Isolation,Identification And Genetic Evolution Analysis Of Porcine Delta Coronavirus In Shandong Province

Porcine?coronavirus(PDCoV),also known as Porcine D-type coronavirus,is a single-stranded plus RNA virus with capsule,which belongs to Coronavirus in the family Coronaviridae.PDCoV is a contact intestinal infectious disease which can cause vomiting,watery diarrhea and dehydration in swine.In 2012,the first case of PDCoV infection occurred in Hong Kong.And it began to cause a widespread epidemic in Ohio,USA in 2014.In addition,PDCoV is often co-infected with porcine epidemic diarrhea(PEDV),porcine transmissible gastroenteritis(TGEV)and other swine diarrhea viruses,which brought great challenges to the prevention and treatment of this disease.In recent years,PDCoV has been widely spread in all parts of China,seriously hindering the development of swine industry.but there is no commercial vaccine and special drugs for the disease,and strengthening disease prevention is still an important means to control the epidemic in China.Therefore,the isolation of PDCoV and the analysis of its genomic genetic evolutionary information are helpful for epidemiological investigation,vaccine development and prevention and control.In this study,RT-PCR was used to detect the pathogens of porcine diarrhea disease suspected to be infected with PEDV,TGEV and PDCoV from 1043 clinically diagnosed cases in Taian,Jinan,Jining,Linyi and Zaozhuang from 2019 to 2021.The results showed that the detection rates of PDCoV,PEDV and mixed infection were 12.28%(128/1043),42.1%(439/1043)and 4.21%(44/1043),respectively,in a total of 1043 diarrhea samples from some parts of Shandong province,while TGEV and other diarrhea diseases were not detected.From the detection rate of the pathogen causing pig diarrhea in some areas of Shandong province,PEDV is the main pathogen causing pig viral diarrhea in some areas of Shandong Province,followed by PDCoV.In order to observe the clinical lesions caused by PDCoV,clinical autopsy showed diarrhea emasculation,undigested milk clot in stomach,hyperemic ulcer in gastric wall,hyperemic and bleeding in intestinal tract,hyperemia and enlargement of mesenteric lymph nodes,etc.Histopathological observation showed necrosis and shedding of intestinal villi,severe hyperemia and bleeding of mucosal lamina propria,hyperemia and bleeding of blood vessels in muscle layer and serosal surface,necrosis and shedding of gastric epithelial cells,shortening of villi and other pathological changes.Two porcine?coronaviruses strains were isolated from suspected porcine?coronaviruses collected from swine farms in Taian,Shandong province by cell inoculation and virus isolation,and named as PDCoV SDTA01-2021 and SDTA02-2021.The results showed that TCID₅₀of SDTA01-2021 and SDTA02-2021 strains were 10^-5.19/0.1mL and 10^-5.61/0.1mL respectively.The growth curve of the isolates was plotted.It was found that the titer of the two isolates increased after 8h of infection,and reached the highest titer(2.57×10¹²/mL and2.82×10¹²/ml)after 24 h of infection.Isolates S,E,M and N were cloned and sequenced,and bioinformatics analysis was performed on homology,genetic evolution and sequence alignment with reference strains at home and abroad.The homology of S gene was 97.2%to99.7%.Genetic evolutionary analysis showed that the isolates in this experiment were located in a different branch from foreign strains in the United States and Southeast Asia recorded in Gen Bank,and in the same branch as the Chinese epidemic strains.In particular,it was closest to CH-SDLY52-2021 and CHN-SC2015 strains in China,indicating that two Chinese epidemic strains of PDCoV were successfully isolated from clinical cases in this study.After sequence alignment analysis,it was found that the isolates in this study had the same characteristic deletion mutation point as the PDCoV epidemic strains,that is,there was an aspartic acid(N)deletion in the 52nd position of the S amino acid sequence.In addition.The deletion of aspartic acid(N)at 52 was typical of the domestic epidemic strains.No deletion of amino acid(N)at 52 was found in the S amino acid sequence of the foreign PDCoV strains isolated from the United States and Southeast Asia,and the same mutation sites were not found in E,M and N genes as those of the domestic epidemic strains.In addition,there are other unique mutation loci in the main genes of this isolated strain.There were 49 potential B cell epitopes in S protein,most of which were in S1(1?522aa)region of the protein.This study aims to understand the current prevalence of PDCoV among swines in Shandong Province and clarify the epidemic characteristics of the disease.In addition,we intend to analyze the molecular characteristics,genetic variation and bioinformatics of the structural genes in PDCoV isolates.The above researches will lay an important foundation for vaccine preparation and disease prevention,having important clinical significance and potential application value.