The Persistent Infection Of Rice Ragged Stunt Virus In The Cultured Cells Derived From Nilaparvata Lugens

Rice ragged stunt virus (RRSV), a member of the genus Oryzavirus, is transmitted by the brown planthopper (BPH), Nilaparvata lugens in a persistent propagative manner. RRSV has spread rapidly throughout in southern China and Southeast Asia. Viroplasms, the sites of virus replication and assembly of progeny virions, were formed when RRSV enters the insect cells. To solve the rice production lost caused by rice ragged stunt disease, the route for BPH to transmit RRSV has to be cut off. And this needs us to study the mechanism of RRSV persistent infection in the cultured cells derived from Nilaparvata lugens.This research adopted cultured cells derived from Nilaparvata lugens to study the mechanism of RRSV persistent infection in the cells of vector. By RT-PCR and Western Blot methods, we verified that the rice plant, collected by our lab, had been infected with RRSV. And we proved that there were complete RRSV virions in the rough extract of infected rice plant by negative staining electron microscopy. Using RRSV rough extract of infected rice plant to infect the cultured cells derived from Nilaparvata lugens, and using RT-PCR to test if RRSV successfully infected the cells. Using immunofluorescence and immunoelectronic microscope(IME) techniques to observe RRSV persistent infection in cultured cells derived from Nilaparvata lugens. Immunofluorescence test adopted structural proteins P2, P9 and non-structural protein Pns6, Pns10 antibodies, which have been conjugated to FITC or rhodamine. The result shows that these four antibodies were all marked on its antigens, which were induced by RRSV in cultured cells, and can be co-localized with each other. All the others were located on viroplasm, except P2, which was located in the periphery of viroplasm. Compared with 1 day post-inoculation(dpi), the amount of infected cells and protein expression induced by virus became more after 5 dpi. This shows RRSV can successfully infect cultured cells derived from Nilaparvata lugens and get proliferation in cells, and spread to other cells. IME adopts structural proteins P2, P9 and non-structural protein Pns6, Pns10 antibodies, cross-linked by immunogold. With IEM, we can see there is viroplasm in RRSV-infected cells. There are matrix, mature and immature virions in viroplasm. P2 antibodies reacted specifically with the mature virus particles which distribute on the periphery of viroplasm. P9 reacted with the mature, immature virus particles and viroplasm matrix. Pns6 and Pns10 antibodies reacted specifically with the viroplasm matrix.Those results show RRSV Pns6 and Pns10 are the components of viroplasm matrix.In all, RRSV can successfully develop persistent infection in the cultured cells derived from Nilaparvata lugens, and induce to the formation of viroplasm, which is the place for virus replication and assembly of virions. This essay lays the foundation of further research in interaction between RRSV and the vector Nilaparvata lugens, also it provides theoretical foundation for fundamentally cut off the insects transmission route.