Abstract：Rice gall dwarf virus(RGDV), the genus Phytoreovirus in the family Reoviridae, is transmitted by the leafhopper vector(Recilia dorsalis) in a persistent-propagative manner. Nonstructural protein(Pns12), encoded by segment 12 of RGDV, is one of the components of viroplasm which is the site for viral replication and assembly of progeny virons during viral infection in its insect vector cells. In this study, to investigate the functional role of Pns12 in the formation of viroplasm in its insect vector cells, the polyclonal antibody against Pns12 was prepared and purified, then conjugated to fluorescein isothiocyanate. The immunoglobulin G(IgG) of P8 was purified and conjugated to rhadamine. Immunofluorescence microscopy demonstrated that Pns12 antibodies specifically distributed in the viroplasm matrix during RGDV infection in the vector cells in monolayers (VCMs), while outer capsid protein P8 were accumulated at the periphery of the viroplasm. The viroplasm increased in size over time. Knockdown of Pns12 by RNA interference (RNAi) induced by dsRNAs, targeting Pns12 gene of RGDV, significantly inhibited the formation of viroplasm compared with dsGFP, suggesting that RGDV replication was inhibited. Western blot showed that viral Pns12 and P8 expression reduced in the VCMs treated with dsPns12. Thus, the present study indicated that Pns12 of RGDV played an important role in viroplasm formation and viral replication in its insect vector cells. RNAi induced by dsRNAs derived from the viral genes of viroplasm matrix protein may be an ideal tool for inhibiting the infection and transmission of RGDV by leafhopper vectors.
