Sandeep Bhatia

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.

Highly pathogenic avian influenza H5N1 virus induces cytokine dysregulation with suppressed maturation of chicken monocyte-derived dendritic cells

One of the major causes of death in highly pathogenic avian influenza virus (HPAIV) infection in chickens is acute induction of pro‐inflammatory cytokines (cytokine storm), which leads to severe pathology and acute mortality. DCs and respiratory tract macrophages are the major antigen presenting cells that are exposed to mucosal pathogens. We hypothesized that chicken DCs are a major target for induction of cytokine dysregulation by H5N1 HPAIV. It was found that infection of chicken peripheral blood monocyte‐derived dendritic cells (chMoDCs) with H5N1 HPAIV produces high titers of progeny virus with more rounding and cytotoxicity than with H9N2 LPAIV. Expression of maturation markers (CD40, CD80 and CD83) was weaker in both H5N1 and H9N2 groups than in a LPS control group. INF‐α, ‐β and ‐γ were significantly upregulated in the H5N1 group. Pro‐inflammatory cytokines (IL‐1β, TNF‐α and IL‐18) were highly upregulated in early mid (IL‐1), and late (IL‐6) phases of H5N1 virus infection. IL‐8 (CXCLi2) mRNA expression was significantly stronger in the H5N1 group from 6u2009hr of infection. TLR3, 7, 15 and 21 were upregulated 24u2009hr after infection by H5N1 virus compared with H9N2 virus, with maximum expression of TLR 3 mRNA. Similarly, greater H5N1 virus‐induced apoptotic cell death and cytotoxicity, as measured by terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling and lactate dehydrogenase assays, respectively, were found. Thus, both H5N1 and H9N2 viruses evade the host immune system by inducing impairment of chMoDCs maturation and enhancing cytokine dysregulation in H5N1 HPAIV‐infected cells.

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.

Evaluation of antiviral activity of Ocimum sanctum and Acacia arabica leaves extracts against H9N2 virus using embryonated chicken egg model

BackgroundIn the view of endemic avian influenza H9N2 infection in poultry, its zoonotic potential and emergence of antiviral resistance, two herbal plants, Ocimum sanctum and Acacia arabica, which are easily available throughout various geographical locations in India were taken up to study their antiviral activity against H9N2 virus. We evaluated antiviral efficacy of three different extracts each from leaves of O. sanctum (crude extract, terpenoid and polyphenol) and A. arabica (crude extract, flavonoid and polyphenol) against H9N2 virus using in ovo model.MethodsThe antiviral efficacy of different leaves extracts was systematically studied in three experimental protocols viz. virucidal (dose-dependent), therapeutic (time-dependent) and prophylactic (dose-dependent) activity employing in ovo model. The maximum non-toxic concentration of each herbal extracts of O. sanctum and A. arabica in the specific pathogen free embryonated chicken eggs was estimated and their antiviral efficacy was determined in terms of reduction in viral titres, measured by Haemagglutination (HA) and real time quantitative reverse transcription polymerase chain reaction (RT-qPCR) assays.ResultsAll the extracts of O. sanctum (crude extract, terpenoid and polyphenol) and A. arabica (crude extract, flavonoid and polyphenol) showed significant virucidal activity, however, crude extractocimum and terpenoidocimum showed highly significant to significant (pxa0<xa00.001–0.01) decrease in virus genome copy numbers with lowest dose tested. Similarly, therapeutic effect was observed in all three extracts of O. sanctum in comparison to the virus control, nevertheless, crude extractocimum and terpenoidocimum maintained this effect for longer period of time (up to 72xa0h post-incubation). None of the leaves extracts of A. arabica had therapeutic effect at 24 and 48xa0h post-incubation, however, only the crude extractacacia and polyphenolacacia showed delayed therapeutic effect (72xa0h post-inoculation). Prophylactic potential was observed in polyphenolacacia with highly significant antiviral activity compared to virus control (pu2009<u20090.001).ConclusionsThe crude extract and terpenoid isolated from the leaves of O. sanctum and polyphenol from A. arabica has shown promising antiviral properties against H9N2 virus. Future investigations are necessary to formulate combinations of these compounds for the broader antiviral activity against H9N2 viruses and evaluate them in chickens.

Reverse genetics based rgH5N2 vaccine provides protection against high dose challenge of H5N1 avian influenza virus in chicken

An inactivated vaccine was developed using the rgH5N2 virus (6xa0+xa02 reassortant) generated by plasmid based reverse genetics system (RGS) with WSN/33/H1N1 as backbone virus. Following mutation of the basic amino acid cleavage site RRRKKR*GLF to IETR*GLF, the H5-HA (haemagglutinin) gene of the selected donor H5N1 virus (A/chicken/West Bengal/80995/2008) of antigenic clade 2.2 was used along with the N2-NA gene from H9N2 field isolate (A/chicken/Uttar Pradesh/2543/2004) for generation of the rgH5N2 virus. A single dose (0.5xa0ml/bird) of the inactivated rgH5N2 vaccine protected 100% of the vaccinated chickens (nxa0=xa010) on 28(th) dpv (early challenge) and 90% of the vaccinated chickens (nxa0=xa010) on 200(th)xa0dpv (late challenge) against high dose challenge with HPAI virus (10(9) EID50/bird). Challenge virus shedding via oropharynx and cloaca of the vaccinated chickens was detectable by realtime RT-PCR during 1-5xa0dpc and 1-9 days dpc in the early and the late challenge, respectively. The protective level of antibodies (mean HI titrexa0>xa0128) was maintained without booster vaccination for 200 days. The present study provides the experimental evidence about the extent of protection provided by a reverse genetics based vaccine for clade 2.2 H5N1 viruses against challenge with high dose of field virus at two different time points (28xa0dpv and 200xa0dpv). The challenge study is uniquely different from the previous similar experiments on account of 1000 times higher dose of challenge and protection at 200xa0dpv. The protection and virus shedding data of the study may be useful for countries planning to use H5 vaccine in poultry especially against the clade 2.2 H5N1 viruses.

Cross-sectional study indicates nearly a quarter of sheep population in Karnataka state of India is infected with ovine herpesvirus 2

AbstractnIn a cross-sectional study, prevalence of ovine herpesvirus 2 (family: Herpesviridae, subfamily: Gammaherpesvirinae, genus Macavirus and species: Ovine herpesvirus2) infection was estimated in sheep population of Karnataka state in India. Based on the three stage cluster sampling method, whole blood samples (356) of sheep were collected from 11 sheep-dense districts of the state. The samples were tested for presence of OvHV-2 genome by recommended hemi-nested polymerase chain reaction (PCR) test. The true prevalence of OvHV-2 infection in sheep population of Karnataka was 24.44xa0%. Of the 11 district surveyed, highest true prevalence of 42.42xa0% (CI 25.56–59.29) was found in Raichur followed by Tumkur (39.02xa0%, CI 24.09–53.96). Inverse distance weighted interpolation of prevalence indicated that OvHV-2 prevalence within a given district is not uniform and there are areas of varied prevalence. The nucleotide sequence of the 422xa0bp DNA fragment, amplified in PCR, matched 99xa0% with OvHV-2 reference sequence and other sequences reported from India. Grouping of OvHV-2 sequences obtained from Karnataka with those from Andhra Pradesh, Tamil Nadu and Jammu and Kashmir in the neighbour joining tree indicated a close relationship among the OvHV-2s circulating in India. This is the first study in the country where systematic screening of sheep population of a state for the presence of OvHV-2 infection has been carried out, which indicated a widespread prevalence calling for an urgent need for policy measures to prevent economic losses due to the disease in susceptible cattle and buffalo species.

In-silico search of virus-specific host microRNAs regulating avian influenza virus NS1 expression

Avian influenza is a highly contagious viral infection caused by avian influenza virus type A of the family Orthomyxoviridae primarily affecting the avian species. The non-structural protein 1 (NS1) encoded by the NS1 gene of the virus is critical in establishing the infection. NS1 protein acts to suppress the virus-induced host interferon response and also inhibit Protein kinase R activation thereby helping the virus to establish the infection. MicroRNAs (miRNA) are small regulatory endogenous non-coding RNAs of ~22 nucleotides in length located within introns of coding and non-coding genes, exons of non-coding genes or inter-genic regions. miRNAs can target the gene at various sites and effectively reduce or shut down its expression. In this study, set of differentially expressed chicken miRNA identified by deep sequencing H5N1 infected and SPF chicken lung were computationally analyzed, to identify targets in the NS1 gene. 300 differentially expressed miRNAs were then analyzed individually for target sites in gi|147667147|gb|EF362422.1| influenza A virus (A/chicken/India/NIV33487/06(H5N1)) segment 8, complete sequence using RNAhybrid 2.2. The analysis yielded gga-miR-1658* as the potential miRNA which is targeting the NS1 gene of H5N1 genome.

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.