CHARACTERIZATION OF AN UNUSUAL ADENOVIRUS STRAIN, SIBU97

DAVID PERERA

A thesis submitted

in fulfillment of the requirements for the degree of Doctor of Philosophy

INSTITUTE OF HEALTH AND COMMUNITY MEDICINE

UNIVERSITI MALAYSIA SARAWAK

2005

Abstract

An adenovirus isolate, SIBU97, associated with an unexplained cluster of deaths of

children with fatal acute myocardial ysfunction in Sarawak. was characterized and

compared with other human adenovirusesSequence comparisons with exenBank of portions

of the SIBU97 genome that include the El, E2, and E3 transcript regions showed that it

shared >94% identity to human species B adenoviruses and simian adenovirus type 21. A

comparison of restriction digest profiles of SIBU97 with prototype species B adenoviruses

and several European Ad21 variants revealed profiles generated with Bam H1, Sma 1 and

Kpn 1 that differed from the other adenoviruses but were closely related to prototype Ad21.

Analysis of the VA RNA showed that SIBU97 is a species B1 adenovirus and different from

other prototype human adenoviruses. Phylogenetic analysis of the hexon and fiber genes

suggest that SIBU97 is closely related to prototype Ad21 with a 99.1% and 98.9% DNA

identity to both these genes respectively. Differences between SIBU97 and prototype Ad21

occurred in 5 of 7 hypervariable regions in the hexon gene and this accounted for 73% of the

differences observed between the two viruses within this gene. A comparison of the E3

region of SIBU97, prototype Ad21 and several European Ad21 variants showed that

SIBU97 is closely related but different from these viruses.

The amino acid sequence of the fiber knob of SIBU97 revealed a unique AATSSK

motif that differentiated it from prototype Ad21. This motif was also shown to overlap with

a previously identified receptor binding region on the Ad5 fiber knob. A predicted structure

of the SIBU97 fiber knob, modeled after a published structure of the Ada fiber knob,

localized this motif to the putative EG loop of the SIBU97 knob structure. Recombinant

fiber knob of SIBU97 was successfully cloned and purified and used to screen human heart

i

and brain cDNA libraries using a commercial T7 phage display system. Ten fiber-

interacting phage clones including 7 clones from the brain cDNA library and 3 clones from

the heart cDNA library were sequenced. Sequence data of the cDNA revealed three different

molecules that interacted with the SIBU97 fiber knob: (1) A nucleolar-related kinase

protein, (2) Microtubule-associated protein 4 (MAP4) and (3) Nuclear phosphoprotein,

Nopp 140. These proteins suggest a intracellular trafficking and nuclear targeting role for

the SIBU97 fiber. Additionally, Nopp 140 could potentially serve as a cell surface receptor

for SIBU97. These findings suggest that SIBU97 is a species BI adenovirus and could

potentially represent a new variant of Ad21. Screening of fiber-interacting proteins suggest

that besides serving as the primary attachment molecule during virus infection, this

molecule may also be active in several intracellular processes.

ii

Abstrak

Isolat adenovirus, SIBU97, yang mempunyai kaitan dengan punca kematian yang tidak

dikenalpasti sekelompok kanak-kanak yang mengidap penyakit akut miokardial disfungsi di Sarawak telah

dicirikan dan dibanding dengan adenovirus manusia yang lain. Perbandingan jujukan dengan GenBank

mendapati sebahagian genom SIBU97 termasuk transkrip El, E2 dan E3 menunjukkan Tanya mempunyai

>94% persamaan dengan adenovirus manusia spesies B dan adenovirus type 21 simian. Perbandingan

profil `restriction digest' SIBU97 dengan prototaip adenovirus spesies B dan beberapa varian Ad21 dari

Eropah menunjukkan profil SIB U97 yang dihasilkan dengan Bam HI, Sma 1 dan Kpn 1 berbeza dengan

adenovirus yang lain tetapi mempunyai persamaan yang rapat dengan prototaip Ad21. Analisa VA RNA

menunjukkan yang SIBU97 ialah adenovirus spesies B1 clan berbeza dari protolaip adenovirus manusia

yang lain. Analisis filogenetik dari gen hexon dan gen fiber mencadangkan yang SIBU97 mempunyai

persamaan yang rapat dengan prolotaip Ad21 clan mempunyai identiti sebanyak 99. 1% dun 98.9% duri

segi persamaan DNA untuk gen hexon dan fiber musing-musing. Perbezaan diantaru SIBU97 dan

prototaip Ad21 berlaku pada 5 dari 7 kawasan hipervariasi di gen hexon dan ini merupakun perbezaan

sebanyak 73% diuntara keduu virus tersebut di gen ini. Perbandingan kawasan E3 dari SIBU97,

prototaip Ad21 dan beberapa varian Ad21 duri Eropah menunjukkan yang SIBU97 mempunvai

persamaan yang rapat tetapi berbeza dari virus-virus tersebut.

Jujukan asid amino dari knob fiber SIB U97 mendapali adanya satu motif yang unik iailu AATSSK

yang membezakannya dengan prototaip Ad21. Mot if ini juga didapati beradu pada kawasan knob fiber di

mana penglibatan dengan receptor pengikat Ads yang sudah diterbitkan. Struktur ramalan knob fiber

SIBU97, selepas dibandingkan dengan struktur knob fiber Ada yang telah diterbitkan, menempaikan motif

ini di loop EG di struktur knob SIBU97. Rekombinan knob fiber SIBU97 lelah beijaya diklon, ditulenkan

dan diguna untuk mengimbas dua perpustakaan cDNA Juni jantung dan otak manusiu rnenggunakan

system 'T7 phage display' yang dikomersilkan. Sebunyak 10 klon faj yang berinteraksi dengan fiber

iii

SIB U9 7 termasuk 7 klon dari perpustakaan cDNA otak dan 3 klon dari perpustakaan cDNA jantung telah

dijujukkan. Data dari jujukan ini menunjukkan adanya 3 molekul yang berbeza yang berinteraksi dengan

knob fiber SIB U97: (1) Nukleolar yang berkait dengan protein kinase, (2) Mikrotubul yang ada hubungan

dengan `Microtubule-associated protein 4' (MAP4) dan (3) Nuklear fosfoprotein, Nopp140. Kesemua

protein ini mencadangkan adanya peranan `intracellular trafficking' dan `nuclear targeting' bagi fiber

SIBU97. Seterusnya Nopp140 mungkin berpotensi bertindak sebagai receptor untuk SIBU97 pada

permukaaan sel. Semua penemuan ini mencadangkan yang SIBU97 adalah adenovirus spesies BI yang

mungkin berpotensi mewakili varian barn dari Ad21. Pengimbasan protein : fiber-interacting'

mencadangkan iaitu disamping bertindak sebagai molekul utama dalam pelekatan primer semasa infksi

virus, molekul inijuga mungkin aktifdidalam beberapa proses intraselular.

iv

Acknowledgements

Firstly, a big `Thank You' to my supervisor Prof. Jane Cardosa. She has been and

continues to be instrumental in guiding both my academic and mental formation here in

UNIMAS. During the many years here, she has been like a second `mother' to all of us in the lab

with her concern not only for our academic success but for our general well being. A huge thank

you also goes to Phaik Hooi who has shown great support in the completion of this thesis. She

has offered unselfish help in so many ways that it would be impossible to mention them all.

However, her gift of a computer deserves special mention and 1 will always be grateful. To other

senior staff members past and present namely Dr. Sim, Dr. Donald, and Dr. Charley, thank you

for all your advice and support. To all other members of the institute, students, support staff and

office staff, of whom there are too many to mention, thank you for all you help. I feel that we

have such a fun bunch of people at the institute and this has made it a pleasure to be a part of this

fantastic team. Thanks also to UNIMAS who have been patient and supportive of my endeavors

here. Finally, to my wife Catherine, this as much a success for her as it is for rne.

This research was funded in part by the National Science Foundation Scholarship from

the Ministry of Science, Technology and Innovation (MOSTI) and various research grants (IRPA

and National Biotechnology Directorate).

V

TABLE OF CONTENTS

Abstract

Abstrak

Acknowledgements

Table of contents

List of figures

List of tables

Chapter 1. Literature review.

1.1 The Sarawak 1997 hand, foot, and mouth disease epidemic

1.2 An overview of adenoviruses

1.2.1 Morphology and biophysical properties

1.2.2 Classification

1.2.3 Clinical syndromes

1.2.4 Genome organization and replication

1.3 Adenovirus receptors and viral tropism

1.4 Adenovirus entry into host cells

1.5 Laboratory detection of adenoviruses

1.5.1 Direct detection

1.5.1.1 Electron microscopy and immunoelectron microscopy

1.5.1.2 Antigen detection

1.5.1.3 Polymerase chain reaction (PCR)

1.5.2 Indirect examination (cell culture)

1.5.3 Serology

i

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I

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4

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7

II

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vi

1.6 Adenoviruses as vectors 21

1.6.1 First-generation vectors 21

1.6.2 Second- and third-generation vectors 22

1.7 Application of adenovirus vectors 23

1.7.1 Gene therapy 23

Chapter 2. Statement of the problem. 25

Chapter 3 Materials and Methods 27

3.1 General methods 27

11.1 Virus specimens and culture 27

3.1.2 Nucleic acid extraction 27

3.1.3 Primers and PCR 28

3.1.4 Purification of DNA fragments from agarose gels 29

3.1.5 Cloning 30

3.1.5.1 Preparation of inserts 30

3.1.5.2 Cloning with the Zero Blunt PCR Cloning Kit (Invitrogen Carlsbad, CA, USA) 31

3.1.5.3 Cloning into other plasmid vectors 31

3.1.5.4 Screening for inserts 33

3.1.6 Software 34

3.2 Cloning the SIBU97 genome 34

3.2.1 Purification of virus particles 34

3.2.2 Extraction of viral DNA 35

3.2.3 Cloning 36

3.2.4 Sequencing and analysis 36

3.3 Analysis of the VA RNA gene region 36

vii

3.3.1 Virus specimens

3.3.2 PCR and primers

3.3.3 TaqI restriction digest profiles

3.3.4 Electrophoresis

3.4 Restriction digest profiles of adenovirus genomes

3.4.1 Nucleic acid extraction

3.4.2 Restriction enzyme digestion

3.4.3 Electrophoresis

3.5 Detection of species B adenoviruses by PCR

3.5.1 Virus specimens

3.5.2 Extraction of viral DNA

3.5.3 Primer design and PCR of hexon gene flanks

3.5.4 PCR and sequencing of complete SIBU97 hexon gene

3.5.5 Cloning and sequencing hexon gene flanks

3.5.6 Comparison of PCR assays

3.5.7 Additional GenBank sequences for hexon phylogenetic analysis

3.6 Analysis of the fiber gene

3.6.1 Cloning and sequencing the fiber genes of adenovirus isolates

3.6.2 Cloning a truncated fiber gene of SIBU97

3.6.3 Expression of truncated fiber protein

3.6.4 SDS-PAGE and Western blot

3.6.5 Large scale preparation of truncated fiber protein

3.6.6 Screening for the SIBU97 fiber-binding proteins

3.6.6.1 Amplification of the human cDNA libraries

36

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viii

3.6.6.2 Titering the amplified lysate

3.6.6.3 Biopanning

3.6.6.4 PCR amplification of bound phage

3.6.6.5 Picking individual plaques for screening

3.6.6.6 Detection of bound phage by ELISA

3.6.6.7 Selection of bound phage

3.6.7 Additional GenBank sequences for fiber phylogenetic analysis

3.7 Analysis of the E3 region

3.7.1 PCR amplification and sequencing of the E3 region

49

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53

Chapter 4. Results and discussion: Identification and characterization of SIBU97 56

4.1 Cloning and sequencing the SIBU97 genome 56

4. 1.1 Preparation of viral DNA 56

4.1.2 Cloning the genome of SIBU97 56

4.1.3 Sequencing and comparison of SIBU97 sequence to other adenoviruses in GenBank 58

4.2 Analysis of the VA RNA gene region 61

4.2.1 VA RNA PCR of all species B adenoviruses and comparison with local adenovirus

isolates 63

4.2.2 TagI profiling of the VA RNA PCR amplicon 64

4.3 Restriction digest profiles of SIBU97 and comparisons with prototype species B adenoviruses

and other clinical isolates 64

4.3.1 Digestion with Smal 66

4.3.2 Digestion with BamHl 70

4.3.3 Digestion with Kpnl 73

4.4 Analysis of the hexon gene. 73

ix

4.4.1 Phylogenetic analysis of complete hexon gene sequence 73

4.4.2 Analysis of the hypervariable regions in the hexon protein of species B adenoviruses 75

4.5 Discussion 77

4.5.1 The genome of SIBU97 is similar to human adenovirus species B genomes 77

4.5.2 SIBU97 is a member of species B I human adenoviruses 79

4.5.3 SIBU97 is similar to Ad21 80

4.5.4 SIBU97 is possibly an Ad21 variant 82

Chapter 5. Results and discussion: Further characterization of SIBU97: Fiber and E3

region. 84

5.1 Analysis of the fiber gene 84

5.1.1 Phylogenetic analysis of the SIBU97 fiber gene and other human adenovirus fiber genes 84

5.1.2 Amino acid alignment of the fiber knob region 86

5.1.3 Predicted structure of the SIBU97 fiber knob 86

5.2 Analysis of the E3 gene region 89

5.2.1 Sequence analysis of the E3 gene region 89

5.2.2 Phylogenetic analysis of the E3 gene region 92

5.3 Discussion 92

Chapter 6. Results and discussion: Detection of species B adenoviruses by PCR. 97

6.1 Detection of Species B adenoviruses by PCR 97

6.1.1 PCR of the left and right ends of the hexon gene 97

6.1.2 Analysis of the primer binding site 98

6.1.3 Construction of primers for a novel nested PCR and comparison of PCR primer pairs for

species B adenovirus detection 100

6.1.4 Utility of the modified nested PCR 105

X

6.2 Discussion 110

Chapter 7. Results and discussion: Bio-panning for the cellular receptor of SIBU97. 112

7.1 Preparation of recombinant fiber protein of SIBU97 112

7.2 Biopanning for proteins that bind the fiber of SIBU97 116

7.2.1 Screening of phage displayed peptides expressed from human brain and heart cDNA

libraries 116

7.2.2 Sequencing analysis of selected phage clones that bound truncated recombinant fiber

protein of SIBU97 118

7.3 Discussion 119

7.3.1 Nopp140 is a potential receptor for the fiber of SIBU97 123

7.3.2 The fiber of SIBU97 is involved in virus trafficking in the host cell 124

7.3.3 The fiber knob of SIBU97 may interact with nuclearpore-interacting protein motifs 127

Chapter 8. General discussion and conclusion. 129

8.1 Adenoviruses from the Sarawak 1997 outbreak are Ad21 variants 129

8.2 Adenovirus detection 130

8.3 Cardiac tropism of adenoviruses 132

8.4 Adenovirus pathogenesis 134

8.5 Conclusion 136

References 137

Appendices

List of publications generated from the 1997 Sarawak outbreak

X1

LIST OF FIGURES

Figure 1.1 Structure of adenovirus. 3

Figure 1.2 Gene layout and transcription of the Mastadenovirus genus. 8

Figure 1.3 Characteristics of E3 and E4 gene products. 12

Figure 4.1 SIBU97 viral DNA extracted from purified virus particles. 57

Figure 4.2 Cloned Bam HI fragments of the SIBU97 genome. 59

Figure 4.3 Schematic gene organization of different cloned SIBU97 genome fragments. 60

Figure 4.4 Separation of Ad VA RNA PCR products by agarose gel electrophoresis. 65

Figure 4.5 Taq I digestion profiles of the larger VA RNA PCR products. 67

Figure 4.6 Sma I digestion of prototype species B adenoviruses and other clinical adenovirus

isolates. 68

Figure 4.7 Adenovirus Sma I digestions separated on 2% agarose gels. 69

Figure 4.8 Baml digestion of prototype species B adenoviruses and other clinical adenovirus

isolates. 71

Figure 4.9 Adenovirus Bam HI digestions separated on 2% agarose gels. 72

Figure 4.10 Kpn I digestion of prototype species B adenoviruses and other clinical adenovirus

isolates. 74

Figure 4.11 Phylogenetic analysis of complete hexon gene sequences of human adenoviruses. 76

Figure 4.12 Alignment of partial hexon amino acid sequence of adenoviruses serotypes

2,3,4,7,16, 21, 34, 35 and local adenovirus isolate, SIBU97. 78

Figure 5.1 Phylogenetic analysis of human adenovirus fiber genes. 85

Figure 5.2 Amino acid alignment of the human adenovirus knob region. 87

Figure 5.3 Structure of a monomeric fiber knob of Ad5 and a similar predicted structure for

xii

SIBU97. 88

Figure 5.4 Phylogenetic analysis of the E3 gene region. 91

Figure 6.1 PCR amplification of the left and right ends of the hexon gene for species B

adenoviruses. 99

Figure 6.2 Alignment of left end region of hexon gene sequences. 101

Figure 6.3 Alignment of right end region of hexon gene sequences. 102

Figure 6.4 PCR amplification of primary clinical material using the modified Cardosa nested-

PCR primer sets. 109

Figure 7.1 PCR amplification of truncated fiber gene of SIBU97 and cloning into pET30a

expression vector. 113

Figure 7.2 Expression of recombinant fusion protein of truncated fiber of SIBU97. 114

Figure 7.3 Purification of truncated fiber protein of SIBU97. 115

Figure 7.4 SDS-PAGE and western blot analysis of purified truncated fiber protein of SIBU97. 117

Figure 7.5 Monitoring biopanning of human brain and heart cDNA libraries by PCR. 120

Xlll

LIST OF TABLES

Table 1.1 Properties of human adenovirus serotypes of species A to F 6

Table 3.1 List of primers for PCR and sequencing of the E3 region 55

Table 4.1 Comparison of cloned SIBU97 genome sequence with sequences deposited in GenBank 62

Table 5.1 Comparison of SIBU97 E3 genes with several adenovirus isolates and species B

prototype strains. 90

Table 6.1 Number of mismatches in primers used in various PCR assays 104

Table 6.2 Detection limits for various PCR assays 106

Table 6.3 Numbers of mismatches between primers examined in this study and their respective

binding sites on the hexon genes of selected adenovirus prototypes 107

Table 7.1 Representive ELISA screening results for phage clones from SIBU97 fiber biopanning

experiments. 121

Table 7.2 BLAST search results of human cDNA expressing phage clones that bound truncated

recombinant fiber protein of SIBU97. 122

xiv

Chapter 1. Literature review.

1.1 The Sarawak 1997 hand, foot, and mouth disease epidemic

Sarawak is one of the states that form Malaysia and is situated on the island of

Borneo that also includes the Malaysian state of Sabah and the Indonesian province of

Kalimantan. In mid-1997, the state experienced a large outbreak of hand, foot, and

mouth disease (HFMD) that has since been shown to be caused by viral strains of sub-

genogroup B3 human enterovirus type 71 (HEV71) (McMinn et a]., 2001). Infection by

the virus is often asymptomatic or may manifest as a mild self-limiting illness often

characterized by the presence of lesions on the palms, soles, and oral mucosa (Minor et

al., 2000). However, HEV71-associated severe neurological complications have also been

reported (Lum et a]., 1998; Komatsu et al., 1999; McMinn et al., 2001; Fujimoto et a].,

2002).

The 1997 outbreak in Sarawak was associated with an unexplained cluster of

deaths of children presenting with fatal acute myocardial dysfunction and an unexpected

increase in children with acute flaccid paralysis (AFP) (Cardosa et al., 1999). A total of

34 child deaths attributed to this syndrome were recorded over a period of 4-5 months.

Although the HFMD outbreak was clearly caused by HEV71, obtaining a consensus on

the aetiology of cases involving paralysis and fatal cardiac outcomes has proven difficult

(AbuBakar eta]., 2002; Lum eta]., 2002).

Laboratory investigations on 20 of the recorded 34 children who died and 8

surviving children with AFP detected an adenovirus in ten of the children who died and

five of the AFP cases (Cardosa et al, 1999). In the same study, enteroviruses (echovirus

type 25 in one case and HEV71 in two cases) were detected in three of the children who

1

died but none in the AFP cases tested. All the three enterovirus positive cases also had

an adenovirus. All the AFP samples were submitted to the poliovirus surveillance

laboratory at the Institute for Medical Research in Kuala Lumpur but no poliovirus

infection was reported. Initial investigations of the adenovirus genome sequence

obtained by PCR from some of the cases showed a strong homology (>80%) to species B

adenovirus sequences deposited in GenBank. A similar untypable adenovirus isolated

from a child with AFP in Singapore also produced almost identical adenovirus sequence

from its PCR product. The Singaporean adenovirus was neutralized by antisera to Adll,

Ad34, and Ad35, all species B adenoviruses. Separate neutralization experiments on the

Malaysian adenovirus isolates done later showed that the Malaysian adenovirus isolates

were neutralized by antisera to Ad21, Ad50, and Adl l (Ooi eta]., 2003).

The association of a species B adenovirus with child deaths and AFP during an

outbreak of HEV71 HFMD raised uncertainties about the aetiological agent responsible

for these deaths during the Sarawak 1997 outbreak. In an effort to further understand

this adenovirus, a study to characterize the virus was undertaken and this forms the

subject matter for this Ph. D thesis.

1.2 An overview of adenoviruses

1.2.1 Morphology and biophysical properties

Adenoviruses have a characteristic morphology (Stewart et a]., 1993), with an

icosahedral capsid with a diameter of 70-90 nm. The virion is composed of a number of

different structural proteins. These include three major proteins, hexon (II), penton base

(III), and a knobbed fiber (IV) along with several minor proteins, VI, VIII, IX, IIla, and

2

ý, Igk"w*A.

. 1

: s:

4

w

S " .

. .. .. ý ýý

_" r1 " ý "" ý

ýp ý

Ilk ý, �ý,. Iý"

ý

( " , ! , " t: ins Corc pn4cins C'emcnl pn4cins

VIII on ý IX

i . ýý " VII ý Illo

Pýnt,. n IIýý: u \I� 7 ý 1

Figure 1.1. Structure of adenovirus. "The locations of the capsid and cement components are reasonably well defined. In contrast, the disposition of the core components and the virus DNA is largely conjectural. Reproduced from Fig. I (Russell, 2000).

. 1

3

1Va2 (Figure 1.1) The virus capsid is formed by 2: 52 capsomers from which 210

capsomers (the herons) are symmetrically arranged so that each is surrounded by six

other capsomers. The icosahedral arrangements of the c: ipsid proteins produce a virion

with 20 triangular faces and 12 vertices. The fiber extends from the 12 vertices of the

virion structure. The vertex capsomers, that include the Penton base together with their

fiber projections, are called pentons. Hatt fiber consists of a tail region that anchors it to

the vertex capsomer, a shaft of variable length that protrudes from the capsid surface

and a terminal knob (Hierholzer, 1989). The virus genome is a linear, double-stranded

DNA with a terminal protein ('1'P) attached covalently to the 5' termini (Rekosh of : it,

1977), which have inverted terminal repeats. rI'l1e virus I)NA approximately between : i: 11-

45 kb pairs, is intimately associated with the highly basic protein VII and a small

peptide termed mu (Anderson il nl., 1989). Another protein, V, is packaged with this

DNA-protein complex and appears to provide a structural linli to the capsid via protein

VI (Matthews and Russell, 1995). The virus also contains a virus-encoded protease

(Webster et a!., 1989), which is necessary for processing of some of the structund

proteins to produce mature infectious virus.

1.2.2 Classification

'T'ile taxononnical classification of adenoviruses are ; overned by the

international Committee on 'hasonoºm' of, Viru. ýcs (I(TV). Recent aamendnients invulviii

adenoviruses are included in 7th' report of the IC1'V rind can be accessed from thee w(A)

address http: //www. ncbi. nlm. nih. gov/I("I'V.

Human adenoviruses n iv members of the family Adenoviridae. This loinnly

includes the genera, Mastadenovirus (from mammals) and Aviadenovirus (from hinds)

4

and two recently described genera, Atadenovirus (from ruminants, snakes, mai"supinis)

(Benko and Harrach, 1998) and 5iadenovirus (fioin frogs, poultry) (I)<ividson and

Harrach, 2002). To date, 51 different serotyhes of human adcnoviruses h; tvc, hoen

recognized (Schnurr and Dondero, 1993; De Jong et raL, 1999). These are grouped into six

species on the basis of their hemagglutinating properties, biophysical and biochemical

criteria: species A, I3 (subdivided into species I11 : 1111 132), t', 1), I';, and i" (Wadell, I9ýti I).

A complete listing of adenovirus serotypes of each species and some of their biolo-ical

properties are listed in Table 1.1.

1.2.3 Clinical syndromes

Human adenoviruses are responsible for a wide rouge of clinic, il manifest; itions,

such as pneumonia, cystitis, conjunctivitis, diarrhea, hepatitis, myocarditis, and

encephalitis (Wadell, 2000). Disease may vary from mild and self-Iimited to severe,

disseminated, and fatal. However, its most common association is with respiratory

conditions. Adenovirus-related respiratory infections are predominantly caused by

adenoviruses from species 1> (Ad; ý and Ad7), species (' (Ad 1, A(12, and A(15) and species Iý;

(Add) (Wadell, 2000). Large outbreaks of acute respiratory disease have been reported in

military training camps (Barraza cat Q, 1999; Kolavic-Gray et al., 2002; Iiyan et : i1.,

2002). The serotypes most commonly associated with these outbreaks were AdI and Ad 7,

with Ada, Ad14, and Ad21 less frequently detected. A few fatal cnscs have also been

reported among military trainees (Ryan et a!., 2001).

ý\denoviruses are also important causes of respiratory illness in infants and

children. Adenovirus can cause severe, often fatal pneumonia or hroncholitis. Severe

acute respiratory illness has been noted in association with Ad1 through AdM, Ad 19,

Table 1.1 Properties of human adenovirus serotypes of species A to Fa

Homology (°�)DNA

Species Serotype Intragenic Intergenic G+C Number of Haemagglutination Length of Oncogenicity in (%) Sma 11, fragments pattern' fibers (nm) newborn hamsters

A 12,18.31 48-69 8-20 48 4-5 IV' 28-31 High (tumours in most animals in 4 months)

BI 3.7.16.21.50, ' 89-94 9-20 51 8-10 I 9-11 Weak (tumours in few B2 11.14,34,35 animals in 14-18

months)

L2,5.6 99-100 10-16 58 10-12 III 23-31 Nil

J

D 8-10.13.15. 94-99 4-17 58 14-18 II 12-13 Nil 1 -1

.19.20.22- 30.32.33736. 37.3&39.42- 47.48-49.5 1e

E 1 4-23 58 16-19 III 1 i Nil

F 40.41 62-69 15-22 52 9-12 IV 28-33 Nil

1, A partial reproduction from Wadell (2000) with additional published data. The restricted DNA fragments were analysed on 0.8-1.2`% agarose slab gels. DNA fragments smaller than 400 bp were not resolved.

J. Complete agglutination of monkey erythrocytes: II. complete agglutination of rat erythrocytes. 111. parallel agglutination of rat erythrocytes (fewer receptors). IV, agglutination of rat erythrocytes discernible only after addition of heterotypic antisera. 'Described by De Jong et al. (1999). 'Only restriction digest analysis has been performed with ad32-Ad39 and Ad42-4 7 .

Restriction digest analysis of _ßd48. Ad-19. and Ad5l published by De Jong et al. (1999).

Ad21, Ad35, and others (flierholzer, 19, ' 9), but outbreaks of severe infections in heaItby

children are not common and only more frequently reported with Ad 7, followed by \d3

and Ad21 (James et al., 1979; Kajon et al., 1990; Li cat al, 1996; Ilon(,, et al, 201)1;

Carballal et al, 2002; Kim et al, 2003).

Viral infections of the heart are important causes of morbidity and moº"tality in

children and adults. 1!: nteroviruses, especially the coxsackievirus ß family, have been

considered the most common cause of acute mvocarditis (din of 'i!, 1990; ßaboonian 0

al, 1997; Pauschinger et al, 1999). Recent, evidence however, suggests that adenoviruses

genoºnes are commonly found in hearts of affected children and could be an imporkuit

cause of myocarditis (Pauschinger et al., 1999; Bowles of al, 2003).

1.2.4 Genome organization and replication

The Nlastadenovirus genome is divided into . 1 different transcription regions: Iß: 1,

E2, E3 and 14. Figure 1.2 is a schematic representation of the gene layout and the

different transcription events that follow adenovirus replication. The adem)virus

infectious cycle can be clearly defined into two phases. The first or `early phase, includes

the entry of the virus into the host cell and the relocation of the virus gem me to the

nucleus, followed by the selective transcription and translation of the early genes. These

early genes are arranged in four cassettes of' transcripts (termed E1, H, 2, H, ý)' and I; I) in

the adenovirus genome. The early events modulate the functions of the cell so as to

facilitate the replication of the virus UN, and the resultant ta"anscription and

translation of the late genes. There; ifter, Lite transeript. ion ensues, with five cassettes of

transcripts (termed I, l to l0) resulting- from a complex series of splicing events.

7

100K, 33K, pVIII P. L4

EIB 55K, 19K

EIA 243R, 289R

10 º

EIA EIB

19K

Ilia

III, pVII, V

0 L1

VA RNAs 1&2

-00.

`/ql 'IL 1

Ndll

pVI, II, Pr No

L2

nK

:. \'15S1 vat 11 ý

ý

Nß fP7P

Pd

E1 E2B

ý IX

ý IVa2

E2B

Pol F

pTP

& am

L3

J l:! -JC

D

1? I"; 12.5K CR1

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Sill,

22K

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DBP4

E3

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E4

E4

orfs 1-6/7

- 4

Figure 1.2. Gene layout and transcription of the Mastadenovirus genus. The genome is represented by a central blue horizontal line marked at 5 kbp intervals. Protein-encoding regions are shown as block arrows. Cyan denotes genus-common genes, other colours indicate related genus-specific genes. El, E3 and E4 regions are shaded violet while the E2A and E2B regions are indicated in white. The early transcripts (EIA, El B, E2A, E2B, E3 and E4) are labelled in green, the late (LI, L2, L3, L4 and L5) in blue. Arrows indicate the direction of transcription. Modified from Fig. 2 (Davison et. al., 2003) and Fig. 2 (Russell, 2000).

fiber 0 L5

E3 12.5K, 6.7K, gpl9K, ADP. RIDaß. 14.7K

14.7K

12.5KCR1- (M RIO-p

LulRID. i

fl 1111CRI-u1 CR1-i+t 0

CRI-11 U

8

The early phase events in adenovirus replication begins with the interaction of

the fiber knob domain and specific host cell receptors located on the surface of permissive

cells. This is followed by entry of the virus into the cell cytoplasm and the transportation

of the virus genome to the cell nucleus. A more detailed discussion of these events will be

presented in later sections of this chapter.

Upon entry of the viral genome into the cell nucleus, early traanscription events

take place. The 1 l gene products can be subdivided further into I "AA and 1"A l'), The 1, l, A

proteins are primarily concerned with modulating cellular metabolism to make the cell

more susceptible to virus replication (Davison et. aL, 200: 1). These proteins interfere with

the processes of cell division (IKeblusek et ; il, 1999) and with the regulation of NF-i: lý

(Shisler eta]., 1996) and p53 (Hale and Braithwaite, 1999). The transcription factor, NP

KB, is involved in pathways of innate and adaptive immune systems while the

transcription factor, p53, plays a role ill induction of cell apoptosis. Interference of these

processes by the I,: 1A proteins helps the virus overcome normal cell defensive

mechanisms to protect against disruption to cellular metabolism and to remove defective

cells.

Phe 1lß 19I' gene product is concerned with prolonging cell survival by

interacting and ablating members oI, the I>axfamily, which induce ipopIosis rand necrosis

(Ilan et a1., 199G). The E I B : ), 5K gene product h. ºs been shown to interact directly with

p53 (Grand etal, 19)9), possibly to regulate p53 level in the cell, and addit, ionallv has in

effect on late virus mRNA transcription (Ilarada and Berk, 1999). (Both the 1; 1 1 and Iý', 111

gene products also seem to function in promoting oncogenesis and Iransfornintion

(kIamnahirnn (t; ll, 199)).

()