Atul Pateriya

Novel Molecular Beacon Probe-Based Real-Time RT-PCR Assay for Diagnosis of Crimean-Congo Hemorrhagic Fever Encountered in India

Crimean-Congo hemorrhagic fever (CCHF) is an emerging zoonotic disease in India and requires immediate detection of infection both for preventing further transmission and for controlling the infection. The present study describes development, optimization, and evaluation of a novel molecular beacon-based real-time RT-PCR assay for rapid, sensitive, and specific diagnosis of Crimean-Congo hemorrhagic fever virus (CCHFV). The developed assay was found to be a better alternative to the reported TaqMan assay for routine diagnosis of CCHF.

Ovine herpesvirus type 2 infection in captive bison in India

MALIGNANT catarrhal fever (MCF) is a fatal disease of wild and domestic ruminants, with severe and widespread inflammatory and degenerative changes in affected animals. It typically has a short, dramatic clinical course, characterised primarily by high fever, severe depression, swollen lymph nodes, salivation, diarrhoea, dermatitis, neurologic disorders and ocular lesions often leading to blindness (Plowright and others 1990). MCF is increasingly being recognised as the cause of significant economic losses in several …

Detection and molecular characterization of naturally transmitted sheep associated malignant catarrhal fever in cattle in India

Malignant catarrhal fever (MCF) is a fatal herpesvirus infection of domestic and wild ruminants, with a short and dramatic clinical course characterized primarily by high fever, severe depression, swollen lymph nodes, salivation, diarrhea, dermatitis, neurological disorders, and ocular lesions often leading to blindness. In the present study, fatal clinical cases of sheep associated malignant catarrhal fever (SA-MCF) were identified in cattle in the state of Karnataka. These cases were initially presented with symptoms of diarrhea, respiratory distress, conjunctivitis, and nasal discharges. Laboratory diagnosis confirmed the detection of ovine herpesvirus-2 (OvHV-2) genome in the peripheral blood samples of two ailing animals. The blood samples collected subsequently from sheep of the neighboring areas also showed presence of OvHV-2 genome indicating a nidus of infection in the region. The positive test results were further confirmed by nucleotide sequencing of the OIE approved portion of tegument gene as well as complete ORF8 region of the OvHV-2 genome. Phylogenetic analysis based on the sequence of the latter region indicated close genetic relationship with other OvHV-2 reported elsewhere in the world.

Production and characterization of monoclonal antibodies against nucleoprotein of avian influenza virus.

The present study was carried out with an aim to develop anti-nucleoprotein (anti-NP) monoclonal antibodies (MAbs) for use in immunodiagnostic testing for detection of avian influenza virus (AIV) antigen or antibodies. The NP gene of AIV, cloned in pET vector, was expressed in Escherichia coli BL 21 strain to produce a 6x-His tagged recombinant NP (rNP) antigen of ∼61 kDa molecular weight as soluble fraction. The rNP antigen was detected in soluble fraction of bacterial cell lysate with anti-His HRPO conjugate and reacted with the reference AIV antibody positive serum in immunoblotting. The rNP was used to immunize BALB/c mice to produce hybridoma secreting anti-NP MAbs. Out of 11 anti-NP MAbs produced, 8D2-H5, 8D2-H9, and 6D11-A7 were of IgM isotype and 5D10-C9 and 5D10-F11 were of IgG2b type, while 3F3-D2, 7D2-C9, 7D2-G7, and 7D2-G8 were of IgG1 isotype. The MAbs 3F3-D2 and 7D2-G8 showed high intensity positive reaction with rNP and a low intensity reaction with H5N1 virus in Western blot analysis. The anti-NP MAbs produced in the present work may be valuable in developing a competitive ELISA or immunochromatographic strip test-based assays for the rapid diagnosis of avian influenza.

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.

A two dose immunization with an inactivated reassortant H5N2 virus protects chickens against lethal challenge with homologous 2.3.2.1 clade and heterologous 2.2 clade highly pathogenic avian influenza H5N1 viruses

The present study was aimed at generating a reassortant vaccine candidate virus with clade 2.3.2.1 Hemagglutinin (HA) and its evaluation in a challenge study for protection against homologous (2.3.2.1 clade) and heterologous (2.2 clade) highly pathogenic avian influenza (HPAI) H5N1 viruses. Plasmid-based reverse genetics technique was used to rescue a 5 + 3 reassortant H5N2 strain containing the modified HA of H5N1 (clade 2.3.2.1), the Neuraminidase (NA) of H9N2, the Matrix (M) of H5N1 and the internal genes of A/WSN/33 H1N1. In addition, another 6 + 2 reassortant virus containing modified HA from H5N1 (clade 2.3.2.1), the NA from H9N2 and the internal genes of A/WSN/33 H1N1 was also rescued. The 5 + 3 reassortant H5N2 virus could grow to a higher titer in both MDCK cells and chicken eggs compared to the 6 + 2 reassortant H5N2 virus. The vaccine containing the inactivated 5 + 3 reassortant H5N2 virus was used in a two-dose immunization regime which protected specific pathogen free (SPF) chickens against two repeated challenges with homologous 2.3.2.1 clade and heterologous 2.2 clade HPAI H5N1 viruses. The 5 + 3 reassortant H5N2 virus based on clade 2.3.2.1 generated in this study can be effective in protecting chickens in the case of an outbreak caused by antigenically different clade 2.2 HPAI H5N1 viruses and opens the way to explore its applicability as potential vaccine candidate especially in the Asian countries reporting these clades frequently. The study also indicates that sequential immunization can broaden protection level against antigenically diverse strains of H5N1 viruses.

Neuraminidase inhibitors susceptibility profiles of highly pathogenic influenza A (H5N1) viruses isolated from avian species in India (2006–2015)

We tested 65 highly pathogenic avian influenza (HPAI) A(H5N1) viruses, isolated from avian species in India between 2006 and 2015, for susceptibility to the FDA approved neuraminidase (NA) inhibitors (NAIs), oseltamivir and zanamivir using a phenotypic fluorescence-based assay. The overall incidence of resistant variants among HPAI A(H5N1) viruses was 7.69% (5/65). The NA inhibition assay identified 3 viruses resistant to oseltamivir (N294S substitution, N2 numbering) and 2 cross-resistant to oseltamivir and zanamivir (E119A or I117V+E119A substitutions), all of which belonged to hemagglutinin (HA) clade 2.2 (5/17) and predominantly circulated in Indian poultry during 2006-2010. In comparison to E119A substitution alone, viruses with I117V+E119A double substitutions showed greater reduction in susceptibility to both oseltamivir and zanamivir. The NAI resistance-associated NA markers, identified in this study, were as a result of naturally occurring mutations. Of note, 48 viruses of HA clade 2.3.2.1 that circulated in Indian poultry during 2011-2015 were susceptible to both oseltamivir and zanamivir. It is essential to monitor NAI susceptibility among human and avian HPAI A(H5N1) viruses that would provide baseline data to develop strategies for pandemic preparedness and therapeutic interventions.