Establishment Of Taqman Fluorescence Quantitative PCR Method For Porcine Astrovirus Type Ⅰ And Preliminary Study On Protein Components Of NsP1a-1,nsP1a-3 And NsP1a-4

Astrovirus（Ast V）is a kind of single stranded plus RNA virus.Porcine Astrovirus and human Astrovirus have high homology and gene recombination,which has the ability of gene recombination,and is a potential zoonosis pathogen.Porcine astrovirus diarrhea mainly occurs in piglets and immunodeficient individuals.Porcine astrovirus non-structural proteins are cleaved from two polyproteins,nsP1a and nsP1ab.Currently,the function of each protein component of nsP1a is not clear,and this study will preliminarily explore each protein component of nsP1a.It will lay the experimental foundation for the subsequent study on the corresponding escape mechanism of porcine astrovirus to innate immunity.A pair of specific primers and a Taqman probe were designed based on the gene sequence of PAst V1 in Gen Bank to establish a Taqman quantitative PCR method for porcine astrovirus type I.The results showed that the sensitivity limit of this method was 3.89×10¹copies/μL,100 times higher than that of conventional PCR.The coefficient of variation of intra-group and inter-group repeatability experiments was less than 2%,and there was no cross-reaction with other porcine enterovirus.The detection rate of 30 random clinical samples was70%,higher than that of conventional PCR（56.7%）.A total of 170 fecal samples were collected from 6 pig farms in Guangxi in 2021.The overall positive rate of PORcine astrovirus TYPE I was 44.1%,and the positive rate of piglet population was 57.7%.Bioinformatics software was used to analyze the proteolytic site of porcine astrovirus nsP1a,establish the tertiary structure model of each protein component,and predict the function of each protein component.The prediction showed that the hydrolytic sites of nsP1a were A171/Q172,A408/A409,and Q565/T566.According to the tertiary structure model,it was found that 97—134AA of nsP1a/1 had leucine zipper structure,and it was speculated that nsP1a/1 was the DNA binding protein of the virus,which played a related role in the replication and transcription of viral genes.216—237AA of nsP1a/2 was similar to the structure of FLGD cap hook,suggesting that nsP1a/2 was the membrane protein of the virus.The structure of 21—157AA of nsP1a/3 was consistent with that of human astrovirus serine protease,indicating that nsP1a/3was a viral serine protease.The 26—77AA of nsP1a/4 is similar to the c-terminal coiled helix domain of CIN85 protein,which is a signal connector protein.It is speculated that nsP1a/4 has the ability to mediate signal pathway.The prokaryotic expression recombinant plasmids nsP1a/1-p ET-32a,nsP1a/3-pCold-I and nsP1a/4-p ET-32a were successfully constructed and transformed into escherichia coli BL21（DE3）competent cells.The recombinant proteins nsP1a/1,nsP1a/3 and nsP1a/4 were induced by IPTG.All of them were identified as inclusion bodies by SDS-PAGE.The purified recombinant protein was immunized with healthy New Zealand white rabbits,and polyclonal antibodies against nsP1a/1,nsP1a/3 and nsP1a/4 were successfully prepared.The polyclonal antibodies were identified by WB with specific recognition ability.The eukaryotic expression recombinant plasmids pCAGGS-MCS-nsP1a/1,pCAGGS-MCS-nsP1a/3 and pCAGGS-MCS-nsP1a/4 were successfully constructed and transfected into 293T cells by liposome,respectively.The results showed that the recombinant proteins nsP1a/1,nsP1a/3 and nsP1a/4could be expressed in 293T cells.The m RNA levels of IL-6 and TNF-αwere detected by q PCR at different time points and compared with the no-load control group.The experimental results showed that TNF-αin nsP1a/1 group was gradually upregulated at 6—36h（0.2—3.3 times of the no-load control group）,but the multiple was low.Il-6 at each time point was similar to that of the no-load control group,and the change trend of TNF-αand IL-6 m RNA was not consistent with that of PK-15 after PAst V1 infection,indicating that nsP1a/1overexpression did not cause significant changes in inflammatory factors,and it was speculated that nsP1a/1 was not involved in the inflammatory response caused by PAst V1 infection.TNF-αm RNA levels in nsP1a/3 and nsP1a/4 groups increased with time,peaked at 24h,and then decreased at 36h,which was consistent with the change trend of TNF-αm RNA in PK-15 cells infected with PAst V1.Il-6 was inhibited at 24h in the nsP1a/3 group and at 6—36h in the nsP1a/4 group,in contrast to the up-regulation of il-6 promoted by PAst V1infection in pk-15 cells.These results indicate that nsP1a/3 and nsP1a/4 are involved in inflammatory response,and it is speculated that the overexpression of nsP1a/3 and nsP1a/4 affects NF-κB signaling pathway and interferes with the transcription of il-6 pro-inflammatory cytokines.pCAGGS-MCS no-load,pCAGGS-MCS-nsP1a/1,pCAGGS-MCS-nsP1a/3,pCAGGS-MCS-nsP1a/4 were transfected into PK-15 cells,and PAst V1 was infected 24h later（0.01MOI）.Taqman fluorescent quantitative PCR was used to detect the virus RNA copy number at 12h and 24h after infection and compared with the no-load control group.The results showed that the rate of virus replication in nsP1a/1 cells was 29.8%after 12—24 hours,which was higher than that in nsP1a/3 group（18.8%）,nsP1a/4 group（13.1%）and no-load control group（20.8%）.The viral load was 3.89×10^4.72copies/μL at 12h in nsP1a/4overexpressing group.Significantly higher than that in nsP1a/1 group(3.89×10^2.83copies/μL),nsP1a/3 group(3.89×10^3.28copies/μL)and no-load control group(3.89×10^2.83copies/μL).The results showed that nsP1a/1 promoted viral replication,and the prediction of nsP1a/1 as viral DNA protein in experiment 2 was preliminatively verified.The overexpression of nsP1a/4 is beneficial to virus infection and can interfere and inhibit the transcription of IL-6,and has the potential to mediate signaling pathway in protein structure.It was preliminarily screened that nsP1a/4 plays a relevant role in the innate immune escape mechanism of PAstV1.