Establishment Of An Animal Model Of Co-infection Of PEDV And PDCoV In Piglets And Its Synergistic Pathogenicity Study

Porcine epidemic diarrhea virus(PEDV)and porcine deltacoronavirus(PDCoV)are the main porcine intestinal coronaviruses causing acute diarrhea in piglets.Coinfection of PEDV and PDCoV is common in pigs,which seriously affects the healthy development of pig industry.However,the clinical results of the interaction between PEDV and PDCoV are not clear.In this study,we inoculated PEDV and PDCoV into 4-day-old healthy piglets in different order,and established an animal model of coinfection of PEDV and PDCoV through weight change,diarrhea and histopathological evaluation.To study the pathogenic effect of co-infection of PEDV and PDCoV on piglets by detecting fecal detoxification,viral load in tissues,histopathological examination,cytokine expression and the composition and structure of colon microbial community.Finally,the involvement of PEDV and PDCoV in the induction of inflammatory factor expression by co-infecting porcine dendritic cells(DC)in vitro was analyzed.This study settles the foundation for the study of the pathogenic mechanism of the co-infection with PEDV and PDCoV.1.Establishment of animal model of co-infection of PEDV and PDCoV in pigletsThirty 4-day-old healthy piglets were randomly selected and divided into six groups: Control,PDCoV,PEDV,PEDV/ PDCoV(PEDV first infected for 12 h followed by PDCoV infection),PDCoV-PEDV(PDCoV and PEDVsimultaneously infected),and PDCoV/ PEDV(PDCoV first infected for 12 h followed by PEDV infection)with five piglets in each group.Piglets in the PDCoV group were infected orally with virus 5 × 107 TCID50 / head,piglets in the PEDV group were infected orally with virus 1 × 108 TCID50 / head,co-infected group piglets were orally administered PDCoV 5 × 107 TCID50 and PEDV 1 × 108 TCID50 / head,piglets in the control group were orally administered DMEM.During the trial,the piglets were observed daily for clinical symptoms,and the piglet body weight and diarrhoea were recorded.At day 3 post infection,three piglets from each group were selected for necropsy to observe the pathological anatomy of the piglets,and heart,liver,spleen,lung,kidney,small intestine(duodenum,jejunum and ileum),large intestine(colon,cecum and rectum)tissues were collected,the tissue distribution of the virus was determined by q RT-PCR,histopathological examination of the small intestine using H & E staining,and calculation of the ratio of villus height to crypt depth(VH: CD)of the small intestine.Showed that,compared with the PDCoV or PEDV alone infection group,the diarrhea of piglets co-infected with PEDV and PDCoV in the 3 groups was more severe,prolonged diarrhea time,and the body weight gain was significantly slowed.Piglets in the group co-infected with PEDV and PDCoV were selected for further testing,and the results showed that the co-infected group had significantly thinner small intestines,intestinal villus atrophy,necrosis,and sloughing,and the degree of intestinal villus atrophy in the piglets was significantly higher than that in the group infected with alone.q RT-PCR results showed higher viral loads of PEDV and PDCoV in small intestinal tissues.The above results indicated that we successfully established an animal model of co-infection of PEDV and PDCoV in piglets.2.Synergistic pathogenicity of PEDV and PDCoV co-infection to pigletsBased on the successful establishment of the animal model of co-infection of PEDV and PDCoV in piglets,the pathogenicity of co-infection of PEDV and PDCoV was studied.After PEDV and PDCoV co-infection of piglets,the content of virus in the feces and small intestinal tissues of piglets was detected by q RT-PCR,the distribution of virus in the small intestinal tissues of piglets was detected by immunohistochemistry,the content of inflammatory factors in the serum of piglets was determined by ELISA,and 16 S r RNA sequencing was applied to analyze structural changes in the colonic microbiota composition of piglets.Showed that PDCoV load in feces and small intestinal tissue was significantly higher(p < 0.01)and mainly distributed to the villus epithelial cells of the small intestine in co-infected piglets compared with PEDV or PDCoV alone infected group,but viral load of PEDV was not significantly different among the infected groups.At 3 days post viral infection(dpi),the secretion of inflammatory factors(IL-6,IL-8 and TNF-α)was significantly higher(p < 0.01)in the serum of PEDV and PDCoV co-infected than infected alone.16 S r RNA sequencing of colonic contents from 3 dpi piglets revealed significant alterations in colonic microbial structure in both PEDV and PDCoV co-infected and singly infected piglets compared to controls.At the genus level,the abundance of the genera Mitsuokella(p < 0.001)and Collinsella and Collins(p < 0.05)was significantly increased in the co-infected group compared to the singly infected group.Spearman correlation analysis showed a positive correlation between the Mitsuokella and TNF-α or IL-6 secretion.The above results suggested synergistic pathogenicity in piglets co-infected with PEDV and PDCoV.3.Effect of PEDV and PDCoV co-infection of DCs on the expression of inflammatory factorsOn the basis that PEDV and PDCoV co-infection was able to aggravate intestinal injury and inflammatory factor expression in piglets,the effect of PEDV and PDCoV co-infected DCs on the expression of inflammatory factors was further carried out.Immature DCs were isolated and induced from porcine peripheral blood,and DCs were co infected with PEDV and PDCoV to investigate the infection of PEDV and PDCoV on DCs and the effect on the expression of inflammatory factors by techniques such as q RT-PCR and indirect immunofluorescence detection(IFA).The results showed that both PEDV and PDCoV significantly increased viral copys at 3-12 h post infection(hpi)in DC and began to decrease by 24 hpi;Virus titers in the supernatant continued to rise at 3-24 hpi and were highest at 24 hpi.IFA results showed that PEDV with PDCoV antigen was detected in both the singly infected group and the co-infected group.Compared with the group infected alone,the levels of inflammatory cytokines(IL-6,IL-1β,TNF-α and IL-8)were significantly higher.The above results demonstrate that PEDV and PDCoV can co-infect DCs in vitro and significantly promote inflammatory cytokine expression.In summary,this study successfully established an animal model of PEDV and PDCoV co infection in piglets,and found that there was a synergistic pathogenic effect of PEDV and PDCoV,which could aggravate intestinal tissue injury in piglets;Meanwhile,PEDV and PDCoV can co infect DCs in vitro and significantly promote inflammatory cytokine expression.This study lays the foundation for the study of the pathogenic mechanism of PEDV and PDCoV co-infection.