Genome sequence analysis of Tamana bat virus and its relationship with the genus Flavivirus.

Tamana bat virus (TABV, isolated from the bat Pteronotus parnellii) is currently classified as a tentative species in the genus FLAVIVIRUS: We report here the determination and analysis of its complete coding sequence. Low but significant similarity scores between TABV and member-viruses of the genus Flavivirus were identified in the amino acid sequences of the structural, NS3 and NS5 genes. A series of cysteines located in the envelope protein and the most important enzymatic domains of the virus helicase/NTPase, methyltransferase and RNA-dependent RNA polymerase were found to be highly conserved. In the serine-protease domain, the catalytic sites were conserved, but variations in sequence were found in the putative substrate-binding sites, implying possible differences in the protease specificity. In accordance with this finding, the putative cleavage sites of the TABV polyprotein by the virus protease are substantially different from those of flaviviruses. The phylogenetic position of TABV could not be determined precisely, probably due to the extremely significant genetic divergence from other member-viruses of the family FLAVIVIRIDAE: However, analysis based on both genetic distances and maximum-likelihood confirmed that TABV is more closely related to the flaviviruses than to the other genera. These findings have implications for the evolutionary history and taxonomic classification of the family as a whole: (i) the possibility that flaviviruses were derived from viruses infecting mammals rather than from mosquito viruses cannot be excluded; (ii) using the current criteria for the definition of genera in the family Flaviviridae, TABV should be assigned to a new genus.

Complete coding sequence of the Alkhurma virus, a tick-borne flavivirus causing severe hemorrhagic fever in humans in Saudi Arabia.

To date, tick-borne flaviviruses responsible for hemorrhagic fever in humans have been isolated in Siberia (Omsk hemorrhagic fever virus), India (Kyasanur Forest disease virus, KFDV), and in Saudi Arabia (Alkhurma virus, ALKV). Prior to this study, only partial coding sequences of these severe pathogens had been determined. We report here the complete coding sequence of ALK virus, which was determined to be 10,248 nucleotides (nt) long, and to encode a single 3,416 amino acid polyprotein. Independent analyses of the complete polyprotein and the envelope protein provided genetic and phylogenetic evidence that ALKV belongs to the tick-borne flavivirus group, within which it is most closely related to KFDV. Analysis of structural genes, genetic distances, and evolutionary relationship indicate that ALKV and KFDV derived from a common phylogenetic ancestor and constitute two genetic subtypes of the same virus species according to current genetic criteria of classification.

Evidence for recombination in natural populations of dengue virus type 1 based on the analysis of complete genome sequences.

Recombination events are known to occur in non-segmented RNA viruses like polioviruses or alphaviruses. Analysis of the subgenomic sequences of dengue virus type 1 (DENV-1) structural genes has recently allowed the identification of possible recombination breakpoints. Because DENV is a major human pathogen, this discovery might have important implications for virus pathogenicity, vaccine safety and efficiency, or diagnosis and, therefore, requires clear confirmation. We report the complete sequence determination of one Asian and two African strains of DENV-1 isolated from human patients. Rigorous sequence analysis provided strong evidence for the occurrence of intragenomic recombination events between DENV-1 strains belonging to different lineages. Singapore S275/90 strain appears to be the evolutionary product of a recombination event between viruses belonging to two distinct lineages: one lineage includes an African strain isolated in Abidjan (Ivory Coast) and the other includes isolates from Djibouti and Cambodia. The 'Recombination Detection Program', bootscanning and analysis of diversity plots provided congruent results concerning the existence of a two-switch recombination event and the localization of recombination breakpoints. Thus, the 5' and 3' genomic ends of the Singapore S275/90 strain were inherited from a Djibouti/Cambodia lineage ancestor and an internal fragment located in the envelope/NS1 region originated from an Abidjan lineage ancestor.

Strategies for the sequence determination of viral dsRNA genomes.

The genetic study of viruses having dsRNA genomes is hampered by the technical difficulty of complete sequence determination of dsRNA. Optimised methods are described here for sequencing dsRNAs, which meet three different situations: (1) genomes that can be obtained in fairly high amounts (>20 ng per separated segment); (2) genomes with limited amounts of RNA that can be detected by electrophoretic gel separation and staining; (3) genomes that cannot be detected by electrophoretic gel separation and staining. These methods include improved Single Primer Amplification Technique protocols, an adaptation of the SMART methodology, and a new method permitting the selective enzymatic removal of dsRNA segments. Strategies permitting adaptation of these protocols to the full-length determination of dsRNA viral genomes are described. Each of the protocols is described for sequence determination of a chosen dsRNA virus.

Sequence determination and analysis of the full-length genome of colorado tick fever virus, the type species of genus Coltivirus (Family Reoviridae).

The Colorado tick fever virus (CTFV) is the type species of genus Coltivirus, family Reoviridae. Its genome consisting of 12 segments of dsRNA was completely sequenced. It was found to be 29,174 nucleotides long (the longest of all Reoviridae genomes characterized to date). Conserved sequences at the 5' end (SACUUUUGY) and at the 3' end (WUGCAGUS) of the 12 segments were identified. The analysis of the putative proteins deduced from the nucleotide sequences permitted to identify functional motifs. In particular, the VP1 was identified unambiguously as the viral RNA dependent RNA pylmerase (RDRP) (VP1pol), with a GDD located at a similar position to Reoviridae RDRPs. In other genes, RGD cell-binding, NTPAse, single strand binding protein and kinase motifs were identified. Comparison with Reoviridae proteins showed significant similarities to RDRPs (CTFV-VP1) and sigma C protein of orthoreovirus (CTFV-VP6). Similarities to nonviral enzymatic proteins, such as methyltransferases, NTPAses, RNA replication factors, were also identified.

Complete sequence determination and genetic analysis of Banna virus and Kadipiro virus: proposal for assignment to a new genus (Seadornavirus) within the family Reoviridae.

Arboviruses with genomes composed of 12 segments of double-stranded (ds) RNA have previously been classified as members or probable members of the genus Coltivirus within the family REOVIRIDAE: A number of these viruses have been isolated in North America and Europe and are serologically and genetically related to Colorado tick fever virus, the Coltivirus type species. These isolates constitute subgroup A of the coltiviruses. The complete genome sequences are now presented of two Asian arboviruses, Kadipiro virus (KDV) and Banna virus (BAV), which are currently classified as subgroup B coltiviruses. Analysis of the viral protein sequences shows that all of the BAV genome segments have cognate genes in KDV. The functions of several of these proteins were also indicated by this analysis. Proteins with dsRNA-binding domains or with significant similarities to polymerases, methyltransferases, NTPases or protein kinases were identified. Comparisons of amino acid sequences of the conserved polymerase protein have shown that BAV and KDV are only very distantly related to the subgroup A coltiviruses. These data demonstrate a requirement for the subgroup B viruses to be reassigned to a separate new genus, for which the name Seadornavirus is proposed.

Phylogeny of the genus flavivirus using complete coding sequences of arthropod-borne viruses and viruses with no known vector.

Attempts to define the evolutionary relationships and origins of viruses in the genus Flavivirus are hampered by the lack of genetic information particularly amongst the non-vectored flaviviruses. Using a novel protocol for sequence determination, the first complete coding sequence of St Louis encephalitis virus and those of two representative non-vectored flaviviruses, Rio Bravo (isolated from bat) and Apoi (isolated from rodent), are reported. The encoded polyproteins of Rio Bravo and Apoi virus are the smallest described to date within the genus FLAVIVIRUS: The highest similarities with other flaviviruses were found in the NS3 and NS5 genes. The proteolytic cleavage sites for the viral serine protease were highly conserved among the flaviviruses completely sequenced to date. Comparative genetic amino acid alignments revealed that p-distance cut-off values of 0.330-0.470 distinguished the arthropod-borne viruses according to their recognized serogroups and Rio Bravo and Apoi virus were assigned to two distinct non-vectored virus groups. Within these serogroups, cladogenesis based on the complete ORF sequence was similar to trees based on envelope and NS5 sequences. In contrast, branching patterns at the deeper nodes of the tree were different from those reported in the previous study of NS5 sequences. The significance of these observations is discussed.

Molecular diagnosis of group B coltiviruses infections.

The group-B of genus Coltivirus encompasses isolates from humans, ticks or mosquitoes collected in Indonesia and China. Subgroup-B1 includes the strain JKT/dsR-7075 and subgroup-B2 strains JKT/dsR-6423, JKT/dsR-6969, JKT/dsR-7043 and the Banna virus. Data are described for the PCR-based diagnosis of infection by group B coltiviruses. Sets of primers were designed from the sequences of the 7th, 9th and 12th viral segments and RT PCR assays were developed. Consensus primers permitted the detection of all known isolates of subgroup 1 or 2. Viral strains could be characterised further using primers specific for type B2a or B2b, or based on the length of the amplification products. All primers gave negative results when using RNAs from Orbiviruses or Group-A coltiviruses. These primers permitted the detection of Group-B coltiviruses-RNA in infected mouse blood at the acute stage of the disease. Accordingly, they could be used for the diagnosis of infection in humans.

Serologic and molecular diagnosis of Colorado tick fever viral infections.

Molecular and serologic methods usable for the biological diagnosis of Coltivirus infection are reported. We designed a multiplex reverse transcription-polymerase chain reaction system that allowed the simultaneous and specific amplification of three genomic segments from as little as 0.01 plaque-forming units. Another system in the S2 viral segment permitted the differential diagnosis of American and European viral isolates. We also discuss some improvements of previous ELISAs, and the results obtained with paired sera from Colorado tick fever (CTF) virus-infected individuals. Western blot analysis was developed that allowed the detection of antibodies to a 38-kD viral protein in all tested sera. It also enabled the detection of anti-CTF virus antibodies in ELISA-negative sera. Specific IgM antibodies against a synthetic viral peptide could be detected in sera at the acute stage of the infection. Together, these results should permit the diagnosis of Coltivirus infection at any stage of the pathology.

Comparative sequence analysis of American, European and Asian isolates of viruses in the genus Coltivirus.

In this study, the basis for the classification of virus isolates grouped within the genus Coltivirus, family Reoviridae, is discussed. Sequences of dsRNA segments from American (segments 9-12), European (segment 12) and Asian (segments 7-12) isolates were characterized and polythetic criteria were defined for their taxonomic classification. These criteria (including sequence analysis) permitted the different species to be distinguished and classified into two groups. In both groups, subgroups were defined according to the degree of homology between the genomic sequences. American and European isolates are classified within group A, which includes subgroups A1 (Colorado tick fever virus species) and A2 (Eyach virus species). Asian isolates are classified in group B, which includes subgroups B1 (JKT-7075 virus species) and B2 (JKT-6423 virus species). The proteins encoded by the sequenced genomic segments were analysed. This allowed the identification of dsRNA binding domains in the proteins encoded by segment 8 of subgroup B1 isolates and segment 12 of subgroup B2 isolates. A conserved pattern of amino acids in segment 7 of group B isolates matched sequences found in the catalytic domains of protein kinases.