Genetic analysis of two viroceptor genes of orf virus.

In the present study, we analyzed the chemokine-binding protein (CBP) and the GM-CSF/IL-2 inhibition factor (GIF) of orf virus (ORFV) isolates of sheep and goat origin from different geographical regions of India. Both are immunomodulatory proteins known for their unique strategy of establishing short-term immunity and re-infection in their host. The GIF gene is highly conserved, whereas the CBP gene is highly variable. Both the proteins have conserved potential N-glycosylation sites. The GIF protein contains the "WDPWV" motif responsible for receptor activation. In addition, the SUSHI/short consensus repeats (SCR) domain is reported for the first time in ORFV. Both proteins could potentially be used as immunotherapeutic agents in inflammatory diseases related to the overexpression of specific cytokines.

Comparative sequence and structural analysis of the ORF095 gene, a vaccinia virus A4L homolog of capripoxvirus in sheep and goats.

Sheeppox and goatpox are important transboundary animal viral diseases of sheep and goats caused by sheeppox virus (SPPV) and goatpox virus (GTPV), respectively, of the genus Capripoxvirus, family Poxviridae. Among the proteins encoded by the capripoxvirus (CaPV) genome, ORF095 (vaccinia virus A4L homolog) is an immunodominant virion core protein that plays a pivotal role in virus assembly and morphogenesis. In the present study, sequence analysis of the ORF095 genes of 27 SPPV and GTPV isolates or field samples from different geographical regions of India was performed, and structure was prediction was done by homology modeling. A multiple sequence alignment of different CaPV isolates revealed that CaPV-A4L is highly conserved, with several species-specific signature residues, namely A93, A216, A315, G136 and G146 in GTPV, G47, A63, A168 and A276 in SPPV, and G48 and C98 in lumpy skin disease virus (LSDV). Phylogenetically, the CaPV isolates were separated into three major clusters, GTPV, SPPV and LSDV, based on the complete coding sequence of the CaPV-A4L gene. Genus-specific clustering of poxviruses was observed in phylogenetic analysis based on A4L protein homologs of chordopoxviruses. A secondary structure prediction showed the presence of six α-helices and one ß-sheet as well as some coils. The signature residues identified here are potentially useful for genotyping, and the predicted characteristics of the CaPV-A4L protein make it an ideal candidate for use as an immunogenic or diagnostic antigen for the development of immunoassays in  the sero-evaluation of CaPV in target hosts.

Serological profiling of rabies antibodies by enzyme-linked immunosorbent assay and its comparative analysis with rapid fluorescent focus inhibition test in mouse model.

AIM: In this study, we have used enzyme-linked immunosorbent assay (ELISA) as an alternative test to replace the cumbersome rapid fluorescent focus inhibition test (RFFIT) to ascertain the immune status of immunized mice against rabies virus. MATERIALS AND METHODS: Rabies is a devastating disease worldwide caused by rabies virus. Proper usage of pre- or post-exposure rabies vaccine can prevent the disease transmission. In this study, mice were immunized with Vero cell-adapted inactivated rabies vaccine. RFFIT was used as a test to determine the serum neutralizing titers in infected/vaccinated mice. Seroprofiling of mice sera was done in vitro by ELISA. RESULTS: Twenty-one days post-immunization, both ELISA and RFFIT assays indicated similar antibody levels in mice sera that were immunized with Vero cell-adapted inactivated rabies vaccine. Both the tests were correlated, and the linearity was verified by the regression line (R²=0.979). CONCLUSION: In this study, we profiled the serological status of Vero cell-adapted inactivated rabies vaccine through ELISA in mice model that correlated well with the OIE gold standard test RFFIT.

Prokaryotic expression, purification and evaluation of goatpox virus ORF117 protein as a diagnostic antigen in indirect ELISA to detect goatpox.

Goatpox is an economically significant transboundary viral disease of goats that is caused by goatpox virus (GTPV). This study describes the prokaryotic expression of the GTPV ORF117 protein, a homologue of vaccinia virus A27L, and evaluation of its diagnostic potential in ELISA. The GTPV ORF117 gene was cloned into the pET32a vector to express recombinant ORF117 protein (rA27L) in E. coli BL21-CodonPlus (DE3)-RIPL. The bacterial expression of the protein was confirmed by western blot analysis using anti-GTPV polyclonal antibodies that detected rA27L, which is ~ 35 kDa in size. rA27L was affinity purified under native conditions and used to assess the antibody response in an optimized indirect ELISA. The purified antigen specifically reacted with anti-GTPV and anti-SPPV serum in ELISA. A preliminary screening of random and purposive serum samples (n = 520) from sheep and goats using this optimized ELISA gave a positivity rate of 19.4 % with a diagnostic specificity of 88.7% and diagnostic sensitivity of 98.5% when compared to the gold standard serum neutralization test. Our results suggest that the indirect ELISA based on the rA27L protein has potential for serosurveillance and seromonitoring of GTPV in goats.

Baculovirus expression and purification of peste-des-petits-ruminants virus nucleocapsid protein and its application in diagnostic assay.

Peste-des-petits-ruminants (PPR) is a contagious and highly devastating disease of small ruminants. For control of endemic PPR, adequate supply of affordable and reliable diagnostics is critical for effective surveillance, along with the use of highly efficacious live vaccines that are currently available. The nucleocapsid (N) protein of PPR virus (PPRV) is an important candidate antigen for developing specific diagnostic, as it is a major viral protein being highly immunogenic and conserved among the structural proteins. In the present study, we expressed the N protein of PPRV (Sungri/96 strain), in baculovirus expression system and purified using affinity column chromatography. The recombinant protein reacted well with PPRV anti-N monoclonal antibodies and PPRV-specific polyclonal antiserum, suggesting that the expressed protein was authentic and in native form. The recombinant protein was evaluated as antigen in the diagnostic ELISA as reference positive control in place of whole virus antigen. The utility of recombinant PPRV N protein circumvents the need to use live PPRV antigen in the routinely used diagnostics targeting 'N' protein of PPRV, thus allowing large-scale field application of the test.

Molecular evidence and phylogenetic analysis of orf virus isolates from outbreaks in Tripura state of North-East India.

This study describes the first confirmed report of contagious ecthyma in Black Bengal goats from Tripura state, a North-Eastern state of India situated at the Indo-Bangladesh border. Outbreaks were characterized by the high rates of morbidity (58-67%), low mortality (8-10%) and case fatality (11-15%). The etiology of the outbreaks was confirmed as orf virus (ORFV) by standard virological/serological and molecular techniques including sequence analysis of B2L, a major envelop protein gene of genus Parapoxvirus. Sequence and phylogenetic analysis based on B2L gene of ORFV isolates from Tripura revealed that they were closely related to each other and also to other Indian isolates, in particular to ORFV-Shahjahanpur 82/04 isolate from North India. They revealed several specific nucleotide/amino acid substitutions, namely G299A (G100D), G660A, C705T, C795T (N267D) and G872A (R291H) which may be of notable epidemiological significance. This report necessitates the systematic investigation of orf outbreaks in susceptible populations including wild species particularly at transboundary regions by use of rapid diagnostics to control the infection by deploying an effective vaccine/therapeutics and better managemental practices.

Genetic analysis of L1R myristoylated protein of Capripoxviruses reveals structural homogeneity among poxviruses.

Sheeppox virus (SPPV) and goatpox virus (GTPV) are members of the genus Capripoxvirus (CaPV) of the family Poxviridae. CaPVs are responsible for important contagious diseases of small ruminants that are enzootic to the Indian sub-continent, Central and Northern Africa and the Middle East. In the present study, the sequence and phylogenetic analysis of the L1R gene of sixteen CaPV isolates (seven SPPV and nine GTPV) from India were performed along with 3D homology modeling of the L1R protein. L1R is a myristoylated protein responsible for virion assembly and being present on intracellular mature virion (IMV) surface, it is also a potent target for eliciting neutralizing antibodies. Sequence analysis of CaPV L1R gene revealed an ORF of 738bp with >99% and >96% identity within species and between species, respectively, at both nucleotide as well as amino acid levels. Phylogenetic analysis displayed distinct clusters of members of genus Capripoxvirus, as GTPV, SPPV and LSDV. L1R at the protein level showed various species-specific signature residues that may be useful for future grouping or genotyping of CaPV members. CaPV L1R was predicted to possess myristoylation motif GAAASIQTTVNTLNEKI and a potential N-glycosylation site at amino acid residue 50 (Asn). Despite of different host specificity in poxviruses, comparative sequence analysis of L1R proteins revealed highly conserved nature with presence of myristoylation motif (GXXXS) and six cysteine residues forming three disulfide bonds among all poxviruses. The conserved and immunogenic nature of the CaPV L1R gene may prove to be a potential candidate/target for developing molecular diagnostics including recombinant protein based assays and prophylactics for the control of CaPV diseases in tropical countries like India.

Capripox outbreak in a mixed flock of sheep and goats in India.

Generally, capripoxvirus infections are host specific in nature and occasionally infect more than one species. In this study, an investigation was carried out from an outbreak of capripox in a mixed flock of sheep and goats which occurred in 2013 in the State of Jammu & Kashmir. The genetic analysis of P32, RPO30 and GPCR genes revealed that both goats and sheep were infected with goatpox virus.

Loop-mediated isothermal amplification assay for rapid and sensitive detection of sheep pox and goat pox viruses in clinical samples.

A Loop-mediated isothermal amplification (LAMP) assay targeting the highly conserved DNA polymerase gene of capripox virus genome was developed and evaluated for rapid detection of sheep pox and goat pox viruses. The optimized LAMP assay is found specific and sensitive for amplification of target DNA with a diagnostic sensitivity and specificity of 96.6% and 100% respectively compared to quantitative PCR. The detection rate of LAMP, PCR and Q-PCR assays is found to be 81.5%, 67% and 83% respectively. This LAMP assay has the potential for rapid clinical diagnosis and surveillance of sheep pox and goat pox in field diagnostic laboratories.

Isolation of bluetongue virus-1 from cattle in India and phylogenetic analysis of the complete coding sequence of the segment-2 gene.

The isolation of BTV-1 serotype from cattle in India and its phylogenetic relationship based on VP2 gene has been reported. Virus (JBP42/12/Ind) is isolated in BHK-21 cell line from blood sample tested positive for BTV antigen in sandwich ELISA from asymptomatic cattle. Full length VP2 gene of cattle isolate was amplified, cloned and sequenced. On BLAST analysis virus isolate was identified as BTV-1 serotype. Phylogenetic tree based on complete VP2 coding region segregated Indian isolates, Australian isolates and African/European isolates in three distinct clusters. Segregation of Indian BTV-1 isolates at close proximity to the monophyletic cluster of Australian BTV-1 isolates indicates the present isolate as "eastern topotype' of BTV. Multiple alignments of VP2 gene nucleotide sequences suggest that, Indian BTV-1 isolate is more closely related to Australian BTV-1 isolates; where 14.1% to 14.4% and 6.8% to 7.4% divergence was observed at nucleotide and deduced amino acid sequence level respectively.

Characterization of cytopathogenicity of classical swine fever virus isolate induced by Newcastle disease virus.

Classical swine fever virus (CSFV), the causative agent of classical swine fever, belongs to the family Flaviviridae and genus Pestivirus. Some pestiviruses exhibit cytopathic effect in cell culture but exact phenomenon is unknown. Over expression of NS2-3 gene, presence of defective interfering particle and exaltation of Newcastle disease virus (END) phenomenon could be the reasons of cytopathogenicity. In the present study, a CSFV isolate exhibiting cytopathic effect (CPE) in Madin-Darby Canine Kidney (MDCK) cell line was characterized. To characterize cytopathogenicity of such isolate, END test was carried out. Interference of Newcastle disease virus (NDV) in MDCK adapted CSFV was confirmed by RT-PCR and virus neutralization test. Absence of CPE and NDV specific nucleic acid after neutralization confirmed the induction of CPE by NDV. Further, identity of the CSFV isolate in MDCK cell line by immunoperoxidase test, immunoblotting and RT-PCR post NDV neutralization established the virus replication without CPE (non-cytopathic isolate). Findings suggest that, there could be a chance of mixed infection of both CSFV and NDV in the piglet from which the sample was collected for virus isolation.

Prevalence of Peste-des-petits-ruminant virus antibodies in cattle, buffaloes, sheep and goats in India.

The present study describes the prevalence of Peste-des-petits-ruminant virus (PPRV) antibodies in cattle, buffaloes, sheep and goats carried out during the period 2011 using the serum samples randomly collected from different villages of five states of India. A total of 1,498 serum samples [n = 605 (cattle); n = 432 (buffaloes); n = 173 (sheep); n = 288 (goats)] were collected from 52 districts in five states (Andhra Pradesh, Gujarat, Jammu and Kashmir, Maharashtra and Rajasthan) of India and were screened for PPRV-specific antibodies by using PPR monoclonal antibody-based competitive ELISA kit. Analysis of 1,498 samples indicates that an overall seroprevalence of 21.83 % with 11.07 % in cattle, 16.20 % in buffaloes, 45.66 % in sheep and 38.54 % in goats. This report presents the results of PPRV-specific antibodies in situations where the subclinical, inapparent or nonlethal or recovery of infection was suspected in cattle, buffaloes, sheep and goats. The presence of PPRV antibodies demonstrate that bovines are exposed to PPRV infection and it implies the importance of cattle and buffaloes as subclinical hosts for the virus besides widespread presence of the disease in sheep and goats. Further, the study showed that the prevalence of PPRV antibodies in apparently healthy livestock under natural situation, 21.83 % of the animals were protected from PPRV re-infection. This inturn help in the implementation of disease control strategies such as vaccination in that particular geographical area.

Investigation of an outbreak of infectious pustular balanoposthitis in cattle breeding bulls at a frozen semen bank.

Infectious pustular balanoposthitis (IPB) is one of the reproductive disorders caused by bovine herpesvirus 1 (BoHV1) that can be transmitted through artificial insemination. A herd of 63 breeding bulls at a frozen semen bank in Odisha state in India experienced a suspected outbreak of IPB, with 11 bulls showing clinical signs of the infection. Clinical signs were noticed in two bulls initially and a few days after in the other nine animals. Serum samples from 53 bulls were examined for anti-BoHV1 antibodies using a virus neutralisation test (VNT) and a competitive enzyme-linked immunosorbent assay (cELISA); the remaining ten bulls were not included in the study because it was difficult to restrain them at that time. Paired serum samples were collected 21 days apart from ten clinically affected bulls (the eleventh clinically affected bull was not included in the study for the reason stated above). In the neutralisation test, the paired serum samples showed a two- to fourfold increase in anti-BoHV1 antibody titre; in the cELISA, the paired samples were also found positive for anti-BoHV1 antibodies. Serum samples from 43 in-contact bulls were collected about day 22 after the first observation of clinical infection in the herd. Among these serum samples, a total of 30 were found positive for anti-BoHV1 antibodies in the VNT and a total of 30 were found positive in cELISA. Ten samples were positive in one test but not the other and 25 tested positive in both tests. In all, 35 serum samples from in-contact bulls tested positive in either one or both of the two types of test. An overall agreement of 76.74% was found in detection of anti-BoHV1 antibodies in the two tests. Sensitivity was higher than specificity in detection of anti-BoHV1 antibodies in the serum samples. The glycoprotein C region of the genomic DNA of BoHV1 was amplified from semen samples by polymerase chain reaction. The findings from the outbreak indicate that continuous monitoring of breeding bulls at frozen semen banks is warranted to avoid the risks associated with artificial insemination.

Development of loop-mediated isothermal amplification assay for specific and rapid detection of camelpox virus in clinical samples.

In this study, development of loop-mediated isothermal amplification (LAMP) assay based on ankyrin repeat protein gene (C18L) for specific and rapid detection of camelpox virus (CMLV) was carried out. The assay was optimized using viral genomic DNA (gDNA) extracted from density gradient purified CMLV and standard control recombinant DNA plasmid containing the target, which resulted in reliable amplification at 62°C for 60 min. The amplified LAMP product was identified by agarose gel electrophoresis and subsequent direct visualization under UV light or observation by naked-eye for the presence of turbidity and color change following the addition of SYBR Green I dye and hydroxy naphthol blue (HNB). The analytical specificity of LAMP and conventional PCR assays was evaluated using other related poxviruses namely buffalopox, goatpox, sheeppox, and orf viruses, which revealed only a specific amplification of CMLV. The LAMP assay was 10-fold more sensitive than the conventional PCR. Further, the assay was evaluated with DNA extracted from the cell culture isolates of CMLV (n=11) and clinical samples (n=23). These results proved that the developed LAMP is a simple, specific, sensitive, rapid and economical diagnostic tool for detection of CMLV from clinical materials.

TaqMan hydrolysis probe based real time PCR for detection and quantitation of camelpox virus in skin scabs.

The study describes the development of TaqMan hydrolysis probe based real time PCR (rt-PCR) assay targeting the ankyrin repeat protein (C18L) gene sequences for the detection and quantitation of camelpox virus (CMLV) nucleic acid and its comparison with established conventional and SYBR green rt-PCR assays. The assay was specific with an efficiency of 99.4%. The analytical sensitivity was 4 × 10¹ and 0.35 in terms of copy number and picogram of virus genomic DNA, respectively. The assay was linear with an acceptable intra (0.9-2.83% and 0.9-2.3%) and inter-assay (0.46-2.3% and 0.9-3.3) variations, when standard plasmid DNA and genomic DNA from purified CMLV respectively were tested. The assay was rapid, specific and sensitive as that of SYBR green and 1000 times more sensitive than the conventional PCR. It is suitable for the detection of CMLV nucleic acid directly from clinical samples. Further, the assay was evaluated using cell culture adapted CMLV isolates (n=11) and clinical samples (n=23) from camels and humans suspected of camelpox. This is an improved technique over conventional and SYBR green rt-PCR methods for the detection and quantitation of CMLV from skin scabs.

Enhanced proinflammatory cytokine activity during experimental bluetongue virus-1 infection in Indian native sheep.

Bluetongue disease has been causing variable morbidity and mortality in sheep in India and other countries of the world. The experimental infection with Indian BTV-1 strain induced mild to sub-acute infection in native sheep. There were low induction of IL-12, IFN-γ and TNF-α cytokine mRNA expressions determined by real time RT-PCR in draining lymph nodes (DLN), spleen, and PBMCs during the initial stages of infection (8 days post inoculation, DPI) and higher around 15 DPI. The reduced pro-inflammatory cytokine (IFN-γ and TNF-α) responses during the initial stage of infection (8 DPI) was also accompanied by similarly decreased T-cell populations and overt clinical symptoms. Later up regulation of these cytokines and substantial increase in the proportion of CD8(+) T-cells occurred with reduction of clinical signs and disappearance of BTV-1 antigen from tissues as determined by immunohistochemistry and RT-PCR. Thus there is definite involvement of pro-inflammatory cytokines and CD8(+) T cell activity in disease induced by BTV-1 strain.

Picobirnavirus detection in bovine and buffalo calves from foothills of Himalaya and Central India.

The present study describes detection of picobirnavirus (PBV) in faecal samples from bovine and buffalo calves employing the polyacrylamide gel electrophoresis (PAGE). A total of 136 faecal samples from buffalo (n = 122) and cow calves (n = 14) exhibiting clinical signs of diarrhoea and from healthy calves were collected during 2007-2010 from subtropical (central India) and tarai area of western temperate Himalayan foothills (Uttarakhand). The dsRNA nature of the virus was confirmed by nuclease treatment (RNase A, RNaseT1 and DNase 1). PAGE results confirmed 3.67% (5/136) positivity for PBV, showing a typical genomic migration pattern with two discrete bands with size of approximately 2.4 and 1.7 kbps for the larger and smaller segments, respectively. Among the five PBV samples identified, three were from buffalo calves and one from cow calf exhibiting clinical signs of acute diarrhoea, while one sample from non-diarrhoeic buffalo calf also showed the presence of PBV. None of the samples showed dual infection of rotavirus and PBV. The preliminary findings indicate sporadic incidences of PBV in bovine calves and emphasize the need for the development of better diagnostics for early detection and genetic characterization of these emerging isolates of farm animals of economic significance.

Prevalence of classical swine fever virus in India: a 6-year study (2004-2010).

A study was undertaken regarding the prevalence of classical swine fever virus (CSFV) antibodies and antigens in sera and suspected tissue samples of domestic pigs. The samples were received between January 2004 and September 2010. A total of 594 serum samples from 12 states and 287 tissue samples from 13 states of India were tested using commercial enzyme-linked immunosorbent assay (ELISA) kits. The mean prevalence of CSFV antibodies in suspected sera was 63.3% (376/594), whereas 76.7% (220/287) of the suspected samples were found to contain CSFV antigen. The high prevalence of CSFV antibodies suggests that the disease is endemic in the country. This baseline data will be of use in the formulation of control and eradication programmes.

Verotoxic Escherichia coli (STEC) from beef and its products.

In the present investigation, out of 27 (24.10%) strains of Escherichia coli isolated from 112 beef samples comprising raw meat (45), kabab (36) and kofta (31), 9 (33.33%) belonging to 7 different serotypes were verotoxic as tested by vero cell cytotoxic assay. Serotype O145 was the predominant STEC in raw meat. Interestingly, one STEC-O157 strain was also detected. All the STEC strains were positive for Stx genes by polymerase chain reaction showing stx2 (77.78%) to be most predominant followed by stx1 (22.22%). Phenotypic enterohaemolysin production on washed sheep blood agar supplemented with CaCl2 revealed 6 (66.67%) STEC strains to be positive. Presence of STEC in cooked beef products, viz., kabab and kofta appeared to be a matter of concern and potential threat to public health.