Construction Of A Full-length Infectious Clone Of Bovine Leukemia Virus And Its Biological Characteristics

Bovine leukemia virus(BLV)belongs to the family Retroviridae and is the etiologic agent of enzootic bovine leukosis(EBL),which mainly infects cattle,zebu and buffalo.After being infected by BLV,the host carries the virus for life,triggering a dysregulation of its immune system and a reduced immune response,which increases its risk of infection with other pathogens.In addition to this,BLV infection will cause significant economic losses to the cattle industry by leading to reduced performance in dairy and beef cattle,increased costs for treatment and prevention of the disease,and restricted exports.As of today,there are no commercially available BLV vaccines for cattle in the world.Economic BLV control and decontamination strategies are still lacking.Therefore,in this study,a BLV reverse genetics operating system was established and two BLV-infectious clones with phenotypic differences were constructed based on it.Further,the biological characteristics of the two rescue viruses were investigated by site-directed mutagenesis,homologous recombination,indirect fluorescent assay(IFA),syncytium formation assay,RT-FRET-qPCR,and reverse transcriptase activity assay.The main study components and findings are as follows.1.Construction of a full-length infectious clone of BLVFour pairs of primers were designed based on the whole genome DNA sequence of BLV provirus published in GenBank(Accession number:AB987702,MH170030,LC080656)to amplify the pro virus genome of BLV17985 and BLV2723 preserved in our laboratory.Segmentally amplified BLV provirus genomic nucleic acid fragments were cloned into the pCAGGS vector using overlap PCR with homologous recombination.They were named as pCAGGS-BLV17985 and pCAGGS-BLV2723,respectively.The recombinant plasmids carried the full-length sequence(8739 bp)of the BLV provirus genome,including the long terminal repeat(LTR)(1-528 bp,8212-8739 bp),structural genes env(4828-6375 bp),gag(631-1812 bp),pol(2324-4882 bp),pro(1806-2345 bp),and non-structural genes tax(48754878 bp,7269-8194 bp),rex(4828-4878 bp,7269-7688 bp),G4(433-505 bp,7088-7332 bp),and R3(4828-4878 bp,7040-7123 bp)were composed,consistent with the BLV provirus genome structural features.2.Rescue and identification of recombinant virus and infection in vivoFour cells,HeLa,MDBK,Tb 1 Lu,and Vero,were transfected with pCAGGSBLV17985 for syncytium formation assay.The expression of BLV-p24 antigen in Vero and Tb 1 Lu cells was further observed using IFA.Subsequently,virus rescue and passaging were performed on Tb 1 Lu cells,and the stability of the rescued virus on this cell line was monitored by IFA with RT-FRET-qPCR and its reverse transcriptase activity was determined.In the animal infection assay,sheep were inoculated with the same copy number of rBLV17985 and wild virus,and virus replication in sheep was monitored by FRET-qPCR.Peripheral blood mononuclear cells(PBMC)were isolated from sheep and co-cultured with blank Tb 1 Lu to confirm infection.The formation of antibodies to the virus in sheep was detected using the collected sheep serum as IFA primary antibody.The results showed that the constructed pCAGGS-BLV17985 could successfully rescue the replicating virus on Tb 1 Lu cells in vitro and in vivo,and the biological properties of the rescued virus in sheep were similar to those of the wild virus.3.Biological characterization of rBLV17985 and rBLV2723 strainsIn the previous study,another infectious clone of BLV2723,pCAGGS-BLV2723,was constructed using the same construction method as pCAGGS-BLV17985.rBLV17985 and rBLV2723 were found to have significantly different replication abilities in vitro when verifying the stability of the rescue virus.A total of three mutations were found in the whole genome of rBLV 17985 and rBLV2723 proviruses by sequencing,all located in the pol gene,A2898G,C3072T,and T4094C,corresponding to mutated amino acids Q966R,T1024I,and S1365P,respectively.To investigate the key amino acid sites that regulate the replication ability of the virus,mutant recombinant viruses with three nucleotide sites,rBLV17985 and rBLV2723,named rBLV17985-Q966R,rBLV17985-T1024I,rBLV17985-S1365P,rBLV2723-R966Q,rBLV2723-I1024T,rBLV2723-P1365S,were constructed using targeted mutagenesis and homologous recombination techniques,respectively,and were serially passaged in Tb 1 Lu cells.The results of reverse transcriptase activity assay,RT-FRET-qPCR and IFA assay showed that the in vitro replication ability of recombinant virus rBLV17985Q966R was not significantly different compared with rBLV17985,while the in vitro replication ability of recombinant virus rBLV2723 was significantly reduced with the rest of mutant recombinant viruses.Thus,1024 and 1365 are the key amino acid sites regulating viral replication,and the replication ability of the virus is normal when they are both T and S.Mutation of either of these amino acids will result in the inability of BLV to replicate efficiently in vitro.In this study,infectious clones of BLV were constructed and viruses were rescued and characterized,which is the first successfully established BLV reverse genetics operating system in China.The biological characteristics of two phenotypically distinct viruses were further analyzed,and key amino acid loci regulating viral replication were identified.Our study provides a good platform for basic research of BLV.