The effect of genetic variation in hemagglutinin gene of H9N2 influenza viruses in diagnosis using RT-PCR

Genetic variations in the hemagglutinin gene of influenza viruses are an important characteristic. These variations may cause limitation of RT-PCR assay for detection of influenza subtypes. In this study, a total of 6 samples, suspected to type A influenza virus, were isolated from industrial birds’ trachea in North Khorasan Province and RT-PCR analysis were performed. Results showed that all of the 6 suspected samples were detected positive as type A influenza viruses. These isolates were then determined their subtypes using RT-PCR H9N2 specific primers. The result showed that 4 out of 6 isolates were positive as H9N2 and other two isolates did not have any bond on gel electrophoresis and considered negative. For further evaluation, all of the hemagglutinin genes of viruses were sequenced and confirmed as H9N2 viruses using BLAST software in Gene Bank. It was also found, that primers binding sites with hemagglutinin genes in two last viruses were mismatched in three nucleotide acids with 3' end of forward and reverse primers. These factors may reduce the sensitivity of RT-PCR tests and cause false negative results. Therefore, it would recommend updating RT-PCR primers and protocols in a timely manner.