Double and quadruple deletion mutant of EHV-1 is highly attenuated and induces optimal immune response.

Equid alphaherpesvirus 1 (EHV-1) infection causes significant health problems in equines. The EHV-1 infection leads to abortion storm in mares, respiratory disease and myeloencephalopathy. Despite the wide use of vaccines, the outbreaks of EHV-1 infections keep occurring globally, suggesting the need for the development of improved vaccines. Gene deletion attenuated mutant viruses could be a good candidate for the development of modified live vaccines. Here, we report the generation of mutant EHV-1 by deleting virulence (glycoprotein E & internal repeat 6; IR6) and immune evasive (pUL43 & pUL56) associated genes either individually or in combinations; and comprehensive evaluation of mutants through in vitro characterization followed by in vivo study in murine model to adjudge the attenuation of the virus and immune responses generated by mutants vis-à-vis wild type (wt) virus. The EHV-1 mutants with deletion of IR6 and gE genes (vToH-DMV) and four genes (i.e., gE, IR6, pUL43 and pUL56) (vToH-QMV) revealed a significant reduction in plaque size with minimal loss in replication efficiency in comparison to the wt virus. Further, in vivo studies showed virus attenuation adjudged through significant reduction in clinical signs, weight loss, gross and histopathological lesions in comparison to wt virus also revealed improved immune responses estimated through serum neutralization and flow cytometric analysis of CD4 + and CD8 + cell populations. Thus it can be concluded that EHV-1 mutants viz. vToH-DMV and vToH-QMV (novel combination) are promising vaccine candidates and qualify to be studied for adjudging the protective efficacy with wt virus challenge.

Pathological and immunological protection induced by inactivated reverse genetics-based H3N8 equine influenza vaccine candidate in murine model.

Equine influenza (EI) is an important viral respiratory disease of equines caused by influenza A virus (IAV). The antigenic drift in IAVs necessitates regular updating and harmonization of vaccine strain with the circulating virus. The reverse genetics-based recombinant viruses could be easy instrument in generating vaccine against circulating virus in a quick and effective manner. Present study has been envisaged to evaluate the immunogenicity and protective efficacy of inactivated recombinant equine influenza virus (rgEIV) vaccine candidate having six segments from H1N1 virus (A/WSN/33/H1N1) and HA (hemaglutinin) and NA (neuraminidase) segments from H3N8 equine influenza virus [(A/eq/Jammu-Katra/06/08) of clade 2 of Florida sublineage] generated through reverse genetic engineering. BALB/c mice were immunized with inactivated rgEIV adjuvanted with aluminium hydroxide gel and challenged with H3N8 virus (A/eq/Jammu-Katra/06/08). The protective efficacy was evaluated through serology, cytokine profiling, clinical signs, gross and histopathological changes, immunohistochemistry and residual virus quantification. Immunizations induced robust humoral immune response as estimated through hemagglutination inhibition assay (HAI). The antibodies were isotyped and the predominant subclass was IgG1. The vaccine candidate produced mixed Th1 and Th2 responses through stimulation of IFN-γ, IL-2, IL-4 and IL-6 expression. Immunization protected mice against challenge as reflected through reduction in clinical signs and body weight loss, early recovery, mild pathological changes (gross and histopathological lesions) as evident through scoring of lesions, low residual virus in nasopharynx and lungs quantified through egg titration and quantitative reverse transcriptase PCR (qRT-PCR). The study demonstrates that inactivated recombinant EIV generated through reverse genetic approach provides equivalent protection to that observed with inactivated whole H3N8 EIV vaccine. Keywords: equine influenza; reverse genetics; vaccine; pathology; murine model.

Phage therapy for treatment of virulent Klebsiella pneumoniae infection in a mouse model.

OBJECTIVES: Klebsiella pneumoniae is an important emerging pathogen of humans and animals leading to serious clinical consequences. Increased antibiotic use has promoted the emergence of carbapenem-resistant and extended-spectrum ß-lactamase (ESBL)-producing K. pneumoniae strains. Recently, phage therapy has gained momentum as a possible alternative against emerging antimicrobial resistance. This study was performed to assess the therapeutic effects of a novel lytic phage (VTCCBPA43) in a pneumonic mouse model in order to explore the efficacy of phage therapy against virulent K. pneumoniae infection. METHODS: The tailed phage VTCCBPA43 was assessed for its growth kinetics, in vitro host range, and temperature and pH sensitivity. Protein constituents were analysed by SDS-PAGE and nLC-MS/MS. Therapeutic efficacy was observed 2 h post-challenge with virulent K. pneumoniae in a BALB/c mouse model. RESULTS: Phage VTCCBPA43 was found to be highly temperature-tolerant (up to 80 °C). It was most active at pH 5, had a burst size of 172 PFU/mL and exhibited a narrow host range. It was identified as a KP36-like phage by shotgun proteomics. Following intranasal application of a single dose (2 × 109 PFU/mouse) post-challenge with virulent K. pneumoniae, the presence of biologically active phage in vivo and a significant reduction in the lung bacterial load at all time points was observed. A reduction in lesion severity suggested overall beneficial effects of VTCCBPA43 phage therapy in the pneumonic mouse model. CONCLUSION: This research represents the first in vivo evidence of effective phage therapy against K. pneumoniae infection by the intranasal route.

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

BACKGROUND: Equine 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. RESULTS: The 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. CONCLUSIONS: Various 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.