Construction Of European PRRSV Reverse Genetic Operation Platform And Analysis Of Its Transcription Characteristics

Porcine Reproductive and Respiratory Syndrome(PRRS)caused by Porcine Reproductive and Respiratory Syndrome Virus(PRRSV),is one of the biggest problems the global pig industry is facing.The virus has the characteristics of high mutation degree and fast mutation speed.The spread of the disease has caused incalculable losses to the global pig industry.Effective prevention and control of porcine reproductive and respiratory syndrome is very important for the healthy development of the pig industry.The transcription process of PRRSV subgenomic RNA(sg RNA)is discontinuous,and a series of subgenomic sg RNA with the same 5 'and 3' ends are formed during the transcription process.Starting from the special transcriptional mechanism of PRRSV,a new idea may be found for the prevention and control of PRRSV.Currently,it has been found that the transcription process of PRRSV is regulated by numerous transcriptional regulatory sequences(TRS)in the genome,including leader-TRS in 5 'UTR and body-TRS in the genome.According to regional distribution and genetic differences,PRRSV is divided into two genotypes,namely European(type I)and American(type II).The two genotypes of PRRSV not only have low homology in nucleic acid,but also have obvious regional distribution differences.In this paper,the reverse genetic technology of RNA virus and high-throughput sequencing technology were mainly used to analyze the similarities and differences in transcriptional regulation of European and American strains from the transcriptional mechanism.Based on the previous study of HuN4 strain in the American strain,the LLV-1 strain of PRRSV isolates from Europe preserved in our laboratory was studied.In this study,the establishment of the reverse genetic system of the European type strain,the analysis of sgm RNA high-throughput sequencing results,the rescue of chimeric strains in Europe and America,and the expression of exogenous genes were carried out,the results were as follows:(1)The reverse genetic platform of European PRRSV LLV-1 strain was successfully established.Connected two fragments of PRRSV LLV-1 keeped in our laboratory into the pc DNA3.1 plasmid by using restriction enzymes San D I and Xba I and recivied recombinant plasmid pc DNA3.1-LLV-1 include LLV-1 gene.Transfer the plasmid pc DNA3.1-LLV-1 in the BHK-21 cell.48 h after transfection,transfer the cell supernatant into Marc-145 cells,passage the cell 3 generation in blind,CPE was appeared in the cells.PRRSV GP5 could be dectectioned from cell supernatant genes through the RT-PCR,proved the European PRRSV LLV-1 was reversed successfully.(2)Preliminary analysis of transcriptional characteristics of European PRRSV sg RNA.Collect the samples of Marc-145 cells and PAM cells infected with LLV-1 strain after 48 h for high-throughput sequencing.By analyzing the results,it was found that there were 163 valid B-TRS sites in the genome of PRRSV LLV-1.The 6 bases constituting the B-TRS sequence can be any combination,but the combination of L-TRS(TTAACC)is preferred.The higher the composition similarity with L-TRS,the higher the transcriptional abundance of its mediated sg RNA.Statistics the number of B-TRS in ORF and its transcriptional abundance,the results showed that the transcriptional ratio of ORF2-7 transcripton in the two cells was same basically.The virus transcripon abundance ratio in Marc-145 cells was 3.4 : 1 : 3 : 4.3 : 7.3 : 35.8,and the virus transcripon abundance ratio in PAM cells was 3.3 : 1 : 2.8 : 3.7 : 7.9 : 30.1.Compared with the American PRRSV HuN4,the transcripon abundance of ORF3 in the American type was basically the same as that of ORF2 and ORF4,while the transcripon abundance of ORF3 in the European type was significantly lower than that of ORF2 and ORF4.LLV-1 has a high transcriptional abundance of all transcripons in Marc-145 cells,while HuN4 strain has a higher transcriptional abundance in PAM cells,suggesting that HuN4 has a stronger pathogenic effect on PAM.(3)Constructed chimeric strain LLV-1-1-HuN4 s successfullyThe structural protein gene(ORF2-ORF6)sequence in LLV-1 genome was replaced with the corresponding structural protein sequence of the American PRRSV HuN4 strain,and the chimeric virus was successfully rescued by transfection with the same method.The proliferation and the cytokines IL-8 and IL-10 of chimeric virus were detected,and the results showed that chimeric virus had similar proliferation characteristics with the parental strain,and the protein in Americam type could has normal fuction on the European PRRSV gene.(4)The stable insertion sites of European PRRSV were analyzed.The positions between ORF7 and 3'UTR and between ORF1 b and ORF2 a were selected as the insertion sites of foreign protein genes,and GFP was used as the target gene to save the virus.The transcription of GFP genes was detected by RT-PCR.The results showed that when GFP was inserted in two sites,the stable was poor,and the exogenous gene was lost in the third or fourth generation.Compared with ORF1 b and ORF2 a,it is more stable that GFP was inserted before 3'UTR.RT-PCR showed that the expression level was higher when GFP inserted between ORF7 and 3'UTR than that between ORF1 b and ORF2 a.(5)Analyze the differences in the expression of exogenous genes induced by B-TRS in European and American strains.In order to increase the stability of exogenous gene expression,GFPS with TRS6 and its flanking sequence of European and American strains were inserted between ORF7 and 3'UTR,respectively.GFP gene introduced without TRS as a control to save the virus,and the transcription of GFP m RNA was detected by RT-PCR.The results showed that the recombinant virus of the American strain B-TRS and its flanking sequence inserted in front of the exogenous gene had the highest expression stability and could be transmitted to the 6th generation at least.In summary,this paper established the European PRRSV reverse genetic platform successfully,analyzed the transcriptional characteristics of the European and American PRRSV,and preliminarily proved that the American PRRSV has a higher transcriptional abundance of subgenes in PAM cells.The chimeric strains were constructed,and it was proved that the structural genes of the American strains could function normally in the European PRRSV,indicating the potential of recombination between the two strains.The stability of the inserted genes at different insertion sites was analyzed when the European PRRSV was used as an exogenous gene expression vector,and it was confirmed that the American PRRSV TRS6 had a better function in guiding the expression of exogenous genes in the European PRRSV genome.