Study On The Isolation,Identification And Host Infection Characteristics Of Porcine Epidemic Diarrhea Virus In Some Areas Of Central China

Porcine epidemic diarrhea(PED)is an acute and contact infectious disease caused by porcine epidemic diarrhea virus(PEDV),which can cause vomiting,diarrhea,and dehydration in pigs of all ages,and cause the high mortality of suckling piglets.The PEDV genome has the characteristics of continuous mutation and recombination.In recent years,various PEDV strains have appeared frequently and are widespread,which has brought huge losses to the global pig industry.Continuous molecular epidemiological investigations of PEDV can clarify the epidemiological characteristics and evolutionary laws of PEDV to provide clues for clinical prevention and control.Central China is an important pig breeding and transportation base in China.In this study,we focused on the case of piglet diarrhea in pig farms in some areas of central China.Firstly,the study of pathogen detection,sequence determination and analysis,virus isolation and identification were carried out to lay the foundation for revealing the epidemiological rules and molecular evolution characteristics of PEDV.At present,most studies have used passage cell lines such as African monkey kidney cells(Vero)and porcine small intestinal epithelial cells(IPEC)as models to explore the infection,immunity and pathogenesis mechanisms of PEDV.However,it is difficult to reflect the actual status of PEDV infection in animals.Therefore,this study used 2-day-old piglets as the infection model to systematically determine the distribution of PEDV clinical isolates in different tissues and cells of infected piglets,and used transcriptomics to reveal the host response changes in the jejunum and spleen tissues of the piglets after PEDV infection.Finally,this study initially explored the susceptibility of PEDV to porcine alveolar macrophages,and studied the role of clathrin and sialic adhesin-mediated endocytosis in the entry of PEDV in alveolar macrophages.This study can lay the foundation for the study of clinical prevention and control and pathogenesis mechanism of PED.1.Phylogenetic analysis of PEDV in some areas of central ChinaIn this study,PCR detection was carried out on piglets suspected of diarrhea in Henan and some central areas from 2015 to 2016,and it was proved that PEDV was the main pathogen causing diarrhea in piglets.The PEDV positive samples were sequenced,and 14 PEDV S gene full-length sequences were successfully obtained.Sequence analysis confirmed that the PEDV epidemic strains in these areas mainly belong to the G2a and G2b genotypes.Several variant PEDV strains were found with special insertion characteristics in S gene.Among them,four PEDV strains have 3 nucleotide deletions in S2 gene;one PEDV(HNAY)has a unique 21 base insertion in S1 gene,and the insertion sequence is derived from N Gene,located in the flexible domain of S1 A protein.The whole genome recombination analysis of the PEDV strains revealed that the HNAY strain had recombination event,and its parent strains were the HNZZ47 and GDS28 strains of the early epidemic PEDV strains in China.This study reported for the first time a new PEDV strain with 21 base insertions in the S1 gene,which provides a reference for clinical diagnosis,early warning,and prevention and control of PEDV infection.2.Isolation and animal experiments of PEDV epidemic strainsIn order to assess the pathogenicity differences of different PEDV clinical strains,the PEDV positive samples were inoculated with Vero cells,and three PEDV strains were successfully isolated.Cell infection experiments found that all three strains of viruses can replicate effectively in Vero cells,and there were obvious membrane fusion and multinucleate syncytia.Animal infection experiments showed that 2-day-old piglets infected with all three PEDV strains showed typical PEDV symptoms and pathological changes such as diarrhea and vomiting at 12-24h after challenge.Among them,the HNAY strain with 21 base insertions in the S1 gene caused piglets to die in shorter days.Immunohistochemical analysis showed that the HNAY strain had a wider infection tropism to the intestinal tissues of piglets.In this study,three clinical PEDV strains were successfully isolated,and the in vivo and in vitro infection characteristics of different strains were compared,and it was found that the HNAY strain has more serious pathogenicity to piglets.3.PEDV distribution in infected piglets and transcriptomic analysis against of PEDV-infected tissuesIn order to explore the infection characteristics of PEDV to other organs,tissues or cells outside the intestine,the PEDV HB isolate was used as a representative to infect 2-day-old piglets,and the dynamic distribution of the virus in the body after PEDV infection was measured systematically,which proved that the epidemic strain of PEDV can not only infect intestinal tissues,but it can also effectively infect other tissues and organs such as lung,spleen,and peripheral blood,which proves that PEDV has the pan-tropic characteristics of causing multi-organ and multi-cell infection.Further transcriptome sequencing and analysis of the jejunum and spleen of PED V-infected piglets revealed that 541 genes were up-regulated and 540 genes were down-regulated after PEDV-infected jejunum;GO analysis,KEGG analysis and interaction network analysis found innate immune response and inflammatory response play a potential role in PEDV infection in the intestine.Similarly,PEDV infection in the spleen lead to changes in the expression of 392 genes,of which 162 genes were up-regulated and 230 genes were down-regulated;further GO analysis,KEGG analysis and interaction network analysis showed the potential role of related genes in the immune system process and defense response in PEDV infection of the spleen.This research lays a foundation for in-depth exploration of PEDV-induced multi-organ infections and host defense mechanisms in piglets.4.Infection characteristics and entry mechanism of PEDV in primary alveolar macrophagesIn order to clarify the infection tropism of PEDV on macrophages,we first used immunohistochemistry to detect the distribution of the virus in lung tissue,and isolated primary porcine alveolar macrophages(PAM),and systematically determined the infection characteristics of PEDV in PAM,Results showed that PEDV can infect PAM but can not replicate effectively,without causing obvious cytopathic changes.Viruses possessed persistent infectivity from intracellular and extracellular PAM infected with PEDV.In order to further explore the entry pathways of PEDV in PAM,experiments with protein colocalization,gene knockdown,and specific inhibition were used to determine that PEDV enters PAM through clathrin-mediated endocytosis;cell surface sialic adhesin was also involved in PEDV entry in PAM.This study lays the foundation for revealing the molecular mechanism of PEDV infection and entry in PAM.