of a vaccine prepared from 53 kDa truncated hepatitis E virus capsid protein expressed in insect cells. Vaccine 2001, 20(5-6):853-857.
188
46. Kaur R, Gur R, Berry N, Kar P: Etiology of endemic viral hepatitis in urban North India.Southeast Asian J Trop Med Public Health 2002, 33(4):845-848.
47. Okamoto H: Genetic variability and evolution of hepatitis E virus. Virus Res 2007, 127(2):216-228.
48. Lu L, Li C, Hagedorn CH: Phylogenetic analysis of global hepatitis E virus sequences: genetic diversity, subtypes and zoonosis. Rev Med Virol 2006,16(l):5-36.
49. Jameel S, Zafrullah M, Ozdener MH, Panda SK: Expression in animal cells and characterization of the hepatitis E virus structural proteins. J Virol 1996, 70(1):207-216.
50. Zafrullah M, Ozdener MH, Kumar R, Panda SK, Jameel S: Mutational analysis of glycosylation, membrane translocation, and cell surface expression of the hepatitis E virus ORF2 protein. J Virol 1999,73(5):4074-4082.
51. Tsarev SA, Emerson SU, Reyes GR, Tsareva TS, Legters LJ, Malik lA , Iqbal M, Purcell RH: Characterization of a prototype strain of hepatitis E virus. Proc Natl Acad Sci U SA 1992, 89(2):559-563.
52. Meng XJ, Purcell RH, Halbur PG, Lehman JR, Webb DM, Tsareva TS, Haynes JS, Thacker BJ, Emerson SU: A novel virus in swine is closely related to the human hepatitis E virus. Proc Natl Acad Sci USA 1997, 94(18):9860-9865.
53. Graff J, Zhou YH, Torian U, Nguyen H, St Claire M, Yu C, Purcell RH, Emerson SU: Mutations within potential glycosylation sites in the capsid protein of hepatitis E virus prevent the formation of infectious virus particles. J Virol 2008, 82(3): 1185-1194.
54. Pelham HR, Munro S: Sorting of membrane proteins in the secretory pathway. Cell 1993, 75(4):603-605.
55. Torresi J, Li F, Locarnini SA, Anderson DA: Only the non-glycosylated fraction of hepatitis E virus capsid (open reading frame 2) protein is stable in mammalian cells. J Gen Virol 1999, 80 ( Pt5):l 185-1188.
56. Panda SK, Nanda SK, Zafrullah M, Ansari IH, Ozdener MH, Jameel S: An Indian strain of hepatitis E virus (HEV): cloning, sequence, and expression of structural region and antibody responses in sera from individuals from an area of high-level HEV endemicity. J Clin Microbiol 1995, 33(10):2653-2659.
57. Torresi J, Johnson D: Hepatitis A and E Infection in International Travellers. Curr Infect Dis Rep 2011.
58. Robinson RA, Burgess WH, Emerson SU, Leibowitz RS, Sosnovtseva SA, Tsarev S, Purcell RH: Structural characterization of recombinant hepatitis E virus ORF2 proteins in haculovirus-infected insect cells. Protein Expr Purif 199S, 12(l):75-84.
59. Carl M, Isaacs SN, Kaur M, He J, Tam AW, Yarbough PO, Reyes GR: Expression of hepatitis E virus putative structural proteins in recombinant vaccinia viruses. Clin Diagn Lab Immunol 1994, l(2):253-256.
60. Tyagi S, Jameel S, Lai SK: The full-length and N-terminal deletion of ORF2 protein of hepatitis E virus can dimerize. Biochem Biophys Res Commun 2001, 286(1):214-221.
61. Tsarev SA, Emerson SU, Tsareva TS, Yarbough PO, Lewis M, Govindarajan S, Reyes GR, Shapiro M, Purcell RH: Variation in course of hepatitis E in experimentally infected cynomolgus monkeys. J Infect Dis 1993,167(6): 1302-1306.
62. Tsarev VN, Ushakov RV, Vaneeva NP, lastrebova NE, Ushakova TV: [The development of a test system for determining antibodies to Bacteroides melaninogenicus using solid- phase immunoenzyme analysis]. Klin Lab Diagn 1993(5):11-15.
63. McAtee CP, Zhang Y, Yarbough PO, Fuerst TR, Stone KL, Samander S, W illiams KR: Purification and characterization of a recombinant hepatitis E protein vaccine candidate by liquid chromatography-mass spectrometry. J Chromatogr B Biomed Appl 1996,685(0:91-104 .
64. Zhang M, Yi Y, Liu C, Bi S: [Expression of full-length hepatitis E virus structural gene using haculovirus expression system]. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi 2 0 0 2 ,16(4):354-356.
65. Li F, Riddell MA, Seow HF, Takeda N, Miyamura T, Anderson DA: Recombinant subunit ORF2.1 antigen and induction of antibody against immunodominant epitopes in the hepatitis E virus capsid protein. J Med Virol 2000, 60(4):379-386.
66. Li TC, Yamakawa Y, Suzuki K, Tatsumi M, Razak MA, Uchida T, Takeda N, Miyamura T: Expression and self-assembly of empty virus-like particles of hepatitis E virus. J Virol 1997,71(10):7207-7213.
189
67. Yarbough PO, Tam AW, Fry KE, Krawczynski K, McCaustland KA, Bradley DW, Reyes GR: Hepatitis E virus: identification of type-common epitopes. J Virol 1991, 65(ll):5790-5797 .
68. Grabow WO, Favorov MO, Khudyakova NS, Taylor MB, Fields HA: Hepatitis E seroprevalence in selected individuals in South Africa. 7 Virol 1994, 44(4):384-388.
69. Parvez K, Purcell RH, Emerson SU: Hepatitis E virus ORF2 protein over-expressed hy haculovirus in hepatoma cells, efficiently encapsidates and transmits the viral RNA to naive cells. VirolJ 2D\\, 8(1): 159.
70. Xing L, Li TC, Mayazaki N, Simon MN, Wall JS, Moore M, Wang CY, Takeda N, Wakita T, Miyamura T et al: Structure of hepatitis E virion-sized particle reveals an RNA- dependent viral assembly pathway. 7 Bm/ Chem 2010, 285(43):33175-33183.
71. Emerson SU, Clemente-Casares P, Moiduddin N, Arankalle VA, Torian U, Purcell RH: Putative neutralization epitopes and broad cross-genotype neutralization of Hepatitis E virus confirmed hy a quantitative cell-culture assay. 7 Gen Virol 2006, 87(Pt 3):697-704.
72. Yamada A, Sako A, Nishimura S, Nakashima R, Ogami T, Fujiya K, Tsuda N, Asayama N, Yada T, Shirai K et al: [A case of HIV coinfected with hepatitis B virus treated hy entecavir]. Nippon Shokakibyo Gakkai Zasshi 2009,106(12): 1758-1763.
73. Zafrullah M, Ozdener MH, Panda SK, Jameel S: The ORF3 protein of hepatitis E virus is a phosphoprotein that associates with the cytoskeleton. 7 Virol 1997,71(12):9045-9053.
74. Xing L, Kato K, Li T, Takeda N, Miyamura T, Hammar L, Cheng RH: Recombinant hepatitis E capsid protein self-assemhles into a dual-domain T = 1 particle presenting native virus epitopes. Virology 1999, 265(l):35-45.
75. Kar-Roy A, Korkaya H, Oberoi R, Lai SK, Jameel S: The hepatitis E virus open reading frame 3 protein activates ERK through binding and inhibition of the MAPK phosphatase. 7 Biol Chem 2004, 279(27):28345-28357.
76. Ratra R, Kar-Roy A, Lai SK: The ORF3 protein of hepatitis E virus interacts with hemopexin hy means of its 26 amino acid N-terminal hydrophobic domain II. Biochemistry 2008, 47(7): 1957-1969.
77. Chandra V, Kar-Roy A, Kumari S, Mayor S, Jameel S: The hepatitis E virus ORF3 protein modulates epidermal growth factor receptor trafficking, STAT3 translocation, and the acute-phase response. Journal of virology 2008, 82(14):7100-7110.
78. He S, Miao J, Zheng Z, Wu T, X ie M, Tang M, Zhang J, Ng MH, Xia N: Putative receptor- binding sites of hepatitis E virus. The Journal of general virology 2008, 89(Pt l):245-249.
79. Williams TP, Kasorndorkbua C, Halbur PG, Haqshenas G, Guenette DK, Toth TE, M eng XJ: Evidence of extrahepatic sites of replication of the hepatitis E virus in a swine model. 7 Clin Microbiol 2001, 39(9):3040-3046.
80. Ippagunta SK, Naik S, Jameel S, Ramana KN, Aggarwal R: Viral RNA hut no evidence of replication can he detected in the peripheral blood mononuclear cells of hepatitis E virus-infected patients. 7 Viral Hepat 2010.
81. Aggarwal R, Kamili S, Spelbring J, Krawczynski K: Experimental studies on suhclinicalhepatitis E virus infection in cynomolgus macaques. 7 Infect Dis 2001, 184(11): 1380- 1385.
82. Sriram B, Thakral D, Panda SK: Targeted cleavage of hepatitis E virus 3' end RNAmediated hy hammerhead rihozymes inhibits viral RNA replication. Virology 2003,312(2):350-358.
83. Huang F, Zhou J, Yang Z, Cui L, Zhang W, Yuan C, Yang S, Zhu J, Hua X: RNAinterference inhibits hepatitis E virus mRNA accumulation and protein synthesis in vitro. Vet Microbiol 2 0 1 0 ,142(3-4):261-267.
84. Kamar N, Izopet J, Cintas P, Garrouste C, Uro-Coste E, Cointault O, Rostaing L: Hepatitis E virus-induced neurological symptoms in a kidney-transplant patient with chronic hepatitis. Am 7 Transplant 2 0 1 0 ,10(5):1321-1324.
85. Feagins AR, Opriessnig T, Guenette DK, Halbur PG, Meng XJ: Inactivation of infectious hepatitis E virus present in commercial pig livers sold in local grocery stores in the United States. Int J Food Microbiol 2 0 0 8 ,123(l-2):32-37.
86. Bouwknegt M, Frankena K, Rutjes SA, Wellenberg GJ, de Roda Husman AM, van der Poel WH, de Jong MC: Estimation of hepatitis E virus transmission among pigs due to contact-exposure. Vet Res 2008, 39(5):40.
190
87. Feagins AR, Opriessnig T, Guenette DK, Halbur PG, Meng XJ: Detection andcharacterization of infectious Hepatitis E virus from commercial pig livers sold in local grocery stores in the USA. J Gen Virol 2007, 88(Pt 3):912-917.
88. Matallana-Surget S, Douki T, Meador JA, Cavicchioli R, Joux F: Influence of growthtemperature and starvation state on survival and DNA damage induction in the marine bacterium Sphingopyxis alaskensis exposed to UV radiation. J Photochem Photobiol B 2 0 1 0 ,100(2):51-56.
89. Nuanualsuwan S, Mariam T, Himathongkham S, Oliver DO: Ultraviolet inactivation of feline calicivirus, human enteric viruses and coliphages. Photochem Photobiol 2002, 76(4):406-410.
90. Giese AC: Living with the sun’s ultraviolet rays.. Plenum
Press, New York 1976.91. Nicholson WL, Schuerger AC, Setlow P: The solar UV environment and bacterial spore
UV resistance: considerations for Earth-to-Mars transport hy natural processes and human spaceflight. Mutat Res 2005, 571(l-2):249-264.
92. Borio L, Inglesby T, Peters CJ, Schmaljohn AL, Hughes JM, Jahrling PB, Ksiazek T, Johnson KM, Meyerhoff A, O'Toole T et al: Hemorrhagic fever viruses as biological weapons: medical and public health management. JAMA 2002, 287(18):2391-2405.
93. Lytle CD, Sagripanti JL: Predicted inactivation of viruses of relevance to hiodefense hy solar radiation. J Virol 2005,79(22): 14244-14252.
94. Rzezutka A, Cook N: Survival of human enteric viruses in the environment and food. FEMS Microbiol Rev 2004, 28(4):441-453.
95. W ei H, Zhang JQ, Lu HQ, Meng JH, Lu XX, X ie W: [Construction and screening of hepatitis E virus-specific phage antibody combinatorial library]. Xi Bao Yu Fen Zi Mian YiXue Za Zhi 2 0 0 3 ,19(5):473-475, 485.
96. Schlauder GG, Mushahwar IK: Genetic heterogeneity of hepatitis E virus. J Med Virol 2001,65(2):282-292.
97. Teo CG: Much meat, much malady: changing perceptions of the epidemiology of hepatitis E. Clin Microbiol Infect 2010,16(l):24-32.
98. Geng J, Wang L, Wang X, Fu H, Bu Q, Zhu Y, Zhuang H: Study on prevalence and genotype of hepatitis E virus isolated from Rex Rabbits in Beijing, China. J Viral Hepat 2010 .
99. Zhao K, Liu Q, Yu R, Li Z, Li J, Zhu H, Wu X, Tan F, Wang J, Tang X: Screening of specific diagnostic peptides of swine hepatitis E virus. Virol J 2009, 6:186.
100. Johne R, Plenge-Bonig A, Hess M, Ulrich RG, Reetz J, Schielke A: Detection of a novel hepatitis E-like virus in faeces of wild rats using a nested hroad-spectrum RT-PCR. J Gen Virol 2010, 91(Pt 3):750-758.
101. Takahashi M, Nishizawa T, Sato H, Sato Y, Jirintai, Nagashima S, Okamoto H: Analysis of the full-length genome of a hepatitis E virus isolate obtained from a wild hoar in Japan that is classifiable into a novel genotype. The Journal of general virology 2011, 92(Pt 4):902-908.
102. Zhao C, Ma Z, Harrison TJ, Feng R, Zhang C, Qiao Z, Fan J, Ma H, Li M, Song A et al: A novel genotype of hepatitis E virus prevalent among farmed rabbits in China. J Med Virol 2009, 81(8):1371-1379.
103. Geng JB, Fu HW, Wang L, Wang XJ, Guan J, Chang YB, Li LJ, Zhu YH, Zhuang H, Liu QH et al: [Hepatitis E virus (HEV) genotype and the prevalence of anti-HEV in 8 species of animals in the suburbs of Beijing.]. Zhonghua Liu Xing Bing Xue Za Zhi 2010, 3I(l):47-50.
104. Bilic I, Jaskulska B, Basic A, Morrow CJ, Hess M: Sequence analysis and comparison of avian hepatitis E viruses from Australia and Europe indicate the existence of different genotypes. J Gen Virol 2009, 90(Pt 4):863-873.
105. Haqshenas G, Shivaprasad HL, W oolcock PR, Read DH, Meng XJ: Genetic identification and characterization of a novel virus related to human hepatitis E virus from chickens with hepatitis-splenomegaly syndrome in the United States. J Gen Virol 2001, 82(Pt 10):2449-2462.
106. Banks M, Heath GS, Grierson SS, King DP, Gresham A, Girones R, Widen F, Harrison TJ: Evidence for the presence of hepatitis E virus in pigs in the United Kingdom. Vet Rec2004,154(8):223-227.
191
107. Purdy MA, Khudyakov YE: Evolutionary history and population dynamics of hepatitis E virus. PLoS One 2010, 5(12):el4376.
108. Reuter G, Fodor D, Katai A, Szucs G: [Molecular detection of hepatitis E virus in nonimported hepatitis case-identification of a potential new human hepatitis E virus lineage in Hungary]. Orv Hetil 2 0 0 5 ,146(47):2389-2394.
109. Goens SD, Perdue ML: Hepatitis E viruses in humans and animals. Anim Health Res Rev 2004, 5(2): 145-156.
110. Hakze-van der Honing RW, van Coillie E, Antonis AF, van der Poel WH: First isolation of hepatitis E virus genotype 4 in Europe through swine surveillance in the Netherlands and Belgium. PloS one 2011, 6(8):e22673.
111. Tanaka Y, Takahashi K, Orito E, Karino Y, Kang JH, Suzuki K, Matsui A, Hori A, Matsuda H, Sakugawa H et al: Molecular tracing of Japan-indigenous hepatitis E viruses. J Gen Virol 2006, 87(Pt 4):949-954.
112. Drummond AJ, Ho SY, Phillips MJ, Rambaut A: Relaxed phylogenetics and dating with confidence. PLoS Biol 2006, 4(5):e88.
113. Emerson SU, Purcell RH: Hepatitis E virus. Rev Med Virol 2003,13(3): 145-154.114. Kwo PY, Schlauder GG, Carpenter HA, Murphy PJ, Rosenblatt JE, Dawson GJ, Mast EE,
Krawczynski K, Balan V: Acute hepatitis E hy a new isolate acquired in the United States. Mayo Clin Proc 1997 ,72(12):1133-1136.
115. Pina S, Buti M, Cotrina M, Piella J, Girones R: HEV identified in serum from humans with acute hepatitis and in sewage of animal origin in Spain. J Hepatol 2000, 33(5):826- 833.
116. Takahashi K, Kang JH, Ohnishi S, Hino K, Miyakawa H, Miyakawa Y, Maekubo H, Mishiro S: Full-length sequences of six hepatitis E virus isolates of genotypes HI and IV from patients with sporadic acute or fulminant hepatitis in Japan. Intervirology 2003, 46(5):308-318.
117. Peron JM, Mansuy JM, Poirson H, Bureau C, Dupuis E, Alric L, Izopet J, Vinel JP: Hepatitis E is an autochthonous disease in industrialized countries. Analysis of 23 patients in South-West France over a 13-month period and comparison with hepatitis A. Gastroenterologie clinique et biologique 2006, 30(5):757-762.
118. Meng S, Zhan S, Li J: Nuclease-resistant douhle-stranded DNA controls or standards for hepatitis B virus nucleic acid amplification assays. Virol J 2009, 6:226.
119. Lees D: International Standardisation of a Method for Detection of Human Pathogenic Viruses in Molluscan Shellfish. Food and Environmental Virology 2010, 2(3): 146-155.
120. Li D, Baert L, De Jonghe M, Van Coillie E, Ryckeboer J, Devlieghere F, Uyttendaele M: Inactivation of murine norovirus 1, coliphage phiX174, and Bacillus fragilis phage B40- 8 on surfaces and fresh-cut iceherg lettuce hy hydrogen peroxide and UV light. Applied and environmental microbiology 2011, 77(4):1399-1404.
121. Banks M, Bendall R, Grierson S, Heath G, Mitchell J, Dalton H: Human and porcine hepatitis E virus strains, United Kingdom. Emerging infectious diseases 2004, 10(5):953- 955.
122. Martin M, Segales J, Huang FF, Guenette DK, Mateu E, de Deus N, Meng XJ: Association of hepatitis E virus (HEV) and postweaning multisystemic wasting syndrome (PMWS) with lesions of hepatitis in pigs. Vet Microbiol 2007,122(1-2): 16-24.
123. Hirano M, Ding X, Li TC, Takeda N, Kawabata H, Koizumi N, Kadosaka T, Goto I, Masuzawa T, Nakamura M et al: Evidence for widespread infection of hepatitis E virus among wild rats in Japan. Hepatol Res 2003, 27(1): 1-5.
124. Meng XJ, Wiseman B, Elvinger F, Guenette DK, Toth TE, Engle RE, Emerson SU, Purcell RH: Prevalence of antibodies to hepatitis E virus in veterinarians working with swine and in normal blood donors in the United States and other countries. Journal of clinical microbiology 2002, 40(1):117-122.
125. Dalton HR, Stableforth W, Thurairajah P, Hazeldine S, Remnarace R, Usama W, Farrington L, Hamad N, Sieberhagen C, Ellis V et al: Autochthonous hepatitis E in Southwest England: natural history, complications and seasonal variation, and hepatitis E virus IgG seroprevalence in blood donors, the elderly and patients with chronic liver disease. Eur J Gastroenterol Hepatol 2 0 0 8 ,20(8):784-790.
126. Aggarwal R, Manadan AM, Poliyedath A, Sequeira W, Block JA: Safety of etanercept in patients at high risk for mycobacterial tuberculosis infections. J Rheumatol 2009, 36(5):914-917.
192
127. Khuroo MS, Kamili S, Jameel S: Vertical transmission of hepatitis E virus. Lancet 1995, 345(8956): 1025-1026.
128. Kruttgen A, Seheithauer S, Hausler M, Kleines M: First report of an autochthonous hepatitis E virus genotype 3 infection in a 5 month old female child in Germany. J Clin Virol 2011, 50(2): 175-176.
129. Thapa R, Pramanik S, Biswas B, Mallick D: Hepatitis E virus infection in a 7-year-old hoy with glucose 6-phosphate dehydrogenase deficiency. J Pediatr Hematol Oncol 2009, 31(3):223-224.
130. Balayan MS, Andjaparidze AG, Savinskaya SS, Ketiladze ES, Braginsky DM, Savinov AP, Poleschuk VF: Evidence for a virus in non-A, non-B hepatitis transmitted via the fecal- oral route. Intervirology 1983, 20(1):23-31.
131. Boeeia D, Guthmann JP, Klovstad H, Hamid N, Tatay M, Ciglenecki I, Nizou JY, Nicand E, Guerin PJ: High mortality associated with an outbreak of hepatitis E among displaced persons in Darfur, Sudan. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 2006, 42(12):1679-1684.
132. Guthmann JP, Klovstad H, Boeeia D, Hamid N, Pinoges L, Nizou JY, Tatay M, Diaz F, Moren A, Grais RF et al: A large outbreak of hepatitis E among a displaced population in Darfur, Sudan, 2004: the role of water treatment methods. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 2006, 42(12): 1685- 1691.
133. Tei S, Kitajima N, Takahashi K, Mishiro S: Zoonotic transmission of hepatitis E virus from deer to human beings. Lancet 2003, 362(9381):371-373.
134. Colson P, Borentain P, Queyriaux B, Kaba M, Moal V, Gallian P, Heyries L, Raoult D, Gerolami R: Pig liver sausage as a source of hepatitis E virus transmission to humans. The Journal of infectious diseases 2010, 202(6):825-834.
135. Legrand-Abravanel F, Kamar N, Sandres-Saune K, Lhomme S, Mansuy JM, Muscari F, Sallusto F, Rostaing L, Izopet J: Hepatitis E virus infection without reactivation in solid- organ transplant recipients, France. Emerg Infect Dis 2 0 1 1 ,17(l):30-37.
136. Wiehmann O, Sehimanski S, Koch J, Kohler M, Rothe C, Plentz A, Jilg W, Stark K: Phylogenetic and case-control study on hepatitis E virus infection in Germany. J Infect Dis 2008,198(12): 1732-1741.
137. Kim YM, Jeong SH, Kim JY, Song JC, Lee JH, Kim JW, Yun H, Kim JS: The first case of genotype 4 hepatitis E related to wild hoar in South Korea. Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology 2011, 50(3):253-256.
138. Yazaki Y, Mizuo H, Takahashi M, Nishizawa T, Sasaki N, Gotanda Y, Okamoto H: Sporadic acute or fulminant hepatitis E in Hokkaido, Japan, may he food-horne, as suggested hy the presence of hepatitis E virus in pig liver as food. J Gen Virol 2003, 84(Pt 9):2351-2357.
139. Lewis HC, Boisson S, Ijaz S, Hewitt K, Ngui SL, Boxall E, Teo CG, Morgan D: Hepatitis E in England and Wales. Emerg Infect Dis 2008,14(1):165-167.
140. De Silva AN, Muddu AK, Iredale JP, Sheron N, Khakoo SI, Pelosi E: Unexpectedly high incidence of indigenous acute hepatitis E within South Hampshire: time for routine testing? JMed Virol 2008, 80(2):283-288.
141. Mitsui T, Tsukamoto Y, Hirose A, Suzuki S, Yamazaki C, Masuko K, Tsuda F, Endo K, Takahashi M, Okamoto H: Distinct changing profiles of hepatitis A and E virus infection among patients with acute hepatitis, patients on maintenance hemodialysis and healthy individuals in Japan. J Med Virol 2 0 0 6 ,78(8):1015-1024.
142. Peron JM, Mansuy JM, Izopet J, Vinel JP: [Hepatitis E virus: an emerging disease]. Sante 2006,16(4):239-243.
143. Amon JJ, Drobeniuc J, Bower WA, Magana JC, Escobedo MA, Williams IT, Bell BP, Armstrong GL: Locally acquired hepatitis E virus infection, El Paso, Texas. Journal of medical virology 2006,78(6):741-746.
144. Nakamura M, Takahashi K, Taira K, Taira M, Ohno A, Sakugawa H, Arai M, Mishiro S: Hepatitis E virus infection in wild mongooses of Okinawa, Japan: Demonstration of anti-HEV antibodies and a full-genome nucleotide sequence. Hepatol Res 2006, 34(3).T37-140.
145. Li TC, Miyamura T, Takeda N: Detection of hepatitis E virus RNA from the bivalve Yamato-Shijimi (Corbicula japonica) in Japan. Am J Trop Med Hyg 2007,76(1): 170-172.
193
146. Arankalle VA, Joshi MV, Kulkarni AM, Gandhe SS, Chobe LP, Rautmare SS, Mishra AC, Padbidri VS: Prevalence of anti-hepatitis E virus antibodies in different Indian animal species. J Viral Hepat 2001, 8(3):223-227.
147. Yarbough PO: Hepatitis E virus. Advances in HEV hiology and HEV vaccine approaches. Intervirology 1999, 42(2-3):179-184.
148. Wang YC, Zhang HY, Xia NS, Peng G, Lan HY, Zhuang H, Zhu YH, Li SW, Tian KG, Gu WJ et al: Prevalence, isolation, and partial sequence analysis of hepatitis E virus from domestic animals in China. J Med Virol 2002, 67(4):516-521.
149. Halbur PG, Kasorndorkbua C, Gilbert C, Guenette D, Potters MB, Pureell RH, Emerson SU, Toth TE, Meng XJ: Comparative pathogenesis of infection of pigs with hepatitis E viruses recovered from a pig and a human. J Clin Microbiol 2001, 39(3):918-923.
150. Chobe LP, Lole KS, Arankalle VA: Full genome sequence and analysis of Indian swine hepatitis E virus isolate of genotype 4. Vet Microbiol 2 0 0 6 ,114(3-4):240-251.
151. Bouwknegt M, Engel B, Herremans MM, Widdowson MA, Worm HC, Koopmans MP, Frankena K, de Roda Husman AM, De Jong MC, Van Der Poel WH: Bayesian estimation of hepatitis E virus seroprevalence for populations with different exposure levels to swine in The Netherlands. Epidemiology and infection 2 0 0 8 ,136(4):567-576.
152. Rutjes SA, Lodder WJ, Bouwknegt M, de Roda Husman AM: Increased hepatitis E virus prevalence on Dutch pig farms from 33 to 55% hy using appropriate internal quality controls for RT-PCR. J Virol Methods 2 0 0 7 ,143(1): 112-116.
153. Wibawa ID, Muljono DH, Mulyanto, Suryadarma IG, Tsuda F, Takahashi M, Nishizawa T, Okamoto H: Prevalence of antibodies to hepatitis E virus among apparently healthy humans and pigs in Bali, Indonesia: Identification of a pig infected with a genotype 4 hepatitis E virus. J Med Virol 2004, 73(l):38-44.
154. Pina S, Jofre J, Emerson SU, Pureell RH, Girones R: Characterization of a strain of infectious hepatitis E virus isolated from sewage in an area where hepatitis E is not endemic. Appl Environ Microbiol 1998, 64(ll):4485-4488 .
155. Hussaini SH, Skidmore SJ, Riehardson P, Sherratt LM, Cooper BT, O'Grady JG: Severe hepatitis E infection during pregnancy. J Viral Hepat 1997, 4(l):51-54.
156. Chauhan A, Jameel S, Dilawari JB, Chawla YK, Kaur U, Ganguly NK: Hepatitis E virus transmission to a volunteer. Lancet 1993, 341(8838): 149-150.
157. Arora NK, Nanda SK, Gulati S, Ansari IH, Chawla MK, Gupta SD, Panda SK: Acute viral hepatitis types E, A, and B singly and in combination in acute liver failure in children in north India. J Med Virol 1996, 48(3):215-221.
158. Clayson ET, Innis BL, Myint KS, Narupiti S, Vaughn DW, Giri S, Ranabhat P, Shrestha MP: Detection of hepatitis E virus infections among domestic swine in the Kathmandu Valley of Nepal. Am J Trop Med Hyg 1995, 53(3):228-232.
159. Nanda SK, Ansari IH, Aeharya SK, Jameel S, Panda SK: Protracted viremia during acute sporadic hepatitis E virus infection. Gastroenterology 19 9 5 ,108(l):225-230.
160. Jameel S: Molecular hiology and pathogenesis of hepatitis E virus. Expert Rev Mol Med 1999,1999:1-16.
161. Aggarwal R, Kini D, Sofat S, Naik SR, Krawczynski K: Duration of viraemia and faecal viral excretion in acute hepatitis E. Lancet 2 0 0 0 ,356(9235):1081-1082.
162. Dawson J, Sedgwick AD, Edwards JC, Lees P: The monoclonal antibody MEL-14 can block lymphocyte migration into a site of chronic inflammation. Eur J Immunol 1992, 22(6): 1647-1650.
163. Favorov MO, Fields HA, Purdy MA, Yashina TL, Aleksandrov AG, Alter MJ, Yarasheva DM, Bradley DW, Margolis HS: Serologic identification of hepatitis E virus infections in epidemic and endemic settings. J Med Virol 1992, 36(4):246-250.
164. Khuroo MS, Kamili S, Dar MY, M oecklii R, Jameel S: Hepatitis E and long-term antibody status. Lancet 1993, 341(8856):1355.
165. Haagsma EB, van den Berg AP, Porte RJ, Benne CA, Vennema H, Reimerink JH, Koopmans MP: Chronic hepatitis E virus infection in liver transplant recipients. Liver Transpl 2 0 0 8 ,14(4):547-553.
166. Kamar N, Guitard J, Ribes D, Esposito L, Rostaing L: A monocentric observational study of darhepoetin alfa in anemic hepatitis-C-virus transplant patients treated with ribavirin. Exp Clin Transplant 2008, 6(4):271-275.
194
167. Kamar N, Mansuy JM, Cointault O, Selves J, Abravanel F, Danjoux M, Otai P, Esposito L, Durand D, Izopet J et al: Hepatitis E virus-related cirrhosis in kidney- and kidney- pancreas-transplant recipients. Am J Transplant 2008, 8(8): 1744-1748.
168. Kamar N, Selves J, Mansuy JM, Ouezzani L, Peron JM, Guitard J, Cointault O, Esposito L, Abravanel F, Danjoux M et al: Hepatitis E virus and chronic hepatitis in organ- transplant recipients. N EnglJ Med 2008, 358(8):811-817.
169. Dalton HR, Bendall RP, Keane FE, Tedder RS, Ijaz S: Persistent carriage of hepatitis E virus in patients with HIV infection. N EnglJ Med 2009, 361(10): 1025-1027.
170. Mandai K, Chopra N: Acute transverse myelitis following hepatitis E virus infection. Indian Pediatr 2006, 43(4):365-366.
171. Sood A, Midha V, Sood N: Guillain-Barre syndrome with acute hepatitis E. Am J Gastroenterol 2000, 95(12):3667-3668.
172. Loly JP, Rikir E, Seivert M, Legros E, Defrance P, Belaiehe J, Moonen G, Delwaide J: Guillain-Barre syndrome following hepatitis E. World J Gastroenterol 2009,15(13):1645- 1647.
173. Kamani P, Baijal R, Amarapurkar D, Gupte P, Patel N, Kumar P, Agal S: Guillain-Barre syndrome associated with acute hepatitis E. Indian J Gastroenterol 2005, 24(5):216.
174. Fong F, Illahi M: Neuralgic amyotrophy associated with hepatitis E virus. Clin Neurol Neurosurg 2009,111(2): 193-195.
175. Rianthavorn P, Thongmee C, Limpaphayom N, Komolmit P, Theamboonlers A, Poovorawan Y : The entire genome sequence of hepatitis E virus genotype 3 isolated from a patient with neuralgic amyotrophy. Scand J Infect Dis 2010, 42(5):395-400.
176. Kamar N, Bendall RP, Peron JM, Cintas P, Prudhomme L, Mansuy JM, Rostaing L, Keane F, Ijaz S, Izopet J et al: Hepatitis E virus and neurologic disorders. Emerg Infect Dis 2011, 17(2): 173-179.
177. Kamar N, Abravanel F, Mansuy JM, Peron JM, Izopet J, Rostaing L: [Hepatitis E infection in dialysis and after transplantation]. Nephrol Ther 2010, 6(2):83-87.
178. Gupta DN, Smetana HF: The histopathology of viral hepatitis as seen in the Delhi epidemic (1955-56). Indian J Med Res 1957, 45(SuppL): 101-113.
179. Hamid SS, Jafri SM, Khan H, Shah H, Abbas Z, Fields H: Fulminant hepatic failure in pregnant women: acute fatty liver or acute viral hepatitis? J Hepatol 1996, 25(l):20-27.
180. McCormick JB: Clinical, epidemiologic, and therapeutic aspects of Lassa fever. Med Microbiol Immunol 1986,175(2-3):153-155.
181. Longer CF, Denny SL, Caudill JD, M iele TA, Asher LV, Myint KS, Huang CC, Engler WF, LeDue JW, Binn LN et al: Experimental hepatitis E: pathogenesis in cynomolgus macaques (Macaca fascicularis). 7 /n/^crDw 1 9 9 3 ,168(3):602-609.
182. Arankalle VA, Chadha MS, Banerjee K, Srinivasan MA, Chobe LP: Hepatitis E virus infection in pregnant rhesus monkeys. Indian J Med Res 1993, 97:4-8.
183. Navaneethan U, Al Mohajer M, Shata MT: Hepatitis E and pregnancy: understanding the pathogenesis. Liver Int 2008,28(9):1190-1199.
184. Kasorndorkbua C, Guenette DK, Huang FF, Thomas PJ, Meng XJ, Halbur PG: Routes of transmission of swine hepatitis E virus in pigs. 7 Clin Microbiol 2004, 42(11):5047-5052.
185. Dalton HR, Keane FE, Bendall R, Mathew J, Ijaz S: Treatment of Chronic Hepatitis E in a Patient With HIV Infection. Ann Intern Med 2011,155(7):479-480.
186. Aggarwal R: Hepatitis E: Historical, contemporary and future perspectives. Journal of gastroenterology and hepatology 2011, 26 Suppl l(April 1979):72-82.
187. Kageyama T, Kojima S, Shinohara M, Uehida K, Fukushi S, Hoshino FB, Takeda N, Katayama K: Broadly Reactive and Highly Sensitive Assay for Norwalk-Like Viruses Based on Real-Time Quantitative Reverse Transcription-PCR. Society 2003, 41(4): 1548- 1557.
188. Kolasa K: Food Hypersensitivity: Diagnosing and Managing Food Allergies and Intolerance. Journal of Nutrition Education and Behavior 2010,42(2):142.el47-142.el47.
189. Dawson GJ, Chau KH, Cabal CM, Yarbough PO, Reyes GR, Mushahwar IK: Solid-phase enzyme-linked immunosorbent assay for hepatitis E virus IgG and IgM antibodies utilizing recombinant antigens and synthetic peptides. 7 Virol Methods 1992, 38(1):175- 186.
190. Pavan S, Varma K, Kumar A, Kapur N, Durgapal H, Aeharya K, Panda SK: HEV replication involves alternating negative and positive sense RNA synthesis. 2010.
195
191. Li T-C, Suzaki Y, Ami Y, Tsunemitsu H, Miyamura T, Takeda N: Mice are not susceptible to hepatitis E virus infection. The Journal of veterinary medical science / the Japanese Society of Veterinary Science 2 0 0 8 ,70(12): 1359-1362.
192. Martelli F, Caprioli A, Zengarini M, Marata A, Fiegna C, Di Bartolo I, Ruggeri FM, Delogu M, Ostanello F: Detection of hepatitis E virus (HEV) in a demographic managed wild hoar (Sus scrofa scrofa) population in Italy. Vet Microbiol 2 0 0 8 ,126(l-3):74-81.
193. Nanda SK, Yalcinkaya K, Panigrahi AK, Aeharya SK, Jameel S, Panda SK: Etiological role of hepatitis E virus in sporadic fulminant hepatitis. J Med Virol 1994, 42(2):133-137.
194. Varma SP, Kumar A, Kapur N, Durgapal H, Aeharya SK, Panda SK: Hepatitis E virus replication involves alternating negative- and positive-sense RNA synthesis. J Gen Virol 2011,92(Pt 3):572-581.
195. Huang R, Nakazono N, Ishii K, Li D, Kawamata O, Kawaguchi R, Tsukada Y: Hepatitis E virus (87A strain) propagated in A549 cells. J Med Virol 1995 ,47(4):299-302.
196. Huang R, Li D, W ei S, Li Q, Yuan X, Geng L, Li X, Liu M: Cell culture of sporadic hepatitis E virus in China. Clin Diagn Lab Immunol 1999, 6(5):729-733.
197. Tanaka T, Takahashi M, Kusano E, Okamoto H: Development and evaluation of an efficient cell-culture system for Hepatitis E virus. The Journal of general virology 2007, 88(Pt 3):903-911.
198. Okamoto H: Efficient cell culture systems for hepatitis E virus strains in feces and circulating blood. Reviews in medical virology 2011, 21(1): 18-31.
199. Nickerson CA, Goodwin TJ, Terlonge J, Ott CM, Buchanan KL, Uieker WC, Emami K, LeBlanc CL, Ramamurthy R, Clarke MS et al: Three-dimensional tissue assemblies: novel models for the study of Salmonella enterica serovar Typhimurium pathogenesis. Infection and immunity 2001, 69(11):7106-7120.
200. Nauman EA, Ott CM, Sander E, Tucker DL, Pierson D, Wilson JW, Nickerson CA: Novel quantitative hiosystem for modeling physiological fluid shear stress on cells. Appl Environ Microbiol 2007, 73(3):699-705.
201. Kageyama T, Kojima S, Shinohara M, Uchida K, Fukushi S, Hoshino FB, Takeda N, Katayama K: Broadly reactive and highly sensitive assay for Norwalk-like viruses based on real-time quantitative reverse transcription-PCR. J Clin Microbiol 2003, 41(4): 1548- 1557.
202. Klaus DM: Clinostats and bioreactors. Gravit Space Biol Bull 2 0 0 1 ,14(2):55-64.203. Eaton B, Gangluff D, Mengel M: Fetal alcohol spectrum disorders: flying under the
radar. J Ark Med Soc 2 0 1 1 ,107(12):260-262.204. Deveic Z, Rayikanti BA, Hevia JP, Popenko NA, Karimi K, Wong BJ: Nasal tip projection
and facial attractiveness. Laryngoscope 2011,121(7): 1388-1394.205. Shrestha MP, Scott RM, Joshi DM, Mammen MP, Jr., Thapa GB, Thapa N, Myint KS,
Fourneau M, Kuschner RA, Shrestha SK et al: Safety and efficacy of a recombinanthepatitis E yaccinc. N Engl J Med 2007, 356(9):895-903.
206. Casas M, Cortes R, Pina S, Peralta B, Allepuz A, Cortey M, Casal J, Martin M:Longitudinal study of hepatitis E virus infection in Spanish farrow-to-finish swineherds. Vet M /cra^io/2 0 1 1 ,148(l):27-34.
207. Satou K, Nishiura H: Transmission dynamics of hepatitis E among swine: potential impact upon human infection. BMC Vet Res 2007, 3:9.
208. Martinez-Martinez M, Diez-Valearee M, Hernandez M, Rodriguez-Lazaro D: Design and Application of Nucleic Acid Standards for Quantitative Detection of Enteric Viruses by Real-Time PCR. Food Environ Virol 2011, 3(2):92-98.
209. Hundesa A, Bofill-M as S, Maluquer de Motes C, Rodriguez-Manzano J, Bach A, Casas M, Girones R: Development of a quantitative PCR assay for the quantitation of bovine polyomavirus as a microbial source-tracking tool. J Virol Methods 2 0 1 0 ,163(2):385-389.
210. Hernroth BE, Conden-Hansson AC, Rehnstam-Holm AS, Girones R, Allard AK: Environmental factors influencing human viral pathogens and their potential indicator organisms in the blue mussel, Mytilus edulis: the first Scandinavian report. Applied and environmental microbiology 2 0 0 2 ,68(9):4523-4533.
211. Hundesa A, Maluquer de Motes C, Albinana-Gimenez N, Rodriguez-Manzano J, Bofill-M as S, Sunen E, Rosina Girones R: Development of a qPCR assay for the quantification of porcine adenoviruses as an MST tool for swine fecal contamination in the environment. J Virol Methods 2009,158(1-2): 130-135.
196
212. Diez-Valcarce M, Kovac K, Cook N, Rodnguez-Lâzaro D, Hernandez M; Construction and Analytical Application of Internal Amplification Controls (lAC) for Detection of Food Supply Chain-Relevant Viruses hy Real-Time PCR-Based Assays. Food Analytical Methods 2011, 4(3):437-445.
213. da Silva AK, Le Saux JC, Parnaudeau S, Pommepuy M, Elimelech M, Le Guyader PS: Evaluation of removal of noroviruses during wastewater treatment, using real-time reverse transcription-PCR: different behaviors of genogroups I and II. Appl Environ Microbiol 2007, 73(24):7891-7897.
214. Svraka S, Duizer E, Vennema H, de Bruin E, van der Veer B, Dorresteijn B, Koopmans M: Etiological role of viruses in outbreaks of acute gastroenteritis in The Netherlands from 1994 through 2005. Journal of clinical microbiology 2007, 45(5):1389-1394.
215. Loisy F, Le Cann P, Pommepuy M, Le Guyader F: An improved method for the detection of Norwalk-like caliciviruses in environmental samples. Lett Appl Microbiol 2000, 31(6):411-415.
216. Baert L, Wobus CE, Van Coillie E, Thackray LB, Debevere J, Uyttendaele M: Detection of murine norovirus 1 hy using plaque assay, transfection assay, and real-time reverse transcription-PCR before and after heat exposure. Applied and environmental microbiology 2 0 0 8 ,74(2):543-546.
217. Ivo MDANCIDBFMRABFMMBPVPK: Multicenter Collaborative Trial Evaluation of a Method for Detection of Human Adenoviruses in Berry Fruit. Food Anal Methods 2011.
218. D ’Agostino M, Cook N, Di Bartolo I, Ruggeri F, Berto A, Martelli F, Banks M, Vasickova P, Kralik P, Pavlik I et al: Multicenter Collaborative Trial Evaluation of a Method for Detection of Human Adenoviruses in Berry Fruit. Food Analytical Methods 2012, 5(1): 1-7.
219. Williams SB, Prasad SM, Weinberg AC, Shelton JB, Hevelone ND, Lipsitz SR, Hu JC: Trends in the care of radical prostatectomy in the United States from 2003 to 2006. BJUInt 2 0 1 1 ,108(l):49-55.
220. Jothikumar N, Cromeans TL, Robertson BH, Meng XJ, Hill VR: A broadly reactive one- step real-time RT-PCR assay for rapid and sensitive detection of hepatitis E virus. Journal of virological methods 2 0 0 6 ,131(1):65-71.
221. Hundesa A, Maluquer de Motes C, Albinana-Gimenez N, Rodriguez-Manzano J, Bofill-M as S, Sunen E, Rosina Girones R: Development of a qPCR assay for the quantification of porcine adenoviruses as an MST tool for swine fecal contamination in the environment. Journal of virological methods 2009,158(1-2): 130-135.
222. Prado T, Silva DM, Guilayn WC, Rose TL, Caspar AM, Miagostovieh MP: Quantification and molecular characterization of enteric viruses detected in effluents from two hospital wastewater treatment plants. Water research 2011, 45(3):1287-1297.
223. MeCreary C, Martelli F, Grierson S, Ostanello F, Nevel A, Banks M: Excretion of hepatitis E virus hy pigs of different ages and its presence in slurry stores in the United Kingdom. The Veterinary record 2 0 0 8 ,163(9):261-265.
224. de Deus N, Seminati C, Pina S, Mateu E, Martin M, Segales J: Detection of hepatitis E virus in liver, mesenteric lymph node, serum, bile and faeces of naturally infected pigs affected hy different pathological conditions. Vet Microbiol 2007,119(2-4): 105-114.
225. Emerson SU, Arankalle VA, Purcell RH: Thermal stability of hepatitis E virus. J Infect Dis 2 0 0 5 ,192(5):930-933.
226. Takahashi M, Tanaka T, Azuma M, Kusano E, Aikawa T, Shibayama T, Yazaki Y, M izuo H, Inoue J, Okamoto H: Prolonged fecal shedding of hepatitis E virus (HEV) during sporadic acute hepatitis E: evaluation of infectivity of HEV in fecal specimens in a cell culture system. Journal of clinical microbiology 2007, 45(ll):3671-3679.
227. Lorenzo FR, Tanaka T, Takahashi H, lehiyama K, Hoshino Y, Yamada K, Inoue J, Takahashi M, Okamoto H: Mutational events during the primary propagation and consecutive passages of hepatitis E virus strain JE03-1760F in cell culture. Virus Res 2 0 0 8 ,137(l):86-96.
228. Goodwin TJ, Schroeder WF, W olf DA, Moyer MP: Rotating-wall vessel coculture of small intestine as a prelude to tissue modeling: aspects of simulated microgravity. Proc Soc Exp Biol Med 1993, 202(2): 181-192.
229. Chopra V, Dinh TV, Hannigan EV: Three-dimensional endothelial-tumor epithelial cell interactions in human cervical cancers. In Vitro Cell Dev Biol Anim 1997, 33(6):432-442.
197
230. Crabbe A, De Boever P, Van Houdt R, Moors H, Mergeay M, Cornells P: Use of the rotating wall vessel technology to study the effect of shear stress on growth behaviour of Pseudomonas aeruginosa PAOl. Environ Microbiol 2 0 0 8 ,10(8):2098-2110.
231. Straub TM, Honer zu Bentrup K, Orosz-Coghlan P, Dohnalkova A, Mayer BK, Bartholomew RA, Valdez CO, Bruckner-Lea CJ, Gerba CP, Abbaszadegan M et al: In vitro cell culture infectivity assay for human noroviruses. Emerg Infect Dis 2007,13(3):396-403.
232. Navran S: The application of low shear modeled microgravity to 3-D cell hiology and tissue engineering. In: Biotechnology Annual Review. Edited by El-Gewely MR, vol. Volume 14: Elsevier; 2008: 275-296.
233. Emerson SU, Anderson, D., Arankalle, V.A., Meng, X.J., Purdy, M.„ Schlauder GG, Tsarev,S.A.,: Hepevirus, Elsevier/Aeademie Press edn. London, ; 2004.
234. Chopra V, Fadl AA, Sha J, Chopra S, Galindo CL, Chopra AK: Alterations in the virulence potential of enteric pathogens and hacterial-host cell interactions under simulated microgravity conditions. J Toxicol Environ Health A 2006, 69(14): 1345-1370.
235. Walker JS, Carter RC, Klein F, Snowden SE, Lincoln RE: Evaluation of factors related togrowth of Rift Valley fever virus in suspended cell cultures. Applied microbiology 1969,17(5):658-664.
236. Rainbow AJ, Mak S: Functional heterogeneity of virions in human adenovirus types 2and 12. Journal of virology 1970, 5(2):188-193.
237. Zand V, Salem-Milani A, Shahi S, Akhi MT, Vazifekhah S: Efficacy of different concentrations of sodium hypochlorite and chlorhexidine in disinfection of contaminated Resilon cones. Med Oral Patol Oral CirBucal 2 0 1 2 ,17(2):e352-355.
238. Sabbah S, Springthorpe S, Sattar SA: Use of a mixture of surrogates for infectious hioagents in a standard approach to assessing disinfection of environmental surfaces. Applied and environmental microbiology 2010, 76(17):6020-6022.
239. Thurston-Enriquez JA, Haas CN, Jacangelo J, Riley K, Gerba CP: Inactivation of felinecalicivirus and adenovirus type 40 hy UV radiation. Appl Environ Microbiol 2003, 69(l):577-582.
240. Benarde MA, Israel BM, Olivieri VP, Granstrom ML: Efficiency of chlorine dioxide as a h2Lctcr\c\dc. Applied microbiology 1965,13(5):776-780.
241. Thurston-Enriquez JA, Haas CN, Jacangelo J, Gerba CP: Chlorine inactivation of adenovirus type 40 and feline calicivirus. Applied and environmental microbiology 2003, 69(7):3979-3985.
242. Meng J, Dai X, Chang JC, Lopareva E, Pillot J, Fields HA, Khudyakov YE: Identification and characterization of the neutralization epitope(s) of the hepatitis E virus. Virology 2001,288(2):203-211.
243. Bouwknegt M, Lodder-Versehoor F, van der Poel WH, Rutjes SA, de Roda Husman AM: Hepatitis E virus RNA in commercial porcine livers in The Netherlands. Journal of food protection 2007,70(12):2889-2895.
244. Jo SH, Back SB, Ha JH, Ha SD: Maturation and survival of Cronohacter hiofilms on silicone, polycarbonate, and stainless steel after UV light and ethanol immersion treatments. J Food Prot 2010, 73(5):952-956.
245. Matallana-Surget S, Douki T, Meador JA, Cavicchioli R, Joux F: Influence of growth temperature and starvation state on survival and DNA damage induction in the marine bacterium Sphingopyxis alaskensis exposed to UV radiation. J Photochem Photobiol B 2 0 1 0 ,100(2):51-56.
246. Fino VR, Kniel KE: UV light inactivation of hepatitis A virus, Aichi virus, and feline calicivirus on strawberries, green onions, and lettuce. J Food Prot 2008, 71(5):908-913.
247. Nuanualsuwan S, Mariam T, Himathongkham S, Cliver DO: Ultraviolet inactivation of feline calicivirus, human enteric viruses and coliphages. Photochem Photobiol 2002, 76(4):406-410.
248. Fino VR, Kniel KE: UV light inactivation of hepatitis A virus, Aichi virus, and feline calicivirus on strawberries, green onions, and lettuce. J Food Prot 2008, 71(5):908-913.
249. Browning ABaG. In: Phenotypic diversity and cell invasion in host subversion by pathogenic mycoplasmas In: Mycoplasmas, molecular biology, pathogenicity and strategies for control
439-483.
198
250. Xing L, Li TC, Mayazaki N, Simon MN, Wall JS, Moore M, Wang CY, Takeda N, Wakita T, Miyamura T et al: Structure of hepatitis E virion-sized particle reveals an RNA- dependent viral assembly pathway. 7 Rio/ Chem 2010, 285(43):33175-33183.
251. J.A. Baeker AB, F. Martelli, W.H.M. van der Poel: Transmission dynamics of Hepatitis E virus in pigs: estimation from field data and effect of vaccination. Epidemics 2011.
252. Fernandez-Barredo S, Galiana C, Garcia A, Vega S, Gomez MT, Perez-Gracia MT: Detection of hepatitis E virus shedding in feces of pigs at different stages of production using reverse transcription-polymerase chain reaction. 7 Vet Diagn Invest 2006, 18(5):462-465.
253. de Deus N, Casas M, Peralta B, Nofrarias M, Pina S, Martin M, Segales J: Hepatitis E virus infection dynamics and organic distribution in naturally infected pigs in a farrow-to- finish farm. Veterinary microbiology 200%, 132(1-2): 19-28.
254. Keeling MJ, Ross JV: On methods for studying stochastic disease dynamics. 7 R Soc Interface 2008, 5(19):171-181.
255. Di Bartolo I, Martelli F, Inglese N, Pourshaban M, Caprioli A, Ostanello F, Ruggeri FM: Widespread diffusion of genotype 3 hepatitis E virus among farming swine in Northern Italy. Veterinary microbiology 2 0 0 8 ,132(l-2):47-55.
256. Straub TM, Honer zu Bentrup K, Orosz-Coghlan P, Dohnalkova A, Mayer BK, Bartholomew RA, Valdez CO, Bruekner-Lea CJ, Gerba CP, Abbaszadegan M et al: In vitro cell culture infectivity assay for human noroviruses. Emerg Infect Dis 2001,13(3):396-403.
257. Sherwood LJ, Osborn LE, Carrion R, Jr., Patterson JL, Hayhurst A: Rapid assembly of sensitive antigen-capture assays for Marburg virus, using in vitro selection of llama single-domain antibodies, at biosafety level 4 .7 Infect Dis 2 0 0 7 ,196 Suppl 2:S213-219.
258. Fray MD, Mann GE, Charleston B: Validation of an Mx/CAT reporter gene assay for the quantification of bovine type-I interferon. Journal of immunological methods 2001, 249(1- 2):235-244.
259. Srivastava R, Aggarwal R, Jameel S, Puri P, Gupta VK, Ramesh VS, Bhatia S, Naik S: Cellular immune responses in acute hepatitis E virus infection to the viral open reading frame 2 protein. Viral Immunol 2001, 20(l):56-65.
260. Yu C, Boon D, McDonald SL, Myers TG, Tomioka K, Nguyen H, Engle RE, Govindarajan S, Emerson SU, Purcell RH: Pathogenesis of hepatitis E virus and hepatitis C virus in chimpanzees: similarities and differences. Journal of virology 2010, 84(21): 11264-11278.
261. Kamar N, Garrouste C, Haagsma EB, Garrigue V, Pisehke S, Chauvet C, Dumortier J, Cannesson A, Cassuto-Viguier E, Thervet E et al: Factors associated with chronic hepatitis in patients with hepatitis E virus infection who have received solid organ transplants. Gastroenterology 2 0 1 1 ,140(5): 1481-1489.
262. Peterhans E, Jungi TW, Sehweizer M: BVDV and innate immunity. Biologicals .'journal of the International Association of Biological Standardization 2003, 31(2): 107-112.
199
Appendix
Appendix A
A.l Attempted construction of an Interferon knock-out cell line
An Interferon knock out cell line was planned to verify if the IFN-KN cells better
allowed more efficient HEV replication. Before the IFN-KN constructions the IFN
production was evaluated by CAT-BLISA to determine if HEV activates the
interferon cascade in the cells otherwise the IFN-KN was not going to be
performed.
A. 1.1 Introduction
This work is reported in the thesis although the experiment did not produce useful
results, the techniques applied should be described.
1) Attempted Production of interferon knockout PLC/PRF/5 cell line to facilitate in-
vitro replication of HEV.
A.1.1.1 Introduction CAT-ELISA test:
Signal Transducing Activator of Transcription-1 (STATl), regulates the innate
cellular antiviral response through the transcriptional activation of interferon.
Activation of the IFN gene and its respective receptor triggers intracellular signaling
pathway resulting in the activation or expression of distinct but related signaling
pathways, known as the Janus kinase and signal transducer and activator of
transcription pathway (JAK-STAT).
These JAK and STAT proteins are known to perform distinct functions in cytokine
signaling, mediating IFN-dependent biological responses, and inducing an antiviral
state.
201
The simian virus 5 (SV-5) V protein is a specific inhibitor of STATl. The
construction and use of cells constitutively expressing the SV-5 V protein in a
lentivirus vector has been established to enable the propagation of viruses that are
difficult to grow in-vitro [257].
The aim was to construct a STATl knockout (IFN KO) of the hepatocarcinoma cell
line PLC/PRF5 to increase permissivity/sensitivity to HEV infection and to evaluate
the cell line in 3 culture systems (2D, 3D and 3D transferred in to 2D). The
approach was to transfect PLC/PRF5 cells with the SV-5 lentivirus vector to alter
gene expression in the target cell line PLC/PRF/5 such that they no longer produce
IFN, therefore allowing a more efficient replication of HEV.
Type I IFN bioactivity of expressed interferon alpha subtypes was determined using
an Mx/CAT (chloramphenicol acetyltransferase) reporter gene assay developed for
the quantification of IFN I [258]. This assay was performed to check if HEV
stimulates IFN activation.
It is known that a large variety of cells can produce IFN-y. In the liver NK cells and
NKT cells are known to be potent sources of IFN-y [259].
In HBV infection, IFN-y produced in the liver has been shown to recruit
neutrophils, macrophages, NK cells, and NKT cells. NK, NKT, and CD4+ cells that
express a glycoprotein that induces cell death. IFN-y also has non cytopathic
antiviral activity, which is important for HBV and HCV clearance [259]. In patients
with hepatitis A virus, HBV, and HCV infections the CD8+ cytotoxic cells play the
major role in the pathogenesis of viral clearance [273]. However, no increase in
HEV-specific cytokine-producing CD8+ cells was found in patients with hepatitis E
[259] and the CD3+ cells produced less IFN-y- and TNF-«- in response to activation
202
with PMA. Srivastava et al [259] noticed an increased of IFN-y production in
patients with acute hepatitis E and this may be important in the pathogenesis of liver
injury in patients with acute hepatitis E virus [259]. Furthermore, the study
suggested [259] that during the acute phase of hepatitis E infection there is no
detectable HEV 0RF2-specific immune activation of CD4+ and CD8+ cells in the
peripheral blood of those patients. However, the increasing of IFN-y production
with no specific CD8+ cell responses suggests that probably no-specific innate
mechanisms are involved in the activation of NK or NKT cells and this could play a
significant role in hepatitis E pathogenesis [259].
A.2 Material and Methods of CAT-ELISA (enzyme immunoassay for the
quantitative determination of chloramphenicol acetyltransferase (CAT) from E. coli
in transfected eukaryotic cells) test:
A.2.1 Type I IFN bioassay of recombinant HEV-IFN-a
The assay is based on MDBK cells transfected with a plasmid, containing a human
MxA promoter driving the expression of the reporter CAT gene.
MDBK-t2 cells maintained under blasticidin selection were seeded into 96-well
microtitre plates at a density of 2.5x10^ cells/well. Expressed recombinant IFNa
proteins alongside a serial dilution of recombinant porcine IFN-al (R&D Systems,
Abingdon, UK) which served as a standard to calculate the activity of the expressed
protein were added to the cells. Cultures were incubated for 24 hours at 37°C 5%
C02. Lysates were prepared from the MDBK-t2 cultures and the amount of CAT
expression induced by recombinant IFNa was quantified by ELISA using an
enhanced substrate (Roche, Welwyn garden City, UK) [258]. Luminescence was
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read at 405nm using a FLUOstart OPTIMA microplate reader (BMG Labtech,
Aylesbury, UK).
A 3 Results of the CAT ELISA test:
A.3.1 Biological activity of expressed recombinant protein
To confirm that the expressed recombinant proteins are biologically active, the cell
supernatants were analyzed using the Type I IFN bioassay. Addition of cell culture
supernatants to the MDBKt2 reporter cell line alongside quantified commercial
IFNa standards resulted in no expression of CAT enzyme, indicating no induction
of the interferon responsive MX promoter. Figure 1 shows the IFN type I
concentration, measured from each sample (Figure 1).
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12
1 0
8
6
4
2
O
IFN A l p h a C A T E l i s a
---- i
M ocK tran s fe c t not in fectedmedia
HEV positive media
Figure 1: IFNa CAT ELISA, IFNU/ml comparison between MocK positive control cells, not HEV infected supernatant and HEV positive supernatant. (IFNU = type I interferon unit per ml).
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A.4 Discussion of CAT ELISA
This assay demonstrated that HEV positive supernatant was apparently not
activating INF signalling. For this reason, INF-KO cells were not produced.
Yu et al described the pathogenesis of Hepatitis E Virus and Hepatitis C Virus in
Chimpanzees. Result of Yu et al [260] study was that the expression of adaptive
immune-associated genes and immune-specific cell markers, was dramatically
lower in HEV-infected chimpanzees than in HCV-infected chimpanzees [260].
Kamar et al [261] described three-month pegylated interferon-alpha-2 a therapy for
chronic hepatitis E virus infection in a haemodialysis patient. Result obtained in the
study was that after 3-month of Peg-IFN-a-2a treatment. Serum HEV RNA patient
became negative by third week of Peg-IFN-a-2a therapy [261].
Furthermore, literature describes infection with bovine viral diarrhea virus (BVDV),
the virus exists in two biotypes, cytopathic and non-cytopathic [262]. BVDV
cytopathic and non-cytopathic biotypes have specific immune response and only the
non-cytopathic BVDV virus can establish persistent infection [262]. Non-cytopathic
BVDV fails to induce interferon type I in cultured bovine macrophages. Non-
cytopathic BVDV may dispose of a mechanism suppressing a key element of the
antiviral defence of the innate immune system [262]. Since interferon is also
important in the activation of the adaptive immune response, suppression of this
signal may be essential for the establishment of persistent infection and
immunotolérance [262].
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A possible conclusion from these four studies is that probably INF type I probably
does not play a significant role in hepatitis E pathogenesis as also Srivastava et al
[259] suggested.
After this possible explanation, for this study was essential a cell line able to permit
the virus to replicate efficiently and the production of an INF-KO cell line was not
beneficial for the study. The KO cell line would have probably been able to support
HEV replication as the wild type, so there was no point in putting effort in
producing a KO cell line in PLC/PRF-5.
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MATERIAL REDACTED AT REQUEST OF UNIVERSITY
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