Introduction: The reverse transcription polymerase chain reaction (RT-PCR) is one of the most extensively used methods for identification of animals infected with bluetongue virus (BTV). There are several RT-PCR protocols published and several real-time RT-PCR (rtRT-PCR) commercial kits available on the market. Because Poland faced BTV-14 infection in 2012, different protocols were implemented in the country to confirm the RT-PCR results positive for this virus. The article presents a comparative study of several RT-PCR protocols and discusses their diagnostic reliability and applicability.

Bluetongue virus (BTV) genome consists of 10 segmented double-stranded ribonucleic acid (dsRNA). Of these, genome segment 2 (Seg-2), which encodes outer capsid protein VP2 is the primary determinant of virus serotype. In the present study, partial VP2 gene of KRL2/2017 (BTV-16) isolate collected from Kurnool district of Andhra Pradesh was amplified to know the evolutionary relationship of this isolate with previously reported Indian and global BTV-16 isolates. Seg-2 sequence data of KRL2/2017, showed >98% nucleotide identity with other Indian isolates. Comparison of this isolate with BTV-16 isolates across the world, showed higher sequence homology to isolates of Japan followed by Greece and China than to those of isolates from South Africa, Italy, UK, Australia and Indonesia. Phylogenetic analysis suggest that the isolate of the current study belong to eastern topotype similar to the viruses recovered from Japan, Greece and China reflecting their common eastern origin.

To evaluate herd-level risk factors for seropositive status of cattle to 1 or more bluetongue viruses. 110 herds of cattle in Nebraska, North Dakota, and South Dakota. Blood samples were collected before and after the vector season. Samples were tested for antibodies against bluetongue virus by use of a commercially available competitive ELISA. Factors evaluated included descriptors of geographic location and management practices. Trapping of insect vectors was conducted to evaluate vector status on a subset of 57 operations. A multivariable logistic regression model was constructed to evaluate associations. For the full data set, altitude and latitude were associated with risk of having seropositive cattle (an increase in altitude was associated with an increase in risk, and a more northerly location was associated with a decrease in risk of a premise having seropositive cattle). Import of cattle from selected states was associated with an increase in risk of having seropositive cattle. From the subset of herds with data on vector trapping, altitude and latitude were associated with risk of having seropositive cattle, similar to that for the full model. However, commingling with cattle from other herds was associated with a decrease in risk of seropositivity. Findings reported here may be useful in generating additional hypotheses regarding the ecologic characteristics of bluetongue viruses and other vector-borne diseases of livestock. Sentinel surveillance programs are useful for documenting regionalization zones for diseases, which can be beneficial when securing international markets for animals and animal products.

The consequences of inoculation of bluetongue virus (BTV) serotype 11 into 16 susceptible cows either at the time of breeding or at specified stages of pregnancy were studied. The cows were free of BTV or epizootic hemorrhagic disease virus, and none had antibodies to BTV before virus inoculation. A group of 4 cows was mated naturally to a bull reported to shed BTV (CO75B300 strain) in the semen. The bull was suspected of infecting cows at mating with BTV-11, which subsequently transplacentally infected the developing fetuses and induced persistently infected and congenitally malformed progeny. Two groups of 4 pregnant cows were inoculated with an insect-derived strain of BTV-11 (CO75B300), one group by direct deposit into the uterus at estrus, the other, by intradermal and sc administrations. A 90-day fetus was inoculated in utero with virus from the same pool. Four pregnant cows were inoculated with sheep blood-passaged virus of the same BTV-11 strain (CO75B300) by intradermal and sc routes. Three cows were inoculated with BTV-free suspending fluids and ovine erythrocytes by the intrauterine and intradermal-sc routes and were used as in-contact controls.Infection with insect-derived BTV-11 was confirmed in 3 cows of 1 group by virus isolation and by detection of serum antibodies. The 4 cows inoculated with sheep blood suspension of BTV-11 developed viremia and produced antibodies to the virus. None of the cattle had clinical signs of bluetongue, other than 2 cows that had a slight rectal temperature increase on postinoculation day 4.All cows and fetuses that ranged in gestational age from 69 to 217 days appeared grossly normal at necropsy. Antibodies were not detected in fetal blood. Viral antigen was not detected in fetal tissues by inoculation into sheep or by immunofluoerscence, and viral RNA was not detected by use of the polymerase chain reaction. Developmental deformities were not seen in any fetus. The BTV-11 was not transmitted via the bull semen after natural mating. The BTV-11 strain CO75B300, isolated from this bull and passaged either as insect-derived or ovine erythrocyte suspensions, infected 8 cows. However, the virus was not transplacentally transmitted to their fetuses. It was concluded that there was no evidence for congenital BTV-11 infection in this study.

An antigen-capture ELISA was used to detect bluetongue virus (BTV) from blood of infected sheep. A rabbit-origin capture antibody and a mouse-origin detection antibody combined with biotin-avidin amplification were used for the assay. The antigen-capture ELISA could not detect virus directly from the blood of infected sheep because of low virus titer. To enhance detection, virus from infected blood was amplified in cell culture. Virus could then be detected from cell culture supernatant fluids, using the ELISA. This amplification step increased the sensitivity of the assay comparable to that of assays performed in cell culture measuring cytopathic effects. The ELISA procedure was specific for BTV and did not mistakenly identify the antigenically related epizootic hemorrhagic disease virus. The antigen-capture ELISA permitted indirect quantitation and identification of BTV from the blood of infected sheep.

Sheep had viremias that were first detected on day 3 (+/- 1) after infection with several strains of bluetongue virus (BTV) representing United States serotypes 10, 11, 13, and 17. Diphasic peaks of infectivity were attained on days 6 and 10 (+/- 2). Interferon (IFN) was first detected in serum samples on day 5 (+/- 1), and reached greatest concentrations on day 6 (+/- 2), which coincided with the first viremic peak; IFN concentrations then decreased toward zero by day 10 (+/- 2). Interferon peak concentrations induced approximately a 90% decrease in virus titer. The decrease in IFN concentrations by day 9 (+/- 2) corresponded with the second viremic peak on day 10 (+/- 2). Onset of the decrease in detectable concentrations of virus after the second peak of viremia corresponded to the initial detection of serum antibody to BTV by day 10 (+/- 2). Virus titer decreased and antibody production increased until approximately days 21 to 28, when the titers plateaued and virus was not detected. Febrile responses peaked on day 7 (+/- 1) during the peak viremic period. The WBC count was depressed at the time the virus titer increased, but returned to normal values while the sheep were still viremic. Diphasic viremias in BTV-infected sheep were attributed to induction of high concentrations of IFN concurrent with the first virus titer peak, followed by production of antibody to specific BTV strains and a subsequent reduction in viremia at the second virus titer peak.

Bluetongue (BT) is a vector-borne disease of ruminants caused by Bluetongue virus (BTV). Twenty-nine different serotypes of BTV are currently reported throughout the world. The main objective of this study is the development of a subunit vaccine model that could potentially be adapted to provide broad spectrum protection against multiple BTV serotypes, which the conventional vaccines fail to address. To this end, three different BTV proteins (conserved region of viral protein [VP]2, VP5 and NS1) were expressed and purified in an Escherichia coli expression system. The immunogenicity of these proteins was tested in murine models using the MontanideTM ISA 201 VG adjuvant. BALB/c mice were immunised thrice (with individual proteins and a mixture of three proteins) at two-week intervals and were monitored until Day 40 post-infection/vaccination. Protein-specific antibodies directed against the recombinant proteins were detected by indirect enzyme-linked immunosorbent assay. Neutralising antibody (Nab) titres and cross-neutralisation against a range of BTV serotypes (BTV-1, -2, -4, -5, -9, -10, -12, -16, -21, -23 and -24) were determined by serum neutralisation test. The recombinant proteins elicited higher Nab titres compared with the inactivated vaccine group, except for BTV-1, where the inactivated vaccine group elicited higher Nab titres. Additive effect of the three proteins was not observed as the Nab titres generated with a combination of conserved VP2, VP5 and NS1 was similar to those of the individual protein groups. Whilst BTV-12 could only be neutralised by serum raised against the inactivated vaccine group, BTV-5 and -24 could not be neutralised by any of the groups tested. Our cumulative data suggest that the conserved regions of VP2 (cVP2), VP5 and NS1 could play an important part in the novel vaccine design against multiple BTV serotypes. Importantly, given that VP2 was already known to elicit a serotype-specific immune response against BT, we report, for the first time, that the conserved region of VP2 has the ability to induce cross-protective immune response.