Development For Differentiation And Rapid Quantification Of Wild-type And C-strain Vaccine Viruses Of Classical Swine Fever Virus

Classical swine fever (CSF), caused by Classical swine fever virus (CSFV), is a highly contagious disease of pigs. The epidemiological characteristic of CSF has become more complicate resent years in China, above all, the current mode and clinical characteristic of classical swine fever virus has changed a great deal in recent years, which change for inapparent and subvirus infection, there appear several types such as"non-typical CSF","mild-type CSF", and"carrier sow syndrome"according to the clinic signs, which also have different pathological necropsy lesions from CSFV wild-type. The important method to control CSF is vaccination, chinese hog cholera lapinized virus was used extensively at home and abroad because of the characteristic of quick immunity and having no remained virulence, which was the most safe and valid vaccine virus in international, played a great role in controlling classical swine fever virus. For HCLV, the key index is vaccine effectiveness and the main challenge is how to detect quickly and accurately. Besides, large-scale applications of HCLV in China have made it more difficulty to distinguish CSFV wild-type from HCLV infections. It is necessary to establish a specific and accurate method to detect CSFV wild-type virus and HCLV easily and quickly. To solve these problems, this study was divided into three parts:1. 25 strains classical swine fever virus(CSFV)genomic sequences including wild-type and vaccine virus pubulished in GenBank were aligned, and a pair of primers corresponding to the highly conserved regions of CSFV genome was used as a common primer pair in the first round of the RT-nPCR, amplified fragment length 609 bp.Another primers specific for CSFV vaccine virus, respectively, were used for the second round of the RT-nPCR together with the forward common reverse primer, amplified fragment length 237 bp,in order to develop a reverse transcription-multiplex nested polymerse chain reaction for differentiation wild-type and C-strain vaccine virus with the characteristic of sensitivity and specificity. It can completely differentiate wild-type of different subgroups from vaccine virus, which has no specific reaction with bovine viral diarrhea virus and other virus of pigs. This assay can mark detection accurately and rapidly for CSFV, which can prevent it from extending. Besidely, it can differentiate pigs infected with wild-type CSFV from those vaccinated with vaccine virus, which can minimizes the possibility of miss-killed vaccinates during outbreaks.2. In order to development a fluorescence quantitative RT-PCR combined melting curve analysis method that can differentiation of Wild-type and C-Strain Vaccine Viruses of Classical Swine Fever Virus. 25 strains classical swine fever virus(CSFV)genomic sequences including wild-type and vaccine virus published in GenBank were aligned, design one pair of sharing reverse primer of classical swine fever virus and specific forward primer respectively for wild-type and C-strain vaccine virus, and estimate that Tm value is 84.5±0.5℃and 89.5±0.5℃respectively. The melting curve analysis with such advantages as high specificity and sensibility and good repeatability, shows single specific peak. The fluorescence quantitative RT-PCR combined melting curve analysis method developed in this experiment with high amplification efficiency, vast linear range and short detection period, can provide fast and accurate differentiation for the breakout of classical swine fever, Besidely, it can differentiate pigs infected with wild-type CSFV from those vaccinated with vaccine virus, which can minimizes the possibility of miss-killed vaccinates during outbreaks.3. In order to develop a real-time RT-PCR assay for quantitative detection of hog cholera lapinized virus (HCLV). 25 strains classical swine fever virus( CSFV ) genomic sequences including wild-type and vaccine virus pubulished in GenBank were aligned, and a pair of primers corresponding to the highly conserved regions of CSFV genome was designed, amplified fragment length 245 bp, which has no specific reaction with bovine viral diarrhea virus and other virus of pigs. 16 samples were detected, the sensitivity of the assay was 102 copies per reaction, and the assay has good linear relationship in a wide range of 106～102 copies/μL. The method developed in this paper has the advantages of high sensibility, specificity and good stability, which can detect HCLV rapidly and accurately and provides a simple and effective tool for the development of HCLV vaccine and the molecular biological studies on classical swine fever virus.