| The swine reproductive and respiratory syndrome virus is an important pathogen affecting the global pig industry,causing great pressure and economic loss to Chinese pig industry.Porcine reproductive and respiratory syndrome first broke out in the United States in1987 and quickly spread around the world.PRRSV was introduced to the Chinese mainland in 1995.Due to its extremely high variability,PRRSV kept mutating in China.In 2006,a highly pathogenic strain represented by JXA1 appeared in China,which had 1+29 amino acid deletion model in the NSP2 region,with more severe clinical symptoms and higher fatality rate.In 2013,there was another outbreak of NADC-30 strain in China,and the recombinant variability of this strain was higher,which made the clinical prevalence of PRRS more complicated.At present,PRRSV vaccine has attenuated live vaccine and inactivated vaccine,but the immune effect is not very ideal,and there are some disadvantages such as strong virulence,cross recombination with wild strain,and insufficient immune protection of inactivated vaccine.Therefore,the prevention and control of PRRSV in China becomes more complicated.GP5 protein is the main structural protein of PRRSV,which has high variability and immunogenicity,and can induce the production of neutralizing antibody.Studies have shown that glycosylation of PRRSV protein affects virus recognition,invasion,immune escape and virulence of host cells.However,the mechanism of how glycosylated proteins play these roles is not completely clear at present and needs further study.This study started with a PRRSV strain isolated in our laboratory.First,its NSP2 gene fragment was amplified and sequenced,and the sequencing results were compared with the NSP2 gene of various PRRSV strains.Name it CSR1801.Nine pairs of overlapping primers were designed for amplification according to the complete genome sequence of classical strains published in Gene Bank,and the whole genome sequence of CSR1801 was obtained by cutting and splicing the sequencing results of each fragment.Homology analysis showed that CSR1801 had the highest homology with classical strains CH-1a and CH-1R,which were99.0% and 99.1%,respectively.The homology with HP-PRRSV was 94.8%-95.1%.The homology with NADC-30 strains was 83.6% to 83.9%.The homology with NADC-34 was84.7% to 85.7%.The results of genetic evolution analysis showed that CSR1801 belonged to classical PRRSV strain,belonging to the same branch as CH-1a and CH-1R.The results of recombination analysis showed that the CSR1801 strain did not recombine with PRRSV strains commonly found in Chinese pigs.These results indicated that the classical strain of PRRSV had a certain stability and was not easy to recombine with other PRRSV strains.The reverse genetic system of CSR1801 was constructed by homologous recombination.First,the p Bluescript Ⅱ KS(+)(p BSK)vector was modified,and CMV eukaryotic promoter,hepatitis Delta ribozyme(HDV)and BGH terminator sequences were inserted into the vector to ensure the precise cutting of the 3 ’terminal at the transcriptional level.The modified carrier was named p CMV-HB.Five pairs of primers were designed according to the full-length genome sequence of CSR1801,and the amplification was carried out with hi-fi enzyme.Five correctly sequenced fragments of CSR1801 were cloned into p CMV-HB successively to construct the plasmid p CMV-CSR1801-HB,and the 811 base of fragment E was mutated from A to G as a genetic marker.The plasmid p CMV-CSR1801-HB was transfected into MARC-145 cells,and the cell culture was freeze-thawed for 3 times after 5 days of culture.The supernatant was taken by centrifugation and inoculated into MARC-145 cells.After 48 hours of blind transmission,the cell culture was collected for RT-PCR and IFA identification.The results showed that the virus was saved successfully.The plaque formation and multistep growth curves of parent and rescue viruses were compared.The results showed that there was no significant difference in the proliferation of CSR1801 rescue virus and parent virus on MARC-145 cells,and the plaque size and shape were similar.These results indicated that the infectious cloning of the classical PRRSV strain CSR1801 was successfully constructed,which provided a technical platform for further study of the molecular biology and immune mechanism of PRRSV.On the basis of the successful construction of infectious clones of CSR1801,the glycosylation sites of GP5-N34 and GP5-N51 were selected for single point mutation to deglycosylation,and the full-length infectious clones containing single point mutations of the glycosylation sites of N34 and N51 were successfully constructed.MARC-145 cells were transfected and transmitted blind for three generations.Two strains of GP5-N34 R and GP5-N51 R with glycosylation site mutations were successfully saved by RT-PCR and IFA identification.The plaque formation and multi-step growth curves of r CSR1801 and GP5-N34 R and GP5-N51 R glycosylated mutants were compared.The results showed that: There was no significant difference between GP5-N34 R and GP5-N51 R glycosylation mutant and r CSR1801 strain in the multi-step growth curve on MARC-145 cells,and the shape and size of the plaque formed were similar.These results indicated that the mutation of N34 and N51 glycosylation sites in GP5 of CSR1801 strain did not affect the rescue of the virus,the proliferation of the virus in MARC-145 cells and the generation of progeny virions.In this study,whole genome sequencing and analysis of PRRSV,construction of infectious clone and construction of infectious clone with glycosylated site mutation were used.CSR1801 strain was identified as the classical PRRSV strain,infectious cloning of CSR1801 strain was constructed,and the virus was successfully saved,which provided a platform for the study of the pathogenic mechanism of PRRSV and the development of vaccine at the gene level.The full-length infectious c DNA clones containing single point mutations of GP5-N34 and GP5-N51 glycosylation sites were constructed,and there was no significant difference in the replication and proliferation characteristics between the rescued mutant viruses and non-mutated viruses,which provided ideas and basis for subsequent studies on the pathogenicity and immune escape of glycosylation on PRRSV. |
