Development And Validation Of A Multiplex TaqMan Real-time RT-PCR For The Quantitative And Differential Detection Of Wild-type Viruses From C-strain Vaccine Of Classical Swine Fever Virus

Classical swine fever (CSF), caused by Classical swine fever virus (CSFV), is a highly contagious disease of pigs, and one of OIE Listed diseases. In recent years, CSF has changed a lot in clinical manifestations under the pressure of mass vaccination in China, resulting in atypical or chronic forms quite different from acute CSF, and causing great economic losses in pork industry. CSFV, together with Bovine viral diarrhea virus 1 (BVDV-1), BVDV-2, Border disease virus (BDV) and Pestivirus of giraffe, belongs to the genus Pestivirus within the family Flaviviridae. Pigs can be infected with almost any pestivirus species. It is rather difficult to differentiate CSFV from other pestiviruses by conventional serological tests due to cross-reactivity. On the other hand, extensive vaccination with C-strain vaccine makes it difficult to differentiate the animals infected with wild-type viruses from C-strain vaccine vaccinated ones using conventional assays.This study is aimed to develop a multiplex real-time RT-PCR for the quantitative and differential detection of wild-type viruses and C-strain vaccine of CSFV. All genomic sequences of pestiviruses available in GenBank including 34 strains CSFV genomic sequences were aligned. CSFV specific primers and two differently labeled TaqMan probes for the differentiation of wild-type viruses from C-strain vaccine were designed in the 5'-UTR of the viral genome of CSFV. The two TaqMan probes specifically hybridize wild-type CSFV of different subgroups or C-strain vaccine virus, respectively, in the multiplex real-time RT-PCR, with no cross-reaction to other viruses of porcine origin. Completely correct differentiation of wild-type CSFV of different subgroups (1.1, 2.1, 2.2 and 2.3 subgroups) from C-strain vaccine was achieved when testing reference strains and characterized field isolates of CSFV. There was an excellent linear relationship from 10⁶ to 10¹ copies/μL when the positive standards of wild-type and C-strain-type viruses were detected simultaneously by the assay. The sensitivity of the assay for detecting wild-type and C-strain-type vaccine viruses was determined to be 41.8 and 81.5 copies of viral RNA, respectively. The agreements between the multiplex real-time RT-PCR and a multiplex RT-nested PCR for detection of wild-type or C-strain-type viruses were 96.2% (wild-type viruses) and 100% (C-strain-type viruses), respectively, when detecting 152 different samples (including cell cultures and field samples). Thirty-two out of 106 (30.2%) of field samples were detected positive for wild-type viruses by the multiplex real-time RT-PCR.To determine the viral replication kinetics of CSFV in infected pigs, whole blood samples of pigs experimentally infected with 10⁶ TCID₅₀ Shimen strain or contact pigs were quantitated by the multiplex real-time RT-PCR from 0 to 14 days post-infection. The assay was able to detect the viral RNA in the whole blood samples of experimentally infected pigs as early as 2 days post-infection and viral RNA was detectable in contact pigs 3 to 4 days prior to the onset of clinical symptoms.There is an excellent correlation between the titers of C-strain vaccines titrated in rabbits and the RNA copies quantitated by the multiplex real-time RT-PCR when detecting 8 different batches of C-strain vaccines. It was determined that cell cultures containing more than 2.23×10⁷ viral RNA copies (corresponding to 5×10⁴ RID₅₀) can be used for vaccine production.By using the assay, pigs infected with wild-type CSFV can be identified and removed from the vaccinated population, which will help to establish a CSF-free swineherd. Also this assay can be used in the pathogenesis study, vaccine efficacy evaluation and vaccine titration of CSFV.