نقش ضد ویروسی مسیر small interfering RNA در سلول‌ها Spodoptera frugiperda (Sf9) در زمان آلودگی با Autographa californica multiple nucleopolyhedrovirus

Baculovirus are among the most important biocontrol agents, particularly for the control of lepidopteran pests. Their high pathogenicity and specificity make them attractive for pest control applications, particularly on crops where a complex of pests is established. Considering the importance of these viruses in pest control, better understanding of Baculovirus- host insect interaction is of interest. It is thought that siRNA pathway is the most important antiviral response in insects. In this study we assessed the role of siRNA pathway in Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Sf9cells interaction. The Sf9 cell line was maintained as a monolayer at 27°C. To determine the levels of viral DNA accumulation, DNA was extracted from AcMNPV-infected Sf9 cells at 4, 8, 16, 24 and 48 hours post infection (hpi) and quantitative PCR (qPCR) was performed using ie-1 (AcMNPV specific) and Rpl27 (reference) primers. To explore how Ago2 and Dcr2 gene expression altered during AcMNPV infection, the total RNA was extracted from mock and AcMNPV-infected Sf9 cells at 4, 8, 16, 24 and 48 hpi. These RNA samples were subjected to cDNA synthesis followed by RT-qPCR with the ie-1 and Rpl27 primers. Our results showed that the transcript level of Dcr2 and Ago2 significantly increased at 8 hpi and 16 hpi, respectively. Moreover, the expression of Ago2 gene was more than Dcr2 in mock and infected cells. To confirm the role of Ago2 and Dcr2 genes in antiviral response, RNAi was used to silence Dcr2 and Ago2 genes in Sf9 cells using specific double-stranded RNA (dsRNA). DsRNAs were in vitro synthesized using the MEGA Script kit. Also, dsRNA of Green Fluorescent Protein (dsGFP) was generated as control. After validation of gene silencing using RT-qPCR, the cells were infected with AcMNPV. Twenty-four hours later, viral DNA replication in the infected cells was quantified using qPCR. Reduction in the expression levels of Ago2 and Dcr2 genes improved AcMNPV replication (more than two folds) compared to the mock and dsGFP transfected cells. Although Dcr2 was induced earlier than Ago2 in the infected cells, our results showed that Ago2 has stronger effect on AcMNPV replication. In conclusion, the present study provide more evidence for importance of siRNA pathway in insect antiviral defense.