Rapid Detection of "Norwak-like Viruses" by Reverse Transcription-Polyrnerase Chain Reaction (RT-PCR) and Southem Blot Hybridisation (SBH) in Outbreaks of Acute

Gastroenteritis in Eastern Ontario, Canada.

by

SHANNON MEREDITH WIRES

A thesis submitted to the Department of Microbiology and Imrnunology in conformity with the requirements for the degree of Master of Science

Queen's University Kingston, Ontario, Canada

April, 2001

Copyright O Shannon Meredith Wires, 200 1

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Abstract

Objective: Nonvalk-like viruses (NLVs) are estimated to cause millions of infections and

comuni ty outbreaks of acute gastroenteritis world-wide each year. Although NLV-

associated ilhess is self-Iirniting and usually resolves without senous health-related

complications, cornrnunity outbreaks of acute gastroenteritis due to NLVs in institutions,

such as long-term care facilities, require irnmediate attention to control their rapid

transmission. To date, stool specimens received fiom suspected viral outbreaks in

Ontario are referred to rernote sites for electron microscopy (EM) analysis. However.

EM detection of NLVs in stool is challenged by the iow titre and small size of these

vinrses, thus impeding outbreak diagnosis and public health intervention. Behveen

December 1999 and November 2000, a comparative analysis for the detection of NLVs

was undertaken between EM and the reverse transcription-polymerase chain reaction

(RT-PCR) method of Ando et al. (Ando et al., 1995). Methods: Viral RNA was

extracted from stool specimens submitted to the Kingston Public Health Laboratory

during outbreaks of acute gastroenteritis in long-term care and child-care faci li ties. For

the RT-PCR, we used 2 primer sets, G1 and G2, which have the ability to detect

genetically diverse NLVs in stool samples (Ando et al., 1995). Confirmation of the RT-

PCR results was obtained by Southern blot hybridisation (SBH), using 4 DIG-labelled

probe sets, P Z -A, P 1 -B, P2-A and P2-B (Ando et al., 1995). Ninety-eight specimens

fkom 25 outbreaks in Eastern Ontario were analysed- EM was perforrned at two sites, the

Central and Thunder Bay Regional Public Health Laboratones. Results: The RT-

PCR/SBH method was able to identie NLVs as the causative agents in 80% (20/25) of

the outbreaks analysed, whereas EM detected NLVs in oz:y 20% (5/25) o f these

outbreaks. The use of RT-PCR as an initial screen detected NLV involvement in 32 of a

total of 98 stool specimens. SBH detected the presence of NLVs in an additional 19

specimens, in which the RT-PCR amplicons were not visible following ethidium brornide

staining. Two stool sarnples considered NLV-positive upon RT-PCR screening could not

be co-ed by SBH. Eight of the outbreaks were detected with the G1 set of primers, 7

with the G2 set of prirners and 1 outbreak appeared to be dually caused. A tùrther 4

outbreaks, which were not detected with ethidiun brornide staining following RT-PCR,

were found to be positive by SBH, whereas 1 RT-PCR positive outbreak detected with

the G1 primer could not be confirmed by SBH. In keeping with the observations of other

investigators, a distinct winter seasonality of NLV-associated outbreaks was observed.

Conclusion: Norwalk-like viruses are common causes of outbreaks of acute

gastroenteritis in long-term care and child-care facilities in Eastern Ontario. This

combined RT-PCR/SBH procedure is rapid, simple and significantly more sensitive when

compared to EM and can be easiIy adapted to a routine diagnostic setting thus allowing

for timely patient care and management. Moreover, this diagnostic test identifies the

genogroup and antigenic type of the Norwalk-like virus infection, thus allowing public

health officials to participate in local, national and international epidemiological

investigations.

Acknowledgements

At this time, 1 would like to thank my CO-supervisors, Dr. Heather Onyett and Dr.

Perin Sankar-Mistry, for their time, support and guidance during this project. I would

also like to thank the staff at the Kingston Public Health Laboratory for their patience,

expertise and support. 1 am grateful to the Kingston, Ottawa, Central and Thunder Bay

Regional Public Health Laboratories for the udimited access to clinical stool sampIes

from outbreaks of gastroenteritis and the provision of al1 equipment and reagents required

for this research. Dr. Frederick Bail (Thunder Bay Regional Public Health Laboratory) ,

and Joan Stubberfield (Central Public Health Laboratory) deserve my gratitude for their

expert electron microscopy analysis. 1 would also like to thank Olive Hsueh, of the

Ottawa Public Health Laboratory, for her assistance with the Southem blot hybridisation

protocoI.

1 am deeply indebted to fellow graduate student Mamie Fiebig for al1 her

assistance, fiiendship and moral support throughout this project. 1 would also like to

thank the many other graduate students in the Department of Microbiology and

Immunology at Queen's University.

Finally, I would like to thank my fî-iends and family for their unwavering

encouragement and suppoa during my MSc studies and research.

Tabte of Contents

Abstract Page Number -.

11

Acknowledgements

Table of Contents

List of Tables

List of Figures

Abbreviations

1 Introduction

1 2 Taxonomy of Caliciviridae and "Nonvalk-like Viruses"

1.3 NLVs and acute gastroenteritis

1.3.1 Transmission

1.3.2 Clinical syndrome

1.3 -3 Pathology

xii

1.3 -4 Irnrnunity to NLV infection and vaccine development 12

1.4 Epidemiology, outbreak management and control, and prevention 16

1.4.2 Epidemiology of NLV infections 17

1.4.2 Outbreak management and control of acute NLV-associated gastroenteritis

1.4.3 Prevention of NLV infections

1 -5 Considerations for clinical laboratory diagnostics

1.5.1 Electron microscopy (EM) and immune EM

1 S.2 Lrnmunological detection

i -5-3 MoIecular diagnostics

Research objectives

Materials and Methods

S pecimens

RNA extraction ftom stool specimens

RT-PCR with Ando et al- SRSV primers

Agarose gel electrophoresis and ethidiurn bromide staining

Southem blotting

Hybridisation with Ando et al. SRSV primers

Detection ofNLV amplicons following Southern blot hybridisation

Results

RT-PCR of RNA extractions korn stool specimens

Confirmation of RT-PCR results using Southern blot hybndisation

Seasonality of NLV infections in Ontario

Discussion

The use of the RT-PCR method, with gel electrophoresis and ethidiurn bromide (EtBr) staining, for the detection of NLVs in stool specimens

The application of the Southern blot hybridisation (SBH) method for the detection of NLVs arnplicons

Specificity and sensitivity of the RT-PCR and Southern blot hybridisation procedures for detection of NLVs in stool specimens

Adaptation of the RT-PCR and Southern blot hybridisation procedures for use in a routine diagnostic laboratory

T-maround tirne for RT-PCR and SBH detection of NLVs in cornparison with Electron Microscopy

4.6 Cost considerations for the RT-PCR and SBH method in cornparison with Electron Microscopy

4.7 The application of RT-PCR and SBH to epidemiological investigation of NLV outbreaks in Ontario

4.8 Future Work

4-9 Conclusion

5 References

6 Appendiv

Curriculum Vitae

vii

List of Tables

Table 2.1

Table 2.2

Tabie 3.1

Table 3.2

Table 4.1

Table 6.1

Table 6.2

Table 6.3

Table 6.4

Table 6.5

Table 6.6

Table 6.7

Table 6.8

Table 6.9

Table 6.10

RT-PCR pnmers for amplification of diverse NLV genomes

Hybndisation probes for identification of NLV amplicons

Surnrnary of Nonvak Outbreak Data: Electron Microscopy resuIts vs. RT-PCR and Southem Hybridisation

Genogroup and Antigenic Groups Associated with Primer and Probe Detection for NLV Outbreaks in Eastern Ontario

Common nucleotide sequence in NLV probes used in this study (Ando et al., 1995)

EM and RT-PCWSouthern blot hybridisation analysis of stool specimens kom Outbreak A, Kingston, Ontario

EM and RT-PCR/Southem blot hybridisation analysis of stool specimens f?om Outbreak B, Kingston, Ontario

EM and RT-PCWSouthern blot hybridisation analysis of stool specimens fkom Outbreak C, VankIeek Hill, Ontario

EM and RT-PCEUSouthem blot hybridisation analysis of stool specimens fkom Outbreak D, Renfrew, Ontario

EM and RT-PCR/Southem blot hybridisation analysis of stool specimens fkom Outbreak E, Ottawa, Ontario

EM and RT-PCR/Southem blot hybridisation analysis of stool specimens ffom Outbreak F, Gloucester, Ontario

EM and RT-PCWSouthem blot hybridisation analysis of stool specimens fkom Outbreak G, Pembroke, Ontario

EM and RT-PCWSouthern blot hybridisation anaIysis of stool specimens fiom Outbreak H, Ottawa, Ontario

EM and RT-PCR/Southem blot hybridisation analysis of stool specimens fkom Outbreak 1, Ottawa, Ontario

EM and RT-PCWSouthern blot hybndisation analysis of stool specimens from Outbreak J, Ottawa, Ontario

Table 6-1 1

Table 6-12

Table 6.13

Table 6.14

Table 6.15

Table 6.16

Table 6.1 7

Table 6.18

Table 6.19

Table 6.20

Table 6.21

Table 6.22

Table 6.23

Table 6.24

Table 6.25

EM and RT-PCR/Southem blot hybridisation analysis of stool specirnens kom Outbreak K, Perth, Ontario

EM and RT-PCR/Southern blot hybridisation analysis of stool specimens from Outbreak L, Ottawa, Ontario

EM and RT-PCWSouthern blot hybridisation anafysis of stool specimens from Outbreak M, Brockville, Ontario

EM and RT-PCR/Southern blot hybridisation analysis of stool specimens from Outbreak N, Belleville, Ontario

EM and RT-PCWSouthern blot hybndisation analysis of stool specimens Erom Outbreak O, Gloucester, Ontario

EM and RT-PCR6outhem blot hybridisation analysis of stool specimens from Outbreak P, Pembroke, Ontario

EM and RT-PCWSouthern blot hybridisation analysis of stool specimens from Outbreak Q, Merrickville, Ontario

EM and RT-PCEVSouthern blot hybridisation analysis of stool specimens Erom Outbreak R, Ottawa, Ontario

EM and RT-PCWSouthern blot hybridisation analysis of stool specimens from Outbreak S, Gloucester, Ontario

EM and RT-PCEUSouthern blot hybridisation analysis of stool specimens from Outbreak T, Smith's Falls, Ontario

EM and RT-PCWSouthern blot hybridisation analysis of stool specimens from Outbreak U, Ottawa, Ontario

EM and RT-PCR/Southern blot hybridisation analysis of stool specimens from Outbreak V, Ottawa, Ontario

EM and RT-PCR/Southern blot hybridisation analysis of stool specimens korn Outbreak W, Toronto, Ontario

EM and RT-PCR/Southern blot hybridisation analysis of stool specimens from Outbreak X, Thunder Bay, Ontario

EM and RT-PCRlSouthern blot hybridisation analysis of stool specimens fi-om Outbreak Y, Thunder Bay, Ontario

Table 6-26 Cost per reaction ($CDN) of components used in RT-PCR method in a Iaboratory with PCR capabilities, not including labour.

List of Figures

Figure 1.1

Figure 1.2

Figure 1.3

Figure 1.4

Figure 2.1

Figure 3.1

Figure 3.2

Figure 3 -3

Figure 6.1

Negative staining transmission electron microscopy image 3 of NLVs.

The three-dimensional structure of the baculovirus-expressed 4 Norwalk virus capsid.

Organization of the NLV genome (genogroups 1 and 2). 5

Dendogram of predicted phylogenetic relationship arnong 8 30 NLV strains.

Flow chart explaining the procedwe for RT-PCR and 37 Southern blot hybridisation of clinical stool sarnples from Nonvalk-suspected outbreaks.

RT-PCR products visualized on 3% agarose gel stained with ethidium bromide.

RT-PCR products visualized on 3% agarose gel stained with ethidiurn bromide and corresponding Southern blots hybridized with Pl-% and P2-B probe sets. 42

Seasonal distribution of outbreaks of acute gastroenteritis and NLV-associated outbreaks between December 1999 and Novernber 2000 in Ontario, Canada

Southern blot apparatus for transfer of DNA from gel to nylon membrane.

Abbreviations

AGE

bp

CDC

CDN

cDNA

cv

DNA

dNTP.

EIA

ELISA

EtBr

FDA

O a

GE

ICTV

kDa

L

r-rg

ctl

mg

mg/L

ml

Agarose gel electrophoresis

base pairs

Centres for Disease Control and Prevention

Canadian

complementary DNA

Calicivims

deoxyribonucleic acid

deoxynucleoside triphosphate

enzyme irnmunoassay

enzyme-linked imrnunosorbant assay

ethidium bromide

US Food and Dmg Administration

,oram

gastroententis

International Cornmittee on Taxonomy of Viruses

kilodalton

litre

rnicrogram

microlitre

rnilligram

rnilligram per litre

millilitre

xii

m

nt

NLV

ORF

PPm

RIA

RNA

RT

RT-PCR

rVLPs

SPIEM

SSRNA

Taq

USA

UV

v

w/v

nanometer

nucleotide

Norwalk-like virus

open reading k a m e

parts per million

radioimmune assay

ribonucleic acid

room temperature

reverse transcription-polymerase chain reaction

recombinant virus-like particles

solid-phase immune electron rnicroscopy

single-stranded RNA

Themus aquaticu DNA polymerase

United States of America

ultra violet

volt

weight per volume

-. . Xlll

1 Introduction

Nonvalk-like viruses (NLVs) are common human pathogens, estimated to be

responsible for millions of infections and cornrnunity outbreaks of acute gastroenteritis

each year, in many regions of the world (Green, 2000). Although NLV-associated

gastroenteritis is self-limiting and usually resolves without serious health-related

complications, the explosive nature of transmission coupled with significant resultant

morbidity makes NLV infection an important public health concem. However, public

health monitoring and intervention face a challenge due to difficulties in NLV detection.

Traditionally, the NLVs have been detected in stool samples using electron microscopy

(EM), therefore limiting the availability of this diagnostic approach to specialised centres.

In addition, detection of NLVs by EM is dependent on viral titres in stool specimens and

is difficult due to the lack of a morphologically definitive structure of the individual virus

particle. Due to the considerable antigenic diversity within NLVs, identification by

immunological methods also poses a substantial challenge. However, the successfül

cloning and characterisation of NLVs genornes by Jiang et al. and Lambden et al. paved

the way for the development of molecular diagnostic approaches, thus greatly enhancing

our understanding of NLV infection and epidemiology (Jiang et al., 1990; Jiang et al.,

1993 and Lambden er al., 1993). These molecular diagnostic methods, which include

reverse transcription coupled with the polyrnerase chain reaction (RT-PCR) and So uthem

blot hybridisation (SBH), allow for greater sensitivity for detection of diverse NLVs in

stool specimens (Ando et al., 1995). Thus, it is increasingly evident that the use of these

molecular methods in diagnostic laboratories for the investigation of outbreaks of acute

gastroenteritis will allow for improved public health monitoring and outbreak

management.

1.1 Discovery of Norwalk Virus

The search for a Wal aetiological agent for acute gastroenteritis began in the late

1 96Os, as the majority of cases of infectious acute gastroenteritis arnong paediatric and

adult populations were noted to be of unknown, non-bacterial aetiology (Kapikian, 2000).

Animal studies, organ culture and standard tissue-culture methods were unsuccessFui in

propagating agent@) responsible for the majority of these cases, thus lirniting the

availability of matenal for analysis and characterisation of the probable aetiological

agent(s).

In 1972, a 27 nrn viral particle was visualised by immune electron microscopy

(IEM) in stools of volunteers who had agreed to ingest prepared filtrates of faecal

suspensions from a 1968 outbreak of acute gastroenteritis onginating in an elementary

school in Nonvalk, Ohio Sapikian et al., 1972). This viral particle, subsequently referred

to as the "Nonvalk agent", was confinned as the entenc pathogen following the

appearance of symptoms in the volunteers and the observation of a specific, heightened

antibody response to the viral particle when the paired acute and convalescent sera fiom

volunteers were used for immune electron microscopy of stool specimens (IEM)

(Kapikian et al., 1972). These virus particles, among the smallest viruses known, were

found to be in low titres in stool specimens and were characterised by an arnorphous

surface morphology and ragged, ill-defined outline (Kapikian et al.. 1 972).

Figure 1.1 Negative staining transmission electron microscopy image of NLVs. Note the lack of distinct rnorphology and appearance of a ragged edge (F.P. Williams, U.S. EPA, URL: http:llwww.epa.govlnerlcwwwlnorwalkhtm).

Morphologically sirnilar viruses were subsequently detected by EM in other

gastrointestinal outbreaks involving clinically indistinguishable syrnptoms. Most of these

viruses were narned after the geographical setting where the outbreaks occurred: for

exarnple, Norwalk, Montgomery County and Hawaii agents in the USA (Thornhill et al.,

1977), the Wollan and Ditchling agents in the UK (Paver et al,, 1973, Appleton et al.,

1977), and the Paramalta agent in Australia (Christopher et al., 1978). Initially,

observations of micrographs of IEM suggested that these agents were antigenic variants

within a single group of viruses; however, upon fùrther analysis it was deterrnined that

these viruses clearly belonged to more than one group, collectively referred to as either

the "small round structured viruses" or "Nonvalk-like viruses" (NLVs).

1.2 Taxonomy of Caliciviridae and "Norwalk-li ke viruses"

The Nonvalk-like viruses (NLVs) are simple viruses, 27 nm to 35 nm in diameter,

belonging to the Caliciviridae family (Kapikian et al., 1996). The non-enveloped,

icosahedral capsid is composed of 80 dimers of a singIe 58 to 62 kDa protein, which is

rather unusuai for animai viruses (Kapikian et al., 1996 and Jiang et al., 2000). These

viruses possess a positive-sense polyadenylated single-stranded RNA genome, with three

open reading *es (ORFs) (Jiang et al,, 1992 and Kapikian et al., 1996).

Figure 1.2 The three-dimensional structure of the baculovirus-expressed Norwalk virus capsid has been determined to a resolution of 2.2 nm using electron cryomicroscopy and cornputer image processing techniques. The empty capsid, 38.0 nrn in diameter, exhibits T=3 icosahedral symmetry and is composed of 90 dimers of the capsid protein. The striking features of the capsid structure are arch-like capsorneres formed by dimers of the capsid protein and large hollows at the icosahedrai 5- and 3-fold axes (Prasad et al., 1996).

In August 1999, the International Cornmittee on Taxonomy of Viruses (ICTV)

approved several changes to the Caliciviridae family of positive-sense RNA viruses

(Green et al., 2000). The family Calicl'vividae consists of a number of viruses, whose

hosts inciude pigs, cats, sea lions and hurnans. These vimses share cornmon features: a

single major structural protein in an icosahedral capsid with 32 cup-shaped depressions

on its surface and the presence of a small protein (VPg) of approximately 10-12x1 o3 kDa

instead of a methylated cap at the 5' end terminus of the virion RNA (Green et al., 2000).

MI NLV genomes are organised with a protease and RNA-dependent RNA polymerase

gene (ORF1) that overIaps and precedes a single structural capsid protein gene (ORF2);

the extent of this Carne-shift varies between NLV genogroups (Clarke and Lambden,

2000). The small ORF3 encodes for a protein, which is hypothesised to interact with the

RNA and capsid protein during encapsulation, and has been detected as a 32-kDa

irnmuno-reactive soluble protein in stools of NLV-infected volunteers (Clarke and

Larnbden, 2000).

ORF 1, 1788aa , - Z n - - - , . -+ . -

ORF 3. 21 1aa < . Genogroup 1 5' . , . _. - - . . , _ _ - , .- - - 2 . . ,.

. - 1 - - _ _ - ._. _ . _ _ _ - 7708b

Heliiase proteas, ~ o l h e r a s e

ORF 1, 1699aa . - . . ORF 3,268aa

-, - . -. '..' . - - - . . . . - - - .

Genogroup 2 5 ' - .- . _ . - _ . .. . :> . ., .- 1 3 . - -,-

A - 7. : .: - - -.- - - 7555b

ProtJase Po/r_rase ORF 51

Figure 1.3 Organisation of the NLV genome (genogroups 1 and 2). The open reading Crames (ORO predicted by sequence analysis and the number of amino acids (aa) in the protein products are indicated. The positions of the helicase, protease and RNA polymerase motives are shown (Jiang et al., 1993; Lambden et al., 1993; Caul, 1996).

Initially, the caliciviruses (CVs) were grouped based on the host species of origin

(Green et aL, 2000). However, when it was discovered that viruses genetically and

antigenically related to San Miguel sea lion vims (SMSV) could be detected in diverse

animal hosts and enteric CVs geneticaily related to NLVs were discovered in swine, host

range was deterrnined to be an ineffective criterion for defining genus (Green et al.,

2000). Advances in sequence and phylogenetic analysis of enteric CVs subsequently

provided a fiamework for the development of a definition of genus (Green et al., 2000).

For example, charactensation of the genomes of the Sapporo, Plymouth and Manchester

vimses demonstrated a major clifference in the reading b e usage between the classical

human CVs and NLVs respectively, and h l y established these two groups as distinct

entities (Green et al., 2000). Thus, the NLVs and the "Sapporo-like viruses" have been

recognised as two separate genera in the Caliciviridae farnily (Green et al., 2000).

The genus narnes 'Worwalk-like virus" and "Sapporo-like virus" were proposed

in the Seventh Report of the ICTV, but these are considered provisional narnes and will

be re-examined at the next meeting of the Caliciviridae Shidy Group (Green et al., 2000).

This proposa1 ends the confùsion resulting fiom the interchangeable use of the terms

"small round structured vinises" and 'Norwalk-like viruses" for the description of the

same group of viruses. The application o f a cryptogram nomenclature has been proposed

for the description of CV strains identified in epiderniological studies, which would use

the host of origidgenus abbreviatiodspecies abbreviation/virus namelyear of

occurrence/country of origin. Thus, the Southampton virus would be designated as

HuCV/NLV/Southarnpton/199 1RK (Green et al., 2000). The ICTV does not address

classification issues beiow the species level, thus the establishment of the cryptogam as

the standard for CV strain designation would be decided by CV researchers (Green et al.,

2000).

Within the NLV genus, a remarkable genetic and antigenic diversity has been

observed. Three genogroups have been descnbed which reproducibly group together on

a distinct branch of the phylogenetic tree and are sufficiently close in arnino acid and

nucleotide sequences to be distinguished fiom genetic clusters falling outside the group.

The genogroups comprise several prototype strains, which were the first reference strains

to have the entire open-reading fiame 2 (ORF2) region sequenced and to be detemined

genetically distinct fkom other reference strains. Genogroup 1 (GI) indudes genetic

clusters represented by the prototype viruses Nonvalk (NV), Southampton (SOV), Cruise

Ship (CSV), Desert Storm (DSV) and outbreak 3 18. Genogroup II (GII) compt-ises

genetic clusters with prototype vimses Snow Mountain (SMV), Hawaii (HV), Toronto

(TV), Bristol (BV), and outbreaks 290,269,273, 539 and 378. Finally, genogroup 111

comprises a single genetic cluster of 2 bovine vinises with prototype virus Jena (JV).

These classifications are based on analysis of the clustering pattern of strains in a

phylogenetic tree and the pair-wise sequence distances of the strains using four regions of

the ORF2 gene.

The NLV have also been ciassified based on antigenicity observed using solid-

phase immune electron microscopy (SPIEM). The prototype strains for SPIEM-based

antigenic characterisation were Taunton virus &JK- l), Nonvalk w - 2 ) , Hawaii (UK-3)

and Snow Mountain (UK-4). This classification scheme is limited by the presence of

cross-reactive antibody responses and by a lack of reproducibility between laboratories;

nevertheless, the SPIEM results indicate a remarkable antigenic diversity arnong strains

and a world-wide distribution of strains with similar antigenicity. The relationship

between the SPIEM-based antigenic types and the genetic clusters previously described is

yet to be deterrnined; however, a tnily unified NLV classification scheme should account

for both genetic and antigenic characterisation of NLV strains. (Ando et

al., 2000)

Genogroup 1 UKZ-8 - L m - 1 2

l CKI-6

m l - 7

sov DSV

UKI -1 UKI-4 1 925

Genogroup 2 I

Figure 1.4 Dendogram of predicted phylogenetic relationship among 30 NLV strains. The length of the abscissa to the connecting node is proportional to the genetic distance between sequences. @SV) Desert Storm virus; (SOV) Southampton virus; (BRV) Bristol virus; (HWA) Hawaii agent; (NV) Norwaik virus; (SMA) Snow Mountain agent; (TV24) Toronto virus; (OTH, KY89,1283 and 925) Japanese strains; (UKl-UK4) antigenic groups as identified by solid-phase immune electron microscopy (Ando et al., 1995).

Caliciviridae taxonomy is constantly evolving due to the accumulation of new

data relating to the genetic and biologic characterisation of CVs. Further classification

research is currently Iimited by the inability to propagate most CVs in culture, thus

restricting examination of several important features traditionally used for classification,

including protein synthesis in infected cells, antigenic relationships, physicochemical

properties and ce11 tropism. The development of efficient ce11 culture systems and

infectious cDNA clones of CV genera would provide critical information for the

classification of these vinses. Furthemore, the establishment of a cornputer network

"CaliciNet" will facilitate collaboration amongst NLV researchers, allow improved

tracking of NLV strains and clar ie epidemiological features of NLVs. (Green, 2000)

1.3 NLVs and Acute gastroenteritis

With the development of sensitive molecular methodologies for virus detection,

NLVs are now recognised as the major cause of epidemic, non-bacterial acute

gastroenteritis world-wide (Ando et al., 2000). It is estimated that NLVs are responsible

for 95% of nonbacterial, acute gastroententis outbreaks, many of which are associated

with contaminated food and water (Green, 2000). NLVs are responsible for millions of

infections each year in both developing and developed nations, resulting in significant

costs to their health budgets due to management and control within populations and the

consequent loss in person-power productivity (Green, 2000). However, the degree to

which NLV infection is endemic is unknown because those affected by acute

gastroenteritis do not always seek medical attention (Becker et al., 2000).

1.3.1 Transmission

NLVs are transmitted to hurnans via the oral route thus these viruses exhibit acid

stability and retain infectiousness after passage through the stomach to the small intestine

(Caul, 1996). The explosive nature of many NLV outbreaks suggests that the infection is

often acquired fiom a common source such as NLV contaminated food or water

(Kapikian et al., 1996). Transmission of NLV infection is pnmarily via the faecal-oral

route and is facilitated by an infectious dose estirnated to be as low as 10-100 virus

particles (Schaub and Oshiro, 2000). In volunteers who ingested NLV infected material,

viral shedding was observed to occur maxirnally for 72 hours in both symptomatic (90%)

and asymptomatic (50%) persons, but, could be detected for up to 13 days following

NLV infection (Okhuysen et al., 1995). This extended viral shedding fi-om convalescent

and asymptomatic persons could contribute to the propagation and continuation of such

NLV outbreaks (Okfiuysen et al-, 1995).

The most comrnon source of NLV infection is food-borne contamination: m v s

have been implicated in up to 41% of food-associated infections analysed by

epidemiological and diagnostic methods (Deneen et al., 2000). Food may be

contaminated, either by contact with environmental sources or by contact with infected

food handlers (Becker et al., 2000). In one study, it was found that 18% of food-

associated NLV outbreaks involved healthy, asymptomatic food-handlers who reported

acute gastroenteritis among members of their household, suggesting either the possibility

of person-to-person transmission, coupled with unhygienic food-handling practices

(Deneen et al., 2000).

Direct and indirect person-to-person transmission (via contaminated

environmental surfaces) has been described by Caceres et al. and Russo et al. as

important transmission routes in hospitals and long-term care facilities, affecting both

patients and staff (Caceres et al., 1997 and Russo et al., 1997). Furthemore, the

detection of NLVs in vomitus and the projectile nature of NLV-associated vomiting are

consistent with the possibility of airborne spread (Russo et al., 1997). Person-to-person

transmission was reported during an outbreak involving a college football garne: one

team sufferïng 6.om food-borne NLV-associated, acute gastroenteritis transmitted the

virus to the opposing team. This may have occurred through faecal-oral and aerosol

transmission of vomitus during the intense physical and bare hand contact, which is

characteristic of this game (Becker et al., 2000).

The identification of a single NLV strain responsible for 60 outbreaks in at least 8

countries on 5 continents during the 1995-1996 season, also suggests that the circulation

of NLV strains may involve currently &own patterns of transmission which allow for

the sudden ernergence and rapid global spread of NLV strains (Noel et al., 1999).

1.3.2 Clinical Syndrome

In studies using volunteers who ingested NLV infected material, incubation

pex-ïods were observed to range fiom 10 to 5 1 hours following ingestion of NLVs, with

the acute gastroenteritis lasting &om i2 to 60 hours. Clinical manifestations include

nausea, vomiting, diarrhoea, abdominal crarnps, headache, fever, chills, anorexia and

myalgias; however, being a self-limiting illness, symptoms were usually resolved without

S ~ ~ O U S complications. Nevertheless, severe gastroenteritis requiring medical intervention

for dehydration and electrolyte imbalance has been recently associated with NLVs,

especially in elderly or irnmuno-compromised patients (Green, 2000). Though deaths

due to NLV gastroenteritis of debilitated elderly patients have been docurnented, these

deaths are considered to be mainly due to a pre-existing condition. Antiviral therapy for

NLV infection is currently unavailable, but oral administration of bismuth subsalicylate

(a gastrointestinal anti-inflamrnatory dmg) following the onset of symptoms, has been

shown to reduce the duration of the acute gastroenteritis ilhess and the severity and

duration of abdominal crarnps. (Kapikian et al., 1996).

1.3.3 Pathology

Biopsies of the jejunum from volunteers, who were adrninistered oral Notwalk or

Hawaii virus and subsequently developed acute gastroenteritis, indicated that NLVs

target manire enterocytes of the small intestine, resulting in histopathological lesions

(Kapikian er al., 1996 and Clarke and Larnbden, 2000). Although the epithelial cells of

the mucosa in the proximal small intestine remained intact, there was a broadening and

blunting of the villi and microvilli, cytoplasmic vacuolisation and an infiltration of the

lamina propria by mononuclear cells (Kapikian et al., 1996). Furthemore, ultrastructure

studies by transmission EM have reveaied alterations in the rough and smooth

endoplasmic reticulum with an accompanying increase in multi-vesiculate bodies (Caul,

1996). This pathology was also observed in those volunteers who were administered

NLVs but who did not develop any signs or symptoms of acute gastroententis.

Furthemore, the enzyme levels in the brush border of the small intestine (trehelase and

alkaline phosphatase) were markedly reduced and a transient malabsorption of fat, D-

xylose, and lactose was noted. A significant delay in gastric emptying was observed in

al1 NLV infected volunteers and has been hypothesised to be responsible for the nausea

and vomiting associated with NLV illness. (Kapikian et al., 1996) The site of virus

replication has not been identified by observation of virus or viral antigen in intestinal

cells, though the virus has been detected in both stool and vomitus (Estes et ul., 2000).

1.3.4 Immunity to NLV Infection and Vaccine Development

lmmunity to NLVs is poorly understood. Resistance to NLV infection has been

observed in individuals lacking antibody, while the presence of specific antibody for

NLVs is not found to provide long-term protection (Kapikian et al., 1996). h developed

countries, 65% of individuals possess antibody to NLVs by 1 1-15 years of age (Matsui

and Greenberg, 2000); however, these adults consistently demonstrate a high

susceptibility to NLVs, to the extent that in some outbreaks, more that 80% of adults c m

become il1 (Kapikian et al., 1996). It has been suggested that natural resistance to NLV

infection may not be widespread in the general population despite serum antibody

presence or, that these viruses are particularly efficient in evading host immune defences

(Matsui and Greenberg, 2000). In developing countries, a trend toward higher rates of

infection arnong young children is observed along with an antibody prevalence rate of

75-100% in the first five years of life, which has been seen to provide some protection

against subsequent NLV infection (Matsui and Greenberg, 2000). It is plausible that the

6equency with which these children are exposed to NLVs, as a result of poor sanitation

and hygienic conditions, may result in the developrnent of continuous short-term natural

imrnunity to the virus (Matsui and Greenberg, 2000).

Short-terrn immunity folIowing initial NLV infection has been observed to be

serotype-specific and protective 6 to 14 weeks after the original NLV infection. Matsui

and Greenberg noted that a recent illness with NV would protect individuals korn

subsequent challenge with Montgomery County v i r ~ ~ s (MCV), but did not protect against

challenge with Hawaii virus (HV) (Matsui and Greenberg, 2000). In contrast, long-term

immunity deviates Ei-om the traditional pattern: volunteers who developed acute

gastroenteritis following oral NLV administration also succumbed to gastroenteritis when

challenged 27 to 42 months later, while volunteers who did not develop acute

gastroenteritis following oral NLV administration were also resistant to subsequent

challenge (Kapikian et al., 1996). Analysis of senun antibody did no t shed light on this

difference in susceptibility: in fact, volunteers who were susceptible to NLV infection

demonstrated significantly higher s e m IgG titres for NLVs than those who were

resistant (Johnson et al., 1990). Moreover, the presence of high titres of NLV-specific

faecal IgA in volunteers prior to NV challenge seems to correlate with the likelihood for

development of clinical illness and the failure of protection to subsequent NV exposure

(Okhuysen et al., 1995). In contrast, volunteers who were resistant to NLV illness and

demonstrated low or undetectable antibody titres pnor to NLV challenge, failed to

seroconvert following challenge and resisted NLV-associated illness with rechallenge

(Baron et al., 1984). It is possible that a geneticaIly-determined variation in intestinal

viral receptors or other cellular mechanisms may be responsible for long-tem resistance

to infection, while short-term transient immunity is rnediated by serurn antibody

(Kapikian et al., 1996). Support for the presence of a intestinal viral receptor was

presented by White et al., who used competition experirnents to demonstrate that the

binding of recombinant NLV virus-like particles (rVLPs) was specific in differentiated,

- hurnan intestinal Caco-2 cells and involved the C-terminal region of the virus capsid

(White et al., 1996).

Molecular cloning ofNLVs using the baculovirus expression system have

allowed for the production of large yields of recombinant virus-like particles (rVLPs),

which spontaneously assemble fkom 180 identical 58 kDa NLV capsid proteins to form

particles comparable in morphology and antigenicity to native NLVs (Jiang et al. 1993

and Bal1 et al., 1998). The rVLPs could be used for the study of short-tem and long-

term imrnunity, as they are safe and imrnunogenic when ingested by expenmental rnice

and human volunteers (Matsui and Greenberg, 2000 and Estes et al., 2000). Phase 1 trials

in oral administration of rVLPs in hurnans have revealed stimulation of a predominant

IgG2 subclass response and serurn IgA antibodies, which is similar although smaller in

magnitude to the serological response following NLV challenge (Estes et al., 2000). Co-

administration of rVLPs with mucosal adjuvant may improve the magnitude of this

rVLPs-related serological response (Estes et al., 2000). It is hypothesised that the rVLPs

bind to intestinal receptors ancilor are taken up by Peyer' s patches and endocytosed,

processed and presented to the mucosal immune system (Bal1 et al., 1998). Since NLV

infection is generally believed to be localised in the intestine, induction of local imrnunity

by rVLPs may be significant for protection against Uifection (Ball et al., 1998).

As the widespread incidence and clinical significance of NLV infection continue

to be recopised, the need for an efficacious, cost-effective vaccine is imminent (Estes er

nL, 2000). The need for an NLV vaccine was particularly evident during Operation

Desert Storm, when an outbreak of NLV-associated acute gastroenteritis affected the

performance of military personnel and interfered with tactical operations, thus

endangering the lives of the soldiers (Estes et al., 2000). Thus, the development of a

NLV vaccine and its use could be justified particularly in settings where individuals rnay

be at risk of serious consequences; for example, the elderly in long-term care Facilities,

immuno-cornpromised individuals and travellers to endemic areas (Green, 2000, Estes et

al., 2000).

The curent candidate vaccines for NLVs are the orally administrated rVLPs, and

edible transgenic plants, including potato, tobacco and possibly banana (Estes et al., 2000

and Matsui and Greenberg, 2000). The rVLPs are deemed excellent candidates for a

NLV vaccine for a number of reasons. These recombinant particles (i) can be produced

and puified on a large scale, (ii) are stable at the low pH of the stomach, (iii) are capable

of IyophiIization with long-term storage at 4OC once reconstituted in water or buffer, (iv)

do not induce tolerance with multiple oral doses and (v) are potentially targeted to the

Peyer's patches in the gastrointestinal tract due to their particulate nature (Ball et aL,

1998 and Estes et al., 2000). The transgenic plants produce self-assembled recombinant

NLV capsid protein, which stimulates humoral and mucosal immunity in mice without

requiring an adjuvant (Matsui and Greenberg, 2000). Human trials are essential, and will

be required to determine if the immune response stimulated by these oral vaccines is

protective, since the mouse model cannot simulate NLV-associated illness (Matsui and

Greenberg, 2000). The potential advantages of such oral vaccines lie in their ease of

delivery, rninor side effects and their potential for production of immunity localised at

mucosal surfaces (Estes et al., 2000). Although these candidate vaccines are promising,

vaccine development is harnpered by several serious challenges: (i) patterns of immune

protection to NLVs are poorly understood in humans, (ii) numerous antigenic types of

NLVs exist involving complex cross-protection, (iii) the development and significance of

mucosal irnrnunity to NLVs is unclear, (iv) the lack of an in vitro propagation system

prevents analysis of the presence of neutralising antibodies, and (v) no adequate animal

model currently exists to assess NLV challenge (Estes et al., 2000). Furthermore, the

development of an effective NLV vaccine may not be beneficial for certain NLV high

risk groups, such as elderly and irnrnuno-compromised patients, who may not be able to

mount a protective immune response following immunisation.

1.4 Epidemiology, Outbreak Management and Control, and Preveotion

Despite major public health advances to improve the quality and safety of food,

water and sanitation, acute gastroenteritis remains one of the most common illnesses in

North Arnerica (Glass et al., 2000). Since the incidence and prevalence of an enteric

pathogen is dependent upon the ability to effectively diagnose the agent, the importance

of NLVs in acute gastroenteritis was recognised only afler major advances in molecular

biology allowed for the development of novel diagnostic approaches for NLV detection

in clinical and epidemiological studies (Glass et al., 2000). It is now clear that the

majority of acute gastroenteritis outbreaks affecting patients of al1 ages in the United

States, the United Kingdom, Australia, Japan and the Netherlands are attributable to

caliciviruses (Glass et al., 2000).

1 Al Epidemiology of NLV infections

Environmental surveys have found that caliciviruses are ubiquitous and stable in

the environment, thus providing an accessible source of virus for potential infection

(Green, 2000). It is hypothesised that either the large-scale food-borne transmission of a

single strain or the introduction of new strains h-om a non-human reservoir is responsible

for the emergence of epidemic strains of caliciviruses. The animal reservoir hypothesis

has recently been supported by the discovery of NLV-like sequences in stool specimens

fiom pigs and calves. The genetic distances between these human and animal NLVs are

similar to that between NLV genogroups 1 and II. In addition, the epidemic spread of

NLVs within human populations resembles the rapid spread of another calicivirus, the

Vesicular Exanthema of Swine Virus W S V ) , following its reintroduction into the

Arnerican swine population h-om an oceanic (opaleye perch, marine marnmals) reservoir

via the feeding of swine with VESV-contarninated fish products. Thus, the occasional

widespread epidemics caused by a single strain of NLV may be the result of an

introduction of strains fkom a zoonotic reservoir. Furthemore, the stability of discrete

genetic lineages in animal NLVs is yet undetermined, allowing for the possibility of the

existence of a common pool of viruses circulating arnong humans and animals. (Smith et

al., 1998, Van der Poe1 et al., 2000)

Although it was once believed that NLV infections rnainly affect school-age

children and adults, a NLV outbreak in Finland involved al1 age groups, whereas studies

in Australia and Japan found NLVs to have caused outbreaks of acute gastroententis in

babies (Green et al., 1993, Marshall et al., 1997, Kukkilla et al., 1999, houke et ai., 2000

and Nakata et al., 2000). Furthemore, recent advances in laboratory diagnostics have

demonstrated the presence of NLVs with the sarne fiequency as rotavirus in faecal

specimens fkom Finnish children with diarrhoea (Monroe et al., 2000).

NLV outbreaks cornmonly occur in camps, recreation areas, elementary schools,

cruise ships, nursing homes, colleges, restaurants, small families and cornmunity settings

(Kapikian et al., 1996). In fact, NLVs are currently recognised as the most cornmon

cause of outbreaks of gastroenteritis in restaurants and institutions such as nursing homes

and hospitals (Van der Poe1 et al., 2000). NLV outbreaks at long-term care facilities and

hospitals are especially problematic due to the considerabIe morbidity and mortality

associated with acute gastroenteritis in immuno-compromised and elderly patients

(Augustin et al., 1995 and Marx et al., 1999). Furthemore, these outbreaks involved

high attack rates, prolonged transmission and infection, problematic outbreak

management and control, and significant resultant absenteeisrn due to NLV illness in

medical personnel (Augustin et al., 1995 and Marx et al., 1999)- Identification of NLV

cases may be harnpered in long-tenn care facilities, as faecal sample collection for NLV

detection becomes increasingly difficult due to the use of super-absorbent incontinence

pads (Augustin et al-, 1995).

The impact of NLVs on food-related disease is underscored by the observation

that these viruses are the major cause of food-related, acute gastroenteritis outbreaks in

the United Kingdom (Kapikian et al., 1996 and Mead et al-, 1999). NLVs have also been

recognised as the most common cause of al1 food-borne illnesses (66.6%) and

hospitalisations (32.9%) and account for 7% of food-related deaths in the USA (Mead er

al., 1999)- Furthemore, the Centre for Disease Control in the United States estimates

that 40% of al1 NLV-associated illness is food-borne (Mead et a l , 1999). The major

food-borne vehicles of NLV infection include salads, sandwiches, cold cooked rneats,

melon, fruit salad and other cold or Eesh foods (Kapikian et al., 1996). NLVs are also

the most cornmon cause of outbreaks of acute gastroententis following ingestion of raw

or steamed shellfish (Kapikian et aL, 1996). Although shellfish can concentrate NLVs

fiom contaminated water through bivalve feeding, NLV contaminated shellfish do not

necessarily originate fi-om ocean beds contaminated with human waste, suggesting

currently unidentified modes of transmission therein (Smith et al., 1998). The trend

towards increased consumption of prepared, cold and fresh produce and restaurant meals

in western society makes NLV contamination an increasingly important food safety issue

oeneen et al., 2000). Furthermore, increases in international trade in food products and

international rapid travel increases the potential for NLV contamination to result in

widespread, multinational acute gastroenteritis outbreaks.

Exmination of the results of surveys of NLV outbreaks and sporadic cases in

Canada, the Netherlands, the United States, Japan, the United Kingdom, Australia, and

Denmark by Mounts et al. revealed a marked cold weather seasonality of NLV-

associated disease (Mounts et al., 2000). Although the faecal-oral route is the primary

route of NLV transmission, winter seasonality of NLV infection supports the hypothesis

that airbome spread may serve as a secondary route for NLV transmission (Mounts et ni..

2000). Other vimses, such as rotavirus and influenza, with h o w n airbome transmission

exhibit similar winter seasonality and the increased time spent by populations in indoor

environrnents dunng the winter months rnay facilitate airbome transmission (Mounts er

al., 2000).

As NLVs may be responsible for millions of gastroenteritis episodes per year in

individual countries, this considerablc disease burden is clearly expensive to society in

terrns of the cost of outbreak management, medical intervention and loss of productivity

(Green, 2000). For instance, a NLV outbreak caused by contaminated municipal water in

a small Finnish town affecting over 50% of the 4860 inhabitants, was responsible for the

loss of 8OO working days and a total cost of outbreak intervention and medical care of

approximately $300 000 US4 (Kukkula et al., 1999). In addition, a NLV outbreak in an

Australian hospital involving 18 patients and 14 staff, cost $7600 for nursing staff sick

leave and $10 600 for bed closures in addition to the cost of outbreak control measures

(Russo et al., 1997).

As previously mentioned, epidemics of acute gastroenteritis due to NLVs are a

major cause of acute morbidity arnong USA military forces and were the single most

common cause of disability of USA troops deployed in the Persian Gulf during Operation

Desert Shield (McCarthy et al., 2000 and Monroe et al., 2000). Deployed military

personnel are at increased risk o f sporadic and epidemic acute gastroenteritis due to

crowded conditions that facilitate rapid person-to-person transmission and the difficulty

in maintaining high levels of sanitation during combat activities (McCarthy et al., 2000).

Outbreak control is thus extremely limited, allowing NLV outbreaks to persist over

several weeks (McCarthy et al., 2000). Despite the use of intravenous rehydration fluids,

military operational activities were comprornised by the inability of key personnel to

perform cntical duties and the increased risk of accidents among sick soldiers (McCarthy

et al., 2000). Thus, NLV vaccine development is likely the best solution to prevent

NLV-associated disability in the military environment (McCarthy et al., 2000).

1.4.2 Outbreak Management and Control of Acute NLV-associated Gas troenteritis

General methods for prevention of the spread of NLV Uection such as fiequent

and effective hand-washing, hygienic preparation of food and proper disposa1 and

disuifection of contaminated material, may reduce transmission within a family or an

institution (Kapikian et al., 1996). Strict infection control rneasures are required to

respond to NLV outbreaks in long-tem care facilities and hospitals: (i) patient

movements should be limited; (ii) no patients should be admitted to or discharged from

the affected wards until NLV symptoms have ceased for at least 48 hours; (iii) visitors

should be restricted to immediate farnily members; (iv) staff should Wear long-sleeved

gowns and gloves when attending affected patients and should irnrnediately remove

gowns and gloves when finished; (v) staff should wash or disinfect hands afier contact

with each patient; (vi) staffing for wards should be individualised and staff rnovement

between wards restricted; (vii) affected staff should not work until asymptomatic for 48

hours; (viii) environmental surfaces in affected wards should be cleaned frequently with

100-200 ppm disinfectant containing sodium hypochlonte; (ix) soiled linens should be

handled as biohazardous material and washed separately f?om other linens and (x) wards

should remain closed until patients and staff are fiee from signs or symptoms of acute

gastroenteritis for 5 days (Russo et al., 1997). Effective outbreak control is also

dependent on rapid collection and transport of chical specimens to the laboratory for

identification of the infectious agent. Furthemore, timely notification of health officials

and prompt irnplementation of infection control measures, including dedication of staff to

the outbreak area until the outbreak is over and immediate sick leave for staff with signs

or syrnptoms of acute gastroenteritis, is essential (Russo et al-, 1997 and Marx et al.,

1999).

1.4.3 Prevention of NLV Infection

The USA Environmental Protection Agency has expressed concems about NLV

contamination of cornrnunity and recreation water systems because NLVs are ubiquitous

in the environment, can pass through simple water filters, remain infectious despite

standard levels of chlorine and require a small inoculum to cause the disease (Monroe et

al., 2000). NLV outbreaks have also been directly associated with consumption of

contaminated drinking water or the recreational use of contarninated water (Schaub and

Oshiro, 2000). In 1979, an outbreak due to NLVs caused illness in 78% of teenagers at a

recreational camp and was deterrnined to be associated with well-water contaminated by

runoff fiom the camp's sewage treatment facility (Baron et al., 1984). Another NLV-

related outbreak was confirmed by RT-PCR to result fiom NLV-contarninated municipal

water in Finland in Much 1998: although the source of contamination was unknown, the

municipal chlorination of water was found to be inadequate for destroying NLVs

(Kukkula et al., 1999). This outbreak affected over 50% of the population and resulted in

considerable economic costs despite the relative mildness of NLV infection, thus

highlighting the need to consider viruses in the quality assessrnent and surveillance of

drinking water (Kukkula et al., 1999).

Direct monitoring of these water sources is irnpractical due to the large sample

size required to detect virus particles, the kequency of water testing required and the lack

of diagnostic techniques to detect and quanti@ al1 potential pathogens (Schaub and

Oshiro, 2000). Water safety indicators, such as the total and faecal coliform counts, are

cwrently used to identiQ potential contamination events; however the use of these

indicators is inherently problematic as potential infectious agents Vary in size,

physiology, susceptibility to disinfection, and reçponse to environmental Factors (Schaub

and Oshiro, 2000). For instance, bacteria and vimses do not necessarily respond to

disinfection treatment in an identical fashion,

Currently, the treatrnent of drinking water includes coagulation, settling, sand or

multi-media filtration and disinfection, which typically remove protozoa, bacteria and

large viruses (Schaub and Oshiro, 2000). However, caliciviruses continue to cause

outbreaks associated with drinking water thus indicating that these viruses may require

special consideration by public health officids (Grant et ai., 1999 and Schaub and

Oshiro, 2000). Although NLVs are resistant to inactivation following standard water

treatment with 3.75 to 6.25 mg/L chlorine, these vimses are inactivated by the 10 mg/L

chlorine treatment nomally used to treat the water supply after contamination has been

detected (Kapikian et al., 1996). Further studies are required to develop analytical

rnodels for the identification and quantification of NLVs in water sarnples and to

determine the efficacy of water disinfection practices for virus removal (Grant et al.,

1999 and Schaub and Oshiro, 2000).

The USA Food and Dmg Administration @DA) has introduced guidelines on

hygiene practices, contamination screening and the exclusion of infected food handlers

kom the workplace in response to the identification of NLV-associated, acute

gastroententis outbreaks resulting from ingestion of foods contaminated at their source or

by food handlers (Monroe et ai., 2000). For example, a USA study found that four

separate outbreaks of acute gastroenteritis were epidemiologically linked to a single

bakery and the kosting made by a bakery worker who was il1 with vomiting and

diarrhoea during his work shift (Deneen et al,, 2000). Moreover, as a result of the

globalisation of the food market, multinational outbreaks of NLV gastroenteritis can and

will occur, as was demonstrated by a multinational outbreak of caiicivirus-related

gastroenteritis associated with faecally contarninated raspbemes from Slovenia (Monroe

et al., 2000). These NLV outbreaks underscore the importance of the ability to link

outbreaks of NLV-associated, acute gastroenteritis to contarninated food using sequence

analysis and the necessity for public health policy development for tracing, screening,

and recall of contaminated food products at a national and international Ievel (Glass et

al., 2000).

1.5 Considerations for Clinical Laboratory Diagnostics

Research into the role of NLVs in human iIlness has progressed slowly due to the

extreme difficulty in detecting these vinises (Monroe et al., 2000). As mentioned earlier,

these viruses have resisted cultivation in cell-tissue and organ cultures, and nurnerous

attempts to induce NLV illness in a variety ofexperimental animais have al1 failed

(Monroe el al., 2000). Current diagnostic techniques rely on EM visualisation of the

virus particle, detection of NLV antigen by ELISA, detection of viral RNA matenal from

stool specimens by RT-PCR or detection of NLV-specific antibody from paired acute and

convalescent serum specimens (Ando et al., 1995 and Jiang et al., 1992). The time of

collection for stools is critical to the identification of NLVs, as detection rates are greatly

reduced when the sample is collected more than 72 hours following the onset of

syrnptoms (Kapikian et aL, 1996). Timing of s e m collection is complex as IgG

detection requires paired (acute and convalescent) sera taken at appropriate intervals,

whereas IgM and IgA detection require single serum samples taken 6-8 days after

exposure (Brinker et al., 1998, 1999).

1.5.1 Electron Microscopy (EM) and Immune EM

Although EM of stool material fiom a patient with gastroenteritis may reveal

NLV particles, EM is of lïmited diagnostic value as it is dependent on the concentration

of viral particles (Xo6 particles per rnL) in the individual specimen; most often, NLV

particles are present in rather low concentration such that they are rarely detected

(Kapikian et al., 1996). However, when NLVs are present in adequate titre for EM

detection, the reproducible arnorphous appearance of NLVs and the lack of discernible

organised .geometric symrnetry demand considerable expertise for definitive

identification by the EM technoIogist (Caul, 1996). A distinct advantage of EM is its

potential "catch-all" role for the detection of viral agents in stools, whether they be new

undetermined antigenic variants of NLVs or other acute gastroenteritis pathogen (Caul,

1996).

Immune eiectron microscopy (IEM) yields better results; however, IEM is a

specialised, labour-intensive technique that also requires relatively large amounts of

antigen to be present in stools (Kapikian et al., 1996). IEM involves incubation of the

stool sample with the patient's serum, negative staining of the antigen-antibody

complexes with phosphotungstic acid and their visualisation by EM (Kapikian et al.,

1996). However, dernonstration of an antibody response to particles in stool does not

establish an aetiological relationship, unless a four-fold nse in antibody titre is

demonstrated between acute and convalescent sera reactions (Kapikian et al., 1996). The

requirement of convaIescent senun for E M analysis Iimits the use of IEM for the

provision of prompt diagnosis. In addition, both EM and IEM are not practical in a

routine diagnostic settuig as very few diagnostic laboratones are equipped with electron

microscopes on site, requiring specimens to be forwarded to reference centres thus

increasing the tum-around-time for delivery of rapid results.

1.5.2 Immunologicai Detection

In the past, immuno-diagnostic reagents for NLV detection were obtained fiom

volunteer specimens, thus limiting the use of these tests outside of the research field

(Monroe et al., 2000)- More recently, the development of the baculovirus expression

system for the production of NLV rVLPs allows for virtually an unlimited supply of

highly purïfied NLV capsid protein, providing reagents for sensitive and specific

irnmunologic assays for diagnosis of NLV infections (Jiang et al., 1992, 2000). These

rVLPs cm be applied directly as NLV antigen for antibody detection or used to produce

hyperimmune monoclonal antibodies in laboratory animals for use in antigen detection

(Henmann et al., 1995 and Jiang et aL, 2000). Although the antigen-detection enzyme

imrnunoassay (EIA) was found to be as sensitive as molecular methods for detection of

homologous strains of NLVs, low detection rates were obtained when this EIA was used

to detect NLVs from a variety of clinical outbreak samples (Jiang et al., 2000). It was

hypothesised that the high specificity of the ELA prevents detection of the diverse

circulating NLV strains (Jiang et al., 2000). The creation of rVLPs for al1 antigenic

clusters of the NLVs may ùnprove detection by EIA though coverage of al1 antigenic

groups may not be a very practical approach in a routine diagnostic setting at this time.

However, the recent identification of an epitope cornrnon to most genogroup 1 NLVs by

Haie et al. may allow for the development of a broadly cross-reactive EIA or enzyme-

linked irnrnunosorbant assay (ELISA) for detection of G l NLVs in stool (Hale et al-,

2000).

Detection of NLV-specific antibody, using radioimmune assays (RIA), EIA or

ELISA, is more sensitive tban IEM for diagnosis of NLV infection (Kapikian et al-,

1996). However, the immune diagnostic assays for IgM and IgG antibody detection are

not appropriate for timely diagnosis in outbreak situations, as seroconversions are noted

6-8 days and 12 days post infection respectively (Brinker et al., 1998, 1999).

Furthemore, these immunoassays are not practical due to their high specificity, which

would require the use of NLV antigen reagents representative of the circulating strains,

udess a common cross-reacting antigen could be found (Glass et al., 2000).

1.5.3 Molecular Diagnostics

The cloning and sequencing of the Norwalk and Southampton viruses revealed

the genomic organisation of the hurnan caliciviruses and led to the development of

sensitive and specific molecular diagnostic tests which could also assist in

epiderniological analysis (Monroe et al., 2000). These diagnostic advances have

demonstrated great sequence diversity in circulating NLV strains and provided sequence

data on more than 100 NLV strains world-wide (Noel et al., 1999). Specifically, the

reverse-transcription polymerase chain reaction and probe hybridisation techniques have

proved exceptionally useful (Monroe et al., 2000). These techniques are especially

usefil for epidemiological investigations because the identification of genetic sequences

among specimens collected &om patients in different locations can either link or separate

the sources of NLV contamination in ambiguous outbreaks (Glass er al., 2000).

Furthemore, molecular epidemiology is a powefi l tool for the investigation of point-

source outbreaks related to foodstuffs, food handiers, and water supplies and also to

monitor the spread of NLVs within a semi-closed environment (Caul, 1996). These

applications are based on the genetic prïnciple that strains with the same sequence have a

clona1 origin and that they accumulate sequence changes with continued passage (Noel er

al., 1999). However, we do not as yet understand the interactions that regulate the rate of

introduction of these genetic changes; thus, elucidation of the latter would aid in

understanding the dynamics of the spread of closely related NLV strains (Noel et al.,

1999). Moreover, monitoring of these genetic changes is essential since the function of

the RT-PCR method for viral detection is dependent on the presence of complementary

nucleotides between the primers and probes and the viral sequence (Glass et al., 2000).

1.6 Research Objectives

As outbreaks of NLVs in long-tenn care facilities and hospitals are often

prolonged, with high attack rates among patients and staff, rapid diagnosis and prompt

initiation of outbreak control measures are critical and essential (Augustin et al., 1995)-

The availability of new NLV diagnostic assays and the recognition of the importance of

NLVs in outbreaks of acute gastroententis require that research-based diagnostic tools be

applied in local public health and hospital laboratones (Glass ef al., 2000). At present,

when NLVs are suspected in outbreaks of acute gastroenteritis, EM analysis is ~erforrned

at two sites in Ontario: the Central Public Health Laboratory (CPHL) in Toronto and

Thunder Bay Regional Public Health Laboratory (TBRPKL). Thus, it was felt that the

adaptation of RT-PCR on-site in local public health laboratories rnight allow for

improved efficiency and sensitivity in NLV detection.

Ando et aL, at the Centre for Disease Control and Prevention (CDC, Atlanta,

Georgia) developed NLV-specific RT-PCR prkners and hybïidisation probes for routine

diagnosis of NLV infection, which are designed to broadly detect strains previously

classified into the 4 SPIEM-based antigenic groups (Ando et al., 1995). These primers

ampli@ a 123 nucleotide (nt) region of the RNA polymerase gene with 8 1 unique nt

when the primers are excluded (Ando et al., 2000). Southem blot hybridisation with 4

sets of probes allows classification of NLVs into 4 genetic groups: P 1 -A, P l -B, P2-A and

P2-B (Ando et ul., 1995). CDC investigators have used these rnolecular tools to conduct

epidemiological investigations, linking outbreaks at distant locations to common sources

of NLV contamination and identifjring the common NLV circulating strains. As a

consequence, these probes and primers were selected for detection of NLVs in outbreaks

of acute gastroenteritis in Ontario.

To this end, the objectives of this research project are identified below:

The RT-PCR and Southem blot hybridisation detection of NLVs fi-om clinicaI stool

sarnples kom outbreaks of acute gastroenteritis in long-term care Facilities in Ontario

using the primers and probes developed by Ando et al., 1995.

Cornparison of these RT-PCR results with those of standard EM perfonned off-site

Analysis of the frequency of outbreaks caused by K V genogroups i and 11 in Ontario

Examination of the feasibility of this RT-PCR procedure for routine use in a

diagnostic laboratory

2 Materials and Methods

2.1 Specirnens

A total of 98 stool specimens korn 25 outbreaks of acute gastroententis at long-

tem care facilities and child-care centres in Ontario were submitted to the Kingston,

Ottawa and Central Public Health Laboratories for analysis between December 1999 and

November 2000. As per standard protocol, stool sarnples were transferred to two sites,

the Central Public Health Laboratory in Toronto and the Thunder Bay Regional Public

Heaith Laboratory, for detection of vinises by electron microscopy (EM). For the

purposes of this study, these specirnens were analysed by the reverse transcription

polymerase chah reaction (RT-PCR) and Southern blot hybridisation (SBH) methods of

Ando et al. adapted at the Kingston Public Health Laboratory for detection of NLVs

(Ando et al., 1995).

All procedures involving the handling of stools, viral RNA and RT-PCR products

were carried out aseptically using stenle techniques in a Biosafety ~ e v e l II laminar flow

cabinet. Al1 materials in contact with specimens, such as glassware, micropipette tips,

eppendorfs and instruments, were stedised and solutions were autoclaved at 121°C for

20 minutes.

2.2 FtNA Extraction from Stool Specimens

Approximately IrnL of each stool sample was mixed with 5mL of 0.9% w/v NaCl

solution and centnfûged at 4000 rpm for 20 minutes to clariQ the suspension, using a

Baxter Canlab Megafuge 1 .O centrifiige (Heraeus Instruments, Germany). The

supernatant was filtered through a MILLEX@-GP 0.22 p m filter (Millipore, Bedford,

MA) and collected in a sterile tube for viral RNA extraction. Extraction of RNA fiom

k n o m G1 and G2 NLV positive and negative stool filtrates was perforrned alongside the

clinical samples as positive and negative controls. Viral RNA was extracted using the

QIAamp Viral RNA Mini Kit, as per the manufacture's instructions (QIAGEN, Valencia,

CA). Bnefly, the aliquoted AVL bufier with carrier RNA was heated to 80°C for 5

minutes, to dissolve any crystals, and cooled to room temperature. For viral lysis, 140pL

of stool filtrate was added to 560 pL AVL buffer with carrier RNA, vortexed for 15

seconds and incubated for 10 minutes at room temperature. The highly denaturing

conditions provided by the AVL buffer ensured inactivation of any RNases and the

carrier RNA lirnited any degradation of viral RNA by residual RNase activity. 560 PL of

96% ethanol was then added to the sample and mixed by vortexing, thus adjusting the

buffering conditions. Together with the activity of the carrier RNA, this change of

buffering conditions allowed for isolation of intact viral RNA and optimum binding of

the viral RNA to the QIAamp silica-gel membrane. The sarnple was then loaded into the

QMarnp spin column, 630pL at a t h e , and centrifuged at 8000rpm for 1 minute using a

Micromax centrifuge (International Equipment Company, Meedham Hts., MA). During

this centrifugation, viral RNA was adsorbed to the QIAamp spin colurnn membrane,

while the salt and pH conditions of the lysate buffer ensured that the membrane did not

retain proteins and other contaminants. Two wash buffers, AW 1 and AW2, were used to

remove residual contaminants, such as proteins, nucleases, and other inhibitors, which

could affect the RT-PCR reaction. 500pL of AWl buffer was added to the spin colurnn

and removed through the membrane by centrifugation at 8000 rpm for 1 minute.

Subsequently, 500pL of AW2 buffer was added to the colurnn and removed through the

membrane by centrifugation at 15000 rpm for 4 minutes. Finally, the RNA was eluted by

adding 60pL of AVE buffer, containing RNase-free water and 0.04% sodium azide for

prevention of microbial growth and contamination with RNases, to the column and

incubating at room temperature for one minute. This incubation was followed by

centrifugation at 8000 rpm to remove the viral RNA into an eppendorftube. The RNase-

ftee viral RNA filtrate was retained for RT-PCR amplification and stored at -20°C.

2.3 RT-PCR with Ando et ai. SRSV Primers

RT-PCR prirners were designed by Ando et al. for detection of genetically diverse

NLVs, and were produced by Cortec DNA Service Laboratories, Inc. (Ando et aL, 1995).

The extracted RNA fiom each sarnple was heated at 90°C for 5 minutes then chilled on

ice for 3 minutes, so as to prevent the formation of secondary structure in the ssRNA. 10

pL of sample RNA, 50 pmoles of the G1-G2 primer for negative-strand cDNA synthesis

and 25 pmoles of either the G1 or G2 primers were added to Ready-To-Go RT-PCR

beads with sufficient DEPC-treated water to make-up a total volume of 50 pL

(Amersham Pharmacia Biotech Inc., Piscataway, NJ). The Ready-To-Go RT-PCR

reaction mixture (50pL) comprised of 2.0 units Taq DNA polymerase, 1OmM Tris-HCI

(pH 9.0), 60mM KCI, 1.5m.M MgC12, 200 pM of each dNTP, FPLCptu-e@, RNAguardB,

Murine Leukernia Virus RT and stabilisers (including RNase/DNase-fiee BSA)

(Amersham Pharmacia Biotech Inc., Piscataway, NJ). The G1-G2 primer for negative-

strand cDNA synthesis annealed at the YGDD motifs of the RNA polymerase region, and

the G1 and G2 primers annealed at the same position between GLPSG and YGDD motifs

of the RNA polymerase gene, delineating a predicted 123 bp product (Ando et OZ., 1995).

For each RNA sarnple, a separate G1 and G2 amplification was performed in order to

determine the genogroup of the NLV infection.

Initially, the RT-PCR reaction tubes were incubated at 42T for 30 minutes to

allow for reverse transcription to occur. This was followed by a 5-minute incubation at

95°C to inactivate the reverse transcriptase and denature the RNA-cDNA strands. PCR

amplification involved 32 cycles, each of:

denaturation (95OC for 60 seconds)

primer annealing (55°C for 60 seconds)

polymerisation (72°C for 60 seconds).

A final extension was perforrned for 5 minutes at 72OC and the sample was stored at 4OC

pnor to gel electrophoresis. For long-tem storage, RT-PCR products were stored at -

Table 2.1 RT-PCR primers for amplification of diverse NLV genomes (Ando et ai., 1995)

Primer 1 Antigenic Croup Protype Virus 1 Identity # Polarity , Sequence

I G1-G2 RT j AI1 I NLVs 1 SRSV33 i negative : tgt cac gat ctc atc atc acc ' G2 1 UK-1, IX-3 , UK-4 1 Snow Mountain 1 SRSV46 / positive , tgg aat tcc atc gcc cac tgg

G l 1 l

IX-2 i Nonvak virus / SRSV48 i positive / gtg aac agc ata aat cac tgg

G1 / LJK-2 1 Nonvalk virus 1 SRSVSO 1 positive 1 gtg aac agt ata aac cat tgg i i G1 I UK-2 1 Norwaik virus 1 SRSV52 1 positive / gtg aac agt ata aac car tgg :

Note: Genogroup (G), Reverse Transcription (RT), Norwalk-like viruses (NLVs), Small Round Sû-uctured Virus (SRSV)

2.4 Agarose Gel Electrophoresis

25 pL of each of the control and clinical RT-PCR products were loaded onto the

wells of a 3% agarose gel with 3 pL of loading buffer (0.25% xylene cyanol, 15% ficoll).

Although 1OpL of RT-PCR product was initially used, it was determined that this volume

be changed to 25pL, as it allowed for irnproved detection following EtBr staining. The

gel was electrophoresed at 80 V for approximately 90 minutes alongside a 50 bp ladder,

stained with ethidium brornide (EtBr) and visually observed under short-wave UV light.

The visual observation of a 123 bp band indicated the presence of NLVs and the

genotype was determined based on the primer set used in the PCR.

2.5 Southern Blotting

To denature the DNA, the gel was soaked in denaturation buffer (0.5M NaOH,

1 S M NaCl) for 1 hour, rinsed with distilled water and neutralised in neutralisation buffer

(OSM Tris-HCl, 1 S M NaCl pH 7.5) for another hour. A Hybond-Nt positively charged

nylon nucleic acid transfer membrane (Arnersharn Pharmacia Biotech Inc., Piscataway,

No, was cut to the size of the gel and was soaked in u1traPUR.E 20xSSC (3.OM NaCl,

0.3M Sodium Citrate, pH 7.0) for 5 minutes (Life Technologies, Paisley, Scotland). A

mark was made in a corner of the membrane to indicate the orientation of the blot. The

bIot apparatus was set up as shown in Figure 6.1 (Appendix), and the DNA transfer was

allowed to proceed overnight. The membrane was then rinsed with 2xSSC to rernove any

gel particles and baked for 2 hours at 80°C to fix the DNA to the membrane. Four blots

were required per specimen to allow for the identification of the NLV genogroup.

2.6 Hybridisation with Ando et al. SRSV Prirners

Six oligonucleotide probes, designed by Ando et al. on the basis of interna1

sequences at the sarne location of the 123 bp NLV RT-PCR products, were produced by

Cortec DNA Service Laboratories, Inc. (Ando et al., 1995). These probes were labelled

at the 3' end with digoxigenin @IG) using the DIG Oligonucleotide 3'-End Labelling

Kit, according to manufacturer's instructions (Roche Diagnostics, Indianapolis, IN). 100

pmoles of each NLV oligonucleotide were added to separate sterile eppendorfhbes on

ice, with 4pL 5x reaction buffer, 4pL CoC12, 1 pL DIG-ddUTP, 1 pL terminal hansferase,

and sufficient DEPC-treated water for a total volume of 20 PL. The reaction tubes were

incubated at 37°C for 15 minutes in a water bath and then placed on ice. 1 pL of glycogen

solution was mixed with 200pL of 0.2mM EDTA and 2pL of this mixture was added to

the reaction tube to terminate the DIG-labelling reaction. The labelled O ligonucleotide

was precipitated with 2SpL of 4M LiCl and 75pL absolute ethanol at -20°C and

maintained ovemight at -20°C overnight. The reaction tubes were centrifuged at 15 000

rpm for 20 minutes, using a Micromax centrifuge (International Equipment Company,

Meedham Hts., MA). The pellet was washed with 70% ethanol (at -20°C), dried under

vacuum and dissolved in 40pL DEPC-treated water. DIG-labelled oligonucleotide

probes were stored at -20°C.

Following Southern bIotting and immobilisation of DNA, the nylon membrane

was incubated in standard pre-hybndisation buffer (SxSSC, 0.1% (w/v) N-Iaurosarcosine,

0.02% (w/v) SDS, 1% (w/v) blocking reagent) at 68OC for 1 hour. Hybridisation

solutions containing oligonucleotide probes (stored at -20°C) were heated at 95°C for 5

minutes and cooled on ice so as to denature the probe pnor to bybridisation; probes were

repeatedly used for a maximum of 10 times (or less if the quality of the signal for the

positive control detection was too weak for adequate visualisation). Four probe sets were

used to detect the presence of diverse NLVs: Pl-A, P 1-B, P2-A and P2-B (Ando et al.,

1995). Hybridisation of the membrane was performed overnight at 4g°C, using 20mL of

hybridisation solution buffer (SxSSC, 0.1% (w/v) N-laurosarcosine, 0.02% (w/v) SDS,

1% (w/v) blocking reagent, and 100 pmoles of DIG-labelled oligonucleotide probe). To

remove unbound antibody, the membrane was subjected to four washes: two washes in

2xSSC, O.l%SDS solution for 15 minutes each, followed by two washes in 0-IxSSC,

O. l%SDS solution for 15 minutes each.

Table 2.2 Hybridisation probes for identification of NLV amplicons, Ando et al.

Probe P2-B P2-A PI-B

1 Pl-A 1 UK-2 1 Nonvaikvirus 1 SRSV69 ] negative 1 aca tcg ggt gat agg cct gt 1 Note: Small Round Stmctured Virus (SRSV)

Antigenic Group UK-1, UK-3, UK-4

Pl-A P 1-A

2.7 Detection of NLV amplicons following Southern blot hybridisation

Colourimehic detection of DIG-labelled oligonucleotide probes hybridised to the

membrane was perforrned using the DIG Nucleic Acid Detection Kit and DIG Wash and

Block Buffer Set, according to manufacturer's instructions (Roche Diagnostics,

Indianapolis, IN). Briefly, the membrane was equalised for one minute in washing

buffer, blocked in blocking reagent buffer for one h o u and incubated at room

temperature in 15000 anti-DIG-AP conjugate diluted in biocking reagent buffer for one

hour (Roche Diagnostics, Indianapolis, IN). Unbound antibody was removed with two

15-minute washes in washing buffer. Finally, the membrane was equilibrated in

detection buffer for 2 minutes and incubated overnight in a solution of IOrnL of colour

substrate solution buffer (200pL NBTBCIP in LOmL detection buffer) (Roche

Diagnostics, Indianapolis, IN). The colour reaction was arrested by transfemng the

membrane into 50mL of TE buffer (1Om.M Tris, ImM EDTA). The presence of bands

was obsewed visually and the membrane was dried for storage.

UK- 1 UK-1. weaklv UK-2

Protype Virus Snow Mountain

UK-2 UK-2

Toronto virus Toronto virus

Ldentity # SRSV47

Norwak virus Norwalk virus

SRSV61 1 negative SRSV67 1 neeative

Polarity negative

atg tca ggg cct agt cct gt aca tct ect eae aea cct ea

SRSV63 SRSV65

--

Sequence atg tca ggg gac agg ttt gt

negative neeative

aca tca gga gag tgc cca ct aca tca gct cat aae cca ct

L

0.5-1 -0mL of stool is suspended in 0-9% NaCl, centrïfuged and filtered through a 0.22pm filter

Filtrate is treated with reagents from the Qiagen QIAamp Viral RNA Mini Kit for RNA extraction

- . .- .- - - .

m a i s r e d (5 minutes at 90°C) and 2 RT-PCR reactions are set up with G l and G2 primers 1

I .-.

RT-PCR product is applied to a 3% agarose gel, electrophoresed at 80V and visualised 1

Gel is soaked in denaturation buffer for 1 hour and soaked in neutralisation buffer for 1 hour

n L

DNA bands of gel are transferred to nylon membrane overnight with Southern blot apparatus (four blots are required for each specirnen)

Membrane is washed in 2xSSC, baked at 80°C for 2 hours, pre-hybridised for 1 hour at 6S°C

n

L

Membrane is hybridised at 48OC overnight, using one of 4 probes: Pl-A, P2-A, Pl-B, P2-B

Membrane is washed to rernove unbound probe, equilibrated in washing buffer, and blocked with a 1-hour incubation in blocking reagent

i i

Membrane is incubated in DIG antiiody for 1 hour, then washed nvice in washing buffer and soaked in NBT/BCIP overnight to develop the colour detection

Figure 2.1 Flow chart explaining the procedure for RT-PCR and Southern blot hybridisation of clinical stool samples from Norwalk-suspected outbreaks

3 Results

The objective of this study was to assess the feasibility of a RT-PCR and Southern

blot hybridisation (SBH) procedure as a rapid, sensitive and specific method for detection

of NLVs fkom stool samples for analysis of NLV-related outbreaks. To this end, the RT-

PCR and SBH method of Ando et al. was adapted for use in a routine diagnostic setting

at the Kingston Public Health Laboratory (Ando et al., 1995).

The visual exarnination of RT-PCR products with ethidiurn bromide (EtBr)

staining and W illumination of the 3% agarose gel following electrophoresis, provided

for screening of NLV infection in this study, whereas SBH and colourimetric detection

allowed for simultaneous confirmation and typing of the RT-PCR products.

Confirmation of the RT-PCR result with SBH is essential for reliabie diagnosis: that is, to

ensure NLV-specific amplification (thus elirninate false positives) and to provide

additional sensitivity for detection of minimal amounts of RT-PCR product undetectable

by the naked eye (thus confirm the negative results). As will be demonstrated later, the

combined RT-PCR and SBH procedures shouId be considered as a single diagnostic test

for routine use in a laboratory in the analysis of clinical material subrnitted from

suspected NLV outbreaks.

Ninety-eight stool specirnens received at the Kuigston Public Health Laboratory

from 25 outbreaks of acute gastroenterïtis were analysed by RT-PCR and Southem blot

hybridisation (SBH). These outbreaks occurred in 23 long-term care facilities and 2

child-care centres in Eastern Ontario between December 1999 and Novernber 2000.

Electron Microscopie exarnination was perfonned at Toronto and Thunder Bay sites.

3.1- RT-PCR of RNA extractions from stool specimens

RT-PCR was performed on RNA extractions fkom stool specimens and detection

was based on direct visualisation of a 123bp amplification product following EtBr

stainins and W illumination; the intensity of the band varied between samples and

ranged fiom very faint to clearly discernible.

A s shown in Table 3.1, 17 of the 25 outbreaks were identified as due to NLVs: 4

(23%) were identified by both RT-PCR and EM, 1 (6%) by EM alone and 12 (71%) by

RT-PCR only. Thus, RT-PCR and/or EM detected NLVs in 68% of outbreaks of acute

gastroenteritis. RT-PCR detection rates within individual outbreaks varied fiom 18% (2

of 1 1 specimens in Outbreak P, Table 6.16) to 83% (5 of 6 specimens in Outbreak W,

Table 6.23). Of the 16 RT-PCR positive outbreaks, 8 were detected with the G 1 set of

primers, 7 with the G2 set of prirners (Table 3.1) and a single outbreak, Outbreak A

(Table 6.1 ), was detected using both G 1 and G2 primers. Eight of the 25 outbreaks

(32%) were found to be negative for the presence of NLVs by both RT-PCR and EM, as

shown in Table 3.1. Taken individually, 34 (35%) of 98 specimens were identified as

NLV positive: 26 (27%) were detected by RT-PCR only, 2 (2%) by EM alone and 6 (6%)

by both mettiods.

3.2 Confirmation of RT-PCR Results using Southern Blot Hybridisation

As reported in Table 3.1, Southern blot hybridisation (SBH) revealed 19 (76%)

NLV positive outbreaks: 15 of the 16 RT-PCR positive outbreaks were confirmed by

SBH and 4 additional outbreaks were detected by SBH. Outbreaks B, C, F and T, which

did not show visible RT-PCR amplicons following EtBr staining, were determined to be

positive upon SBH (including the EM positive, RT-PCR negative Outbreak C, Table 6.3),

whereas 1 RT-PCR positive outbreak, Outbreak P, could not be confirmed by SBH

(Table 6.16). Within NLV-positive outbreaks, SBH detection rates of NLVs in submitted

specimens ranged fkom no detection (O of 11 specimens in Outbreak P, 2 of 1 1 were

NLV-positive by RT-PCR) to 100% detection (6 of 6 specimens in Outbreak W, 5 of 6

were NLV-positive by RT-PCR), as reported in Tables 6.16 and 6.23 respectively. Taken

individually, 19 (30%) RT-PCR negative specimens were found to be positive upon SBH

and 2 RT-PCR positive specirnens could not be confirrned by SBH.

The intensity of the SBH detection did not always reflect the intensity of the EtBr

detection: faint and imperceptible G2 bands visualised by EtBr staining were strongly and

clearly detected by SBH, whereas intense G l bands visualised by EtBr staining were

faintly detected or undetected by SBH.

Of the 19 SBH positive outbreaks, 7 (37%) were detected with the Pl-A probe

suggesting infection with the UK-2 antigenic group, 4 (21 %) were detected with the P 1 -B

probe implicating ihe UK-1 antigenic group, and the remaining 2 (1 1%) were detected by

the PZ-B probe thus involving the UK-3 or UK-4 antigenic groups (Table 3.2).

Furthemore, 3 (1 6%) outbreaks involved mixed P 1 -B and PZ-B hybridisation and 3

(16%) outbreaks involved cross-hybridisation between two or more probes, as seen in

Tables 3.1 and 3.2.

Lane 1 - 50pb rnolecular ruler Lane 5 - G2 specimen N2 Lane 2 - G2 negative control Lane 6 - G2 specimen N3 Lane 3 - G2 positive control Lane 7 - G2 specimen N4 (+) Lane 4 - G2 specimen NI (+) Lane 8 - G2 specimen N5

Figure 3.1 RT-PCR products visualised on 3% agarose gel stained with ethidium bromide. Note the ease with which band size determination may be made by using the 50bp molecular ruler and the variation in clarity of the RT-PCR products.

Lane 1 - 50pb molecular d e r Lane 5 - G2 specimen L2 Lane 2 - G2 negative control Lane 6 - G2 specimen L3 (+) Lane 3 - G2 positive control Lane 7 - G2 specimen 0 1 (+) Lane 4 - G2 specimen L1 (+) Lane 8 - G2 specimen 0 2 (+)

Figure 3.2 RT-PCR products visualised on 3% agarose gel stained with ethidium bromide and corresponding Southern blots hybridised with Pl-B and P2-B probe sets. Note that L1 was not detected by visualisation of the gel following ethidium bromide staining, but was detected by SBH using the Pl-B probe. In addition, L3,Ol and 0 3 were detected by the Pl-B probe set and faint cross-hybridisation was observed with the P2-B probe set.

Table 3.1 Summary of Norwalk Outbreak Data: Electron Microscopy results vs. RT-PCR and Southern Eybridisation

PCR data 1 Genogroup ISBH data1 Probe ;

1/5+ f G l , G 2 1 Z5+ ! l ,Pl-B;2,P2-BI

Institution 1 Location 1 # Specimens

Outbreak A ( Kingston 1 5 1 1 1 l 1

EM data -

/ Outbreak B 1 Kingston 1 5

1 Outbreak E / Ottawa

I l

Outbreak G 1 Pembroke 1 4 Outbreak H j Ottawa 1 2

1 Outbreak C I Vankleek Hill / 2 1 1/2+ 1 - 1 NiA 212 - 1, Pl-B; 2. P2-B

j Outbreak D j Renfrew ! 2 / - 1 112+ G l 1/2 + P 1-A

-

NIA j - NIA I 1 -

l I 1 I

- 1 214+ / G1 i 214+ 1 P 1-A

- 1 - I N I A l - i NIA I r

! Outbreak 1 ! Ottawa 1 2 1 1 l

- / 112 + ; G1 ! 1/2 + 1 P 1-A

! Outbreak I . Ottawa i 3 1 - 1 2 / 3 7 / G1 , 2/3+ . Pl-A

i Outbreak K 1 Perth 1 3 - 213 + 1 G1 1 2/3+ P 1-A

1 Outbreak L / Ottawa 1 3 1 213 + / 113 + 1 G2 , 2/3 -4- P 1 -B. P2-B

- f NIA U 5 + 1,Pl-B;2,P2-B

-

Outbreak M 1 BrockviUe

Outbreak N Belleville

1 1 1 &

1 Outbreak P j Pembroke 1 I l I l + U l l i j G1 : - N/A 1

! Outbreak Q j Merrïckville i 2 1 - 212 + ! GI 212 + Pl-A , L

!

Note: - uidicates a negative result for al1 specimens

1 Outbreak F 1 Gloucester

Outbreak R Ottawa 1 2

OutbreakU 1 Ottawa 1 2 1 - 1 - 1 NIA j - N/A

1 Outbreak O i Gloucester 1 2 1 - 2/2+ 1 G2 : 2/2+ , Pl-B,P2-B

4

1 O

- i 1/2+ G2 2 2 + PI -B

Outbreak V 1 Ottawa 1 3

Outbreak W 1 Toronto 6

Outbreak X 1 Thunder Bay 1 7

2 - 1 - 1 NIA ! 112+ i PI-B

- 1 U 4 + G1 2:4 I Pl-A

Outbreak S / Gloucester 1 2 1 - 1 - 1 N/A N/A

Outbreak Y 1 Thunder Bay 1 6 1 - 1 216 -+ G2 216 + P2-B

-

316 +

- l 3110 + G2 1 9 1 1 0 + / Pl-%,PZ-A, 1

i P2-B

NIA ; 213 + i P 1-B / Outbreak T / Smith's Falls 1 3

117 + 1 317 t 1 G2 i 5/7 + P2-B

-

- 1 -

NIA 1 - hrlA ! I

516 + 1 G2 1 616 + i P 1-B

Table 3.2 Genogroup and Antigenic Groups Associated with Primer and Probe Detection for NLV Outbreaks in Eastern Ontario

1 1 1 1 M-Q 1 (2). Perth, Brockville.

Probe Pl-A

Genogroup G l

P 1-B

P2-A P2-B

Antigenic Group UK2

G2

Mixed P 1-B,

G2 G2

P2-B Cross-hyb

Outbreak D, G, 1, J, K,

UK1

G2 1 UKl,UKj,WK4

PI-B, PZ-B Cross hyb Pl-B, P2-A,

Locations Renfrew, Pembroke, Ottawa

UK1 UK3, UK4

G2

Total: 7 F, R, T, W

Total: 2 A, B, C

G2

MemckviIle Gloucester, Ottawa, Smith's

Total: 4 None x, y

Kingston (2), Vankleek HiIl

U K I W ~ ~

Falls, Toronto None

Thunder Bay (2)

CI(I/UK3/LTK4

Total: 3 L, 0 Ottawa, Gloucester

Total: 2 N

Total: 1 Belleville

3.3 Seasonality of NLV infections in Ontario

Sweys of NLV outbreaks and sporadic cases in Canada, the Netherlands, the

United States, Japan, the United Kingdom, Austraiia, and D e m a s k by Mounts et al.

revealed a marked cold weather seasonality of bZV-associated dEsease (Mounts et al.,

2000). Although the NLV outbreaks in Ontario were examined cfuring a single year, the

seasonal distribution of outbreaks supports the previously observed cold weather

seasonality of NLV infection, as shown in Figure 3.3. Of note is the observance of a

relatively large nurnber of outbreaks of acute gastroenteritis in April 2000; however, only

one of these outbreaks was identified as due to NLV infection in accordance with winter

seasonality.

Seasonal Distribution of Outbreaks of AGE and NLV-confirrned Outbreaks from December 1999 to November 2000 in Ontario, Canada

i OAGE outbreaks : NLV-confirmed outbreaks

Figure 3.3 Seasonal distribution of outbreaks of acute gastroenteritis and NLV- associated outbreaks between December 1999 a n d November 2000 in Ontario, Canada.

4 Discussion

Since the discovery of the Norwalk vims in 1972, the significance of Nonvalk-

like viruses (NLVs) as aetiological agents in outbreaks of non-bacterial, acute

gastroenteritis as well as the public health challenges associated with NLV control, have

become increasingly evident. Although NLV-associated acute gastroenteritis is self-

lirniting and usually resolves without serious complications, the explosive nature of

transmission of these viruses, the significant resultant morbidity, and the lost productivity

and financial burden of outbreak management makes NLV infection an important public

health concern. Moreover, it is estimated that NLVs are responsible for up to 95% of

non-bacterial acute gastroenteritis, resulting in millions of infections world-wide.

Outbreaks of acute gastroenteritis due to NLVs comrnonly occur in institutions,

such as long-term care facilities and child-care centres. Two reports from Australia

exemplifi the role of NLVs in outbreaks of acute gastroenteritis in child-care centres: a

six-month outbreak in an early parenting centre and a two-week outbreak at a child-care

centre were identified as due to NLVs by RT-PCR (Marshall et al., 1997; Ferson et al.,

2000). Person-to-person transmission, resulting in illness of staff and household contacts

of syrnptomatic children, was also observed during the child-care centre outbreak (Ferson

et al., 2000). Similar outbreaks were reported in an infant home in Japan, where NLVs

were detected in 25% of outbreaks of acute gastroenteritis (Nakata et al., 2000). A

Canadian study on paediatric patients with acute gastroenteritis at the Hospital for Sick

Children in Toronto found that NLV idection was implicated in 82% of these patients

(Levett et al., 1996).

Outbreaks of acute gastroenteritis due to NLVs in hospitals and long-tem care

facilities are often prolonged, with hiph attack rates among residents and staff, resulting

in significant morbidity and mortality (Augustin et aL, 1995). For instance, an outbreak

of NLV-associated acute gastroenteritis at a long-term care facility in Maryland, which

was epidemiologically linked to an il1 nurse, resulted in a 51% attack rate, three

hospitalisations and two deaths among 121 residents (Rodrigues et al., 1996). In

addition, reports on Ontario outbreaks of NLV-associated acute gastroenteritis revealed

attack rates of 72%, 50% and 36% in three units of a long-tenn care facility over 29 days

(Augustin et ai., 1995). Furthemore, if one takes into account the resultant staff sick

leave, ward closures, hospitalisation of patients requiring intravenous rehydration therapy

and the institution of outbreak control measures, costs to the health care system escalate

drarnatically. As a result, rapid identification of the aetiological agent(s) must be

attempted in outbreaks of acute gastroenteritis in long-term care facilities so as to

promptly implement control measures and avoid inappropriate antibiotic therapy.

The standard approach currently in use at most clinical diagnostic laboratories for

identification of viral pathogenic agents in stool specimens is electron microscopy (EM)

and/or culture. EM analysis allows for definitive identification of a variety of viral

pathogens, such as enteroviruses, adenovinises, rotavirus, astroviruses and NLVs. Due to

the fact that very few Iaboratories have EM capabilities, the use of this method for rapid

identification in an outbreak situation is problematic, as stool sarnples must be processed

off-site in the majority of cases. This results in a delay in the diagnosis of infectious

agents responsible for acute gastroenteritis outbreaks and affects the implementation of

appropriate public hea1t.h control measures. For Eastern Ontario, EM is perforrned at two

locations, the Central Public Health Laboratory in Toronto and the Thunder Bay Regional

Public Health Laboratory, thus requiring transportation and centralised processing o f

clinical stool specimens. Furthemore, EM is a labour intensive diagnostic tool requinng

highly trained tec hnicians to perform the anal y sis, and as mentioned earlier, is dependent

upon the nurnber of virus particles present per millilitre of specirnen; as, low virus

shedding is often observed in the majority of patients with NLV infections, this greatly

harnpers detection of these vinses by EM (Kapikian et al., 1996).

Though the RT-PCR and Southem hybridisation method for detection of NLVs

has been conducted for researcb purposes at some laboratories in Canada (such as the

Hospital for Sick Children in Toronto and the University of Calgary), this methodology

has not been applied for use in a routine diagnostic laboratory (Levett et uL, 1996). It

was therefore a priority to introduce a rapid, sensitive method with the capability of

identiwng the majority of NLVs fiom clinical stool sarnples subrnitted from suspected

viral outbreaks of acute gastroenteritis to the public health laboratory. The availability of

such a method would allow for a faster and more sensitive diagnosis, which would assist

in the timely irnplementation of outbreak control measures. To achieve this goal, the RT-

PCR and Southem blot hybridisation method developed by Ando et al. for detection of

antigenically distinct NLVs (1 995) was undertaken and adapted for routine use at the

Kingston Public Health Laboratory. Stool specimens received at the laboratory for

analysis of outbreaks of acute gastroenteritis in long-terrn care facilities and child care

centres in Ontario, were tested by RT-PCR and Southem blot hybridisation and the

results were compared with EM performed at the Central and Thunder Bay Regional

Public Health Laboratories. Finally, the ease of implementation of this methodology in a

routine diagnostic setting, the tumaround tirne and cost, were also evaluated.

4.1 The use of the RT-PCR method, with gel eIectrophoresis and ethidium bromide (EtBr) staining, for the detection of NLVs in stool specimens

A total o f 98 clinical specimens fkorn 25 outbreaks of acute gastroententis

received at the Kingston Public Health Laboratory were initially analysed by RT-PCR

followed by Southern blot hybridisation. The outbreaks occurred in long-terrn care

facilities and child-care facilities in Eastern Ontario between November 1999 and

December 2000. Examination of these outbreaks as illustrated in Figure 3.3, revealed a

distinct winter-spring seasonality, as has been reported by other researchers (Mounts et

al., 2000).

Of 25 outbreaks, 16 were identified as due to NLVs by RT-PCR (68%): 4 (1 6%)

were detected by both RT-PCR and EM, and 12 (48%) were detected by RT-PCR only

(Table 3.1). A single outbreak (4%) was identified by EM and was not detected by

visualization of the RT-PCR products following ethidium bromide staining (Table 6.3).

The detection rates varied within each individual outbreak, from 18% (2/11 specimens) to

83% (5/6 specimens) of specimens submitted for a particular outbreak (Figure 3.1).

However, in 5 of the 17 RT-PCREM positive outbreaks (3 1%), NLV diagnosis was

based on detection of the virus in a single sample (Table 3.1). Kapikian has reported that

in general, due to the nature of NLV infections, a single positive specimen is sufficient to

confirm diagnosis if clinical-epidemiological suspicion exists (Kapikian eï al., 1996).

Taken individually, of the 98 specimens, 3 4 (35%) specimens were identified as

NLV positive: 6 (6%) were detected by both RT-PCR and EM, 26 (27%) by RT-PCR

only, and 2 (2%) by EM alone (Table 3.1). TO date, EM has been the gold-standard for

detection of NLVs- However, this anaiysis demonstrates that the reliance on EM

detection for diagnosis of NLV outbreaks would have identified NLVs in only 4 of the 16

RT-PCR positive outbreaks (3 1%) and in one other outbreak (Outbreak C), which though

not detected on EtBr staining of RT-PCR products, was found to be positive by SBH

(TabIe 3.1).

Of the 16 RT-PCR positive outbreaks, 8 (50%) were detected with the G 1 set of

primers, 7 (44%) were detected with the G2 set of primes and one was detected by both

G1 and G2 primers (Table 3.2). These results are particularly interesting because

nurnerous studies have reported that the vast majority of outbreaks (over 90%) in the

United States, the United Kingdom, Australia, Japan and the Netherlands have involved

the G2 genogroup (FanWiauster et al., 1998; Wright et al., 1998; Tnouye et al., 2000;

Vinje and Koopmans, 1996). Furthemore, a longitudinal study conducted by Levett et

al. in Toronto, Ontario reported only a single specimen detected with the G1 primer pair

fi-om 7 1 RT-PCR positive specimens (Levett et al., 1 996). This suggests a variable

epidemiological profile of outbreak-related NLV strains circulating in Ontario.

One specimen from Outbreak A was detected with both the GZ and G2 sets of

primers (Table 6.1). This result may be explained by the presence of a mixed infection

involving both genogroups of NLVs in this particular case. Altematively, RT-PCR

detection of this specimen may have involved cross-priming between the Gl and G2 sets

of primes, which was also observed by Ando et al. during the development of these

prirners. The authors explain the observation of cross-pnming based on the fact that the -

G1 and G2 primer sets use the same primer of negative polarity (SRSV33), and the

positive polarity primers share 10 of 21 nucleotides (nt), including five of the first six nt

at the 3'-end (refer to Table 2.1) (Ando et al., 1995).

Of the 25 outbreaks, 8 (32%) were found to be negative for presence of NLVs by

both RT-PCR and EM (Table 3-1). No other aetiological agent (bacterial, parasitic or

viral) was identified as the cause of these outbreaks of acute gastroenteritis, with the

exception of Outbreak U, which occurred at a day-care centre and was found to be due to

rotavirus. The Fact that the RT-PCR method did not detect rotavirus provides evidence

that the RT-PCR primers are specific for NLVs (Ando et al., 1995). As the other

negative outbreaks invoIved explosive transmission of acute gastroenteritis and cIinical

symptoms typical of NLV infection, it is possible that NLVs were not detected due to a

number of variables. Poor timing of sample collection may result in a stool specimen in

which the NLV concentration is absent or too low for both EM and visible RT-PCR

arnplicon detection. A number of other factors rnay also be at play: some of the NLV

strains may not be recognised by the RT-PCR primer sets, degradation of viral RNA may

have occurred due to improper handling during transportation to the laboratory, or

inhibitors present in the stools may have interfered with the RT-PCR ampIification.

These issues highlight the importance of prompt sample collection early on in the clinical

syndrome, during the short penod when viral shedding is at its highest, as well as the

proper transportation and handling of clinical specimens to minimize virus degradation.

As seen in Table 3.1,4 of the RT-PCR negative outbreaks (where no NLV

amplicons were detected by EtBr staining) were detected as due to NLVs by Southem

blot hybridisation (SBH). These results attest to the importance of this dual detection

approach for NLV diagnosis, due to the possibility of reduced RT-PCR amplification.

4.2 The application of the Southern blot hybridisation (SBH) method for the detection of NLV amplicons

Southern blot hybridisation (SBH) revealed NLV implication in a further 3 more

outbreaks, bringing the total to 20 NLV positive outbreaks: 4 RT-PCR negative outbreaks

were positive following SBH and 1 RT-PCR positive outbreak remained unconfirmed by

SBH (Table 3.1). Taken individually, 19 RT-PCR negative specimens were found to be

positive with SBH and 2 RT-PCR positive specimens were unconfirmed by SBH. The

follow-up of al1 RT-PCR negative specimens by SBH demonstrates the additional

sensitivity provided by this approach for detection of NLVs, 2s compared to the mere

visualisation of RT-PCR products following EtBr staining and UV iIlumination.

Furthermore, these results highlight the importance of SBH for the confirmation of RT-

PCR negative specirnens, as small amounts of RT-PCR product could be imperceptible

with EtBr staining of the gel. This reduced RT-PCR amplification could result kom the

presence of minute quantities of viral RNA in the original extraction. The presence of

RT-PCR inhibitors in the extract, or reduced complementary nucleotides between the

primer sets and the target sequence may also contribute to negative results.

The combination of RT-PCR and SBH also allows for the identification of both .

the genogroup and the antigenic group of the NLV strain(s) implicated in an outbreak.

which may aid in the identification of the source of contamination and the confirmation

of the extent of the outbreak. Of the 19 SBH positive outbreaks, 7 outbreaks were

detected with the P 1 -A probe and thus irnplicating involvement of the UK-2 antigenic

group (Table 3.1, 3.2). The predominance of the Pl-A genotype indicates a possible shift

in epiderniology; for example, the G1 genogroup/P 1 -A geno type was identified in a

single case by Levett et al. in Toronto between 199 1 and 1995 (Levett et al., 1996).

Furthermore, the Gl genogroup/P 1 -A genotype was detected in only 4% of outbreaks in

the United States fiom 1996 to 1997 (Fankhauser et ai., 1998). These results therefore

represent a significant departure fiom the NLVs detected by other investigators.

Four outbreaks were detected with the P 1-B probe suggesting infection with the

UK-1 antigenic group, and two by the P2-B probe, thus involving the UKl, UK-3 or UK-

4 antigenic goups (Table 3.2). Levett et al. reported a predominance of the P2-B

genotype during a longitudinal study in Toronto ftom 199 1 to 1995, but found that the

frequencies of NLV subtypes varied fiom year to year, suggestive of a succession of

NLV subtypes in a population over time (Levett et al., 1996). Only sporadic cases were

detected with the P 1-B probe in the Levett et al. study, thus the predominance of the P 1 -

B genotype within the G2 NLVs detected in this study may indicate a shifl in the

genotype of NLVs currently causing outbreaks in Ontario (Levett et al., 1996).

Furthermore, the P2-A genotype was cornmonly detected by Levett ef aL in NLV

outbreaks, whereas, this genotype was not identified in this study. It is possible that these

results could be explained by the developrnent of irnmunity to a specific genotype

throughout a population and subsequent decline in the prevalence of infection with that

genotype, followed by succession of a different genotype (Levett et al*, 1996).

Altematively, novel NLV strains rnay have ernerged in another region and become

introduced to Ontario via displacement of infected persons, or importation of infected

food products.

In Outbreaks A, B and C, hybridisation occurred with both P 1-B and Pz-B probes

(Table 3.2). These 3 mixed outbreaks involved specimens that were detected with

different probe sets without cross-hybridisation, and rnay have been the result of NLV

infection from more than a single source; this additional information may be vital for the

epidemiological investigation and for the identification of the primary source(s) of the

outbreak.

Three G2 outbreaks involved cross-hybridisation between two or more probes:

Outbreaks L and O involved specimens which were detected with both the P l -B and P2-

B probes and Outbreak N involved specimens which were detected with the P 1-B, P2-B

and P2-A probes (Table 3.1). The intensity of the detection varied and was greater with a

particuIar probe: P 1-B for the first two outbreaks and P2-B for the last outbreak. This

cross-hybridisation was not observed by Ando et al. during the development of the

prirners used in this study, nor did any subsequent research using these probes report this

phenomenon (Ando et al., 1995). However, in a longitudinal study fiom 1996 to 1999,

Lritani et al. (using the Ando et al. primers and probes) reported 7 of 40 outbreaks that

involved cross-hybridisation between P 1 -B and P2-A, and between P 1 -B, P2-A and P2-

B. They also reported the existence of dominance between the probe sets used to detect a

single specimen, which resulted in a more intense colour detection of the specimen

following SBH (Iritani et al., 2000). Although this issue of cross-hybridisation appears to

be a recent phenomenon, the presence of comrnon nucIeotides in the Ando et al. probes

may explain this observation: P2-B shares 13 nucleotides with P2-A, P3-B shares I l

nucleotides with P 1-B, and P 1-B shares 1 1 nucleotides with P3-A (Ando et al., 1995).

Table 4.1 Cornmon nucleotide sequence in G2 NLV probes used in this study (Ando et aL, 1995)

P2-A LJK- 1 Toronto virus SRSV6 1 negative atg tca ggg cct agt cct gt P 1-B UK-1, weakly UK-2 Toronto virus SRSV67 negative aca tct ggt gag aga cct ga

Note: Small Round Srructured Virus (SRSV), identically highlighted nucleotides are common

As reported in Table 6.16, Outbreak P was detected by RT-PCR with the G1

primer and visualisation using EtBr staining and W illumination; however, these results

could not be confirrned with SBH. Although it is possible that this detection may have

been due to non-specific amplification, it is unlikely that non-specific amplification

would produce a 123bp product in both specimens within an outbreak. The fact that

these hybridisation probes were developed in the United States (where only 4% of

outbreaks were due to the G1 genogroup) based on six G 1, CTK-2 strains, allows for the

assurnption that the genetic diversity of the G1 genogroup of NLVs circulating in Ontario

rnay Vary eom that present in the United States. Thus, examination of the sequence

diversity of the G1 strains detected in Ontario may confirm this variation and explain the

non-fünction of the probes.

Furthemore, in the case of Outbreak A (detected with both G1 and G2 primer

sets following RT-PCR, electrophoresis and EtBr staining of the gel), the G2 band was

confirmed following SBH with the P 1 -B and P2-B probes, but the G 1 band was not

confirmed wiîh the P 1-A probe set (Table 6.1). This result may confirm the hypothesis

that this outbreak involved a G2 genogroup NLV, of UK1, UK3 or UK4 antigenic group,

which possessed nucleotide sequences compatible with the G1 primer, resulting in cross-

priming. Altematively, this outbreak may have uivolved both the G1 and G2 genogroup

NLVs from independent sources, but the G1 NLV was not detected by the Pl-A probe

due to a lack of nucleotide compatibility. Since another G1 outbreak, Outbreak P, was

similarly undetected by the P 1-A probe, as previously described, this hypothesis is not

improbable. Sequencing of the RT-PCR products fiom these specimens would clariQ

these issues.

4.3 Specificity and sensitivity of the RT-PCR and Southern blot hybridisation procedures for detection of NLVs in stool specimens

It is essential to assess the specificity and sensitivity of a diagnostic test pnor to

its institution in a clinical laboratory setting. The RT-PCR and SB H pnmers and probes

used in this study were assessed by Ando et al. and were found to be both sensitive and

specific for the detection of NLVs in stool specimens (Ando et ni., 1995, 2000). In this

study, it was not possible to calculate statistically relevant values for the specificity and

sensitivity of this RT-PCR and SBH procedure due to the sma1l sarnple size of

specirnens. Nevertheless, the fact that Outbreak U was found to be NLV-negative by this

RT-PCR and SBH procedure, and was subsequently found to involve rotavirus infection,

is evidence of the specificity of this diagnostic test. Furthemore, EM detected NLVs in

only 5 (20%) of the 25 outbreaks analysed in this study, whereas 20 (80%) NLV-

associated outbreaks were identified by either RT-PCR andior SBH; thus this RT-

PCRISBH procedure is definitely more sensitive when compared to EM.

4.4 Adaptation of the RT-PCR and Southern blot hybridisation procedures for use in a routine diagnostic laboratory

One of the objectives of this study was to assess the effectiveness and

irnplementation of a rapid, sensitive and specific diagnostic method for the detection of

NLVs in stool specimens submitted to the public health laboratories for outbreak

analysis. Following the success with the RT-PCR and SBH procedure on stool

specimens fiom outbreaks of acute gastroenteritis in Ontario, the ease of introduction of

this test for routine analysis and the associated costs were considered.

Incorporation of these procedures into the daily routine of a virol&y laboratory

was effortless since most laboratories already possess the basic necessary equipment for

PCR analysis. Furthemore, these diagnostic methods involved relatively simple

procedures and required limited additional training for the laboratory technologists. In

addition, the RT-PCR and Southern hybndisation methods used for this study were not

labour-intensive nor did they require extensive training for technologists as compared to

EM,

The use of the QIAarnp Viral RNA Mini Kit (QIAGEN, Valencia, CA) for the

extraction of viral RNA fiom stool specimens allowed for simple, reliable and efficient

extraction and required equipment currently present in the regional public health

laboratories. As most public health laboratories do have PCR capabilities, the integration

of this RT-PCR method as a part of routine diagnostics should not pose a problem.

Visual analysis of the RT-PCR products following 3% agarose gel electrophoresis

was facilitated by the use of a 50bp molecular marker, as shown in Figures 3.1 and 3.2.

Thus, visualisation of a band at 123bp and at the same relative position as the positive

control using ethidium bromide (EtBr) staining and UV illumination provided a

preliminary diagnosis. Southem blotting required simple equipment and minimal labour.

and was allowed to proceed overnight, allowing this procedure to be easily integrated into

the routine activities of the laboratory. Similarly, hybndisation with NLV probes

involved minimal labour, simple equipment and was allowed to proceed overnight. The

use of the DIG Nucleic Acid Detection Kit for the colourimetric detection of hybridised

RT-PCR products involved minimal persona1 nsk to laboratory technologists and

required no special equipment for handling of material as would be necessary for

radioactive probes. On the whole, results indicated that the RT-PCR and SBH procedure

could be easily incorporated into a routine diagnostic laboratory.

4.5 Turnaround time for RT-PCR and SBH detection of NLVs in cornparison with Electron Microscopy

The RT-PCR method, using visualisation of RT-PCR amplification products with

EtBr staining and UV illumination, allowed for a preliminary diagnosis for NLV-

suspected outbreaks in approxknately 6 hours, with simultaneous processing of up to 10-

15 stools. However, the timing of the submission of stool specimens to the public health

laboratory would affect this tumaround tirne: due to the working day of the technologists,

samples received at the laboratory in the aftemoon would necessitate RT-PCR

processing, such that the initia1 NLV screen result would be available the next rnoming.

This is a significant improvement when compared with the tirne required for EM

diagnosis, which is dependent upon the transportation t h e required to ship the specirnens

to either of the two public health laboratories at Toronto and Thunder Bay. With a RT-

PCR performed on site, the institution c m expect timely detection of the suspected NLV

agent, such that outbreak controT measures may be implemented as soon as possible.

Confimation of the initial RT-PCR results by SBH would require an additional 4

to 5 days, due to the requirement for long incubations for blotting, hybridisation and

detection. However, the use of vacuum or dot blotting techniques and cherniluminescent

detection of hybridised Southern blots would greatly reduce the time required for

confirmation of the RT-PCR results. However, institution of these techniques would

require the procurement of equipment not currently present at the regional public health

laboratories thus increasing the expense of the test. Furthemore, these techniques may

not be required since the preliminary RT-PCR notification is adequate for the irnrnediate

implementation of NLV outbreak control measures.

As a result of the explosive nature and rapid progression of outbreaks and the time

required for diagnosis, public health officials often face the problem of needing to take

outbreak control rneasures before an aetiological agent can be identified (LeBaron et al.,

1990). This fact highlights the importance of using ch ica l symptoms (such as severe

nausea and vomiting, non-bIoody diarrhoea and abdominal cramps with an incubation

period of 24-48 hours and an average duration of illness of 12-60 hours) as suggestive of

a NLV outbreak, Thus, if an epidemiological investigation indicates a possible NLV

aetiology in an outbreak of acute gastroenteritis, public health officials should

presumptively irnplement outbreak control measures in the absence of a NLV diagnosis.

Furthemore, these public heaith measures rnay be continued until no additional acute

gastroenteritis cases are identified, whether or not the aetiological agent is detected. in

order to minimize the likelihood of prolonged transmission within an institutional setting

and the resultant health challenges.

4.6 Cost considerations for the RT-PCR and SBH method in comparison with Electron Microscopy

The implernentation of a new test in a diagnostic laboratory requires that it be

comparable in cost with standard available routine rnethodologies. The total cost of

reagents for processing a single specimen using the RT-PCR and SBK method was

calculated to be approxirnately $80.00 CDN, not including labour or equipment related

costs, the details of which are listed in Table 6.26 in the Appendix. The inclusion of

multiple specimens (which would be typical for determination of NLV infections in

outbreaks of acute gastroenteritis) would reduce the cost to $12.26 CDN or less per

specimen. Currently, electron rnicroscopic (EM) identification of NLV in stool

specirnens costs approximately $0.85 per specimen, not including equipment costs or

labour. Although the costs of EM appear to be negligible, the significant additional

equiprnent costs of EM must be considered: the purchasing of an electron microscope

wouid necessitate a start-up cost of $175 000 to over $250 000 CDN (depending on the

specifications), with annual maintenance costs of approxirnately $20 000 CDN.

Moreover; as shown in the results, the RT-PCR and SBH method geatly surpasses EM

for the detection oENLVs. Also, the cost for transportation of specimens to Toronto and

Thunder Bay wouId be eliminated. Finally, the rapid and sensitive identification of

NLVs as the causative agents in outbreaks of acute gastroenteritis would eliminate the

costs related to further diagnostic testing for other potential aetiological agents.

4.7 The application of RT-PCR and SBH to epidemiological investigation of NLV outbreaks in Ontario

The establishment of RT-PCR and Southern blot hybridisation as the standard

diagnostic method for NLV detection in outbreaks of acute gastroenteritis at Ontario

public health laboratories, will allow the province to better participate at an international

level in epidemiological investigation and surveillance of NLVs, such as the proposed

"Calicinet7'. The importance of such collaboration is highlighted by the outbreak which

occurred due to contaminated well-water at a restaurant in the Yukon Temtory and

infected travellers from al1 over the globe en-route to Alaska. As international travel

grows and trade in food products expands over continents, collaboration amongst public

health agencies the world over will necessitate the implementation of a variety of

standards, one among them being the use of sensitive and specific diagnostic procedures

for detection of infectious agents (Beller et al., 1997).

4.8 Future Work

Future work should involve examination of the sequence diversity of the NLVs

circulating in Ontario, especially the Gl genogroup. This would provide essential

information for the M e r development of prirners and probes necessary for the detection

of the diverse NLV strains circulated in Ontario. In addition, sequencing of the Ontario

NLV strains may provide an explanation for the lack of hybridisation with the P l -A

probe set in Outbreak P, and rnay provide additional epidemiological information.

Moreover, molecular approaches provide a tool to link small, focal gastroenteritis

outbreaks nationally and intemationally through examination of the sequences of the

infecting strains (Monroe et al., 2000). Such linked outbreaks could be then traced to a

single contaminated source (person, food, water) in which virus of the sarne strain could

be identified to c o n f m the causal link (Monroe et al., 2000). Thus, rnolecular detection

and sequencing could improve surveillance and infection control in Ontario and allow the

Ontario Ministry of Health to participate in national and international surveillance, For

prevention of NLV-associated outbreaks of acute gastroenteritis and epidemiological

study.

Due to the surprising heterogeneity of the NLV genome, it is important to

continually monitor the circulation of NLVs in the general population and to reassess,

refine and design new primers as required, to achieve maximum sensitivity of the RT-

PCR detection method (Caul, 1996). Thus, examination of the genetic diversity of NLVs

circulating in Ontario may allow for Eurther development of RT-PCR primer sets with

improved nucleotide compatibility to the viral genorne, for an increased range of

detection for NLVs.

In addition, Mprovement to the current proposed diagnostic system of RT-PCR

and SBH for routine detection of NLVs may involve acquisition of additional equipment

and minor adaptations of procedures for enhanced rapid diasgosis of larger sample

numbers. For example, the use of vacuum or dot blotting would reduce the time required

for Southem blotting, and the use of cherniluminescent detection of DIG-labelled

hybridisation probes would reduce the tirne required for detection of NLV amplicons.

The development of practical, effective diagnostic tests for NLVs, such as a

broadly-reactive RT-PCR assay or an immunoassay that recognises al1 circulating strains

has been problematic (Green, 2000). Moreover, there remain several serious challenges

to the study of NLVs, including the incredible genetic diversity of NLVs, the need for a

method to grow the virus in vitro or an effective animal model, and the detemination of

the aetiology of currently undiagnosed gastroenteritis and its relation to Ccdiciviriclrre

(Monroe et al., 2000). Nevertheless, the RT-PCR and Southern hybridisation procedure

of Ando et al- has been found to be rapid, simple and sensitive as compared to EM, thus

is acceptable for routine detection of NLVs in stool specimens until such a time as a more

effective detection method is developed.

4.9 Conclusion

Norwalk-like Wuses (NLVs) are an important cause of outbreaks of acute

gastroenteritis in Eastern Ontario. This RT-PCR and Southern blot hybridisation

procedure is appropriate for use in a clinical diagnostic laboratory for detection of NLVs,

as it is simple, rapid and significzmtly more sensitive when compared to EM, thus

allowing for timely patient care and outbreak management. Southern blot hybndisation

is essential to confirrn positive ET-PCR results and to detect amplicons not visible with

ethidiurn bromide staining of the gel, as it increases the sensitivity of the procedure. RT-

PCR combined with Southern blmt hybridisation is usehl for classification of the NLVs

into genogroups, as well as for their Wher differentiation into antigenic types. This is

essential for NLV outbreak analysis, as it allows one to determine whether or not the

spread of infection is in fact fiom a single source. Moreover, the classification of NLV

strains causing outbreaks of acut~e gastroenteritis in Ontario will allow pubIic health

officials to participate in local, national and international epidemiological studies. This

will ultimately clariQ the importance of Norwalk-like viruses in hurnan illness and public

health.

5 References

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6 Appendix: Southem blot apparatus

0.75 kg weight absorbent paper towels

2,3MM Whatman paper

positively-charged nylon membrane gel plastic wrap, cut around membrane 3MM Whatman paper wick platform

tray filled with 20xSSC

Figure 6.1 Southern blot apparatus for transfer of DNA from gel to nylon membrane

6 Appendix: EM and RT-PCR/Southern blot hybridisation analysis of stool specimens from outbreaks of acute gastroenteritis in Eastern Ontario between December 1999 and November 2000

Table 6.1 EM and RT-PCR/Southern blot hybridisation analysis of stool specimens from Outbreak A, Kingston, Ontario

Stool Specirnen Al A2 A3

Table 6.2 EM and RT-PCWSouthern blot hybridisation analysis of stool specimens from Outbreak B, Kingston, Oritario

Date Collected 0711 1/99

A4 A5

1 Stool Specimen 1 Date Collected 1 EM Result 1 RT-PCR Result 1 Hybridisation Detection 1

06/12/99 06/ 12/99

EM Result Neeative

10/12/99 16/12/99

Negative Neeative

BI B2

Table 6.3 EM and RT-PCR/Southern blot hybridisation analysis of stool specimens from Outbreak C, Vankleek Hill, On tario

RT-PCR Result G l and G2 ~ositive

Negative Negative

83 B4 B5

Eybridisation Detection P 1 -B ~ositive

Negative Negative

02/12/99 02/ 12/99

Table 6.4 EM and RT-PCWSouthern blot hybridisation analysis of stool specimens from Outbreak D, Renfrew, Ontario

Negative P2-B ~osit ive

Negative Negative

02/ 1 2/99 021 12/99 13/12/99

-

Stool Specimen Cl C2

L ~ t o o l Specimen 1 Date Collected 1 EM Result 1 RT-PCR Result 1 Hybridisation Detection 1

Negative Negative I

Negative Negative Neg ative Negative Negative

Date Collected 09/12/99 0911 2/99

Negative Negative

D l

Pl-B positive Negative

Negative Negative Negative

EM Result Negative Positive

Negative P2-B positive

Negative

1 9/ 12/99

RT-PCR Result Nega tive Negative

Hybridisation Detection 1 P 1-B positive P2-B positive

Nega tive D2 Negative 1 110 1/00

Nega tive Negative G1 positive P 1-A positive

Table 6.5 EM and RT-PClUSouthern blot hybridisation analysis of stooi specimens from Outbreak E, Ottawa, Ontario

1 E5 20/ 12/99 1 Negative 1 Negative Negative

Stool Specimen El E2 E3 E4

Table 6.6 EM and RT-PClUSouthern blot hybridisation analysis of stool specimens from Outbreak F, Gloucester, Ontario

RT-PCR Result Negative Negative

Date Collected 13/ 12/99 13/12/99

Hybridisation Detection Negative Negative

13/12/99 19/ 12/99

F2 29/12/99 1 Negative ] Negative Negative 1

EM Result Negative Negative

~ t o o l Specimen 1 Date Collected F1 1 29/12/99

Table 6.7 EM and RT-PCRISouthern blot hybridisation analysis of stool specimens from Outbreak Gy Pembroke, Ontario

Negative Negative

EM Result Neeative

Negative Negative

Table 6.8 EM and RT-PCR/Southern blot hybridisation analysis of stool specimens from Outbreak H, Ottawa, Ontario

Negative Negative

RT-PCR Result Negative

Hybridisation Detection P 1 -A ~osit ive

Stool Specimen Cr1 ~2 G3 G4

Hybridisation Detection Pl-B ~osi t ive

Date Collected 05/0 1/00 OSiOl/OO 06/0 1/00 1 6/0 1 /O0

Table 6.9 EM and RT-PCR/Southern blot hybridisation analysis of stool specimens from Outbreak 1, Ottawa, Ontario

EM Result RT-PCR Result Ne~ative 1 G 1 ~osit ive

Hybridisation Detection Negative

H2

Negative Negative Negative

Stool Specimen Hl

10/0 1/00 1 Negative 1 Negative Negative 1

EM Result Negahve

Date Coliected 07/0 1/00

G 1 positive Negative Negative

RT-PCR Result Negative

Hybridisation Detection P 1 -A positive

Negative

P 1 -A positive Negative Negative

RT-PCR Result G1 positive

Negative

EM Result Negative Negative

Stool Specimen 11 12

Date Collected 12/01/00 15/01/00

Table 6.10 EM and RT-PCWSouthern blot hybridisation analysis of stool specimens from Outbreak J, Ottawa, Ontario

Table 6.11 EM and RT-PCWSouthern blot hybridisation analysis of stool specimens from Outbreak K, Perth, Ontario

' Stool Specimen J1 52 53

Table 6.12 EM and RT-PCR/Southern blot hybridisation analysis of stool specimens from Outbreak L, Ottawa, Ontario

Date ColIected 1610 1/00 19/0 1/00 03/02/00

Stool Specimen K1 K2 K3

EM Result Negative Negative Nega tive

Date Collected 13/0 1/00 1410 1/00 1 810 1 /O0

Table 6.13 EM and RT-PCWSouthern blot hybridisation analysis of stool specimens €rom Outbreak M, Brockville, Ontario

Stool Specimen L1 L2

1 L3 2710 1/00 1 Positive 1

RT-PCR Result Negative Gl positive G1 positive

EM Result Negative Negative Negative

Date Collected 2710 1/00 2710 1/00

EM Result Positive Neeative

G2 positive

M 4 07/02/00 1 Negative ( G1 positive P 1 -A positive 1

Hybridisation Detection 1 Negative

P 1 -A positive P 1 -A positive

P 1-B, P2-B positive

-stool Specimen Ml M2 M3

RT-PCR Result G1 positive G1 positive Negative

RT-PCR Result Negative Nenative

Hybridisation Detection ' Pl-A positive Pl-A positive

Negative

Hybridisation Detection Pl-B positive

Neeative

Date Collected 02/02/00 02/02/00 03/02/00

EM Result Negative Negative Neeative

RT-PCR Result Gl positive

Negative Nenative

- - --

Hybridisation ~ e t e c t i o n l P 1 -A positive

Negative Negative

Table 6.14 EM and RT-PCRISouthern blot hybridisation analysis of stool specirnens from Outbreak N, Belleville, Ontario

Stool Specirnen N1 N2 N3 N4

Table 6.15 EM and RT-PCWSouthern blot hytridisation analysis of stool specimens from Outbreak O, Gloucester, Ontario

N7 N8 N9 NI0

Date Collected 1 1 /02/00 11/02/00 1 1/02/00 1 1/02/00

Table 6.16 EM and RT-PCWSouthern blot hybridisation analysis of stool

13/02/00 14/02/00 1 5/02/00 12/03/00

specimens from Outbreak P, Pembroke, Ontario

EM Result Nega tive Negative Negative Negative

N5 N6

Hybridisation Detection ' P 143, P2-B positive PI-B, P2-B positive

Negative Negative

1 1/02/00 13/02/00

Nega tive Negative Negative

Stool Specimen 0 1 0 2

RT-PCR Result G2 positive

Negative Negative

G2 positive

Date CoIlected 22/02/00 22/02/00

EM Result Negative Negative

P6

Table 6.17 EM and RT-PCWSouthern blot hybridisation analysis of stool specimens from Outbreak Q, Merrickvitle, Ontario

Hybridisation Detection P 1-B, P2-B, P2-A positive

P l -B. P2-B positive Negative

P 1 -B, P2-B, PZ-A positive Negative Negative Negative Negative

G2 positive

RT-PCR Result G2 positive G2 positive

P8 P9 Pl0 Pl 1

Pl-B, P2-B, P2-A positive Pl-B positive

- -

PI-B positive P2-B positive

P 1-B, P2-B, P2-A positive Negative 1 Negative

P7 O 1/03/00 / Negative Negative Negative i

O 1 /03/00 1 Negative

Pz-B positive

13/03/00 1 Negative 13/03/00 [ Positive 13/03/00 1 Negative 13/03/00 1 Negative

Negative

Hybridisation Detection Pl-A positive P 1 -A positive

Negative

Negative G1 positive

Negative Negative

RT-PCR Result GI positive G1 positive

Negative Negative Negative Negative

EM Result Negative

Stool Specimen Ql

Date Collected 16/02/00

4 2 17/02/00 1 Negative

Table 6.18 EM and RT-PCR/Southern biot hybridisation analysis of stool specimens from Outbreak R, Ottawa, Ontario

Table 6.19 EM and RT-PCWSouthern blot hybridisation analysis of stool specimens from Outbreak S, Gloucester, Ontario

Stool Specimen Rl R2

TabIe 6.20 EM and RT-PCWSouthern blot hybridisation analysis of stool specimens from Outbreak T, Smith's Falls, Ontario

Date Collected 23/03/00 24/03/00

EM Result Negative Negative

Hybridisation Detection Neaative Negative

RT-PCR Result G2 positive Negative

EM Result 1 RT-PCR Result Negative 1 Negative Negative 1 Negative

Stool Specimen Si S2

Table 6.21 EM and RT-PCWSouthern blot hybridisation anaIysis of stool specimens from Outbreak U, Ottawa, Ontario

Hybridisation Detection P I -B positive P 1 -B positive

Date Collected 03/04/00 05/04/00

1 T3

EM Resutt Negative Negative

RT-PCR Result Negative Negative

Stool Specimen TI T2

1 7/04/00 ] Negative 1 Pl-B positive 1 Negative

Table 6.22 EM and RT-PCR/Southern blot hybridisation analysis of stool specimens from Outbreak V, Ottawa, Ontario

Hybridisation Detection Negative

Pl-B positive

Date Collected 1 1/04/00 17/04/00

Stool Specimen U l U2

1 Stool Specimen 1 Date Collected 1 EM Result 1 RT-PCR Result 1 Hybridisation Detection

1 VI. 1 12/04/00 ( Negative 1 Negative Negative I

Date Collected 17/04/00 17/04/00

EM Result Neaative Negative

-

V2

RT-PCR Result 1 Hybridisation Detection

25/04/00 Negative

Negative

I V3 26/04/00 / hregative

Negative

Negative Negative

Negative Negative

Negative Negative

Table 6.23 EM and RT-PCIUSouthern blot hybridisation analysis of stool specimens from Outbreak W, Toronto, Ontario

W6 03/05/00 1 Positive 1 G2 positive P 1-B positive 1

Stool Specimen W1 W2 W3 W4 WS

Table 6.24 EM and RT-PCRISouthern blot hybridisation analysis of stool specimens from Outbreak X, Thunder Bay, Ontario

Date Collected 03/05/00 03/05/00 03/05/00

EM Result Negative Positive Neeative

03/05/00 03/05/00

RT-PCR Result G2 positive G2 positive Neeative

Stoot Specimen X1

Hybridisation Detection P 1 -B positive P 1 -B positive Pl-B oositive

Negative Positive

X2 X3 X4

Table 6.25 EM and RT-PCR/Southern blot hybridisation analysis of stool specimens from Outbreak Y, Thunder Bay, Ontario

t

Date Collected ?/11/00

X5 X6 X7

G2 positive G2 ~osi t ive

?/11/00 ?/ 1 1 /O0 ?/11/00

- P 1 -B positive P 1 -B ~osit ive

EM Result Negative

?/11/00 ?/11/00 ?Il 1/00

- S ~ O I Specimen YI Y2 Y3 Y4

Negative Positive Neeative

Y5 Y6

RT-PCR Result Negative

Negative Negative Ne~ative

Date Collected ?/l 1/00 ?/11/00 ?Il 1/00 ?/11/00

Hybridisation Detection Negative

Negative G2 positive Negative

?/l 1/00 ?/l 1/00

Negative P2-B positive P2-B ~ositive

Negative G2 positive G2 ~osi t ive

EM Result Negative Negative Negative Neeative

P2-B positive P2-B positive P2-B ~ositive

Negative Negative

RT-PCR Result G2 positive Negative

G2 positive Negative

Hybridisation Detection P2-B positive

Negative P2-B positive

Neeative Negative Negative

Negative Negative

Appendix: Cost analysis of RT-PCR and Southern blot hybridisation procedure reagents

Table 6-26 Cost per reaction ($CDN) of components irsed in RT-PCR method in a laboratory witb PCR capabilities and not including labour.

1 Sterile tubes 1 0.34

b

Cornponent

MILLEX@-GP 0.22um filter

Cost per Reaction ($CDN)

6.99

QUIamp Viral RNA Mini Kit (QIAGEN) Eppendorps (0SmlL and 1 SmL)

Ready-to-go RT-PCR beads (Arnersham Pharmacia B io tec h) Ando et al. NLV primers

Ando et al. NLV hybridisation probes pro ces sin^ of controls

3 -44 0-33 7.67 0.1 1 OSO* 3 7.24*

Molecular grade agarose (Gibco BL) TBE buffer

2.16* 0.67*

Ethïdium bromide 0.02* 50bp ruler (Arnersham f harmacia Biotech)

Hybond-N+ positively charged nucleic acid transfer membrane Whatman 3 filter paper

DIG Oligonucleotide 3'-End Labelling Kit DIG Wash and Block Buffer Set DIG Nucleic Acid Detection Kit

sarnples were processed at a tirne.

4.12* 1.92* 0.52* 3.17" 1 .go* 9.00*

Absolute ethanol TOTAL

0.06* 80.3 Olspecimen

Note: * Cost for one sample m per gel, the cost would be substantially lower if multiple