S. C. Dubey

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.

Avian Influenza (H5N1) Virus of Clade 2.3.2 in Domestic Poultry in India

South Asia has experienced regular outbreaks of H5N1 avian influenza virus since its first detection in India and Pakistan in February, 2006. Till 2009, the outbreaks in this region were due to clade 2.2 H5N1 virus. In 2010, Nepal reported the first outbreak of clade 2.3.2 virus in South Asia. In February 2011, two outbreaks of H5N1 virus were reported in the State of Tripura in India. The antigenic and genetic analyses of seven H5N1 viruses isolated during these outbreaks were carried out. Antigenic analysis confirmed 64 to 256-fold reduction in cross reactivity compared with clade 2.2 viruses. The intravenous pathogenicity index of the isolates ranged from 2.80–2.95 indicating high pathogenicity to chickens. Sequencing of all the eight gene-segments of seven H5N1 viruses isolated in these outbreaks was carried out. The predicted amino acid sequence analysis revealed high pathogenicity to chickens and susceptibility to the antivirals, amantadine and oseltamivir. Phylogenetic analyses indicated that these viruses belong to clade 2.3.2.1 and were distinct to the clade 2.3.2.1 viruses isolated in Nepal. Identification of new clade 2.3.2 H5N1 viruses in South Asia is reminiscent of the introduction of clade 2.2 viruses in this region in 2006/7. It is now important to monitor whether the clade 2.3.2.1 is replacing clade 2.2 in this region or co-circulating with it. Continued co-circulation of various subclades of the H5N1 virus which are more adapted to land based poultry in a highly populated region such as South Asia increases the risk of evolution of pandemic H5N1 strains.

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.

Emergence of amantadine-resistant avian influenza H5N1 virus in India

This study reports the genetic characterization of highly pathogenic avian influenza (HPAI) virus (subtype H5N1) isolated from poultry in West Bengal, India. We analyzed all the eight genome segments of two viruses isolated from chickens in January 2010 to understand their genetic relationship with other Indian H5N1 isolates and possible connection between different outbreaks. The hemagglutinin (HA) gene of the viruses showed multiple basic amino acids at the cleavage site, a marker for high virulence in chickens. Of greatest concern was that the viruses displayed amino acid substitution from serine-to-asparagine at position 31 of M2 ion channel protein suggesting emergence of amantadine-resistant mutants not previously reported in HPAI H5N1 outbreaks in India. Amino acid lysine at position 627 of the PB2 protein highlights the risk the viruses possess to mammals. In the phylogenetic trees, the viruses clustered within the lineage of avian isolates from India (2008–2009) and avian and human isolates from Bangladesh (2007–2009) in all the genes. Both these viruses were most closely related to the viruses from 2008 in West Bengal within the subclade 2.2.3 of H5N1 viruses.

Outbreak of avian influenza virus H5N1 in India

SIR, — The highly pathogenic avian influenza (hpai) virus subtype h5n1 has become endemic in poultry populations in south-east Asia since 2003. During the second week of July 2007, an unusual mortality of 82 per cent was reported in a flock of 132 chickens on a poultry farm in Manipur, northeast

Serological evidence of West Nile virus infection in wild migratory and resident water birds in Eastern and Northern India.

To assess West Nile virus (WNV) infection in wild resident and migratory birds, we tested 3887 samples from 1784 birds belonging to 119 identified species within 30 families collected during 2008-10 from 13 states in India. The serum samples were tested for WNV antibodies initially by a competition ELISA and subsequently by a micro-plaque reduction neutralization test (Micro-PRNT), whereas tracheal and cloacal swabs were subjected to real-time RT-PCR for the detection of the WNV RNA. Twenty six birds (2.46%) out of 1058 tested showed evidence of flavivirus antibodies by ELISA. End point neutralization antibody determinations for WNV and Japanese encephalitis virus (JEV) showed that of the 22 ELISA positive sera, WNV-specific neutralizing antibodies were detected in 17 samples representing nine species of wild birds (residents: Purple swamphen, Little cormorant, Little egret, Black ibis and Spot-billed duck; residents with winter influx: Common coot and Mallard; migratory birds: Ruff and Purple heron), and two samples were positive for both WNV and JEV antibodies. The WNV-specific antibodies were most commonly detected in Mallards and Common coots. WNV genomic RNA was not detected by real-time RT-PCR. The results in this study suggest that wild resident birds are infected occasionally and wild migratory birds rarely with WNV. Additionally, our study provides evidence of WNV infection in eastern and northern India for the first time.

Avian influenza virus (H5N1) in chickens in India

In India, outbreaks of avian influenza H5N1 in chickens were previously confirmed in February 2006 (in commercial and backyard units), July 2007 (in a single backyard unit) and January 2008 (mainly in backyard units). All these outbreaks were recorded in different geographical areas and were

Phylogenetic evidence of multiple introduction of H5N1 virus in Malda district of West Bengal, India in 2008

Outbreaks of H5N1 avian influenza virus were reported in 15 districts of West Bengal State in India in early 2008 and subsequent re-occurrence in 5 districts in December, 2008 to May, 2009. We have sequenced complete genome of 12 viruses isolated from early 2008 outbreak and from recurrent outbreak and determined the phylogenetic relationship between the viruses isolated from the two outbreaks. One of the virus isolated in early 2008 from Malda district (A/chicken/West Bengal/81760/2008) clustered with Korean and Russian isolates of 2006 in European-Middle Eastern-African (EMA) 3 sub-lineage of sub-clade 2.2, whereas other viruses showed close genetic relationship with 2007-2009 isolates of Bangladesh. Nucleotide sequence analysis revealed that the PB1-F2 protein expression might be completely abolished due to mutated start codon ((95)ATG(97)→(95)ACG(97)) in this isolate but in all other isolates it was completely expressed. Hence, we conclude that there were two separate introductions of H5N1 viruses in Malda district and this H5N1 virus was not epidemiologically dominant as the viruses isolated subsequently from the same district and region did not share close relationship with this virus. The failure of this virus to spread to adjoining areas suggests that the culling and disposal operations initiated by Government of India were effective.

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.

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.