Construction Of Bacterial Artificial Chromosomes For Swine Pseudorabies Virus

Pseudorabies(PR)is caused by pseudorabies virus(PRV),and it is an acute infectious disease that can infect most mammals.Porcine is the natural and reservoir host of PRV,and PRV is characterized by neurological symptoms in piglets,pregnant swine abortion or having stillbirths.PR once prevailed all over the world and caused great economic losses in pig industry in many countries.Since the 1980 s,by the immunization of Bartha-K61 vaccines,most pig herds in China have PR effectively controlled.However,since the second half of 2011,the sudden arise of PRV variants in Heilongjiang,Jiangsu,Henan and other provinces in China,which have many insertions and deletions compared to previous PRV strains and belong to genotype II,caused great economic losses in pig industry.PRV genome can be as long as 142 kb,which makes it hard to manipulate or reform genes.The traditional homologous recombination(HR)method to gain recombinant virus is low-efficiency and spends long time,but constructing the infectious clone by using the Bacterial Artificial Chromosome(BAC)makes the PRV large genome easy to have genes deleted,inserted or site-mutated in bacteria.In the study we first inserted the GFP cassette into pBACloxp vector,generating pBAC-GFP;then we used the PRV variant strain PRV HLJ8 as a template,and constructed the upstream homologous arm Us4-Us6 and downstream homologous arm Us2-Us1,and cloned them to pBAC-GFP vector separately,thus generating pBAC-GFP62.Then we co-transfected PRV HLJ8 genome and the linearized transfer vector pBAC-GFP62 into Vero cells and obtained PRV recombinant virus vBAC-HLJ8 containing pBAC.The vBAC-HLJ8 circular genome was extracted,and then it was electrotransfected to E.coli.DH10 B.By the method of PCR to screen the positive BAC clones,the identified positive BAC clone was transfected to Vero and we obtained the resBAC-HLJ8.The viral growth curve indicated resBAC-HLJ8 was replicating slower than PRV HLJ8.The result showed that we have constructed PRV variant strain HLJ8 BAC.With the same method,PRV attenuated vaccine strain Bartha-K61 BAC was constructed.To improve recombinant virus screen efficacy,while we co-transfected the transfer vector and PRV HLJ8 genome,we also co-transfected pCas9 s which have different gRNAs that are targeting homologous arms or targeting genes inside homologous arms.Our study proved that whether by single gRNA incision or double gRNA incisions inside of homologous arms,they can both improve the recombinant virus efficiency.And double gRNA incisions can be more effective in promoting large DNA fragment insertion.The successful construction of PRV variant strain HLJ8 BAC and PRV attenuated vaccine strain Bartha-K61 BAC lays a foundation to further manipulate and modify PRV genome quickly and correctly.Meanwhile,the HR efficiency can be greatly improved by CRISPR/Cas9,and as a result it shortens the time to gain the recombinant virus greatly.The discovery not only reduces the time to gain recombinant virus,but provides a good solution for the construction of other recombinant DNA viruses,which is of important scientific significance.