K. Rajukumar

Isolation and pathotyping of H9N2 avian influenza viruses in Indian poultry.

A total of 1246 faecal and tissue samples collected/received from 119 farms located in various states of India were processed for isolation of avian influenza viruses (AIV) during 2003-2004 as part of a program to monitor AIV infection in Indian poultry population. Avian influenza virus was isolated for the first time in India from poultry farms with history of drop in egg production, respiratory illness and increased mortality in Haryana state. A total of 29 H9N2 AIV isolates were obtained from the states of Punjab, Haryana, Uttar Pradesh, Gujarat, and Orissa and Union Territory Delhi. Subtyping was done by HI, RT-PCR and neuraminidase inhibition assay. Pathotyping of six representative isolates by intravenous pathogenicity index (0.0/3.0) in 6-8 weeks old chicken, trypsin dependency in cell culture and HA cleavage site analysis (335RSSR*GLF341) confirmed that these isolates are low pathogenic. Nucleotide sequence analysis of the HA gene showed that the Indian isolates are very closely related (95.0-99.6%) and shared a homology of 92-96% with H9N2 isolates from Germany and Asian regions other than that of mainland China. Deduced amino acid sequences showed the presence of L226 (234 in H9 numbering) which indicates a preference to binding of alpha (2-6) sialic acid receptors. Two of the six isolates had 7 glycosylation sites in the HA1 cleaved protein and the remaining four had 5 sites. Phylogenetic analysis showed that they share a common ancestor Qa/HK/G1/97 isolate which had contributed internal genes of H5N1 virus circulating in Vietnam. Further characterization of Indian H9N2 isolates is required to understand their nature and evolution.

Genetic analysis of H9N2 avian influenza viruses isolated from India

H9N2 avian influenza viruses are endemic in domestic poultry in Asia and are grouped into three major sublineages represented by their prototype strains A/Duck/Hong Kong/Y280/97 (Y280-like), A/Quail/Hong Kong/G1/97 (G1-like) and A/Chicken/Korea/38349-p96323/96 (Korean-like). To understand the genetic relationship of Indian viruses, we determined the partial nucleotide sequence of five H9N2 avian influenza viruses isolated from chicken in India during 2003–2004 and compared them with H9N2 sequences available in GenBank. Deduced amino acid sequence analysis revealed that four isolates shared an R–S–S–R/G motif at the cleavage site of HA, representing low pathogenicity in chickens, while one virus harbors an R–S–N–R/G motif at the same position. All the viruses maintained the human-like motif 226Lysine (H3 numbering) at the HA receptor binding site. Phylogenetic analysis showed that 50% of the genes (HA, NA, NP and M) were similar to G1-like viruses, whereas the remaining genes of the Indian isolates formed a separate, not yet defined, sublineage in the Eurasian lineage. Our finding provides evidence of a novel reassortant H9N2 genotype of G1-like viruses circulating in India.

An Enzyme-linked Immunosorbent Assay using Immonoaffinity-purified Antigen in the Diagnosis of Caprine Paratuberculosis and Its Comparison with Conventional ELISAs

An enzyme-linked immunosorbent assay (APA-ELISA) using an immunoaffinity-purified antigen was developed and compared with the unabsorbed and absorbed ELISA procedures, using a crude antigenic preparation, for its efficacy in detecting antibodies in goat sera against Mycobacterium avium paratuberculosis. Serum samples from 89 goats belonging to three different flocks, two with a history and evidence of paratuberculosis and one without it, were subjected to each ELISA, which had been standardized on known positive sera from goats experimentally infected with paratuberculosis. Faecal culture, faecal examination and histopathology were used as indicators of infection. The diagnostic sensitivities of the unabsorbed, absorbed and APA-ELISA were 81.8%, 77.3% and 77.3% and the specificities were 90.6%, 93.7% and 96.8%, respectively. The positive predictive values of APA-ELISA (94.4%) was the highest, followed by absorbed ELISA (80.9%) and unabsorbed ELISA (72.0%). The negative predictive values for APA-ELISA, absorbed ELISA and unabsorbed ELISA were 93.0%, 92.7% and 93.8%, respectively. The results indicated the value of APA-ELISA in avoiding the need to absorb individual test sera with Mycobacterium phlei and giving more consistent results than the absorbed ELISA. The APA-ELISA was also better than the other two procedures in terms of specificity and positive predictive values.

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.

Isolation of a lytic bacteriophage against virulent Aeromonas hydrophila from an organized equine farm.

A bacteriophage (VTCCBPA6) against a pathogenic strain of Aeromonas hydrophila was isolated from the sewage of an organized equine breeding farm. On the basis of TEM analysis, phage belonged to family Myoviridae. PCR amplification and sequence analysis of gp23 gene (encoding for major capsid protein) revealed phylogenetic resemblance to T4 like virus genus. Protein profiling by SDS‐PAGE also indicated its resemblance to T4 like phage group. However, the comparison of its gp23 gene sequence with previously reported phages showed similarity with T4‐like phages infecting Enterobacteriaceae instead of Aeromonas spp. Thus, to our knowledge, this report points toward the fact that a novel/evolved phage might exist in equine environment against A. hydrophila, which can be potentially used as a biocontrol agent.

Pathology of Equine Influenza virus (H3N8) in Murine Model

Equine influenza viruses (EIV)—H3N8 continue to circulate in equine population throughout the world. They evolve by the process of antigenic drift that leads to substantial change in the antigenicity of the virus, thereby necessitating substitution of virus strain in the vaccines. This requires frequent testing of the new vaccines in the in vivo system; however, lack of an appropriate laboratory animal challenge model for testing protective efficacy of equine influenza vaccine candidates hinders the screening of new vaccines and other therapeutic approaches. In the present investigation, BALB/c mouse were explored for suitability for conducting pathogenecity studies for EIV. The BALB/c mice were inoculated intranasally @ 2×106.24 EID50 with EIV (H3N8) belonging to Clade 2 of Florida sublineage and monitored for setting up of infection and associated parameters. All mice inoculated with EIV exhibited clinical signs viz. loss in body weights, lethargy, dyspnea, etc, between 3 and 5 days which commensurate with lesions observed in the respiratory tract including rhinitis, tracheitis, bronchitis, bronchiolitis, alveolitis and diffuse interstitial pneumonia. Transmission electron microscopy, immunohistochemistry, virus quantification through titration and qRT-PCR demonstrated active viral infection in the upper and lower respiratory tract. Serology revealed rise in serum lactate dehydrogenase levels along with sero-conversion. The pattern of disease progression, pathological lesions and virus recovery from nasal washings and lungs in the present investigations in mice were comparable to natural and experimental EIV infection in equines. The findings establish BALB/c mice as small animal model for studying EIV (H3N8) infection and will have immense potential for dissecting viral pathogenesis, vaccine efficacy studies, preliminary screening of vaccine candidates and antiviral therapeutics against EIV.

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.

Anti-proliferative role of recombinant lethal toxin of Bacillus anthracis on primary mammary ductal carcinoma cells revealing its therapeutic potential

Bacillus anthracis secretes three secretary proteins; lethal factor (LF), protective antigen (PA) and edema factor (EF). The LF has ability to check proliferation of mammary tumors, chiefly depending on mitogen activated protein kinase (MAPK) signaling pathway. Evaluation of therapeutic potential of recombinant LF (rLF), recombinant PA (rPA) and lethal toxin (rLF + rPA = LeTx) on the primary mammary ductal carcinoma cells revealed significant (p < 0.01) reduction in proliferation of tumor cells with mean inhibition indices of 28.0 ± 1.37% and 19.6 ± 1.47% respectively. However, treatment with rPA alone had no significant anti-proliferative effect as evident by low mean inhibition index of 3.4 ± 3.87%. The higher inhibition index observed for rLF alone as compared to LeTx is contrary to the existing knowledge on LF, which explains the requirement of PA dependent endocytosis for its enzymatic activity. Therefore, the plausible existence of PA independent mode of action of LF including direct receptor mediated endocytosis or modulation of signal transduction cascade via unknown means is hypothesized. In silico protein docking analysis of other cellular receptors for any plausibility to play the role of receptor for LF revealed c-Met receptor showing strongest affinity for LF (H bond = 19; Free energy = −773.96), followed by nerve growth factor receptor (NGFR) and human epidermal growth factor receptor (HER)-1. The study summarizes the use of rLF or LeTx as therapeutic molecule against primary mammary ductal carcinoma cells and also the c-Met as potential alternative receptor for LF to mediate and modulate PA independent signal transduction.

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.