The complete nucleotide sequence of fowl adenovirus type 8

Davor Ojkic1 and Éva Nagy1

Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, N1G 2W1 Ontario, Canada1

Author for correspondence: Éva Nagy. Fax +1 519 824 5930. e-mail enagy{at}ovc.uoguelph.ca


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The fowl adenovirus type 8 (FAdV-8) genome was sequenced and found to be 45063 nucleotides in length, the longest adenovirus (AdV) genome for which the complete nucleotide sequence has been determined so far. No regions homologous to early regions 1, 3 and 4 (E1, E3 and E4) of mastadenoviruses were recognized. Gene homologues for early region 2 (E2) proteins, intermediate protein IVa2 and late proteins were found by their similarities to protein sequences from other AdVs. However, sequences homologous to intermediate protein IX and late protein V could not be identified. Sequences for virus-associated RNA could also not be recognized. Two regions of repeated sequences were found on the FAdV-8 genome. The shorter repeat region contained five identical and contiguous direct repeats that were each 33 bp long, while the longer repeat region was made of 13 identical and contiguous, 135 bp long repeated subunits.


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Adenoviruses (AdVs) in the genus Aviadenovirus of the family Adenoviridae have a worldwide distribution and can infect many avian species (Monreal, 1992 ). Aviadenoviruses are subdivided into three groups. Group I viruses have been isolated from both healthy and sick birds and their significance as primary pathogens has not been clearly determined. Even the strains within the same serotype can vary in pathogenicity, which appears to be determined by differences in the fibre protein alone (Pallister et al., 1996 ). On the other hand, viruses that belong to groups II and III are the causative agents of diseases, such as egg drop syndrome (EDS) (McFerran, 1996 ), turkey haemorrhagic enteritis (HE) and chicken splenomegaly (Pierson & Domermuth, 1996 ).

Mastadenoviruses have been extensively investigated and their genomic organization has been well characterized. Although aviadenoviruses have been studied to a lesser extent than mastadenoviruses, the complete nucleotide sequences from chicken embryo lethal orphan (CELO) virus (Chiocca et al., 1996 ), EDS virus (Hess et al., 1997 ) and HE virus (Pitcovski et al., 1998 ) have been reported. Partial sequence data for fowl adenovirus type 8 (FAdV-8) and type 10 (FAdV-10) are published (Cao et al., 1998 ; Sheppard et al., 1998a , b ). Surprisingly, sequence comparison of aviadenoviruses with mastadenoviruses could not identify early regions E1A and E3 in EDS virus or E1, E3 and E4 in CELO virus. Intermediate protein IX-like (pIX) sequences could also not be found. Although other late regions appeared to be well conserved, protein V (pV) sequences could not be found in either CELO or EDS viruses.

The goal of this study was to determine the complete nucleotide sequence of the genome of fowl adenovirus strain A-2A (ATCC, type 8), designated as FAdV-8, and investigate its genomic organization and relationship with other AdVs. Knowledge of the complete FAdV-8 sequence will also allow us to undertake mutational and functional studies to help explain the aspects of the genomic organization unique to FAdVs.

The plaque-purified virus was propagated in a chicken hepatoma cell line (Alexander et al., 1998 ). A library of cloned, overlapping restriction enzyme DNA fragments obtained from purified virus was used for sequencing (Clavijo et al., 1996 ). The reactions were carried out with an ABI 377 automated sequencer at the University of Guelph Molecular Supercentre. Both strands of the cloned DNA fragments were sequenced and discrepancies between sequences of the two strands were resolved by repeated reactions until a consensus was achieved. The contiguous sequence was compiled from the overlapping sequences using the Laser Gene software package. The FAdV-8 genome was found to be 45063 nucleotides (nt) in length, the longest AdV genome yet determined. There were 48 open reading frames (ORFs) with a potential to encode polypeptides that were at least 100 amino acid (aa) residues long (Fig. 1). By using the GenBank BLAST server (Altschul et al., 1990 ), 26 of these ORFs were shown to share homology with protein sequences from other viruses. In addition, two FAdV-8 ORFs shorter than 100 aa were included because of their similarity to AdV protein VII (ORF17971–18252) and bovine papillomavirus type 1 (BPV-1) E5 oncoprotein (ORF1735–1890).



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Fig. 1. Schematic representation of FAdV-8 ORFs, TR-1 and TR-2. Labelled arrows represent ORFs identified according to their similarities to protein sequences from other viruses and indicate the direction of transcription; unlabelled arrows represent unidentified ORFs, unique to FAdV-8.

 
The FAdV-8 genome contained two regions of repeated sequences (Fig. 1). The shorter repeat region (TR-1) contained five identical and contiguous direct repeats that were each 33 bp long (Cao et al., 1998 ). The exact nucleotide sequence of the longer repeat region (TR-2) was determined by the transposon-based GPS-1 priming system (NEB) and was found to consist of 13 identical and contiguous, 135 bp long direct repeats. The function(s) of these repeats, if any, is not known. Seven 145 bp long tandem repeats were similarly positioned on the hypervirulent FAdV strain (CFA40) genome (M. A. Johnson & C. Pooley, unpublished results), showing 80·4% similarity to FAdV-8 TR-2.

The oncogenic potential of FAdVs, as measured by the ability of the virus to induce tumours following injection into newborn rodents, varies. Six of eleven FAdV strains, including FAdV-8 and CELO virus, can cause tumors in baby hamsters (Dhillon & Jack, 1997 ), demonstrating that these viruses possess genes encoding proteins with oncogenic properties. Previous work in our laboratory examined transcriptional profiles of 16 ORFs located in the FAdV-8 terminal regions. Fifteen of these ORFs were transcribed early, before the onset of viral DNA replication (Cao et al., 1998 ). However, based on the sequence analysis alone, the similarities found between several FAdV-8 polypeptides and proteins with transforming properties from other viruses were too weak and not sufficient to identify FAdV-8 E1 and E4. FAdV-8 ORF847–1335 showed similarity to dUTP pyrophosphatase (dUTPase) sequences. This ORF, also found in CELO virus, showed a weak similarity, 23·7%, to a transforming gene (E4 ORF1) from human AdV type 9 (HAdV-9). FAdV-8 ORF847–1335 was 69·5% similar to its CELO virus counterpart. It has been reported that CELO virus dUTPase has enzymatic activity in vitro, but its transforming potential was not investigated (Weiss et al., 1997 ).

A polypeptide encoded by FAdV-8 ORF1735–1890 showed 36·8% similarity to BPV-1 E5 oncoprotein. BPV-1 E5 protein and its FAdV-8 putative homologue also shared several structural characteristics, including a predicted alpha-helical configuration of their N-terminal ends, which contained 27 hydrophobic aa each, as well as a non-helical configuration of their C-terminal ends. One of two cysteine residues, conserved among transforming papillomavirus E5 proteins, was also present in the FAdV-8 ORF1735–1890 polypeptide. If this FAdV-8 protein indeed possessed transforming properties it might be an avian equivalent of mastadenovirus E1 oncoproteins. FAdV-8 ORF1950–2750 showed 48·1% similarity to CELO virus ORF2.

E4 could also not be identified in the FAdV-8 genome. While the right end of the FAdV-8 genome contained several leftward oriented ORFs, none of these ORFs showed homology to known E4 sequences from other AdVs. FAdV-8 ORF37859–38687 showed 30·3% similarity to a gene located on the right end of the CELO virus genome. This gene, GAM-1, encodes a nuclear protein that has functions similar to the HAdV E1B 19 K protein and can inhibit apoptosis in a functional assay (Chiocca et al., 1997 ).

Three proteins necessary for viral DNA replication, DNA polymerase (DNA pol), terminal protein precursor (pTP) and DNA binding protein (DBP), are encoded by E2. This was the only recognizable early region found on the FAdV-8 genome. E2 proteins were well conserved when compared with their CELO virus counterparts. The percentages of similarities between FAdV-8 and CELO virus DNA pol, pTP and DBP were 71·3%, 70·8% and 61·8%, respectively. The percentages of similarity between FAdV-8 E2 proteins and those from other AdVs are shown in Table 1.


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Table 1. Summary of identified FAdV-8 ORFs and similarity (%) between FAdV-8 proteins and those from other viruses

 
The proteins encoded by the mastadenovirus E3 are involved in counteracting the host immune response. E3 is found between the protein VIII and the fibre protein ORFs. This intergenic region was only 254 nt in length in FAdV-8 and contained ORF29942–30301, partially overlapping the fibre protein gene. ORF29942–30301 has a potential to encode a 120 aa long polypeptide with a molecular mass of 14·1 kDa. However, this arginine-rich protein was not similar to any proteins from other AdVs, making the identity of the E3 on the FAdV-8 genome uncertain.

Genes encoding protein IVa2 (pIVa2) and pIX were grouped as intermediate genes. The activity of the AdV major late promoter (MLP) is enhanced by pIVa2 (Tribouley et al., 1994 ). The FAdV-8 pIVa2 sequence showed 74·7% similarity to its CELO virus counterpart, whereas similarity to pIVa2 sequences from other AdVs varied between 31·4% and 38·5%. Minor capsid component pIX is essential for the packaging of viral DNA (Ghosh-Choudhury et al., 1987 ) and also possesses transcriptional properties (Lutz et al., 1997 ). Sequences homologous to pIX could not be identified in FAdV-8, as reported for other aviadenoviruses and ovine adenovirus 287 (OAdV287; Vrati et al., 1996 ).

AdV late regions are transcribed from the MLP after the onset of viral DNA replication and encode mostly structural proteins. FAdV-8 DNA replication in the hepatoma cells begins at ~10 h post-infection (Alexander et al., 1998 ). An FAdV-8 sequence that exhibits strong similarity (70·9%) to the CELO virus MLP sequence was located between nt 8173 and 8361, with a putative TATA box between nt 8345 and 8351. In general, FAdV-8 late genes were collinear with the corresponding regions of mastadenoviruses. Two core proteins, protein VII and protein X were identified on the FAdV-8 genome, but the sequence for pV, the third core protein found in mastadenoviruses, could not be recognized. The absence of the pV-like sequences was also reported for CELO and EDS viruses and OAdV287. The remaining FAdV-8 late genes were identified at positions similar to those in other AdVs (Fig. 1). FAdV-8 late polypeptides exhibited the most similarity to the CELO virus late proteins. The highest percentage of similarity was found for the hexon gene, 80·0%, whereas the lowest similarity found was for the fibre gene, only 33·9%. It was shown by electron microscopy that FAdVs contain two fibre proteins at each vertex of the virion. CELO virus has a long and a short fibre, whereas FAdV-8 has two fibres of about the same length (Gelderblom & Maichle-Lauppe, 1982 ). In contrast to CELO virus, which has two fibre protein genes, only one fibre gene was identified on the FAdV-8 genome. Interestingly, similarities between FAdV-8 late proteins and corresponding proteins from EDS and HE viruses were approximately similar or even lower than those obtained by comparing late proteins from FAdV-8 with their mammalian counterparts. The percentages of similarity between the FAdV-8 late polypeptides and the corresponding protein sequences from other AdVs are shown in Table 1. MegAlign software (DNASTAR) was used for pairwise sequence alignments.

The short, partially double-stranded virus-associated (VA) RNAs, transcribed by RNA polymerase III, play an important role during the AdV replication cycle. The functions of VA RNAs result from their interaction with protein kinase PKR, facilitating down-regulation of interferon-mediated antiviral response and selective translation of viral mRNAs in infected cells (Ghadge et al., 1994 ). Human AdVs have one or two VA RNA genes located between the pTP and 52K ORFs (Ma & Mathews, 1996 ). CELO and EDS viruses have one, much shorter VA RNA gene, located at map position ~90 (Chiocca et al., 1996 ; Hess et al., 1997 ). Based on sequence analysis and RNA secondary structure prediction a VA RNA gene could not be identified on the FAdV-8 genome. The lack of a VA RNA gene was also recently reported for OAdV287 (Venktesh et al., 1998 ).

In summary, our results are concordant with findings reported for the genomes of other aviadenoviruses. Along with FAdV-8, CELO and EDS viruses lack identifiable E1A, E3 and E4 regions as well as sequences for pV and pIX. FAdV-8, CELO virus and HE virus also lack E1B. However, there appears to be a large difference in the genome size and G+C content among the aviadenoviruses. FAdV-8 so far has the largest genome of any AdV. Group I avian AdVs, CELO virus and FAdV-8, have genomes of 43804 and 45063 nt, respectively, which are much larger than genomes from mastadenoviruses. On the other hand, EDS virus (group III) and HE virus (group II) genomes are much shorter, 33213 and 26263 nt, respectively. The G+C content of FAdV-8 is 53·9%, close to that of CELO virus (54·4%), but much higher than the G+C content of EDS virus (42·5%) and HE virus (34·9%). Existing differences among the aviadenoviruses have led to a proposal that the current AdV classification scheme should be modified (Hess et al., 1997 ; Harrach et al., 1997 ). It was suggested that group I avian AdVs be classified as genus III AdVs, and separated from other avian AdVs such as EDS and HE viruses. The two latter viruses, together with OAdV287, would be classified into AdV genus II. All the remaining mastadenoviruses would be classified into genus I. Our findings support the hypothesis (Chiocca et al., 1996 ) that, as a result of using avian species as their hosts, genes that are involved in interactions of fowl AdVs with their hosts are different from those in their mammalian counterparts.


   Acknowledgments
 
This work was supported by the Natural Sciences and Engineering Research Council of Canada and the Ontario Ministry of Agriculture, Food and Rural Affairs. We thank Dr Peter J. Krell for critical reading of the manuscript.


   Footnotes
 
The GenBank accession number of the sequences reported in this paper is AF083975.


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Received 20 January 2000; accepted 13 March 2000.