Isolation and pathotyping of H9N2 avian influenza viruses in Indian poultry

High Security Animal Disease Laboratory, Indian Veterinary Research Institute, Anand Nagar, Bhopal 462021, Madhya Pradesh, India.
Veterinary Microbiology (Impact Factor: 2.51). 07/2008; 133(1-2):154-63. DOI: 10.1016/j.vetmic.2008.06.013
Source: PubMed


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.

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    • ". These H5N8 viruses were identified by reverse - transcription polymerase - chain reaction ( RT - PCR ) , hemagglutination test , and hemagglutination inhibition ( HI ) test as per the standard protocol ( WHO , 2002 ; Nagarajan et al . , 2009 ; Office International des Epizooties ( OIE ) , 2015 ) . Moreover , these isolates were identified again as H5N8 viruses by nucleotide sequence and a BLAST search of the Influenza Sequence Datebase in GeneBank . Briefly , fecal samples were inoculated into the allantoic cavity of 9 to 10 - day - old specific pathogen free ( SPF ) embryo"
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    ABSTRACT: New reassortant H5N8 highly pathogenic avian influenza viruses were isolated from waterfowl in Southern China. Blast analysis demonstrated that the PB2 gene in these viruses were most closely related to A/wild duck/Shangdong/628/2011 (H5N1), while their NP genes were both more closely related to A/wild duck/Shandong/1/2011 (H5N1) and A/duck/Jiangsu/k1203/2010 (H5N8). However, the HA, NA, PB1, PA, M, and NS genes had the highest identity with A/duck/Jiangsu/k1203/2010 (H5N8). Phylogenetic analysis revealed that their HA genes belonged to the same GsGd H5 clade detected in China in 2010. Therefore, we supposed that these H5N8 viruses might be novel reassortant viruses that have a H5N8 backbone while acquiring PB2 and NP genes from H5N1 viruses. This study is useful for better understanding the genetic and antigenic evolution of H5 avian influenza viruses in Southern China.
    Frontiers in Microbiology 11/2015; 6:1170. DOI:10.3389/fmicb.2015.01170 · 3.99 Impact Factor
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    • "H9N2 is enzootic in birds in India and causes infection with low pathogenicity. H9N2 infection in domestic poultry was reported during 2003–04 in poultry farms with history of reduced egg production and respiratory illness in Punjab, Haryana, Uttar Pradesh, Gujarat and Orissa states of India (Nagarajan et al. 2009). Similar cases of respiratory illness were reported from layer poultry farms in the Sarsa village of Gujarat, India. "
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    ABSTRACT: The present study reports isolation and characterization of H9N2 virus responsible for disease characterized by symptoms including difficulty in respiration, head swelling, nasal discharge, reduced feed intake, cyanotic comb, reduced egg production and mortality. Virus isolation from allantoic fluid inoculated with tracheal aspirates and whole genome sequencing of two isolates were performed on an Ion-Torrent sequencer. Phylogenetic analysis revealed that the two H9N2 isolates are reassortant viruses showing a G1-like lineage for HA, NA and NP, a Hok/49/98-like lineage for PB1 and PA, PK/UDL-01/05-like lineage for PB2, IL/90658/00-like lineage for NS and an unknown lineage for M gene. Analyses of the HA cleavage site showed a sequence of (333PARSSR↓GL340) indicating that these isolates are of low pathogenicity. Isolate 2 has leucine at amino acid position 226, a substitution which is associated with mammalian adaptation of avian influenza virus. Isolate 1 has the S31N substitution in the M2 gene that has been associated with drug resistance as well as R57Q and C241Y mutations in the NP gene which are associated with human adaptation. The result reported here gives deep insight in to H9N2 viruses circulating in domestic poultry of India and supports the policy of active efforts to control and manage H9N2 infections in Indian poultry. Electronic supplementary material The online version of this article (doi:10.1186/2193-1801-3-196) contains supplementary material, which is available to authorized users.
    SpringerPlus 04/2014; 3(1):196. DOI:10.1186/2193-1801-3-196
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    • "who stated that both broiler and SPF-infected groups with H9N2 virus were seroconverted on the hemagglutination inhibition test at 16 DPI giving a geometric mean titer of Log 2 8.2 and Log 2 9.3, respectively. Similarly, the Indian virus isolates of H9N2 gave a positive HI test with H9 reference antisera with titers ranging from -Log 2 4 to Log 2 9 (Nagarajan et al., 2009) "

    Pakistan Veterinary Journal 01/2014; · 1.39 Impact Factor
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