| Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious and often fatal hemorrhagic disease of swine. The outbreak of CSF in China causes a great loss in pig industry. Vaccination is an efficient tool to prevent the outbreak of CSF.Hog cholera lapinized Virus (HCLV), or Chinese(C)-strain vaccine has been used for a long time and played a key role in the control of epizootic CSF. C-strain is derived from CSFV highly virulent Shimen strain after adaptation in rabbit for hundreds of passages.In this study, we focused on the effect of C-strain E2 and 3'UTR on virus replication and pathogenicity to investigate the molecular mechanism of C-strain attenuation. Our study shed light to the development of novel vaccine against CSFV and control of CSF.(1) Using viral genome RNA of CSFV strain Shimen as the template, viral cDNA fragments were amplified by RT-PCR with specific primers. Then, these cDNA fragments were orderly cloned into plasmid pACNR1180 to generate the full length infectious cDNA clone, pSM. Infectious RNA was transcribed in vitro from a linearized pSM, and transfected into PK15 cells to rescue infectious CSFV. The rescued CSFV (vSM) exhibited similar growth characteristics compared to parental virus wtSM.(2) The CSFV strain Shimen E2 and 3'UTR were replaced with the corresponding region of vaccine C strain by reverse genetics based on the infectious cDNA clone. Therefore, the chimeric infectious clone pSM/CE2, pSM/C3'UTR and pSM/CE2/3'UTR were constructed, and the chimeric viruses vSM/CE2, vSM/C3'UTR and vSM/CE2/3'UTR were rescued from in vitro-transcribed viral RNAs, respectively. Compared with the parental vSM, chimeric virus vSM/CE2 exhibited a reduction in replication and plaque formation, and the growth and plaque formation of vSM/3'UTR reduced lightly. However, the growth and plaque formation of vSM/CE2/3'UTR was remarkably reduced, suggesting that the E2 and 3'UTR of C-strain reduced virus replication and plaque formation synergistically. Groups of pigs infected with vSM/CE2, vSM/C3'UTR, and vSM/CE2/3'UTR were survived. The infected pigs presented with no or mild clinical symptoms. In contrast, pigs infected with vSM presented with acute clinical symptoms and died within 11 days post-infection. These results demonstrated that chimeric viruses vSM/CE2, vSM/C3'UTR,and vSM/CE2/3'UTR were attenuated.(3) Chimeric viruses were serially passaged in PK15 cells, and were evaluated in vitro. Compared to parental chimeric viruses, virus titers of both the vSM/CE2-p11 and vSM/CE2/3'UTR-pll were increased approximately 10 fold. The plaque size of vSM/CE2-pll was larger than that of parental vSM/CE2, and the growth and plaque formation of vSM/3'UTR-p11 were similar to its parental chimeric virus. Compared with parental viruses, the pathogenicity of vSM/CE2-pll and vSM/C3'UTR-pll were increased remarkably, and the pathogenicity of vSM/CE2/3'UTR-pll was increased lightly.(4) The genome of vSM/CE2-pll was sequenced. Data showed amino acid substitutions T745I and M979K in E2 ocuuring in the vSM/CE2-p11 genome. Mutant viruses vSM/CE2/T745I, vSM/CE2/M979K, and vSM/CE2/T745I;M979K were constucted by site-directed mutagenesis in the vSM/E2 backbone using reverse genetics. Compared with the parental vSM/CE2, all mutant viruses replicated better in PK15 cells. The results from infected pigs indicated that amino acid substitutions T745I and M979K were responsible for the acquisitions of pathogenicity.(5) Pigs were inocilated with the attenuated chimeric viruses. At 23 days post-inoculation, pigs were challenged with CSFV virulent strain Shimen. All these pig survived and showed no CSF clinical symptoms. These results demonstrated that chimeric CSFVs containing vaccine C strain E2 and/or 3'UTR induced complete protection against CSFV virulent strain lethal challenge.Our results could contribute to understanding the molecular mechanism of C-strain atteunation and the the development of novel vaccine against CSFV. |
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