| Porcine deltacoronavirus(PDCoV)is a new member of the Coronavirus family.PDCoV can cause clinical symptoms such as diarrhea,vomiting,and dehydration in sows and suckling piglets.The mortality rate of suckling piglets can reach 40%,which is one of the serious epidemics that harm the pig industry.In order to investigate the prevalence of PDCoV in Sichuan province,a total of 634 clinical diarrhea samples collected from pig farms in various regions of Sichuan during 2017 to 2019 were tested by RT-PCR and4 PDCoVs were amplified and analyzed.The detection results showed that the positive rate of PDCoV was relatively low in diarrheal pigs and was 13.25%(84/634),but the positive rate of PEDV was high up to 32.18%(204/634).Besides co-infection of PDCoV with PEDV were common in pigs and the positive rate was 7.14%(47/634).Additionally,the chance of PDCoV infection was 2.77 times higher in sucking piglets than sows,and about 3.30 times higher in Spring and Winter than in Summer.The whole gene analysis showed that the homology rate between the four strains of PDCoV was 98.9%-99.3%,and the homology rate with other PDCoV strains was 97.6%-99.0%.There were some insertion-deletion signatures in whole genome sequences of four strains,including a 6-nt deletion in non-structural gene 2 region,a 9-nt insertion in non-structural gene 3 region,a3-nt deletion in S gene region,and a distinguishing 11-nt deletion in the 3'UTR region.Genetic phylogenetic tree analysis showed that the PDCoV strain showed obvious regional characteristics.Four PDCoVs were in the same large branch as other China PDCoVs,but each were in two different small branches.Besides,analysis of the genetic phylogenetic tree of the S gene revealed that one of the sequenced PDCoV formed a large branch with PDCoVs from other countries,indicating the genetic diversity of PDCoV strains in Sichuan.These data advance our understanding of the genetic diversity and evolutionary characteristics of PDCoV in China,and provided a reference for subsequent research on PDCoV virology.In addition,for understanding the prevalence of PDCoV in Sichuan,this study established a reliable indirect ELISA method for detecting PDCoV IgG antibody.The prokaryotic expression system was used to express PDCoV membrane protein(M)as a detection antigen.By optimizing the reaction conditions,an indirect ELISA method named PDCoV-r M-ELISA based on PDCoV M protein was established,and 430 clinical samples were detected by this method.The recombinant expression E.coli strain BL21-pET-32a-M was successfully constructed,and the recombinant M protein was expressed after induction.Western blot assay showed that the recombinant protein has good reactivity.PDCoV-r M-ELISA method could specifically detect PDCoV antibody,no cross reaction with five common pathogen positive sera,suggesting good specificity;The sensitivity of this method was high.PDCoV-r M-ELISA was high reproducible with the coefficients of variation were less than 10%;48 clinic serums were detected using both PDCoV-r M-ELISA and PDCoV antibody detection kits.The results showed that the positive agreement rate was 88.46%,the negative agreement rate was 90.91%,and overall coincidence rate was 89.58%.The results of clinical samples detection showed that the average positive rate of PDCoV antibody was 11.86%,and the results showed that antibodies can also be detected in serum of healthy pigs.Overall an indirect ELISA was established by using the recombinant M protein of PDCoV express in E.coli BL21.PDCoV-r M-ELISA method had good specificity,reproducibility and stability,and could be used for the detection of PDCoV antibody in clinic swine serum samples. |
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