Phylogenetic Analysis of Hemagglutinin Gene and Evaluation of the Viral Shedding of H۹N۲ Avian Influenza Viruses Using Real-time RT-PCR in SPF Chickens

In recent years, the H۹N۲ influenzavirus has been circulating widely in poultry farms causing extensive damage. The hemagglutinin (HA) genes of the two virus isolates of H۹N۲ subtype in specific pathogen-free chickens were studied to determine the shedding rate in the host’s oropharyngeal and cloacal routes and their genetic relationship. The sequence analysis and phylogenetic study of the samples were performed by comparing each isolate with other H۹N۲ isolates in the gene bank. In the present study, the chickens were inoculated with low pathogenic avian influenza virus (LPAIV) (A/Chicken/Iran/ZMT-۱۰۱/۱۹۹۸ [H۹N۲]) through the intranasal route. Oropharyngeal and cloacal swabs were collected from the chickens within ۱-۱۰ days after inoculation. The rate of viral shedding was measured within the previous ۱۰ days by the real-time reverse transcriptase polymerase chain reaction molecular technique. No clinical symptoms were observed during the experiment in the chickens. The results obtained from this technique showed that the main route of shedding for LPAIV was oropharyngeal areas (p <۰.۰۵). Both isolates had a similar proteolytic R-S-S-R sequence at the cleavage site of the HA gene and contained glutamine (Q) amino acid at position ۲۲۶ of the HA receptor-binding site, indicating that these isolates were nonpathogenic. Phylogenetic analysis demonstrated that both isolates belonged to the Eurasian clade. The comparison of these isolates with other isolates in the gene bank showed that they had the greatest similarity with the isolates in clade ۱ and the least homology with the isolates in clade ۴.

کلیدواژه ها:

influenza virus ، H۹N۲ ، Real-time RT-PCR ، Phylogenetic analysis