1 Veterinary Medical Research Institute, Hungarian Academy of Sciences, PO Box 18, H-1581 Budapest, Hungary
2 INRS-Institut Armand-Frappier, Université du Québec, 531 boul. des Praires, Laval, Quebec, Canada H7V 1B7
3 Institute for Zoology, Fish Biology, Fish Diseases, University of Munich, Germany
Correspondence
Peter Tijssen
peter.tijssen{at}inrs-iaf.uquebec.ca
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ABSTRACT |
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The GenBank accession number of the sequence reported in this paper is AY349010.
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MAIN TEXT |
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These viruses are known to infect invertebrates, such as insects and shrimp (Bergoin & Tijssen, 2000), and higher vertebrates, such as birds (Zádori et al., 1995
) and mammals (Brown & Young, 2000
; Truyen & Parrish, 2000
). Historically, the adeno-associated parvoviruses (AAVs) that depend on helper viruses were classified in a separate genus, the genus Dependovirus (Berns et al., 2000
). However, some autonomous avian parvoviruses that are closely related to AAVs (Zádori et al., 1995
) have now also been classified in this genus.
Diseases of reptiles and their pathogens have become a more common subject of veterinary research due to the increased popularity of reptiles as pets. The isolation and successful propagation of a reptilian parvovirus-like virus in tissue culture has been reported (Ahne & Scheinert, 1989), but no studies on the further identification and molecular characterization of this virus have as yet been provided. Several case reports state that parvovirus-like particles have been detected by histopathological examination and electron microscopy in samples from lizards, including bearded dragons (Pogona vitticeps; Kim et al., 2002
), and from snakes such as Aesculapian snake (Elaphe longissima) and four-lined snake (Elaphe quatuorlineata; Heldstab & Bestetti, 1984
), corn snakes (Elapha guttata; Ahne & Scheinert, 1989
) and California mountain kingsnakes (Lampropeltis zonata multicincta; Wozniak et al., 2000
). These parvovirus-like particles were often detected in the presence of adenovirus and herpesvirus or other pathogens, such as picornaviruses (Ahne & Scheinert, 1989
) and Isospora (protozoa) species (Kim et al., 2002
). Reptiles infected by parvovirus-like viruses have shown different clinical signs such as gastroenteritis (Ogawa et al., 1992
; Wozniak et al., 2000
), necrotic duodenum and liver (Heldstab & Bestetti, 1984
; Jacobson et al., 1996
), pneumonia (Ahne & Scheinert, 1989
) and neurological signs (Kim et al., 2002
), but the link between these virus infections and the pathologic features have as yet not been established. The purpose of this study was the molecular characterization of these parvovirus-like viruses from snakes.
Viruses were obtained from a diseased royal python (Python regius) and a boa constrictor (Boa constrictor) and submitted for pathological and microbiological examinations to the University of Hohenheim (described in detail in Ogawa et al., 1992). The viruses were separately isolated from the spleen and liver of the Boa constrictor and the heart, liver and kidney of the Python regius. Tissue specimens were removed aseptically from the carcasses and homogenized. Bacteria-free organ suspensions were inoculated into monolayers of viper heart (VH-2; ATCC CCL 140) and iguana heart (IgH-2; ATCC CCL 108) cells and incubated at 28 °C in minimal essential medium supplemented with 10 % foetal calf serum. The cultures were checked daily for cytopathic effects (CPE). IgH-2 and VH-2 cells inoculated with the tissue homogenates from either boa or python exhibited CPE that were undistinguishable and characterized by rounding of cells by 35 days and cell lysis within 710 days. Both virus cultures investigated by electron microscopy revealed two different particles. Ultrathin sections of infected IgH-2 cells revealed the assembly of icosahedral particles with mean sizes of
75 nm in the nucleus (Fig. 1
A).
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DNA was isolated from virus particles using the SDS/proteinase K method. Electrophoretic analysis of the extracted DNA showed two bands with sizes of approximately 4·4 and 30 kb, which correspond to the sizes of the snake parvovirus and the snake adenovirus genomes, respectively. The ratio of the two virus genomes was approximately equal in the royal python sample, whereas the Boa constrictor isolate contained approximately 10 times more adenovirus than parvovirus DNA.
Digestion of the DNA extracted from the royal python sample with PstI revealed three fragments, of which the 2·7 kb fragment was cloned into PstI-digested pBluescript II KS. The two terminal fragments were cloned into PstI/EcoRV-digested pBluescript II KS and the complete genome was generated as described above. PCR amplification using primers that flank the two PstI sites and sequencing showed that no small fragments were missing (primer pairs 5'-GGACTACAAGGAAGACAAGC-3' plus 5'-ACCATACCTGGCATTGTCTC-3', and 5'-TCGTCGCTTGGAAGCCATTC-3' plus 5'-CTTCTGTTGTCAGGTAATC-3'). The use of the Sure-2 bacterial host and incubation at 30 °C decreased recombination and deletion in the inverted terminal repeats (ITRs) (Tijssen et al., 2003).
Direct cloning of the complete viral DNA was unsuccessful. Therefore, the clone containing the 3' terminus was cut with HindIII, blunt-ended with T4 polymerase for 15 min at 12 °C, then cut with PstI and cloned into the clone containing the 5' terminus, which had been digested by PstI and SmaI. The PstI clone containing the core of the genome was inserted between the two ends. The complete genome of the parvovirus isolated from royal python was sequenced on an ABI310 automated DNA sequencer and found to be relatively short, 4432 nt long (Fig. 2A), with an organization that is typical of the AAVs (Fig. 2B
). Phylogenetic analysis also demonstrated that this new virus belonged to the genus Dependovirus (Fig. 2C
). The ends of the genome were flanked by identical, short ITRs of 154 nt, of which 122 nt could fold up forming a Y-shaped double-stranded hairpin structure with a putative Rep protein binding motif located 17 nt from the terminal resolution site (trs) (Fig. 2D
).
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Two large open reading frames (ORFs) were found in one strand (positive strand), encoding the putative non-structural (Rep1, Rep2) and capsid (VP1, VP2, VP3) proteins, respectively. These ORFs proved to be most similar to those of members of the genus Dependovirus using BLAST homology searches. We named this virus serpentine adeno-associated virus (SAAV) by analogy with ovine and avian (Bossis & Chiorini, 2003) AAV. In the genome, three putative transcriptionally active promoters could be identified from which, by analogy with the mammalian and bird AAVs, the Rep and VP proteins are most likely generated (Fig. 2B
). The left ORF encoded a putative unspliced non-stuctural protein (NS1) of 562 aa (Fig. 2B
), which showed 46·1 % similarity to the goose parvovirus and 49·9 % to the AAV-5 NS1 proteins, respectively. The putative VP1 gene consisted of 2181 nt encoding 726 aa residues. The virion contained three putative capsid proteins. The VP1 gene was found to be more conserved than the NS1 gene, showing 64·9 % similarity to the goose parvovirus and 63·8 % to AAV-5, respectively. Translation of the VP1 and VP3 genes most probably initiates from AUG codons at nt 2030 and 2600, respectively. The translation of the capsid protein VP2 was hypothesized to start from an atypical ACG start codon at nt 2441, as in other dependoviruses.
The most conserved region in the genome was the domain encoding the viral phospholipase A2 within VP1. This capsid enzyme is critical for infection (Zádori et al., 2001; Girod et al., 2002
). The catalytic dyad (HD) and calcium-binding loop (GPG) in these distinct AAVs are equidistant from the initiation codon of VP1, which in turn is 2 nt downstream from the first acceptor site (Fig. 2B
) and about 27 nt upstream from a second, alternative, splice acceptor site. The facultative splice donor, which would also cause a swapping of the C terminus of the Rep products, could use either acceptor site so that translation would start at the initiation codon of either VP1 or VP2/VP3. The phospholipase A2 domain is thus only present in a minority of the structural proteins. The Rep C terminus after splice acceptor site 1 is only 5 aa long and absent after acceptor site 2. For AAV-2, both alternative Rep products have short tails (16 and 7 aa, respectively), whereas for AAV-5 there are no tails because of the polyadenylation of the Rep transcripts within the intron region (Qiu et al., 2002
).
A surprising difference was the small size of the SAAV genome. This was reflected in smaller Rep products, particular at their C termini (Fig. 3). Sequences of two independent clones confirmed this organization. Moreover, the sequence of the boa isolate was identical from nt 1269 to 2305, which overlaps these different regions.
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Members of the genus Dependovirus, except the Muscovy duck and goose parvoviruses, need helper viruses (adeno-, herpes- or papillomaviruses) for efficient replication (Hoggan et al., 1968; Buller et al., 1981
; McPherson et al., 1982
; Walz et al., 1997
). It has been reported, however, that under certain conditions AAV-2 can propagate autonomously in differentiating squamous epithelium (Meyers et al., 2000
). There is no evidence as to whether SAAV can propagate autonomously or not, but so far this parvovirus has only been found with a snake adenovirus, which may be required for, or facilitate, SAAV replication. In this respect, conditions in the host may differ from those in tissue culture. It is tempting to speculate that AAVs isolated from primates and other mammals have a Sauria (Diapsida) origin. The study of additional reptilian parvoviruses may reveal whether the dependoviruses are originally parvoviruses of diapsids (birds, crocodilians, beaked reptiles and squamates), i.e. whether they co-evolved and a host switch to primates occurred only relatively recently. By analogy, a similar host switch from reptiles to ruminants, birds and marsupials is proposed to have occurred for adenoviruses (Farkas et al., 2002
; Benk
& Harrach, 2003
).
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ACKNOWLEDGEMENTS |
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REFERENCES |
---|
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---|
Ahne, W. & Scheinert, P. (1989). Reptilian viruses: isolation of parvovirus-like particles from corn snake Elapha guttata (Colubridae). Zentralbl Veterinarmed B 36, 409412.[Medline]
Benk, M. & Harrach, B. (2003). Molecular evolution of adenoviruses. Curr Top Microbiol Immunol 272, 335.[Medline]
Bergoin, M. & Tijssen, P. (2000). Molecular biology of Densovirinae. Contrib Microbiol 4, 1232.[Medline]
Berns, K. I., Bergoin, M., Bloom, M., Muzyczka, N., Tal, J. & Tattersall, P. (2000). Family Parvoviridae. In Virus Taxonomy. Seventh Report of the International Committee on Taxonomy of Viruses, pp. 311323. Edited by M. H. V. van Regenmortel, C. M. Fauquet, D. H. L. Bishop & 8 others. San Diego: Academic Press.
Bossis, I. & Chiorini, J. A. (2003). Cloning of an avian adeno-associated virus (AAAV) and generation of recombinant AAAV particles. J Virol 77, 67996810.
Brown, K. E. & Young, N. S. (2000). Epidemiology and pathology of erythroviruses. Contrib Microbiol 4, 107122.[Medline]
Buller, R. M., Janik, J. E., Sebring, E. D. & Rose, J. A. (1981). Herpes simplex virus types 1 and 2 completely help adenovirus-associated virus replication. J Virol 40, 241247.[Medline]
Farkas, S. L., Benk, M., El
, P., Ursu, K., Dán, Á., Ahne, W. & Harrach, B. (2002). Genomic and phylogenetic analyses of an adenovirus isolated from a corn snake (Elaphe guttata) imply a common origin with members of the proposed new genus Atadenovirus. J Gen Virol 83, 24032410.
Fédiere, G., Li, Y., Zádori, Z., Szelei, J. & Tijssen, P. (2002). Genome organization of Casphalia extranea densovirus, a new Iteravirus. Virology 292, 299308.[CrossRef][Medline]
Girod, A., Wobus, C. E., Zádori, Z., Ried, M., Leike, K., Tijssen, P., Kleinschmidt, J. A. & Hallek, M. (2002). The VP1 capsid protein of adeno-associated virus type 2 is carrying a phospholipase A2 domain required for virus infectivity. J Gen Virol 83, 973978.
Heldstab, A. & Bestetti, G. (1984). Virus-associated gastrointestinal diseases in snakes. J Zoo Anim Med 5, 118128.
Hoggan, M. D., Shatkin, A. J., Blacklow, N. R., Koczot, F. & Rose, J. A. (1968). Helper-dependent infectious deoxyribonucleic acid from adenovirus-associated virus. J Virol 2, 850851.
Ilyina, T. V. & Koonin, E. V. (1992). Conserved sequence motifs in the initiator proteins for rolling circle DNA replication encoded by diverse replicons from eubacteria, eucaryotes and archaebacteria. Nucleic Acids Res 20, 32793285.[Abstract]
Jacobson, E. R., Kopit, W., Kennedy, F. A. & Funk, R. S. (1996). Coinfection of a bearded dragon, Pogona vitticeps, with adenovirus- and dependo-like viruses. Vet Pathol 33, 429439.
Kim, D. Y., Mitchell, M. A., Bauer, R. W., Poston, R. & Cho, D. Y. (2002). An outbreak of adenoviral infection in inland bearded dragons (Pogona vitticeps) coinfected with dependovirus and coccidial protozoa (Isospora sp.). J Vet Diagn Invest 14, 332334.[Medline]
Koonin, E. V. (1993). A common set of conserved motifs in a vast variety of putative nucleic acid-dependent ATPases including MCM proteins involved in the initiation of eukaryotic DNA replication. Nucleic Acids Res 21, 25412547.[Abstract]
Li, Y., Zádori, Z., Bando, H., Dubuc, R., Fédiere, G., Szelei, J. & Tijssen, P. (2001). Genome organization of the densovirus from Bombyx mori (BmDNV-1) and enzyme activity of its capsid. J Gen Virol 82, 28212825.
McPherson, R. A., Ginsberg, H. S. & Rose, J. A. (1982). Adeno-associated virus helper activity of adenovirus DNA binding protein. J Virol 44, 666673.[Medline]
Meyers, C., Mane, M., Kokorina, N., Alam, S. & Hermonat, P. L. (2000). Ubiquitous human adeno-associated virus type 2 autonomously replicates in differentiating keratinocytes of a normal skin model. Virology 272, 338346.[CrossRef][Medline]
Ogawa, M., Ahne, W. & Essbauer, S. (1992). Reptilian viruses: adenovirus-like agent isolated from royal python (Python regius). Zentralbl Veterinarmed B 39, 732736.[Medline]
Pintel, D. J., Gersappe, A., Haut, D. & Pearson, J. (1996). Determinants that govern alternative splicing of parvovirus pre-mRNAs. Semin Virol 6, 283290.
Qiu, J., Nayak, R., Tullis, G. E. & Pintel, D. J. (2002). Characterization of the transcription profile of adeno-associated virus type 5 reveals a number of unique features compared to previously characterized adeno-associated viruses. J Virol 76, 1243512447.
Saraste, M., Sibbald, P. R. & Wittinghofer, A. (1990). The P-loop a common motif in ATP- and GTP-binding proteins. Trends Biochem Sci 15, 430434.[CrossRef][Medline]
Simpson, A. A., Chipman, P. R., Baker, T. S., Tijssen, P. & Rossmann, M. G. (1998). The structure of an insect parvovirus (Galleria mellonella densovirus) at 3·7 A resolution. Structure 15, 13551367.
Simpson, A. A., Hebert, B., Sullivan, G. M., Parrish, C. R., Zádori, Z., Tijssen, P. & Rossmann, M. G. (2002). The structure of porcine parvovirus: comparison with related viruses. J Mol Biol 315, 11891198.[CrossRef][Medline]
Srivastava, A., Lusby, E. W. & Berns, K. I. (1983). Nucleotide sequence and organization of the adeno-associated virus 2 genome. J Virol 45, 555564.[Medline]
Tijssen, P. & Bergoin, M. (1995). Densonucleosis viruses constitute an increasingly diversified subfamily among the parvoviruses. Semin Virol 6, 347355.[CrossRef]
Tijssen, P., Li, Y., El-Far, M., Szelei, J., Letarte, M. & Zádori, Z. (2003). Organization and expression strategy of the ambisense genome of densonucleosis virus of Galleria mellonella (GmDNV). J Virol 77, 1035710365.
Truyen, U. & Parrish, C. R. (2000). Epidemiology and pathology of autonomous parvoviruses. Contrib Microbiol 4, 149162.[Medline]
Tsao, J., Chapman, M. S., Agbandje, M. & 8 other authors (1991). The three-dimensional structure of canine parvovirus and its functional implications. Science 251, 14561464.[Medline]
Walz, C., Deprez, A., Dupressoir, T., Durst, M., Rabreau, M. & Schlehofer, J. R. (1997). Interaction of human papillomavirus type 16 and adeno-associated virus type 2 co-infecting human cervical epithelium. J Gen Virol 78, 14411452.[Abstract]
Wozniak, E. J., DeNardo, D. F., Brewer, A., Wong, V. & Tarara, R. P. (2000). Identification of adenovirus- and dependovirus-like agents in an outbreak of fatal gastroenteritis in captive born California mountain kingsnakes, Lampropeltis zonata multicincta. J Herpetol Med Surg 10, 47.
Xie, Q., Bu, W., Bhatia, S., Hare, J., Somasundaram, T., Azzi, A. & Chapman, M. S. (2002). The atomic structure of adeno-associated virus (AAV-2), a vector for human gene therapy. Proc Natl Acad Sci U S A 99, 1040510410.
Zádori, Z., Stefancsik, R., Rauch, T. & Kisary, J. (1995). Analysis of the complete nucleotide sequences of goose and muscovy duck parvoviruses indicates common ancestral origin with adeno-associated virus 2. Virology 212, 562573.[CrossRef][Medline]
Zádori, Z., Szelei, J., Lacoste, M. C., Li, Y., Gariépy, S., Raymond, P., Allaire, M., Nabi, I. R. & Tijssen, P. (2001). A viral phospholipase A2 is required for parvovirus infectivity. Dev Cell 1, 291302.[Medline]
Received 1 September 2003;
accepted 18 November 2003.