The P18 Protein Functional Characterization And Establishment Of Reverse Genetics System For Waterfowl Reovirus

Reovirus separated from animal respiratory or intestina system in 1960 s.The parasitifers is very extensive,it can infect people,vertebrates,insects,plants,fungi,poultry,and so on.ARVs cause a range of diseases in chicken,including viral arthritis/tenosynovitis,and are associated with respiratory disease,enteric disease,inclusion body hepatitis,hydropericardium,runting stunting syndrome,malabsorption syndrome,and sudden death.It can be divided into three categories which infect poultry,including avian reovirus(ARV),muscovy duck reovirus(MDRV)and duck reovirus(DRV).The virions of reovirus are nonenveloped,with icosahedral symmetry and a double capsid containing 10 dsRNA segments that can be separated by polyacrylamide gel electrophoresis into 3 size classes: large(L1,L2,L3),medium(M1,M2,M3),and small(S1,S2,S3,S4).It is predicted that the DRV S1 gene encode P10,P18 and ?C protein The P18 protein is corresponding to the ARV P17 protein,whether the function of DRV P18 protein is similar to ARV P17 protein,which requires further exploration.P18 gene was amplified from Novel Duck Reovirus TH11 strain by RT-PCR and subcloned into pCold-TF vector.Recombinant P18 protein was obtained by expression and purification and then used as immunogen to make polyclonal antibodies.Western-blot analysis showed that the polyclonal antibodies have specifically immunogenicity.Indirect immunofluorescence assay and confocal laser scanning microscopy were employed to examine the distribution and subcellular localization of P18 protein at different time points post-infection.The results indicated that P18 protein was in both nucleus and cytoplasma at 6hpi and it is in nucleus in the following time.It only can be examined in nuclear30 hpi and then the cells were collapsed.These different patterns might indicate different stages in infection.These data could lay the foundation for further study on the molecular mechanism of P18 protein transportation to the nucleus.Moreover,recombinant P18 protein might serve as useful antigens for assays for NDRV.Novel Duck reovirus,the prototype strain of species Avian orthoreoviruses(ARVs)is currently an infectious agent for ducks.The investigation of DRV replication and pathogenesis has been hampered by the lack of a reverse genetics system.In this study,we developed a plasmid-based reverse genetics system free of helper viruses and independent of any selection for DRV isolated.In this system,cDNAs corresponding to each of the 10 full-length gene segments are driven by bacteriophage T7 RNA polymerase,which can be supplied transiently by recombinant vaccinia virus.Production of infectious virus was shown by inoculation of cell lysate derived from transfected cells into 10-day embryo.The growth kinetics and infectivity in vivo of recombinant strains were indistinguishable from those of wild-type strain.These viruses grew well and were genetically stable both in vitro and in vivo.Altogether,these results indicate the first successful production of an infectious clone for ARVs.The infectious clone reported will be useful for elucidating the mechanism of genetically engineered,host tropism,viral replication and pathogenesis as well as immunological changes induced by ARVs.It also can be exploited to engineer recombinant reoviruses for vaccine.