N. Mishra

Serological evidence of West Nile virus infection in wild migratory and resident water birds in Eastern and Northern India.

To assess West Nile virus (WNV) infection in wild resident and migratory birds, we tested 3887 samples from 1784 birds belonging to 119 identified species within 30 families collected during 2008-10 from 13 states in India. The serum samples were tested for WNV antibodies initially by a competition ELISA and subsequently by a micro-plaque reduction neutralization test (Micro-PRNT), whereas tracheal and cloacal swabs were subjected to real-time RT-PCR for the detection of the WNV RNA. Twenty six birds (2.46%) out of 1058 tested showed evidence of flavivirus antibodies by ELISA. End point neutralization antibody determinations for WNV and Japanese encephalitis virus (JEV) showed that of the 22 ELISA positive sera, WNV-specific neutralizing antibodies were detected in 17 samples representing nine species of wild birds (residents: Purple swamphen, Little cormorant, Little egret, Black ibis and Spot-billed duck; residents with winter influx: Common coot and Mallard; migratory birds: Ruff and Purple heron), and two samples were positive for both WNV and JEV antibodies. The WNV-specific antibodies were most commonly detected in Mallards and Common coots. WNV genomic RNA was not detected by real-time RT-PCR. The results in this study suggest that wild resident birds are infected occasionally and wild migratory birds rarely with WNV. Additionally, our study provides evidence of WNV infection in eastern and northern India for the first time.

Entry of bovine viral diarrhea virus into ovine cells occurs through clathrin-dependent endocytosis and low pH-dependent fusion

Although mechanisms of bovine viral diarrhea virus (BVDV) entry into bovine cells have been elucidated, little is known concerning pestivirus entry and receptor usage in ovine cells. In this study, we determined the entry mechanisms of BVDV-1 and BVDV-2 in sheep fetal thymus cells. Both BVDV-1 and BVDV-2 infections were inhibited completely by chlorpromazine, β-methyl cyclodextrin, sucrose, bafilomycin A1, chloroquine, and ammonium chloride. Simultaneous presence of reducing agent and low pH resulted in marked loss of BVDV infectivity. Moreover, BVDV was unable to fuse with ovine cell membrane by the presence of reducing agent or low pH alone, while combination of both led to fusion at low efficiency. Furthermore, sheep fetal thymus cells acutely infected with BVDV-1 or BVDV-2 were found protected from heterologous BVDV infection. Taken together, our results showed for the first time that entry of both BVDV-1 and BVDV-2 into ovine cells occurred through clathrin-dependent endocytosis, endosomal acidification, and low pH-dependent fusion following an activation step, besides suggesting the involvement of a common ovine cellular receptor during attachment and entry.

Serological evidence of widespread West Nile virus and Japanese encephalitis virus infection in native domestic ducks (Anas platyrhynchos var domesticus) in Kuttanad region, Kerala, India

Birds can act as reservoirs of West Nile virus (WNV) with a key role in its epidemiology. WNV lineage 1 associated fatal cases of human encephalitis in 2011 and acute flaccid paralysis in 2013 were reported in Alappuzha district, Kerala, India. But no information is available on WNV circulation in domestic ducks, which are abundant, cohabit with humans and occupy wetlands and water bodies in the region. To determine the extent of WNV infection, we investigated 209 sera, 250 oral and 350 cloacal swab samples from local Chara and Chemballi domestic ducks (Anas platyrhynchos var domesticus) in the districts of Alappuzha, Kottayam, Kollam and Pathanamthitta collected during January and March 2015. The serum samples were tested for WNV antibodies first by a competition ELISA and then by a micro virus neutralization test (micro-VNT), while oral and cloacal swabs were subjected to WNV real-time RT-PCR. Ninety five ducks showed evidence of flavivirus antibodies by ELISA. End point neutralizing antibody titre against WNV and Japanese encephalitis virus (JEV) revealed WNV specific antibodies in 24 (11.5%) ducks in 3 districts, JEV specific antibodies in 21 (10%) ducks in 2 districts and flavivirus specific antibodies in 19 (9%) ducks. However, no WNV genomic RNA could be detected. The results of this study demonstrate evidence of widespread WNV and JEV infection in domestic ducks in Kuttanad region, Kerala with a higher seroprevalence to WNV than JEV. Additionally, it highlights the utility of domestic ducks as a surveillance tool to detect WNV/JEV circulation in a region.

Elevated level of pro inflammatory cytokine and chemokine expression in chicken bone marrow and monocyte derived dendritic cells following LPS induced maturation.

The study was designed to characterize and compare chicken bone marrow and peripheral blood monocyte derived dendritic cells (chBM-DC and chMoDC) and to evaluate inflammatory cytokine and chemokine alterations in response upon LPS stimulation. Typical morphology was observed in DCs from 48h of culture using recombinant chicken GM-CSF and IL-4. Maturation of DCs with LPS (1μg/ml) showed significant up regulation of mRNA of surface markers (CD40, CD80, CD83, CD86, MHC-II and DC-LAMP (CD208)), pro-inflammatory cytokines (IL-1β, IL-6, TNF-α (LITAF)), iNOS, chemokine CXCli2 and TLRs4 and 15. Basal level of TLR1 mRNA expression was higher followed by TLR15 in both DCs irrespective of their origin. Expression of iNOS and CXCLi2 mRNA in mature DCs of both origins were higher than other surface molecules and cytokines studied. Hence, its level of expression can also be used as an additional maturation marker for LPS induced chicken dendritic cell maturation along with CD83 and CD40. LPS matured DCs of both origins upregulated IL-12 and IFN-γ. Based on CD40 and CD83 mRNA expression, it was observed that LPS induced the maturation in both DCs, but chMoDCs responded better in expression of surface markers and inflammatory mediator genes.

Molecular characterization of RNA and protein synthesis during a one-step growth curve of bovine viral diarrhoea virus in ovine (SFT-R) cells.

The aim of this study was to determine the kinetics of noncytopathic bovine viral diarrhoea virus (BVDV) multiplication and synthesis of BVDV specific RNA and proteins in ovine cells (SFT-R) during a one-step growth curve. The virus titre and RNA level were determined by focus-forming assay and real time RT-PCR. The RNA synthesis was detected by Northern blot while synthesis of E2 and NS3 proteins was assayed by immunohistochemistry and Western blot. The results showed that synthesis of viral RNA is initiated at 4h, NS3 and E2 proteins are detectable at 6-7h and the replication cycle is complete at 10-12h. Additionally, we provide evidence that NS2-3 protein was cleaved in ovine cells early during infection and in proliferated leukocytes of acutely infected sheep. This study showed that synthesis of BVDV RNA and proteins in ovine cells occurs at similar times as found in bovine cells.

Evidence of a humoral immune response against the prokaryotic expressed N-terminal autoprotease (Npro) protein of bovine viral diarrhoea virus

Bovine viral diarrhoea virus (BVDV) is an economically important pathogen of cattle and sheep belonging to the genus Pestivirus of the family Flaviviridae. Although the BVDV non-structural N-terminal protease (Npro) acts as an interferon antagonist and subverts the host innate immunity, little is known about its immunogenicity. Hence, we expressed a recombinant BVDV Npro-His fusion protein (28 kDa) in E. coli and determined the humoral immune response generated by it in rabbits. The antigenicity of the Npro protein was confirmed by western blot using anti-BVDV hyperimmune cattle, sheep and goat serum, and anti-Npro rabbit serum. When rabbits were immunized with the Npro protein, a humoral immune response was evident by 4 weeks and persisted till 10 weeks post immunization as detected by ELISA and western blot. Despite Npro-specific antibodies remaining undetectable in 80 serum samples from BVDV-infected sheep and goats, BVDV hyperimmune sera along with some of the field cattle, sheep and goat sera with high BVDV neutralizing antibody titres were found positive for Npro antibodies. Our results provide evidence that despite the low immunogenicity of the BVDV Npro protein, a humoral immune response is induced in cattle, sheep and goats only with repeated BVDV exposure.

Expression of Bovine Viral Diarrhea Virus Envelope Glycoprotein E2 in Yeast Pichia pastoris and its Application to an ELISA for Detection of BVDV Neutralizing Antibodies in Cattle

The aim of this article is to express envelope glycoprotein E2 of bovine viral diarrhea virus (BVDV) in yeast Pichia pastoris and its utility as a diagnostic antigen in ELISA. The BVDV E2 gene was cloned into the pPICZαA vector followed by integration into the Pichia pastoris strain X-33 genome for methanol-induced expression. SDS-PAGE and Western blot results showed that the recombinant BVDV E2 protein (72 kDa) was expressed and secreted into the medium at a concentration of 40 mg/L of culture under optimized conditions. An indirect ELISA was then developed by using the yeast-expressed E2 protein. Preliminary testing of 300 field cattle serum samples showed that the E2 ELISA showed a sensitivity of 91.07% and a specificity of 92.02% compared to the reference virus neutralization test. The concordance between the E2 ELISA and VNT was 91.67%. This study demonstrates feasibility of BVDV E2 protein expression in yeast Pichia pastoris for the first time and its efficacy as an antigen in ELISA for detecting BVDV neutralizing antibodies in cattle.

Genetic Analysis and Expression of NS3 Gene of Bovine Viral Diarrhoea Virus 1 from India for Detection of Antibodies in Cattle

Abstract Mishra, N., Pitale, S.S. and Pradhan, H.K. 2008. Genetic analysis and expression of NS3 gene of bovine viral diarrhoea virus 1 from India for detection of antibodies in cattle. J. Appl. Anim. Res., 33: 99–103. Considering the importance of NS3 antigen in diagnosis of bovine viral diarrhea virus (BVDV) infection, we analysed genetically selected Indian isolates in NS3 gene region and generated recombinant NS3 protein in Escherichia coli for its use as ELISA antigen. High degree of conservation was observed when the nucleotide and deduced amino acid sequence of Indian and the reference BVDV 1 isolates were analysed. The helicase domain of NS3 gene of an Indian isolate was cloned into pTriEx-2-Neo expression vector and a recombinant protein of 50 KDa was expressed as cytoplasmic inclusion bodies. The affinity purified protein showed antigenic properties in western blot with BVDV infected cattle serum. When used in ELISA, it could detect and BVDV antibodies in bovine sera. The expression of NS3 recombinant protein showing immunological and diagnostic significance is first such study in India.

Molecular Characterization of Bovine Leukaemia Virus (BLV) Strains Reveals Existence of Genotype 6 in Cattle in India with evidence of a new subgenotype

Bovine leukaemia virus (BLV) causes enzootic leucosis in cattle and is prevalent worldwide. Although recent studies have shown that BLV strains can be classified into 10 distinct genotypes, no information is available regarding the BLV genotype prevalent in cattle in India. To determine the genetic variability in BLV, in this study, 118 adult dairy cows from three states of India were screened for BLV infection by env gp51-specific ELISA and nested PCR. Of the 33 cows found positive by both PCR and ELISA, 10 selected BLV strains were subjected to molecular characterization. Phylogenetic analyses of partial and full-length env gp51 gene sequences of Indian BLV strains and other geographical diverse BLV strains representing all the 10 genotypes revealed that Indian strains belonged to BLV genotype 6. Although Indian strains showed close genetic proximity with the strains circulating in South America, they were classified into a new subgenotype within genotype 6. Alignment of deduced amino acid sequences in gp51 demonstrated substitutions mainly in conformational epitope G, neutralizing domain 2 and linear epitope D, with a novel mutation (threonine to alanine at residue 252) found in D-epitope of all the Indian BLV strains. Although serological evidence of BLV infection in India has been reported earlier, this study on molecular characterization of BLV strains established the existence of BLV genotype 6 in India. Additionally, the results of this study highlight the importance of genetic analysis of geographically diverse BLV strains to understand BLV global genetic diversity and further studies are required to determine BLV genetic diversity and extent of BLV infection in cattle in India.

Development and Evaluation of a Truncated Recombinant NS3 Antigen-based Indirect ELISA for Detection of Pestivirus Antibodies in Sheep and Goats

The aim of this study was to develop an indirect ELISA using the helicase domain of bovine viral diarrhoea virus (BVDV) NS3 protein instead of full-length NS3 protein for detection of BVDV and BDV antibodies in sheep and goats and its validation by comparing its sensitivity and specificity with virus neutralization test (VNT) as the reference test. The purified 50 kDa recombinant NS3 protein was used as the coating antigen in the ELISA. The optimal concentration of antigen was 320 ng/well at a serum dilution of 1:20 and the optimal positive cut-off optical density value was 0.40 based on test results of 418 VNT negative sheep and goat sera samples. When 569 serum samples from sheep (463) and goats (106) were tested, the ELISA showed a sensitivity of 91.71% and specificity of 94.59% with BVDV VNT. A good correlation (93.67%) was observed between the two tests. It showed a sensitivity of 85% and specificity of 86.6% with VNT in detecting BDV antibody positive or negative samples. This study demonstrates the efficacy of truncated recombinant NS3 antigen based ELISA for seroepidemiological study of pestivirus infection in sheep and goats.