| Bovine viral diarrhea virus (BVDV) has a wide range of hosts. It is an important pathogen of cattle worldwide and can also infect pig, sheep, deer, camel and many wild animals. BVDV is caused by bovine viral diarrhea, and characterized bovine viral diarrhea-mucosal disease, reproductive disorders, thrombocytopenia and hemorrhage syndrome, persistent infection and immunotolerance. Pregnant animals between30～120days when the immune system of the fetus has not yet developed well, infected with noncytopathic (NCP) BVDV, will produce stillbirth, fetal malformation, or fetus persistent infected with BVDV. In recent years, several key factors lead to the outbreak of BVDV infection in pigs, including direct or indirect contact with cattle, feeding with offal or milk, inoculation of classical swine fever (CSF) vaccine contaminated with BVDV, etc. Besides, calf serum and cells used for the production of CSF vaccines also increase the risk of BVDV infection. BVDV and classical swine fever virus (CSFV) have cross antigenicity and pigs infected with BVDV displays the CSF-like infection symptoms, so BVDV infection is often mistaken for CSFV infection which bring difficulties to the prevention and control of BVDV and CSFV. Now, there still has not yet effective measures for the prevention and control of BVDV infection. In developed countries, vaccine inoculation, enhancement of monitoring and eradicating persistent infection (PI) and immunotolerance animals are the main measures. While in domestic situation, most of the BVDV infections in pigs and cattle are persistent infection, along with inadequate monitoring and attention, contribute to the high prevalence of BVDV. Consequently, in addition to strengthening the biological safety management, much of fundamental studies also should be done. On one hand, we should investigate the prevalence of BVDV infection in pigs and identify their genetic characteristics. On the other hand, the similarity and differences between pig BVDV and bovine BVDV should be focused which will be helpful for planning an effective prevention method and developping a novel BVDV vaccine.In this study, we isolated and genetically characterized BVDV-2strains from the diseased pigs and CSF vaccine, sequenced the complete genomic sequence of one pig BVDV-2isolate and enriched the genetic information of pig BVDV. Then an infectious cDNA clone of pig BVDV-2was constructed which provides an effective tool for deeply studying pig BVDV and bovine BVDV. In order to explore the mechanism of persistent infection of BVDV, we expressed the nonstructural proteins Npro, NS2and NS3of pig BVDV-2strain SH-28in vitro and explored their functions involved in the antiviral cellular response. A subgenomic infectious clone with Npro gene deletion was also developed by modifying the infectious cDNA clone of SH-28and confirmed its certain role in the innate cellular immunity. The main studies can be divided into the following four parts:1. Isolation and identification of pig BVDV isolatesIn2010, twelve serum samples of pig and a kind of commercial CSF vaccine were sent to our laboratory for BVDV detection. The same as the positive and negative controls, the samples were modulated and passaged on MDBK cells, respectively. Total RNA from the cells inoculated were extracted after72h and detected by RT-PCR with FBVDVI-Ⅱ/RBVDVI-Ⅱ primers directed to the5’ untranslated region (5’UTR) of BVDV. A positive DNA band about288bp for BVDV were amplified in all the thirteen samples. Indirect immunofluorescent assay (IFA) also confirmed that all the thirteen isolates could react well with the monoclone antibody (MAb) Bz-53(against BVDV-2) rather than MAb D89(against BVDV-1). 5’UTR DNA fragment and E2gene from the thirteen isolates were sequenced. The phylogenetic analysis revealed that the thirteen isolates all fell into BVDV-2subgenotype. Moreover, homology comparison demonstrated that the E2genes of the thirteen isolates were highly conserved. It’s supposed that they may have the same origin and the BVDV infections in this pig herd may result from the vaccination contaminated with BVDV.2. Sequencing and analyzation of the complete genomic sequence of pig BVDV-2strain SH-28To further study the genetic characteristics of pig BVDV-2, the complete genomic sequence of a pig BVDV-2strain SH-28was sequenced. Eighteen fragments covered the whole genome of SH-28were PCR amplified and cloned into pGEM-T Simple vector. Each positive clone was sent to a biotechnology company for sequencing with the repetition of three times. To obtain the5’-3’terminal sequence of SH-28, the RNA was cyclized before reverse transcription and12positive clones were sequenced after cloning as above. The retrieved sequences were aligned and spliced using Lasergene software. The genome of SH-28comprises12,279nucleotides (nt) and contains a large open reading frame (ORF) beginning at nucleotide386and ending at nucleotide12073. The phylogenetic analysis of the complete sequences showed that SH-28and HLJ-10(isolated from calf serum, BVDV-2) were classified into the same branch and was most similar to bovine BVDV-2strain XJ-04(92.3%), but was genetically divergent from the pig BVDV-1strains (ZM-95, SD0806)(70.0%,70.1%). While the phylogenetic analysis of5’-UTR indicated that SH-28belonged to BVDV-2a2and both HLJ-10and XJ-04were classified into BVDV-2al. In view of above, we speculate that SH-28is a novel BVDV-2isolate different from the previous BVDV-2strains and may be originated from bovine BVDV-2strain.3. Construction of the infectious cDNA clone of pig BVDV-2strain SH-28To further explore the genomic function and pathogenesis of pig BVDV, an infectous cDNA clone of pig BVDV-2strain SH-28was constructed. T7promoter and a single endonuclease SbfI were inserted in the5’and3’terminal of the SH-28genome, respectively. Infectious cDNA clone pASH28was generated by cloning a full length of cDNA, with a complete genome of SH-28, into the pACYC184plasmid. Pig BVDV-2progeny virons were recovered from this clone after linearization, RNA transcription in vitro and transfection into MDBK cells. Evaluated by indirect immunofluorescence assay and semi-quantitative RT-PCR, the rescued progeny virus vASH yielded similar viral titers as its parental virus SH-28. Datas suggested that pASH28may represent the first infectious cDNA clone for pig BVDV-2which may become valuable for future studies of replication, virulence, and pathogenesis of pig and bovine BVDV. Then, a subgenomic infectious cDNA clone with Npro gene deletion was developed by modifying pASH28and rescued the mutant virus vASH⊿Npro with the deletion of gene Npro successfully. All of the vASH, vASH⊿Npro and parental virus SH-28had good growth characteristics. However, unlike vASH, the virus titer of vASH⊿Npro was lower than SH-28which may be attributed to the indirect effect result from Npro deletion. The mutant virus vASH⊿Npro contructed not only confirms the effectiveness of the infectious cDNA clone of pig BVDV-2, but also lay a foundation for further studying the function of the Npro gene.4. The nonstructural proteins of pig BVDV-2involved in the innate cellular immunityTo probe into the pathogenesis of persistent infection involved in the nonstructural proteins of pig BVDV-2strain in the innate cellular immunity, primers directd to Npro, NS2and NS3genes were designed according to the complete genomic sequence of SH-28known. After amplification and cloning, the recombinant eukaryotic expression plasmids pEGFP-Npro, pEGFP-NS2and pEGFP-NS3were established, respectively. It showed that GFP labeled protein and the nonstructural proteins were fusion-expressed successfully confirmed by Western blot. Results indicated that infection of pig BVDV-2strain SH-28or in vitro expression of nonstructural protein Npro inhibited the production of type I interferon (IFN-I) induced by poly(IC) in MDBK cells while NS2 and NS3had no effect on it. This may mean that Npro play an important role in the process of interfering the innate cellular immuity by pig BVDV-2. Further detected the effect of the nonstructural proteins on the antiviral proteins induction by real-time PCR, we found that overexpression of Npro down-regulated the expression of ISG15, OAS and Mx1significantly (P≤0.05) rather than PKR. While both of NS2and NS3proteins had no influence on the four antiviral proteins induction. Infected with the mutant virus vASH⊿Npr0, the expression of ISG15, OAS and Mxl were up-regulated significantly (P≤0.05). Consequently, this confirmed that Npro played an important role in the process of interfering the innate cellular immunity by pig BVDV-2through both of OAS and Mx1pathways and the help of ISG15protein. |
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