Phylogenetic analysis of Hemagglutinin genes and evaluation of the viral shedding of H9N2 avian influenza viruses with Real Time RT-PCR in SPF chicken

Document Type: Original Articles


1 Department of Microbiology, Fars science and Research branch, Islamic Azad University, Fras, Iran

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

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

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


During the last several years, H9N2 AIVs have been circulating widely in poultry farms and caused tremendous losses, especially in Iran. Nowdays, 17 and 9 different hemagglutinin (HA) and neuraminidase (N) serotypes, have been identified among avian influenza viruses. HA genes of two virus isolates of H9N2 subtype in specific pathogen-free (SPF) chickens were studied to determine the shedding rate in the host’s Oropharyngeal (OP) and cloacal (CL) routes and their genetic relationship. The sequence analysis and phylogenetic study of the samples were performed by comparing each isolate with the other H9N2 isolates in the gene bank. In the present study, chickens were inoculated with LPAIV (A/chicken/Iran/ZMT-101/1998(H9N2)). Oropharyngeal and Cloacal swabs were collected from chickens from 1 to 10 days after the inoculation. The rate of viral shedding was measured within 10 days by the RRT-PCR (Real-Time Reverse Transcriptase Polymerase Chain Reaction) molecular technique. No clinical symptoms were observed during the experiment. The results obtained from this technique showed that the main route of shedding for LPAIV was in OP areas (p<0.05). Both isolates had a similar proteolytic R-S-S-R sequence at the cleavage site of 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 indicated that both isolates belonged to the Eurasian clade. The comparison of these isolates with the 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.