Construction Of Bacterial Artificial Chromosome Recombinant Virus Of Feline Herpesvirus Type 1 CH-B Strain And Its Culture Characteristics

Feline viral rhinotracheitis(FVR)is a common feline upper respiratory tract infection caused by feline herpesvirus type 1(FHV-1).For a long time,it has been difficult to obtain mutant strains of FHV-1 by the traditional method of homologous recombination because of the large genome of FHV-1 with a nucleotide length of about 135 kb,which can encode about 74 proteins,and it is unfavorable to perform operations such as deletion or mutation of its target gene.In this study,we proposed to construct the whole genome of FHV-1 CH-B strain isolated from Changchun City as infectious bacterial artificial chromosome(BAC)and establish the reverse genetics system of FHV-1 CH-B strain in order to modify the whole genome of FHV-1CH-B strain with the help of E.coli plasmids and improve the efficiency of constructing The genome-wide modification of the FHV-1 CH-B strain with the help of E.coli plasmids will improve the efficiency of recombinant virus construction and lay the foundation for further research on its gene function and novel vaccines.In this study,the upstream homologous arm US2-A and the downstream homologous arm US2-B containing the Loxp locus were amplified from the partial gene sequences on both sides of the US2 region of the FHV-1 CH-B strain,and the obtained US2-A and US2-B and the green fluorescent marker gene CMV-EGFP were sequentially cloned into the p UC119 vector to construct the intermediate transfer vector p UC119-A-Loxp-EGFP-B.The recombinant viral transfer vector p UC119-A-Loxp-EGFP-B was constructed by ligating the p Belo BAC11 vector to p UC119-A-Loxp-EGFP-B after Sph I digestion.p UC119-A-Loxp-EGFP-BAC-Loxp-B was constructed by cotransfecting F81 cells with the FHV-1 CH-B strain and the recombinant viral transfer vector.The recombinant feline herpesvirus r-FHV-1-BAC with the exogenous gene EGFP-BAC was purified after four rounds of phospho screening.r-FHV-1-BAC was blindly transmitted to F81 cells for 15 generations,but EGFP was still normally expressed,and the exogenous large gene fragment inserted in the US2 region of FHV-1 and some essential genes of FHV-1 were identified by PCR,which showed that The results showed that the recombinant virus r-FHV-1-BAC was purified and genetically stable.The r-FHV-1-BAC was infected with F81 cells,and the cyclized genome of r-FHV-1-BAC was extracted at the right time and immediately electrotransformed into DH10 B receptor cells to obtain FHV-1 CH-B strain BAC transformants.The in vitro culture characteristics of the parental virus FHV-1 CH-B strain and the purified recombinant virus r-FHV-1-BAC revealed that the proliferation rate of r-FHV-1-BAC was slightly slower than that of FHV-1 CH-B strain and the cryptic period was prolonged,but the morphology and size of the phagocytic spots of the two strains were similar.The above results demonstrated that the deletion of the FHV-1 US2 gene and here the insertion of the exogenous large gene fragment EGFP-BAC reduced the replication and proliferation ability of the FHV-1 CH-B strain on F81 cells.The purified r-FHV-1-BAC provided materials for the construction of an artificial chromosome system for the bacteria of FHV-1 CH-B strain,and provided a strong condition for obtaining a gene deletion vaccine for FHV-1 CH-B strain in the future,which laid the foundation for the establishment of a technical platform for reverse genetic manipulation of FHV-1 CH-B strain.