Basic Research On The Construction Of Reverse Genetic System Of Bovine Respiratory Syncytial Virus

BRSV are members of the Pneumovirus genus within the subfamily Pneumovirinae of family Paramyxoviridae.The BRSV genome is a single-stranded negative-sense RNA with about 15 000 nt,encoding 11 proteins.These proteins include two nonstructural proteins and nine structural proteins.BRSV infection is widely distributed around the world,and the relevant investigation demonstrated that BRSV is prevalent in China.BRSV infection can cause secondary bacterial infections,leading to respiratory disease complex that is the main cause of increased morbidity and mortality in cattle,and has brought huge economic losses to cattle industry.Vaccination remains a powerful means for preventing and controlling the BRSV infection.The research on viral gene structure and function based on reverse genetic technology is the basis of the research on the pathogenesis,immune mechanism,host and tissue tropism of BRSV.In this study,the full-length genome of LJX31 was sequenced from a nasal swab sample of a cow with BRSV nucleic acid,and the sequence homology and evolutionary tree analysis were carried out.On this basis,the genetic elements of BRSV reverse genetic system was constructed to generate BRSV reverse genetic system,which laid a foundation for the pathogenesis of BRSV and other related research.The sequencing and analysis of LJX31 full-length genome revealed that the Chinese BRSV has a genome with length of 15150 nt,including a 45-nt leader region,a 161-nt tailer region.Nucleotides 46 to 14989 of the consensus sequence spans the NS1,NS2,N,P,M,SH,G,F,and M2 genes,of which,L gene is the largest BRSV gene,composed of 6573 nt,NS2 gene is the smallest gene,composed of 492 nt.For the NS1,N,P,M,SH,G,F,M2 genes,their sequence is 527,492,1202,869,950,466,840,90 and 960 nt in length,respectively.Compared with Atue51908 strain,the sequence identity of complete LJX31 genome was 96.4% contributed by 13 insertions and 3 gaps,revealing considerable divergence.For the NS1,NS2,N,P,M,SH,G,F,M2 and L genes compared to the strain Atube51908 sequence,nucleotides identities were in the range of 92.1% to 98.5%.The homology and genetic evolution analysis of the coding gene sequence of G protein the displayed the closest nt sequence identity(95.1%)of LJX31 to the American V41 strain,suggesting that LJX31 might originate from the United States.In order to construct the reverse genetic operating system of LJX31,the eukaryotic expression plasmids of genome-length cDNA,N,P,L and M2-1 genes should be constructed respectively.Meanwhile,in order to increase the adaptability of the virus to cells,the eukaryotic expression plasmids of BRSV receptor protein CX3CR1 were constructed in this study.Susceptible cells were transfected with the full genome-length cDNA plasmid and the support plasmids expressing the BRSV viral protein N,P,L,M2-1 and BRSV receptor protein CX3CR1.In this study,full-length genome cDNA plasmids PVK-LJX31 and PCI-LJX31 based on T7 promoter and CMV promoter were constructed simultaneously.The coding genes of N,P and M2-1 proteins were cloned directly into the PCI-neo vector,and the expression plasmids PCI-neo-N,PCI-neo-P and PCI-neo-M2-1 were constructedrespectively.The coding gene of L protein was optimized and cloned into p3xflag-CMV vector to construct the expression plasmid p3xflag-YH-L.All the plasmids,including PVK-LJX31,PCI-LJX31,PCI-neo-N,PCI-neo-P,PCI-neo-M2-1 and p3xflag-YH-L,were correctly digested and sequenced,indicating that all the plasmids as the genetic elements of reverse genetic system of LJX31 were successfully prepared.The expression of PCI-neo-N,PCI-neo-P,PCI-neo-M2-1 and p3xflag-YH-L plasmids was confirmed by IFA.The results showed that the expression products in the transfected cells could react with the corresponding antibody,indicating that all the helper plasmids could be expressed in the transfected cells.In addition,the functions of cis-acting elements and regulatory regions of LJX31 genome and four support plasmids,PCI-neo-N,PCI-neo-P,PCI-neo-M2-1 and p3xflag-YH-L,were verified by microgenome plasmid pok(-T7)-W.The results showed that after transfection of pok(-T7)-W plasmid into cells,the cells infected with BRSV could express green fluorescent protein,indicating the cis-acting elements and regulatory regions of LJX31 genome are functional.However,when pok(-T7)-W plasmid was co-transfected with PCI-neo-N,PCI-neo-P,PCI-neo-M2-1 and p3xflag-YH-L auxiliary plasmids,the expression of green fluorescent protein could not be observed in the cells,indicating that the ability of PCI-neo-N,PCI-neo-P,PCI-neo-M2-1,p3xflag-YH-L auxiliary plasmids to form RNP complex or the function of RNP complex is defective.In summary,the full-length genome sequencing and its analysis of BRSV LJX31 were carried out,and the construction genetic elements of reverse genetic operation system of LJX31 was structed to establish LJX31 infectious clone,which laid a solid foundation for the establishment of reverse genetic platform of BRSV.