Phylogenetic Analysis of Hemagglutinin Gene and Evaluation of the Viral Shedding of H9N2 Avian Influenza Viruses Using Real-time RT-PCR in SPF Chickens

AUTHORS

P. Fazel 1 , 2 , M. J. Mehrabanpour 3 , * , M. K. Shahkarami 4

1 Department of Microbiology, Fars Science and Research Branch, Islamic Azad University, Shiraz, Iran

2 Department of Microbiology, Shiraz Branch, Islamic Azad University, Shiraz, Iran

3 Department of Virology, Razi Vaccine and Serum Research Institute, Shiraz Branch, Agricultural Research, Education and Extension Organization (AREEO), Shiraz, Iran

4 Department of Human Viral Vaccine, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran

How to Cite: Fazel P, Mehrabanpour M J, Shahkarami M K. Phylogenetic Analysis of Hemagglutinin Gene and Evaluation of the Viral Shedding of H9N2 Avian Influenza Viruses Using Real-time RT-PCR in SPF Chickens, Arch Razi Inst. 2020 ; 75(3):e109646. doi: 10.22092/ari.2019.125477.1308.

ARTICLE INFORMATION

Archives of Razi Institute: 75 (3); 339-348
Published Online: October 01, 2020
Article Type: Research Article
Received: March 03, 2019
Accepted: June 11, 2019
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Abstract

In recent years, the H9N2 influenzavirus has been circulating widely in poultry farms causing extensive damage. The hemagglutinin (HA) genes of the two virus isolates of H9N2 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 H9N2 isolates in the gene bank. In the present study, the chickens were inoculated with low pathogenic avian influenza virus (LPAIV) (A/Chicken/Iran/ZMT-101/1998 [H9N2]) through the intranasal route. Oropharyngeal and cloacal swabs were collected from the chickens within 1-10 days after inoculation. The rate of viral shedding was measured within the previous 10 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 <0.05). 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 226 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 1 and the least homology with the isolates in clade 4.

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© 2020, Author(s). Razi Vaccine and Serum Research Institute.
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