Construction Of Jev Infection Clones And The Functional Studies Of ITG αv β3 In The Jev Replication

Japanese encephalitis (JE) is a zoonosis caused by the Japanese encephalitis virus (JEV) with a mosquito media. JEV is mainly spread in the cycle with"the pigs-mosquitoes-people", including pigs are JEV propagation amplification storage reservoir, people is final host of JEV. In the majority of patients who had been infected by JEV showed a latent infection, infected patients who have been even after effective cure will be accompanied by irreversible brain inflammation and seriously nervous system damage. JEV have brought a huge economic loss to the pig industry in China. Despite the molecular biology, immunology, and clinical treatment of JEV have been made some progress, and a large number of vaccines popularity make the JE incidence in the control within a certain range, but the molecular mechanisms of JEV infection is not well clear.A third of the integrins are known to identify different viral RGD sequence, including alphav beta3. Now it has been confirmed that it is the function receptor of foot-and-mouth virus, adenovirus, and some flaviviruses. The envelope protein E protein of JEV comprises three regions, wherein the RGD sequence of the domain III, has been speculated that is the main portion of the receptor binding, mediates membrane fusion of virus and host cells.In this study, we constructed JEV infection clones, and conducted a series of biological identification, repeatedly passaged confirmed that rescued viral particles can be stably inherited. At the same time,we studied the influence of integrin av 03 in the JEV replication, to explore the possibility as a the JEV functional receptor.Lay the foundation for JEV invasion mechanism research and providing an ideal target for new drug development for future anti-JEV virus.The main contents are as follows:1. Construction of JEV infection clonesUsing site-directed mutagenesis to transform JEV strain infectious clone RP-9, in the rearmost end of the 3’-UTR of RP-9 mutated into a NotI restriction sites, followed by the BamHI and NotI replace the 3 half-molecules into vaccine strain SA14-14-2, to get a new infectious clone molecular RP-9". The same method, the green fluorescent protein (eGFP) was inserted into the 3’-UTR of RP-9", to get the infectious clone molecule RP-9"-eGFP. We transfected two infectious clone plasmids in BHK-21 cells, obtained two virus particles. We amplified E, NS4A of two virus particles by RT-PCR, the sequencing analysis confirmed that the E gene fragments belonging to the RP-9, the NS4A gene fragments belonging to SA-14-14-2, indicating successfully get the chimeric virus. And continuously passaged for 20 generations, the two virus can be stably inherited.We measured virus titer of V-RP-9,V-RP-9" and V-RP-9"-eGFP with the same generation in the BHK-21 cells, the results show that the presence of three viral titer of the same generation and plaque size certain differences in the titers. the titers (logPFU/mL) were 6.3,6.5,8.5. The sizes of the plaque were 1,0.5,0.2mm respectively.V-RP-9, V-RP-9" and V-RP-9"-eGFP was respectively through the brain and intraperitoneal injected with 4-week-old Balb/C mice, neurovirulence and neuroinvasiveness were 0.28,0.91,2.18 and 1.98,2.91,3.98 respectively, indicating that neurovirulence and neuroinvasiveness of the V-RP-9" and V-RP-9"-eGFP than the V-RP-9 were weaker, the neurovirulence and neuroinvasiveness of the V-RP-9"than those V-RP-9"-eGFP were stronger.2. the functional studies of integrin αv/β3 in the JEV replicationFirst designed αv β3 gene shRNA,the transfected BHK-21 and Hela cells to cut integrin protein expression, Western blot results show significant silencing effect in 45h. the plaque test infected with 1.0 MOI JEV results show JEV plaque of down-regulation of integrin group significantly reduced. Real-time PCR also showed that αv/β3 down-regulated expression influence JEV replication. αv/β3 antibody blocking test confirmed αv/β3 influence JEV replication. Further, in order to confirm the β3 influences JEV replication, in CHO cells (lacking endogenous human β3) overexpression β3, results show obviously enhanced JEV infection.To research av/β3 influence JEV replication in what stage in the viral replication cycle, αv β3 affect JEV replication through Binding this process is confirmed by Real-time PCR and plaque assay.Constructed E protein/EIII domain and integrin protein eukaryotic expression plasmids to study the interaction of the JEV E protein with integrin αv/β3.Co-immunoprecipitation results show that the the Domain3 of E protein and β3 exist interaction. Immunofluorescence colocalization observed αv,β3 and E proteins located in the cell membrane.RGD peptide plaque assay further confirmed RGD influences JEV replication. RGD sequence on the E gene was mutated into RGT, there also had a relatively weak impact in the replication of JEV, indicate that in addition to αv β3, there may be other coreceptor involved in JEV replication.