| Current research shows that porcine epidemic diarrhea virus(PEDV),porcine transmissible gastroenteritis virus(TGEV),and porcine δ coronavirus(PDCo V)are common pathogens that cause viral diarrhea in pigs.They all allow pigs of different growth stages.Acute watery diarrhoea and vomiting occurred in the group.Dehydration resulted in severe death,especially in suckling piglets.Since these three pathogens are indistinguishable in clinical symptoms,it is particularly important to detect pathogens in suspected porcine viral diarrhea cases.To investigate the prevalence of viral diarrhea in pigs in Henan Province,the study investigated the infection of diarrhea coronaviruses(PDCo V,TGEV,and PEDV)in pigs in some areas of Henan Province,and investigated 25 of them for PEDV-positive disease.ORF3,N,and M genes were amplified and sequenced.Eighteen PEDVpositive S genes were also amplified and sequenced,and were sequenced with classical strain CV777 and the domestic and foreign epidemic strains.Genetic evolution analysis.1.Epidemiological investigation of diarrhea coronavirus in pigs in parts of Henan ProvinceRT-PCR was performed on 255 diarrheal disease samples collected from pig farms in some areas of Henan Province from 2017 to 2018 to analyze the prevalence of PDCo V,PEDV and TGEV in Henan herds.The results showed that among 255 diarrheal diseases,115 were positive for PEDV,accounting for 45.10%,68 were positive for TGEV,26.67% were positive,47 were positive for PDCo V,and 18.43% were positive.The prevalence of TGEV,PEDV,and new PDCo V is present in most pig farms in Henan Province.Moreover,there were different mixed infections among the three diarrhea coronaviruses.Among them,the combined infection rate of PEDV and PDCo V was 14.12%,the mixed infection rate of PEDV and TGEV was 5.49%,and the mixed infection rate of PDCo V and TGEV was 2.35%.The positive rate of three pathogenic mixed infections of PEDV and PDCo V and TGEV was 1.96%.The positive rates of coronavirus infection were also different in different regions where diarrheal diseases were collected.Among them,the positive rates of diarrhoea coronavirus infection in diarrheal diseases submitted to Anyang,Hebi,Xinxiang,Luohe,Sanmenxia and Xinyang regions were up to 100%.In the category of infected coronaviruses,only PEDV infection was detected in Hebi,Luoyang,Sanmenxia,and Zhengzhou.Two to three kinds of coronavirus infections were found in other regions.Overall,PEDV infection in Henan was more serious.And there are relatively many scattered areas.The positive rates of coronavirus at different time points were also different.The highest detection rate was in January.PEDV was as high as 96.43% in January.The PDCo V detection rate was as high as 82.14% in January,and PDCo V,TGEV and No PEDV was detected.In addition,the coronaviruses have different infections in different stages of pigs.PDCo V mainly infects suckling piglets,the positive rate is as high as 48.19%,followed by nursery pigs 7.14%,but the sow infection rate is only 2.59%;The positive rates of PEDV in suckling piglets and nursery pigs and sows were 75.90%,35.71% and 27.59%,respectively,indicating that PDCo V and PEDV are the most serious infections in suckling piglets.The analysis of the infection rates of immunized and non-immune pig farms found that the positive rate of infection of PDCo V,PEDV and TGEV in unvaccinated pig farms was high,being 20.88%,54.40% and 34.07%,respectively.However,the positive rates of PEDV and TGEV in infected pig farms also reached 21.92% and 8.22%,respectively.This result provides epidemiological information for the prevention and control of swine diarrhea coronavirus infection in Henan Province.2.Phylogenetic analysis of PEDV S geneAccording to the survey results of swine diarrhea coronavirus infection in Henan Province,select PEDV-positive materials from representative regions,different times,and different farms to amplify the full length of S gene,and then analyze the genetic evolution of S gene after sequencing.A total of 18 S gene sequences were detected in this study.The fragment size was approximately 4150 bp.The nucleotide and deduced amino acid homology between the gene sequences was 95.3 to 100% and 95.2 to 100%,respectively.The nucleotide and amino acid homology between the strain CV777 was 93.8-95.8% and 93.2-96.3%.The results of phylogenetic analysis of S gene sequences showed that most of the strains in this study were in the same branch as the domestic epidemic strains in previous years,two Korean strains,and three American strains.CH-hb-13/2017,CH-HBS-21/2017,CH-Jiyuan2/2017,CH-PDSYX-G37/2018,CH-PDSLS-3L/2018,CH-zmdG9/2017 and three Japanese strains(MYZ-1/JPN /2013,KCH-2-JPN-2014,and OKY-1-JPN-2014)belong to one branch,indicating that the six strains in this study are closely related to Japanese strains.3.Genetic and evolutionary analysis of PEDV ORF3,N,and M genesIn addition,phylogenetic analysis of ORF3,N,and M genes was performed on 25 PEDV strains of Henan.The ORF3 gene sequence of the PEDV strain was found to consist of 675 nucleotides,all of which were virulent strains;the nucleotide and deduced amino acid homology with the classical strain CV777 was 95.2% to 97.5% and 95.1%,respectively.~96.9%.The deduced amino acid sequence compared with CV777 showed that there were three point mutations(CH-ZKG14/2017,CH-XYBSQ-G30/2018,CH-XYBSQO-G29/2018)at point 80.That is,the 80 th bit changes from V to F,there are 9 in the 182th(CH-ZMDO-G9/2017,CH-ZMDO-G10/2017,CHPDS25LSO-2L/2018,CH-PDSYXO-G34/2018,CH-PDSYXO-G37/2018,CH-XYBSQ-G30/2018,CH-XYBSQO-G29/2018,CH-ZK-G14/2017,CH-LH-1/2017)mutations occur,that is,from Q to H;The phylogenetic tree of the nucleotide sequence of the ORF3 gene showed that most of the strain strains in this study belonged to the same strain as the strains in other regions of China,the strains of Henan in previous years,the American strain,the Korean strain,and the Japanese strain..The nucleotide and deduced amino acid homology between the N gene of the PEDV strain in the study was 96.2 to 100% and 93.8 to 99.8%,respectively;the homology to the nucleotide and deduced amino acid of the classical strain CV777 was 94.7 respectively.~95.8%,93.2~96.8%.Phylogenetic analysis revealed that most of the strains in this study were closely related to the strains CH HNAY 2015,CH HNLH 2015,and CH HNZZ 47-2016 in previous years in Henan.The nucleotide and amino acid sequence identities of the 25 strains of M strains in Henan were 97.1 to 100% and 95.6 to 99.6%,respectively.The nucleotide and deduced amino acid homologies between the strains and the classical strain CV777 were 97.7~ 98.2% and 95.6 to 98.7%.The deduced amino acid alignment results showed that there were 4 amino acid changes,and there were 6 at position 17(CH-AY-1/2017,CH-HB-13/2017,CH-JY2/2017,CH-JY3/ 2017,CH-NY-1,CH-NY-7)E mutation to Q;at position 46,except CH-ZKFG-374,CH-ZKFG-377,CH-ZKLYM-370 and CHZKLY-373 In addition,V-A occurred in 21 other strains;at position 196,G-S occurred at CH-XYBsq-G29 and CH-XYBSQ-G30;and at position 218(except CH-XYBsq-G29 and CH-XYBSQG30)A-S.The genetic phylogenetic tree showed that the PEDV strain in Henan Province was not in the same branch as the classical strain CV777,indicating that the reason that the outbreak of porcine epidemic diarrhea and immunization vaccine in pig farms is still difficult to control may be related to the variation of most PEDV Henan epidemic strains. |
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