ABSTRACT
From adenovirus-like viruses originating from a fish and a snake species, a conserved part of the adenoviral DNA polymerase gene was PCR amplified, cloned and sequenced. Phylogenetic analysis showed that the snake adenovirus is closely related to the members of the proposed genus Atadenovirus, whereas the fish isolate seems to represent a separate cluster, likely a new genus.
Adenoviruses are medium-sized, nonenveloped, double-stranded DNA viruses commonly infecting humans and a wide variety of wild and domestic mammals and birds (32). Many members of the two traditional (Avi- and Mastadenovirus) genera of the family Adenoviridae are well characterized, providing a rapidly increasing number of full genomic sequences (6). Occurrence of adenovirus infection was repeatedly described in different species of lower classes of vertebrates (14). The complete DNA sequence and analysis of an adenovirus isolated from frog (Rana pipiens) (10) were recently published (13). Adenovirus-like particles were also observed in several fish and reptile species; however, none of these viruses have ever been characterized at the molecular level.
Genomic study of a snake adenovirus (SnAdV) isolate originating from a corn snake (Elaphe guttata) and a fish isolate from white sturgeon (Acipenser transmontanus) was initiated in order to get data concerning their relatedness to each other and to the better studied mammalian, avian, and amphibian adenoviruses.
The SnAdV strain 145/88 isolated from corn snake (26) was grown on the VH2 (Russell's viper heart) cell line, while the white sturgeon adenovirus (WSAdV) isolate (19) was propagated on an established epithelial cell line, WSS-2, prepared from the spleen of white sturgeon (18). When the cytopathic effect was maximal, the cells were frozen (−75°C) and thawed three times and the cell debris was sedimented by low-speed centrifugation. The virions were concentrated from the supernatant in a Beckman XL-90 ultracentrifuge. Because of the low quantity of virion resulting from the poor replication efficiency of these viruses on the available cell lines, no further purification steps, such as banding on density gradient, could successfully be performed. The viral DNA was extracted with the phenol-chloroform method and precipitated in ethanol as described previously (3).
For the confirmation that these isolates were adenoviruses, approximately 10 ng of DNA from each strain was subjected to PCR in a 50-μl reaction volume with primers specific for the hexon gene sequence of mastadenoviruses (27), aviadenoviruses (20), or atadenoviruses (11). Since no amplification of the hexon gene fragment was achieved with either strain or any primers, another, more degenerate primer pair was tested for the detection of the most conserved region of the adenoviral DNA polymerase gene (33) from the early region E2B. Using the nucleotide ambiguity symbols recommended by the International Union of Pure and Applied Chemistry-International Union of Biochemistry and Molecular Biology Biochemical Nomenclature Commission, the sequence of the primers, named adenovirus E2B forward and reverse, can be described as follows: AdVE2B-F, 5′-TCMAAYGCHYTVTAYGGBTCDTTTGC-3′; and AdVE2B-R, 5′-CCAYTCHSWSAYRAADGCBCKVGTCCA-3′. Both of the samples gave a positive reaction with these primers. The amplification products of approximately 470 and 430 bp in size from the fish and snake samples, respectively, were excised from the gel, purified (22), and cloned into pBluescript II KS phagemid (Stratagene). The viral inserts were sequenced from two directions with T3 and T7 primers. The genome fragments between the primers proved to be 415 and 373 nucleotides long and were submitted to GenBank under accession numbers AY082700 (SnAdV) and AY082701 (WSAdV).
The 138- and 124-amino-acid (aa)-long predictable protein sequences were compared to the corresponding region of other adenoviruses, and both of them proved to be new and distinct from all the previously known adenoviral sequences. The alignment in Fig. 1 was arranged according to the proposed new classification of adenoviruses (6), and the members of the existing and proposed genera are separated by lines. Residues that are conserved through the whole family or within several genera are highlighted. The SnAdV sequence shared most amino acid residues with the members of the atadenovirus genus. For phylogenetic analyses, the highly variable regions that seemed not to be homologous and therefore unsuitable for such calculations were removed, and the remaining 106-aa-long sequence alignment was submitted for distance matrix and parsimony analyses by the PHYLIP program package as described previously (16).
The phylogenetic tree based on the distance matrix analysis showed the clear separation of five distinct clusters (Fig. 2). Four groups corresponded to the two established and two proposed genera, while the WSAdV appeared in a separate branch and formed a fifth cluster. Interestingly, the SnAdV fell in the group of the proposed new genus Atadenovirus (9). The same five major clusters resulted from the parsimony analysis.
For several decades, the family Adenoviridae was known to contain only two genera composed for the allocation of adenoviruses isolated from mammals or birds. The results of recent molecular genetic studies indicated that at least four different types of genomic organization exist among adenoviruses; therefore, the establishment of two novel genera is pending (4, 12). One of them, the genus Atadenovirus, was proposed to contain unusual ruminant adenoviruses together with the so-called egg drop syndrome virus (officially named duck adenovirus type 1), which used to be an exceptional member in the genus Aviadenovirus (17). More recently, a newer candidate atadenovirus was described (33) from a marsupial species, the brushtail possum (Trichosurus vulpecula). Several other nonconventional avian adenoviruses (namely, the serologically indistinguishable isolates causing hemorrhagic enteritis in turkeys, marble spleen disease in pheasants, or splenomegaly in chickens) referred to as turkey adenovirus type 3 (TAdV-3) were also considered exceptions among aviadenoviruses. Complete genome comparison and analysis proved that TAdV-3 (30) is a close relative of an adenovirus isolated from frog (13). The proposed name of this fourth adenovirus genus is Siadenovirus (12), referring to the putative sialidase gene present in its members on the left-hand end of their genome. With the proposed new genera, the nonconventional members of the Avi- and Mastadenovirus genera may be reclassified.
The occurrence of these exceptional viruses in birds and mammals, however, remains an intriguing question. The candidate siadenovirus isolated from birds (30) is obviously closely related to the frog adenovirus (13), while, based on the results of the present study, atadenoviruses seem to share common origin with SnAdV. Our preliminary data concerning the genomic organization of the corn snake isolate 145/88 (S. L. Farkas, unpublished data) indicate the presence of a homologue of the gene p32K (21, 34), a protein that apparently exists only in atadenoviruses.
Previously, we attempted to explain the concurrent presence of genetically distant adenoviruses in certain ruminant and avian hosts by hypothesizing several interclass host switches during the adenoviral evolution (5). According to this hypothesis, members of the mastadenovirus and aviadenovirus genera contain the adenoviruses that evolved together with their present hosts, while TAdV-3 might have emerged when an adenovirus from a yet-unknown but likely amphibian origin made a host switch to birds. Similarly, the reptilian origin of atadenoviruses was also assumed (15), and their occurrence in different ruminant and bird species, as well as in a marsupial, was also considered the result of several independent host switches from reptiles.
Adenovirus infection has been reported in numerous reptilian species (23, 24), including different snakes (29, 31). It would be interesting to compare these viruses at the DNA sequence level and to clarify their genetic relationship to the corn snake isolate and to other atadenoviruses.
Based on our present data, the WSAdV isolate seems to be the first member of a fifth adenovirus genus. The genome organization of WSAdV is of interest not only because of its potential applicability as a gene delivery vector in fishes but also from the point of evolution of adenoviruses. Recently, the common origin of adenoviruses and tectiviruses was hypothesized based on the striking similarity found in the architecture of the major capsid protein of human adenovirus type 2 and the phage PRD1 (7), even though no sequence homology was demonstrated between the two viruses. If this hypothesis is correct, detectable sequence similarity between the phages and adenoviruses of lower vertebrate species should be expected. WSAdV likely represents the most ancient type of adenovirus known to date. The presence of adenovirus-like particles has been described in additional fishes (8, 25, 28). The isolation and characterization of these tentative fish adenoviruses would certainly provide interesting data for the confirmation of the fifth genus and for the evolutionary pathway of adenoviruses in general. If one accepts the above theory on the common origin of adeno- and tectiviruses, the occurrence of primitive adenoviruses or intermediates between adenoviruses and tectiviruses (1, 2) in invertebrate animals cannot be excluded.
Alignment of the partial amino acid sequence of the DNA polymerase gene from 24 adenovirus types. Abbreviation of the adenovirus types is as follows (complemented with the serotype number where appropriate): B, bovine; C, canine; D, duck; F, fowl; fish, white sturgeon; Fr, frog; H, human; M, murine; O, ovine; P, porcine; Po, brushtail possum; snake, corn snake; T, turkey; and TS, tree shrew adenovirus. From each of the six human adenovirus species (A to F), only one representative serotype was included. Amino acid residues conserved in the members of at least two genera are printed in boldface. Regions applicable and selected for the phylogenetic analyses are boxed.
Phylogenetic tree calculated by distance matrix analysis. The total length of the edited alignment after removal of the obviously nonhomologous parts was 106 aa. Unrooted tree, the fish adenovirus was chosen as outgroup. Bootstrap values (from 100 data sets) show the high reliability of the tree topology regarding the clustering of the five proposed genera (printed in boldface).
ACKNOWLEDGMENTS
We are indebted to Blaine Parker (Columbia River Intertribal Fish Commission, Portland, Oreg.), who provided the WSAdV samples, and Bill Shewmaker (Clear Springs Foods, Inc., Research Division, Buhl, Idaho), who assisted with the cell culture work.
This work was partially supported by Hungarian Scientific Research Fund grants OTKA T030073, T034461, and A312 and a grant from the New Zealand Ministry of Agriculture and Forestry.
FOOTNOTES
- Received 15 March 2002.
- Accepted 24 June 2002.
- Copyright © 2002 American Society for Microbiology
