| Objective: Porcine reproductive and respiratory syndrome virus(PRRSV)and Severe Acute Respiratory Syndrome-Coronavirus-2(SARS-Co V-2)belong to the order Nidovirales and have similar genome structure and mechanisms of infection and replication.The nucleocapsid(N)protein and the nuclear localization signal(NLS)it carried were of great importance for viral transcription and replication.In the first part of this paper,the whole genome of the virus was amplified and sequenced,and molecular epidemiological methods were applied to analyze the genetic recombination and mutation of PRRSV epidemic wild strains and representative strains after multiple transmissions,with the aim of understanding the patterns of genomic recombination and mutation of the viruses in the condominium,which could provide an important basis for the prevention and control of these diseases.Based on the findings of the first part,the second part of this paper investigated the effects of natural mutations in the NLS region of N protein on the nucleoplasmic shuttle of N protein and the transcription and replication of viral genes,aiming to analyze the effects of natural mutations in the NLS region on the nucleoplasmic transport mechanism of these viruses,which might provide new ideas and basis for the screening of antiviral drug targets.Methods: Virus identification and isolation from lesioned tissues of pigs with highly pathogenic PRRS events.The genomes of the epidemic wild strain and the representative strain were amplified by RT-PCR using Primer5.0,and positive clones were obtained by ligation and transformation,and positive plasmids were extracted and sent to biotechnology companies for sequencing,and the whole genome sequence of PRRSV was obtained by splicing.The full sequences of the classical representative strains of PRRSV were downloaded from Gene Bank,and the recombinant and SNP analyses were performed on the whole genome sequences of the epidemic wild strains and the representative strains of the epidemic after multiple passages using RDP4 and SIMPLOT software respectively.The classical PRRSV strain(NLS unmutated),HP-PRRSV vaccine strain(NLS mutated),and HP-PRRSV epidemic representative strain(NLS mutated)were used as the subjects of the study.The virus titer was determined by applying the virus null spot assay,and Marc-145 cells were infected with MOI=0.1.Firstly,Western-blot method was applied to analyze the three strains respectively from protein level The expression of N protein was monitored by Western-blot at the protein level during 48 h of infection(20h,24 h,28h,32 h,36h,40 h,44h,48h),and the differences in N protein expression were compared and analyzed for different strains at the same time and different times of the same strain,respectively.The N protein localization of the three viruses was monitored within 48 h(4h,8h,12 h,16h,20 h,24h,28 h,32h,36 h,40h,44 h,48h),and the differences in N protein localization were compared and analyzed for different strains at the same time and at different times of the same strain.All three strains were infected with Marc-145 cells at MOI=0.1,and the expression of the N protein gene and the N protein entry receptor alpha(Karyopherin alpha,KPNA)were investigated by Real-time PCR within 48 h(4h,8h,12 h,16h,20 h,24h,28 h,32h,36 h,40h,44 h,48h).The expression of N protein gene,N protein entry receptor alpha(KPNA),Karyopherin beta1(KPNB1)and seven receptors of three subtypes of Karyopherin alpha(KPNA)were examined simultaneously,and the differences in the expression of N protein and eight receptors were statistically compared and analyzed at the same time and different times of the same strain,respectively.Results: In this study,a wild strain of PRRSV(SD2020)was isolated from the tissues of clinically affected animals.After RT-PCR amplification,ligation,transformation,sequencing and splicing,we successfully obtained the whole genome sequence of the wild strain(SD2020)and the representative strain(JXA1 PX)after multiple passages.The whole genome sequence of SD2020 was 15,386 bp long(sequence submitted to Gene Bank registration number: MW408254)and JXA1 PX was 15,287 bp long;the homology analysis based on the whole genome sequence showed that SD2020 was 94%homologous to the representative strain of PRRSV type 2 and only 58% homologous to the representative strain of PRRSV type 1,and SD2020 was the strain of PRRSV type 2.The results of recombination and SNP analysis showed that SD2020 was a recombinant virus,whose genome was formed by the recombination of the classical PRRSV strain VR2332 and the highly pathogenic HP-PRRSV strain JXA1-like(which is derived from the evolutionary mutation of JXA1,hereafter referred to as JXA1-like)strain CH-YY with 15SC2;the results of point mutation analysis showed that The results of the point mutation analysis showed that JXA1 PX underwent continuous deletion and mutation of the gene sequence during the transmission process,and that the amino acid site K46 R was mutated in PRRSV compared to HP-PRRSV and was stably inherited in JXA1-like strains,altering the N protein NLS sequence.Compared with JXA1-R N protein expression at 24 hpi and JXA1 N protein expression at 32 hpi,VR2332 N protein expression started at 36 hpi and was significantly lower than that of JXA1-R and JXA1 at the same time,with statistically significant differences.The results of cellular immunofluorescence experiments showed that the nucleoplasmic shuttle of N protein was observed in all strains within 48 h of infection,and the intranuclear localization of N protein was obvious at 36 hpi and 48hpi;compared with JXA1-R and JXA1,VR2332 N protein was expressed later and the amount of nucleoplasmic shuttle was involved,especially in the nucleus,later than JXA1-R and JXA1 in the same period.Real-time PCR results showed that the expression of the N protein gene increased with increasing infection time in all strains,showing a clear temporal effect,which further confirmed the results of Western-blot and cellular immunofluorescence experiments.The results of real-time monitoring of the expression of the eight receptors during the infection of the three strains of viruses showed that the consistent pattern was that the expression of KPNA3,KPNA4 and KPNA5 did not change with the increase of virus infection time and the change of N protein gene expression,compared with the expression levels of KPNB1,KPNA1,KPNA2,KPNA6 and KPNA7 genes The expression levels of KPNA1 and KPNB1 in VR2332 were significantly lower than those of JXA1 and JXA1-R at48 hpi,and the difference was statistically significant.Conclusion: The clinical epidemic strain SD2020 underwent genetic recombination,and the recombinant genes were derived from the classic PRRSV strain(VR2332),HP-PRRSV vaccine strain(CH-YY),and HP-PRRSV wild strain(15SC2).HP-PRRSV epidemic represented a strain with continuous gene sequence deletions and mutations during the transmission process,the natural mutation in the NLS region of the N protein resulted in the enhanced nucleoplasmic shuttling ability of the N protein.The possible mechanism was that the natural mutation of the 46 th amino acid in the NLS region caused the N protein to interact with the affinity of the nuclear transport receptors KPNA1 and KPNB1 increased. |
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