Full genome sequencing of the bluetongue virus-1 isolate MKD20/08/Ind from goat in India

ABSTRACT This communication reports full genome sequencing of the bluetongue virus-1 (BTV-1) isolate MKD20/08/Ind from goat in northern India. The total BTV-1 genome size was found to be 19,190 bp. A comparison study between the Indian isolate and other global isolates revealed that it belongs to the 'Eastern' BTV topotype. The full genome sequence of BTV-1 will provide vital information on its geographical origin and it will also be proved useful for comparing the Indian isolate with global isolates from other host species.

Restriction enzyme analysis of VP7 gene of Indian isolates of bluetongue virus.

The genome segment 7 of two Indian isolates of bluetongue virus (BTV) from Avikanagar (BTV-1-western India) and Hyderabad (BTV-Untyped Hyderabad-southern India) was amplified by RT-PCR using two sets of VP7 specific primers. A sequence of 1137 bp comprising the complete coding sequence of the VP7 gene from Avikanagar isolate and a 1154 bp full-length sequence from BTV-UT Hyderabad isolate were amplified. Further, restriction enzyme digestion of these full-length amplicons, using EcoRI, PstI and TaqI revealed that genome segment 7 from both isolates were different from each other by absence of any EcoRI site in the BTV-UT Hyderabad isolate. There were also variations in the number and position of restriction sites for TaqI enzyme in these two isolates. Taql has two sites in the Avikanagar isolate whereas four sites in BTV-UT Hyderabad. The restriction digestion fragments obtained after PstI digestion were differentiated on the basis of their distinct sizes in both isolates. Comparison of their in silico restriction profiles with that of other isolates from different countries revealed that the two Indian isolates belonging to different parts of India had variations in their VP7 gene which was also distinguishable from at least some isolates from Australia and South Africa. Hence the restriction enzyme (RE) based analysis might be a useful tool in determining the genetic diversity in genome segment 7 and also for tracing their evolutionary relationships.

Restriction analysis of conserved and variable regions of VP2 gene of Indian isolates of bluetongue virus serotype 1.

Bluetongue virus (BTV) is a member of Orbivirus genus in family Reoviridae. The virus genome is composed of 10 double-stranded RNA segments. The RNA segment L2 encodes an outer capsid viral protein VP2, which is the main determinant of neutralization and serotype-specific immune response. BTV serotype 1 (BTV-1) specific novel primer pair was designed using VP2 gene sequences available in GenBank to amplify 1240-1844 bp region because two hypervariable and three conserved regions have been reported within these 604 nucleotides. This primer pair successfully amplified cell culture adapted six Indian isolates of BTV-1 from different geographical regions of the country. The 604 bp PCR product of VP2 gene of all six BTV-1 yielded two fragments of 273 and 331 bp when digested with Taq1 restriction enzyme. This indicated that there is only one TaqI site at 1513 bp (within 1240-1844 bp region) of VP2 gene of BTV-1 Indian isolates. The in silico restriction analysis revealed that in BTV-1 South African isolate (BTV-1SA) there is no TaqI site while in BTV-1 Australian isolates (BTV-1AUS), there are two TaqI sites (at 1513 and 1567 bp) within 1240-1844 bp region of VP2 gene. The earlier reported VP2 gene based primer pair for BTV-1 was used in the present study to amplify 2242-2933 bp region of six BTV-1 Indian isolates as three conserved regions have been reported within these 691 nucleotides. The digestion of 691 bp PCR products with XmnI yielded three fragments of 364, 173 and 154 bp with all the six Indian isolates of BTV-1 suggesting that there are two XmnI sites within 2242-2933 bp region of VP2 gene. A single XmnI site was observed in silico in BTV-1AUS and BTV-1SA isolates at different positions within this region. The in vitro and in silico restriction profile analyses of partial VP2 gene sequences using TaqI and XmnI restriction enzymes indicated a close relationship of Indian isolates of BTV-1 with BTV-1AUS isolates but not with BTV-1SA isolate.

Lengua Azul: Aislamiento y caracterización del virus e identificación de vectores en el noreste argentino / Bluetongue: isolation and characterization of the virus and identification of vectors in northeastern Argentina

To establish if BTV was circulating in Argentina, 94 bovines from the Santo TomÚ and Ituzaingó Departments of Corrientes Province were sampled every 30-60 days during 14 months. Red blood cells from those animals that showed seroconvertion with a c-ELISA were processed for virus isolation by inoculation in embryonated chicken eggs and cell cultures. Cells with CPE were positive by direct and indirect immunofluorescence with BTV specific reagents. These samples examined by electron microscopy showed virus particles with BTV morphological characteristics. Blood samples and tissue culture supernantants were positive by RT-PCR technique with primers corresponding to the segment 3 of the BTV genome. Haematophagous insects were captured in one farm using light traps and Culicoides insignis Lutz was the predominant species detected. This is the first isolation of BTV in Argentina from northeastern bovines without any disease symptom.

RT-PCR and its detection limit for cell culture grown bluetongue virus 1 using NS1 gene group specific primers.

RT-PCR was standardised for the detection of bluetongue viral RNA using highly expressed non structural protein 1 gene as the target gene with specific primers targeted to 274 bp of 5' end of NS1 gene. PCR product was consistently obtained in 30 PCR cycles. Further, detection limit of RT-PCR was estimated using serial 10 fold dilutions of BHK 21 cells grown BTV 1. The study suggested that RT-PCR can be used for detection of BTV in Indian conditions with the sensitivity limit of 10 infectious particles of the virus. The study suggested that this technique may be used as a tool for sensitive detection of BTV in carrier/reservoir animals, insect vectors and certification of animals and their germ- plasm for export and import purposes.

Detection of bluetongue virus genome segment 6 sequences by RT-PCR.

Reverse transcription-PCR (RT-PCR) technique was adopted to amplify a 101 basepair nucleotide sequence of bluetongue virus (BTV) genome segment 6. The specificity of the amplicon was determined by its approximate size in 3% agarose gel electrophoresis, digestion with restriction enzyme MspI, dot-blot hybridization and cycle sequencing. The technique was found to be suitable for detection of bluetongue virus in infected cell culture and clinical samples.