Isolation And Pathogenicity Of Porcine Sapelovirus

Porcine sapelovirus(PSV)is a single-stranded,positive-sense,and nonenveloped RNA virus belonging to the genus Sapelovirus in the family Picornaviridae.PSV is the causative agent of pneumonia,diarrhea,polioencephalomyelitis,reproductive failure of sow but frequently asymptomatic.PSV was first detected in England in 1960 and subsequently reported in China,Spain,South Korea,Brazil,the United States,India,France and other countries.An outbreak of diarrhea and polioencephalomyelitis in pigs occurred in a farm that lies in Shanghai,China in December 2009 and PSV was isolated.It was also the first report of PSV in China and then it had been sequentially reported in domestic.In recent years,there has been reported diarrhea and polioencephalomyelitis caused by PSV and it results in economic losses on pig-breeding industry.We obtained two viruse isolates which could proliferate on swine testis(ST)cells steadily during the isolation of pig diarrhea samples in clinical.Through RT-PCR detection we proved that these two isolates were not porcine epidemic diarrhea virus(PEDV),porcine transmissible gastroenteritis virus(TGEV),porcine rotavirus(PoRV),or porcine deltacoronavirus(PDCoV).Finally these two isolates were confirmed as PSV through next-generation sequencing and they were named PSV-JXXY-a2 and PSV-JXXY-c respectively.Then,sequence analysis showed that PSV-JXXY-a2 and PSV-JXXY-c had the same genomic organization with the length of 7564nt excluding the poly(A)tail.Phylogenetic tree analysis showed that two isolates were classified into the China cluster,which were more closely related to the South Korea PSV cluster.We carried out the prevalence investigation of PSV in eastern China:the PSV positive rate in diarrhea samples was 44.93%(62/138)while it was 27.66%(13/47)in asymptomatic samples.The total positive rate was 40.54%(75/185)and PSV could co-infect with PEDV with the rate of 17.30%(32/185).For sero-epidemiological investigation,the positive rate was 80.23%(207/258)and 79.85%of the tested serum titer was over than 43.These data indicated that PSV had a high prevalence in eastern China.The biological characteristics showed that PSV was characterised as a microsphaeric virion with a diameter of approximately 30nm.It could infect various cell lines such as ST,PK15,IPEC-J2,293T and BHK-21.PSV still remained infectivity at 53? for 1h,but was inactivated completely at 54? for only 6 min.Further investigation was conducted to detect the pathogenicity of PSV-JXXY-a2.One-day-old piglets were mock inoculated or inoculated with PSV-JXXY-a2 and PSV-inoculated piglets developed watery diarrhea with the viral RNA detected in most of the organs.We could also isolate PSV again from diarrhea samples.In addition,PSV VP 1 genome was amplified and cloned into pET-28a(+)and pGEX-4T-1 and the prokaryotic expression system of PSV VP 1 protein was successfully established.The expressed his-VP 1 was purffied and immunized to BALB/c'mice in order to obtain serum of polyclonal antibody against VP1 protein.Finally,we determined to know the effect of PSV infection on IFN-I signaling pathway in its susceptible cell lines.Results showed that the levels of IFN-? mRNA were increased in PSV-infected ST cells.However,PSV impeded SeV-mediated activation of IFN-? in a dose-dependent manner in 293T cells.Furthermore,PSV 2A protein played an important role in inhibiting the IFN-? promoter activity.In conclusion we investigated the prevalence investigation of PSV in eastern China and two isolated were obteined and identified.Then,we sequenced the whole genome and established phylogenetic tree.Biological characteristics and pathogenicity were conducted as well.These results will provide basic data for further:research of PSV.