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Abstract Introduction Hemagglutination (HA) and hemagglutination inhibition (HI) assays are conventionally used for detection and identification of influenza viruses. HI assay is also used for detection of antibodies against influenza viruses. Primarily turkey or chicken erythrocytes [red blood cells (RBCs)] are used in these assays, as they are large, nucleated, and sediment fast, which makes it easy to determine the titer. Human influenza viruses agglutinate RBCs from chicken, human, and guinea pig, but not from horse. Human influenza viruses bind preferentially to sialic acid (SA) linked to galactose (Gal) by α 2, 6 linkage (SA α 2, 6-Gal), whereas avian influenza (AI) viruses bind preferentially to SA α 2, 3-Gal linkages. With this background, the present study was undertaken to study erythrocyte binding preferences and receptor specificities of AI viruses isolated from India. Materials and methods A total of nine AI virus isolates (four subtypes) from India and three reference AI strains (three subtypes) were tested in HA and HI assays against mammalian and avian erythrocytes. The erythrocytes from turkey, chicken, goose, guinea pig and horse were used in the study. The receptor specificity determination assays were performed using goose and turkey RBCs. The amino acids present at 190 helix, 130 and 220 loops of the receptor-binding domain of the hemagglutinin protein were analyzed to correlate amino acid changes with the receptor specificity. Results All tested highly pathogenic avian influenza (HPAI) H5N1 viruses reacted with all five types of RBCs in the HA assay; AI H9N2 and H5N2 viruses did not react with horse RBCs. For H5N1 viruses guinea pig and goose RBCs were best for both HA and HI assays. For H9N2 viruses, guinea pig, fowl and turkey RBCs were suitable. For other tested AI subtypes, avian and guinea pig RBCs were better. Eight isolates of H5N1, one H4N6 and one H7N1 virus showed preference to avian sialic acid receptors. Importantly, two isolates of HPAI H5N1, H9N2 and H11N1 viruses showed receptor specificity preference to both avian and mammalian sialic acid (α-2, 3 and α-2, 6) receptors. Conclusions Use of different types of RBCs resulted in titer variations in HA and HI assays. This showed that RBCs giving optimum HA and HI titers would increase sensitivity of detection and would be more appropriate for identification and antigenic analysis of AI viruses. Analysis of 16 amino acids in the receptor-binding domain of the hemagglutinin of HPAI H5N1 viruses revealed that the only variation observed was in S221P amino acid position. Two H5N1 viruses showed S221P amino acid change, out of which only one H5N1 virus showed preference to α 2, 6 sialic acid receptor. One H5N1 virus isolate with amino acid S at 221 position, showed preference to α 2,3 as well as α 2,6 sialic acid receptors. This indicated that factor(s) other than S221P mutation in the hemagglutinin are probably involved in determining receptor specificity of H5N1 viruses. This is the first report of receptor specificity and erythrocyte binding preferences of AI viruses from India.

Abstract Introduction More than 70 outbreaks of the highly pathogenic avian influenza (HPAI) H5N1 have been reported in poultry in the western and north-eastern parts of India. Therefore, in view of the recent HPAI H5N1 outbreaks in poultry, active AI surveillance encompassing wild, resident, migratory birds and poultry was undertaken during 2009–2011 in the State of West Bengal. Methods A total of 5722 samples were collected from West Bengal; 3522 samples (2906 fecal droppings + 616 other environmental samples) were from migratory birds and 2200 samples [1604 tracheal, cloacal swabs, environmental samples, tissue samples + 596 blood (serum)] were from domestic ducks and poultry. All tracheal, cloacal and environmental samples were processed for virus isolation. Virus isolates were detected using hemagglutination assay and identified using hemagglutination inhibition (HI) and reverse transcriptase polymerase chain reaction (RT-PCR) assays. Sequencing and phylogenetic analysis of partial region of the hemagglutinin and neuraminidase genes was done. Intravenous pathogenicity index assays were performed in chickens to assess pathogenicity of AI virus isolates. Serum samples were tested for detection of antibodies against AI viruses using HI assay. Results A total of 57 AI H9N2, 15 AI H4N6 and 15 Newcastle Disease (NDV) viruses were isolated from chickens, from both backyard and wet poultry markets; AI H4N6 viruses were isolated from backyard chickens and domestic ducks. Characterization of AI H9N2 and H4N6 viruses revealed that they were of low pathogenicity. Domestic ducks were positive for antibodies against H5 and H7 viruses while chickens were positive for presence of antibodies against AI H9N2 and NDV. Conclusions In the current scenario of HPAI H5N1 outbreaks in West Bengal, this report shows presence of low pathogenic AI H9N2 and H4N6 viruses in chickens and domestic ducks during the period 2009–2011. This is the first report of isolation of H4N6 from India. Antibodies against AI H5 and H7 in ducks highlight the probable role of domestic ducks in the transmission of AI viruses. Human infections of H9N2 have been reported from China and Hong Kong. This necessitates implementation of prevention and control measures to limit the spread of AI viruses.

Abstract Background The world has recently overcome the first influenza pandemic of the 21st century caused by a novel H1N1 virus (pH1N1) which is a triple reassortant comprising genes derived from avian, human, and swine influenza viruses and antigenically quite different from seasonal H1N1 strains. Although the case fatality rates have decreased in many developed countries, the situation is still alarming in many developing countries including India where considerable numbers of new cases are appearing everyday. There is still a high morbidity and mortality of susceptible adult as well as young population without having underlying health issues due to the influenza infection. Results To achieve a better understanding of the risk posed by the pH1N1 and to understand its pathogenicity, we studied the host gene expression response to Indian isolate of pH1N1 infection and compared it with seasonal H1N1 infection. The response was studied at four different time points (4, 8, 16 and 24 h) post infection (hpi) in A549 cells using microarray platform. We found that pH1N1 induces immune response earlier than seasonal H1N1 viruses, but at the later stages of infection there is a suppression of host immune responses. The infection with pH1N1 resulted in considerable decrease in the expression of cytokine and other immune genes namely IL8, STAT1, B2 M and IL4 compared to seasonal H1N1. Conclusion We propose that the inability to induce strong innate immune response could be a reason for the high transmissibility, pathogenicity and mortality caused by pH1N1 virus.

Abstract Background To understand the molecular mechanism of host responses to highly pathogenic avian influenza virus infection and to get an insight into the means through which virus overcomes host defense mechanism, we studied global gene expression response of human lung carcinoma cells (A549) at early and late stages of infection with highly pathogenic avian Influenza A (H5N1) virus and compared it with a reverse genetics modified recombinant A (H5N1) vaccine virus using microarray platform. Results The response was studied at time points 4, 8, 16 and 24 hours post infection (hpi). Gene ontology analysis revealed that the genes affected by both the viruses were qualitatively similar but quantitatively different. Significant differences were observed in the expression of genes involved in apoptosis and immune responses, specifically at 16 hpi. Conclusion We conclude that subtle differences in the ability to induce specific host responses like apoptotic mechanism and immune responses make the highly pathogenic viruses more virulent.

Abstract Background A focal H5N1 outbreak in poultry was reported from Manipur, a north-eastern state, of India, in 2007. The aim of this study was to genetically characterize the Manipur isolate to understand the relationship with other H5N1 isolates and to trace the possible source of introduction of the virus into the country. Results Characterization of the complete genome revealed that the virus belonged to clade 2.2. It was distinctly different from viruses of the three EMA sublineages of clade 2.2 but related to isolates from wild migratory waterfowl from Russia, China and Mongolia. The HA gene, had the cleavage site GERRRRKR, earlier reported in whooper swan isolates from Mongolia in 2005. A stop codon at position 29 in the PB1-F2 protein could have implications on the replication efficiency. The acquisition of polymorphisms as seen in recent isolates of 2005–07 from distinct geographical regions suggests the possibility of transportation of H5N1 viruses through migratory birds. Conclusion Considering that all eight genes of the earlier Indian isolates belonged to the EMA3 sublineage and similar strains have not been reported from neighbouring countries of the subcontinent, it appears that the virus may have been introduced independently.

Background: To understand the molecular mechanism of host responses to highly pathogenic avian influenza virus infection and to get an insight into the means through which virus overcomes host defense mechanism, we studied global gene expression response of human lung carcinoma cells (A549) at early and late stages of infection with highly pathogenic avian Influenza A (H5N1) virus and compared it with a reverse genetics modified recombinant A (H5N1) vaccine virus using microarray platform. Results: The response was studied at time points 4, 8, 16 and 24 hours post infection (hpi). Gene ontology analysis revealed that the genes affected by both the viruses were qualitatively similar but quantitatively different. Significant differences were observed in the expression of genes involved in apoptosis and immune responses, specifically at 16 hpi. Conclusion: We conclude that subtle differences in the ability to induce specific host responses like apoptotic mechanism and immune responses make the highly pathogenic viruses more virulent. [ABSTRACT FROM AUTHOR]