Studies On Pig Bovine Viral Diarrhea Virus In Some Regions Of China

1. Isolation and Identification of a pig Bovine Viral Diarrhea Virus. Virus were isolated from BVDV positive sample from the piglet and used to study pig bovine viral diarrhea virus. BVDV positive sample from the piglet were inoculated in MDBK cells and a pig BVDV SD0803were obtained. In the study, cell culture, direct immunofluorescence assay, PCR amplification of5’-UTR, electron microscopy and TCID50were performed, moreover, bioinformatics software were used to analyze the characteristics of molecular evolution. The results showed that the virus strain hadn’t caused cytopathic effect (CPE) until thirteenth passage on MDBK cell. SD0803infected cells showed strong fluorescent signal. DNA fragment of307bp and428bp were amplified by PCR from5’-UTR and Npro of SD0803.Under electron microscopy, virus particles were near round and enveloped with size of50nm in diameter. Virus titer of SD0803was10-6.5TCID5o·0.2mL-1. The homology comparison and phylogenetic analysis of5’-UTR and Npro between SD0803and representative BVDV isolates showed that SD0803shares the same group with BVDV-1reference isolates; thereby the isolate belongs to BVDV-1. However, SD0803have an obvious distance from the subtypes of BVDV-1and formed a separate group according to the phylogenetic relationship and this showed that the isolate might be a new subtype of BVDV-1. So a pig BVDV SD0803was successfully isolated and identified, and belongs to NCP BVDV-1and was a probably new subtype of BVDV-1.2. Sequencing and Bioinformation Analysis for complete genome of pig Bovine viral diarrhea virus SD0803strain. Complete genome of Bovine viral diarrhea virus (BVDV) SD0803isolate were sequenced and used to study swine BVDV. RT-PCR assay was respectively applied to obtain10fragments of SD0803strain. Sequence of both5’-and3’-ends were performed with the Rapid-amplification of cDNA ends (RACE). All fragments were spliced with Seqman software and the complete genome of the isolate obtained was12272nt. The genomic RNA contained5’-untranslated region (5’-UTR),3’-untranslated region (3’-UTR), and one open reading frame (ORF) encoding a large polyprotein of3899amino acids but not exogenetic genomic sequences. Complete genome of BVDV SD0803strain was successfully obtained. The study on molecular epidemiology of pig BVDV in China, to some extent, was promoted.The purpose of this present study was to analyze the complete genome of Bovine viral diarrhea virus SD0803strain and predict the structure-function relationship. Bioinformationatic softwares such as MEGA4.1, DNAStar, I-TASSER Server, CLC RNA Workbench were applied to analyzed the complete genome. The results showed:SD0803isolate belongs to BVDV-1and the highest homology identity was83.2, compared with ZM-95, the homology identity of each gene is42.7%～91.7%with pestivirus strains. There were three high variable domains four functional ones (A、B、C、D) and two oligopyrimidine-rich in5’-UTR. There were one high variable and one conserved domain and contains3stem-loop structures in3’-UTR. The structural proteins contain14Potential N-linked glycosylation sites. There are50.9%amino acids (AAs) forming coil,8.6%AAs forming P-sheet, and the remainders posing helix. Tertiary structure possessed replicas’structures.3. Establishment of Nested-PCR for Detecting pig Bovine Viral Diarrhea Viruses. Two pairs of primers were designed according to these genomic sequences of BVDV-1, BVDV-2, CSFV and a new member of the pestivirus genus, and a Nested-PCR were developed for the differentiation of pig BVDVs and CSFV. This PCR assay could respectively amplify a198bp fragment from BVDV NADL, pig BVDV, but not from CSFV, porcine reproductive and respiratory syndrome virus (PRRSV). Sensitivity was determined as0.195fg RNA. And in the field trail of100pig specimen,36%of them were positive. These results showed that the Nested-PCR with high sensitivity and specificity provided a new and alternative tool for the detection of pig BVDVs and CSFV.4. Molecular epidemiological characterization of pig bovine viral diarrhea virus in some region of China. Known about molecular epidemiological characterization of pig bovine viral diarrhea virus in some region of China and primary explore of evolution of pig BVDV and the relationship among BVDV from bovine and vaccines, to establish the base of prevent and control pig BVDV. Samples from Beijing, Henan, Xinjiang, Shandong, Jiangxi, Zhejiang, Anhui, Hunan, Guangxi, Jiangsu and Shanghai and so on were detected by Nested-PCR. Also, Bovine serums come from cattle farm; commercial fetal serum made in China and imported fetal serum, live vaccine for Classical swine fever virus, PRRSV, PRV, and MDBK cells. The results showed, the positive rate of pig were11.88%(146/1229) and was increasing tendency. Serum from cattle farm26.0%(130/500), commercial domestic fetal serum96.4%(53/55); imported fetal serum100%. BVDV were not detected in vaccine for PRRSV and PRV. The sequences of BVDV5’-UTR of different source were analyzed by Bioinformatics software. The results revealed, all sequences but FS1(commercial domestic fetal serum), belonged to BVDV-1; among serum from cattle farm4isolates were BVDV-1m,2isolates BVDV-1b,1one BVDV-1a. among imported fetal serum,2ones BVDV-1b.1one BVDV-1c,3ones BVDV-1m,1one BVDV-1p,1one BVDV-2a and1one can’t be ensured. Among CSF live vaccine,2strains were BVDV-1m,2ones BVDV-1o,1one BVDV-1k,1one BVDV-1b.20pig BVDV strains,1strainBVDV-1a,6ones BVDV-1b,6ones BVDV-1m,1one BVDV-1o, and6ones can’t be ensured. The homology between pig BVDV and serum from cattle farm was87.3%～100%, and commercial domestic fetal serum77.1%～98.8%. imported fetal serum80.5%～97.5%. there are mutant sites in of5’-UTR of all BVDV strain and not regular.5. Development of real-time fluorescence quantitative PCR assay for detection of Bovine viral diarrhea virus in pigs. According to the conserved gene sequence of5’-UTR of BVDVs and CSFV, pairs of primer and specific TaqMan probe were designed. The positive standard plasmids were used as quantitative template to make the standard curves, through the method was optimized. Specificity, sensitivity, and conformity were tested. By sensitivity analysis, the FQ-PCR indicated that a minimum of10copies of plasmid DNA was detected. The assay exhibited reproducibility and specificity, and all negative controls such as classical swine fever virus (CSFV) and porcine reproductive and respiratory syndrome virus (PRRSV) showed negative detection. As a result of the sensitivity and specificity of the assay with a relatively rapid and simple procedure, the real-time FQ-PCR will be used as a routine assay for the diagnosis of pig BVDV infection and CSFV vaccine polluted by BVDVs.6. Expression and Antibody Preparation of E2Protein of Pig Bovine Viral Diarrhea Virus. To provide the method about the diagnosis on pig bovine viral diarrhea virus, sE2gene, which removed C-terminal transmernbrane of E2gene of BVDV SD0803isolate (sE2), and sE2protein were got and anti-sE2antibody was prepared. sE2gene which was amplified from SD0803strain by PCR was cloned into pGEX-4T-1and transformed into BL21(DE3). After induction with IPTG, the recombinant protein was obtained. Rabbits were immunized with the purified protein as antigen, and antiserum was acquired. The polyclonal antibodies were analyzed by indirect ELISA, Western-blot and indirect immunofluorescence assay. The antiserum titer was determined by indirect ELISA and was1:256000. The results of Western-blot and indirect immunofluorescence assay confirmed the antibodies reacted with specifically to the protein expressed. A recombinant SD0803sE2protein and the specific antibodies have been obtained.