| Bovine viral diarrhea virus(BVDV),together with classical swine fever virus(CSFV),and Border disease virus(BDV)belongs to Pestivirus genus within the familily Flaviviridae.The virus infection results in diverse clinical sysdromes such as mucosal disease,immune tolerance,abortion,congenital malformation,and gastroenteric disorders.In addition to cattle,BVDV also infects more than 40 species such as swine,sheep and camels.Persistent infected(PI)cattle is an important virus carrier.Virus shedding will continiuity occurred in PI animals,which promotes the virus transmission.Pestivirus infection leads to signficant economic losses to the cattle industry worldwide.BVDV are grouped into cytopathic(CP)and noncytopathic(NCP)biotypes,based on their ability to kill cultured cells.Only NCP-BVDV can establish persistent infection in the fetus when infecting a dam early in gestation.The measures for the control of BVDV infection is largerly dependending on effective rapid detection methods,efficient vaccines for the prevention purpose and antiviral drugs for the treamtents under some conditions.However,an rapid BVDV detection technologies suit for clinical use by the famer is still not available in China.In addition,NCP-BVDV could not be tittered with a conventional method as used for CP-BVDV,which impeded the identification of antiviral drugs targeting NCP-BVDV virus strains.Therefore,ultimate aim of this study is to develop BVDV detection strips for rapid detection of virus or serum in livestocks.The BVDV virus was first purified using TFF followed by SEC on a Superose 6 10/300 GL column and used for immunization of BALB/C mice.Monoclonal antibodies(mAbs)against BVDV were generated.We finally got five clones of hybridoma cell lines that could stablely secrete anbitodies named as 9G6D6,14G5F2,19A3H8,28H11G4 and 31D11C11.The titer of antibody for ascites was 1.28x105,1.28x105,1.02x106,1.28x105and 2x103 based on the method of immunostaining.Ig types and subtypes were identified by mouse antibody isotyping Kit,all the light chain subtypes were all Kappa and the heavy chain were 1gG1.None of them showed reactivity with CSFV.In addition,19A3H8 showed neutralization activity,with a titer of 1:6400.Unfortunately,these mAbs were not suit for the generation of BVDV detection strip.Another goals was to deveope a simple method for titration of BVDV and that could be applied for antiviral drugs screening.With above mAb we established an immunoperoxidase assay.The capability of this method in the application of antiviral drugs screening was confirmed by using two known antiviral inhibitors Ribavirin and Ammonium Chloride.The results was further validated by real-time PCR.In addition,with immunoperoxidase assay we found that phospholipase C signaling inhibitor U73122 also affected BVDV replication in vitro.In order to obtain an active BVDV Erns protein,both prokaryotic expression system and eukaryotic expression system were employed.N-terminal BVDV Erns was cloned into pET-28a plasmid and got pET28a-N-Ems,which was used to express N-terminal BVDV Erns in Ecoli BL-21(DE3)pLysS.The purified protein showed reactivity with BVDV serum with ELISA.Further identification of this ELISA kit is ongoing.In addition,BVDV E2 and Erns was cloned into plasmid pEGFP-N1 and generated pEGFP-N1-E2 and pEGFP-N1-Erns.However,only Erns could be expressed at high level in transfected 293T cells.In summary,we have generated mAbs against BVDV and established an immunoperoxidase assay applicated for antiviral drugs screening by titration of virus.The expressed Erns protein with E.coli could be potentially used for the generation of an ELISA kit for BVDV serum detection.The successful expression of Erns protein in 293T cells would provide us a resource for the screening of mAb against Erns protein,and further study is ongoing. |
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