The nucleotide sequence of the RNA segment 3 of bluetongue virus (BTV) serotype 2 (Ona-A) from North America was determined to be 2772 nucleotides containing a single large open reading frame of 2703 nucleotides (901 amino acid). The predicted VP3 protein exhibited general physiochemical properties (including hydropathy profiles) which were very similar to those previously deduced for other BTV VP3 proteins. Partial genome segment 3 sequences, obtained by polymerase chain reaction (PCR) sequencing, of BTV isolates from the Caribbean were compared to those from North America, South Africa, India, Indonesia, Malaysia and Australia, as well as other orbiviruses, to determine the phylogenetic relationships amongst them. Three major BTV topotypes (Gould, A.R. (1987) Virus Res. 7, 169-183) were observed which had nucleotide sequences that differed by approximately 20%. At the molecular level, geographic separation had resulted in significant divergence in the BTV genome segment 3 sequences, consistent with the evolution of distinct viral populations. The close phylogenetic relationship between the BTV serotype 2 (Ona-A strain) from Florida and the BTV serotypes 1, 6 and 12 from Jamaica and Honduras, indicated that the presence of BTV serotype 2 in North America was probably due to an exotic incursion from the Caribbean region as previously proposed by Sellers and Maaroof ((1989) Can. J. Vet. Res. 53, 100-102) based on trajectory analysis. Conversely, nucleotide sequence analysis of Caribbean BTV serotype 17 isolates suggested they arose from incursions which originated in the USA, possibly from a BTV population distinct from those circulating in Wyoming.
Bluetongue virus (BTV) is the 'type' species of the genus Orbivirus within the family Reoviridae. The BTV genome is composed of ten linear segments of double-stranded RNA (dsRNA), each of which codes for one of ten distinct viral proteins. Previous phylogenetic comparisons have evaluated variations in genome segment 3 (Seg-3) nucleotide sequence as way to identify the geographical origin (different topotypes) of BTV isolates. The full-length nucleotide sequence of genome Seg-3 was determined for thirty BTV isolates recovered in the eastern Mediterranean region, the Balkans and other geographic areas (Spain, India, Malaysia and Africa). These data were compared, based on molecular variability, positive-selection-analysis and maximum-likelihood phylogenetic reconstructions (using appropriate substitution models) to 24 previously published sequences, revealing their evolutionary relationships. These analyses indicate that negative selection is a major force in the evolution of BTV, restricting nucleotide variability, reducing the evolutionary rate of Seg-3 and potentially of other regions of the BTV genome. Phylogenetic analysis of the BTV-4 strains isolated over a relatively long time interval (1979-2000), in a single geographic area (Greece), showed a low level of nucleotide diversity, indicating that the virus can circulate almost unchanged for many years. These analyses also show that the recent incursions into south-eastern Europe were caused by BTV strains belonging to two different major-lineages: representing an 'eastern' (BTV-9, -16 and -1) and a 'western' (BTV-4) group/topotype. Epidemiological and phylogenetic analyses indicate that these viruses originated from a geographic area to the east and southeast of Greece (including Cyprus and the Middle East), which appears to represent an important ecological niche for the virus that is likely to represent a continuing source of future BTV incursions into Europe.