Effects Of PRRSV Guizhou Isolates GZZJ11 And GZBJ12 On Helicases DDX6 And Mov10

Porcine reproductive and respiratory syndrome（PRRS）is caused by porcine reproductive and respiratory syndrome virus（PRRSV）.It is commomly known as blue ear disease and its characteristics are mainly reproductive failure in slow and respiratory problems in pig of all ages.At present,PRRSV multi-type strains of pig farms are more common,mainly PRRSV vaccine strains,wild strains,recombinant strains and NADC30-like strains.The diversity of strains makes the prevention and control of the disease difficult.Since the first report in China,the disease has experienced for more than 20years in China,but it has not been effectively controlled,which has brought unpredictable economic losses to the development of China’s pig industry.In this experiment,two PRRSV strains were successfully isolated from pig farms suspected of PRRSV infection,the whole gene sequence analysis was conducted to explore the effect of PRRSV isolates on helicases DDX6 and Mov10 at mRNA level.This study will provide a reference for preliminary understanding of PRRSV infection and strain variation in Guizhou,and it also lays a foundation for further study of the relationship between helicase DDX6,Mov10 and PRRSV proliferation.1.Isolation and identification of PRRSV isolates GZZJ11 and GZBJ12 from Guizhou.The test was performed to isolate and identify PRRSV virus in organs of suspected PRRSV-infected pigs.The diseased material was ground,centrifuged,and sterilized and filtered,and then inoculated into Marc145 cells for virus isolation;the isolated virus was identified by CPE observation,RT-PCR,IFA and Western blotting.The results showed:（1）After the GZZJ11 strain was blindly transmitted to the sixth generation on Marc145 cells,obvious CPE such as agglomeration and shrinkage of Marc145 cells was observed,and the same CPE could be observed after GZBJ12 was blindly transmitted to the third generation.（2）After the GZZJ11 strain and GZBJ12 strain were blindly transmitted to the sixth generation and the third generation respectively,RT-PCR detected 372 bp N gene band and931 bp NSP2 gene band.（3）IFA and Western blotting were used to identify the virus by rabbit polyclonal antibodies against N protein of PRRSV type 2 gene.The results of IFA showed that specific green fluorescence could be seen in the infected virus group;Western blotting showed that specific bands appeared at 15 kDa in the PRRSV infected group,but no specific bands were found in the non-infected group.（4）The TCID₅₀0 of the 6th generation cell culture of GZZJ11 was 10^-2.2/0.1 mL,and that of the 3rd generation cell culture of GZBJ12 was 10^-4.67/0.1 mL.In this test,two PRRSV strains were successfully isolated and identified as GZZJ11 and GZBJ12,which laid the foundation for further research on PRRSV related characteristics.2.Whole gene sequence analysis of PRRSV Guizhou isolates GZZJ11,GZBJ12PrimerSelect software was used to design 13 pairs of primers for the whole gene conserved region of PRRSV.The 13 gene fragments of two PRRSV strains were RT-PCR,cloned into pMD-19T vector,sequenced and spliced.The whole gene sequences of two PRRSV strains were obtained（polyA tail was removed）.Lasergene software package and MEGA7.0 were used to compare and analyze the whole gene sequences of two PRRSV strains.（1）The target bands of 13 genes were detected by RT-PCR,and the full-length genomic sequences of GZZJ11 and GZBJ12 strains were 15323 bp and 14963 bp,respectively.Full-gene nucleotide alignment showed that the homology of the whole gene sequence of the two strains was about 60%with genotype 1,over 82%with genotype 2,and between 98.8%and 99.2%with the representative strains of HP-PRRSV,JXA1,HUN4 and TJ.（2）Variation analysis of NSP2,NSP9,ORF3,ORF4 and ORF5 genes in two strains showed that NSP2 gene had different degrees of variation,among which NSP2 gene had the greatest variation.There were 24 and 23 amino acid mutations in the NSP2 gene of GZZJ11and GZBJ12 strains,respectively,with 1+29 amino acid discontinuous deletions.The deletion location was consistent with that of HP-PRRSV.In addition to 30 discontinuous amino acid deletions,120 amino acid deletions were found downstream of the NSP2 gene of GZBJ12 strain,suggesting that GZBJ12 strain may evolve into HP-PRRSV TJ attenuated vaccine strain TJM.（3）The results of virulence site analysis indicated that the two strains were located at position 151 of the virulence site of ORF5,and the mutations at this site were mutated with the mutations of the NADC30-like strains CHsx1401 and FJXS15 isolated from China.Consistently,it is also identical to the virulence sites of the newly isolated TJnh1501,GDHY,and XJu-1 strains.（4）Based on the results of genetic evolutionary tree of nucleotide sequence of NSP2,ORF5 and whole gene,the genetic evolutionary relationship between GZZJ11 and TJnh1501 and HENZZ-8 genotype 2 strains was relatively close,and that between GZBJ12 and GDHY and XJu-1 genotype 2 strains was relatively close,which was basically consistent with the results of homology analysis of nucleotide sequence of whole gene.The test results provide a reference for preliminary understanding of PRRSV infection and strain variation in Guizhou.1.Effect of PRRSV Guizhou isolates GZZJ11 and GZBJ12 on helicases DDX6 and Mov10.The DDX6 and Mov10 gene CDS regions were amplified from Marc145 cells by RT-PCR,and the DDX6 gene CDS region was cloned and bioinformatic analysis.Effects of PRRSV isolated from Guizhou province on helicase DDX6 and Mov10 genes after infection of Marc145 cells at different time periods were detected by q-PCR at the mRNA level.（1）The full length CDS region of DDX6 gene was 1452 bp and encoded 483 amino acids.The DDX6 gene encoding protein contains 4 motifs and containing DEAD-like helicase superfamily domain and C-terminal domain.The main secondary structures areα-helix（37.27%）and irregular crimp（36.44%）.The results of homology and phylogenetic tree analysis showed that Marc145 cells and human DDX6 had the highest homology and the closest phylogenetic relationship.（2）The results of q-PCR showed that the mRNA level of DDX6 gene increased after 6 hours of infection of Marc145 cells by PRRSV,and the difference was significant at 36 h.The transcription of Mov10 gene in the Marc145 cells infected with PRRSV at 0 h,6 h,36 h and 72 h.The level is decreased,and the transcription level of the Mov10 gene is elevated in other periods.This indicates that Guizhou isolate PRRSV has a certain influence on the transcription levels of helicase DDX6 and Mov10.The experiment provides a reference for further investigation of the relationship between the helicases DDX6,Mov10 and PRRSV proliferation.