| Classical swine fever (CSF) is a highly contagious, multisystem hemorrhagic swine disease, caused by classical swine fever virus (CSFV). The major measure for prevention and control of CSF in mainland China is through massive vaccination of "C-strain" vaccine. However, the sporadic outbreak of CSF still happens in mainland China, distributed in large areas, and becoming more and more complicated.Under such circumstances, it is of necessity to differential detect wild-type viruses infected animals from vaccinated, investigate the status of persistent or silent infection in swine farms, enhance the quality of produced "C-strain" vaccine through certain methods. The present study is aiming to establish certain assays for differential detection of "C-strain" vaccine and wild-type viruses of classical swine fever virus and bovine viral diarrhea virus (BVDV), and an alternative method for potency test of ST cell-line produced "C-strain" vaccine that can be beneficial for the prevention and control of CSF in mainland China.(1) The potency test of "C-strain" vaccine based on rabbit fever test could be significantly influenced by individual differences or/and experimental environment. In the present study, tested vaccines were serial diluted and infected permissive cells; RT-nestPCR, RT-PCR and antigen capture ELISA were used to detect the virus particles produced in the infected cells. The results can represent the virus titer of the tested vaccine, so as to be the indicator of the potency of tested vaccine. The potency test results of the12batches of ST cell-line produced "C-strain" vaccines and3batches of bovine testicular cells produced "C-strain" vaccines tested by both of the alternative method established in this study and the rabbit fever test were subjected to parallel comparison. And it indicated that the positive dilution of10-6～10-7of tested vaccines correspond to2.6×104～3.0×104RID/dose, the positive dilution of10-4and lesser of tested vaccines correspond to7.0×103～8.0×103RID/dose. The potency test results of the alternative method correspond to the results of rabbit fever test, can indicate the quantity of infectious virus particles in vaccine.(2) One pair of outside primers and one pair of inside primers were designed in the conservative regions to encompass the T-rich insertion exclusively existing in the3’end region of "C-strain" vaccine viral genome. Thus, the amplified fragment of "C-strain" vaccine was longer than that of wild-type viruses. The detection limit of the RT-nestPCR established in this study to "C-strain" vaccine and wild-type virus Shimen strain was0.032pg and0.045pg of viral RNA respectively. The results of specificity tests showed that the RT-nestPCR assay can effectively detect different genotypes of CSFV, and the detection results of other non-CSFV pathogens were negative. The detection results of400tonsil tissue samples collected from clinical healthy pigs indicated that4samples were "C-strain" vaccine positive and12samples were wild-type virus positive. All vaccines were "C-strain" vaccine positive and free of wild-type viruses contamination in the detection of14batches of commercialized "C-strain" vaccine. The RT-nestPCR developed in this study can effectively differentiate "C-strain" vaccine and wild-type viruses of classical swine fever virus.(3) Three TaqMan MGB probes and corresponding primer pairs were designed in the conservative regions of "C-strain" vaccine and wild-type viruses of CSFV and BVDV viral genome respectively through alignment of complete genome sequences of47CSFV strains,7BVDV strains and3border disease virus (BDV) strains. The concentration of sense and antisense primers, probes in the reaction mix and annealing temperature were optimized according to the orthogonal experimental design. The results of specificity tests showed this method can simultaneously differential detect "C-strain" vaccine and different genotypes of wild-type viruses of classical swine fever virus and BVDV, the detection results of other non-CSFV pathogens were negative. The results of reproducibility tests indicated that the inter-and intra-group coefficient variation (CV) was no more than1%. The detection limit of this assay to "C-strain" vaccine and wild-type virus Shimen strain of CSFV and BVDV_Oregon was31.4copies/μL,15.8copies/μL,32.5copies/μL respectively. The results of the detection of400clinical samples showed that4samples were "C-strain" vaccine positive,12samples were wild-type virus positive,22samples were BVDV positive. All vaccines were "C-strain" vaccine positive and free of wild-type viruses and BVDV contaminations in the detection of14batches of commercialized "C-strain" vaccines. The agreement rate of this assay with other detection methods were more than95%. The assay established in this study can differential detect "C-strain" vaccine and wild-type viruses of CSFV and BVDV, and can also provide an effective tool for quality monitoring of vaccine production.(4) In order to investigate the status of silent infection of CSFV in breeding swine farms in mainland China,400tonsil tissue samples collected from clinical healthy pigs in breeding swine farms in northern China were subjected to detection of wild-type CSFV using the RT-nestPCR and triplex real-time RT-PCR developed in the present study, and positive samples were subjected to virus isolation in which11isolates were successfully obtained. The phylogenic analysis of E2sequences indicated that all11isolates belonged to subgenotype2.1a. Certain mutations specific to the11isolates were observed in the alignment and analysis of the deducted amino acid sequence of E2protein.This study can be benficial for the prevention and control of CSFV in mainland China. An alternative method for potency test of ST cell-line produced "C-strain" vaccine was developed and of the ability to replace the potency test methods based on animal experiments and provide potential benefits for improving quality of produced "C-strain" vaccine. |
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