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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    • "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) "
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    ABSTRACT: The study was performed to investigate the immunosuppressive effect of aflatoxin on the pathogenesis of H9N2 AI virus in SPF chickens. The experiment was carried out on 110 unvaccinated day old SPF chicks. They were divided into four groups of 25 birds each. Group I was kept as a non-treated and non-infected control; group II was intranasally infected with H9N2 AI virus at the 4th week of age; group III was fed on a diet containing 0.75 ppm aflatoxin from day one through the entire experiment period and group IV was fed on diet containing 0.75 ppm aflatoxin as group III and infected intranasally with H9N2 AI virus at the 4th week of age. Five chicks were kept as contact control (without infection) to group II and group IV. Five chickens from each group were slaughtered at 4th, 9th, 14th, 20th and 27th DPI Serum was collected from all slaughtered birds (5 serum samples/group/slaughter time) for serology (HI). Specimens from nasal conchae, trachea, lungs, liver, kidneys, bursa of Fabricius, thymus, spleen, pancreas and brain were collected from slaughtered birds for histopathology and immunohistochemistry. The histopathological lesions were more severe and persist till the end of the experiment in group IV. Using immunoperoxidase technique viral antigens were detected in the nasal conchae, trachea, lungs, thymus, kidneys and brain in group II while in group IV it extended further to the pancreas and bursa of Fabricius. In conclusion, the immunosuppression caused by aflatoxin increased the severity of lesions and allowed the virus to be disseminated to more organs.
    Pakistan Veterinary Journal 01/2014; 34(2):234-238. · 1.39 Impact Factor
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    • "The partial sequencing and phylogenetic analysis of HA and NA genes of AI H9N2 showed that H9N2 viruses belonged to G1 lineage and were similar to H9N2 viruses from Iran, Saudi Arabia and Pakistan (data not shown). H9N2 viruses have been reported from India [19] and Asian countries including Bangladesh, Iran, and Pakistan have reported this virus. The seroprevalence of AI H9N2 and NDV have been reported in emus (Dromaius novaehollandiae) from India [20]. "
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    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.
    Virology Journal 08/2012; 9(1):151. DOI:10.1186/1743-422X-9-151 · 2.18 Impact Factor
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