ENTERIC ADENOVIRUS TYPE 41

Genome organization and specific detectionprocedures

Annika Allard

Department of Virology University of Umeå

Umeå 1992

UMEÅ UNIVERSITY MEDICAL DISSERTATIONS New Series No 332 - ISSN 0346-6612

From the Department of Virology University of Umeå, Umeå, Sweden

ENTERIC ADENOVIRUS TYPE 41

Genome organization and specific detection procedures

Annika Allard

Umeå 1992

Copyright(c) 1992 by Annika Allard

ISBN 91-7174-646-3

Printed in Sweden by Solfjädern Offset AB

Umeå 1992

ENTERIC ADENOVIRUS TYPE 41

Genome organization and specific detection procedures

AKADEMISK AVHANDLING

som för avläggande av doktorsexamen i medicinsk vetenskap via Umeå universitet, offentligen kommer att försvaras i föreläsningssalen, Institutionen för Mikrobiologi, Umeå universitet, lördagen den 28

mars 1992, kl. 10.00

av

Annika Allard

Avdelningen för Virologi Umea universitet

Umeå 1992

ENTERIC ADENOVIRUS TYPE 41 Genome organization and specific detection procedures.

Annika Allard, Department of Virology, University of Umeå, S-901 85 Umeå, Sweden.

ABSTRACT

Enteric adenoviruses (EAd) types 40 and 41 (Ad40 and Ad41) representing subgenus F, are primary pathogens of children being second only to rotaviruses as the most important cause of infantile diarrhea.

The EAds differ from all other adenoviruses in their inability to grow in most conventional established cell lines and have been suggested to be deficient in some early gene functions since they could be complemented by Ad 5 early regions EIA and E1B. In order to search for differences that could explain its characteristic growth restriction, the early regions EIA and E1B of Ad41 (strain D389) were sequenced, analysed and compared with the corresponding regions of Adl2, Ad7, Ad2, and Ad4. As revealed by the analysis of Ad2, three major mRNAs of 9S, 12S and 13S are generated from region EIA. The EIA region of Ad41 encodes two mRNAs corresponding to the 12S and 13S mRNAs. Only the 13S mRNA is transcribed at detectable levels. This mRNA can be translated into a 251 aa putative protein that contains the three highly conserved domains found in all other human adenoviruses and shown to be responsible for many important regulatory functions during infection.

The E1B region of Ad41 encodes three transcripts that correspond to 22S, 14S and 9S mRNA of Ad2. No equivalent to the 13S mRNA of Ad2 E1B is found. In addition the Ad41 14S mRNA exhibits an additional exon of 23 bp created by a donor and an acceptor splice sites not desribed for other adenovirus E1B sequences.

Due to their growth restriction in conventional cultures, rapid diagnostic procedures developed for the enteric adenovirus infections have mainly been aimed at the detection of viral antigens or nucleic acids. This thesis also describes several procedures developed for the general detection of adenoviruses and specific detection of the enteric types in stools specimens. General and specific hybridization assays were developed by use of two BamHI clones obtained from the EIA region of Ad41. One- and two-step PCR procedures were also developed for the general detection of adenoviruses using primers corresponding to highly conserved sequences within the hexon gene. Subgenus F specific one- and two-step PCRs were developed by using primers located in the Ad41 E1B region.

The one-step PCR systems were tested and validated against isolation in tissue culture, DNA restriction enzyme analysis and a commercial latex agglutination test in the study of 60 specimens obtained from children with rotavirus negative diarrhea. The asymptomatic fecal excretion of adenoviruses was evaluated by two-step PCR amplifications on samples from 50 healthy children, 50 healthy adults, and 50 adults suffering from diarrhea.

Finally, a simplified procedure for detection, discrimination and typing of EAd was also designed by combining the one-step PCR amplification of the hexon region with the restriction of the 300 bp product.

Keywords: EAd/ Ad41/ EIA/ E1B/ detection/ PCR

ISBN 91-7174-646-3 ISSN 0346-6612

VAR INTE RÄDD FÖR DET LÅNGSAMMA

FRAMÅTSKRIDANDET, VAR BARA RÄDD FÖR

STILLASTÅENDET.

Kinesisk vishet

CONTENTS

1. ABSTRACT 3

2. PAPERS IN THIS THESIS 5

3. INTRODUCTION 6

3:1 Classification, disease and epidemiology 63:2 Structural characteristics of adenoviruses 73:3 Adenovirus productive infection of cultured human cells 103:4 Organization of the viral genome 103:5 Organization of the EIA and E1B regions 123:6 The proteins of the EIA region: characteristics and functions 143:7 The proteins of the E1B region: characteristics and functions 16

4. ENTERIC ADENOVIRUSES 18

4:1 Growth restriction in cell cultures 184:2 Genetic variability 194:3 Epidemiology 204:4 Pathogenesis and clinical manifestations 214:5 Immunity 224:6 Laboratory diagnosis 23

5. PURPOSE OF THIS THESIS 24

6. RESULTS AND DISCUSSION 25

6:1 Regulatory elements in the EIA gene 256:2 Identification of the EIA signal sequence 296:3 Regulatory elements in the Ad41 E1B and pIX genes 346:4 The E1B transcription map of Ad41 366:5 Detection of adenoviruses by hybridization 386:6 Polymerase chain reaction for detection of

adenoviruses 39

1

6:7 Detection of adenoviruses in stools of childrenwith diarrhea 46

6:8 Detection of persistent adenovirus infections 476:9 A simplified procedure for detection and discrimination

of enteric adenoviruses 48

7. GENERAL SUMMARY 50

8. CONCLUDING REMARKS 52

9. ACKNOWLEDGEMENTS 53

10. LITERATURE CITED 56

11. PAPERS I-VI 68

2

1. ABSTRACT

Enteric adenoviruses (EAd) types 40 and 41 (Ad40 and Ad41) representing subgenus F, are primary pathogens of children being second only to rotaviruses as the most important cause of infantile diarrhea.

The EAds differ from all other adenoviruses in their inability to grow in most conventional established cell lines and have been suggested to be deficient in some early gene functions since they could be complemented by Ad5 early regions EIA and E1B. In order to search for differences that could explain its characteristic growth restriciton, the early regions EIA and E1B of Ad41 (strain D389) were sequenced, analysed and compared with the corresponding regions of Adl2, Ad7, Ad2, and Ad4. As revealed by the analysis of Ad2, three major mRNAs of 9S, 12S and 13S are generated from region EIA. The EIA region of Ad41 encodes two mRNAs corresponding to the 12S and 13S mRNAs. Only the 13S mRNA is transcribed at detectable levels. This mRNA can be translated into a 251 aa putative protein that contains the three highly conserved domains found in all other human adenoviruses and shown to be responsible for many important regulatory functions during infection.

The E1B region of Ad41 encodes three transcripts that correspond to 22S, 14S and 9S mRNA of Ad2. No equivalent to the 13S mRNA of Ad2 E1B is found. In addition the Ad41 14S mRNA exhibits an additional exon of 23 bp created by a donor and an acceptor splice sites not described for other adenovirus E1B sequences.

Due to their growth restriction in conventional cultures, rapid diagnostic procedures developed for the enteric adenovirus infections have mainly been aimed at the detection of viral antigens or nucleic acids. This thesis also describes several procedures developed for the general detection of adenoviruses and specific detection of the enteric types in stools specimens. General and specific hybridization assays were developed by use of two BamHI clones obtained from the EIA region of Ad41. One- and two-step PCR procedures were also developed for the general detection of adenoviruses using primers corresponding to highly conserved sequences within the hexon gene. Subgenus

3

F specific one- and two-step PCRs were developed by using primers located in the Ad41 E1B region.

The one-step PCR systems were tested and validated against isolation in tissue culture, DNA restriction enzyme analysis and a commercial latex agglutination test in the study of 60 specimens obtained from children with rotavirus negative diarrhea. The asymptomatic fecal excretion of adenoviruses was evaluated by two-step PCR amplifications on samples from 50 healthy children, 50 healthy adults, and 50 adults suffering from diarrhea.

Finally, a simplified procedure for detection, discrimination and typing of EAd was also designed by combining the one-step PCR amplification of the hexon region with the restriction of the 300 bp product.

4

THIS THESIS IS BASED ON THE FOLLOWING ARTICLES WHICH WILL BE REFERRED TO BY THEIR ROMAN NUMERALS

Annika Allard and Göran Wadell (1988). Physical Organization of the Enteric Adenovirus Type 41 Early Region 1A.Virology 164, 220-229.

Annika Allard and Göran Wadell (1992). The E1B Transcription Map of the Enteric Adenovirus Type 41.Virology, in press.

Annika Allard, Göran Wadell, Magnus Evander and Kristina Lindman (1985). Specific Properties of Two Enteric Adenovirus 41 Clones Mapped within Early Region 1A.Journal of Virology 54, 145-150.

Annika Allard, Rosina Girones, Per Juto and Göran Wadell (1990). Polymerase Chain Reaction for Detection of Adenoviruses in Stool Samples.Journal of Clinical Microbiology 28, 2659-2667.

Annika Allard, Bo Albinsson and Göran Wadell (1992).Detection of Adenoviruses in Stools from Healthy Persons and Patients by Two-step Polymerase Chain Reaction.Journal of Medical Virology, in press.

Annika Allard, Adriana Kajon and Göran Wadell (1992).A Straight-forward Procedure for Discrimination and Typing of Enteric Adenoviruses after Detection by PCR.Submitted for publication.

3. INTRODUCTION

Human adenoviruses are some of the best characterized of all mammalian viruses. They were first isolated by Rowe et al. (1953) from adenoids in an attempt to evaluate new tissues for growth of polio viruses. Due to the slowly progressive cytopathic effect, they were designated adenoid-degenerating (AD) agents. Adenoviruses were soon established as etiologic agents of acute respiratory disease by Hilleman and Werner (1954) and have thereafter been described also in association with gastrointestinal, urinary, and ocular infections. They are also an increasing problem as opportunistic pathogens of immunocompromised patients.The adenoviruses have become powerful models to study cell transformation, eukaryotic gene regulation, DNA replication and more recently the immunology of virus infections.

3:1 Classification, disease and epidemiology

The adenoviruses (Ad) constitute a family of ubiquitous DNA-viruses (the Adenoviridae) that infect humans and a wide range of animal species. According to their host range two genera have been defined, Mastadenovirus which comprises all the mammalian viruses and Aviadenovirus which comprises adenoviruses with an avian host (Classification and nomenclature of viruses, 1991).There are 47 known human adenovirus serotypes (Green et al. 1979; Wigand and Adrian, 1987; Hierholzer et al. 1988a,b) which have been classified into six subgenera, A to F, based on their haemagglutination properties (Rosen, 1960), oncogenicity in newborn hamsters (Huebner et al. 1967), G+C content, DNA genome homologies (Green et al. 1979), and structural proteins by SDS- polyacrylamide electrophoresis (Wadell, 1979; Wadell et al. 1980).

Subgenus A consists of Adl2, Adl8 and Ad31 which represent only 0.5% of the reported typed virus isolates. The majority of the isolates belonging to this subgenus have been recovered from stools and most of them in cases of pediatric gastrointestinal disease. Ad31 has also been recovered from immunocompromised patients (Johansson et al. 1991).

6

Subgenus B consists of Ad3, 7, 11, 14, 16, 21, 34 and 35 which have been subclassified into 2 clusters of DNA homology: B1 comprises Ad3, 7, 16 and 21 which cause primarily outbreaks of respiratory disease. B2 comprises Adll, 14, 34 and 35 which cause persistent infections of the urinary tract. Adll, 34 and 35 have also been reported to be serious pathogens in immunocompromised patients.Subgenus C contains Adi, 2, 5 and 6. The first three represent more than one half of all isolates reported to WHO (1980). The characteristic latent infection of lymphoid tissue described for adenoviruses is mostly associated with these types (Fox et al. 1977).Subgenus D consists of 23 well established serotypes and five recently described serotypes; candidates Ad43, 44, 45, 46 and 47 (Hierholzer et al. 1988a). They show a predilection for infecting the eye. Serotypes 8, 19 and 37 are frequently associated with epidemic outbreaks of keratoconjunctivitis. Adenovirus strains corresponding to serotypes 43 to 47 have so far only been isolated from patients with AIDS (Hierholzer et al. 1988a).Subgenus E contains only one type: Ad4 which has been associated with both epidemic follicular conjunctivitis and respiratory disease.Subgenus F: This subgenus contains the two so called enteric or fastidious adenoviruses (EAds) serotypes 40 and 41. After rotavirus, the EAd are now recognized as the second most commonly identified agents in stools of infants and young children with gastroenteritis (Uhnoo et al 1983; Horwitz 1990).

The properties of all 47 human adenovirus serotypes are described in Table 1.

3:2 Structural characteristics of adenoviruses

The adenovirus particle or virion is a non-enveloped icosahedron with a diameter of around 65-80 nm (Home et al. 1959). It is composed of at least 10 structural proteins most of which are encoded in one multiply -spliced transcription unit (Fig. 1). The virus capsid is composed of 252 capsomers. The 240 hexons are arranged symmetrically so that each one is surrounded by 6 other capsomers. Each hexon is formed from 3 molecules of polypeptide II (Horwitz et al. 1970). The 12 vertices of the virion contain a capsomer (the penton) out of which a spike (the fiber) projects. The penton is composed of

7

TABL

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I ?■£ B 2r « ® >« 5 i °0c -o .2 c1 8 . — C/3

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■d ? ü ® p ç p ao w > o ± >: 2 » ö u £ ö i î - ï £w X 03 u

8

dMem

bers

of

subg

enus

B

are

divi

ded

into

two

clus

ters

of

DNA

hom

olog

y ba

sed

on pr

onou

nced

di

ffere

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in

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y po

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with

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d47.

f P

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d VI

of

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show

ed

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rent

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ecul

ar m

ass

of 45

and

22 kD

a, re

spec

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y. P

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e V

of Ad

30

show

ed

an ap

pare

nt m

olec

ular

mas

s of

48.5

kDa.

probably 5 copies of polypeptide III (van Oostrum and Burnett, 1985). The fiber is glycosylated and is composed of 2 molecules of polypeptide IV. It is this structure that is believed to be important in the attachment of the virion to cells. Polypeptides Ilia, VI, VIII, and IX connect hexons and vertex capsomers into a dense capsid. This capsid contains a nucleoprotein core composed of polypeptides V and VD and the viral DNA (Nermut, 1984). The adenovirus particle is resistant to low pH, bile, and many proteolytic enzymes, which enables the virus to grow to high titers in the human gut.

Polypeptid« SDS-gel S tructu ra l Unit

ÏÏ---

ma

m ---vnr

• Hexon

• Pentonbase - Perip«• Fiber• Ter mil• Core protein I

- Hexon-associated protein

• Core protein II

, Hexon associated protein- Protein specific for

groups of nine hexons

Fig. 1. A tentative model for the location of the proteins in the Ad2 virion based on protein-protein cross-linkage studies. The polypeptide composition o f the virion proteins are shown in a stained exponential 16% SDS-polyacrylamide gel. Adopted from Philipson (1983).

Adenoviruses contain 13% DNA and 87% protein, have no membranes or lipids, and are therefore stable in solvents such as ether and ethanol (Green and Pina, 1963). The genome is a linear duplex DNA which has been completely sequenced and consists of 35.937 ± 9 base pairs (Roberts et al, 1986b). Ad DNA has the unusual feature of a virus-coded 55-kd terminal polypeptide (TP) covalently linked to dCMP at each 5'end of the linear genome (Rekosh et al. 1977). Adenovirus DNA has inverted terminal redundancies (Wolfson and

9

Dressier, 1972), and the 100- to 140-base-pair length of these repeats varies with the serotype (van Ormondt and Galibert, 1984).

3:3 Adenovirus productive infection of cultured human cells

The virus growth, which takes about 24 h to complete, is strictly regulated (reviewed by Pettersson and Roberts, 1986). During infection, the virion attaches via fiber to specific receptors on the cell surface and is internalized either by endocytosis or by direct penetration (reviewed by Svensson, 1985). The capsid is then stripped away and the DNA core is transported to the nucleus where the viral genes are transcribed. The viral genes are expressed in two broadly defined phases: "early" which is the period prior to viral DNA replication at about 7 h postinfection, and "late", which follows the initiation of viral DNA replication (Akusjärvi et al. 1986). Early genes carry out a number of functions that usurp the cell and prepare it for the efficient synthesis of viral DNA and proteins (Akusjärvi et al. 1986; Esche, 1986). During the early phase, mainly host cell mechanisms are used to synthesize viral mRNA and proteins, and host cell macromolecular synthesis continues essentially unabated. During the late phase, host cell DNA, mRNA, and protein synthesis are shut off, and the cell becomes devoted to the synthesis of viral macromolecules. Late viral genes encode mainly virion structural proteins which are synthesized in the cytoplasm and transported to the nucleus where the virion is assembled. Eventually, the cell dies, and the virions are released by unknown mechanisms.

3:4 Organization of the viral genome

Most of our knowledge about the molecular biology of adenovirus has been derived from studies on the replication of Ad2 and the very closely related Ad5 (both group C) in suspension cultures of human HeLa or KB cells. The organization of the Ad2 genome is illustrated in Figure 2. By convention, the 36 000 bp genome is divided into 100 map units (360 base pairs per unit), and the two strands are denoted r and /, for rightward and leftward transcription, respectively. The split arrows in Figure 2 depict the exons in the spliced mRNAs. All the transcription units except those for proteins IX and IVa2 are

10

organized such that multiple overlapping spliced mRNAs are produced from a common pre-mRNA precursor.

42-S4K 22K48-68K S5K (X

75K

Fig. 2. Transcriptional organization oftheAd.2 genome. The genome is divided, into 100 map units. The r-strand is rightward-transcribed into RNA and the 1- strand leftward. The direction o f transcription is indicated by arrows. The capped 5 'ends o f the cytoplasmic RNA indicate the positions o f transcriptional promoters, while the arrowheads represent the 3'poly adénylation sites. Gaps in the arrows indicate intervening sequences, which have been removed from the cytoplasmic RNA by splicing. The RNA shown in bold lines can be detected early in infection before the onset o f DNA replication (regions EIA, E1B, E2A, E3, E4; also the late promoter at 16.5 units is active early in infection, leading to transcription to 39 units). The light lines represent intermediate RNAs synthesized at early as well as at late times in the infection cycle (E2A, E2B, polypeptide IX). The double-lined arrows indicate late RNA species. Correlation o f mRNAs with encoded proteins are based on cell-free translation o f selected RNA species and RNA mapping data. Proteins are designated by their molecular weights in kilodaltons (K) or by roman numerals (virion components). Adopted from Sussenbach (1984).

11

3:5 Organization of the EIA and ElB regions

The early genes are grouped in six transcription units denoted EIA, ElB, E2A/B, E3A/B, and E4, and LI (early). Among these blocks of viral genes, EIA and ElB are the first to be transcribed. They have thus been defined as "immediate-early" genes (reviewed in Berk, 1986b), and represent also the transforming region of the adenovirus genome (Fig. 2). EIA messenger RNAs have been deteced as early as 30 min after infection (Berk et al. 1979). Proteins are translated from three major mRNAs sedimenting at 13 S (1.1 kb), 12 S (0.9 kb) and 9 S (0.6 kb). Two additional mRNA species with respective sedimentation coefficients of 10 S and 11 S have been detected (Stephens and Harlow, 1987; Ulfendahl et al. 1987). All mRNAs, except the 9 S transcript, are translated in the same reading frame. They have the same 5' and 3' ends, but they are differentially spliced (Fig. 3). Unlike the other EIA transcripts which accumulate at the early stage of infection, the 9 S mRNA is preferentially transcribed and accumulated at late stages of infection (Berk and Sharp, 1978).

AAAA„

AAAA„

26 87 104

11 s H B / X B H M H B B H B ______aaaa„

Fig. 3. Splicing events and gene products o f the Ad2 EIA regions. The solid line at the top o f the Figure is the left-hand end o f the adenovirus linear genome, from nucleotide 500 to 1600. The Ad2 EIA mRNAs are shown as solid lines and the protein coding sequences as black, grey, and hatched boxes, representing the alternative reading frames. Alternative splicing occurs between one single acceptor site at nucleotide 1226 and two possible donor sites at nucleotides 1111 and 973, respectively. The 10S and IIS mRNAs possess two additional donor and acceptor sites: one donor site is common with the 9S transcript, the second donor site is either common with the 12S or 13S species. Adopted from Boulanger and Blair (1991).

12

The ElB region encodes three major mRNA species, 22 S, 13 S and 9 S (Fig. 4). The ElB transcriptional unit is activated by the 13S mRNA product of the EIA region (Dery et al. 1987), generating the two major overlapping mRNAs of 22 S and 13 S in infected cells. The ElB 9 S mRNA is transcribed from an independent promoter and encodes a structural component of the adenovirus capsid, present in groups of nine hexons, which is referred to as polypeptide IX (Boulanger et al. 1979; Furcinitti et al. 1989). The polypeptide IX gene has been termed an intermediate gene since it is expressed at around 6-8 h after infection, before other structural polypeptides of the virus can be detected (Spector et al. 1978). Two minor mRNAs of 14 S and 14.5 S (Virtanen and Pettersson, 1985) coding for proteins which share N-termini with the major p55 protein (Anderson et al. 1984) have also been described but as yet no function has been ascribed to them in infection or transformation.

AAAA„

I I 9 S

Fig. 4. Map o f the Adi ElB region. ElB promoter controls a rightward transcript that starts at nucleotide 1699 (+1) in the Ad2 genome. Black, grey and hatched boxes represent the three alternative reading frames. Synthesis of the virion structural polypeptide IX begins at the intermediate stage o f infection, from the ElB 9S mRNA. Adopted from Boulanger and Blair (1991).

13

3:6 The proteins of the EIA region: characteristics and functions

EIA expresses two major proteins early after infection of 289 amino acids (289R) and 243R derived from 13S and 12S mRNAs, respectively. As shown in Fig 5, the 289R contains 3 domains CD1, CD2 and CD3 which are conserved among all adenovirus types analysed so far (Kimelman et al. 1985; Moran and Mathews, 1987). The 13S and 12S mRNAs are spliced such that the 289R and 243R proteins are identical except for the 46 amino acids that constitute the CD3 region and are unique to the 289R protein.

Nuclear locali* at ton

Enhancer R eprettton

Tranter o ttonai Aetivatton

E 1H< I 3 S - 2 H W I > 4q g o 120 139 196i i _____ _C O I * IC D 2 1 CDS

Im m ortahtation

r a t Cooperation

Coll DNA S ynthettt and Proliferation

Fig. 5. Schematic representation o f the adenovirus 2 region ElA-encoded 289R and 243R proteins. CD1, CD2, and CD3 indicate conserved domains 1, 2 and3. The 289R and 243R proteins are identical except that 243R lacks CD3 because this region is spliced out o f the mRNA. Various functions that have been ascribed to the 289R and 243R proteins are listed at the left; the bars above and below the proteins indicate regions where mutations abrogate the Junctions in question. Adopted from Gooding and Wold (1990).

The 289R and 243R proteins are localized to the nucleus via specific nuclear localization signals, one of which is a pentapeptide KRPRP located at the C- terminus of either protein (Lyons et al. 1987).

14

The EIA proteins carry out a number of key regulatory functions. The 289R has a primary, if not exclusive, transactivation function that induces transcription of the other early regions and also EIA itself. The major transactivation activity is contained in the CD3 domain (Lillie et al. 1987; Moran and Mathews, 1987). The mechanism of 289R transactivation appears to involve the activation of a number of preexisting cellular elements in the promoters of the viral transcription units (Berk, 1986a). Certain cellular genes, e.g. heat shock proteins (Simon et al. 1988) and /3-tubulin (Stein and Ziff, 1984) are also transactivated by EIA.CD3 also exhibits the consensus sequence postulated for metal-binding domains that characterize a group of proteins which interacts with DNA, and are involved in the regulation of gene expression (Berg, 1986).

The EIA proteins have been found to be highly phosphorylated and recent data have shown that the DNA binding activities of transcription factors are positively regulated by phosphorylation, and that the EIA 289R protein may activate some cellular kinase involved in this process (reviewed in Berk, 1989).

EIA products interact with at least 3 cellular proteins, the pl05 retinoblastoma product (Rb) (Whyte et al. 1988) that appears to be a negative regulator of DNA replication (Howe et al. 1990), p300, a 300 kD protein (Harlow et al. 1986), and pl07, a 107 kD host cell protein that have been suggested to be important for transformation. The Rb gene functions as an antioncogene and binding of the EIA proteins to the Rb protein may prevent dividing cells from returning to Go rather than stimulate resting cells to enter the cell cycle (Wang et al. 1991). By binding to either pl05 Rb or p300, EIA products also induce cellular DNA synthesis (Smith and Ziff, 1988; Howe et al. 1990). Since the SV40 large T antigen and the human papillomavirus-16 E7 transforming proteins also bind to pl05 Rb, a common mechanism of transformation is suggested for adenovirus and these two other DNA tumor viruses (DeCaprio et al. 1988).The EIA proteins can also repress transcription controlled by some enhancers (Lillie et al. 1987; Schneider et al. 1987) and collaborate with E1B or ras to cause full cell transformation (Ruley, 1983; Moran and Mathews, 1987). These properties also seem to be confined to the CD1 and CD2 domains common to

15

both 289R and 243R (Moran et al. 1986a,b; Kuppuswamy and Chinnadurai, 1987; Lillie et al. 1987; Murphy et al. 1987; Velcich and Ziff, 1988).

Very scarce mRNAs of 1 IS, 10S, and 9S encoding proteins of 217R, 171R and 55R can also be found in infected cells but their significance is not known (Stephens and Harlow, 1987; Ulfendahl et al. 1987).

3:7 The proteins of the E1B region: characteristics and functions

The E1B region of adenoviruses is located immediately to the right of EIA and is also transcribed in a rightwards direction (Fig. 2). There are five differently spliced transcripts in Ad 2/5 (Virtanen and Pettersson, 1985) which presumably give rise to separate protein products. Only two proteins have been identified, one of 19 kD (pl9) and the other of 55 kD (p55), which are unrelated in amino acid sequence and are synthesized from the 22S mRNA template using AUG codons in different reading frames (Bos et al. 1981). The pl9 protein is also translated from the 13 S mRNA and thus the level of this protein increases as infection progresses. The 13S mRNA can also encode an 8 kD species (p8) related to the N-terminal region of the 55 polypeptide, although this truncated product has not been detected in infected cells.

Both p55 and pl9 proteins are post-translationally modified. The pl9 protein has been reported to be covalently linked to lipid, but only a minor fraction of intracellular pl9 protein is fatty acylated in infected cells (McGlade et al. 1987). The pl9 protein has been shown to be located in an intracelluar membrane (probably the endoplasmic reticulum), the nucleus (White et al. 1984) and also in the plasma membrane of adenovirus transformed human cells (Persson et al. 1982; Smith et al. 1989). The most well-documented function of the E1B 19kD protein is to protect viral and cellular DNA from degradation that is induced as a consequence of viral infection. The pl9 protein therefore plays an essential role in the viral replication cycle in that it is indispensable for maintaining the integrity of viral and cellular DNAs (Stillman, 1986). However, since several classes of immune effector cells cause lysis by a mechanism involving a specialized form of DNA degradation, it may be that the 19 kD protein has some protective role in preventing Tc cell, natural killer (NK) cell, or cytokine

16

induced cell death (Shiroki and Toth, 1988; Shiroki et al. 1990; Gooding et al. 1991).The pl9 protein has also been found to associate with vimentin-containing intermediate filaments and the nuclear lamina, causing disruption of these structures (White and Cipriani, 1989, 1990). Such effects may have important implications for viral pathogenicity and transformation. It is possible that anchorage-independent growth of Ad-transformed cells results from such altered organization of intermediate filaments induced by pl9.

The p55 protein occupies a predominantly nuclear location in infected cells (Samow et al. 1982; Scugart et al. 1985). It has been found to form a molecular complex with the p34 protein of Ad5 E4 region and appears to collaborate with this protein to facilitate the transport of viral mRNA from the nucleus and to inhibit the transport of cellular mRNA (Babiss et al. 1985; Leppard and Shenk, 1989; Bridge and Ketner, 1990).The p55 has also been shown to exist in complex with the cellular oncoprotein p53 (Bishop, 1985) in Ad2 and Ad5 transformed cells (Samow et al. 1982; Zantema et al. 1985), but not in Adl2, Ad7 and Ad9 transformed cells (Boulanger and Blair, 1991). This is probably due to an alteration in the binding site which remains to be defined. It has also been concluded that the formation of the p55-p53 complex is determined by an as yet undefined property of p53 (Braithwaite and Jenkins, 1989). As reported by Finlay et al. (1989) the complex between Ad5 E1B p55 and p53 could release cells from controlled growth in an analogous fashion to the ElA-pl05-Rb complex.It has been recently demonstrated that p53 forms a complex with the transforming E6 proteins of HPV16 and 18 (Wemess et al. 1990) stressing the potential importance of its interaction with oncoproteins of DNA tumor viruses in general.

An altered distribution of both p55 and pl9 proteins has been reported as infection proceeds in human cells (Rowe et al. 1983; White et al. 1984). The proteins were found to be present in a cytoplasmic location early in infection and to accumulate in the nucleus late in infection.

17

4. ENTERIC ADENOVIRUSES

The introduction of electronmicroscopy (EM) for direct examination of stool specimens led to the discovery of a new group of adenoviruses that failed to grow in human embryo kidney cells or in most heteroploid conventional cell lines. They were first observed by Flewett et al. (1975) in association with a hospital outbreak of infectious diarrhea in children.In a study of 200 antigenically related fastidious adenoviruses isolated from cases of infantile diarrhea deJong et al. (1983) described two new distinct serotypes that exhibited no relationship to the known human adenoviruses species either by neutralization or haemagglutination inhibition tests. They were however, identical in haemagglutination inhibition assays. The new serotypes were called Ad40 (with reference strains Dugan and Hovi X) and Ad41 (with reference strain Tak). The DNA restriction site maps of the reference strain Tak was very different from that of Dugan (Kidd et al. 1983) supporting the idea that the variants should be considered as two distinct species. Analysis of the virion polypeptides by SDS-polyacrylamide electrophoresis (Wadell et al. 1980) revealed that this so-called enteric adenoviruses (EAd) were distincly different from the other adenoviruses and a more complete restriction enzyme analysis of both serotypes showed that they exhibited unique cleavage profiles with no resemblance to those of the established types (Wadell et al. 1986). By liquid hybridization van Loon et al. (1985) determined the DNA homology between Ad40 and Ad41 to be 62-69%. Their common characteristics such as the association with gastroenteritis, cross reaction in immunological tests, restricted host cell range and structural polypeptide profiles justified their classification within only one new subgenus F.With the development of techniques for definite identification, the role of the enteric adenoviruses in the etiology of gastroenteritis and particularly infantile diarrhea has been confirmed by many workers (Uhnoo et al. 1984; Brown et al. 1984a; Kidd et al. 1986; Kim et al. 1990; Cruz et al. 1990; Lew et al. 1991).

4:1 Growth restriction in cell cultures

The enteric adenoviruses differ from all other adenoviruses by their inability to grow in human embryonic kidney cells or in most heteroploid cell lines. They

18

can grow efficiently, however, in Graham 293 cells (an Ad5-transformed human embryonic kidney cell line) thus behaving like adenovirus host-range mutants deficient in early gene functions.van Loon et al. (1987b) postulated that the lack of growth of Ad41 on certain cell lines could be due to a relative incapacity of the Ad41 EIA gene to transactivate other Ad41 early genes. However, it has been shown that Ad40 DNA replicates in KB18 cells which express the Ad2 E1B gene, suggesting a defective Ad40 E1B function (Mautner et al. 1989).It has been recently shown that Ad40 can propagate efficiently and produce plaques on A549 cells (Hashimoto et al. 1991). Replication of both Ad40 and Ad41 has also been achieved with variable success on several human heteroploid epithelium cells such as HeLa, Hep-2, Chang conjunctional, human amnion and tertiary cynomologous monkey kidney cells, tCMK (deJong et al. 1983; Albert, 1986; Perron-Henry et al. 1988; Witt and Bousquet, 1988; Pieniazek et al. 1990a). In most cases the CPE resembled that of other adenovirus species but some strains did not produce a definite progressive CPE. It has also been reported that a more efficient growth of Ad41 in HeLa, Hep-2 and human intestine cells (HI407) can be obtained by keeping the serum concentration in the maintenance medium between 0.2 and 1 % (Pieniazek et al. 1990b).

4:2 Genetic variability

The heterogeneity of adenovirus DNA has been well documented for several serotypes belonging to other subgenera. A recent study by van der Avoort et al. (1989) demonstrated that the enteric types 40 and 41 also exhibit a considerable genomic variability. By DNA restriction enzyme analysis of 48 strains of Ad40 and 128 strains of Ad41 isolated between 1971 and 1986 from various countries the existence of 11 genome types of Ad40 and 24 genome types of Ad41 was revealed. With a set of 9 restriction enzymes more than 70 cleavage sites in Ad40 DNA and more than 80 sites in Ad41 DNA were screened.

19

4:3 Epidemiology

Information on the impact and world distribution of enteric adenovirus infections has been gained from seroepidemiological surveys, the study of outbreaks and prospective studies.EAds have been found in the stools of young children with acute gastroenteritis in Europe, Asia, Latin and North America (deJong et al. 1983). In a serological survey conducted by Kidd et al. (1983), neutralizing antibodies to Ad40 and Ad41 were found in one third of the analysed sera from children of the UK, New Zealand, Hong-Kong and the Gambia.EAds are pathogens primarily of infants as suggested by the finding that the presence of specific antibodies gradually rose through childhood from 20% of infants less than 6 months of age to 50% of children and young adults. Only 10% of people over 70 were positive.EAds have been detected in the stools of infants with acute diarrhea in several studies conducted in industrialized countries (Madeley et al. 1977; Appelton et al. 1978; Vesikari et al. 1981; Yolken et al. 1982; Uhnoo et al. 1984; Cevenini et al. 1987), with an incidence of infection varying between 4 and 17% suggesting that EAds are a frequent cause of viral diarrhea second only to rotaviruses. There are a few studies on the incidence of adenovirus diarrhea in developing countries. Leite et al. (1985) found an infection rate of 2% for EAds during a 2-year survey of infantile gastroenteritis in Brazil and Kidd et al.(1986) reported a 6.5% rate in a 7-month study in Johannesburg, South Africa. In a more recent investigation Tiemessen et al. (1989) detected Ad40 and Ad41 in 13.2% of 310 children with diarrhea in a rural South African environment. A similar detection (12%) for EAds was observed by Puerto et al. (1989) in Mexico. EAds usually cause sporadic infantile gastroenteritis but they have also been implicated in outbreaks of diarrhea in different settings in the UK (Flewett et al. 1975; Richmond et al. 1979) and Japan (Chiba et al. 1983).No characteristic seasonal variations has been observed with EAd-associated gastroenteritis. In most studies Ad40 and Ad41 were found throughout the year (Middleton et al. 1977; Vesikari et al. 1981; Uhnoo et al. 1984; Brandt et al. 1985; Herrmann et al. 1988) but in South Africa (Kidd et al. 1986; Tiemessen et al. 1989) a higher occurrence of EAds was recorded during the summer months.

20

The relative frequency of Ad40 and Ad41 over long time periods has not been well studied although some reports have documented temporal shifts from one predominating type to the other (Willcocks et al. 1988; Ushijima et al. 1988) which may reflect a change in immunity of the population at risk.

4:4 Pathogenesis and clinical manifestations

Enteric adenoviruses are excreted in large amounts, up to 10^ particles per gram of faeces at the acute stage of the diarrheal disease indicating an active multiplication of the virus in the gastrointestinal tract (Retter et al. 1979).In a fatal case of Ad41 gastroenteritis crystalline arrays of virus particles were demonstrated in the duodenal mucosa suggesting active replication in this site (Whitelaw et al. 1977).The demonstration of a significant serum antibody response to EAd in children with adenovirus gastroenteritis provided suggestive evidence of causality by diarrheal disease (Chiba et al. 1983; Uhnoo et al. 1984).The question of whether EAd can infect the respiratory tract, like most other adenovirus types, and produce symptoms remains to be answered. Attempts to identify EAds in nasopharyngeal secretions of infected individuals has been unsuccessful (Petrie et al. 1982; Wadell et al. 1987). The most prominent feature of enteric adenovirus infection is diarrhea. Other symptoms associated with the disease include vomiting, low grade fever, and dehydration. The course of the illness is usually mild, but can be more severe. The incubation period is approximately 7 to 8 days and virus excretion in the stools lasts 10 to 14 days (Uhnoo et al. 1984).Uhnoo et al. (1984) carried out a detailed clinical study of 56 children with adenovirus gastroenteritis of whom 33 has enteric adenovirus infection. The mean duration of diarrhea in children with Ad40 and Ad41 infection was 8.6 and 12.2 days, respectively. Prolonged diarrhea was common, particularly in association with Ad41, with one third of the patients having symptoms for 14 days or more. Vomiting and fever were mild and lasted for a median of 2 days. Dehydration was mostly mild and isotonic in nature. One third of the patients required hospitalization. Upper respiratory symptoms were found in 21% of the patients. The clinical characteristis of enteric Ad infections were also found to differ from those of rotavirus and bacterial infections. Enteric adenoviruses

21

caused a more protracted (mean 10.2 days) but milder infection than did rotavirus with a reduced frequency of vomiting and a more moderate elevation of fever. However, a recent study found that diarrheal disease associated with EAds could be as severe as that seen with rotavirus (Kotloff et al. 1989). Other authors have also reported long-lasting diarrhea in association with EAd infection (Zissis et al. 1981; Yolken et al. 1982). There is a considerable discrepancy in the frequency of observation of associated respiratory illness. Both Yolken et al. (1982) and Kotloff et al. (1989) found comparably higher rates. Enteric adenoviruses have been reported to be also associated with lactose and gluten intolerance in a few cases (Mavromichalis et al. 1977; Uhnoo et al. 1984).

4:5 Immunity

Only a few reports have been published on the immune response to enteric adenoviruses. The studies by Kidd et al. (1981, 1983) and Shinozake et al.(1987) demonstrated that the proportion of sera with neutralizing antibodies against EAd increased with age; the level of positivity was more than double in children 2 to 4 years than in children less than 2 years of age. The low seropositivity found in sera from old people might indicate a decrease in immunity to infection among the elderly. In a Japanese study (Shinozake et al. 1987) 20% of serum samples from pregnant women and their cord samples had antibodies to Ad40 and Ad41. Fifteen percent of sera from newborns were positive. These observations demonstrate the existence of passively transferred neutralizing antibodies to enteric adenoviruses. Whether these transplacentally aquired antibodies can confer protection during infancy remains to be investigated. Reinfections with enteric adenoviruses have not been described to date. It is not known whether circulating antibodies or local immune response in the intestine are the major mediators of protection. The role of cell-mediated immunity in either protection or recovery from enteric adenovirus infection also needs to be investigated.

22

4:6 Laboratory diagnosis

Although suitable cell lines have been established, the isolation of the enteric adenoviruses in tissue culture is still troublesome and blind passages might be required before any evidence of CPE is observed. Therefore most specific diagnostic procedures aim at the detection of viral antigens or nucleic acids. Electron microscopy has been the method of choice for evaluating unknown causative agents of viral diarrhea, but the fact that several other adenovirus serotypes are often shed in stools for several months with no particular clinical significance (Brandt et al. 1985; Kidd et al. 1982) stresses the need for diagnostic tests specific for Ad40 and Ad41.

The identification of enteric adenoviruses can be performed directly on fecal specimens by immune electron microscopy (Svensson et al. 1983), solid-phase immune-electron microscopy (Wood and Bailey, 1987) or type specific ELISAs (Johansson et al. 1980, 1985; Singh-Naz and Naz, 1986; Herrmann et al. 1987). In addition several nucleic acid hybridization assays using both radioactive- and peroxidase labeled probes have been developed for the general detection and identification of the enteric types (Stålhandske et al. 1985; Takiff et al. 1985; Kidd et al. 1985; Niel et al. 1986; Hammond et al. 1987). DNA restriciton enzyme analysis has been found to be a useful technique for the identification of enteric adenoviral DNA extracted from infected cells (Brown et al. 1984b; Kidd, 1984; Kidd et al. 1984, 1986).

23

5. PURPOSE OF THIS THESIS

• To analyse the early region El of the enteric adenovirus type 41 and compare it with the corresponding regions of those already characterized types in order to search for differences that could explain the characteristic growth restriction of this serotype.

• To develop new sensitive procedures for the general detection of adenovirus and the specific detection of the enteric types 40 and 41.

24

6. RESULTS AND DISCUSSION

Organization of the Ad41 El region

6:1 Regulatory elements in the EIA gene (paper I)

The EIA region of Ad41 is slightly shorter than the corresponding regions of other adenovirus serotypes, comprising 1350 basepairs. The inverted terminal repeat (ITR) at the 5'end of the Ad41 genomic DNA consists of 163 nucleotides, being similar to the ITR of Adl2 (subgenus A) and the closely related Ad40, and longer than the ITRs of adenoviruses of subgenera B, C, and E. The terminal sequences are biologically important because the origin and termination of DNA replication are situated in these regions. Initiation of DNA replication takes place at the two ends of the viral genome with about the same frequency (Lechner and Kelly, 1977). After each initiation event, a daughter strand is synthesized in the 5' to 3'direction, displacing one of the parental strands. Upon completion of the first daughter strand, the displaced parental strand serves as template for the synthesis of a second daughter strand. Initiation of DNA replication occurs by the covalent association of the 5' terminal nucleotide of the nascent chain, dCMP, to the viral terminal protein. This process requires the presence of the terminal protein, the viral 140-kD DNA polymerase, the viral 72 kD DNA-binding protein (DBP) and a cellular protein, nuclear factor I (NFI). A 10 bp sequence conserved in all human Ad serotypes is also essential for initiation to occur (Tamanoi and Stillman, 1983). This sequence may represent the binding site for one of the viral proteins involved in initiation (Rijnders et al. 1983). In Ad41 this 10 bp conserved sequence lies between nucleotides 9-18. Adjacent to this conserved domain is the domain containing a specific binding site for NFI. The consensus sequence recognized for NFI, (T)TGG(A/C)N5GCCAA, appears to consist of two symmetrical half sites separated by a spacer of five nucleotides. The tentative recognition site in the Ad41 origin, CTGGAAACGAGCCAA (nt 25-39), (Fig. 6) differs from the canonical sequence in only one nucleotide. The NFI has been shown to be identical to transcription factor CTF, which is responsible for selective

25

recognition of eukaryotic promoters that contain the sequence CCAAT (Jones et al. 1987).

Nuclear factor III (NFIII) is another DNA-binding protein that enhances activity of the adenovirus origin of replication (Prujin et al. 1987,1988). NFIII recognizes the octamer/decanucleotide sequence ATGCAAAT(NA). This sequence is shared with a number of cellular promoters of important genes such as those for human interferon beta and immunoglobulin Vr or Vjj . A sequence in Ad41 very similar to this octamer is found at nt 41-48 and reads ATGATAAT(GA). An identical sequence is also found in the same position in the Ad2 origin (Fig. 6).

The EIA origin also has two potential binding sites for the ATF/CREB factor. The binding site (consensus GTGACGT(A/C)(A/G)) is identical to a sequence found in promoters that are induced by cAMP, such as that of the somatostatin gene. In addition, this sequence has been shown to mediate cAMP inducibility and to bind the 43 KD phosphoprotein CREB (Montiminy and Bilezikjian,1987). Three sites with the sequence GTGACGT in the Ad5 EIA promoter have been shown to interact with the ATF/CREB factor (Hardy and Shenk, 1988; Lin and Green, 1988). This sequence is also found at two positions, nt 78-84 and nt 118-124 in the Ad41 origin. A third potential site for ATF/CREB binding (GTGACGG) is also found in the 3'end of the ITR at positions 158-163 in the Ad41 genome (Fig. 6).

The adenovirus EIA transcriptional control region contains a complex array of regulatory elements. For example, the Ad5 has an enhancer region which is composed of at least three distinct enhancer elements. Enhancer element I is repeated at -300 and - 200 relative to the EIA capsite at +1, and shares sequence similarities with elements in several eukaryotic enhancer regions (Hearing and Shenk, 1983). Element I specifically enhances EIA transcription in virus-infected HeLa cells. The sequence of adenovirus enhancer element I (consensus sequence AGGAAGTGACA) is highly conserved among different serotypes. In Ad41 the tentative enhancer element I is represented by two 11-bp repeats CGGAAGTTGAA and CGGAAGTGACG, located 188 and 288 bp respectively, upstream the suggested capsite for initiation of transcription.

26

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Recently a cellular enhancer-binding protein, termed EF-1A has been identified (Bruder and Hearing, 1989), that binds to the EIA enhancer I of Ad5 in vitro. EF-1A binds to two adjacent and related sequence motifs (CGGAAGT), one of which is the upstream copy of enhancer element I in Ad5. In Ad41a a putative binding site for EF-1A is found at the same position (nt 153-159) in the upstream copy of enhancer element I together with a similar sequence (CGGAAAT) at position 185-191 (Fig. 6).

A second enhancer element, element II, is located between the two copies of element I in Ad5 and modulates the activity of all other early transcription units (E1B, E2, E3, and E4) in cis (Hearing and Shenk, 1986). The enhancer element n in Ad5 is arranged as two sets of direct repeats that are inverted relative to each other. The consensus for this repeated sequence is (C/G)GCG(A/T)AA. Enhancer mutants having the most dramatic effect on element n function have a deletion of two or more copies of this repeated sequence. Mutants having a less dramatic effect on element II function present a deletion in only one copy of this sequence (Hearing and Shenk, 1986). By sequence comparison only, we could not identify sequences in the Ad41 EIA origin that could correspond to the enhancer element II.

A third enhancer element, the E2F-binding site (concensus TTTCGCG(C/G), is repeated at positions -285 and -220 in Ad5. E2F DNA-binding is induced by adenovirus infection and the E2F site at -285 has been shown to be responsible for the ElA-mediated stimulation of the EIA gene (Kovesdi et al. 1986,1987). Neither binding position can be identified in Ad41 only from sequence information.

In general, in the proximal promoter region, TATA and CAAT boxes are evident and DNA sequences surrounding these boxes together with the capsite are highly conserved among human adenoviruses (van Ormondt and Galibert, 1984). The putative TATA-box in Ad41 is found 31 bp upstream of the initiation of transcription as TATTTA, and the capsite is found as CCACTCTT with the cap position at nt 440. The CAAT box of Ad41 EIA is probably

28

located at position 369 as CAAAGT, 70 bp upstream of the transcription initiation site.

6:2 Identification of the EIA signal sequence (paper D

Three major mRNA transcripts (13S, 12S and 9S) and two minor transcripts (10S and 1 IS) are transcribed from the EIA unit in Ad2. By sequence analysis of Ad41 only two mRNAs can be predicted corresponding to the EIA 12S and 13S of Ad2. The splicing events which generate these mRNAs take place within their coding regions. The donor and acceptor splice sites observed in Ad41 resemble consensus sequences. No specific splice signals can be found in Ad41 for the 9S, 10S and 1 IS mRNAs. To investigate the structures of the EIA mRNAs the technique of cDNA-PCR (polymerase chain reaction) was utilized (unpublished data). cDNA was made using hexamer primers followed by PCR amplification using a primer pair which flanks the 5' and 3'ends of the Ad41 EIA gene. Only one product of 850 bp could be identified, corresponding in size to the 13S mRNA. Sequencing analysis confirmed the splice donor and acceptor sites at nt positions 1027 and 1106, giving evidence for the existence of a 13S mRNA product (Fig. 7). These results corroborate previous data presented by van Loon et al. (1987a) showing that only one EIA mRNA species is synthesized in Ad41 transformed cells. It has also been shown that the transcriptional activation function of the Ad41 EIA protein(s) is less active than that of the Ad5 EIA proteins in HeLa-cells (van Loon et al. 1987b). This might be the result of the absence or low transcriptional levels of one or several EIA products.

The Ad41 13S mRNA product can be translated into a 251-aa putative protein. The 13S mRNA of Ad2 codes for a protein of 289aa, which has separate domains that induce cell DNA synthesis (CD1), mitosis (CD2), transformation and transcriptional repression (CD1 + CD2) or transcriptional activation (CD3). These three conserved domains could also be detected in Ad41 in a comparison of the Ad41 251-aa protein with the corresponding peptides of Adl2, Ad7, Ad5, and Ad4 (Fig. 8).

29

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Ad41 Ad4 Ad 5 Ad7 Ad 12

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Fig. 8. Alignment o f deduced polypeptide sequences coded by EIA 13S mRNAs from Ad41, Ad4, Ad5, Ad7, and Adl2. The deduced amino acid sequences o f the different adenoviruses are aligned to identify the conserved regions CS1-CS3, by introducing gaps in the appropriate places. The alignment is adjusted to obtain a maximium of homologous amino acids within the three conserved regions, which are boxed in the Figure. The occurrence o f identical amino acids in the sequence o f the Ad4, Ad5, Ad7, and Adl2 EIA 13S polypeptide are indicated by dots underneath the corresponding amino acids in the Ad41 EIA 13S polypeptide.

The short second conserved region, CD2, spans 19 amino acids in Ad41 (amino acids 95-114) and shows a homology of more than 60% with the other four

31

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serotypes compared. The CD2 region is particularly acidic in all five serotypes, with a-helix structures in both ends. Of eight conserved amino acids, Ad41 differs only in one, which represents a conversion from aspartic acid to glycine at position 108. Ad41 exhibits a unique insertion of alanine at position 111, which can not be found in any of the other analyzed serotypes.

The CD3 region is adjacent to CD2 in Ad5, whereas in the other serotypes compared there is an intervening stretch of 15-22 amino acids residues between CD2 and CD3. The number of conserved amino acids in CD3 is very high. Eighteen out of 48 amino acids, are common to all five serotypes compared, including four specific cystein residues responsible for possible metal-binding (amino acids positions 151, 154, 168, and 171 in Ad41).Adenovirus types 4,5,7 and 12 have an additional set of five conserved amino acids which is not shared by Ad41.A theoretical colorimetric plot was made to compare the tentative structure in the three conserved regions of the 13S translation products of Ad41, Ad4, Ad5, Ad7, and Adl2, according to Robson et al. (1983)(Fig 9a). A similar plot was also made to compare the amino acid side-chain properties of the same set of conserved protein domains, to assess the degree of similarity in acidic and basic properties (Fig 9b).

Fig. 9. A: Outline o f protein structures according to Robson et al. (1983). Comparative analysis of the conserved sequences 1-3 (CS1-CS3) within the EIA 13S mRNA product o f Ad41, Ad4, Ad5, Ad7, and Adl2. B: Amino acid chain properties. Comparative analysis o f the same set o f adenovirus types. The conserved sequences CSI, CS2 and CS3 o f AD41 are located between amino acid residues 38 and 71, 95 and 114, and 136 and 186, respectively. In all five types CS1 has a quite hydrophilic character with a predominance o f a-helix structures. The C-terminal portion o f CS2 consists o f a cluster o f acidic amino acids that preceeds a stretch o f hydrophobic residues. This structure is reminiscent o f the acidic character and o f the negatively charged amphipathic a- helices observed in many activating region sequences o f eukaryotic transcriptional activators (Ptashne et al. 1988). CS3 exhibits a hydrophobic character with abundant ß-sheet structures and this domain is also considerably more basic than EIA proteins overall.

One property that does appear to depend on sequences outside the three highly conserved domains is the rapid nuclear localization of the EIA products (Lyons et al. 1987). The EIA C-terminus in every adenovirus serotype sequenced

33

consists of a short stretch of predominantly basic amino acid residues, not strictly conserved, but similar to SV40 and polyoma T-antigen internal sequences required for nuclear localization. Adenovirus types 41, 5, 7, and 12 contain three basic amino acids in the 3'end, and Ad4 contains as many as five.

6:3 Regulatory elements in the Ad41 E1B and pIX genes (paper II)

The nucleotide sequence of the Ad41 strain D389 E1B region was determined. When compared to the corresponding region of the Ad41 prototype strain (Tak) the degree of homology was close to 100%. In the following section the presence of regulatory elements in the Ad41 E1B promotor region will be discussed based on the sequence comparison with the corresponding region of Ad2/5.

In contrast to the EIA promoter, the regulatory region of the Ad2 E1B transcriptional unit appears to be a rather short and simple eukaryotic polymerase II promoter, requiring only two sequence elements for proper regulation, the TATA box and a binding site for the transcription factor Spi (Wu et al. 1987). A sequence [consensus (G/T)(G/A)GGCG(G/T)(G/A) (G/A)(C/T)] known to be the high affinity binding site for the Spi transcription factor (Briggs et al. 1986) is localized between nucleotide -49 to -38 in Ad5. A similar sequence (TCCGCGGGTA) is found in the Ad41 E1B promoter region at positions -36 to -27 (Fig. 10).Adjacent to the predicted Spi site in Ad41 a TATA-box motif is found as TATATAA at position 1387. The E1B promoter is stimulated 5 to 10-fold by EIA expression in Ad5, although there is no evidence for an ElA-specific target sequence. However, mutants with an interrupted TATA box were found to be less responsive to activation by EIA (Wu et al. 1987). This suggests that EIA gene products may not interact with specific sequences, but may act on the E1B promoter via host cell factors, e.g. Spi and TATA box-binding factors.

A sequence, TGCATGGCG, between nucleotides -65 and -57 is present in the coding strand of Ad5. The complementary sequence, CGCCATGCA, has a strong homology to the CAAT-box promoter element identified in other viral promoters, such as the HSV-1 TK promoter (Graves et al. 1986). It might

34

therefore be postulated that this sequence in the E1B promoter is a binding site for a CAAT-box binding factor. Such a sequence is not found in the Ad41 E1B promoter region; neither is the consensus pentanucleotide sequence CCAAT that many eukaryotic promoters possess between 60 and 80 bp upstream the initiation site (Chodosh et al. 1988).

Ad 5

■ w -

ni (CAAT) Spi TATA c o p s ite

0 M - ^ ----------1n t 1702

Ad 41in Spi TATA c a p s i te

■L 1n t 1414

Fig. 10. Location o f the putative binding sites for cellular regulatory factors in the E1B region in Ad41 as compared to Ad5.

By footprinting experimens with crude nuclear extracts from infected cells, Parks et al. (1988), detected four additional protein-binding sites (designated I through IV) in the E1B control region of Ad5, located between positions -250 and -120. This distal region was sufficient for stimulation of transcription in vitro but only in the absence of the Spi binding site. In the region between positions -254 and -235 in Ad5, site I is similar to sequences found 5' to all of the adenovirus type 2 and 5 early genes, in two late promoters of these two serotypes, and 5' to the adenovirus type 12 EIA and E1B genes. The site I consensus sequence, GTGGGCG(T/C)NG is not found 5' to the Ad41 E1B gene.Site II-like sequences (consensus GTGG(A/T)(A/T)(A/T)G) are found in the distal control regions of E1B genes in adenovirus types 12, 7, 5 and 4, (subgenus A,B,C, and E) but not in Ad41. The site II contains the "core"

35

sequence found in many enhancers, which has been implicated directly in enhancer function (Hearing and Shenk, 1983; Weiher et al. 1983).Site HI in the E1B promoter region between positions -177 and -157 in Ad5 (CCCCATGCA) is similar to sequences in the control region of the adenovirus type 5 E3 gene, E1B of Ad4 and the SV40 and polyomavirus enhancers. In SV40 this sequence is protected from DNase I digestion and mutations in this site reduce the enhancer activity (Zenke et al. 1986). In Ad41 a sequence (CCCCATGCA) similar to the one in Ad5 is found between positions -211 to - 204 (Fig. 10).Site IV contains a sequence like the enhancer core (TGTGGTTA in Ad5). Similar sequences are found in analogous positions in the control regions of the Ad4 and Adl2 E1B genes as well as in the Ad41 E1B gene (-122 TGTCCTTA - 115)(Fig. 10). Deletions of the adenovirus type 12 sequences involving site IV drastically reduce E1B expression from transfected cells (Bos and ten Wolde- Kraamwinkel, 1983).

The adenovirus pIX gene promoter is remarkably similar to the E1B promoter, in that it requires consensus Spi and TATA-box protein binding sites (Babiss and Vales, 1991). In the Ad41 pIX promoter a tentative Spl-binding site is found at positions 3150-3160 (GGGGCGGAGC), 10 nucleotides upstream a TATA-box motif.

6:4 The E1B transcription map of Ad41 (paper II)

The transcription map of the Ad41 E1B is similar to the E1B maps of other human adenoviruses. The mRNAs from the E1B region of the Ad41 strain D389 were studied by Northern blot, PCR-cDNA analysis, and primer extension.Three fragments could be detected in the hybridization of an Ad41 ElB-specific probe to mRNA transferred to mRNA affinity filters. The fragments corresponded in size to the 14S, 22S, and the 9S mRNAs of the Ad2 E1B gene. The experiment was carried out with RNA isolated every sixth hour for up to 42 hours post infection. Synthesis of E1B specific mRNAs was not detected until 18h post infection.Ad41 E1B transcripts corresponding to Ad2 14S, 22S, and 9S mRNAs were also identified by cDNA-PCR and sequencing analysis but no 13S mRNA

36

equivalent was detected. In Ad2 the 22S and 13S mRNAs are abundant, whereas the 14S is only a minor species.

By primer extension analysis the 5'end, the capsite, of the E1B mRNAs was located at nucleotide position 1414 as a T. The capsite of the pIX mRNA was located at several different positions (at least five), all representing A-residues between positions 3188 and 3197 in the genomic DNA-sequence. This results suggest that the pIX mRNA uses more than one position as a capsite.

The main differences noticed in the transcription map of Ad41 E1B are the relative abundance of a 14S mRNA and the presence in it of an additional small exon of 23 bp, created by a donor and an acceptor splice site located at positions not seen in other E1B mRNAs of previously analysed human adenovirus. (Fig 7). The sequences around the additional splice sites do conform to splice consensus sequences suggesting that they are formed by a conventional splicing mechanism.cDNA sequencing of both Ad 12 (Virtanen et al. 1982) and the enteric Ad40 (Steinthorsdottir and Mautner, 1991) did also reveal a 14S mRNA but not a 13S mRNA.In Ad2 the 14S mRNA encodes a 16.5-18K protein product which overlaps with the 35K E1B polypeptide but lacks a 340 amino acid long internal segment which is deleted in a splicing event (Anderson et al, 1984). In Ad2 this 16.5- 18K protein can not substitute the 55K protein in RNA transport or host shutoff during infection (Babiss and Ginsberg, 1984; Babiss et al. 1985; Pilder et al. 1986). Since a function has not yet been ascribed to this protein in infection or transformation, it is very difficult to explain the abundance of the 14S mRNA in Ad41. A protein product from this mRNA with a coding potential of 122 amino acids corresponding to the Ad2 16.5-18K protein can be predicted but the protein has not been demonstrated. However, a product translated from an Ad40 E1B 13S or a 14S mRNA has been identified in Ad40 infected KB16 cells by immunoprécipitation with antisera raised to a synthetic oligopeptide corresponding to the N- and C-terminal sequences of the putative protein (Mautner et al. 1990).The possible implications of the presence of the additional exon at positions 2471-2493 in Ad41 remain to be determined.

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In Ad40-infected cells a novel type of E1A-E1B cotranscript was found (Steinthorsdottir and Mautner, 1991). This cotranscript consisted of a short fragment from the 5'end of EIA linked to the E1B region 4-5 nt downstream of the major capsite. The splice sites around the E1A-E1B junction in the cotranscripts were found not to be formed by a conventional splicing mechanism. No such cotranscripts were found in Ad41 infected cells by cDNA- PCR analysis.

Detection of adenoviruses

6:5 Detection of adenoviruses by hybridization (paper m )

The genome of the Ad41 strain D389 was cloned and the cleavage sites in the genome for restriction endonucleases BamHI, EcoRI, Hpal, Nrul, Pvul, and Sail were mapped. Eight of the 11 possible BamHI fragments were isolated and cloned into the pBR322 vector. Two of these clones representing the left (BamHI-J) and the right portion (BamHI-H) of the EIA gene, respectively, were found to be particularly useful for the detection of adenoviruses.The BamHI-J clone located between map units 0.3-1.8 in the Ad41 genome contains highly conserved DNA with homology to members of all six human adenovirus subgenera. This was confirmed by both dot blot and Southern blot hybridizations in which the BamHI-J clone was ^-P-labeled and used as probe against genomic DNA from Adl2, Ad31 (subgenus A), Ad3, Ad7 (subgenus B), Ad2, Ad5, (subgenus C), Adl3, Adl9 (subgenus D), Ad4 (subgenus E) and Ad40 and Ad41 (subgenus F). However, the hybridization signals were weak with the DNA from the members of subgenus A, both by dot-blot and Southern blot detection, indicating a lower homology between subgenus A and subgenus F serotypes in this particular region.The second clone, BamHI-H, located between map units 1.8-3.6 in the Ad41 genome, turned out to be specific for enteric adenoviruses but showed a higher sensitivity in the detection of Ad41. This was confirmed by dot-blot and Southern blot hybridization using the -^P-labeled BamHI-H clone as a probe against the same collection of adenoviruses as described above.

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The unique properties of these two different clones make them suitable as selective probes for the diagnosis of adenovirus infections, since adenoviruses in general, EAd, and in particular Ad41 could be detected. However, a higher sensitivity can be achieved by the use of the different PCR systems that will be discussed in the following sections.

6:6 Polymerase chain reaction for detection of adenoviruses (papers IV and V)

The polymerase chain reaction (PCR) is a technique by which DNA can be amplified by enzymatic synthesis (Mullis and Faloona, 1987; Saiki et al. 1985,1988) (Fig 11). The target DNA is denatured by heating (92-95°C). Two sequence specific oligonucleotides with opposite direction, flanking a predetermined stretch, are allowed to anneal to the single-stranded DNA at 30- 70°C. A heat-stable DNA polymerase starts to synthesize a new strand (72°C), by using the annealed oligonucleotides as primers. The size of the resulting amplification product is determined by the distance between the oligonucleotide primers. The three steps in the cycle: dénaturation, annealing, and extension typically go through 30-40 cycles and the amount of amplification product increases exponentially, since the product itself functions as a target for annealing and subsequent extension. The amplification products can be visualized by gel electrophoresis followed by ethidium bromide staining or by hybridization as in the other systems.

The PCR method for amplification could either be designed to specifically detect enteric adenoviruses or to amplify adenovirus types representing all human subgenera.

6:6:1 Strategy for design and use of oligonucleotide primers for PCR detection of adenovirus DNA.

• Select the target for amplification* A specific subgenus e.g. F, representing enteric adenoviruses* A specific serotype e.g. Ad40 and Ad41* All human adenoviruses representing subgenus A-F

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• Select the genome region to be amplified* EIA or E1B for specific identification of adenoviruses* The coding region of the hexon gene for identification of adenoviruses

in general

• Compare the DNA sequence of the selected region between different adenovirus types* for type-specific primers find regions of low similarity* for general primers find region with high similarity

• Check if the selected primer sequence can be found in any other adenovirus sequence

• Check if the selected primer sequence can be found in DNA sequences of other virus families

• Check the primer construction concerning:* GC/AT content* 3'end* internal homologies* dissociation temperature

• Combine two primers making up a pair considering:* homologies between the two primers* a balanced dissociation temperature* the distance between the primers, which determines the size of the

amplification product

• Perform a PCR with the primer pair using genomic adenovirus DNA* optimize the PCR conditions concerning buffer, Mg^+ concentration,

temperature, number of cycles* check the specificity of the primer pair* check the sensitivity of the primer pair

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• Perform a PCR under routine conditions with a pilot panel of clinical samples.

Principles of PCR

Cycle 1Step 1. Dénaturation of target DNA

Step 2. Annealing of primers —»_________________________

Step 3. Elongation of DNA

Cycle 2Step 1. Dénaturation of DNA

Step 2. Annealing of primers

Step 3. Elongation of DNA

Fig. 11. Polymerase chain reaction: Principles o f the method.

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6:6:2 Design of general primers for amplification of adenovirus DNA (papers IV and V)

The hexon gene which codes for the main structural component in the adenovirus capsid, was selected as a target for evaluation of general primers. The DNA sequences of the open reading frames of the hexon genes of Ad2 (Akusjarvi et al. 1984), Ad5 (Kinloch et al. 1984), Ad40 (Toogood et al. 1989) and Ad41 (Toogood and Hay, 1988), were examined to locate suitable target sequences for detection. The most conserved region of the hexon genes in the types compared was found in the first 300 nucleotides of the reading frame. It codes for the basal part of the 0-barrel forming PI domain within the hexon (Roberts et al. 1986a). Two short regions that show a 100% homology between the four serotypes, were selected as primer sequences. The primers were named hexAA1885 and hexAA1913 referring to the sequence positions in the Ad2 hexon region (paper IV). This pair of primers have now been shown to amplify human adenovirus DNA from 28 different serotypes including 52 different genome types (unpublished data), representing all six subgenera of human adenovirus (A-F).

A two-step amplification PCR system for detection of all adenovirus subgenera was also developed, with the aim of further increasing the sensitivity of the method (paper V). Another two conserved nucleotide sequences located within the amplimer created by hexAA1885/hexAA1913 were identified and arranged in a nested fashion to direct a second PCR step. The specificity of these primers nehexAA1893 and nehexAA1905, which flank a region between nucelotides 18937-19079 in Ad2 was tested against 12 different human adenovirus types representing all six subgenera. Amplified products were detected in all cases. The Ad2 sequence was used as a reference sequence. In matching this sequence to Ad40 and Ad41 sequences, 2 mismatches and 1 mismatch, respectively, to the upstream primer nehexAA1893, and 3 mismatches and 4 mismatches, respectively, to the downstream primer nehexAA1905 were revealed. These mismatches did not seem to affect the amplification efficiency of the two EAd DNAs.

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p o r c i n e I o v i n e

■ 1 2 3 T 2 3 4 5 I+ — m

3 0 0 b p

Fig. 12. PCR amplification ofDNA from bovine adenovirus types 1,2,3,4,6,7,8, and 9, porcine adenovirus types 1,2, and 3, and ovine adenovirus types 2,3,4, and 5, by using the general hexon primers hexAA1885 and hexAA1913. Agarose gel electrophoresis o f amplified products shows the characteristic 300 bp amplimer. Lane m = molecular weight standards (4>X 174 Haelll digest).

The general hexon primers hexAA1885 and hexAA1913 were also evaluated in the amplification of viral DNA extracted from cells infected with bovine, ovine and porcine adenoviruses (unpublished data). The amplified products exhibited the same sizes as the amplimers obtained from human adenovirus templates

43

(Fig. 12). The sequence homology in the hexon region between human and animal adenoviruses suggests that the general hexon primers hexAA1885 and hexAA1913 may serve as a useful pair for diagnosis of adenovirus infections in veterinary practice. When the different animal DNAs were tested with the internal hexon primers nehexAA1893 and nehexAA1905, only bovine Ad9 was amplified.In a comparison of the general hexon primers hexAA1885 and hexAA1913 with the corresponding sequences in the hexon gene of bovine adenovirus type 3 (Hu et al. 1984), two and four mismatches, respectively, can be found which did not seem to affect the efficiency of primer annealing. Further comparisons are not possible since no hexon genes of other bovine adenoviruses or porcine and ovine adenoviruses, have been sequenced so far.

6:6:3 Design of type-specific primers for amplification of adenovirus DNA (papers IV and V)

To detect the two enteric adenoviruses, Ad40 and Ad41, subgenus-specific primers were selected from the E1B gene. The specific pair of primers for the two EAds, 41AA142 and 41AA358, flanks the region between nucleotides 1421 and 3608 in Ad41 and the corresponding region in Ad40, creating an amplimer of 2000 bp. When the specificity of these primers was tested against 18 different human adenovirus types, representing all six subgenera, amplification was only detected when the two EAds were used as templates (paper IV).To increase the sensitivity in the detection of EAds, a pair of primers was designed to be used in a two-step amplification (paper V). This pair of primers, ne41AA206 and ne41AA356, is located between the primer pair 41AA142 and 41AA358 and flank the region between nucleotide 2061 and 3584 in Ad41 and the corresponding region in Ad40, creating an amplimer of 1500 bp. The EAd internal primers were shown to specifically amplify DNA from only Ad40 and Ad41 when tested on a collection of 12 different human adenovirus serotypes, representing subgenera A-F. The disadvantage with the two sets of primers used in the detection of EAds is the large size of the amplimers that are obtained. The length of the product to be synthesized reduces the efficiency of the PCR 10 to 100-fold compared to the hexon-primed PCR. Therefore, a two-step amplification is recommended for detection of enteric adenoviruses. To

44

distinguish between the two enteric adenovirus types by a single-step PCR reaction, type-specific primers for Ad40 were selected from the EIA region (paper IV). The pair of primers, 40AA45 and 41AA129, only amplified Ad40- specific DNA when tested on DNA from 18 different human adenovirus types representing all subgenera. The reverse primer 41AA129 binds to both Ad40 and Ad41 EIA regions, however, the specificity results from the lack of homology with Ad41 of the upstream primer 40AA45, due to a deletion in the Ad41 genome at that primer position (van Loon et al. 1987a).

6:6:4 Pretreatment of stool specimens used In the clinical studies (paper IV)

Human adenoviruses cause respiratory, gastrointestinal, urinary and ocular diseases, which means that virus particles can be isolated from different sources. The enteric adenoviruses are excreted in large amounts in feces at the acute stage of the diarrheal disease, suggesting that stool samples are suitable as targets for diagnosis of Ad40 and Ad41 infections. However, it is vital to distinguish between the enteric and other adenoviruses since some types i.e. members of subgenus C may be shed asymptomatically in stools for years after the primary infection and asymptomatic fecal shedding after respiratory infections also has been demonstrated for members of subgenus B (Fox et al. 1977). In view of these facts, stool specimens were selected as the target source to design a PCR detection system for adenovirus DNA.

All fecal specimens used in the studies were collected in a special transport medium and clarified by centrifugation for 5 min at 8.800 x g. In order to make the viral DNA template available for initiation of the PCR mediated amplification in the clinical specimens, a disruption of the adenovirus capsid is required. The chosen disruptive treatment was an incubation of the clarified stool suspension with 0.5 M NaOH for 15 min at 37°C, followed by neutraliztion with HC1 at a final concentration of 0.5 M. All PCR-reactions were performed in a 5011 aliquot of alcaline treated sample, diluted 10 times in water. The dilution was required to reduce the effect of inhibiting factors present in the stools, that may affect the Taq-polymerase.

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6:7 Detection of adenoviruses in stools of children with diarrhea (paper IV)

The excretion of adenovirus particles in stools from 60 children with rotavirus- negative diarrhea, less than 3,5 years old, was investigated by the PCR technique. Both the EAd-specific and the general primers were employed in PCRs to amplify adenovirus DNA.The PCR method was validated against isolation in tissue culture, DNA restriction enzyme analysis, and a commercial latex agglutination test (Adenolex, Orion Diagnostica, Finland) evaluated by Grandien et al. (1987). Twenty of the 60 specimens were found to be positive by demonstration of typical cytopathic effect in tissue culture and by the agglutination test performed on cell cultured supernatants. Only 2 of these samples were Adenolex positive when the test was performed directly on stools. Sixteen samples were identified and typed as adenoviruses by DNA restriction site analysis.PCR was performed on all 60 specimens both directly on diluted pretreated stool samples and on viral DNA extracted from cells inoculated with the same stool sample in parallel. When the general hexon primers (hexAA1885/hexAA1913) were used in a one-step amplification of 30 cycles, 27 out of 60 infected cell cultures were found positive. When the number of cycles was increased to 35, all together 51 samples yielded amplification products. Only 13 specimens turned out to be positive when PCR was performed directly on stools.By use of selective primers for enteric adenoviruses, 16 of the 60 cell cultured samples were found to exhibit amplification products by a one-step PCR of 30 cycles, whereas 4 were detected directly in stool samples. No further samples became positive when the number of cycles was raised to 35.None of the 60 specimens were found positive by PCR when the adenovirus type 40-specific primer pair was used.

In these experiments the PCR method was shown to be more sensitive than the agglutination method for the direct detection of adenoviruses in stools. A cell passage of the samples was necessary before a significant number of specimens became Adenolex positive.In a comparison of the procedures involving cell culturing, a cytopathic effect was seen in 30 out of the 60 infected cultures of which 20 were confirmed to be adenovirus positive by Adenolex. In PCR performed on viral DNA extracted

46

from cell cultured stools, 85% of the samples became positive using the general hexon primers, but only 27% of the samples yielded sufficient amounts of DNA to allow typing by restriction enzyme analysis. In this study Ad41, Adi, Ad2, Ad3, and Ad5 were the isolated types.

Altogether, 16 out of 60 specimens contained EAd-specific DNA. Considering that all children were under 40 months of age and suffering from diarrhea, this figure is not surprising.

6:8 Detection of persistent adenovirus infections (paper V)

In addition to acute disease, adenoviruses also cause persistent infections characterized by prolonged and intermittent fecal excretion. Approximately half of the infants and young children infected with Adi, Ad2, Ad3 or Ad5 excrete viruses for long periods, sometimes years (Fox et al. 1977).The results presented in paper IV, indicated that adenovirus particles were commonly shed in stools without being identified as the cause of illness. To further investigate the persistence of adenoviruses, 2 two-step PCR amplification assays, including two sets of general and EAd-specific primers, respectively, were used in the evaluation of 150 stool specimens from three groups including 50 healthy children, 50 healthy adults, and 50 adults suffering from diarrhea.When the two sets of general hexon primers were used, 25 of the 50 specimens from healthy children (mean age 21 months) were found positive by two-step PCR amplification. Nine of the 50 specimens from healthy adults (mean age 32 years) turned out to be positive whereas 12 of the 50 specimens from sick adults (mean age 31 years) gave amplified products, using the two sets of general hexon primers in a nested fashion. All 150 specimens were found to be negative when assayed to amplification by both the one-step PCR using hexon primes and the two-step PCR using the two sets of EAd-specific primers.The finding of adenovirus particles in 50% of the healthy children is noteworthy but not so surprising since both respiratory and enteric adenovirus infections are common during the first years of life.Adenovirus particles occured at around 20% in stools of both healthy and sick adults. The two-step PCR amplification is a very sensitive method and can

47

easily pick up small numbers of adenovirus particles. Therefore, adenoviruses should not always be identified as the cause of illness when they are isolated from or detected in the stools of an individual with disease. No amplified products were found using the two sets of EAd specific primers. This confirms the notion that infections caused by enteric adenoviruses are rarely found in adults. Information on possible persistence of enteric adenoviruses is scanty. Our experience is that EAds are shed in high amounts during the acute stage of illness, whereas the excretion declines quite rapidly after recovery. This could also explain the absence of EAd-specific products in the stools of healthy children.

6:9 A simplified procedure for detection and discrimination of enteric adenoviruses (paper VI)

As already discussed, many adenovirus types with no proven role in diarrheal diasese can be present in stools. Consequently, the capacity to discriminate the subgenus F adenoviruses from all others is an essential requirement for a diagnostic method. Except for a commercially available test that allows the identification of either Ad40 and Ad41 by the use of monoclonal antibodies (Wood et al. 1989), all other diagnostic tests with a discriminating capacity are not widely available due to limited access to reagents.The developed system is based on the amplification of the highly conserved sequence of the hexon gene by PCR using the primers hexAA1885 and hexAA1913.Adenovirus strains of 19 different serotypes, including several different genome types, representing all six subgenera, were used as targets for amplification of the corresponding conserved regions of the hexon gene. All 35 DNAs used in the study were successfully amplified rendering a product of 301 bp. The amplimers were subsequently subjected to restriction analysis with several enzymes with a four or five-base pair recognition site. By TaqI restriction of the 301 bp amplimers, subgenus F (Ad40 and Ad41) DNA could be discriminated from DNAs of adenoviruses belonging to all other subgenera. Discrimination between Ad40 and Ad41 was subsequently achieved by cleavage with either Cfol, HinPI, Maelll, MvnI and/or Rsal.

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The high degree of conservation of the region used as a PCR template reduces the likelihood of existence of restriction enzyme site polymorphism along the 301 bp sequence. Although of a different origin, the strains of Ad40 (strain DAB) and Ad41 (strain D389) employed in this study yielded the same restriction profiles as deduced from the published sequence data of strains Dugan and Tak, respectively (Toogood et al. 1989; Toogoood and Hay, 1988). However, since genomic variability has been shown to be important also in subgenus F (van der Avoort et al. 1989), it is highly advisable to use a set of two or more endonucleases in the analysis of amplified products. The amplimer typing protocol should therefore start with a Taq I restriction to recognize the enteric types and be subsequently supplemented with analysis with two or more endonucleases yielding distinctive profiles in agarose gels.

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7. GENERAL SUMMARY

V The control region of the Ad41 EIA gene resembles the corresponding regions in Ad2 and Ad5 exhibiting analogous binding sites for NFI and NFni, ATF/CREB factors, CAAT-box and TATA-box binding factors, and also 3 of the 4 enhancer elements found in Ad2.

V The Ad41 EIA DNA signal sequence encodes two mRNAs of sizes corresponding to the Ad2 12S and 13S mRNAs. Only the Ad41 13S product is transcribed at a detectable level in infected 293 and Hep2 cells.

V The translated putative product of the Ad41 13S mRNA contains 3 highly conserved domains also found in all other human adenoviruses analysed so far. These domains are responsible for many important regulatory functions during adenovirus infection.

V The Ad41 E1B and pIX regulatory regions are both simple polymerase n promoters with a TATA-box and a Spi transcription factor binding site.

V The Ad41 E1B region encodes 3 transcripts corresponding to Ad2 14S, 22S and 9S mRNAs, but no equivalent to the 13S mRNA, one of the major species transcribed in Ad2, is found.

V The main differences noticed in the Ad41 E1B transcription studies are the relative abundance of a 14S mRNA and the presence in it of an additional small exon of 23 bp, created by a donor and an acceptor splice sites located at positions not seen in other E1B mRNAs of previously analysed human adenoviruses.

V Two BamHI-clones, both located in the Ad41 EIA region were found to be useful probes for the detection of adenoviruses in general (BamHI-J) and for the specific detection of enteric adenoviruses (BamHI-H) in hybridization assays.

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V Systems for the detection of adenoviruses in general, and the specific detection of Ad40 and Ad41 by PCR were designed and found to be useful for the study of the excretion of adenoviruses in stools.

V Two-step PCRs using general primers or EAd specific primers were shown to be more sensitive than one-step PCRs for the detection of adenovirus DNA in stool specimens.

V A simplified procedure for detection, discrimination and typing of enteric adenoviruses was designed by combining PCR and restriction enzyme analysis.

V When the PCR systems were tested on a collection of 60 stool specimens from children with rotavirus negative diarrhea (<3.5 years old), 85% were found to be adenovirus positive using general primers in a one-step PCR performed on DNA extracted from cells infected with the fecal samples. 27% were Ad41 positive. None was Ad40 positive.

V By a one-step PCR performed directly on stool samples from the sameset of 60 children, 27% were adenovirus positive using general primes. 6.7% were Ad41 positive. None was Ad40 positive.

V Out of 50 examined healthy children (<3 years old) 50% wereadenovirus positive using general primers in a two-step PCR performed directly on stool specimens. None was EAd positive.

V Out of 50 examined adults (mean age 31 years) with diarrhea 24%were adenovirus positive using general primers in a two-step PCR performed directly on stool specimens. None was EAd positive.

V Out of 50 examined healthy adults (mean age 32 years) 18% wereadenovirus positive using general primers in a two-step PCR performed directly on stool specimens. None was EAd positive.

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8. CONCLUDING REMARKS

The absence (or low transcription levels) of the EIA 12S mRNA found in Ad41 might partly explain the restricted growth exhibited by this type.

The abundant transcription of the 14S mRNA and the apparent absence of a 13S mRNA exhibited by Ad41 E1B are characteristics also presented by the other fastidious adenovirus type 40.

The one-step PCR amplification of the hexon region primed by hexAA1885 and hexAA1913 is highly recommendable for routine detection of adenoviruses in stool samples. Discrimination and typing of the enteric types are easily achieved by subsequent restriction analysis of the amplification products.

A significant frequency of asymptomatic shedding has been demonstrated for some adenoviruses by several authors and was also confirmed in this thesis. The capacity to discriminate the enteric types is therefore an essential requirement for any diagnostic method intended to be used in the study of viral diarrheas in order to establish the etiology of the disease.

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9. ACKNOWLEDGEMENTS

This work was carried out at the Department of Virology, Umeå University. I wish to express my sincere gratitude to all friends and colleagues, each in their own way, who have helped and supported me during these years.

In particular I would like to thank:

Göran Wadell, my supervisor, for introducing me and guiding me through the field of adenoviruses.

Bo Albinsson for being my right-hand man (sometimes my brain), and my conscience at bad moments when I wanted to counterfeit my results.

Katrine Isaksson for skilfully and patiently typing this thesis and for being a great friend.

Rolf Sjöberg for help with photography.

My collaborators at other departments in other countries. Rosina "My goodness" Girones, Willy Spaan, and Maria Benkö.

Per Juto for collaboration and for teaching me how to discriminate between different "kremlor".

Magnus Evander for friendship and support during all these years, and for always having answers to my questions (for still being the only one who has ever invited me for canned frog's leg together with whisky).

Karin Edlund for sharing my interests in late nights and early Martinis, Kickan Lindman for sharing my interest in "snus", Johan "S" Forssell for sharing my interest in Bach, Adriana Kajon for sharing a countless number of salads and pies and for great help and support during these last months, Olov Grankvist for sharing my interest in E1B sequencing (a long time ago), Li "Q-G" Quan-gen,

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Mei Ya-fang, Gunnar Sundell and everyone else who has been at the lab for a longer or shorter period.

All the people at "Landstinget".

The Ohlsson sisters, Helena and Monika, and the other "ladies" who convinced me that poker is sometimes as important as running gels.

My dear never-ending supporting "old" friends Mia Norlén and Pia Lundberg.

My parents and my brother for their encouragement and everlasting support without understanding what I have been doing (who does?).

Last, but definitely not least, Mats, for his help, love, support, and patience during all these years (många blev det).

d

V March 28" 1992 7

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Livet är bara den ena jävla saken efter den andra.

Elben Hubbard

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10. LITERATURE CITED

Akusjärvi, G., P. Aleström, M. Pettersson, M. Lager, H. Jömvall, and U.Pettersson. (1984). The gene for the adenovirus 2 hexon polypeptide. J. Biol. Chem. 259:13976-13979.

Akusjärvi, G., U. Pettersson, and R.J. Roberts. (1986). Structure and function of the adenovirus-2 genome, in Adenovirus DNA, the Viral Genome and Its Expression, Doerfler. W., Ed., Martinus Nijhoff, Boston. pp53-95

Albert, M.J. (1986). Enteric adenoviruses: Brief review. Arch. Virol. 88:1-17.Anderson, C.W., R.C. Schmitt, J.E. Smart, and J.B. Lewis. (1984). Early

region IB of adenovirus 2 encodes two coterminal proteins of 495 and 155 amino acid residues. J. Virol. 50:387-396.

Appleton, H., M. Buckley, M.H. Robertsson, and B.T. Thom. (1978). A search for faecal viruses in newborn and other infants. J. Hyg. Camb. 81:279-283.

Babiss, L.E., and H.S. Ginsberg. (1984). Adenovirus type 5 early region lb gene product is required for efficient shutoff of host protein synthesis. J. Virol. 50:202-212.

Babiss, L.E., and L.D. Vales. (1991). Promoter of the adenovirus polypeptide IX gene: similarity to E1B and inactivation by substitution of the simian virus 40 TATA element. J. Virol. 65:598-605.

Babiss, L.E., H.S. Ginsberg, and J.E. Jr Darnell. (1985). Adenovirus E1B proteins are required for accumulation of late viral mRNA and for effects on cellular mRNA translation and transport. Mol. Cell. Biol. 5:2552-2558.

Berg, J.M. (1986). Potential metal-binding domains in nucleic acid binding proteins. Science 232:485-487.

Berk, A.J. (1986a). Adenovirus promoters and EIA transactivation. Annu. Rev. Genet. 20:45-79.

Berk, A.J. (1986b). Functions of adenovirus EIA. Cancer Surv. 5:367-389.Berk, A.J. (1989). Regulation of eukaiyotic transcription factors by post-

translational modification. Biochim. Biophys. Acta 1009:103-109.Berk, A.J. and P.A. Sharp (1978). Structure of the adenovirus 2 early mRNAs.

Cell 14:695-711.Berk, A.J., F. Lee, T. Harrison, J. Williams, and P.A. Sharp. (1979). Pre-

early adenovirus 5 gene product regulates synthesis of early viral messenger RNAs. Cell 17:935-944.

Bishop, J.M. (1985). Viral oncogenes. Cell 42:23-38.Bos, J.L., and H.C. ten Wolde-Kraamwinkel. (1983). The Elb promoter of

Ad2 in mouse L tk' cells is activated by adenovirus region EIA. EMBO J.2:73-76.

Bos, J.L., L.J. Polder, R. Bernards, P.I. Schrier, P.J. van den Elsen, A.J. van der Eb, and H. van Ormondt. (1981). The 2.2 kb Elb mRNA of human Ad 12 and Ad5 codes for two tumor antigens starting at different AUG triplets. Cell 27:121-131.

Boulanger, P., P. Lemay, G.E. Blair, and W.C. Russell. (1979). Characterization of adenovirus protein IX. J. Gen. Virol. 44:783-800.

Boulanger, P.A., and G.E. Blair. (1991). Expression and interactions of human adenovirus oncoproteins. Biochem. J. 275:281-299.

56

Braithwaite, A.W., and J.R. Jenkins. (1989). Ability of p53 and the adenovirus E1B 58-lrilodalton protein to form a complex is determined by p53. J. Virol. 63:1792-1799.

Brandt, C.D., H.W. Kim, W.J. Rodriquez, J.O. Arrobio, B.C. Jeffries, E.P.Stallings, C. Lewis, A.J. Miles, M.K. Gardner, and R.H. Parrott. (1985). Adenoviruses and pediatric gastroenteritis. J. Infect. Dis. 151:437-443.

Bridge, E., and G. Ketner. (1990). Interaction of adenoviral E4 and E1B products in late gene expression. Virology 174:345-353.

Briggs, M.R., J.T. Kadonaga, S.P. Bell, and R. Tjian. (1986). Purification and biochemical characterization of the promoter-specific transcription factor, Spi. Science 234:47-52.

Brown, M., M. Petrie, and P. Middleton. (1984a). Diagnosis of fastidious enteric adenoviruses Ad40 and Ad41 in stool specimens. J. Clin. Microbiol. 20:334-338.

Brown, M., M. Petrie, and P.J. Middleton. (1984b). Silver staining of DNA restriction fragments for the rapid identifiction of adenovirus isolates: application during nosocomial outbreaks. J. Vir. Meth. 9:87-98.

Bruder, J.T., and P. Hearing. (1989). Nuclear factor EF-1A binds to the adenovirus EIA core enhancer element and to other transcriptional control regions. Mol. Cell. Biol. 9:5143-5153.

Cevenini, R., R. Mazzaracchio, F. Rumpianesi t al. (1987). Prevalence of enteric adenoviruses from acute gastroenteritis: a five year study. Eur. J. Epidemiol. 3:147-150.

Chiba, S., S. Nakata, I. Nakamura, K. Taniguchi, S. Urasawa, K. Fujinaga, and T. Nakao. (1983). Outbreak of infantile gastroenteritis due to type 40 adenovirus. Lancet 2:954-957.

Chodosh, L.A., A.S. Baldwin, R.W. Carhew, and P.A. Sharp. (1988). Human CCAAT-binding proteins have heterologous subunits. Cell 53:11-24.

Classification and nomenclature of viruses. (1991). Fifth report of the international committee on taxonomy of viruses. R.I.B. Francki, C.M. Fauquet, D.L. Knudson, and F. Brown, eds. Archives of Virology, supplementum 2, pp 140-144.

Cruz, J.R., P. Caceres, F. Cano, J. Flores, A. Bartlett, and B. Torun. (1990). Adenovirus type 40 and 41 and rotavirus associated with diarrhea in children from Guatemala. J. Clin. Microbiol. 28:1780-1784.

DeCaprio, J.A., J.W. Ludlow, J. Figge, J-Y. Shew, C-M. Huang, W-H. Lee,E. Marsilio, E. Paucha, and D.M. Livingston. (1988). SV40 large tumor antigen forms a specific complex with the product of the retinoblastoma susceptibility gene. Cell 54:275-283.

deJong, J.C., R. Wigand, A.H. Kidd, G. Wadell, J.G. Kapsenberg, C.J.Muzerie, A.G. Wermenbol, and R.G. Firtzlaff. (1983). Candidate adenoviruses 40 and 41: fastidious adenoviruses from human infant stool. J. Med. Virol. 11:215-231.

Dery, C.V., C.H. Herrmann, and M.B. Mathews. (1987). Repsonse of individual adenovirus promoters to the products of the EIA gene. Oncogene 2:15-23.

Esche, H. (1986). Early proteins by the adenovirus genome: functional aspects, in Adenovirus DNA, the Viral Genome and Its Expression, Doerfler, W., Ed., Martinus Nijhoff, Boston. 223-245.

Finlay, C.A., P.W. Hinds, and A.J. Levine. (1989). The p53 proto-oncogene can act as a suppressor of transformation. Cell 57:1083-1093.

57

Flewett, T.H., A.S. Bryden, H. Davies, and C.A. Morris. (1975). Epidemic viral enteritis in a long-stay children's ward. Lancet 1:4-5.

Fox, J.P., C.E. Hall, and M.K. Cooney. (1977). The Seattle Virus Watch. VII.Observations of adenovirus infections. Am. J. Epidemiol. 105:362- 386.

Furcinitti, P.S., J. van Oostrum, and R.M. Burnett. (1989). Adenovirus polypeptide IX revealed as capsid cement by difference images from electron microscopy and crystallography. (1989). EMBO J 8:3563- 3570.

Gooding, L.R., and S.M. Wold. (1990). Molecular mechanism by which adenoviruses counteract antiviral immune defenses. Critical Rev. Immunol. 10:53-71.

Gooding, L.R., L. Aquino, P.J. Duerksen-Hughes, D. Day, T.M. Horton, S. Yei, and W.S.M. Wold. (1991). The E1B 19,000-molecular-weight protein of group C adenoviruses prevents tumor necrosis factor cytolysis of human cells but not of mouse cells. J. Virol. 65:3083- 3094.

Grandien, M., C-A., Pettersson, L. Svensson, and I. Uhnoo. (1987). Latex agglutination test for adenovirus diagnosis in diarrheal disease. J. Med. Virol. 23:311-316.

Graves, B.J., P.F. Johnson, and S.L. McKnight. (1986). Homologous recognition of a promoter domain common to the MSV LTR and the HSV tk gene. Cell 44:565-576.

Green, M., and M. Pina. (1963). Biochemical studies on adenovirus multiplication. IV. Isolation, purification, and chemical analysis of adenovirus. Virology 20:199-207.

Green, M., J. Mackey, W. Wold, and P. Ridgen. (1979). Thirtyone human adenovirus serotypes (Adl-Ad31) from five groups (A-E) based upon DNA genome homologies. Virology 93:482-492.

Hammond, G., C. Hannan, T. Yeh, K. Fischer, G. Mauthe, and S.E. Straus.(1987). DNA hybridization for diagnosis of enteric adenovirus infection from directly spotted fecal specimens. J. Clin. Microbiol. 25:1881-1885.

Hardy, S., and T. Shenk. (1988). Adenoviral control regions activated by EIA and the cAMP response element bind to the same factor. Proc. Natl. Acad. Sci. USA 85:4171-4175.

Harlow, E., P. Whyte, B.R. Franza Jr, and C. Schley. (1986). Association of adenovirus early-region 1A proteins with cellular polypeptides. Mol. Cell. Biol. 6:1579-1589.

Hashimoto, S., N. Sakakibara, H. Kumai, M. Nakai, S. Sakuma, S. Chiba, and K. Fujinaga. (1991). Fastidious human adenovirus type 40 can propagate efficiently and produce plaques on a human cell Une, A549, derived from lung carcinoma. J. Virol. 65:2429-2435.

Hearing, P., and T. Shenk. (1985). Sequence-independent autoregulation of the adenovirus type 5 EIA transcription unit. Mol. Cell. Biol. 5:3214- 3221.

Hearing, P., and T. Shenk. (1986). The adenovirus type 5 EIA enhancer contains two functionally distinct domains: one is specific for EIA and the other modulates all early units in cis. Cell 45:229-236.

Herrmann, J.E., D.M. Perron-Henry, and N.R. Blacklow. (1987). Antigen detection with monoclonal antibodies for diagnosis of adenovirus gastroenteritis. J. Infect. Dis. 155:1167-1171.

58

Herrmann, J.E., N.R. Blacklow, D.M. Perron-Henry, E. Clements, D.N.Taylor, and P. Echeverria. (1988). Incidence of enteric adenoviruses among children in Thailand and the significance of these viruses in gastroenteritis. J. Clin. Microbiol. 26:1783-1786.

Hierholzer, J.C., R. Wigand, L.J. Anderson, T. Adrian, and J.W.M. Gold.(1988a). Adenovirus from patients with AIDS: a plethora of serotypes and a description of five new serotypes of subgenus D (types 43-47). J. Infect. Dis. 158:804-813.

Hierholzer, J.C., R. Wigand, and J.C. deJong. (1988b). Evaluation of human adenoviruses 38, 39, 40, and 41 as new serotypes. Intervirology 29:1- 10.

Hilleman, M.R., and J.H. Werner. (1954). Recovery of new agents from patients with acute respiratory illness. Proc. Soc. Exp. Biol. Med. 85*183-188

Home, R.W., S. Bonner, A.P. Waterson, and P. Wildy. (1959). The icosahedral form of an adenovirus. J. Mol. Biol. 1:84-86.

Horwitz, M.S. (1990). Adenoviruses. B.N. Fields, D.M. Knipe, R.M. Chanock, M.S. Hirsch, J.L. Melnick, T.P. Monath, B. Roizman. eds., Virology. New York: Raven Press, pp 1723-1740.

Horwitz, M.S., J.V. Jr Maizel, and M.D. Scharff. (1970). Molecular weight of adenovirus type 2 hexon plypeptide. J. Virol. 6:569-571.

Howe, J.A., J.S. Mymryk, C. Egan, P.E. Branton, and S.T. Bayley. (1990).Retinoblastoma growth suppressor and a 300-kDa protein appear to regulate cellular DNA synthesis. Proc. Natl. Acad. Sci. USA. 87:5883-5887.

Hu, S-L., W.W. Hays, and D.E. Potts. (1984). Sequence homology between bovine and human adenoviruses. J. Virol. 49:604-608.

Huebner, R.J. (1967). Perspectives in Virology. Voi 5. M. Pollard ed.Academic Press, New York, pp 147-166.

Johansson, M.E., I. Uhnoo, A.H. Kidd, C.R. Madely, and G. Wadell. (1980). Direct identification of enteric adenovirus, a candidate new serotype, associated with infantile gastroenteritis. J. Clin. Microbiol. 12:95- 100.

Johansson, M.E., I. Uhnoo, L. Svensson, C-A. Pettersson, and G. Wadell.(1985). Enzyme-linked immunosorbent assay for detection of enteric adenovirus 41. J. Med. Virol. 17:19-27.

Johansson, M.E., M. Brown, J.C. Hierholzer, Å. Thömer, H. Ushijima, andG. Wadell. (1991). Genome analysis of adenovirus type 31 strains from immunocompromised and immunocompetent patients. J. Infect. Dis. 163:293-299.

Jones, K.A., J.T. Kadonaga, P.J. Rosenfeld, T.J. Kelly, and R. Tjian. (1987).A cellular DNA-binding protein that activates eukaryotic transcription and DNA replication. Cell 48:79-89.

Kidd, A.H. (1984). Genome variants of adenovirus 41 (subgroup G) from children with diarrhea in South Africa. J. Med. Virol. 14:49-59.

Kidd, A.H., I.L. Chrystie, J.E. Banatvala, and G. Hawkins. (1981). Infection by fastidious enteric adenoviruses in childhood. Lancet 2:371-372

Kidd, A.H., B.P. Cosgrove, R.A. Brown, and C.R. Madeley. (1982). Faecal adenoviruses from Glasgow babies. J. Hyg. Camb. 88:463-474.

Kidd, A.H., J.E. Banatvala, and J.C. de Jong. (1983). Antibodies to fastidious adenoviruses (species 40 and 41) in sera from children. J. Med. Virol. 11:333-341.

59

Kidd, A.H., F.E. Berkowitz, P.J. Blaskovic, and B.D. Schoub. (1984).Genome variants of human adenovirus 40 (subgroup F). J. Med. Virol. 14:235-246.

Kidd, A.H., E.H. Harley, and M.J. Erasmus. (1985). Specific detection and typing of adenovirus types 40 and 41 in stool specimens by dot-blot hybridization. J. Clin. Microbiol. 22:934-939.

Kidd, A.H., A. Rosenblatt, T.G. Besselaar, M.J. Erasmus, C.T. Tiemessen,F.E. Berkowitz, and B.D. Shoub. (1986). Characterization of rotaviruses and subgroup F adenoviruses from acute summer gastroenteritis in South Africa. J. Med. Virol. 18:159-168.

Kim, K-H., J-M. Yang, S-I. Joo, Y-G. Cho, R.I. Glass, and Y-J. Cho. (1990). Importance of rotavirus and adenovirus types 40 and 41 in acute gastroenteritis in Korean children. J. Clin. Microbiol. 28:2279-2284.

Kimelman, D., J.S. Miller, D. Porter, and B.E. Roberts. (1985). Eia regions of the human adenoviruses and of the highly oncogenic simian adenovirus 7 are closely related. J. Virol. 53:399-409.

Kinloch, R., N. Mackay, and V. Mautner. (1984). Adenovirus hexon: sequence comparison of subgroup C serotypes 2 and 5. J. Biol. Chem. 259:6431-6436.

Kotloff, K.L., G.A. Losonsky, J.G. Morris, S.S. Wasserman, N. Sing-Naz, and M.M. Levine. (1989). Enteric adenovirus infection and childhood diarrhea: an epidemiologic study in three clinical settings. Pediatrics 84:219-225.

Kovesdi, I., R. Reichel, and J.R. Nevins. (1986). Identification of a cellular transcription factor involved in EIA trans-activation. Cell 45:219- 228.

Kovesdi, I., R. Reichel, and J.R. Nevins. (1987). Role of adenovirus E2 promoter binding factor in ElA-mediated coordinate gene control. Proc. Nad. Acad. Sci. USA. 84:2180-2184.

Kuppuswamy, M.N., and G. Chinnadurai. (1987). Relationship between the transforming and transcriptional regulatory functions of adenovirus 2 Eia oncogene. Virology 159:31-39.

Lechner, R.L., and T.J. Jr Kelly. (1977). The structure of replicating adenovirus 2 DNA molecules. Cell 12:1007-1020.

Leite, J.P.G., H.G. Pereira, R.S. Azeredo, and H.G. Schatzmayr. (1985).Adenoviruses in faeces of children with actue gastroenteritis in Rio de Janerio, Brazil. J. Med. Virol. 15:203-209.

Leppard, K.N., and T. Shenk. (1989). The adenovirus E1B 55 kd protein influences mRNA transport via an intranuclear effect on RNA metabolism. EMBO J. 8:2329-2336.

Lew, J.F., C.L. Moe, S.S. Monroe, J.R. Allen, B.M. Harrison, B.D.Forrester, S.E. Stine, P.A. Woods, J.C. Hierholzer, J.E. Herrmann, N.R. Blacklow, A.V. Bartlett, and R.I. Glass. (1991). Astrovirus and adenovirus associated with diarrhea in children in day care settings. J. Infect. Dis. 164:673-678.

Lillie, J.W., P.M. Loewenstein, M.R. Green, and M. Green. (1987). Functional domains of adenovirus type 5 Eia proteins. Cell 50:1091- 1100.

Lin, Y-S., and M.R. Green. (1988). Interaction of a common cellular transcription factor, ATF, with regulatory elements in both Eia- and cyclic AMP-inducible promoters. Proc. Natl. Acad. Sci. USA. 85:3396-3400.

60

Lyons, R.H., B.Q. Ferguson, and M. Rosenberg. (1987). Pentapeptide nuclear localization signal in adenovirus Eia. Mol. Cell. Biol. 7:2451-2456.

Madeley, C.R., B.P. Cosgrave, E.J. Bell, and R.J. Fallon. (1977). Stool viruses in babies in Glasgow. J. Hyg. Camb. 78:261-273.

Mautner, V., N. Mackay, and V. Steinthorsdottir. (1989). Complementation of enteric adenovirus type 40 for lytic growth in tissue culture by E1B 55K function of adenovirus types 5 and 12. Virology 171:619-622.

Mautner, V., N. Mackay, and K. Morris. (1990). Enteric adenovirus type 40: expression of E1B mRNA and proteins in permissive and nonpermissive cells. Virology 179:129-138.

Mavromichalis, J., N. Evans, A.S. McNeish, A.S. Bryden, H.A. Davies, and T.H. Flewett. (1977). Intestinal damage in rotavirus and adenovirus gastroenteritis assessed by D-zylose malabsorption. Arch. Dis. Child. 52:589-591.

McGlade, J., M.L. Tremblay, S-P. Yee, R. Ross, and P.E. Branton. (1987) Acylation of the 176R (19-kilodalton) early region IB protein of human adenovirus type 5. J. Virol. 61:3227-3234.

Middleton, P.J., H.T. Szymanski, and M. Petrie. (1977). Viruses associated with acute gastroenteritis in young children. Am. J. Dis. Child. 131:733-737.

Montminy, M.R., and L.M. Bilezikjian. (1987). Binding of a nuclear protein to the cyclic-AMP response element of the somatostatin gene. Nature (London) 328:175-178.

Moran, E., and M.B. Mathews. (1987). Multiple functional domains in the adenovirus EIA gene. Cell 48:177-178.

Moran, E., T. Grodzicker, R.J. Roberts, M.B. Mathews, and B. Zerler.(1986a). Lytic and transforming functions of individual products of die adenovirus EIA gene. J. Virol. 57:765-775.

Moran, E., B. Zerler, T.M. Harrison, and M.B. Mathews. (1986b).Identification of separate domains in the adenovirus EIA gene for immortalization activity and the activation of virus early genes. Mol. Cell. Biol. 6:3470-3480.

Mullis, K.B., and F.A. Faloona. (1987). Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Meth. Enzymol. 155:335-350.

Murphy, M., B. Opalka, R. Sajaczkowski, and H. Schulte-Holthausen. (1987).Definition of a region required for transformation in Eia of adenovirus 12. Virology. 159:49-56.

Nermut, M.V. (1984). The architecture of adenoviruses. In: H.S. Ginsberg, ed., The Adenoviruses. New York and London: Plenum Press, pp 5- 34.

Niel, C., S.A. Gomes, J.G. Leite, and H.G. Pereira. (1986). Direct detection and differentiation of fastidious and nonfastidious adenoviruses in stools by using a specific nonradioactive probe. J. Clin. Microbiol. 24:785-789.

Parks, C.L., S. Baneijee, and D.J. Spector. (1988). Organization of the transcriptional control region of the Elb gene of adenovirus type 5. J. Virol. 62:54-67.

Perron-Henry, D.M., J.E. Herrmann, and N.R. Blacklow. (1988). Isolation and propagation of enteric adenoviruses in Hep-2 cells. J. clin. Microbiol. 26:1445-1447.

Persson, H., M.G. Katze, and L. Philipson (1982). Purification of a native membrane-associated adenovirus tumor antigen. J. Virol. 42:905-917.

61

Petrie, M., S. Krajden, N. Dowbnia, and P.J. Middleton. (1982). Enteric adenoviruses. Lancet 1:1074-1075.

Pettersson, U., and R.J. Roberts. (1986). Adenovirus gene expression and replication: a historical review. In: Cancer cells, vol. 4. DNA tumor viruses. M. Botchan, T. Grodzicker, and P.A. Sharp, eds.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. pp 37-57.

Philipson, L. (1983). Structure and assembly of adenoviruses. Curr. Top.Microbiol. Immunol. 109:1-52.

Pieniazek, D., N.J. Pieniazek, D. Macejak, J. Coward, M. Rayfield, and R.B. Luftig. (1990a). Differential growth of human enteric adenovirus 41 (Tak) in continuous cell lines. Virology 174:239-249.

Pieniazek, D., N.J. Pieniazek, D. Maceeak, and R.B. Luftig. (1990b). Enteric adenovirus 41 (Tak) requires low serum for growth in human primary cells. Virology 178:72-80.

Pilder, S., M. Moore, J. Logan, and T. Shenk. (1986). The adenovirus E1B- 55K transforming polypeptide modulates transport or cytoplasmic stabilization of viral and host cell mRNAs. Mol. Cell. Biol. 6:470- 476.

Prujin, G.J.M., W. van Driel, R.T. van Miltenburg, and P.C. van der Vliet.(1987). Promoter and enhancer elements containing a conserved sequence motif are recognized by nuclear factor HI, a protein stimulating adenovirus DNA replication. EMBO J. 6:3771-3778.

Prujin, G.J.M., R.T. van Miltenburg, J.A.J. Claessens, and P.C. van der Vliet.(1988). Interaction between the octamer-binding protein nuclear factor in and the adenovirus origin of DNA replication. J. Virol. 62:3092- 3102.

Ptashne, M. (1988). How eukaryotic transcriptional activators work. Nature (London) 335:683-689.

Puerto, F.I., G.G. Polanco, M.R. Gonzales, J.E. Zavala, and G. Ortega.(1989). Role of rotavirus and enteric adenovirus in acute paediatric diarrhoea at an urban hospital in Mexico. Trans. Royal Soc. Trop. Med. Hyg. 83:396-398.

Rekosh, D.M.K., W.C. Russell, A.J.D. Bellet, and A.J. Robinson. (1977). Identification of a protein linked to the ends of adenovirus DNA. Cell 11:283-295.

Retter, M., P.J. Middleton, S.J. Tam, and M. Petrie. (1979). Enteric adenovirus: detection, replication and significance. J. Clin. Microbiol. 10:574-578.

Richmond, S.J., E.O. Caul, S.M. Dunn, C.R. Ashley, and S.K.R. Clarke.(1979). An outbreak of gastroenteritis in young children caused by adenovirus. Lancet 1:1178-1180.

Rijnders, A.W.M., B.G.M. van Bergen, P.C. van der Vliet, and J.S. Sussenbach. (1983). Specific binding of the adenovirus terminal protein precursor-DNA polymerse complex to the origin of DNA replication. Nucl. Acid. Res. 11:8777-8789.

Roberts, M.M., J.L. White, M.G. Grutier, and R.M. Burnett. (1986a). Three- dimensional structure of the adenovirus major coat protein hexon. Science 232:1148-1151.

Roberts, R.J., G. Akusjärvi, P. Aleström, R. Gelinas, T.R. Gingeras, D.Sciaky, and U. Pettersson. (1986b). A concensus sequence for the adenovirus-2 genome, in: Adenovirus DNA, the Viral Genome and Its Expression. Doerfler, W. Ed., Martinus Nijhoff, Boston, p 1-51.

62

Robson, Douglas and Garnier. (1983). Comp, in biol. sc. Eds. Geison and Barett. Elsevier press, pp 132-142.

Rosen, L. (1960). Hemagglutination inhibition technique for typing adenovirus. Am. J. Hyg. 71:120-128.

Rowe, W.P., R.J. Heubner, L.K. Gilmore, R.H. Parrott, and T.G. Ward.(1953). Isolation of a cytopathogenic agent from human adenoids undergoing spontaneous degeneration in tissue culture. Proc. Soc. Exp. Biol. Med. 84:570-573.

Rowe, D.T., F.L. Graham, and P.E. Branton. (1983). Intracellular localization of adenovirus type 5 tumor antigens in productively infected cells. Virology 129:456-468.

Ruley, H.E. (1983). Adenovirus early region 1A enables viral and cellular transforming genes to transform primary cells in culture. Nature 304:602-606.

Saiki, R.K., S.S. Scharf, F. Faloona, K.B. Mullis, G.T. Horn, H.A. Erlich, and N. Amheim. (1985). Enzymatic amplification of b-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230:1350-1354.

Saiki, R.K., D.H. Gelfand, S. Stoffel, S.J. Scharf, R. Higuchi, G.T. Horn, K.B. Mullis, and H.A. Erlich. (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487-491.

Samow, P., Y.S. Ho, J. Williams, and A.J. Levine. (1982). Adenovirus Elb- 58kd tumor antigen and SV40 large tumor antigen are physically associated with the same 54 kd cellular protein in transformed cells. Cell 28:387-394.

Schneider, J.F., F. Fisher, C.R. Goding, and N.C. Jones. (1987) Mutational analysis of the adenovirus Eia gene: the role of transcriptional regulation in transformation. EMBO J. 6:2053-2060.

Schugart, K., E. Bause, and H. Esche. (1985). Structure and expression of adenovirus type 12 E1B 58K protein in infected and transformed cells: studies using antibodies directed against a synthetic peptide. Virus Res. 3:41-56.

Shinozake, T., K. Araki, H. Ushijima, and R. Fujii. (1987). Antibody response to enteric adenovirus types 40 and 41 in sera from people in various age groups. J. Clin. Microbiol. 25:1679-1682.

Shiroki, K., and M. Toth. (1988). Activation of the human beta interferon gene by the adenovirus type 12 E1B gene. J. Virol. 62:325-330.

Shiroki, K., H. Kato, and S. Kawai. (1990). Tandemly repeated hexamer sequences within the beta interferon promoter can function as an inducible regulatory element in activation by the adenovirus E1B 19- kilodalton protein. J. Virol. 64:3063-3068.

Simon, M.C., T.M. Fisch, B.J. Benecke, J.R. Nevins, and N. Heintz. (1988).Definition of multiple, functionally distinct TATA elements, one of which is a target in the hsp70 promoter for EIA regulation. Cell 52:723-729.

Singh-Naz, N., and R.K. Naz. (1986). Development and application of monoclonal antibodies for specific detection of human enteric adenoviruses. J. Clin. Microbiol. 23:840-842.

Smith, D.H., and E.B. Ziff. (1988). the amino-terminal region of the adenovirus serotype 5 Eia protein performs two separate functions when expressed in primary baby rat kidney cells. Mol. Cell. Biol. 8:3882-3890.

63

Smith, K.J., P.H. Gallimore, and R.J.A. Grand. (1989). The expression of Adl2 E1B 19K protein on the surface of adenovirus transformed and infected human cells. Oncogene 4:489-497.

Spector, D.J., M. McGrogan, and H.J. Raskas. (1978). Regulation of the appearance of cytoplasmic RNAs from region 1 of the adenovirus 2 genome. J. Mol. Biol. 126:395-414.

Stein, R., and E.B. Ziff. (1984). HeLa cell b-tubulin gene transcription is stimulated by adenovirus 5 in parallel with viral early genes by an Ela-dependent mechanism. Mol. Cell. Biol. 4:2792-2801.

Steinthorsdottir, V. and V. Mautner. (1991). Enteric adenovirus type 40: E1B transcription map and identification of novel E1A-E1B cotranscripts in lyrically infected cells. Virology 181:139-149.

Stephens, C., and E. Harlow. (1987). Differential splicing yields novel adenovirus 5 EIA mRNAs that encode 30 kd and 35 kd proteins. EMBO J. 6:2027-2035.

Stillman, B. (1986). Functions of the adenovirus E1B tumour antigens. Cancer Surv. 5:389-404.

Stålhandske, P., R. Hypiä, A. Allard, P. Halonen, and U. Pettersson. (1985). Detection of adenovirus in stool specimens by nucleic acid spot hybridization. J. Med. Virol. 16:213-218.

Sussenbach, J.S. (1984). The structure of the genome, in "The Adenoviruses".H.S. Ginsberg, ed. Plenum Press. New York, London, pp 35-124.

Svensson, U. (1985). Role of vesicles during adenovirus 2 internalization into HeLa cells. J. Virol. 55:442-449.

Svensson, L., G. Wadell, I. Uhnoo, M. Johansson, and C.H. von Bonsdorff.(1983). Cross-reactivity between enteric adenoviruses and adenovirus type 4: analysis of epitopes by solid-phase immune electron microscopy. J. Gen. Virol. 64:2517-2520.

Takiff, H.E., M. Seidlin, P. Krause, J. Rooney, C. Brandt, W. Rodriguez, R.Yolken, and S. Straus. (1985). Detection of enteric adenoviruses by dot-blot hybridization using a molecularly cloned viral DNA probe. J. Med. Virol. 16:107-118.

Tamanoi, F., and B.W. Stillman. (1983). Initiation of adenovirus DNA replication in vitro requires a specific DNA sequence. Proc. Natl. Acad. Sci. USA. 80:6446-6450.

Tiemessen, C.T., F.O. Wegerhoff, M.J. Erasmus, and A.H. Kidd. (1989). Infection by enteric adenoviruses, rotaviruses, and other agents in a rural african environment. J. Med. Virol. 28:176-182.

Toogood, C.I.A., and R.T. Hay. (1988). DNA sequence of the adenovirus type 41 hexon gene and predicted structure of the protein. J. Gen. Virol. 69:2291-2301.

Toogood, C.I.A., R. Murali, R.M. Burnett, and R.T. Hay. (1989). The adenovirus type 40 hexon: sequence, predicted structure and relationship to other adenovirus hexons. J. Gen. Virol. 70:3203-3214.

Uhnoo, I., G. Wadell, L. Svensson, and M. Johansson. (1983). Two new serotypes of adenoviruses causing infantile diarrhoea. Dev. Biol. Stand. 53:311-318.

Uhnoo, I., G. Wadell, L. Svensson, and M. Johansson. (1984). Importance of enteric adenoviruses 40 and 41 in aucte gastroenteritis in infants and young children. J. Clin. Microibol. 20:365-372.

Ulfendahl, P.J., S. Linder, J-P. Kreivi, K. Nordqvist, C. Svensson, H.Hultberg, and G. Akusjärvi. (1987). A novel adenoviurs-2 EIA

64

mRNA encoding a protein with transcription activation properties. EMBO J. 6:2037-2044.

Ushijima, H., Y. Eshita, K. Araki et al. (1988). A study of adenovirus gastroenteritis in the Tokyo area. Eur. J. Ped. 147:90-92.

van der Avoort, H.G.A.M., A.G. Wermenbol., T.P.L. Zomerdijk, J.A.F.W.Kleijne, J.A.A.M. van Asten, P. Jensma, A.D.M.E. Osterhaus, A.H. Kidd, and J.C. deJong. (1989) Characterization of fastidious adenovirus types 40 and 41 by restriction enzyme analysis and by neutralizing monoclonal antibodies. Vir. Res. 12:139-158.

van Loon, A.E., T.H. Rozijn, J.C. de Jong, and J.S. Sussenbach. (1985).Physicochemical properties of the DNAs of teh fastidious adenoviurs species 40 and 41. Virology 140:197-200.

van Loon, A.E., M. Ligtenberg, A.M.C.B. Reemst, J.S. Sussenbach, and T.H.Rozijn. (1987a). Structure and organization of the left-terminal DNA regions of fastidious adenovirus types 40 and 41. Gene 58:109-126.

van Loon, A.E., P. Gilardi, M. Perricaudet, T.H. Rozijn, and J.S. Sussenbach.(1987b). Transcriptional activation by the EIA regions of adenovirus types 40 and 41. Virology 160:305-307.

van Oostrum, J., and R.M. Burnett. (1985). The molecular composition of the adenovirus type 2 virion. J. Virol. 56:439-448.

van Ormondt, H., and F. Galibert. (1984). Nucleotide sequences of adenovirus DNAs. Curr. Top. Microbiol. Immunol. 110(2):73-142.

Velcich, A., and E. Ziff. (1988). Adenovirus Eia ras cooperation activity is separated from its positive and negative transcription regulatory functions. Mol. Cell. Biol. 8:2177-2183.

Vesikari, T., M. Maki, H.K. Sarkkinen, P.P. Arstila, and P.E. Halonen.(1981). Rotavirus, adenovirus and nonviral enterophatogens in diarrhoea. Arch. Dis. Child. 56:264-270.

Virtanen, A., and U. Pettersson. (1985). Organization of early region IB of human adenovirus type 2: identification of four differently spliced mRNAs. J. Virol. 54:383-391.

Virtanen, A., U. Pettersson, J.M. Le Moullec, P. Tiollais, and M. Perricaudet.(1982). Different mRNAs from the transforming region of highly oncogenic and non-oncogenic human adenoviruses. Nature (London) 295:705-707.

Wadell, G. (1979). Classification of human adenoviruses by SDS- polyacrylamide gel electrophoresis of structural polypeptides. Intervirology 11:45-57.

Wadell, G. (1990). In: Zuckerman A.J., J.E. Banatvala, J.R. Pattison, eds., Principles and practice of clinical virology, 2nd ed. London. John Wiley and Sons Ltd. Adenoviruses, pp 267-287.

Wadell, G., M-L. Hammarskjöld, G. Winberg, T.M. Varsanyi, and G. Sundell. (1980). Genetic variability of adenoviruses. Ann. NY Acad. Sci. 354:16-42.

Wadell, G., A. Allard, M. Evander, and Q-G. Li. (1986). Genetic variability and evolution of adenoviruses. Chem. Scripta 26B:325-335.

Wadell, G., A. Allard, M. Johansson, L. Svensson, and I. Uhnoo. (1987).Enteric adenoviruses. In G. Bock and J. Whelan eds., Novel Diarrhoea viruses, Chichester: John Wiley and Sons, pp 63-91.

Wang, H-G.H., G. Draetta, and E. Moran. (1991). EIA induces phosphorylation of the retinoblastoma protein independently of direct physical association between the EIA and retinoblastoma products. Mol. Cell. Biol. 11:4253-4265.

65

Weiher, H., M. König, and P. Grass. (1983). Multiple point mutations affecting the simian virus 40 enhancer. Science 219:626-631.

Wemess, B.A., A.J. Levine, and P.M. Howley. (1990). Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science 248:76- 79.

White, E., and R. Cipriani. (1989). Specific disruption of intermediate filaments and the nuclear lamina by the 19-kDa product of the adenovirus E1B oncogene. Proc. Natl. Acad. Sci. USA. 86:9886- 9890.

White, E., and R. Cipriani. (1990). Role of adenovirus E1B proteins in transformation: altered organization of intermediate filaments in transformed cells that express the 19-kilodalton protein. Mol. Cell. Biol. 10:120-130.

White, E., S.H. Blose, and B.W. Stillman. (1984). Nuclear envelope localization of an adenovirus tumor antigen maintains the integrity of cellular DNA. Mol. Cell. Biol. 4:2865-2875.

Whitelaw, A., H. Davies, and J. Parry. (1977). Electron microscopy of fatal adenovirus gastroenteritis. Lancet 1:361.

Whyte, P., K.J. Buchkovich, J.M. Horowitz, S.H. Friend, M. Raybuck, R.A.Weinberg, and E. Harlow. (1988). Association between an oncogene and an anti-oncogene: the adenovirus EIA proteins bind to the retinoblastoma gene product. Nature (London) 334:124-129.

Wigand, R., T. Adrian, and F. Bricout. (1987). A new human adenovirus in subgenus D: candidate for human adenovirus type 42. Arch. Virol. 94:283-286.

Willcocks, M.M., M.J. Carter, F.R. Laidler, and C.R. Madeley. (1988).Restriction enzyme analysis of faecal adenoviruses in Newcastle upon Tyne. Epidem. Infect. 101:445-458.

Witt, D.J. and E.B. Bousquet. (1988). Comparison of enteric adenovirus infection in various human cell lines. J. Virol. Methods 20:295-308.

Wolfson, J., and D. Dressier. (1972). Adenovirus DNA contains an inverted terminal repetition. Proc. Natl. Acad. Sci. USA. 79:2221-2225.

Wood, D.J., and A.S. Bailey. (1987). Detection of adenovirus types 40 and 41 in stool specimens by immune electron microscopy. J. Med. Virol. 21:191-199.

Wood, D.J., K. Bijlsma, J.C. de Jong, and C. Tonkin. (1989). Evaluation of a commercial monoclonal antibody-based enzyme immunoassay for detection of adenovirus types 40 and 41 in stool specimens. J. Clin. Microbiol. 27:1155-1158.

World Health Organization. (1980). Viral Respiratory Diseases. Technical Report, series no 642.

Wu, L., D.S.E. Rosser, M.C. Schmidt, and A. Berk. (1987). A TATA box implicated in EIA transcriptional activation of a simple adenovirus 2 promoter. Nature (London) 326:512-515.

Yolken, R.H., F. Lawrence, F. Leister, H.K. Takiff, and S.E. Strauss. (1982). Gastroenteritis associated with enteric type adenovirus in hospitalized infants. J. Pedaitr. 101:21-26.

Zantema, A., P.I. Schrier, A. Davis-Olivier, T. van Laar, R.T.M.J. Vaessen, and A.J. van der Eb. (1985). Adenovirus serotype determines association and localization of the large E1B tumor antigen with cellular tumor antigen p53 in transformed cells. Mol. Cell. Biol. 5:3084-3091.

66

Zenke, M., T. Grundström, H. Matthes, M. Wintzerith, C. Schatz, A.Wildeman, and P. Chambon. (1986). Multiple sequence motifs are involved in SV40 enhancer function. EMBO J. 5:387-397.

Zissis, G., J.P. Lambert, and L. Fonteyne. (1981). Enteric adenoviruses and diarrhoea, Abst. no 27, presented at Int. Symp. Recent Adv. Enteric Infect., Brigge, Belgium, August 1981.

67