Establishment And Application Of The Method Of CVB3 Virus RNA Rescue

The family Picornaviridae belongs to the order Picornavirales and currently consists of 80 species grouped into 35 genera.Members of this family have a linear single stranded,positive sense RNA genome of 7.1 to 8.9 kilobases,which is polyadenylated.Their genomes consist of a single open-reading frame,which encodes a polyprotein.This is then cleaved co-and post-translationally by viral proteases to give the viral structural and non-structural proteins.Thus,it is a practical way to rescue virus by transfecting infectious genome RNA of Picornaviruses,and it may be an important approach that can alter the conventional virus isolation and evaluate the characteristic of virus replication.Coxsackievirus B3(CVB3)belongs to the genus Enterovirus in the Picornaviridae family.CVB3 is one of the most important pathogens causing viral myocarditis.CVB3 was choose as model virus in this study.The method of virus RNA rescue was successfully established.The optimum condition of RNA extraction,transfection reagent,and amount of RNA transfection were determined in this study.Moreover,the RNA rescue method was evalued with CVB3、EV71、CA16、ECH03、HRV16、HRV86 and EMCV virus.The results demonstrated that RNA rescue could be used to virus isolation of picornaviridae family.In order to enhance the efficiency of RNA rescue,RNA was extracted from titer of 7.15×105PFU/ml CVB3 by eluting three times.The counts of plaques rescuing virus of each eluting RNA were 150±15、11.67±2.08、1.67±0.58,respectively.In order to improve the efficiency,293T cell,HeLa cell and Vero cell were used to rescue virus by RNA transfection.The RNA of 7.15×105 PFU CVB3 were extracted and transfected into three cells respectively,cytopathic effect(CPE)was observed to judge the replicating efficiency.After RNA transfection for 24 hours,293T cells showed completely CPE,and HeLa cell and Vero cell had no CPE.After transfection for 48 hours,the percentage of CPE in both HeLa cell and Vero cell were about 20%,but 72 hours,these two cells showed completely CPE.After RNA from 7.15×104 PFU CVB3 was transfected for 48h,CPE showed in all 293T cells.HeLa cells appeared completely CPE after RNA transfection for 72h.However,Vero cells only appeared 50%CPE.After RNA transfection of 7.15×103 PFU CVB3 for 48h,293T cell appeared 100%CPE,but after transfection for 72h,25%CPE appeared in HeLa cells,and no change appeared in Vero cells.The results also showed that CVB3 virus was the highest rescuing efficiency in 293T cells.It demonstrated that RNA could replicate efficiently to rescue virus in 293T cells.Different viruses vary greatly in their susceptible cells.It is very importance and difficult for the viologist to choose the most sensitive cell cultures to isolate an unknown suspected virus.Thus,a general method of rescuing picornavirus dependent on 293T cells was successfully established and rescued 7 picornavirus including CVB3,EV71 and CA16,ECH03,HRV16,HRV86 and EMCV.To overcome the tissue cytotoxicity of heart and pancreas and understand fully virus replication in CVB3-induced myocarditis model,we established CVB3-induced myocarditis model.Mice were inoculated intraperitonedally with CVB3 7.15×103 PFU virus in 0.1 ml.Each group had 6 mice.Serum,heart and pancreas were respectively collected at 1,3,5,7,9,11,13 and 15 days post-inoculation(dpi).Dynamic changes of CVB3 in the pancreas,heart and serum of CVB3-infected mice were evaluated using RNA rescue.Cardiac histopathology analysis showed a rapid increase in myocardial inflammation at 5 days post-inoculation(dpi),with a peak at 9 dpi,and followed’ by a slow decrease to nearly normal level.The pancreatic pathological changes were aggravated with the prolongation of infection time.CVB3 showed similar replication trends in the serum,heart,and pancreas of CVB3-infected mice,but virus titer was different from each other.Virus replication was markedly increased at 3 dpi and reached the peak at 5-7 dpi,then gradually decreased after 9 dpi and kept at very low level.The titer peak of CVB3 was observed on day 5 at 826.6±28.0 PFU/mg in the pancreas,day 3 at 24.4±1.9 PFU/mg in the heart,and day 3 at 10953.6±807 PFU/mL in the serum,respectively.Our results indicate that RNA rescue can be used as an efficient approach for the recovery of CVB3 from heart,pancreas and serum of CVB3-infected mice.Our results indicate that CVB3 induced damage to heart and pancreas directly,and at the late infection stage,the injury was caused by inflammatory.in conclusion,a RNA recue method of picornavirus was established and evaluated with 7 virus from picornvirus family in this study.The approach was efficient assessed on virus replication of CVB3 in the CVB3-infected myocarditis model.The dynamics of CVB3 virus replication in the serum,heart,and pancreas of CB3-inoculated mice were assessed by RNA rescue.