Epidemiological Investigation And Viral Antigen Gene Genetic Evolution Analysis Of PEDV,PDCoV,and PKV In Pig Farms In Shandong Province

Porcine epidemic diarrhea virus(PEDV)is the causative agent of porcine epidemic diarrhea(PED),which has a significant impact on the morbidity and mortality of pigs,especially for newborn piglets,with a very high mortality rate;Porcine deltacoronavirus(PDCoV)is a porcine intestinal coronavirus,which can cause diarrhea and dehydration in piglets,with clinical manifestations similar to PEDV,affecting the survival rate of newborn piglets and bringing greater economic losses to farms;PEDV and PDCoV infections in adult pigs are often asymptomatic or mild,and can be cured without treatment.However,adult pigs can continue to carry poisonous detoxification during the infection period,and pigs are also transferred across regions and traded for transportation,which makes its harmful effects more insidious and PEDV and PDCoV infections in pig farms more difficult to control and remove.Porcine kobuvirus(PKV)is a newly discovered viral pathogen in recent years,which was first found in diarrhea feces,and there is no report of successful isolation of PKV up to now,but PKV has a high detection rate in pigs with diarrhea symptoms,and there is also mixed infection with PEDV,so it is presumed that PKV is associated with pig diarrhea disease,and whether it is pathogenic to the host.Pathogenicity also needs further study.In order to gain a comprehensive understanding of the prevalence and viral genetic variation of PEDV,PDCoV and PKV in pig farms in Shandong Province,and to provide theoretical basis and technical support for the prevention and control of diarrhoeal diseases in pig farms,we carried out the following works:Firstly,from September 2020 to July 2021,we collected a total of 1268 adult pig samples from nine major pig breeding areas in Shandong Province(Jinan,Dezhou,Weifang,Binzhou,Jining,Heze,Liaocheng,Dongying and Zibo)that were outwardly healthy(without typical digestive/respiratory clinical signs);After sample processing and total RNA extraction,RT-PCR was performed for the three pathogens using detection primers;Subsequently,we then amplified the full-length sequences of the antigen genes of the identified PEDV,PDCoV and PKV positive samples using primers;Finally,we performed nucleotide and amino acid(aa)sequences of the S gene of four PEDV strains,the S gene of one PDCoV strain and the VP1 gene of two PKV strains;Respectively,using the sequences of the strains published in Gen Bank as a reference,homology analysis and genetic evolution analysis,we also performed N-glycosylation site prediction for PEDV,PDCoV S protein and PKV VP1 protein.In addition,we used viral macrogenomic technology to perform species abundance analysis and whole genome sequencing on 3A2 and 6C3 lung tissue samples,respectively,to provide a preliminary discussion on the application of macrogenomic technology in clinical testing in swine farms.The results found that among the 1268 samples collected from Shandong Province,the detection rate of PEDV was 4.42%(56/1268);the detection rate of PDCoV was 0.47%(6/1268);the detection rate of PKV was 1.18%(15/1268);among them,the positive samples for PEDV were from Weifang,Dezhou and Jinan farms and slaughterhouses,the positive PDCoV samples were from Weifang farms and the positive PKV samples were from Dezhou and Jinan farms and slaughterhouses.The overall positive rate for PEDV mixed with PDCoV(from Weifang)was 0.24%(3/1268);The overall positive rate for PEDV mixed with PKV(from Dezhou)was0.07%(1/1268);No mixed PDCoV and PKV samples were detected,no triple mixed PEDV,PDCoV and PKV samples were detected.The above test results indicate that even healthy looking pigs may carry one or more viruses and there is a risk of viral detoxification,among the three pathogens,PEDV is the most prevalent in healthy looking adult pigs in our province and there are mixed infections with other pathogens.Based on the phylogenetic analysis of the S gene of the PEDV antigen,we found that the prevalent PEDV strains CHN-SDC65-2020,CHN-SDG12-2021 and CHN-SDG38-2021 in Shandong belong to subgroup GII-a,CHN-SDG58-2021 belongs to the S-INDEL group.Compared with the Chinese vaccine strain CV777,the four PEDV endemic strains showed amino acid mutations aa522 A→S,aa554 T→S,aa599 G→S,aa638 Q→E,aa599 G→S and aa638 G→S in the COE gene and at several amino acid sites in the epitope region.aa638Q→E,aa769L→S and aa771D→S in the SS6 region;22-23 N-glycosylation sites were predicted for the S protein in the four PEDV endemic strains.The test results showed that the prevalent strains CHN-SDC65-2020,CHN-SDG12-2021 and CHN-SDG38-2021 were more closely related to the PEDV type GII variant strains isolated at home and abroad in recent years,while the prevalent strain CHN-SDG58-2021 was genetically distinct and more closely related to the strains of the S-INDEL branch represented by the USA/OH851 strain.The four prevalent PEDV strains have low homology with the Chinese vaccine strain CV777 and have significant differences in potential N-glycosylation sites;The four prevalent PEDV strains have substitutions,insertions and deletions in the S-protein amino acid sites,the amino acid mutation sites are mainly concentrated in the S1 region.The epidemic PDCoV strain CHN-SDG25-2021 from Shandong identified in this study belongs to the Chinese branch in terms of genetic evolution,but up to 21 amino acid mutations were detected in its S protein compared to the first Chinese PDCoV strain,HKU15-44,from Hong Kong;The nucleotide/amino acid sequence homology of CHN-SDG25-2021 with the S gene of the Chinese reference strain was 96.8%-98.3% and 96.6%-99.1%,respectively,while the nucleotide/amino acid homology with the foreign reference strain was 95.9%-97.9% and96.7%-98.4%,respectively;Compared with the US/Japan/Korea and Laos/Vietnam/Thailand strains,there were two unique amino acid substitutions on the CHN-SDG25-2021 S protein,N→I at aa510 and K→E at aa534,and its S protein was predicted to have 18 N-glycosylation sites.The test results showed that the prevalent strain CHN-SDG25-2021 shows clear regional characteristics,with CHN-SDG25-2021 being more closely related to other Chinese PDCoV strains than to strains from Southeast Asia,the United States,Japan and Korea,and genetically evolutionarily similar to the Chinese strain.In addition,amino acid differential analysis of the PDCoV S protein showed that the mutation sites in the prevalent strain CHN-SDG25-2021 were mainly concentrated in the S1 region.PKV has three antigenic genes,VP0,VP1 and VP3,and previous studies have reported that the VP1 gene is the most mutable of the three antigenic genes.Phylogenetic analysis based on the PKV VP1 gene revealed that PKV can be divided into four branches: Chinese group I,Chinese group II,Thai-Japanese group and Hungarian group,and the prevalent strains of CHN-SDC71-2020 and CHN-SDG14-2020 identified in this study were in the Chinese group II branch;The nucleotide homology of CHN-SDC71-2020 and CHN-SDG14-2020 ranged from81.4% to 85.8% and amino acid homology from 87% to 92.9% with the Chinese reference strain VP1 gene,while the nucleotide homology ranged from 80.4% to 84.8% and amino acid homology from 85% to 90.6% with the Thai/Japanese/Hungarian reference strain;In addition,compared with the Thai,Japanese and Hungarian reference strains and previous PKV strains isolated in China,the CHN-SDC71-2020 VP1 protein had five amino acid mutations and the CHN-SDG14-2020 VP1 protein had nine amino acid mutations;One N-glycosylation site was predicted for the VP1 protein of both PKV endemic strains.The results showed that the PKV strains CHN-SDC71-2020 and CHN-SDG14-2020 found in this study were closely related and both belonged to the Chinese group II branch in terms of genetic evolution,but their nucleotide and amino acid sequences differed significantly from those of other strains in the Chinese group II branch,suggesting that PKV is undergoing rapid antigenic mutation.Through macro-genome sequencing and data analysis of swine lung tissue sample 3A2,we identified 12 RNA virus families(including Orthomyxoviridae,Herpesviridae,Adenoviridae,etc.)and 19 RNA viruses(including influenza A virus,human adenovirus type C,hepacivirus C,among others)in sample 3A2;Through macro-genome sequencing and data analysis,we obtained the sequences of eight gene fragments(including PB1,PB2,HA,NA,etc.)of an influenza A virus strain(named A/swine/shandong/6C3/2019)from sample 6C3,and the phylogenetic evolutionary tree of HA and NA genes showed that it belonged to the avian-like H1N1 subtype of swine influenza virus.The above test results indicate that there is a mixture of viruses infecting the lung tissue of pigs;The presence of human adenovirus indicates that pigs are potential hosts for zoonotic pathogens;In addition,the preliminary application of macro-genomic technology in this study indicates that macro-genomic technology can help to identify viruses present in pig farms and provide an important reference for pig farm disease diagnosis.In summary,the results of this study indicate that PEDV,PDCoV and PKV are also prevalent in healthy pig herds on the exterior of pig farms in Shandong Province,and that mixed infections exist;Phylogenetic analysis indicates that most of the PEDV,PDCoV and PKV strains prevalent in Shandong Province are derived from genetic mutations of Chinese strains,while a few PEDV strains prevalent in Shandong Province may have been imported from abroad;The results of this study revealed that,compared with the prevalent strains in other provinces in China,the PEDV,PDCoV and PKV prevalent strains in Shandong Province have different degrees of amino acid mutations,deletions and insertions in their antigenic genes,indicating that the PEDV,PDCoV and PKV strains in Shandong Province exhibit evolutionary diversity and uniqueness;Differences in the potential sites of N-glycosylation of endemic strains may have an impact on the antigenicity of the virus;This study also shows that macro-genomic techniques can be used in the diagnosis of clinical samples from pig farms,contributing to a comprehensive understanding of the mixed infections of pathogenic microorganisms present in pig farms.This study elucidated the prevalence of PEDV,PDCoV and PKV in adult outwardly healthy pig herds in Shandong Province,analysed the genetic evolution of antigenic genes of PEDV,PDCoV and PKV prevalent strains in Shandong Province,applied macrogenomic techniques to reveal the mixed infection of viruses in pig lung and obtained the whole genome sequences of virulent strains,providing epidemiological reference and theoretical basis for the prevention and control of pig diarrhoea in Shandong Province.It suggests that the surveillance of healthy pigs on the exterior of pig farms should be strengthened,and the development of multiple polyvalent vaccines to interrupt the mixed infection of multiple viruses and reduce the risk of epidemic.