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Not AvailableAn outbreak of highly pathogenic avian influenza A (H5N1) has recently spread to poultry in 9 Asian countries. H5N1 infections have caused >52 human deaths in Vietnam, Thailand, and Cambodia from January 2004 to April 2005. Genomic analyses of H5N1 isolates from birds and humans showed 2 distinct clades with a nonoverlapping geographic distribution. All the viral genes were of avian influenza origin, which indicates absence of reassortment with human influenza viruses. All human H5N1 isolates tested belonged to a single clade and were resistant to the adamantane drugs but sensitive to neuraminidase inhibitors. Most H5N1 isolates from humans were antigenically homogeneous and distinct from avian viruses circulating before the end of 2003. Some 2005 isolates showed evidence of antigenic drift. An updated nonpathogenic H5N1 reference virus, lacking the polybasic cleavage site in the hemagglutinin gene, was produced by reverse genetics in anticipation of the possible need to vaccinate humans.Not Availabl

Molecular characterization of H5N1 avian influenza virus isolated in Tripura, 2011

Not AvailableIn this study, an avian influenza virus isolated from chicken in Tripura in 2011 was characterized by sequencing all the 8 segments of the virus. The HA cleavage site possessed multi-basic amino acids suggesting highly pathogenic avian influenza. Based on sequence data, virus is sensitive to commonly used anti-influenza drugs. The virus belonged to clade 2.3.2.1 and is closely related to 2011 virus isolated in Bangladesh. The multiple amino acid substitutions in the HA antigenic sites could be the cause of antigenic diversity between clades 2.2.2 and 2.3.2.1 reported earlier. Co-circulation of multiple clades of H5N1 virus in neighbouring countries highlights the need for improved avian influenza surveillance in India. Molecular characterization of H5N1 avian influenza virus isolated in Tripura, 2011. Available from: https://www.researchgate.net/publication/270887731_Molecular_characterization_of_H5N1_avian_influenza_virus_isolated_in_Tripura_2011 [accessed Jun 3, 2017].Not Availabl

Antigenic characterization of H5N1 highly pathogenic avian influenza viruses isolated from poultry in India

Not AvailableHighly pathogenic avian influenza (HPAI) is a major health concern worldwide. In this study, we focused on antigenic analysis of HPAI H5N1 viruses isolated from poultry in India between 2006 and 2015 comprising 25 isolates from four phylogenetic clades 2.2 (1 isolate), 2.2.2.1 (1 isolate), 2.3.2.1a (17 isolates) and 2.3.2.1c (6 isolates). Seven H5N1 isolates from all four clades were selected for production of chicken antiserum, and antigenic analysis was carried out by hemagglutination inhibition (HI) assay. HI data indicated antigenic divergence (6-21 fold reduction in cross-reactivity) between the two recently emerged clades 2.3.2.1a and 2.3.2.1c. These two clades are highly divergent (21-128 fold reduction in HI titre) from the earlier clades 2.2 /2.2.2.1 isolated in India. However, a maximum of 2-fold and 4-fold reduction in cross-reactivity was observed within the isolates of homologous clades 2.3.2.1c and 2.3.2.1a, respectively. The molecular basis of inter-clade antigenic divergence was examined in the haemagglutinin (HA) antigenic sites of the H5N1 virus. Amino acid changes at 8 HA antigenic sites were observed between clades 2.3.2.1a and 2.3.2.1c, whereas 20-23 substitutions were observed between clades 2.3.2.1a/2.3.2.1c and 2.2/2.2.2.1. Therefore, a systematic analysis of antigenic drift of the contemporary field isolates is a pre-requisite for determining the suitable strain(s) for vaccine candidature.Not Availabl

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Not AvailableThe type specific Matrix 1 (M1) protein of an Indian H5N1 avian influenza virus (AIV) was expressed as a histidinetagged fusion protein in a prokaryotic expression system and characterized. The M1 gene was amplified by reverse transcription PCR using appropriately designed primers and cloned into the expression vector, pET28a ( + ). The orientation and reading frame of the recombinant expression construct (pET-M1) was confirmed by sequence analysis. The derived amino acid sequence homology between M1 of AIV H5N1 and other reference AIV subtypes was found to be 93.7% to 99.2%. The 33kDA recombinant M1 (rM1) protein was expressed as inclusion body after induction with 1 mM IPTG in E. coli BL21 (DE3)pLysS cells. The protein was purified to near homogeneity by affinity chromatography using Ni- NTA agarose column. The yield of the purified rM1 was found to be 2 mg/100 ml of induced culture. The rM1 was found to react specifically with H1-H15 AIV subtype specific sera in Western blot. The results indicated that the purified rM1 protein could be used as antigen for detection of type specific AIV antibodies by immunoassays. Prokaryotic expression and characterisation of recombinant M1 protein of an Indian H5N1 avian influenza virus. Available from: https://www.researchgate.net/publication/283134347_Prokaryotic_expression_and_characterisation_of_recombinant_M1_protein_of_an_Indian_H5N1_avian_influenza_virus [accessed Jun 3, 2017].Not Availabl

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Not AvailableLow pathogenic avian influenza H9N2 and highly pathogenic avian influenza H5N1 viruses continue to co-circulate in chickens. Prior infection with low pathogenic avian influenza can modulate the outcome of H5N1 infection. In India, low pathogenic H9N2 and highly pathogenic H5N1 avian influenza viruses are co-circulating in poultry. Herein, by using chickens with prior infection of A/chicken/India/04TI05/2012 (H9N2) virus we explored the outcome of infection with H5N1 virus A/turkey/India/10CA03/2012 natural PB1 gene reassortant from H9N2. Four groups (E1-E4) of SPF chickens (n = 6) prior inoculated with 10(6) EID50 of H9N2 virus were challenged with 10(6) EID50 of H5N1 natural reassortant (PB1-H9N2) virus at days 1 (group E1); 3 (group E2); 7 (group E3) and 14 (group E4) post H9N2 inoculation. The survival percentage in groups E1-E4 was 0, 100, 66.6 and 50%, respectively. Virus shedding periods for groups E1-E4 were 3, 4, 7 and 9 days, respectively post H5N1 challenge. Birds of group E1 and E2 were shedding both H9N2 and H5N1 viruses and mean viral RNA copy number was higher in oropharyngeal swabs than cloacal swabs. In group, E3 and E4 birds excreted only H5N1 virus and mean viral RNA copy number was higher in most cloacal swabs than oral swabs. These results indicate that prior infection with H9N2 virus could protect from lethal challenge of reassortant H5N1 virus as early as with three days prior H9N2 inoculation and protection decreased in groups E3 and E4 as time elapsed. However, prior infection with H9N2 did not prevent infection with H5N1 virus and birds continue to excrete virus in oropharyngeal and cloacal swabs. Amino acid substitution K368E was found in HA gene of excreted H5N1 virus of group E3. Hence, concurrent infection can also cause emergence of viruses with mutations leading to virus evolution. The results of this study are important for the surveillance and epidemiological data analysis where both H9N2 and H5N1 viruses are co-circulating.Not Availabl

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Not AvailableIn view of the tenacity of viruses in the feathers, many workers have suggested the importance of using feathers as the diagnostic material for epidemiological surveillance of highly pathogenic avian influenza (HPAI) H5N1 virus infections. In this study, we have compared the efficiency of two different processing methods, immersion method and trituration method for elution of HPAI H5N1 virus from the feathers of avian influenza negative ducks in terms of virus isolation in 9-to 11-day-old embryonated specific pathogen free chicken eggs and viral RNA detection by haemagglutinin gene based real-time RTPCR. The recovery of virus by immersion method in terms of percent infectivity in 3 replicates was 96.67, 93.33 and 96.67, whereas in trituration method, percent infectivity was 26.67, 20 and 16.67. In real-time RTPCR, viral RNA could be detected in 17 out of 18 samples by immersion method and from only 2 out of 18 samples by trituration method. The study revealed that the immersion method gave higher viral infectivity percentage and could also be easily detected by real-time RTPCR. We conclude that immersion method of virus elution could be useful for processing of shed duck feathers present in the environment for epidemiological screening against HPAI H5N1 virus infections. Evaluation of elution methods for recovery of highly pathogenic avian influenza (H5N1) virus from infected duck feathers. Available from: https://www.researchgate.net/publication/294123021_Evaluation_of_elution_methods_for_recovery_of_highly_pathogenic_avian_influenza_H5N1_virus_from_infected_duck_feathers [accessed Jun 5, 2017].Not Availabl

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Not AvailableThe H9N2 viruses can cause asymptomatic to clinical diseases in chickens under field conditions. Therefore, the present study was designed to assess the pathogenicity of Indian H9N2 avian influenza virus in experimentally infected specific pathogen free (SPF) chickens. The intravenous pathogenicity index of H9N2 virus isolated from Bareilly, Uttar Pradesh was found to be 0.00/3.00 suggesting its low pathogenicity in chickens. For infectivity study, six SPF chickens were inoculated intranasally with 100 μl of 106 EID 50 of A/Chicken/India/04TI05/2012 (H9N2) avian influenza virus. The sham phosphate buffered saline inoculated SPF chickens served as negative control. All the six H9N2 virus inoculated chickens got infected and virus could be isolated from oropharyngeal and cloacal swabs. More viral isolations were obtained from oropharyngeal swabs (38) as compared to cloacal swabs (16). Viral RNA was detected through 1 to 9 days post infection (dpi) by RT-qPCR. High viral RNA copy number (1.56x 103-1.2x 105) was detected in oropharyngeal swabs than cloacal swabs (1.15x1034.81x103). Clinically, all the H9N2 virus and sham inoculated birds did not show any overt signs of illness. All the experimental chickens seroconverted and the geometric mean haemagglutination inhibition titers at 6 and 12 and 30 dpi were 27.5, 28.33 and 27.66, respectively. This study indicates that the H9N2 virus isolated from a natural outbreak was of low pathogenicity under experimental conditions. Continuous shedding by the infected birds by oral and cloacal routes without any clinical signs might cause undetected spread of the virus under field conditions.Not Availabl

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Not AvailableWe report the infectivity of a crow derived influenza A (H5N1) virus (A/crow/India/01TR01/2012) in chickens and its pathological and genetic characterization. Histopathological changes and immunohistochemistry staining of internal organs and skeletal muscle were consistent with influenza A virus infection. Real time RT-PCR and virus isolation results demonstrated the systemic spread of the virus in chickens with 100% mortality. Comparatively higher level of virus shedding was detected in oropharyngeal swab (7.63×106 viral RNA copy) than in cloacal swab (6.66 × 106 viral RNA copy). Concentrations of viral antigen in kidney, lungs, brain, spleen and large intestine were higher compared to pancreas and skeletal muscle. No genetic change was observed on interspecies transmission of the virus. The study revealed that the crow derived H5N1 virus is able to kill the poultry, underlining the need for close monitoring of presence of virus in poultry near crow roosting areas so that further transmission to other avian and mammalian hosts can be prevented. Experimental inoculation of a crow derived influenza A (H5N1) virus in chickens and its pathological and genetic characterization. Available from: https://www.researchgate.net/publication/301629631_Experimental_inoculation_of_a_crow_derived_influenza_A_H5N1_virus_in_chickens_and_its_pathological_and_genetic_characterization [accessed Jun 6, 2017].Not Availabl

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Not AvailableAvian influenza caused by avian influenza viruses (AIV) has become a serious threat to poultry industry worldwide due to the huge economic loss and incidence of human infections in many countries around the world. India has been experiencing intermittent outbreaks of both H9N2 and H5N1 since 2003 and 2006, respectively. Retrospective testing of samples collected from West Bengal during outbreak of H5N1 virus in 2008 revealed mixed infection of both viruses within the same host. In this study, we report the molecular and pathogenic characterization of an H9N2 AIV isolated from a chicken co-infected with H5N1 virus. The sequencing of all the eight genes of the H9N2 virus was carried out. Phylogenetic analysis of HA gene of the H9N2 virus indicated grouping with G1 lineage viruses. The PA, NS, Matrix, PB1, PB2 and NP genes were closely related to H7N3 isolate from Karachi and the NS and polymerase genes did not group with any known lineage of H9N2 viruses. This confirmed that the H9N2 virus was a reassortant between G1and an unknown lineage. The absence of multiple basic amino acids in H9N2 virus (RSSR*GLF) indicated low pathogenicity of the virus in chickens. Molecular analysis revealed the presence of unique amino acids and susceptibility to oseltamivir and amantadine group of antivirals. The intra venous pathogenicity index of the H9N2 virus was found to be 0.325 H9N2. The result indicated that the H9N2 virus is low pathogenic to poultry. These findings corroborate the need for continued surveillance of Indian poultry to monitor the reassortant and evolutionarily divergent avian influenza viruses which might cause huge loss to the Indian poultry industry.Not Availabl

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Not AvailableHighly Pathogenic Avian Influenza (HPAI) H5N1 virus is a threat to animal and public health worldwide. Till date, the H5N1 virus has claimed 402 human lives, with a mortality rate of 58% and has caused the death or culling of millions of poultry since 2003. In this study, we have designed three siRNAs (PB2-2235, PB2-479 and NP-865) targeting PB2 and NP genes of avian influenza virus and evaluated their potential, measured by hemagglutination (HA), plaque reduction and Real time RT-PCR assay, in inhibiting H5N1 virus (A/chicken/Navapur/7972/2006) replication in MDCK cells. The siRNAs caused 8- to 16-fold reduction in virus HA titers at 24 h after challenged with 100TCID50 of virus. Among these siRNAs, PB2-2235 offered the highest inhibition of virus replication with 16-fold reduction in virus HA titer, 80% reduction in viral plaque counts and 94% inhibition in expression of specific RNA at 24 h. The other two siRNAs had 68–73% and 87–88% reduction in viral plaque counts and RNA copy number, respectively. The effect of siRNA on H5N1 virus replication continued till 48h (maximum observation period). These findings suggest that PB2-2235 could efficiently inhibit HPAI H5N1 virus replication.Not Availabl