| There was an outbreak which was called "Mystery Fever Disease Syndrome" or "High Fever Disease" in pig herds in China in2006. In the beginning, it was happened in Jiangxi province. Then it spread to almost all the mainland covered more than20provinces. Tremendous loss was made via this desease to the pig industry in China. The symptom of this disease includes the anorexia, the high body temperature, constipation and diarrhoea, absorption in gestation sows and sometimes with nervous symptom, secretion with the nose especially with the eyes. The most sensitive stages were the gestation sows, the finishing and growing pigs. The desease has very highly incidence and the mortality. The purpose of this study was to investigate the epidemic situation of the PRRSV and CSFV in recent China, including uncover the "mystery" of the disease or syndrome eventually. We also sequencd and analysed the ful genome of one predominant PRRSV strain in China. On the base of these knwoledge, we generated a novel marker virus for the control of PRRS by using the reverse genetics. We also developed a PRRSV vector vaccine candidate and explored the possibilities of the foreign gene can stably exist in the PRRSV genome. These findings enhance our knowledge of the genetic diversity of pocine viral pathogens in China and contribute to the development effective strategies for swine disease control.1. Molecular epidemiology of CSFV field isolates in parts of ChinaTo gain an insight into the molecular epidemiology of classical swine fever in China, we analyzed the E2gene of103Chinese CSFV isolates. Clinical samples were collected between January2008and March2011. CSFV was detected in103out of these samples by RT-nested PCR and were selected for sequencing. Further analysis based on E2sequences revealed that all Chinese isolates belonged to subgroupsl.l,2.1and2.2. CSFV isolate of genogroup3was not found. The most significant correlation between genetic and geographical distribution for the isolates in the study, especially for the subgroup2.1strains, was they take up the widest area, since these viruses existed throughout the mainland of China. These findings enhance our knowledge of the genetic diversity of Chinese CSFV isolates, and may contribute to the development of reliable diagnostic tests, the epidemiological surveillance, and the development of effective strategies for disease control.2. Molecular epidemiology of PRRSV field isolates in parts of ChinaA total of1196clinical samples of dead or sick pigs obtained from15different provinces of China during2006-2010were detected for PRRSV. The ORF5genes of some field isolates were amplified and sequenced for further understanding of the molecular epidemiology and the genetic diversity of PRRSV in China. PRRS viruses were identified by RT-PCR from clinical samples. Sequences of ORF5of69PRRSV positive samples was amplified and analyzed with other17strains available on the GenBank of NCBI. A total of247samples were positive and the positive rate was20.7%. Sequence analysis showed that the69isolates in this study belong to the Type2PRRSV strains. They were related closely to the highly pathogenic PRRSV (HP-PRRSV) with94.0%-100%amino acid sequence identities. The phylogenetic analysis indicated that all these Type2PRRSV strains in China were further divided into five subgenotypes and subgenotype IV showed most highly identity with HP-PRRSV. Although cross-cutting phenomenon exists in the genetic relationship of PRRSV isolates obtained from different areas, there were no obvious relations between the distribution of PRRSV and the region. The PRRSV was widespread and HP-PRRSV was the predominate strain and the genetic evolution was relatively stable in China during2006-2010. These findings enhance our knowledge of the genetic diversity of Chinese PRRSV isolates, and may contribute to the development of reliable diagnostic tests, the epidemiological surveillance, and the development of effective strategies for disease control.3. Genomic sequencing and phylogenetic analysis of predominant PRRSV in ChinaTo gain an insight into the molecular characteristics and the genetic variety of porcine reproductive and respiratory syndrome virus (PRRSV) in China, we sequencd and analysed the ful genome of one Chinese isolate. Primers were designed according to ATCC VR-2332and HP-PRRSV JX143strain. The full genome of the PRRSV strain JS0922, which was isolated from Jiangsu province in2009, was amplified and then sequenced. The genomic sequence was obtained by splicing of correct sequencing and was further aligned with other reference strains. The results showed that JS0922fell into Type2PRRSV. Even though it showed significant differences from the strains isolated before the year2006, that the homology was only89%compared with BJ-4, it shared more than98%homology with HP-PRRSV. JS0922also had different characteristics variously that ORF3and ORF4varied greatly while ORF6revealed more conservative. Analysis showed that JS0922had general molecular characteristics with HP-PRRSV, but also existed a few amino acids mutations which differed from HP-PRRSV. In summary, JS0922strain presented a few new genomic features, but still fell into a same branch of HP-PRRSV and belonged to the current predominant PRRSV in China.4. Study on the N-terminal non-essential domain of PRRSV N protein non-overlapping regionTo define the precise non-essential domain of porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid (N) protein non-overlapping region, we studied a full-length infectious clone p7PNX, which overlapping region of ORF6and ORF7had been separated. Primers were designed according to the sequenc of the p7PNX. We focused on the N protein and constructed a series of N-terminal mutations, right behind the start codon of ORF7using reverse genetics. Transfection and virus rescue assay indicated that residues2-7at the N terminus were non-essential for virus viability. The series of mutants showed stable inheritance in cell culture. Growth kinetics and viral plaque assay in vitro showed that the virological characteristics of the mutant virus were samiliar with the wide type and the mutants replicated well as its parental strain. This was the first time of the investigation of N-terminal non-essential domain on N protein non-overlapping region of PRRSV. Besides, this study opens new pathways for the further elucidating the structure-function relationship of PRRSV N protein and may enable the development of novel gene marker vaccines.5. Development of marker virus and the preliminary immunization study in the structural protein N region of porince reproductive and respiratory syndrome virusCurrent PRRSV vaccines fail to provide serological differentiation between vaccinated and infected pigs and new strains of the virus have been isolated. Therefore, new, more efficacious vaccines developed using novel approaches are required. Using reverse genetics, a marker virus (v7APMa) was generated with a nucleocapsid protein that differs from the wild-type strains (vAPRRS, type2PRRSV). v7APMa shows stable inheritance in cell culture and the virological characteristics of the marker virus in vitro showed that v7APMa replicates well as its parental strain. In the pig model, the v7APMa marker virus induced a similar level of anti-N protein antibodies. The results also showed that all animals vaccinated with vAPRRS and v7APMa developed neutralizing antibody after14days post-infection, and both IL-4and IFN-y in serum samples were markedly induced compare with those in the control group. The v7APMa marker virus induced robust antibodies against the marker peptide, starting from14days post-infection, while the vAPRRS and the control groups didn’t. Following challenge with HP-PRRSV JX143at28d, piglets in the control group showed classical PRRSV clinical signs, such as rise in temperature, decreased appetite, palpebral edema, runny nose and cough. Two piglets dead at11d and14d respectively. Meanwhile, piglets inoculated with vAPRRS and v7APMa, showing lighter clinical signs, lower viremia and no death. These indicates that v7APMa and vAPRRS could provide protection against virulent PRRSV JX143challenge in pigs. This approach, using a rationally designed marker virus, opens up a new avenue, for further development of PRRSV marker vaccines.6. Construction and virological characteristics of PRRSV vector vaccine candidateTo develop a porcine reproductive and respiratory syndrome virus(PRRSV) vector vaccine candidate and to explore the possibilities of the foreign gene can stably exist and play function between ORF4and ORF5of PRRSV genome, we insert ORF4of EAV(equine arteritis virus) to gain a chimeric virus. By using PCR, enzyme digestion and sequencing analysis, the recombinant pAPRRS(EAV4) were selected and purified. After transfection of the recombinant plasmids into BHK-21cells, the recombinant viruses vAPRRS(EAV4) were obtained. The chimeric viruses showed stable inheritance in cell culture. The replication and translation of PRRSV genome was confirmed by RT-PCR and immunofluorescence assay. It was further demonstrated by Northern-blot and the sequencing of subgenome that the transcription of EAV ORF4was not detected. Growth kinetics and viral plaque assay in vitro showed that the virological characteristics of the chimeric viruses were samiliar with the wide type and the mutants replicated well as its parental strain. The chimeric virus vAPRRS(EAV4) should be useful for studying on function of the arterivius structural protein and developing novel PRRSV vector vaccine. |
