Construction And Characterization Of Chimeric Classiccal Swine Fever Viruses Based On Shimen Strain And C-strain

Classical swine fever, a highly contagious and devastating disease of swine, is caused by classical swine fever virus (CSFV), a member of the genus Pestivirus within the Flaviviridae family. CSFV is a small enveloped virus with a single positive-sense RNA strand of about 12.3-kb. CSFV genome is flanked by 5’and 3’untranslated regions and contains a single ORF encoding a polyprotein of 3898 amino acids. The polyprotein is post-translationally processed by both cellular and viral proteases to form 12 mature viral proteins:Npro, C, Erns, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B. Proteins C, Erns, El and E2 are the structural components. With the development of CSFV infectious clones (ICs) based on different virus strains, significant progress has been made in understanding the functional aspects of major viral proteins in viral replication and their interaction with host cells.Of note is the possible development of recombinant marker vaccine based on the vaccine C-strain based infectious clone. However, the C-strain has low replication in cells as compared with strain Shimen. Our laboratory has generated a recombinant C-strain based CSFV. We found that the recombinant virus has low titers probably because of its low adaption to cell cultures. This study was attempted to generate a series of chimeric CSFVs having different combinations of structural and non-structural proteins based on the infectious clones of Shimen strain and C-strain and then to dissect the roles of different proteins in viral replication in cultured ST and PK-15 cells.I. Comparison of replication potential of the recombinant classical swine fever viruses of low and high passages and Shimen strain in cultured cellsAt the same level of MOI (multiplicity of infection), the recombinant C-strain viruses, either low or high passage viruses RecCpp40 and RecCpp80, showed lower replication than the Shimen strain. However, there was no significant difference of genomic RNA levels among the three viruses sampled at 4 time points during the culture (P>0.05). The high passage RecCpp80 displayed enhanced in vitro replication with the progeny virus particles 30 times more than RecCpp40 at 96 hours post-infection (hpi)(P<0.01). Estimation of extracellular virus particles at 96 hpi revealed that release of progeny virus particles was proportional to replication. The Shimen strain showed faster replication and released more progeny virus particles than viruses RecCpp40 and RecCpp80 (P<0.05). Strain Shimen was also superior to RecCpp80 in intercellular diffusion as determined by the size of plaques, being less affected by agarose coverage. Under both conditions of regular culture and agarose covered growth, there were large and diffusing plaques of cells infected with strain Shimen. Therefore, we suppose that both viral replication and potential of intercellular viral spread contribute to the difference between strain Shimen and vaccine C-strain in cell adaptability.Ⅱ. Construction of chimeric viruses based on infectious clones of C-strain and Shimen strain and their replication in cultured cells and rabbit fever patternsTo dissect the molecular basis governing the significant difference of replication among CSFV strains, we constructed a series of chimeric ICs containing different genes encoding structural and non-structural proteins from both C-strain IC and Shimen strain IC constructed in this study. The recombinant Shimen strain and 11 chimeric viruses were successfully rescued. Viral growth was enhanced when the C-strain was chimerized either with the first half (containing 5’-UTR and genes coding for structural proteins) or the second half (containing genes coding for non-structural proteins and 3’-UTR) of the Shimen strain. The chimeric virus containing the genes encoding the non-structural proteins of strain Shimen had replication potential similar to the RecCpp80 virus. Chimerization of the C-strain with NS2-NS4B from Shimen greatly enhanced viral replication, while that of Shimen strain with NS2-NS4B from C-strain had lower replication than the parent Shimen strain, indicating the significant role of NS2-NS4B in viral growth in cultured cells. Replacement of NS4A-3’-UTR or NS5A-3’-UTR did not improve replication of the chimeric C-strain virus. In order to identify the role of nonstructural proteins in adaptation of C-strain to rabbit, selected chimeric viruses were tested for fever response. The C-strain regions of both the first and second halves, as defined above, contained some elements that were sufficient to induce fever in rabbits. Chimeric C-strain with NS2-NS4B from Shimen also induced fever. However, replacement of C-strain NS4A-5’-UTR or NS2-NS4 region in the Shimen strain backbone did not induce fever. Thus, both UTR regions and the structural protein region are important for fever induction by the C-strain.Ⅲ. Roles of the transmembrane regions of NS2 in classical swine fever virus replicationIn order to address the roles of the NS2 protein in viral replication, we PCR-amplified 6 fragments covering the corresponding transmembrane (TM) regions from Shimen strain genome and cloned 12 eukaryotic expression plasmids with gfp fusions. Confocal microscopy showed that all putative NS2 TM segments were localized in the endoplasmic reticulum, showing reticular pattern throughout the cytoplasm of ST cells. To examine the effect of putative TM segments on in vitro viral replication,12 mutated infectious clones of the Shimen strain were generated by deletion of individual TM segments or by alanine insertion. Rescue experiments revealed that loss of any one of the putative NS2 TM regions resulted in defective virus replication and assembly. Alanine insertion in TM1 did not affect virus replication and assembly while insertion in TM3 or TM6 hampered the virus assembly with delayed production of progeny virus requiring 3-4 continuous passages. Insertion of TM2, TM4 and TM5 resulted in defective viral RNA replication and abortive production of the progeny virus. These results indicate that NS2 TM regions play an important role in CSFV replication. Further research is needed to decipher the mechanisms.To conclude, we have elucidated the roles of non-structrual proteins NS2-NS4B and NS2 TM regions in viral replication in cultured cells by means of the reverse genetic strategy in combination with gene replacements between C-strain and Shimen strain of different cell adaptability. We found that the UTR and structural protein regions contribute to fever induction in rabbits. These findings will certainly help facilitate our further investigation into the mechanisms of CSFV replication as well as the development of improved C-strain-based live vaccine.