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Phylogenetic evidence for homologous recombination within the family Birnaviridae

School of Biological Sciences, The University of Hong Kong, Hong Kong SAR

Correspondence
Frederick Chi-Ching Leung
fcleung{at}hkucc.hku.hk

	ABSTRACT

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Birnaviruses are bi-segmented double-stranded RNA (dsRNA) viruses infecting insects, avian species and a wide range of aquatic species. Although homologous recombination is a common phenomenon in positive-sense RNA viruses, recombination in dsRNA viruses is rarely reported. Here we performed a comprehensive survey on homologous recombination in all available sequences (>1800) of the family Birnaviridae based on phylogenetic incongruence. Although inter-species recombination was not evident, potential intra-species recombination events were detected in aquabirnaviruses and infectious bursal disease virus (IBDV). Eight potential recombination events were identified and the possibility that these events were non-naturally occurring was assessed case by case. Five of the eight events were identified in IBDVs and all of these five events involved live attenuated vaccine strains. This finding suggests that homologous recombination between vaccine and wild-type IBDV strains may have occurred; the potential risk of mass vaccination using live vaccines is discussed. This is the first report of evidence for homologous recombination within the family Birnaviridae.

A supplementary figure and supplementary sequence data are available with the online version of this paper.

	INTRODUCTION

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Members of the family Birnaviridae are non-enveloped, bi-segmented double-stranded RNA (dsRNA) viruses infecting insects, avian species and a wide range of aquatic species (Delmas et al., 2005). Birnaviruses can be classified into five phylogenetic clusters, namely tellina virus 1 (TV-1), blotched snakehead virus (BSNV), Drosophila X virus (DXV), infectious bursal disease virus (IBDV) and aquabirnaviruses (AQBV) (Nobiron et al., 2008). The genome segment A of birnaviruses (3 kb) mainly encodes a pVP2-4-3 polyprotein and the genome segment B (2.8 kb) encodes an RNA-dependent RNA polymerase (RdRp) named VP1 (Delmas et al., 2005). Homologous proteins of birnaviruses share only 20 to 50 % amino acid identity (Da Costa et al., 2003; Nobiron et al., 2008). Intra-species diversity of IBDV and AQBV has been characterized extensively, while the diversity of DXV, BSNV and TV-1 is currently unknown. AQBV can be classified into two serotypes and ten subserotypes (A1–A9 and B1) (Hill & Way, 1995), which are correlated with the seven genogroups that are classified based on the genome segment A (Nishizawa et al., 2005). IBDV can be classified into two serotypes. Serotype-I strains can be phenotypically classified into classical, variant, very virulent, attenuated and Australian lineages (Van den Berg, 2000). Serotype-II strains are non-pathogenic and less characterized (Van den Berg, 2000). Evidence for genome segment reassortment among IBDV strains has been documented (Gao et al., 2007; Le Nouen et al., 2006; Wei et al., 2006, 2008). However, the possibility of homologous recombination among IBDV or AQBV strains was not addressed previously.

Recombination is a relatively common phenomenon in positive-sense RNA viruses (Chare & Holmes, 2006) but it appears to be rare in negative-sense RNA viruses. It was explained by the unavailability of their negative-sense RNA genomes for template-switching due to encapsidation by ribonucleoprotein complex (Chare et al., 2003). In birnaviruses, the genomic materials are associated with unique threadlike ribonucleoprotein complexes independent of the capsid (Hjalmarsson et al., 1999), in contrast with the observation that the dsRNA segments are free within virions in viruses of the family Reoviridae. Nonetheless, recombination in dsRNA viruses has not been studied systematically, except for some sporadic reports of recombination events in rotavirus (Phan et al., 2007). Recombination not only complicates the genotyping systems of viruses, it may also affect analyses of the molecular clock, demographic processes or natural selection. Therefore, the extent of recombination in dsRNA viruses deserves further investigations. Here we performed a conservative yet comprehensive survey on potential homologous recombination within the family Birnaviridae, at both inter- and intra-species level.

	METHOD

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Datasets.
All sequences of the family Birnaviridae (n=1881) were downloaded from GenBank in December, 2007. Alignments of pVP2-4-3 polyprotein and RdRp coding sequences were generated using CLUSTAL W based on their amino acid sequences. For inter-species alignments, full-length coding sequences of all birnavirus reference strains were used. For intra-species alignments of IBDV and AQBV, alignments were generated from different genomic regions depending on the availability of the GenBank seqeunces. With these overlapping alignments, we aimed to maximize the number of sites and taxa being analysed. All datasets are summarized in Table 1.

Screening for potential recombinants.
Four methods implemented in the recombination detection program (RDP) v2.0, RDP, GENECONV, MaxChi and Chimaera (Martin et al., 2005), were used for screening of potential recombinants as described by Ma et al. (2007). Phylogenetic incongruence between different genome regions (sequences in Supplementary Material S1, available in JGV Online) was further analysed using Bootscan implemented in Simplot v3.5.1 (Lole et al., 1999).

Identification of breakpoints.
Breakpoint locations were preliminarily estimated based on the distribution of informative sites supporting the two incongruent topologies which maximizes the ² value (Robertson et al., 1995); this method was implemented in Simplot. To locate the breakpoint with statistical significance, we used a likelihood method implemented in LARD (Holmes et al., 1999). Exhaustive searches for a single breakpoint, rather than heuristic searches for multiple breakpoints, were used to enhance the precision of our estimation. Therefore, for recombinants with multiple breakpoints, the alignments were trimmed so that only one breakpoint was estimated at a time. Significance of the estimated breakpoints was assessed using a likelihood ration test (LRT) (Holmes et al., 1999). To investigate whether the mosaic genome structure was a result of convergent evolution, the proportion of informative sites that are third-codons was calculated (Suzuki et al., 1998).

Identification of the closest parental strains.
For recombinants with multiple breakpoints, genome regions with the same parental ancestry (i.e. parental regions) were concatenated manually. The closest parental strains are defined as strains that share the highest nucleotide sequence identity with the potential recombinant. For each of the parental regions, a ML phylogeny was constructed for selected taxa using PHYML as described above.

	RESULTS AND DISCUSSION

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Lack of evidence for recent inter-species recombination
RDP results suggest that there is no significant evidence of recombination in datasets ALL-A and ALL-B. ML phylogenies were then constructed for different genome regions. Although discordant topologies were barely observed, most of these phylogenies were poorly resolved (data not shown). This observation is likely to be caused by the relatively high divergence among birnaviruses, e.g. 19 % amino acid similarity for VP3 (Nobiron et al., 2008), which could seriously affect the accuracy of alignment. Such high divergence prevents us from confidently drawing a conclusion about the possibility of any ancient inter-species recombination events among these birnaviruses. Nonetheless, our results suggest that these birnaviruses were not originated from recent inter-species recombination events.

Phylogenies of AQBV and IBDV
AQBV genome segment A (Fig. 1a) can be classified into seven lineages (designated A-I to A-VII) as described previously (Nishizawa et al., 2005). The phylogeny of AQBV genome segment B (Fig. 1b) is compatible to that of its genome segment A (i.e. no apparent reassortment), thus the lineages were named after those in the segment A phylogeny as B-I, B-II, B-III and B-VII.

View larger version (49K): [in this window] [in a new window]   Fig. 1. Classification of AQBV (a and b) and IBDV (c and d) segments A (a and c) and B (b and d). ML phylogenies were constructed for selected taxa from datasets specified in Table 1. Lineage names are indicated on the right. For segment B phylogenies, the segment A genotype of the taxa are also indicated. The lineages that are well-supported with bootstrap values >80 % are indicated with asterisks. The serotype-II IBDV strains in (c) were omitted for simplicity.

Identification of intra-species recombination
Eight recombinants were identified from both genome segments of AQBV and IBDV (Table 2). Only recombination events identified by both Bootscan and LARD analyses with statistical significance (P<0.001 in ² test and LRT) are included in Table 2. For all recombinants, the majority (71 to 94 %) of the informative sites are third-codons (Table 2), suggesting that the detected mosaic genome structures were likely to be a result of recombination rather than convergent evolution due to selection such as substitutions in VP2 during tissue culture adaptation.

View larger version (65K): [in this window] [in a new window]   Fig. 2. Phylogenetic incongruence between the parental regions of recombinants (indicated by ‘Query’) in (a) to (g). The left column shows Bootscan (bottom plot) and LARD (upper bar) results for the recombinants. Major and minor parental strains are indicated with grey and black lines or bars, respectively. Outgroups in Bootscan are represented by dotted lines. Four taxa were used in Bootscan (indicated on top of the plot), while the outgroups were excluded in LARD. The middle and right columns refer to the ML phylogenies based on the major and minor parental regions, and the recombinants are highlighted with black circles. All phylogenies were rooted with the indicated outgroups except (g), in which HENAN was absent from the phylogeny since the sequence of its 5' region was not available. Bars, number of substitutions per site.

Recombination in both genome segments of IBDV
Six potential IBDV recombinants were identified (Table 2). Interestingly, all potential recombination events in genome segment A involved the A-ATT lineage, which mainly consists of live attenuated vaccine strains. In particular, strain KK1, KSH, SHh and 849VB were identified as potential recombinants between lineage A-VV and A-ATT. Their closest A-ATT parental strain was identified as D78, which is a widely distributed live attenuated vaccine (Nobilis Gumboro D78 Live, Intervet). The two Korean potential recombinant strains, KK1 and KSH, were previously identified as divergent vvIBDV strains (Kwon et al., 2000; Mardassi et al., 2004). Breakpoints of these two potential recombinants were estimated at almost identical locations (Table 2), suggesting that they were likely to have originated from the same recombination events. Strain KSH and KK1 were isolated in 1992 and 1997, respectively (Kwon et al., 2000). The divergence between them could be explained by the 5-year period between their isolation. The closest A-VV parental strain of KSH is SH.92, which is a Korean vvIBDV isolated in 1992 (Kim et al., 2004). Strain SH.92 is geographically and temporally correlated with KSH, suggesting that KSH and KK1 may be originated from recombination events between a Korean vvIBDV and a live vaccine strain during the early 1990s.

Potential recombinant 849VB is a European vvIBDV that was isolated from Belgium in 1987 (Van den Berg et al., 1996). It is geographically and temporally consistent with its closest A-VV parental strain, D6948, which was isolated from the Netherlands in 1989. Moreover, its genome segment B is almost identical to other European vvIBDVs that were isolated during the late 1980s (Le Nouen et al., 2006), suggesting that 849VB might have originated from recombination between early European vvIBDVs and live vaccine strains (Fig. 2d). In contrast, potential recombinant SHh and its closest A-VV parental strain HLJ-5 were both isolated from China (Fig. 2e), suggesting that recombination between Chinese vvIBDVs and live vaccine strains may have occurred.

Potential recombinant ViBursaCE is a tissue culture attenuated vaccine strain derived from a non-cloned master seed (Lohmann Animal Health). Our results suggest that it may be a potential recombinant between lineage A-VAR and A-ATT (Fig. 2f). Its closest A-VAR parental strain is Variant E, which is an American antigenic variant. Its closest A-ATT parental strain is strain-CT, which is a French vaccine strain (Rhone-Merieux). Its genome segment B (GenBank accession number EU162092 [GenBank] ) shares over 99.8 % nucleotide sequence identity with strain-CT (GenBank accession number AJ310186 [GenBank] ) (Table 2; Supplementary Fig. S1d). Taken together, ViBursaCE may have originated from a vaccine strain, and its genome segment A may have recombined with a variant strain. Since the genome sequence strains of ViBursaCE were obtained from samples directly taken from the commercial vaccine vials (personal communication, Dr D. J. Jackwood, Ohio State University), the potential recombination events may have occurred during propagation of the vaccine.

The genome segment B of Harbin-1 was identified as a potential recombinant between strains within the B-NVV2 lineage. Its closest parental strains were identified as Gx and HENAN (Fig. 2g). It should be noted that the full-length sequence of HENAN is not available; therefore, the closest parental strain in dataset IBDV-B1, 2.73, was used in the Bootscan and LARD analyses (Fig. 2g). The genome segment A of Harbin-1, Gx and HENAN (GenBank accession numbers EF517528 [GenBank] , AY444873 [GenBank] and AJ878901 [GenBank] ) were clustered within the A-VV lineage and thus were identified as Chinese vvIBDVs (Supplementary Fig. S1c). In particular, the corresponding parental region of Harbin-1 and Gx shares over 99.8 % nucleotide identity (Table 2). Taken together, these data suggest that Harbin-1 may be originated from strains closely related to Gx and its genome segment B may have recombined with strains closely related to HENAN. This result suggests that recombination between the genome segment B of Chinese vvIBDVs has occurred.

Possibilities that these potential recombination events were non-naturally occurring
Despite the apparent phylogenetically incongruent signals within the genomes of potential recombinants, care has to be taken to interpret whether these signals were non-naturally occurring, i.e. as a result of recombination in cell culture coinfected with multiple strains or PCR recombination in the presence of multiple templates. In both scenarios, these in vitro recombinants might not be picked up by direct sequencing of the PCR product, since they only represent a minor portion of the PCR amplicon and the resulting sequence is a consensus sequence of the overall population of amplicons (assuming the recombinants have no selection advantage in replication and PCR amplification). However, if the amplicons were cloned, these in vitro recombinants could be picked up as rare genotypes among the parental genotypes. In the cases of Ab and 20Gld, the sequences were obtained using a direct PCR sequencing strategy (Blake et al., 2001; Romero-Brey et al., 2004). In addition, for 849VB, six independent clones were sequenced although the amplicons were cloned before sequencing (Van den Berg et al., 1996). Therefore, these three potential recombinants are not likely to have been derived in vitro. Nonetheless, we recognize the uncertainty in these published sequences and confess that our interpretation may not be conclusive if non-random errors were introduced during data processing, before the sequences were submitted to GenBank, e.g. accidental mixing of files belonging to different viruses during sequence assembly, leading to in silico recombination (de Silva & Messer, 2004). However, we consider such non-random sequencing errors as individual events and these should not be overstated.

In the cases of KK1 and KSH, a single recombination pattern was observed for two strains collected at different time points. In addition, for ViBursaCE, two independent but almost identical sequences were deposited in GenBank (EU162089 [GenBank] and EU162088 [GenBank] ). Considering that the possibility of having two independent artefacts for an identical recombination pattern is negligible, we can confidently accept that KK1, KSH and ViBursaCE are naturally occurring recombinants. However, due to the lack of information for Harbin-1 and SHh, we cannot determine whether these recombinant signals were artefacts. Nonetheless, given the conservativeness of our methods, the phylogenetic incongruence within these genomes is definitive. Even if these incongruent phylogenetic signals were non-naturally occurring, their possible biased effects on further phylogenetic analyses should not be overlooked.

Exchange of genetic materials between IBDV vaccine and field strains
Our findings suggest that homologous recombination between live vaccines and wild-type IBDV strains may have occurred. Mass vaccination of farmed animals may increase the probability of emergence of chimeric viruses resulting from recombination or reassortment. Despite the promising efficacy of live vaccines, there are considerable controversies over the potential risks of reversion or generation of chimeric viruses (Seligman & Gould, 2004). Such concerns have been highlighted by the fact that a high proportion (over 50 %) of polioviruses involved in vaccine-associated paralytic poliomyelitis are recombinant (Guillot et al., 2000). While the vast majority of these recombinants did not show dramatically altered phenotypes, a few exceptions with altered virulence were documented (Li et al., 1996; Martin et al., 2002). In the case of IBDV, virulence and tropism are primarily determined by critical residues on VP2 and partially by VP1 (Brandt et al., 2001; Hon et al., 2006). Vaccine strains may acquire these virulence factors through intra-serotypic recombination with wild-type strains. Although we cannot exclude the possibility that chimeric viruses with dramatically altered phenotypes may have resulted from recombination, such theoretical risk may be remote at this point, based on the fact that no inter-species or inter-serotypic recombination was detected in this study. However, continuous surveillance of field strains is recommended for early detection of any recombinants with dramatically altered phenotypes.

Implications and conclusions
This is the first report of homologous recombination within the family Birnaviridae. Homologous recombination in segmented dsRNA viruses is thought to be rare, since replication of their genomes occurs within the capsid after packaging of plus strands (Patton & Spencer, 2000). A prerequisite of recombination is co-packaging of different molecules of the same genome segment into a capsid, which could be rare, if not impossible (Worobey & Holmes, 1999). However, the non-virion-associated form of RdRp of infectious pancreatic necrosis virus (IPNV) was shown to behave as a functional replicase (Xu et al., 2004). Therefore, we cannot exclude the possibility that replication, and thus recombination, may happen outside the capsid before packaging. In addition, previous studies showed that birnaviruses can be recovered from mRNA (Mundt & Vakharia, 1996; Yao & Vakharia, 1998), implying that full-length positive strand RNA is recognizable by viral RNA polymerase; this is similar to the observation in positive strand RNA viruses. This observation suggests that replication of birnaviruses may be different from other dsRNA viruses, while its role in homologous recombination of birnaviruses deserves further investigation. In summary, this study demonstrated that not all birnaviruses are clonal. These recombinants may mislead the phylogenetic-based genotyping system. Here, we suggest a routine screening for recombination before any phylogenetic analysis was performed on the newly sequenced birnaviruses.

	ACKNOWLEDGEMENTS

 
We sincerely thank Dr D. J. Jackwood of The Ohio State University for useful information about their GenBank sequences.

	REFERENCES

TOP ABSTRACT INTRODUCTION METHOD RESULTS AND DISCUSSION REFERENCES

 
Blake, S., Ma, J. Y., Caporale, D. A., Jairath, S. & Nicholson, B. L. (2001). Phylogenetic relationships of aquatic birnaviruses based on deduced amino acid sequences of genome segment A cDNA. Dis Aquat Organ 45, 89–102.[Medline]

Brandt, M., Yao, K., Liu, M., Heckert, R. A. & Vakharia, V. N. (2001). Molecular determinants of virulence, cell tropism, and pathogenic phenotype of infectious bursal disease virus. J Virol 75, 11974–11982.[Abstract/Free Full Text]

Brown, M. D. & Skinner, M. A. (1996). Coding sequences of both genome segments of a European ‘very virulent’ infectious bursal disease virus. Virus Res 40, 1–15.[CrossRef][Medline]

Chare, E. R. & Holmes, E. C. (2006). A phylogenetic survey of recombination frequency in plant RNA viruses. Arch Virol 151, 933–946.[CrossRef][Medline]

Chare, E. R., Gould, E. A. & Holmes, E. C. (2003). Phylogenetic analysis reveals a low rate of homologous recombination in negative-sense RNA viruses. J Gen Virol 84, 2691–2703.[Abstract/Free Full Text]

Da Costa, B., Soignier, S., Chevalier, C., Henry, C., Thory, C., Huet, J. C. & Delmas, B. (2003). Blotched snakehead virus is a new aquatic birnavirus that is slightly more related to avibirnavirus than to aquabirnavirus. J Virol 77, 719–725.[CrossRef][Medline]

Delmas, B., Kibenge, F. S. B., Leong, J. C., Mundt, E., Vakharia, V. N. & Wu, J. L. (2005). Birnaviridae. In Virus Taxonomy, pp. 561–569. Edited by C. M. Fauquet, M. A. Mayo, J. Maniloff, U. Desselberger & A. L. Ball. London: Academic Press.

de Silva, A. & Messer, W. (2004). Arguments for live flavivirus vaccines. Lancet 364, 500[Medline]

Gao, H. L., Wang, X. M., Gao, Y. L. & Fu, C. Y. (2007). Direct evidence of reassortment and mutant spectrum analysis of a very virulent infectious bursal disease virus. Avian Dis 51, 893–899.[CrossRef][Medline]

Guillot, S., Caro, V., Cuervo, N., Korotkova, E., Combiescu, M., Persu, A., Aubert-Combiescu, A., Delpeyroux, F. & Crainic, R. (2000). Natural genetic exchanges between vaccine and wild poliovirus strains in humans. J Virol 74, 8434–8443.[Abstract/Free Full Text]

Guindon, S. & Gascuel, O. (2003). A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52, 696–704.[Abstract/Free Full Text]

Hill, B. J. & Way, K. (1995). Serological classification of infectious pancreatic necrosis (IPN) virus and other aquatic birnaviruses. Annu Rev Fish Dis 5, 55–77.[CrossRef]

Hjalmarsson, A., Carlemalm, E. & Everitt, E. (1999). Infectious pancreatic necrosis virus: identification of a VP3-containing ribonucleoprotein core structure and evidence for O-linked glycosylation of the capsid protein VP2. J Virol 73, 3484–3490.[Abstract/Free Full Text]

Holmes, E. C., Worobey, M. & Rambaut, A. (1999). Phylogenetic evidence for recombination in dengue virus. Mol Biol Evol 16, 405–409.[Abstract]

Hon, C. C., Lam, T. Y., Drummond, A., Rambaut, A., Lee, Y. F., Yip, C. W., Zeng, F., Lam, P. Y., Ng, P. T. & Leung, F. C. (2006). Phylogenetic analysis reveals a correlation between the expansion of very virulent infectious bursal disease virus and reassortment of its genome segment B. J Virol 80, 8503–8509.[Abstract/Free Full Text]

Kim, S. J., Sung, H. W., Han, J. H., Jackwood, D. & Kwon, H. M. (2004). Protection against very virulent infectious bursal disease virus in chickens immunized with DNA vaccines. Vet Microbiol 101, 39–51.[CrossRef][Medline]

Kwon, H. M., Kim, D. K., Hahn, T. W., Han, J. H. & Jackwood, D. J. (2000). Sequence of precursor polyprotein gene (segment A) of infectious bursal disease viruses isolated in Korea. Avian Dis 44, 691–696.[CrossRef][Medline]

Le Nouen, C., Rivallan, G., Toquin, D., Darlu, P., Morin, Y., Beven, V., de Boisseson, C., Cazaban, C., Comte, S. & other authors (2006). Very virulent infectious bursal disease virus: reduced pathogenicity in a rare natural segment-B-reassorted isolate. J Gen Virol 87, 209–216.[Abstract/Free Full Text]

Li, J., Zhang, L. B., Yoneyama, T., Yoshida, H., Shimizu, H., Yoshii, K., Hara, M., Nomura, T., Yoshikura, H. & other authors (1996). Genetic basis of the neurovirulence of type 1 polioviruses isolated from vaccine-associated paralytic patients. Arch Virol 141, 1047–1054.[CrossRef][Medline]

Lole, K. S., Bollinger, R. C., Paranjape, R. S., Gadkari, D., Kulkarni, S. S., Novak, N. G., Ingersoll, R., Sheppard, H. W. & Ray, S. C. (1999). Full-length human immunodeficiency virus type 1 genomes from subtype C-infected seroconverters in India, with evidence of intersubtype recombination. J Virol 73, 152–160.[Abstract/Free Full Text]

Ma, C. M., Hon, C. C., Lam, T. Y., Li, V. Y., Wong, C. K., de Oliveira, T. & Leung, F. C. (2007). Evidence for recombination in natural populations of porcine circovirus type 2 in Hong Kong and mainland China. J Gen Virol 88, 1733–1737.[Abstract/Free Full Text]

Mardassi, H., Khabouchi, N., Ghram, A., Namouchi, A. & Karboul, A. (2004). A very virulent genotype of infectious bursal disease virus predominantly associated with recurrent infectious bursal disease outbreaks in Tunisian vaccinated flocks. Avian Dis 48, 829–840.[CrossRef][Medline]

Martin, J., Samoilovich, E., Dunn, G., Lackenby, A., Feldman, E., Heath, A., Svirchevskaya, E., Cooper, G., Yermalovich, M. & Minor, P. D. (2002). Isolation of an intertypic poliovirus capsid recombinant from a child with vaccine-associated paralytic poliomyelitis. J Virol 76, 10921–10928.[Abstract/Free Full Text]

Martin, D. P., Williamson, C. & Posada, D. (2005). RDP2: recombination detection and analysis from sequence alignments. Bioinformatics 21, 260–262.[Abstract/Free Full Text]

Mundt, E. & Vakharia, V. N. (1996). Synthetic transcripts of double-stranded Birnavirus genome are infectious. Proc Natl Acad Sci U S A 93, 11131–11136.[Abstract/Free Full Text]

Nishizawa, T., Kinoshita, S. & Yoshimizu, M. (2005). An approach for genogrouping of Japanese isolates of aquabirnaviruses in a new genogroup, VII, based on the VP2/NS junction region. J Gen Virol 86, 1973–1978.[Abstract/Free Full Text]

Nobiron, I., Galloux, M., Henry, C., Torhy, C., Boudinot, P., Lejal, N., Da Costa, B. & Delmas, B. (2008). Genome and polypeptides characterization of Tellina virus 1 reveals a fifth genetic cluster in the Birnaviridae family. Virology 371, 350–361.[Medline]

Patton, J. T. & Spencer, E. (2000). Genome replication and packaging of segmented double-stranded RNA viruses. Virology 277, 217–225.[CrossRef][Medline]

Phan, T. G., Okitsu, S., Maneekarn, N. & Ushijima, H. (2007). Evidence of intragenic recombination in G1 rotavirus VP7 genes. J Virol 81, 10188–10194.[Abstract/Free Full Text]

Posada, D. & Crandall, K. A. (1998). MODELTEST: testing the model of DNA substitution. Bioinformatics 14, 817–818.[Abstract/Free Full Text]

Robertson, D. L., Hahn, B. H. & Sharp, P. M. (1995). Recombination in AIDS viruses. J Mol Evol 40, 249–259.[CrossRef][Medline]

Romero-Brey, I., Batts, W. N., Bandin, I., Winton, J. R. & Dopazo, C. P. (2004). Molecular characterization of birnaviruses isolated from wild marine fishes at the Flemish Cap (Newfoundland). Dis Aquat Organ 61, 1–10.[CrossRef][Medline]

Seligman, S. J. & Gould, E. A. (2004). Live flavivirus vaccines: reasons for caution. Lancet 363, 2073–2075.[CrossRef][Medline]

Suzuki, Y., Gojobori, T. & Nakagomi, O. (1998). Intragenic recombinations in rotaviruses. FEBS Lett 427, 183–187.[CrossRef][Medline]

Tamura, K., Dudley, J., Nei, M. & Kumar, S. (2007). MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24, 1596–1599.[Abstract/Free Full Text]

Van den Berg, T. P. (2000). Acute infectious bursal disease in poultry: a review. Avian Pathol 29, 175–194.[CrossRef][Medline]

Van den Berg, T. P., Gonze, M., Morales, D. & Meulemans, G. (1996). Acute infectious bursal disease in poultry: immunological and molecular basis of antigenicity of a highly virulent strain. Avian Pathol 25, 751–768.[CrossRef][Medline]

Wei, Y., Li, J., Zheng, J., Xu, H., Li, L. & Yu, L. (2006). Genetic reassortment of infectious bursal disease virus in nature. Biochem Biophys Res Commun 350, 277–287.[CrossRef][Medline]

Wei, Y., Yu, X., Zheng, J., Chu, W., Xu, H., Yu, X. & Yu, L. (2008). Reassortant infectious bursal disease virus isolated in China. Virus Res 131, 279–282.[CrossRef][Medline]

Worobey, M. & Holmes, E. C. (1999). Evolutionary aspects of recombination in RNA viruses. J Gen Virol 80, 2535–2543.[Free Full Text]

Xu, H. T., Si, W. D. & Dobos, P. (2004). Mapping the site of guanylylation on VP1, the protein primer for infectious pancreatic necrosis virus RNA synthesis. Virology 322, 199–210.[CrossRef][Medline]

Yao, K. & Vakharia, V. N. (1998). Generation of infectious pancreatic necrosis virus from cloned cDNA. J Virol 72, 8913–8920.[Abstract/Free Full Text]

Received 10 May 2008; accepted 28 July 2008.

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