Porcine Reproductive And Respiratory Syndrome Virus ZJ-JX/2015 Virus Establishment Of Strain Reverse Genetic System

Porcine reproductive and respiratory syndrome virus(PRRSV)is a spherical,capsulated,single-stranded RNA virus that causes reproductive disorders in pregnant sows and respiratory diseases in piglets.Neither the attenuated live vaccine nor the inactivated vaccine can provide safe and effective immune protection for pigs.Therefore,the research and development of the new vaccine is one of the key measures to prevent and control the diseases.Reverse genetics to construct recombinant virus technology is an important technology platform for the research of novel vaccines,and it can also be applied to the research of PRRSV protein function and pathogenic mechanism.In this study,the whole genome of clinical isolates of PRRSV/ZJ-JX/2015 was sequenced,and Sequence alignment analysis was performed using CLC Sequence Viewer 8 and Mega 7.0 software.Genome-wide nucleotide evolution analysis indicated that PRRSV/ZJ-JX/2015 strain belonged to genotype II HP-PRRSV.Amino acid comparison analysis showed that there were 38 significant differences in amino acid sequence of NSP2,and there were significant differences in amino acid sequence of GP5 at 59,114 and 164,compared with the 2015 strains in East China.In this study,the genome of PRRSV/ZJ-JX/2015 strain was divided into four segments for segmented amplification,and the genomic c DNA of PRRSV/ZJ-JX/2015 strain was constructed into plasmid p SMART-BAC and plasmid p WSK-29 by one-step homologous recombination technique.Two full-length infectious clones of PRRSV/ZJ-JX/2015 were obtained.By site-directed mutagenesis,a meaningless mutation(C-G)was introduced at 4821 of the genome to form the Bst B I restriction site as a genetic marker for identification.CMV promoter-based p SMART-BAC-CMV-r PRRSV plasmid was transfected into BHK-21 and 293T cells,respectively,and the recombinant viruses(BHK-CMV-r JX15 and 293T-CMV-r JX15)were rescued.The recombinant virus(BHK-T7-r JX15)was rescued by transfecting a T7 promoter-based plasmid p WSK-29-T7-r PRRSV into BHK-21(T7)cells(a stable cell line carrying the T7 promoter polymerase gene).The results showed that the recombinant virus BHK-CMV-r JX15 appeared obvious lesions 48 hours after MARC-145 cells were uploaded to F2 generation,and the lesions tended to be stable after continuous passage to F5generation.The recombinant virus 293T-CMV-r JX15 was transmitted to the F4 generation in MARC-145 cells,and showed obvious lesions at 48 h after passage,and the lesions tended to be stable at the F6 generation.The recombinant virus BHK-T7-r JX15 showed obvious lesions 48 hours after MARC-145 cells were uploaded to F6,and the lesions tended to be stable 48 hours after passage to F8.The three recombinant viruses infected MARC-145 cells,respectively.Western boltting and IFA detection results showed that PRRSV N protein could be detected in the cell precipitation infected by the recombinant virus,and there was a specific green fluorescence signal in the nucleus of living cells,and the fluorescence signal was strong in the cytologic lesions.The 544 bp fragment containing the genetic marker site was amplified by PCR method,and verified by Bst B I enzyme digestion.The 270 bp and 274 bp fragments could be cut out.Sequencing analysis showed that the sequence of this site was(C-G)nonsense mutation.After plaque purification,the recombinant virus TCID₅₀was determined,and the results showed that the F5 generation of BHK-CMV-r JX15 was10^5.8,the F6 generation of 293T-CMV-r JX15 was 10^4.2,and the F8 generation of BHK-T7-r JX15was 10^3.6.The results of one-step growth curve showed that BHK-CMV-r JX15 and 293T-CMV-r JX15 strains had more similar growth rules with their parents.In this study,the reverse genetic recombinant virus rescue system of PRRSV was successfully constructed,which provided a technical platform for the research on live attenuated virus vaccine,pathogenic mechanism and virus biology of PRRSV.