Richa Sood

Revelation of Influencing Factors in Overall Codon Usage Bias of Equine Influenza Viruses

Equine influenza viruses (EIVs) of H3N8 subtype are culprits of severe acute respiratory infections in horses, and are still responsible for significant outbreaks worldwide. Adaptability of influenza viruses to a particular host is significantly influenced by their codon usage preference, due to an absolute dependence on the host cellular machinery for their replication. In the present study, we analyzed genome-wide codon usage patterns in 92 EIV strains, including both H3N8 and H7N7 subtypes by computing several codon usage indices and applying multivariate statistical methods. Relative synonymous codon usage (RSCU) analysis disclosed bias of preferred synonymous codons towards A/U-ended codons. The overall codon usage bias in EIVs was slightly lower, and mainly affected by the nucleotide compositional constraints as inferred from the RSCU and effective number of codon (ENc) analysis. Our data suggested that codon usage pattern in EIVs is governed by the interplay of mutation pressure, natural selection from its hosts and undefined factors. The H7N7 subtype was found less fit to its host (horse) in comparison to H3N8, by possessing higher codon bias, lower mutation pressure and much less adaptation to tRNA pool of equine cells. To the best of our knowledge, this is the first report describing the codon usage analysis of the complete genomes of EIVs. The outcome of our study is likely to enhance our understanding of factors involved in viral adaptation, evolution, and fitness towards their hosts.

Genetic and antigenic characterization of H5N1 viruses of clade 2.3.2.1 isolated in India

The recurrent circulation of highly pathogenic avian influenza (HPAI) H5N1 in Indian poultry since 2006 resulted in emergence of the viruses of distinct antigenic clades of haemagglutinin (HA) with the majority of the H5N1 outbreaks since 2011 belonging to clade 2.3.2.1. The present study was aimed to characterize the antigenic profile of a collection of H5N1 HPAI viruses of clade 2.3.2.1 isolated in India by applying antigenic cartography, serological data and phylogenetic analysis. Eleven H5N1 viruses (2 of clade 2.2 and 9 of clade 2.3.2.1) were selected based on genetic analysis and were further characterized by antigenic cartography analysis based on cross HI (hemagglutination inhibition) data. This study highlights the intercladal antigenic differences between clades 2.3.2.1 and 2.2 and the intracladal antigenic divergence among the clade 2.3.2.1 viruses. Five viruses of clade 2.3.2.1 were also studied for analysis of glycosylation pattern of Hemagglutinin (HA) gene and the growth kinetics analysis in MDCK cells in which the viruses CL03485/H5N1 and 03CL488/H5N1 showed better replication kinetics than other viruses. The study presents a baseline data of antigenicity and other factors that can be used in the selection of suitable H5 vaccine strains or HA donor viruses to develop H5 vaccine strains by reverse genetics or other methods for control of currently circulating H5N1 viruses in Indian region.

Development of a capsid based competitive inhibition enzyme-linked immunosorbent assay for detection of bovine immunodeficiency virus antibodies in cattle and buffalo serum.

The aim of this study was to develop a more specific and sensitive competitive inhibition ELISA (CI-ELISA) than the currently used indirect ELISA for detection of antibodies to bovine immunodeficiency virus (BIV) in cattle and buffaloes. Murine monoclonal antibodies (MAbs) were generated against a recombinant capsid (CA) protein of bovine immunodeficiency virus. Of the 13 anti-CA MAbs developed, MAb-9G10 was selected for CI-ELISA based on the maximum inhibition (98%) obtained with reference BIV antibody positive serum. Based on the distribution of percent inhibition of known negative sera (n=50), a cut-off value was set at 40% inhibition. The MAb-based CI-ELISA showed much higher agreement (concordance: 95.4%) than the indirect ELISA (concordance: 77.8%) with Western blot. Out of 672 sera of cattle and buffaloes tested by CI-ELISA from four states of India, 22% (113/516) of cattle and 19% (30/156) of buffalo were sero-positive for BIV with an overall seroprevalence of 21% (143/672) in India.

Single-chain fragment variable antibody against the capsid protein of bovine immunodeficiency virus and its use in ELISA.

Recombinant antibody specific for the capsid (CA) protein of bovine immunodeficiency virus (BIV) was generated in the form of single-chain fragment variable (ScFv) using the phage display technique for affinity selection. The variable heavy (V(H)) and variable light (V(L)) chain gene fragments were recovered from cells of CA-specific hybridoma (9G10) described previously. The V(H) and V(L) DNA fragments were assembled through a flexible linker DNA to generate ScFv fragment which was cloned in a phagemid expression vector to express ScFv protein. The specific reactivity of the expressed ScFv to the CA antigen was confirmed by Western blot, and the ScFv fragment was used to develop a competitive inhibition ELISA for detection of antibodies to BIV in cattle and buffalo. The recombinant antibody was shown to be more than four times sensitive than its parent monoclonal antibody (MAb, 9G10) by testing of spiked samples of reference positive sera. The improved sensitivity of the recombinant antibody-based ELISA was confirmed by the detection of a larger proportion of animals with BIV antibody by it than by the MAb-based ELISA.

Bovine immunodeficiency virus: a lentiviral infection

The bovine immunodeficiency virus (BIV) is a lentivirus which is known to infect cattle worldwide. Though serological and genomic evidence of BIV in cattle has been found throughout the world, isolation of the virus has been reported only from few places. Very little is known about its impact on animal health status, pathogenesis and mode of transmission. BIV is considered generally non-pathogenic and is not known to cause any serious disease in cattle. BIV is genetically and antigenically related to Jembrana disease virus (JDV), the cause of an acute disease in Bali cattle (Bos javanicus) and human immunodeficiency virus, the cause of acquired immunodeficiency syndrome in human. Therefore, it is important to monitor the presence of BIV in cattle to keep vigil over its possible evolution in its natural host to emerge as pathogenic lentivirus like JDV. Differentiation of BIV infection in cattle from the acutely pathogenic JDV is important for diagnosis of the latter. Currently, BIV is considered as a safe model for understanding the complex genome of lentiviruses. Further research on BIV is indeed needed to elucidate its possible role in animal health as well as for insight into the molecular mechanisms adopted by related lentiviruses.

Sheep associated malignant catarrhal fever: an emerging disease of bovids in India.

Malignant catarrhal fever (MCF) is a fatal lymphoproliferative disease affecting bovids, cervids and other ruminant species caused by viruses belonging to subfamily Gammaherpesvirinae, genus Macavirus. Among the 10 MCF viruses known to cause the disease, alcelaphine herpesvirus 1 (AlHV-1) and ovine herpesvirus 2 (OvHV-2) are the two most widely prevalent causative organisms. The AlHV-1 naturally infects wildebeest and causes wildebeest associated MCF (WA-MCF) in cattle in regions of African sub-continent. The OvHV-2 is prevalent in all varieties of domestic sheep as a sub-clinical infection and causes sheep associated MCF (SA-MCF) in susceptible ruminants in most regions of the world. In India, the detection of cases of SA-MCF in cattle and OvHV-2 infection in sheep during the last decade has established the presence of the virus in native sheep of the country. The present review presents up to date information on various aspects of SA-MCF and its causative agent OvHV-2 with special reference to Indian scenario.

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 …

Genetic and codon usage bias analyses of polymerase genes of equine influenza virus and its relation to evolution

BackgroundEquine influenza is a major health problem of equines worldwide. The polymerase genes of influenza virus have key roles in virus replication, transcription, transmission between hosts and pathogenesis. Hence, the comprehensive genetic and codon usage bias of polymerase genes of equine influenza virus (EIV) were analyzed to elucidate the genetic and evolutionary relationships in a novel perspective.ResultsThe group - specific consensus amino acid substitutions were identified in all polymerase genes of EIVs that led to divergence of EIVs into various clades. The consistent amino acid changes were also detected in the Florida clade 2 EIVs circulating in Europe and Asia since 2007. To study the codon usage patterns, a total of 281,324 codons of polymerase genes of EIV H3N8 isolates from 1963 to 2015 were systemically analyzed. The polymerase genes of EIVs exhibit a weak codon usage bias. The ENc-GC3s and Neutrality plots indicated that natural selection is the major influencing factor of codon usage bias, and that the impact of mutation pressure is comparatively minor. The methods for estimating host imposed translation pressure suggested that the polymerase acidic (PA) gene seems to be under less translational pressure compared to polymerase basic 1 (PB1) and polymerase basic 2 (PB2) genes. The multivariate statistical analysis of polymerase genes divided EIVs into four evolutionary diverged clusters - Pre-divergent, Eurasian, Florida sub-lineage 1 and 2.ConclusionsVarious lineage specific amino acid substitutions observed in all polymerase genes of EIVs and especially, clade 2 EIVs underwent major variations which led to the emergence of a phylogenetically distinct group of EIVs originating from Richmond/1/07. The codon usage bias was low in all the polymerase genes of EIVs that was influenced by the multiple factors such as the nucleotide compositions, mutation pressure, aromaticity and hydropathicity. However, natural selection was the major influencing factor in defining the codon usage patterns and evolution of polymerase genes of EIVs.

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.

Development of single-chain Fv against the nucleoprotein of type A influenza virus and its use in ELISA

Single chain fragment variable (ScFv) antibodies specific to the nucleoprotein (NP) of avian influenza virus (AIV) were developed using a phage display system. The variable heavy (VH) and the variable light (VL) chain gene fragments were derived from spleen cells of Balb/c mouse immunized with a recombinant NP (rNP) antigen (∼63 kDa) of H5N1 influenza virus. The VH and the VL DNA fragments were assembled through a flexible linker DNA to generate ScFv DNA that was cloned subsequently in a phagemid to express ScFv protein in Escherichia coli cells. The specific reactivity of the ScFv with the rNP antigen and viral antigen (H5N1) was confirmed by Western blot and ELISA. A competitive inhibition ELISA (CI-ELISA) was developed using the rNP and the anti-NP ScFv for detection of type-specific antibodies to AIV in chicken sera. The ScFv based CI-ELISA was compared with hemagglutination inhibition (HI) test and agar gel immunodiffusion (AGID) test over 850 sera. Sensitivity of the CI-ELISA was 100% with HI and AGID and specificity was 98.7% with HI and 100% with AGID.