Establishment Of A Novel Vero Cell Line For The Efficient Production Of Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea,which is also known as PED,is a kind of severe infectious diseases occurred in pigs.And the porcine epidemic diarrhea virus(PEDV)is the causative agent of the PED.PEDV can infects pigs of all ages,and causing watery diarrhea and vomiting accompanied by anorexia and depression.PEDV has become a worldwide-spread disease in recent years,and it was reported all over the world like the United States,Canada,South Korea,Japan and China.The outbreaks of PEDV cause a significant damage to the pork industry of the world every year.It becomes more and more important to establish some effective strategies to diagnose this disease,and to prevent the infection of PEDV among pigs.Vaccination of sows is now the best ways to control and eliminate the outbreaks of PEDV.The newborn piglets can get antibodies to the PEDV from the colostrums and milk of the sows.As a consequence,the efficient production of the vaccine to PEDV is in urgent need to control the dissemination of PEDV.The Vero cell line is the most widely-used platform for the production of vaccines,such as the influenza vaccines,the Dengue fever vaccines as well as PEDV vaccine.As we know,the Vero cell line is not the natural host cell of PEDV,and which causes the inefficient propagation of PEDV in Vero cell line.The vaccine against the PEDV we get from Vero cell line usually achieves low virus titer,and the protection effect of this vaccine is not of great satisfaction.The establishment of a novel Vero cell line for the efficient vaccine production of PEDV is badly urged to offer a better protection of the PEDV in newborn piglets.Porcine aminopeptidase N(pAPN)has been identified as the natural host cellular receptor of PEDV.It is expressed on the villous epithelial cells of small intestine of pigs predominantly,which causes the tissue tropism of the PEDV.Overexpression of pAPN in nonpermissive cell lines of PEDV,such as ST cell line,293T and MDCKs,enable the efficient propagation of PEDV.It is reported that it is the density in the cell membrane rather than the enzymatic role of pAPN receptor that contributes to the efficient replication of PEDV.In our research,fisrtly,we amplified the CDS of pAPN from the cDNA of small intestine tissues of pig and inserted it into a modified plasmid pcDNA3.1(+)-puro for expression of pAPN in eukaryocyte.Then we screened a Vero cell line which can express the natural host cellular receptor of PEDV,named as apclone 11.Finally,we sowed the equal dose of PEDV in Vero-con and apclone 11,and our results showed that the overexpression of pAPN facilitated the propagation of PEDV significantly in Vero cell line.As a result,the apclone 11 could be used as a novel Vero cell line for the efficient vaccine production of PEDV.Hetergeneous nuclear ribonucleoprotein A1(hnRNP Al)is involved in pre-mRNA splicing in the nucleus and translational regulation in the cytoplasm.In the recent study,some researchers found that hnRNP A1 also participates in the transcription and replication of a coronavirus RNA virus.The overexpression of hnRNP Al can facilitates the replication of MHV in DBT cell line.In our research,we amplified the CDS of hnRNP Al from the cDNA of Vero cell line and inserted it into the plasmid pcDNA3.1(+)for overexpression of hnRNP Al in eukaryocyte.Then we screened a Vero cell line which can overexpress hnRNP A1.Finally,we sowed the equal dose of PEDV in Vero-con and hnclone 39,and our results showed that the overexpression of hnRNP A1 didn't facilitate the replication of the PEDV in Vero cell line significantly.Although we couldn't use the hnclone 39 as a modified Vero cell line for the efficient vaccine production of PEDV,we still could use it as a cell line to study the impact of overexpressing hnRNP Al in Vero cell line on other virus.In this study,we also established some useful methods to identify and diagnose PEDV at the same time,including(1)the RT-PCR specially identifying the M Gene of PEDV;(2)and a RT-PCR to differentiate the vaccine PEDV and the field PEDV;(3)a Real-time quantitative RT-PCR to quantity the copies of PEDV.