Functional Analysis Of IFITM3 In Antiviral Infection With Muscovy Duck Reovirus

Muscovy duck reovirus disease is caused by infection with Muscovy duck reovirus(MDRV),which can result in significant economic losses to Muscovy duck breeding industry.Main clinical symptom of the disease is grey or yellowish-white necrosis that occurs on the liver and spleen.In order to reduce the effects of MDRV on Muscovy duck breeding,we should have a more comprehensive understanding of the pathogenesis of MDRV.More investigations should be performed to provide more effective prevention and control of the disease.Innate immunity is the first barrier of host defense against viral infection.A previous study in our laboratory has provided preliminary evidence that MDRV infection induces an effective antiviral immune response involving critical interferons.Moreover,we have found that the MDRV infection could induce significant expression of several key ISGs including IFITM3.However,whether such ISGs play a specific role in innate immunity is still poorly understood.In this study,we focused on the functioning of IFITM3 in innate immune response to MDRV infection.In order to study the role of IFITM3 in anti-MDRV infection,we first performed experiments to detect IFITM3 expression in 293T cells and Muscovy duck embryo fibroblasts(MDEF)in vitro and Muscovy ducks in vivo.Then RNA interference was used to address the host-pathogen relationship.293T cell lines stably expressing shRNA targeting IFITM3 were generated and the interference efficiency was tested by RT-PCR and quantitative real-time PCR.We found that IFITM3 silencing affects the replication of MDRV.In addition,293T cells transfected with human or anas IFITM3 were infected with MDRV,and the mRNA expression of virus gene was analysed.The results are shown as follows:(1)The expression of endogenous IFITM3 in 293T cells,MDEF and Muscovy ducks was significantly increased after MDRV infection.(2)Anas IFITM3 expression vector was successfully constructed,and named as pFlag-CMV-anas-IFITM3.(3)293T cells overexpressing Muscovy duck IFITM3 were infected with MDRV for 24 h.We found that the mRNA expression of virus P10 was much less in IFITM3 overexpressing cells than that in the control cells.The results indicate that Muscovy duck IFITM3 has the function of inhibiting MDRV infection and replication.(4)The mRNA expression of virus P10 was lower in 293T cells overexpressing human IFITM3 than that in the control cells,suggesting that human IFITM3 can also inhibit MDRV replication.(5)293T cells expressing shRNA targeting IFITM3 or luciferase control were infected with MDRV for 24 h.We observed that expression of virus P10 was much higher in IFITM3-silencing cells than that in the control cells,which suggests that lack of IFITM3 expression can promote the proliferation of MDRV.Taken together,these results establish that IFITM3 plays a key role in anti-MDRV infection and replication.These experiments provide strong evidence that IFITM3 might be a useful tool for control of Muscovy duck reovirus disease.