MRC Virology Unit, Church Street, Glasgow G11 5JR, UK1
Veterinary Medical Research Institute, Hungarian Academy of Sciences, H-1143 Budapest, Hungary2
Author for correspondence: Andrew Davison. Fax +44 141 337 2236. e-mail a.davison{at}vir.gla.ac.uk
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Abstract |
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Introduction |
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Even within this enlarged scheme, the taxonomic position of one avian virus, turkey adenovirus 3 (TAdV-3; also known as haemorrhagic enteritis virus in turkeys or marble spleen disease virus in pheasants), is problematic. Analysis of the complete genome sequence indicates that this virus is not closely related in genome organization or predicted protein sequences to any of the three genera described above (Pitcovski et al., 1998 ). However, the marginally closer relationship of certain proteins to those of OAdV-287, plus a small, A+T-rich genome, led to the tentative designation of TAdV-3 as an atadenovirus (Benkó & Harrach, 1998
).
In this paper, we report the complete genome sequence of frog adenovirus (FrAdV-1). We show that FrAdV-1 is a relative of TAdV-3 and use genome comparisons to update our understanding of the genetic content of TAdV-3 and to derive new insights into the genetic content and evolution of adenoviruses. Moreover, reconsideration of adenovirus phylogeny leads us to propose a fourth genus containing these two viruses.
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Methods |
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A lyophilized stock of FrAdV-1 (ATCC VR-896) was resuspended in 1 ml basal medium. Virus was passaged in TH-1 cells at 3031 °C, starting with an initial inoculum of 0·10·2 ml added to a 25 cm2 flask. Infected cells grew more slowly than uninfected cells and were subcultured approximately once a week, usually with the addition of uninfected cells at a ratio of 5:1 followed by replating at a density sufficient to permit further growth of the cells. Virus was isolated from infected cells and DNA was extracted from purified virions by proteolytic treatment and phenol extraction (Davison et al., 1993 ).
DNA sequencing.
Random fragments of FrAdV-1 DNA were generated by sonication, cloned into bacteriophage M13mp19 and sequenced by conventional autoradiography procedures or by using an ABI Prism 377 DNA sequencer. Data were compiled by using Stadens sequence analysis program (Staden, 1987 ).
Authentic FrAdV-1 genome terminal fragments were not represented in the M13 library, presumably because the presence of residual terminal protein residues attached to the 5' ends of virion DNA prevented ligation to the vector. Terminal fragments were instead isolated by treating an SphI digest of viral DNA with terminal transferase in the presence of dCTP (to tail the 3' ends of fragments with dC residues), annealing with pUC18 tailed at the PstI site with dG residues and obtaining appropriate clones by transformation of E. coli.
To obtain the finished sequence, each nucleotide was determined an average of 12·3 times and the entire sequence was obtained on both strands. The sequence has been deposited with the GenBank library under accession number AF224336.
DNA sequence and phylogenetic analyses.
DNA sequence analyses were carried out with the GCG package (version 9.1). Phylogenetic calculations were performed with the PHYLIP program package version 3.572c (Felsenstein, 1989 ) on manually edited forms of amino acid sequence alignments produced by CLUSTAL W. PROTPARS was used for parsimony analysis, and PROTDIST (Dayhoffs PAM matrix) followed by FITCH (global rearrangements) was used for distance matrix analysis. For bootstrap analysis, SEQBOOT (with 100 datasets output) preceded the above calculations and, after performing them, CONSENSE was used to calculate the consensus tree. Phylogenetic relationships were visualized using TreeView (Page, 1996
) as implemented previously (Harrach & Benkó, 1998
). Applicable alignments, their edited format and the calculated trees are available in Newick and graphical formats (http://www.vmri.hu/~harrach).
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Results and Discussion |
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The DNA sequence obtained is 26163 bp in size and has a G+C content of 37·9%. The inverted repeat at the genome termini (ITR) is only 36 bp long. The sequence information and the restriction endonuclease cleavage data indicated the presence of a region near the right genome end that comprises a direct repeat of an internal sequence: nucleotides 2551926127 are an exact copy of 2363124239, a region located immediately downstream of the fibre gene. This is equivalent to direct reiteration at the genome terminus of approximately the first third of the 1888 bp region between 23631 and 25518. Moreover, a partial copy of 27 bp of the ITR is located at 2548525511, immediately upstream of this reiteration. This structure indicates that the sequenced genome was originally derived by recombination from one which lacked the repeated sequence and had a size of 25517 bp. All analyses were carried out using the sequence obtained, rather than that of the hypothetical 25517 bp genome, which may or may not have been present in the initial virus stock.
Size heterogeneity of the FrAdV-1 genome was indicated by restriction endonuclease analysis of viral DNA isolated during an independent series of seven passages, starting with the stock obtained from the ATCC. This DNA contained an extra 1·9 kbp near the right end of the genome, a size increment that is consistent with an additional reiteration of 2363125518 (data not shown). This finding also indicated the likelihood that the FrAdV-1 genome as sequenced was present in the ATCC stock.
The 5'-CG dinucleotide is present in the FrAdV-1 genome at a little over half the frequency expected from the mononucleotide composition (Table 1). Four other adenoviruses listed in Table 1
exhibit a level of CG depletion similar to that of FrAdV-1: TAdV-3, canine adenoviruses types 1 and 2 (CAdV-1, CAdV-2) and OAdV-287. CG depletion in each of these genomes is essentially uniform (data not shown). The phenomenon of CG depletion in cellular genomes and in certain other DNA viruses is thought to be due to methylation of cytosine in the CG dinucleotide, followed by deamination to TG and fixation of the mutation by DNA replication. Thus, it is likely that these adenovirus genomes are exposed in their entirety to cytosine methylation at some point during their life-cycles.
Genetic complement
The deduced gene layout of FrAdV-1 is shown in Fig. 1 and details of the genes are listed in Table 2
. Amino acid sequence comparisons indicated that, of the adenoviruses for which sequence data are available, TAdV-3 is the closest relative of FrAdV-1. Data for the TAdV-3 genome are also presented in Fig. 1
and Table 2
. Overall, the two genomes are similar in size, that of FrAdV-1 constituting the smallest yet observed among the family Adenoviridae. They share the same genetic layout and are similar in nucleotide composition (Table 1
).
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The U exon was first identified from an analysis of the human adenovirus type 40 (HAdV-40) sequence as a conserved feature of several mastadenoviruses (Davison et al., 1993 ). It is apparent as a coding region extending from an initiation codon to a splice donor site in the region immediately upstream of, and on the other strand from, the fibre gene. It is now apparent for the first time that a U exon is present in FrAdV-1 and TAdV-3 and also in all aviadenoviruses and atadenoviruses for which data are available. Its genome location in representatives of each genus is shown in Fig. 1
. The alignments shown in Fig. 4
indicate that predicted amino acid sequences are conserved within, but not between, genera. In each case, the U exon is proposed to encode the N terminus of a protein, but downstream exons remain undefined. The current state of analysis indicates that the U exon is present in all sequenced adenoviruses except two mastadenoviruses, murine adenovirus type 1 (MAdV-1) (Meissner et al., 1997
) and bovine adenovirus type 10 (BAdV-10) (K. Ursu, B. Harrach, B. Adair and M. Benkó, unpublished results). These observations are most satisfactorily interpreted as due to inheritance of an ancient exon, with loss in certain lineages. Expression of the U exon is yet to be investigated in detail in any adenovirus genome.
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In addition to proposed splicing in the protein-coding regions of the pTP, DBP, U exon and 33K genes, the initiation codons of most FrAdV-1 and TAdV-3 genes with mastadenovirus counterparts that encode late proteins are preceded close upstream by potential splice acceptor sites, as expected. It is anticipated that these viruses express other coding and non-coding exons that cannot easily be located merely by examination of sequence data. Promoter elements, including the major late promoter, also could not be identified with confidence in the A+T-rich genomes.
Adenovirus evolution
Of the 22 genes proposed for FrAdV-1 and TAdV-3, 16 have counterparts in all sequenced adenoviruses. In general, conserved genes are those involved in DNA replication and virion formation. This constitutes strong evidence that extant adenoviruses evolved from an ancestral virus that contained these genes and was recognizably an adenovirus. Although not a complete gene, the U exon is probably also an ancient feature.
The genome comparisons shown in Fig. 1 highlight the evolution of lineage-specific genes at the genome ends. For example, at the left end, these include the E1A genes in mastadenoviruses, the p32K gene in atadenoviruses and the sialidase-related gene in the fourth genus. Evidence that mastadenoviruses and atadenoviruses may have diverged more recently than the other genera is based on weak similarities between the E1B genes and between one of the E4 genes (encoding the 34K protein). In addition, lineage-specific, unrelated genes have arisen in the E3 region of mastadenoviruses and in FrAdV-1 and TAdV-3. It is quite possible that the common ancestor also had genes in these regions (perhaps evolutionarily unrelated to those in extant adenoviruses), but it is likely that an earlier progenitor lacked them and had a genome of approximately 20 kbp. Additional genes are present at the gene V and IX loci of mastadenoviruses and probably arose at an early stage within that lineage.
The means by which lineage-specific genes have developed is obscure. Gene capture is evidenced by the presence of a deoxyuridine triphosphatase (dut) gene in aviadenoviruses (Chiocca et al., 1996 ; Fig. 1
) and possibly in certain mastadenoviruses and the sialidase-related gene in FrAdV-1 and TAdV-3. Indeed, this may be the major means whereby additional genes have been incorporated, followed by the development of novel functions through divergence. Other possible mechanisms include generation of genes de novo and duplication and divergence of genes that were either captured or generated de novo.
The phylogeny of mastadenoviruses (Fig. 2) is largely similar to that of their hosts, supporting the view that most of these viruses have evolved exclusively with their hosts or have switched hosts only between closely related species. The relationships within the atadenoviruses or the fourth genus do not fit comfortably with this model, however, and instead suggest that certain lineages in both genera may have originated with drastic interspecies transmission events. In exploring this further, it is worthwhile recollecting that Clark et al. (1973)
isolated FrAdV-1 from a renal tumour obtained from a leopard frog (Rana pipiens) by growth on a reptilian cell line (TH-1) known to be particularly susceptible to adenovirus infection. The virus did not cause cytopathic effect on any other reptilian cell line or on cell lines of mammalian, avian, fish or amphibian origin, and did not result in pathology when injected into tadpoles or into chick embryos. Consequently, these workers undertook reisolation experiments to confirm that the virus originated from the tumour. Their ability to isolate the agent repeatedly and only from the tumour tissue constitutes good evidence that FrAdV-1 is indeed a frog adenovirus. Nevertheless, the current lack of additional information about this or other amphibian adenoviruses does leave a grain of doubt. This prompts caution in regard to the proposal that interspecies transmission events are of evolutionary significance within the fourth genus. It does not, however, compromise the important insights gained from the FrAdV-1 sequence into adenovirus phylogeny and gene content.
In conclusion, robust evaluation of the parts played by coevolution and interspecies transmission requires the derivation of a timescale for adenovirus evolution and further sampling of adenovirus genomes, especially in the more sparsely populated genera. It is encouraging that the amount of available adenovirus sequence data is approaching that required for such an evaluation and that studies of other amphibian, reptilian and fish adenoviruses are likely to extend our understanding of ancient events in adenovirus evolution.
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Acknowledgments |
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Footnotes |
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b Present address: Police Forensic Science Laboratory (Dundee), Tayside Police Headquarters, West Bell Street, Dundee DD1 9JU, UK
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References |
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Received 22 May 2000;
accepted 10 July 2000.