High mortality in free-ranging wild boars associated with African swine fever virus in India.

The present study focuses on the pathological and molecular characterization of African swine fever virus (ASFV) associated with an outbreak in wild boars in two national parks in southern India in 2022-2023. Significant mortality was observed among free-ranging wild boars at Bandipur National Park, Karnataka, and Mudumalai National Park, Tamil Nadu. Extensive combing operations were undertaken in both national parks, spanning an area of around 100 km2, originating from the reported epicenter, to estimate the mortality rate. Recovered carcasses were pathologically examined, and ASFV isolates was genetically characterized. Our findings suggested spillover infection of ASFV from nearby domestic pigs, and the virus was equally pathogenic in wild boars and domestic pigs. ASFV intrusion was reported in the Northeastern region of the country, which borders China and Myanmar, whereas the current outbreak is very distantly located, in southern India. Molecular data will help in tracing the spread of the virus in the country.

A retrospective study showing a high rate of seropositivity against SARS-CoV-2 in wild felines in India.

We report a high rate of seropositivity against SARS-CoV-2 in wild felines in India. Seropositivity was determined by microneutralization and plaque reduction neutralization assays in captive Asiatic lions, leopards, and Bengal tigers. The rate of seropositivity was positively correlated with that of the incidence in humans, suggesting the occurrence of large spillover events.

Development and evaluation of recombinant gD protein based ELISA for sero-surveillance of BoHV-1 in India.

Bovine herpes virus-1 (BoHV-1) is responsible for production losses through decreased milk yields, abortions, infertility, and trade restrictions in the bovine population. The disease is endemic in many countries including India. As the virus harbors a unique feature of latency animals once infected with the virus remain sero-positive for lifetime and can re-excrete the virus when exposed to stressful conditions. Hence, identification and culling of infected animals is only the means to minimize infection-associated losses. In this study, an economical indigenous assay for the detection of BoHV-1 specific antibodies was developed to cater to the huge bovine population of the country. The viral structural gD protein, expressed in the prokaryotic system was used for optimization of an indirect ELISA for bovines followed by statistical validation of the assay. The diagnostic sensitivity and specificity of the indirect ELISA were 82.9% and 91.3% respectively. Systematically collected serum samples representing organized, unorganized and breeding farms of India were tested with the indigenously developed assay for further validation.

Genetic and phylogenetic characterization of polycistronic dsRNA segment-10 of bluetongue virus isolates from India between 1985 and 2011.

The smallest polycistronic dsRNA segment-10 (S10) of bluetongue virus (BTV) encodes NS3/3A and putative NS5. The S10 sequence data of 46 Indian BTV field isolates obtained between 1985 and 2011 were determined and compared with the cognate sequences of global BTV strains. The largest ORF on S10 encodes NS3 (229 aa) and an amino-terminal truncated form of the protein (NS3A) and a putative NS5 (50-59 aa) due to alternate translation initiation site. The overall mean distance of the global NS3 was 0.1106 and 0.0269 at nt and deduced aa sequence, respectively. The global BTV strains formed four major clusters. The major cluster of Indian BTV strains was closely related to the viruses reported from Australia and China. A minor sub-cluster of Indian BTV strains were closely related to the USA strains and a few of the Indian strains were similar to the South African reference and vaccine strains. The global trait association of phylogenetic structure indicates the evolution of the global BTV S10 was not homogenous but rather represents a moderate level of geographical divergence. There was no evidence of an association between the virus and the host species, suggesting a random spread of the viruses. Conflicting selection pressure on the alternate coding sequences of the S10 was evident where NS3/3A might have evolved through strong purifying (negative) selection and NS5 through a positive selection. The presence of multiple positively selected codons on the putative NS5 may be advantageous for adaptation of the virus though their precise role is unknown.

Deciphering type-specific neutralizing antibodies to bluetongue virus in goats of Andaman and Nicobar Islands, India.

The presence of antibodies to bluetongue virus (BTV) and the viral antigen is reported recently from the Andaman and Nicobar Islands, a group of islands at the juncture of the Bay of Bengal and the Andaman Sea. A retrospective study was conducted to investigate the presence of neutralizing antibodies to different BTV serotypes in the seroconverted goats of the Islands. Thirty six samples out of 186 serum samples tested were selected on the basis of high antibody titre as predicted in an indirect ELISA. Each of the selected serum samples was used for neutralization of six BTV serotypes (BTV-1, BTV-2, BTV-9, BTV-10, BTV-16 and BTV-23), the most commonly reported serotypes in India. Out of 36 serum samples used in the neutralization study, neutralizing antibodies could be determined in 15 samples. The neutralizing antibodies to BTV-10 were found in more number of the serum samples followed by BTV-1, BTV-2 and BTV-23 and BTV-9 and BTV-16. Many of the serum samples could neutralize more than one BTV serotypes indicating possible widespread superinfections by multiple BTV serotypes in goats in the Islands. Majority of the serum samples used in the neutralization study could not neutralize any of the six BTV serotypes commonly reported from India indicating possible circulation of other BTV serotypes yet to confirm. The present study reveals circulation of multiple BTV serotypes in Andaman and Nicobar Islands where there was no such report available earlier. The findings are laudable as the baseline information for further investigations to identify and characterize the virus and competent vectors and for implementing appropriate suitable control strategies for bluetongue in the Islands and the nearby territories.

An efficient production of hybrid recombinant protein comprising non-structural proteins (NS 1 & NS 3) of bluetongue virus in prokaryotic expression system.

Strategic design and suitable purification techniques are of paramount importance in the production of recombinant proteins, if intended for use in a diagnostic assay. However, there is no single protocol that can be universally adopted for obtaining proteins in requisite quality and quantity across various platforms. In this study, we have targeted proteins of bluetongue virus (BTV), which is the causative agent of an arthropod-borne infectious disease in ruminants. Traditionally, serological diagnosis of the disease has rested upon either virus neutralization test or on an ELISA test that employed a recombinant structural (VP1, VP7) protein. Among the non-structural (NS) proteins of BTV, NS1 and NS3, are preferred candidate antigens in development of immuno-diagnostics as these provide the option for identifying recent/ongoing infection. However, the difficulty in production/purification of recombinant full length NS proteins of BTV in sufficient quantity and quality in various expression systems, due to inherent structural complexities, have restricted their wider applicability as immunodiagnostic reagents. To circumvent the difficulties associated with production/purification, we developed a novel NS1 and NS3 fusion gene (∼1302 bp) encoding for NS1 N-terminus (1M to G252 aa) and NS3 protein containing the N- and C-termini with a deletion of two hydrophobic domains along with intervening variable central domain (118A to A182 aa) of bluetongue virus 23. This construct was cloned, over-expressed and efficiently purified by single step affinity chromatography under unique denaturing/renaturing condition. The purified fusion protein was found suitable for detection of antibodies against BTV in an indirect ELISA (iELISA).

Seroprevalence of bluetongue and presence of viral antigen and type-specific neutralizing antibodies in goats in Tripura, a state at Indo-Bangladesh border of northeastern India.

Bluetongue (BT) is a notifiable multiple species transboundary viral disease of domestic and wild ruminants. Though the disease is enzootic in India, little is known of the disease burden and prevalent serotypes in Tripura, a hilly state of northeastern India sharing a vast porous border with Bangladesh. A surveillance study was conducted to understand the disease burden in goats in Tripura. Serum (n = 1240) and blood (n = 194) samples were collected during the year 2014 to 2017 from all the eight districts of Tripura. The overall prevalence of BT seroconversion was 47.58% whereas the presence of viral antigen was 20.61% at the individual level. Percent seroconversion was found more (50.47 ± 4.00, CI 41.31 to 49.47) in adult goats in comparison to the younger animals where it was 45.39 ± 2.08, CI 42.63 to 58.31. Presence of neutralizing antibodies in selected serum samples (n = 72) was investigated by serum neutralization test (SNT) against six bluetongue virus (BTV) serotypes and BTV-1 was found as most predominant (65.27%) followed by BTV-16 (26.38%), BTV-10 (20.83%), BTV-9 and 23 (13.88%), and BTV-2 (6.94%). To the best of our knowledge, this is the first study conducted in Tripura to investigate the presence of BTV antigen and type-specific neutralizing antibodies in apparently healthy goats.

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.

A coiled-coil motif in non-structural protein 3 (NS3) of bluetongue virus forms an oligomer.

Bluetongue, an arthropod-borne non-contagious hemorrhagic disease of small ruminants, is caused by bluetongue virus (BTV). Several structural and non-structural proteins encoded by BTV have been associated with virulence mechanisms. In the present study, the NS3 protein sequences of bluetongue viral serotypes were analyzed for the presence of heptad regions and oligomer formation. Bioinformatic analysis of NS3 sequences of all 26 BTV serotypes revealed the presence of at least three coiled-coil motifs (CCMs). A conserved α-helical heptad sequence was identified at 14-26 aa (CCM-I), 185-198aa (CCM-II), and 94-116 aa (CCM-III). Among these, CCM-I occurs close to the N-terminus of NS3 and was presumed to be involved in oligomerization. Furthermore, the N-terminus of NS3 (1M-R117 aa) was over-expressed as a recombinant fusion protein in a prokaryotic expression system. Biochemical characterization of recombinant NS3Nt protein revealed that it forms SDS-resistant dimers and high-order oligomers (hexamer and/or octamer) under reducing or non-reducing conditions. Coiled-coil motifs are believed to be critical for NS protein oligomerization and have potential roles in the formation of viroporin ring/pore either with six/eight subunits and this is the first study toward characterization of CCMs in NS3 of bluetongue virus.

Detection of Babesia bigemina infection in cattle from north-eastern India by polymerase chain reaction and its genetic relatedness with other isolates.

A total of 333 blood samples were collected from cattle suspected for haemoprotozoan infections from three states of north-eastern part of India. All the samples were examined for diagnosis of Babesia bigemina infection using PCR for detection of specific DNA. Out of these, 12 (3.60%) samples were found positive for B. bigemina DNA on PCR using the organism-specific primers derived from 18S ribosomal RNA (rRNA) gene of B. bigemina. An expected size of 1124-bp PCR product was visualized on agarose gel electrophoresis with all the 12 samples, and four of the products was further cloned and sequenced. Basic Local Alignment Search Tool (BLAST) analysis of B. bigemina sequences generated in the present study share 99.2 to 99.7% identity at 18S rRNA gene nucleotide sequence level. These Indian B. bigemina sequences were found to be closely related with the cognate gene nucleotide sequences of B. bigemina from Argentina and Kenya where 99.1 to 99.9% and 99.0 to 99.7% nucleotide identities were observed, respectively. Distant relationship of these Indian organisms was observed with few cognate gene sequences from China where more than 7% divergence was observed in the distance matrix.

Design and assessment of a double antigen indirect ELISA for lumpy skin disease surveillance in India.

Lumpy skin disease (LSD), caused by the lumpy skin disease virus of the genus Capripoxvirus, is rapidly emerging across most countries in Asia. Recently, LSD has been linked to very high morbidity and mortality rates. Until 2019, India remained free of LSD, resulting in a lack of locally developed diagnostic kits, biologicals, and other tools necessary for managing the disease in a country with such a large livestock population. Therefore, this study aimed to design and validate an indigenous and cost-effective in-house ELISA for large-scale screening of cattle samples for antibodies to LSDV. The viral major open reading frames ORF 095 and ORF 103 encoding virion core proteins were expressed in a prokaryotic system and the recombinant antigen cocktail was used for optimization and validation of an indirect ELISA (iELISA). The calculated relative diagnostic sensitivity and diagnostic specificity of the iELISA were 96.6 % and 95.1 %, respectively at the cut-off percent positivity (PP≥50 %). The in-house designed double-antigen iELISA was found effective to investigate the seroprevalence of LSDV in various geographical regions of India.

Development of ELISA for the detection of antibodies against VP2 protein of bluetongue virus serotype-1.

Serotype-specific diagnosis of bluetongue virus (BTV) is necessary for sero-surveillance and taking effective control measures. The VP2 is the major serotype determining protein and BTV-1 is the most predominant serotype in India. In the present study, an indirect ELISA (i-ELISA) was optimized for the detection of serotype-specific antibody against BTV-1 serotype. The VP2 protein of BTV-1 was expressed in a prokaryotic expression system and used to optimize i-ELISA to detect VP2 antibodies of BTV-1 in serum samples of both small and large ruminants. Serum samples (n = 363) classified as positive and negative for antibodies to BTV-1 by serum neutralization test (SNT) and also positive and negative for BTV antibodies by c-ELSIA kit (VMRD, USA) were used to determine the cut-off value, diagnostic sensitivity (DSn), and diagnostic specificity (D-Sp) using receiver operating characteristic (ROC) analysis. The percent positivity (PP) value >30.10% was accepted as the cut-off for i-ELISA at which DSn of 99.52% and D-Sp of 99.35% was observed with a 95% confidence interval. Further, there was no cross-reactivity with other available BTV serotypes in the country. The study indicated serotype-specific i-ELISA is sensitive, specific and suitable alternative to tedious SNT method for determining serotype. The assay will also help in the serotype-specific epidemiological studies and implementation of future control strategies including vaccination and selection of suitable serotype as a vaccine candidate.

Development of a rapid lateral flow immunochromatography assay for the detection of group-specific antibodies against VP7 protein of bluetongue virus in multiple species.

Bluetongue virus (BTV), the causative agent of bluetongue disease infects many domestic and wild ruminants. In the present study, colloidal gold nanoparticle-based lateral flow immunochromatography assay (LFIA) was developed to detect the group-specific antibodies to BTV in serum samples of sheep, goats, cattle, and camel. The recombinant VP7 protein of BTV conjugated to colloidal gold nanoparticles (GNPs) was used as a detector reagent. Recombinant streptococcal protein G and monoclonal antibody to BTV group-specific antigen were immobilized as the test and the control line, respectively on a nitrocellulose membrane. The protein G could capture the specific antibodies to BTV present in the serum of multiple ruminant species susceptible to BTV in a common test format and could eliminate the requirement of multiple anti-species antibodies. Upon addition of serum sample, GNP-rVP7 protein-serum complex migrated laterally onto the strip via capillary action and results were analyzed based on appearance of red colour band at test and control line. Serum samples (n = 481) of sheep, goats, cattle, and camel segregated as positive and negative by the commercial competitive-ELISA (c-ELISA) kit were tested in the fabricated LFIA strips to analyze the performance of the assay. In comparison with c-ELISA, the relative diagnostic sensitivity (DSn) of 95.2% with 91.6-97.6 (95%)) confidence interval and relative diagnostic specificity (DSp) of 99.6% 97.8-100.0 (95%) confidence interval were obtained for the optimized LFIA. The agreement between the LFIA and the c-ELISA was excellent as indicated by the kappa coefficient value of 0.949 (SE = 0.0142) with 0.9219 to 0.9779 (95%) confidence interval. The recombinant protein G based LFIA is a sensitive, specific, rapid, one-step test that can be used in the field or poorly equipped laboratories for serological diagnosis and serosurveillance of bluetongue in multiple susceptible species.

Development of recombinant NS1-NS3 antigen based indirect ELISA for detection of bluetongue antibodies in sheep.

Bluetongue is an insect borne (Culicoides) viral disease of small ruminants. The virus blankets the globe with a wide serotypic variation, numbered from 1 to 28. In India 21 different serotypes have been reported to be circulating across the various agro-climatic zones of the country. Non-structural proteins (NSPs) of bluetongue virus have always remained ideal target for differentiation of infected from vaccinated animals. The current study is an extrapolation of our previous work where a novel fusion construct comprising of bluetongue viral segment NS1 and NS3 was successfully cloned, expressed, purified with an efficient strategy for its suitable implementation as a diagnostic antigen. In this study, the applicability of the fusion construct has been further evaluated and optimised for field applicability. The fusion construct used in an ELISA platform projected a relative diagnostic sensitivity and specificity of 98.1% and 95.5% respectively against a pre-established test panel. The rNS1-NS3 ELISA showed substantially good agreement with the commercial BTV antibody detection kit. Finally, the study brings together the diagnostic capability of two NSPs, which can be a handy tool for sero-surveillance of bluetongue.

Genetic characterization and ex-vivo neutralization behavior of bluetongue virus serotype-16 recovered from apparently healthy goat.

Bluetongue virus (BTV) infects almost all the domestic and wild ruminants though the clinical disease is most commonly reported in sheep and some species of deer. Goat and cattle are the most common asymptomatic reservoir of the virus. Full genome sequencing and serological characterization of the virus isolates are emphasized for understanding the phylogenetic relationship and molecular epidemiology of bluetongue (BT). In this study, we report phylogenetic and phenotypic antigenic relationship of a BTV serotype-16 (PDP2/13/Ind) recovered from an apparently healthy goat from the state of Uttarakhand, a hilly terrain of sub-Himalayan India with four other BTV-16 isolates. The full genome sequence data was analyzed and the phylogenetic relationship of the goat isolate with other BTV-16 was established. Phylogenetic analysis revealed cluster of PDP2/13/Ind along with other Indian BTV-16 isolates indicating their close ancestral relationship. A cohesive ancestral relationship, irrespective of the genome segments analyzed, was also observed between Indian and Mediterranean BTV-16. The mean substitution rate of different segments of BTV-16 isolates varied from 3.231 × 10-5 (seg-2) to 1.129 × 10-3 (seg-6) substitutions per site per year. Timescale analysis indicated that all the segments had an older ancestor. No statistically significant geographic structuring of BTV-16 isolates was observed indicating frequent gene flow. The goat isolate shares highest identity (99.5%-99.8%) with G53/ABT/HSR, a BTV-16 recovered from the western part of the country whereas high level of divergence (11.9%-33.3%) at genomic segment level was observed with a Nigerian BTV-16 (NIG1982/10). Phenotypic antigenic relationship (r) of PDP2/13/Ind with other isolate-specific hyperimmune serum (HIS) determined from serum neutralization titer was 0.672 ± 0.058 to 0.948 ± 0.09. On other hand, the calculated 'r' score was 0.636 ± 0.063 to 0.814 ± 0.201 when HIS against PDP2/13/Ind was used to neutralize the other BTV-16 isolates. The percentage antigenic similarity (R) of the PDP2/13/Ind with other BTV-16 isolates was 65.39 ± 5.38-87.67 ± 14.86. Data suggests presence of subtype antigenic variation amongst the BTV-16 isolates recovered from the goats of a geographically restricted area of the state of Uttarakhand, India.

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

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 19,190bp. 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.

Antigenic evidence of bluetongue virus from small ruminant population of two different geographical regions of Odisha, India.

AIM: The aim of the present study was to carry out antigenic detection of bluetongue virus (BTV) among the small ruminant population of two different geographical regions of Odisha (coastal and central) using recombinant VP7 (r-VP-7) based sandwich enzyme-linked immunosorbent assay (s-ELISA). MATERIALS AND METHODS: Blood samples (n=274) were collected from two different geographical pockets of Odisha, which covered mostly the coastal and central regions. Of the total samples under study 185 were from goat and 89 were from sheep. The blood samples were tested for the presence of BTV antigen by r-VP7 based s-ELISA. RESULTS: r-VP-7 s-ELISA detected BTV antigen in 52.43% and 44.94% of the goat and sheep population under study, respectively. This study highlights the antigenic persistence of BTV in the state for the 1(st) time. CONCLUSION: This high antigenic presence in both sheep and goat population suggests an alarming BTV infection in field conditions which warrants more systematic study directed toward isolation and characterization studies as well as the implementation of control strategy for BT in Odisha.

Evaluation of thermo-stability of bluetongue virus recombinant VP7 antigen in indirect ELISA.

This study shows the thermo-stability of lyophilized and purified recombinant VP7 bluetongue virus (BTV) protein in the presence of two sugar stabilizers (trehalose and mannitol) at different temperature. Truncated VP7 protein purified by nickel affinity column was lyophilized in the presence of trehalose and mannitol at 60 mM final concentration and then exposed to different temperature like 4, 25, 37 and 45 °C for various periods like 5 months, 7 weeks, 7 days and 48 h, respectively. After thermal treatment, the reactivity of the protein was evaluated in indirect ELISA. At 4 and 25 °C, the protein was stable up to 5 months and 7 weeks, respectively, irrespective of stabilizers used. At 37 °C, it was stable up to 3 days with both the stabilizers, after which it lost its stability and reactivity. At 45 °C, the protein was stable up to 30 and 24 h with trehalose and mannitol stabilizers, respectively. Both stabilizers found suitable for stability of the protein. However, trehalose appeared to have better stabilizing effect, particularly at higher temperatures than the mannitol. Trehalose could be used as stabilizer for freeze-drying the recombinant VP7 protein if an indirect ELISA kit based on the purified rVP7 protein is supplied to different laboratories of the country for detection of BTV antibody in sheep.

Development of reverse transcription loop mediated isothermal amplification assay for rapid detection of bluetongue viruses.

A single-step reverse transcription loop mediated isothermal amplification (RT-LAMP) assay targeting NS1 - a highly conserved gene among BTV serotypes was optimized and validated with seven serotypes: BTV-1, BTV-2, BTV-9, BTV-10, BTV-16, BTV-21 and BTV-23. The relative sensitivity of the assay was 0.3 TCID50 and no cross reactivity could be observed with foot and mouth disease, peste-des-petits-ruminants, goatpox, sheeppox and orf viruses. The established assay was also assessed by screening of clinical samples and the result is comparable with conventional RT-PCR. The RT-LAMP assay described here could be an additional tool to the existing assays for diagnosis/surveillance of BTV.

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.