A quantitative real-time reverse transcription PCR (qRT-PCR) assay to detect genome segment 9 of all 26 bluetongue virus serotypes.

Bluetongue (BT) is an arboviral disease, which can often be fatal in naïve sheep and white tailed deer, but is usually less severe, or unapparent in other ruminants. Twenty-six bluetongue virus (BTV) serotypes have been recognised so far, two of which (BTV-25 and BTV-26) were recently identified by phylogenetic comparisons of genome-segment/outer-capsid protein VP2 (subsequently confirmed by serological 'virus-neutralisation' assays). Rapid, sensitive, reliable and quantitative diagnostic-assays for detection and identification of BTV represent important components of effective surveillance and control strategies. The BTV genome comprises 10 linear segments of dsRNA. We describe a 'TaqMan' fluorescence-probe based quantitative real-time RT-PCR assay, targeting the highly conserved genome-segment-9 (encoding the viral-helicase 'VP6' and NS4). The assay detected Seg-9 from isolates of all 26 BTV types, as well as from clinical samples derived from BTV-6w and BTV-8w outbreaks (in Europe), BTV-25 from Switzerland, BTV-26 from Kuwait, BTV-1w, BTV-4w and BTV-8w from Spain, BTV-4w, BTV-8, BTV-10 and BTV-16 from Brazil. Assay efficiency was evaluated with RNA derived from the reference strain of BTV-1w [RSArrrr/01] and was 99.6%, detecting down to 4 copies per reaction. Samples from uninfected insect or mammalian cell-cultures, hosts-species (uninfected sheep blood) or vector-insects, all gave negative results. The assay failed to detect RNA from heterologous but related Orbivirus species (including the nine African horse sickness virus [AHSV] and seven epizootic haemorrhagic disease virus [EHDV] serotypes).

Full genome sequencing of Corriparta virus, identifies California mosquito pool virus as a member of the Corriparta virus species.

The species Corriparta virus (CORV), within the genus Orbivirus, family Reoviridae, currently contains six virus strains: corriparta virus MRM1 (CORV-MRM1); CS0109; V654; V370; Acado virus and Jacareacanga virus. However, lack of neutralization assays, or reference genome sequence data has prevented further analysis of their intra-serogroup/species relationships and identification of individual serotypes. We report whole-genome sequence data for CORV-MRM1, which was isolated in 1960 in Australia. Comparisons of the conserved, polymerase (VP1), sub-core-shell 'T2' and core-surface 'T13' proteins encoded by genome segments 1, 2 and 8 (Seg-1, Seg-2 and Seg-8) respectively, show that this virus groups with the other mosquito borne orbiviruses. However, highest levels of nt/aa sequence identity (75.9%/91.6% in Seg-2/T2: 77.6%/91.7% in Seg-8/T13, respectively) were detected between CORV-MRM1 and California mosquito pool virus (CMPV), an orbivirus isolated in the USA in 1974, showing that they belong to the same virus species. The data presented here identify CMPV as a member of the Corriparta virus species and will facilitate identification of additional CORV isolates, diagnostic assay design and epidemiological studies.

Complete genome sequence analysis of a reference strain of bluetongue virus serotype 16.

The entire genome of the reference strain of bluetongue virus (BTV) serotype 16 (strain RSArrrr/16) was sequenced (a total of 23,518 base pairs). The virus was obtained from the Orbivirus Reference Collection (ORC) at IAH, Pirbright, United Kingdom. The virus strain, which was previously provided by the Onderstepoort Veterinary Research Institute in South Africa, was originally isolated from the Indian subcontinent (Hazara, West Pakistan) in 1960. Previous phylogenetic comparisons show that BTV RNA sequences cluster according to the geographic origins of the virus isolate/lineage, identifying distinct BTV topotypes. Sequence comparisons of segments Seg-1 to Seg-10 show that RSArrrr/16 belongs to the major eastern topotype of BTV (BTV-16e) and can be regarded as a reference strain of BTV-16e for phylogenetic and molecular epidemiology studies. All 10 genome segments of RSArrrr/16 group closely with the vaccine strain of BTV-16 (RSAvvvv/16) that was derived from it, as well as those recently published for a Chinese isolate of BTV-16 (>99% nucleotide identity), suggesting a very recent common ancestry for all three viruses.

Genome sequence of a reassortant strain of bluetongue virus serotype 23 from western India.

The full genome sequence (19,177 bp) of an Indian strain (IND1988/02) of bluetongue virus (BTV) serotype 23 was determined. This virus was isolated from a sheep that had been killed during a severe bluetongue outbreak that occurred in Rahuri, Maharashtra State, western India, in 1988. Phylogenetic analyses of these data demonstrate that most of the genome segments from IND1988/02 belong to the major "eastern" BTV topotype. However, genome segment 5 belongs to the major "western" BTV topotype, demonstrating that IND1988/02 is a reassortant. This may help to explain the increased virulence that was seen during this outbreak in 1988. Genome segment 5 of IND1988/02 shows >99% sequence identity with some other BTV isolates from India (e.g., BTV-3 IND2003/08), providing further evidence of the existence and circulation of reassortant strains on the subcontinent.

The genome sequence of a reassortant bluetongue virus serotype 3 from India.

All 10 genome segments (Seg-1 to 10-a total of 19,188 bp) were sequenced from a strain of bluetongue virus serotype 3 (BTV-3) from India (strain IND2003/08). Sequence comparisons showed that nine of the genome segments from this virus group with other eastern topotype strains. Genome Seg-2 and Seg-6 group with eastern BTV-3 strains from Japan. However, Seg-5 (the NS1 gene) from IND2003/08 belongs to a western lineage, demonstrating that IND2003/08 is a reassortant between eastern and western topotype bluetongue viruses. This confirms that western BTV strains have been imported and are circulating within the subcontinent.

The genome sequence of bluetongue virus type 2 from India: evidence for reassortment between eastern and western topotype field strains.

Bluetongue virus type 2, isolated in India in 1982 (IND1982/01), was obtained from the Orbivirus Reference Collection at IAH Pirbright (http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/btv-2.htm#IND1982/01). Full genome sequencing and phylogenetic analyses show that IND1982/01 is a reassortant virus containing genome segments derived from both eastern and western topotypes. These data will help to identify further reassortment events involving this or other virus lineages in the subcontinent.

The genome sequence of bluetongue virus type 10 from India: evidence for circulation of a western topotype vaccine strain.

Bluetongue virus is the type species of the genus Orbivirus in the family Reoviridae. We report the first complete genome sequence of an isolate (IND2004/01) of bluetongue virus serotype 10 (BTV-10) from Andhra Pradesh, India. This isolate, which is stored in the Orbivirus Reference Collection (ORC) at IAH Pirbright, shows >99% nucleotide identity in all 10 genome segments with a vaccine strain of BTV-10 from the United States.

Complete genome sequence of an isolate of bluetongue virus serotype 2, demonstrating circulation of a Western topotype in southern India.

Bluetongue virus serotype 2 (IND2003/02) was isolated in Tiruneveli City, Tamil Nadu State, India, and is stored in the Orbivirus Reference Collection at the Institute for Animal Health, Pirbright, United Kingdom. The entire genome of this isolate was sequenced, showing that it is composed of a total of 19,203 bp (all 10 genome segments). This is the first report of the entire genome sequence of a western strain of BTV-2 isolated in India, indicating that this virus has been introduced and is circulating in the region. These data will aid in the development of diagnostics and molecular epidemiology studies of BTV-2 in the subcontinent.