The Infection Investigation Of Piglet Diarrhea Coronavirus In Parts Of Henan And Pathogenicity Of Porcine Deltacoronavirus In Mice

Coronaviruses is a large family which widely exists in the nature.The infection spectrum is very extensive,including human and many kinds of animals.Coronavirus occurred easily gene recombination in the process of evolution,because of its hosts universality and genome structural characteristics.Therefore a new subtype and new coronavirus appear constantly.The main piglet diarrhea coronavirus include porcine transmissible gastroenteritis virus(TGEV),porcine epidemic diarrhea virus(PEDV)and porcine deltacoronavirus.In 2010,the outbreak variant PEDV cause the rate of piglets reached 100%.Porcine deltacoronavirus(PDCoV)is a newly discovered porcine viral diarrhea virus,the clinical symptoms of piglets is similar to the piglets infected with TGEV and PEDV,can cause severe diarrhea,vomiting and other symptoms.The research investigated the infection of piglet diarrhea coronavirus,the result showed that PEDV and PDCoV were the major pathogens for the piglet diarrhea.We sequenced the genes of PEDV strains in parts of Henan province and compared with other epidemic strain,analyzed the homology and phylogenetic relationship,the result showed that the epidemic strains of PEDV in Henan were close to most epidemic strains rather than CV777.To study the pathogenicity of PDCoV,we infected mice with PDCoV,the result showed that PDCoV could be detected in kidney,lung and intestine,the lung,kidney and intestine have different degrees of pathological changes.This study provide a basis for prevention and control of the diarrhea virus in pars of Henan province,and laid the foundation for further study of PDCoV.The specific research contents are as follows:1.There pairs of primers targeting the S gene of PDCoV,M gene of PEDV and N gene of TGEV were designed,respectively,according to the corresponding gene sequences in GenBank.A total of 148 clinical sample were collected from April 2015 to December 2016 and subjected to detection by RT-PCR.The results showed that positive number of PEDV,PDCoV and TGEV were 54,30 and 3,positive rates were 36.5%,19.6%and 2%.Based on the analysis of different locations of samples,the positive rate of PEDV in Zhengzhou and Anyang was 100%,followed by 85.7%Shangqiu,the positive rate of PDCoV in Zhongmou and Xuchang was 100%.There was a cross infection between PDCoV and PEDV in Sanmenxia and Zhongmou.There was a cross infection of PEDV and TGEV in Puyang,indicated that PEDV and PDCoV were the major pathogens for the piglet diarrhea.Based on the analysis of different season,the positive rate of PEDV was higher in the lower temperature in spring and winter,PDCoV could be detected throughout the year,the highest positive rate in winter,indicated that the piglet diarrhea coronavirus was more frequent in spring and winter with low temperature.2.Several pairs of primers were designed targeting full lenth of M gene,N gene and S gene of PEDV according to GenBank.The M and N genes of PEDV strain,CH-KF-2 strain,CH-SMX strain,CH-KF-1 strain,CH-PY strain and CH-SQ strain in different regions of Henan province were amplified,and the S gene of the CH-SMX strain was amplified.M gene 841bp,N gene full-length 1417bp,S gene full-length 4164bp.We compared the strains with the domestic and foreign strains,analyzed the genetic evolution.The result showed that,homology analysis of M gene,the CH-SMX strain and CH-SQ strain had a high homology with the Korean epidemic strains,which was 99.7%,which indicated that the 4 strains might come from korea.CH-KF-1 strain,CH-KF-2 strain,CH-SMX strain and CH-SQ strain were higher than those of Korean and Japanese strains,which was 99.7%,indicated that the 4 strains might come from Korea or japan.The homology of M gene of strain and Thailand strain was 99.9%,indicated that CH-PY strain might come from Thailand.The homology analysis of S gene showed that the homology of S gene between the CH-SMX strain and the Korean strain was 98.7%,which was the lowest with PEDV homology of the classical CV777 strain.The amino acid mutation of S gene was mainly located in the S1 region,which may be the cause of poor immunity of CV777 vaccine.3.Some research have suggested that PDCoV may have a broad host spectrum,we used a cell adaptive PDCoV strain CH-01 to infect with Balb/C mice,RT-PCR was used to detect the dynamic distribution of PDCoV in mice after 3 days,5 days and 7 days after infection,and analyse pathological change of the main organs.The results showed that PDCoV could be detected in kidneyand intestine of mice after 3 days infection,on the 5 day after infection,it can be detected in lung,kidney and intestine,and in the lungs and intestines of mice after the infection for 7 days.The results of pathological sections showed that the intestinal villi of the mice after infection were partly or completely atrophic and detached,and there are different degrees of lesions in the kidney and lung,consistent with the results of PCR test,indicated that PDCoV can infect mice and have a certain degree of pathogenicity.