Reovirus genomes from plant-feeding insects represent a newly discovered lineage within the family Reoviridae

A complex set of double-stranded RNAs (dsRNAs) was isolated from threecornered alfalfa hopper (Spissistilus festinus), a plant-feeding hemipteran pest. A subset of these dsRNAs constitute the genome of a new reovirus, provisionally designated Spissistilus festinus reovirus (SpFRV). SpFRV was present in threecornered alfalfa hopper populations in the San Joaquin Valley of California, with incidence ranging from 10% to 60% in 24 of 25 sample sets analyzed. The 10 dsRNA segments of SpFRV were completely sequenced and shown to share conserved terminal sequences (5′-AGAGA and CGAUGUUGU-3′) of the positive-sense strand that are distinct from known species of the family Reoviridae. Comparisons of the RNA directed RNA polymerase (RdRp) indicated SpFRV is most closely related (39.1% amino acid identity) to another new reovirus infecting the angulate leafhopper (Acinopterus angulatus) and provisionally designated Acinopterus angulatus reovirus (AcARV). The RdRp of both viruses was distantly related to Raspberry latent virus RdRp at 27.0% (SpFRV) and 30.0% (AcARV) or Rice ragged stunt virus RdRp at 26.2% (SpFRV) and 29.0% (AcARV) amino acid identity. RdRp phylogeny confirmed that SpFRV and AcARV are sister taxa sharing a most recent common ancestor. SpFRV segment 6 encodes a protein containing two NTP binding motifs that are conserved in homologs of reoviruses in the subfamily Spinareovirinae. The protein encoded by SpFRV segment 4 was identified as a guanylyltransferase homolog. SpFRV segments 1, 3, and 10 encode homologs of reovirus structural proteins. No homologs were identified for proteins encoded by SpFRV segments 5, 7, 8, and 9. Collectively, the low level of sequence identity with other reoviruses, similar segment terminal sequences, RdRp phylogeny, and host taxa indicate that SpFRV and AcARV may be considered members of a proposed new genus of the family Reoviridae (subfamily Spinareovirinae), with SpFRV assigned as the type species.     

Hepatitis E virus genotyping based on full-length genome and partial genomic regions

Some genomic regions for hepatitis E virus (HEV) genotyping have been reported to correlate well with the results from the phylogenetic analyses on the basis of the complete genome. However, few studies have systemically investigated the genomic regions for HEV genotyping using a combined phylogenetic and statistical approach. A consensus region for HEV genotyping has not been determined. In this study the nucleotide identities and genetic distances of 24 partial genomic regions and the complete genome sequences of 37 HEV strains were compared statistically. It was demonstrated with both one-way ANOVA and two-way ANOVA that only one genomic region in RNA-dependent RNA polymerase domain (4254–4560 nt) for which there were no significant differences when compared with the full-length genome (P > 0.05). The same four genotypes were identified by phylogenetic analysis based on this statistically predicted region identified as for the complete genome. RT-PCR amplification of HEV strains from all four genotypes confirmed conservation of the flanking primer sites of this region. Serum samples from 20 patients with a clinical diagnosis of hepatitis E were further analyzed by PCR using the same primers, 13 were positive and could be classified into genotype 4. These data strongly suggested that this newly identified region could be used for future HEV genotyping.     

Use of double-stranded RNA templates by the tombusvirus replicase in vitro: Implications for the mechanism of plus-strand initiation

Plus-stranded RNA viruses replicate efficiently in infected hosts producing numerous copies of the viral RNA. One of the long-standing mysteries in RNA virus replication is the occurrence and possible role of the double-stranded (ds)RNA formed between minus- and plus-strands. Using the partially purified Cucumber necrosis virus (CNV) replicase from plants and the recombinant RNA-dependent RNA polymerase (RdRp) of Turnip crinkle virus (TCV), in this paper, we demonstrate that both CNV replicase and the related TCV RdRp can utilize dsRNA templates to produce viral plus-stranded RNA in vitro. Sequence and structure of the dsRNA around the plus-strand initiation site had a significant effect on initiation, suggesting that initiation on dsRNA templates is a rate-limiting step. In contrast, the CNV replicase could efficiently synthesize plus-strand RNA on partial dsRNAs that had the plus-strand initiation promoter "exposed", suggesting that the polymerase activity of CNV replicase is strong enough to unwind extended dsRNA regions in the template during RNA synthesis. Based on the in vitro data, we propose that dsRNA forms might have functional roles during tombus- and carmovirus replication and the AU-rich nature of the terminus could be important for opening the dsRNA structure around the plus-strand initiation promoter for tombus- and carmoviruses and possibly many other positive-strand RNA viruses.