Bluetongue Vaccine Research Articles

PPR (koyun keçi vebası) ve mavidil aşısının merinos ırkı koyunlarda eş zamanlı uygulanabilirliğinin araştırılması

Abstract: Peste des Petits Ruminants (PPR) and Bluetongue (BT), which are among the contagious animal diseases, cause serious economic losses and are included in the notifiable diseases by the World Organization for Animal Health (WOAH). PPR and BT vaccines are widely used in countries where outbreaks occur. In this study, we aimed to examine the neutralizing antibody response of simultaneously administered PPR and BT serotype 4 vaccines in sheep. For this purpose, a study was performed under field conditions in a state-owned breeding farm. All the experimental animals were confirmed seronegative by serum neutralization test (SNT) for antibodies to the PPR virus (PPRV) and BT virus serotype 4 (BTV4). Subsequently, the experimental animals were divided into four groups. Group 1 (Group 1/PPR; lamb n=10, sheep n=10) and group 2 (Group 2/BTV4; lamb n=10, sheep n=10) were vaccinated separately with the PPR and BTV4 vaccines. Group 3 was vaccinated simultaneously with PPR and BTV4 (Group 3/PPR-BTV4; lamb n=10, sheep n=10). Group 4 was unvaccinated and served as the control. In each of the experimental groups, the vaccinations were carried out only once. Post-vaccination blood samples were collected at intervals of 1, 3, and 6 months. A total of 280 post-vaccination blood serum samples were evaluated with SNT for PPRV and BTV4 neutralizing antibodies. When the neutralizing antibody levels between the groups were compared, group 3 did not show any statistically significant difference (p>0.05) from group 1 and group 2. Based on the antibody response of simultaneous vaccination, it is shown that the simultaneous vaccination could be administered on the field, and could be labor and cost-effective.

Computational prediction of highly conserved CD8+ and CD4+ T cell epitopes in bluetongue virus: A promising data for developing broad-spectrum bluetongue vaccine

Abstract Bluetongue (BT) is an economically important arboviral disease of sheep, cattle, goats, and wild ruminants, particularly in America and Europe. However, it has remained uncontrolled due to the evolution of >32 serologically distinct BT virus (BTV) serotypes and the lack of broad-spectrum vaccines. While outer VP2 and VP5 proteins, involved in cell penetration, are less conserved, certain core BTV proteins NS1, NS2, NS3, and VP7 have higher amino acid conservancy. Here, using in silico epitope mapping and multiple sequence alignment, we analyzed all the antigenic BTV proteins for the presence of conserved T cell epitopes recognized by different mammals. We find that mouse Major Histocompatibility Complex-I (MHC-I) conserved epitopes are present in NS1, NS3, and VP7, and MHC-II-conserved epitopes in NS1, NS2, NS3, and VP7; and in bovines, Bovine Leukocyte antigen class-I and II-(BoLA-I & II)-conserved epitopes present in NS1, NS2, NS3, and VP7 proteins. The presence of these conserved epitopes from NS1, NS2, NS3, and VP7 proteins of BTV in the selected mammals, including bovine that is closely related to sheep, we believe they are also likely to be presented by MHCs of the primary natural host sheep. Thus, harnessing this knowledge in developing a pan-BTV vaccine would confer broad-spectrum protection against all the BTV serotypes in sheep and bovines. We are grateful for start-up funding by the Department of Microbiology and Immunology, Faculty of Medicine, Dalhousie University. PPCM wishes to acknowledge support from EU H20:20 grant ‘PALE-Blu’ (project number 727393-2).

Antibody response of sheep to simultaneous vaccination of foot-and-mouth disease, peste des petits ruminants, sheep pox and goat pox, and bluetongue vaccines.

There are many infectious animal diseases in T urkey and generally, vaccination is the primarly control strategy to combat them. However, it is difficult to apply all vaccines in a definite period in the field due to limitations of the labor and finance. Rapid vaccination and effective use of labor can be possible with the help of simultaneous vaccine administrations. The study aims to show the effects of simultaneous foot-and-mouth disease (FMD), peste des petits ruminants (PPR), sheep pox and goat pox (SGP), and bluetongue (BT) vaccine administration on the antibody response of sheep. For this aim, 30 sheep were divided into one experiment and 5 control groups. Blood samples were collected in each group at 0, 30 and 60 days post-vaccination (DPV). Immune response was measured with virus neutralization test (VNT) and, liquid phase blocking ELISA (LPBE) for FMDV; VNT for BTV and PPR. A live virus challenge study was performed to determine the immune response of SGP vaccine. As a result, antibody titers for each vaccine agent decreased on 60 DPV with the simultaneous vaccination except FMD. The difference between means of antibody titer decrease with single and simultaneous vaccinations is significant especially for BTV and PPR vaccines at 60DPV (p<0.05). Briefly, this decreasing immune response of three live vaccines can be explained with the development of the interference, administration of these vaccines from the same injection site, the effect of cytokines, especially IL-10 effect of SGP vaccine. It was concluded that four vaccines can not be used simultaneously in sheep.

Pentavalent Disabled Infectious Single Animal (DISA)/DIVA Vaccine Provides Protection in Sheep and Cattle against Different Serotypes of Bluetongue Virus.

Bluetongue (BT) is a midge-borne OIE-notifiable disease of ruminants caused by the bluetongue virus (BTV). There are at least 29 BTV serotypes as determined by serum neutralization tests and genetic analyses of genome segment 2 encoding serotype immunodominant VP2 protein. Large parts of the world are endemic for multiple serotypes. The most effective control measure of BT is vaccination. Conventionally live-attenuated and inactivated BT vaccines are available but have their specific pros and cons and are not DIVA compatible. The prototype Disabled Infectious Single Animal (DISA)/DIVA vaccine based on knockout of NS3/NS3a protein of live-attenuated BTV, shortly named DISA8, fulfills all criteria for modern veterinary vaccines of sheep. Recently, DISA8 with an internal in-frame deletion of 72 amino acid codons in NS3/NS3a showed a similar ideal vaccine profile in cattle. Here, the DISA/DIVA vaccine platform was applied for other serotypes, and pentavalent DISA/DIVA vaccine for “European” serotypes 1, 2, 3, 4, 8 was studied in sheep and cattle. Protection was demonstrated for two serotypes, and neutralization Ab titers indicate protection against other included serotypes. The DISA/DIVA vaccine platform is flexible in use and generates monovalent and multivalent DISA vaccines to combat specific field situations with respect to Bluetongue.

Alternative methods to reduce the animal use in quality controls of inactivated BTV8 Bluetongue vaccines

The acceptance of serology data instead of challenge for market release of new batches of commercial vaccine is under evaluation by regulatory agencies in order to reduce the use of animals and costs for manufacturers. In this study two vaccines for Bluetongue virus serotype 8 were submitted to quality controls required by the European Pharmacopoeia and tested on sheep in comparison with a commercial inactivated vaccine. Body temperature, antibody titres and viraemia of vaccinated and controls sheep were recorded. In addition IL4 and IFNγ in sera and supernatant derived from in vitro stimulation of blood cells were also quantified using two commercial ELISA kit. The outer-capsid protein VP2 contained in vaccine formulations was quantified using a home-made capture-ELISA. Results obtained indicates that in-lab evaluation of cell-mediated and humoral immune response are useful parameters to predict the efficacy of BTV inactivated vaccines avoiding the challenge phase required to release new batches of vaccines with proven clinical efficacy and safety. The correlation observed between serology data and VP2 protein concentration of final product could be useful in-process control to predict if a new vaccine batch of BTV must be discarded or released to the market.

Comparative Evaluation of T-Cell Immune Response to BTV Infection in Sheep Vaccinated with Pentavalent BTV Vaccine When Compared to Un-Vaccinated Animals.

Recent invasion of multiple bluetongue virus serotypes (BTV) in different regions of the world necessitates urgent development of efficient vaccine that is directed against multiple BTV serotypes. In this experimental study, cell mediated immune response and protective efficacy of binary ethylenimine (BEI) inactivated Montanide™ ISA 206 adjuvanted pentavalent (BTV-1, 2, 10, 16 and 23) vaccine was evaluated in sheep and direct challenge with homologous BTV serotypes in their respective group. Significant (P < 0.05) up-regulation of mRNA transcripts of IFN-α, IL-2, IL-6, IL-12, IFN-γ and TNF-α in PBMCs of vaccinated animals as compared to control (un-vaccinated) animals at certain time points was observed. On the other hand, there was a significant increase in mean ± SD percentage of CD8+ T cells after 7 days post challenge (DPC) but, the mean ± SD percentage of CD4+ T-cell population slightly declined at 7 DPC and enhanced after 14 DPC. Significant differences (P < 0.05) of CD8+ and CD4+T cells population was also observed between vaccinated and unvaccinated sheep. The vaccine also significantly (P < 0.05) reduced BTV RNA load in PBMCs of vaccinated animals than unvaccinated animals following challenge. There were no significant difference (P > 0.05) in cytokine induction, BTV RNA load and CD8+ and CD4+cell count among BTV-1, 2, 10, 16 and 23 serotype challenges except significant increase in mean ± SD percentage of CD8+ in BTV-2 group. These findings put forwarded that binary ethylenimine inactivated montanide adjuvanted pentavalent bluetongue vaccine has stimulated cell mediated immune response and most importantly reduced the severity of BTV-1, 2, 10, 16 and 23 infections following challenge in respective group.

Prospects of Next-Generation Vaccines for Bluetongue.

Bluetongue (BT) is a haemorrhagic disease of wild and domestic ruminants with a huge economic worldwide impact on livestock. The disease is caused by BT-virus transmitted by Culicoides biting midges and disease control without vaccination is hardly possible. Vaccination is the most feasible and cost-effective way to minimize economic losses. Marketed BT vaccines are successfully used in different parts of the world. Inactivated BT vaccines are efficacious and safe but relatively expensive, whereas live-attenuated vaccines are efficacious and cheap but are unsafe because of under-attenuation, onward spread, reversion to virulence, and reassortment events. Both manufactured BT vaccines do not enable differentiating infected from vaccinated animals (DIVA) and protection is limited to the respective serotype. The ideal BT vaccine is a licensed, affordable, completely safe DIVA vaccine, that induces quick, lifelong, broad protection in all susceptible ruminant species. Promising vaccine candidates show improvement for one or more of these main vaccine standards. BTV protein vaccines and viral vector vaccines have DIVA potential depending on the selected BTV antigens, but are less effective and likely more costly per protected animal than current vaccines. Several vaccine platforms based on replicating BTV are applied for many serotypes by exchange of serotype dominant outer shell proteins. These platforms based on one BTV backbone result in attenuation or abortive virus replication and prevent disease by and spread of vaccine virus as well as reversion to virulence. These replicating BT vaccines induce humoral and T-cell mediated immune responses to all viral proteins except to one, which could enable DIVA tests. Most of these replicating vaccines can be produced similarly as currently marketed BT vaccines. All replicating vaccine platforms developed by reverse genetics are classified as genetic modified organisms. This implies extensive and expensive safety trails in target ruminant species, and acceptance by the community could be hindered. Nonetheless, several experimental BT vaccines show very promising improvements and could compete with marketed vaccines regarding their vaccine profile, but none of these next generation BT vaccines have been licensed yet.

A comparison of different vaccination schemes used in sheep combining inactivated bluetongue vaccines against serotypes 4 and 8

Eight different vaccination schemes using four commercially available inactivated Bluetongue vaccines against serotypes 4 and 8 in three different combinations (setting 1–3) were tested under field conditions for their ability to generate a measurable immune response in sheep.Animals of setting 1 (groups A–D) were simultaneously vaccinated using either individual injections at different locations (groups A & D) or double injection by a twin-syringe (groups B & C). For both application methods, a one-shot vaccination (groups C & D) was compared to a boosted vaccination (groups A & B). Sheep of setting 2 (groups E–G) were vaccinated in an alternating, boosted pattern at fortnightly intervals starting with serotype 4 (groups E & F) or vice versa (group G). Group H of setting 3 was vaccinated simultaneously and vaccines were injected individually as a one-shot application. Each group consisted of 30 sheep. The immunogenic response was tested in all sheep (n = 240) by ELISA (IDScreen®Bluetongue Competition), while serum neutralisation tests were performed in five to six sheep from each group (n = 45).All vaccine combinations were well tolerated by all sheep. Of all vaccines and schemes described, the simultaneous double injected boosted vaccination of setting 1 (group B) yielded the highest median serotype-specific titres 26 weeks after the first vaccination (afv) and 100% seropositive animals (ELISA) one year afv. In setting 1, there were no relevant significant differences in the immunogenic response between simultaneously applied vaccines at different sites or at the same injection site. Importantly, a one-shot vaccination induced comparable immunogenicity to a boosted injection half a year afv. Low serotype-specific neutralising antibody levels were detected in settings 2 and 3 and are attributed to diverse factors which may have influenced the measured immunogenicity.

Sero-typing of bluetongue vaccine virus serotypes

Sero-typing of bluetongue vaccine virus serotypes

Bluetongue virus vaccine: conventional to modern approach.

Bluetongue (BT) is an economically important, infectious and non-contagious disease of ruminant animals. BT disease is caused by bluetongue virus (BTV) of the genus Orbivirus (the family Reoviridae). BTV is transmitted by certain species of biting midges of the genus Culicoides. Although originally BT was restricted to African continent, now it is present in all the continents except Antarctica. Conventional BT vaccines such as live attenuated and inactivated vaccines showed different degree of success in BT control. However, conventional vaccines have certain disadvantages of reversion to virulent strain and frequent booster dose requirement. Several BT outbreaks in India and the rest of the world open a new insight for development of better vaccines. The development in molecular biology techniques allowed the development and validation of several modern vaccines such as subunit vaccine, recombinant vector vaccine, disabled infections single cycle (DISC) vaccine, differentiating infected from vaccinated animals (DIVA) approach etc. Most of these vaccines are considered as safer, having better protective immune response and provided cross-protective immunization against more than one serotype. Keywords: bluetongue virus; live vaccine; inactivated vaccine; DISC; recombinant vaccine.

Reassortment of bluetongue virus vaccine serotypes in cattle.

Bluetongue is primarily a disease of sheep in South Africa, while cattle and goats are mostly subclinically infected. The viraemia of bluetongue virus in cattle lasts much longer than in sheep and the role of cattle in the epidemiology of bluetongue in South Africa is poorly understood. Bluetongue virus has a segmented double-stranded ribonucleic acid genome and reassortment of genomes is a common feature. The aim of the study was to investigate whether reassortment occurs between vaccine and field strains when simultaneously administered to cattle. Six cattle between the ages of 6 and 12 months were infected with five strains of modified live vaccine bluetongue virus and a virulent field isolate of bluetongue virus 4. Blood samples were subsequently collected daily from these animals from day 1 to day 39 post-inoculation. Viruses were directly isolated during viraemia from the buffy coat on Vero cells using the plaque forming unit method. Analysis of plaques indicated that no reassortants between virulent field and vaccine strains occurred and the virulent bluetongue virus 4 was identified as the predominant virus strain. However, a reassortant virus between two bluetongue virus vaccine strains was isolated from the buffy coat. Whole genome sequences from the vaccine viruses were compared to the suspected reassortant and it was found that segment 8 exchanged between the bluetongue virus 8 and bluetongue virus 9 vaccine strains. The use of the live-attenuated bluetongue virus multivalent vaccine in South Africa causes circulation of different vaccine serotypes in Culicoides spp. and susceptible hosts and cattle might provide the ideal host for reassortment to occur.

Reassortment of bluetongue virus vaccine serotypes in cattle

Bluetongue is primarily a disease of sheep in South Africa, while cattle and goats are mostly subclinically infected. The viraemia of bluetongue virus in cattle lasts much longer than in sheep and the role of cattle in the epidemiology of bluetongue in South Africa is poorly understood. Bluetongue virus has a segmented double-stranded ribonucleic acid genome and reassortment of genomes is a common feature. The aim of the study was to investigate whether reassortment occurs between vaccine and field strains when simultaneously administered to cattle. Six cattle between the ages of 6 and 12 months were infected with five strains of modified live vaccine bluetongue virus and a virulent field isolate of bluetongue virus 4. Blood samples were subsequently collected daily from these animals from day 1 to day 39 post-inoculation. Viruses were directly isolated during viraemia from the buffy coat on Vero cells using the plaque forming unit method. Analysis of plaques indicated that no reassortants between virulent field and vaccine strains occurred and the virulent bluetongue virus 4 was identified as the predominant virus strain. However, a reassortant virus between two bluetongue virus vaccine strains was isolated from the buffy coat. Whole genome sequences from the vaccine viruses were compared to the suspected reassortant and it was found that segment 8 exchanged between the bluetongue virus 8 and bluetongue virus 9 vaccine strains. The use of the live-attenuated bluetongue virus multivalent vaccine in South Africa causes circulation of different vaccine serotypes in Culicoides spp. and susceptible hosts and cattle might provide the ideal host for reassortment to occur.

From the bluetongue vaccination campaigns in sheep to overimmunization and ovine ASIA syndrome.

The use of vaccines has proven to be very effective in controlling and eradicating infectious diseases, both in veterinary and human medicine; however, vaccines can be also the source of an array of problems caused by procedures such as overimmunization. Bluetongue, an orbiviral disease that affects ruminants, is best controlled by the use of inactivated vaccines. During the last years of the past decade, these vaccines were applied all over Europe to control the spreading of the disease, a goal that was accomplished; however, at the same time, several adverse effects related to the vaccination were reported. Especially in sheep, this vaccination campaign brought out a new cachectic and neurologic disease with harmful consequences for the ovine industry. This disease is now recognized as the ovine version of the autoimmune/inflammatory syndrome induced by adjuvants (ASIA syndrome) and poses an immense challenge in veterinary medicine, immunology, and vaccinology.

Cost distribution of bluetongue surveillance and vaccination programmes in Austria and Switzerland (2007–2016)

Bluetongue virus (BTV) is an emerging transboundary disease in Europe, which can cause significant production losses among ruminants. The analysis presented here assessed the costs of BTV surveillance and vaccination...

Expected Net Benefit of Vaccinating Rangeland Sheep against Bluetongue Virus Using a Modified-Live versus Killed Virus Vaccine.

Recurring outbreaks of bluetongue virus in domestic sheep of the US Intermountain West have prompted questions about the economic benefits and costs of vaccinating individual flocks against bluetongue (BT) disease. We estimate the cost of a BT outbreak on a representative rangeland sheep operation in the Big Horn Basin of the state of Wyoming using enterprise budgets and stochastic simulation. The latter accounts for variability in disease severity and lamb price, as well as uncertainty about when an outbreak will occur. We then estimate the cost of purchasing and administering a BT vaccine. Finally, we calculate expected annual net benefit of vaccinating under various outbreak intervals. Expected annual net benefit is calculated for both a killed virus (KV) vaccine and modified-live virus vaccine, using an observed price of $0.32 per dose for modified-live and an estimated price of $1.20 per dose for KV. The modified-live vaccine’s expected annual net benefit has a 100% chance of being positive for an outbreak interval of 5, 10, or 20 years, and a 77% chance of being positive for a 50-year interval. The KV vaccine’s expected annual net benefit has a 97% chance of being positive for a 5-year outbreak interval, and a 42% chance of being positive for a 10-year interval. A KV vaccine is, therefore, unlikely to be economically attractive to producers in areas exposed less frequently to BT disease. A modified-live vaccine, however, requires rigorous authorization before legal use can occur in Wyoming. To date, no company has requested to manufacture a modified-live vaccine for commercial use in Wyoming. The KV vaccine poses less risk to sheep reproduction and less risk of unintentional spread, both of which facilitate approval for commercial production. Yet, our results show an economically consequential tradeoff between a KV vaccine’s relative safety and higher cost. Unless the purchase price is reduced below our assumed $1.20 per dose, producer adoption of a KV vaccine for BT is likely to be low in the study area. This tradeoff between cost and safety should be considered when policymakers regulate commercial use of the two vaccine types.

Humoral Immune Response and Protective Efficacy of Binary Ethylenimine (BEI) Inactivated Pentavalent Bluetongue Vaccine after Challenge with Homologous Virus in Sheep

Humoral Immune Response and Protective Efficacy of Binary Ethylenimine (BEI) Inactivated Pentavalent Bluetongue Vaccine after Challenge with Homologous Virus in Sheep

Bluetongue Disabled Infectious Single Animal (DISA) vaccine: Studies on the optimal route and dose in sheep

Bluetongue (BT) is a disease of ruminants caused by bluetongue virus (BTV) transmitted by biting midges of the Culicoides genus. Outbreaks have been controlled successfully by vaccination, however, currently available BT vaccines have several shortcomings. Recently, we have developed BT Disabled Infectious Single Animal (DISA) vaccines based on live-attenuated BTV without expression of dispensable non-structural NS3/NS3a protein. DISA vaccines are non-pathogenic replicating vaccines, do not cause viremia, enable DIVA and are highly protective. NS3/NS3a protein is involved in virus release, cytopathogenic effect and suppression of Interferon-I induction, suggesting that the vaccination route can be of importance. A standardized dose of DISA vaccine for serotype 8 has successfully been tested by subcutaneous vaccination. We show that 10 and 100times dilutions of this previously tested dose did not reduce the VP7 humoral response. Further, the vaccination route of DISA vaccine strongly determined the induction of VP7 directed antibodies (Abs). Intravenous vaccination induced high and prolonged humoral response but is not practical in field situations. VP7 seroconversion was stronger by intramuscular vaccination than by subcutaneous vaccination. For both vaccination routes and for two different DISA vaccine backbones, IgM Abs were rapidly induced but declined after 14days post vaccination (dpv), whereas the IgG response was slower. Interestingly, intramuscular vaccination resulted in an initial peak followed by a decline up to 21dpv and then increased again. This second increase is a steady and continuous increase of IgG Abs. These results indicate that intramuscular vaccination is the optimal route. The protective dose of DISA vaccine has not been determined yet, but it is expected to be significantly lower than of currently used BT vaccines. Therefore, in addition to the advantages of improved safety and DIVA compatibility, the novel DISA vaccines will be cost–competitive to commercially available live attenuated and inactivated vaccines for Bluetongue.

Detection of antibodies against Bluetongue virus among domestic ruminants in the highlands of Nepal.

Bluetongue (BT) is one of the most economically important transboundary animal diseases. In recent years, it has been considered a disease related to climate change. A study was undertaken in 2013 in Nepal to measure the prevalence of Bluetongue virus (BTV) infection among domestic ruminants inhabiting the 3 agro-climatic zones with altitudes ranging from 150 to 2,400 metres above sea level. Twelve clusters representing the 3 altitudes were selected. The presence of antibodies against BTV was demonstrated in serum samples of sheep, goats, cattle, buffaloes, yaks/chauries, and chyangra goats (Himalayan goat) of Nepal. For this purpose, a total of 2,084 sera were collected from a population of 202 sheep, 739 goats, 590 cattle, 379 buffaloes, 105 yaks/chauries, and 69 chyangra goats between February 2013 and January 2014. The presence of antibodies against BTV was investigated using competitive enzyme-linked immunosorbent assay (c-ELISA). Of the 2,084 collected sera, 45.20% were positive for BTV antibodies. Species-wise prevalence was 17.82%, 47.50%, 53.05%, 58.05%, 7.62%, and 20.29% in sheep, goats, cattle, buffaloes, yak, and chyangra goats, respectively. Contrary to the general belief, maximum numbers of seropositive cases were recorded in buffaloes followed by cattle, goats, chyangra goats, sheep, and yak/chauries. The samples collected in the post-monsoon period (July-August is the monsoon period) show a seroprevalence higher than the pre-monsoon samples. This study shows the seroprevalence of BT in domestic ruminant population of Nepal at all altitudes. The highest prevalence has been reported in the plains of Terai followed by gradual decline in the mid-hills, and in the high mountains. Furthermore, detection of antibodies against BTV in both small and large ruminants (chyangra goats and yak/chauries) dwelling in high altitudes in the absence of BT vaccination is suggesting vector movement to the highlands as a consequence of warmer climate. These findings suggest that the climatic conditions, even at the higher elevation, are suitable for the survival of biting midges responsible for the transmission of BTV.

Using farmers’ attitude and social pressures to design voluntary Bluetongue vaccination strategies

Understanding the context and drivers of farmers’ decision-making is critical to designing successful voluntary disease control interventions. This study uses a questionnaire based on the Reasoned Action Approach framework to assess the determinants of farmers’ intention to participate in a hypothetical reactive vaccination scheme against Bluetongue.Results suggest that farmers’ attitude and social pressures best explained intention. A mix of policy instruments can be used in a complementary way to motivate voluntary vaccination based on the finding that participation is influenced by both internal and external motivation. Next to informational and incentive-based instruments, social pressures, which stem from different type of perceived norms, can spur farmers' vaccination behaviour and serve as catalysts in voluntary vaccination schemes.

Production of recombinant non-structural protein-3 hydrophobic domain deletion (NS3ΔHD) protein of bluetongue virus from prokaryotic expression system as an efficient diagnostic reagent

Serological diagnostics for bluetongue (BT), which is an infectious, non-contagious and arthropod-borne virus disease of ruminants, are primarily dependent on availability of high quality native or recombinant antigen(s) based on either structural/non-structural proteins in sufficient quantity. Non-structural proteins (NS1-NS4) of BT virus are presumed candidate antigens in development of DIVA diagnostics. In the present study, NS3 fusion gene encoding for NS3 protein containing the N- and C-termini with a deletion of two hydrophobic domains (118A to S141 aa and 162S to A182 aa) and intervening variable central domain (142D to K161 aa) of bluetongue virus 23 was constructed, cloned and over-expressed using prokaryotic expression system. The recombinant NS3ΔHD fusion protein (∼38 kDa) including hexa-histidine tag on its both termini was found to be non-cytotoxic to recombinant Escherichia coli cells and purified by affinity chromatography. The purified rNS3ΔHD fusion protein was found to efficiently detect BTV-NS3 specific antibodies in indirect-ELISA format with diagnostic sensitivity (DSn = 94.4%) and specificity (DSp = 93.9%). The study indicated the potential utility of rNS3ΔHD fusion protein as candidate diagnostic reagent in developing an indirect-ELISA for sero-surveillance of animals for BTV antibodies under DIVA strategy, wherever monovalent/polyvalent killed BT vaccine formulations devoid of NS proteins are being practiced for immunization.