An Investigation Into the Effect of H9n2 Influenza Virus on Apoptosis of Testicular Cells in Chicken Embryo Using Sybr-Green Real-Time PCR

Abstract

Influenza viruses can induce cell death in their host through apoptosis or necrosis.The H9N2 is a subtype of the Avian Influenza Virus (AIV) that can cause severe damage to reproductive organs of laying hens. The present study aimed to investigate the effect of the H9N2 influenza virus on apoptosis of testicular cells in chicken embryos. To this end, A/Chicken/Tehran/ZMT-101/99(H9N2) was inoculated to 210 embryonated fifteen-day-old eggs laid by SPF hens. Then, according to the experiment design, live embryos were dissected on the 19th and 21st days of fetal life and on the 25th day after birth for pathological and molecular studies and evaluation of gene expression in testicular tissue. Dissected tissues were stained with hematoxylin and eosin (HE) and studied under an optical microscope at 400x magnification. For molecular studies, viral RNA was extracted from testicular tissues, replicated by RT-PCR, and finally evaluated H9N2. Then, the genes expression involved in the testicular tissue cells apoptosis was evaluated through real-time PCR. Pathological studies indicated that H9N2 caused lesions in testicular tissue orchitis, seminiferous, and nephritis of the host. Molecular studies also showed that H9N2 replication in the host body increases BAX and Caspase 3 expression and reduces the expression of Bcl-2 and Bcl-XL. These changes in gene expression increased apoptosis and induced cell death in the host. In summary, the study findings suggested that H9N2 can increase the expression of pro-apoptotic genes and reduce the expression of anti-apoptotic genes, resulting in severe destruction of testicular tissue caused by cell apoptosis.