Consensus sequence determination and elucidation of the evolutionary history of a rotavirus Wa variant reveal a close relationship to various Wa variants derived from the original Wa strain.

The consensus nucleotide sequence of a human rotavirus Wa strain, with only a partially known passage history, was determined with sequence-independent amplification and next generation 454® pyrosequencing. This rotavirus Wa strain had the expected genome constellation of G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1 and was designated RVA/Human-tc/USA/WaCS/1974/G1P[8]. Phylogenetic analyses revealed a close relationship to four human rotavirus Wa variants (Wag5re, Wag7/8re, ParWa and VirWa) derived from the original 1974 human isolate. There were rearrangements in the Wag5re- and Wag7/8re variants in genome segments 5 (Wag5re) and 7 and 8 (Wag7/8re), which were not present in WaCS. Pairwise comparisons and a combined molecular clock for the Wa rotavirus genome indicated a close relationship between WaCS and ParWa and VirWa. These results suggest that WaCS is most probably an early cell culture adapted variant from the initial gnotobiotic pig passaged Wa isolate. Evolutionary pressure analysis identified a possible negative selected amino acid site in VP1 (genome segment 1) and a likely positive selected site in VP4 (genome segment 4). The WaCS may be more appropriate as a rotavirus Wa reference sequence than the current composite Wa reference genome.

Identification and genomic characterization of a novel fish reovirus, Hubei grass carp disease reovirus, isolated in 2009 in China.

A novel fish reovirus, Hubei grass carp disease reovirus (HGDRV; formerly grass carp reovirus strain 104, GCRV104), was isolated from diseased grass carp in China in 2009 and the full genome sequence was determined. This reovirus was propagated in a grass carp kidney cell line with a typical cytopathic effect. The total size of the genome was 23 706 bp with a 51 mol% G+C content, and the 11 dsRNA segments encoded 12 proteins (two proteins encoded by segment 11). A nucleotide sequence similarity search using blastn found no significant matches except for segment 2, which partially matched that of the RNA-dependent RNA polymerase (RdRp) from several viruses in the genera Aquareovirus and Orthoreovirus of the family Reoviridae. At the amino acid level, seven segments (Seg-1 to Seg-6, and Seg-8) matched with species in the genera Aquareovirus (15-46 % identities) and Orthoreovirus (12-44 % identities), while for four segments (Seg-7, Seg-9, Seg-10 and Seg-11) no similarities in these genera were found. Conserved terminal sequences, 5'-GAAUU----UCAUC-3', were found in each HGDRV segment at the 5' and 3' ends, and the 5'-terminal nucleotides were different from any known species in the genus Aquareovirus. Phylogenetic analysis based on RdRp amino acid sequences from members of the family Reoviridae showed that HGDRV clustered with aquareoviruses prior to joining a branch common with orthoreoviruses. Based on these observations, we propose that HGDRV is a new species in the genus Aquareovirus that is distantly related to any known species within this genus.

Simian rotaviruses possess divergent gene constellations that originated from interspecies transmission and reassortment.

Although few simian rotaviruses (RVs) have been isolated, such strains have been important for basic research and vaccine development. To explore the origins of simian RVs, the complete genome sequences of strains PTRV (G8P[1]), RRV (G3P[3]), and TUCH (G3P[24]) were determined. These data allowed the genotype constellations of each virus to be determined and the phylogenetic relationships of the simian strains with each other and with nonsimian RVs to be elucidated. The results indicate that PTRV was likely transmitted from a bovine or other ruminant into pig-tailed macaques (its host of origin), since its genes have genotypes and encode outer-capsid proteins similar to those of bovine RVs. In contrast, most of the genes of rhesus-macaque strains, RRV and TUCH, have genotypes more typical of canine-feline RVs. However, the sequences of the canine and/or feline (canine/feline)-like genes of RRV and TUCH are only distantly related to those of modern canine/feline RVs, indicating that any potential transmission of a progenitor of these viruses from a canine/feline host to a simian host was not recent. The remaining genes of RRV and TUCH appear to have originated through reassortment with bovine, human, or other RV strains. Finally, comparison of PTRV, RRV, and TUCH genes with those of the vervet-monkey RV SA11-H96 (G3P[2]) indicates that SA11-H96 shares little genetic similarity to other simian strains and likely has evolved independently. Collectively, our data indicate that simian RVs are of diverse ancestry with genome constellations that originated largely by interspecies transmission and reassortment with nonhuman animal RVs.

Peruvian horse sickness virus and Yunnan orbivirus, isolated from vertebrates and mosquitoes in Peru and Australia.

During 1997, two new viruses were isolated from outbreaks of disease that occurred in horses, donkeys, cattle and sheep in Peru. Genome characterization showed that the virus isolated from horses (with neurological disorders, 78% fatality) belongs to a new species the Peruvian horse sickness virus (PHSV), within the genus Orbivirus, family Reoviridae. This represents the first isolation of PHSV, which was subsequently also isolated during 1999, from diseased horses in the Northern Territory of Australia (Elsey virus, ELSV). Serological and molecular studies showed that PHSV and ELSV are very similar in the serotype-determining protein (99%, same serotype). The second virus (Rioja virus, RIOV) was associated with neurological signs in donkeys, cattle, sheep and dogs and was shown to be a member of the species Yunnan orbivirus (YUOV). RIOV and YUOV are also almost identical (97% amino acid identity) in the serotype-determining protein. YUOV was originally isolated from mosquitoes in China.

Induction of antibody responses to African horse sickness virus (AHSV) in ponies after vaccination with recombinant modified vaccinia Ankara (MVA).

BACKGROUND: African horse sickness virus (AHSV) causes a non-contagious, infectious disease in equids, with mortality rates that can exceed 90% in susceptible horse populations. AHSV vaccines play a crucial role in the control of the disease; however, there are concerns over the use of polyvalent live attenuated vaccines particularly in areas where AHSV is not endemic. Therefore, it is important to consider alternative approaches for AHSV vaccine development. We have carried out a pilot study to investigate the ability of recombinant modified vaccinia Ankara (MVA) vaccines expressing VP2, VP7 or NS3 genes of AHSV to stimulate immune responses against AHSV antigens in the horse. METHODOLOGY/PRINCIPAL FINDINGS: VP2, VP7 and NS3 genes from AHSV-4/Madrid87 were cloned into the vaccinia transfer vector pSC11 and recombinant MVA viruses generated. Antigen expression or transcription of the AHSV genes from cells infected with the recombinant viruses was confirmed. Pairs of ponies were vaccinated with MVAVP2, MVAVP7 or MVANS3 and both MVA vector and AHSV antigen-specific antibody responses were analysed. Vaccination with MVAVP2 induced a strong AHSV neutralising antibody response (VN titre up to a value of 2). MVAVP7 also induced AHSV antigen-specific responses, detected by western blotting. NS3 specific antibody responses were not detected. CONCLUSIONS: This pilot study demonstrates the immunogenicity of recombinant MVA vectored AHSV vaccines, in particular MVAVP2, and indicates that further work to investigate whether these vaccines would confer protection from lethal AHSV challenge in the horse is justifiable.

Sequence analysis of a reovirus isolated from the winter moth Operophtera brumata (Lepidoptera: Geometridae) and its parasitoid wasp Phobocampe tempestiva (Hymenoptera: Ichneumonidae).

A reovirus was isolated from Operophtera brumata (ObRV) and its parasitoid wasp Phobocampe tempestiva. Each of the 10 dsRNA genome segments of ObRV was sequenced and shown to contain a single open reading frame (ORF). Conserved motifs ([+ve] 5'-AAATAAA ...(G)/(T)AGGTT-3') were found at the termini of each segment, with the exception of Seg-6 and Seg-8, where the 5' termini were 5'-AACAAA...-3'. The putative proteins encoded by each segment were compared with those of other members of the family Reoviridae. Phylogenetic comparisons to published sequences for the RNA-dependent RNA polymerase genes from other reoviruses indicated that ObRV is most closely related to members of the genus Cypovirus. However, unlike the cypoviruses, ObRV has a double-layered capsid structure. When the protein encoded by ObRV Seg-10 was expressed (by inserting the open reading frame into a baculovirus expression vector) no 'occlusion bodies' were observed in the recombinant baculovirus infected insect cell cultures. This suggests that unlike the cypoviruses, Seg-10 of ObRV does not contain a polyhedrin gene. Further phylogenetic comparisons also identified relationships between Seg-2 and Seg-10 of ObRV, and genes of Diadromus pulchellus Idnoreovirus 1 (DpIRV1), suggesting that ObRV represents a new species from the genus Idnoreovirus.

Characterisation and partial sequence analysis of two novel cypoviruses isolated from the winter moth Operophtera brumata (Lepidoptera: Geometridae).

The complete nucleotide sequences of genomic segments S5 to S10 from Operophtera brumata cypovirus 18 (OpbuCPV18), and the complete nucleotide sequences of genomic segments S2, S5, S9 and S10 from Operophtera brumata cypovirus 19 (OpbuCPV19) have been determined. Each genome segment contained a single open reading frame (ORF). Conserved motifs 5' (AGUAAA....GUUAGCU) 3' were found at the ends of each segment of OpbuCPV18, whilst conserved motifs 5' (AACAAA....UUUGC) 3' were found at each segment terminus of OpbuCPV19. The putative proteins were compared with those of other members of the Reoviridae family. Phylogenetic analysis using the polyhedrin gene (S10) indicated that OpbuCPV18 was most closely related to Dendrolimus punctatus cypovirus 1, whilst OpbuCPV19 was most closely related to Trichoplusia ni cypovirus 15. In addition, analysis of S2, which encoded a putative RNA-dependant RNA polymerase gene, confirmed OpbuCPV19 belonged to the genus Cypovirus. Following the expression of the ORF from OpbuCPV19 S10, using a baculovirus expression vector, occlusion bodies were observed in insect cell culture. This demonstrated that segment 10 coded for the polyhedrin gene, capable of forming a polyhedral crystalline matrix.

Rapid cDNA synthesis and sequencing techniques for the genetic study of bluetongue and other dsRNA viruses.

The genetic study of double-stranded (ds) RNA viruses by sequence analyses of full-length genome segments, or entire viral genomes, has been restricted by the technical difficulties involved in analyses of dsRNA templates. This paper describes improved methods for sequence-independent synthesis of full-length cDNA copies of dsRNA genes and associated sequencing strategies. These methods include an improved version of the 'Single Primer Amplification Technique' (SPAT - [Attoui, H., Billoir, F., Cantaloube, J.F., Biagini, P., de Micco, P. and de Lamballerie, X., 2000. Strategies for the sequence determination of viral dsRNA genomes. J. Virol. Methods 89, 147-158]), which is described here as 'Full-Length Amplification of cDNAs' (FLAC). They also include the development of direct sequencing methods (without cloning) for the resulting full-length cDNAs. These techniques, which are applicable to any viruses with segmented dsRNA genomes and conserved RNA termini, make it possible to generate sequence data rapidly from multiple isolates for molecular epidemiology studies.

Biological and molecular studies of a cypovirus from the black fly Simulium ubiquitum (Diptera: Simuliidae).

A cypovirus from the black fly Simulium ubiquitum (SuCPV) was isolated and examined using biological and molecular techniques. SuCPV produces small (typically 0.25mum), polyhedral shaped inclusion bodies (polyhedra), in which the virus particles become multiply embedded. SuCPV is the third cypovirus isolated from Diptera, but the first from Simuliidae that has been characterized using molecular analyses. SuCPV has a genome composed of 10 segments of dsRNA, with an electrophoretic migration pattern that is different from those of recent UsCPV-17 and CrCPV-17 isolates from the mosquitoes Uranotaenia sapphirina and Culex restuans, respectively. The SuCPV electropherotype appears to show significant differences from those of the previously characterized lepidopteran cypoviruses. Sequence analysis of SuCPV segment 10 shows that it is unrelated to either of the two CPV isolates from Diptera or to the CPV species for which Seg-10 has been previously characterized from Lepidoptera. A comparison of the terminal regions of SuCPV genome segments to those of CPV-1, 2, 4, 5 14, 15, 16, 17, 18, and 19 also revealed only low levels of conservation. We therefore, propose that SuCPV is classified within a new Cypovirus species, which we have tentatively identified as Cypovirus-20. We have therefore referred to this virus isolate as S. ubiquitum CPV-20 (SuCPV-20).

Detection and characterisation of three novel species of reovirus (Reoviridae), isolated from geographically separate populations of the winter moth Operophtera brumata (Lepidoptera: Geometridae) on Orkney.

Geographically separate populations of winter moth (Operophtera brumata L.) were sampled in heather habitats on the Orkney Isles in order to investigate the prevalence of virus pathogens. Reoviruses were isolated in 11 of the 13 winter moth populations sampled, with 3 novel species being detected. Two species of Cypoviridae (CPV) were isolated, Operophtera brumata CPV18 and O. brumata CPV19, with one host population suffering 46% infection prevalence of OpbuCPV19. A third virus, O. brumata Reovirus (OpbuRV), was isolated from both winter moth and a hymenopteran parasitoid wasp, Phobocampe tempestiva, which is abundant in these populations. This was identified as a non-occluded reovirus, which was clearly able to infect and persist in both the lepidopteran and the hymenopteran host. The genomes of the three viruses were characterised using gel electrophoresis and the virus structure was investigated using transmission electron microscopy. The relationship of these viruses with a baculovirus that also infects winter moth, OpbuNPV, was investigated, as well as the association of OpbuRV with P. tempestiva. The detection of such viruses is discussed with reference to studies of similar viruses in other lepidopteran and hymenopteran host systems.

Molecular and biological characterization of a Cypovirus from the mosquito Culex restuans.

A cypovirus from the mosquito Culex restuans (named CrCPV) was isolated and its biology, morphology, and molecular characteristics were investigated. CrCPV is characterized by small (0.1-1.0 microm), irregularly shaped inclusion bodies that are multiply embedded. Laboratory studies demonstrated that divalent cations influenced transmission of CrCPV to Culex quinquefasciatus larvae; magnesium enhanced CrCPV transmission by approximately 30% while calcium inhibited transmission. CrCPV is the second cypovirus from a mosquito that has been confirmed by using molecular analysis. CrCPV has a genome composed of 10 dsRNA segments with an electropherotype similar to the recently discovered UsCPV-17 from the mosquito Uranotaenia sapphirina, but distinct from the lepidopteran cypoviruses BmCPV-1 (Bombyx mori) and TnCPV-15 (Trichoplusia ni). Nucleotide and deduced amino acid sequence analysis of CrCPV segment 10 (polyhedrin) suggests that CrCPV is closely related (83% nucleotide sequence identity and 87% amino acid sequence identity) to the newly characterized UsCPV-17 but is unrelated to the 16 remaining CPV species from lepidopteran hosts. A comparison of the terminal segment regions of CrCPV and UsCPV-17, an additional method for differentiating various Cypovirus species, revealed a high level of conservation. Therefore, we propose that CrCPV is a member of the Cypovirus-17 group and designate this species as CrCPV-17.

Morphological and molecular characterization of a Cypovirus (Reoviridae) from the mosquito Uranotaenia sapphirina (Diptera: Culicidae).

A novel cypovirus has been isolated from the mosquito Uranotaenia sapphirina (UsCPV) and shown to cause a chronic infection confined to the cytoplasm of epithelial cells of the gastric ceca and posterior stomach. The production of large numbers of virions and inclusion bodies and their arrangement into paracrystalline arrays gives the gut of infected insects a distinctive blue iridescence. The virions, which were examined by electron microscopy, are icosahedral (55 to 65 nm in diameter) with a central core that is surrounded by a single capsid layer. They are usually packaged individually within cubic inclusion bodies (polyhedra, approximately 100 nm across), although two to eight virus particles were sometimes occluded together. The virus was experimentally transmitted per os to several mosquito species. The transmission rate was enhanced by the presence of magnesium ions but was inhibited by calcium ions. Most of the infected larvae survived to adulthood, and the adults retained the infection. Electrophoretic analysis of the UsCPV genome segments (using 1% agarose gels) generated a migration pattern (electropherotype) that is different from those of the 16 Cypovirus species already recognized. UsCPV genome segment 10 (Seg-10) showed no significant nucleotide sequence similarity to the corresponding segment of the other cypoviruses that have previously been analyzed, and it has different "conserved" termini. A BLAST search of the UsCPV deduced amino acid sequence also showed little similarity to Antheraea mylitta CPV-4 (67 of 290 [23%]) or Choristoneura fumiferana CPV-16 (33 of 111 [29%]). We conclude that UsCPV should be recognized as a member of a new Cypovirus species (Cypovirus 17, strain UsCPV-17).

Complete sequence of the genome of two dsRNA viruses from Discula destructiva.

Complete nucleotide sequences were determined for the four dsRNA segments present in isolate 247 of Discula destructiva from South Carolina. The largest dsRNA (dsRNA 1) was 1787 bp in length with a single open reading frame (ORF) that coded for a putative RNA-dependent RNA polymerase (RdRp). The dsRNA 2 was 1585 bp in length with a single ORF that coded for a putative viral coat protein. Both the dsRNA 3 (1178 bp in length) and dsRNA 4 (308 bp) contained single ORFs. However, neither the nucleotide sequence nor the sequence of the putative translation products, showed any similarity with sequences currently available from GenBank. Although distinct, all 4 dsRNAs showed conserved nucleotides at both the 5' and 3' termini. Sequences of the two dsRNAs in an isolate of D. destructiva (331 originating from Idaho) were similar in length to, and shared similarity with, the dsRNA 1 and dsRNA 2 of isolate 247. However, although the putative RdRps of isolates 247 and 331 are closely related, the putative viral coat proteins coded for by the respective dsRNA 2s are distinct. Thus, the dsRNAs in the two fungal isolates appeared to originate from distinct, but related viruses, which we have named D. destructiva virus 1 and D. destructiva virus 2, respectively. Phylogenetic analysis indicated that the two viruses were most closely related to Fusarium solani virus 1 and should be considered members of the genus Partitivirus. Another isolate of D. destructiva (272.1) contains a 12 kb dsRNA in addition to the 4 dsRNAs found in isolate 247. Partial sequence of this 12 kb molecule showed a relationship to other large dsRNA molecules isolated from plants.

Nucleotide sequences of segments 1, 3 and 4 of the genome of Bombyx mori cypovirus 1 encoding putative capsid proteins VP1, VP3 and VP4, respectively.

The complete nucleotide sequences of genomic segments S1, S3 and S4 from Bombyx mori cypovirus 1 (BmCPV-1) have been determined. The segments consisted of 4190, 3846 and 3262 nucleotides encoding putative proteins of 1333, 1239 and 1058 amino acids with molecular masses of approximately 148, 140 and 120 kDa (p148, p140 and p120, respectively). All segments possess a single open reading frame. Homology searches showed that all three proteins have homologies to proteins of Rice ragged stunt virus, a member of the genus Oryzavirus within the family REOVIRIDAE: Partial homologies of p140 to structural proteins in other viruses were also found. The predicted molecular masses and the homologies with structural proteins in other viruses lead us to suggest that S1, S3 and S4 encode the capsid proteins VP1, VP3, and VP4, respectively, of BmCPV-1.