Isolation,Identification,Complete Sequence Analysis And Establishment Of A TaqMan Fluorescence Quantitative PCR For Detection Of Porcine Sapelovirus

Porcine sapelovirus(PSV)can cause multi-system diseases such as enteritis,pneumonia,polio,and reproductive disorders in pigs,and can cause persistent infections in pigs and continuous infection in recovered pigs.PSV can infect pigs of all ages,and is easy to interact with classical swine fever virus(CSFV),porcine reproductive and respiratory syndrome virus(PRRSV),and porcine parvovirus(porcine parvovirus,PPV)and porcine teschovirus(PTV),porcine epidemic diarrhea virus(PEDV),coronavirus(CoV)and porcine circovirus type 2(PCV2)Wait for mixed infections,which increases the difficulty of diagnosis and comprehensive prevention and control,resulting in huge economic losses to pig industry.The current reports on PSV mainly focus on epidemiology,and there are few studies on the rapid detection methods and infection mechanism of the virus.In this experiment,a PSV strain was successfully isolated from the feces of piglets with diarrhea,named PSV HNHB-01;The virus was serially passaged on cells,and full genome sequence determination and genetic evolution analysis of PSV HNHB-01 fifth generation cytotoxicity(P5),30 generation cytotoxicity(P30),60 generation cytotoxicity(P60)and 100 generation cytotoxicity(P100);In addition,a TaqMan real-time fluorescent quantitative PCR detection method was established for the 5'untranslated region(5' UTR)of PSV.The specific test contents are as follows:1.Isolation and identification of PSV.In this study,the positive disease material of PSV was sterilized and inoculated into LLC-PK1 and ST cells,then exogenous trypsin(Trypsin and Pancreatin)was added to promote the viral infection,and the cytopathic condition was continuously observed.After ST cells were inoculated with positive disease materials,the cells became round,clustered,and cytoplasmic granules increased.Then,the virus was collected for PCR identification,and the result was PSV positive;The purified PSV was observed by electron microscope,and spherical particles with diameter of about 25 nm were observed;Using the pig serum prepared in our laboratory,the indirect immunofluorescence test was carried out,and the green fluorescence was observed in the pores of the virus inoculated cells.The results showed that a PSV strain named HNHB-01 was successfully isolated.Then,the virus was purified by plaque in ST cells to obtain a single clone of PSV for subsequent virus passage;TCID50 and qPCR were used to determine the growth curve of PSV on ST cells.The results showed that the viral titer was the highest 24 h after PSV infected ST cells.2.Cell passage,sequencing and genetic evolution of PSV HNHB-01 strain.In order to understand the genetic variation of PSV HNHB-01 virus during cell passage.The virus titers of the P5,P30,P60 and P100 were measured,indicating that the virus titers increased with the increase of passages.After that,the whole genome of PSV HNHB-01 strain was sequenced and the similarity difference was analyzed.A total of 8 amino acid mutations occurred in HNHB-01P5-P100,one of which was in the VP1 gene.The amino acid similarity of HNHB-01P5-P100 is 99.9-100%.This isolate has the highest amino acid similarity with Asian strains,88-90.4%,and the lowest amino acid similarity with American,Indian and European strains,84.5-85.1%;The whole gene sequence of PSV HNHB-01 strain and the genetic evolution analysis of domestic and foreign strains showed that all PSV strains can be divided into two groups,GI and GII.The strains isolated in this experiment are in the same group as the Asian strains.,The genetic relationship is the closest,and the relationship with the European strain and the Indian strain is relatively distant.Sequence analysis of the VP1 gene of PSV HNHB-01 strain showed that only one nucleotide mutation occurred in the VP1 gene of different generations of PSV HNHB-01,causing the amino acid at this position to change from S(serine)to G(glycine),HNHB-01 P5 The amino acid similarity of the VP1 gene of the,30,60 and 100 generations of viruses is 99.9-100%,and the amino acid similarity of other strains in China is 81.2%;The antigenicity of PSV HNHB-01 P5 and P100 VP1 was analyzed by protein software.From PSV P5 to P100,there was only one amino acid mutation in VP1 gene,but the antigenicity was not changed,that is,the VP1 protein of PSV did not change during virus passage.3.Establishment of PSV TaqMan Real-time Fluorescence Quantitative PCR Detection Method.In order to establish the PSV TaqMan real-time fluorescent quantitative PCR detection method,a pair of specific primers were designed with reference to the 5'conserved region of PSV USA-IA-33375-2015 strain(accession number:KX574284),and the target fragment was amplified by PCR.It was cloned into pMD-18T vector to obtain a positive recombinant plasmid.At the same time,design PSV-specific primers and probes,use the constructed positive plasmid as a template to establish a PSV TaqMan fluorescence quantitative PCR detection method,and optimize its amplification system and amplification conditions,and the sensitivity of the method,Specificity and repeatability are verified.The established PSV TaqMan fluorescent quantitative PCR detection method was used to detect 83 samples(44 intestinal samples and 39 fecal samples)collected from pig farms in different regions of Henan from 2018 to 2019.The results show that:The test successfully established a PSV TaqMan fluorescent quantitative PCR detection method,with a detection sensitivity of 3.88×101 copies/?L;No cross-reactivity with porcine epidemic diarrhea virus,porcine transmissible gastroenteritis virus,porcine delta coronavirus and PRRSV,showing good specificity;The coefficient of variation(CV)between batches and batches is less than 1.0%,which has good repeatability.The test results of clinical samples showed that 31 PSV-positive samples(including 22 stool samples and 9 intestinal samples)were detected by this method,and the positive detection rate was 37.3%(31/83),while the normal PCR test for PSV was positive For 10 samples,the detection rate was 12.0%(10/83).The results show that we have successfully established a PSV TaqMan fluorescence quantitative PCR detection method,which has the characteristics of high sensitivity and good specificity,and provides a new method for the rapid clinical detection of PSV.In summary,this experiment successfully isolated a PSV strain and named it HNHB-01.The whole genome of HNHB-01P5,P30,P60 and P100 and the VP1 gene were analyzed for genetic evolution,and the PSV TaqMan real-time was established.Fluorescence quantitative PCR detection method.It provides a data reference for the further study of PSV strains,and at the same time provides an important theoretical basis for the prevention and treatment of PSV.