Isolation,Identification And Infection Mechanism Of Porcine Epidemic Diarrhea Virus CHFJFQ Strain

Porcine epidemic diarrhea(PED)is an acute and highly contagious swine intestinal disease caused by Porcine epidemic diarrhea virus(PEDV),the main transmission route of PEDV is fecal-oral and airborne transmission.PEDV belongs to the order Nidovirales,family Coronaviridae and genus Alphacoronavirus.At present,PEDV is the main pathogen causing diarrhea in piglets,thereby causing huge economic losses to swine industries around the world.PEDV can infect cells from multiple species,suggesting that it may have extensive species tropism and possible cross-species transmission in the natural environment.Although no infection has been reported in humans,but the virus can infect human cells.Therefore,the infection of human cells should be paid attention to be alert to the infection of PEDV in humans.However,the infection mechanism of PEDV remains unclear,and whether porcine aminopeptidase N(p APN,ANPEP,CD13)as the receptor of PEDV is still controversial.Therefore,its receptors need to be further clarified.In addition,the host-dependent factors related to PEDV infection and proliferation require further exploration as well.The discovery of its host-dependent factors will have a significant impact in finding targets for the development of antiviral drugs,vaccines,and breeding of disease-resistant varieties.In this study,a novel PEDV strain was isolated from clinical material(small intestine)by Vero cells and named CHFJFQ.The genome size of this strain is 27953 nt(single-stranded positive-stranded RNA).Based on genome-wide nucleotide evolution analysis,PEDV CHFJFQ was a member of Subgroup Ib(G Ib)and was closely related to PEDV CV777 strain.The full-length of PEDV CHFJFQ S gene is 4149 nt and encodes1382 amino acids.Compared with the PEDV vaccine strain(PEDV CV777,Gen Bank:JN599150.1),its spike(S)protein has 4 amino acid mutations(Ser145Phe,His972 Asn,Leu1199Phe,Ser1221Arg).However,compared with the classic strain(PEDV CV777,Gen Bank: AF353511.1),its S protein has 55 amino acid mutations and R154 deletion,and the multiple site mutations partially explain the reason for the failure of PEDV vaccines.PEDV CHFJFQ was more sensitive to human cells 293 T,293A,mouse L929 cells and African green monkey Vero cells than pig cells ST,IPEC-J2 and PK15.It can proliferate in L929,Vero,293 T,IPEC-J2 and 293 A cells,but does not cause IPEC-J2 cytopathic effects.ST and PK15 cells did not support PEDV-CHFJFQ proliferation,and PK15 cells were not infected by PEDV-CHFJFQ.PEDV CHFJFQ infected piglets caused digestion and absorption disorders,pulmonary hemorrhage,diarrhea,vomiting,dehydration and death,indicating that PEDV CHFJFQ was a virulent strain.Knockout or overexpression of CD13 in ST,293 A or 293 T cells did not affect the proliferation of PEDV,but affected the proliferation of TGEV.These data suggest that porcine and human CD13 are not a receptors for PEDV.To explore whether others coronavirus receptors can be used as PEDV receptors,ACE2,DPP4,and CEACAM1 were knockeout and overexpressed in 293 A cells.We found that the knockout and overexpression of ACE2,DPP4 and CEACAM1 did not affect the proliferation of the virus.These data indicate that human ACE2,DPP4 and CEACAM 1 are not act as the receptors for PEDV.N-acetylneuraminic acid(sialic acid,Neu5Ac)is a PEDV coreceptor.In this study,PEDV was treated with 16 n M,160 n M,1.6 μM,16 μM,160 μM and 1.6 m M sialic acid,respectively,and it was found that different concentrations of sialic acid could inhibit the proliferation of PEDV in 293 T cells.These data indirectly suggest that cell surface sialic acids can promote PEDV infection.However,sialidase treatment of cells can enhance the ability of PEDV to infect cells,which is inconsistent with the conclusion that sialic acid is a PEDV co-receptor.Therefore,we speculate that sialidase induces changes in the viral receptor on the cell surface,which is most likely a sialic acid-binding protein.To explore the effect of sialic acid-binding protein on PEDV proliferation,we overexpressed CD171,CD62 L,CD24,CHF,CD22,CD33 and CD33L3(Siglec15)in 293 T cells,respectively.It was found that overexpression of Siglec15 could enhance the proliferation of PEDV in cells,except for the weak effect of CD171,there was no change.To further determine the effect of Siglec15 on PEDV proliferation.We knocked out Siglec15 in 293 T cells,and found that the knockout of Siglec15 could significantly inhibit the adsorption of PEDV to293 T cells and then affect its proliferation in 293 T cells.To determine whether Siglec15 interacts with PEDV S protein,PEDV-S1 and Siglec15 were co-expressed in 293 T cells,and PEDV-S1 and Siglec15 were found to co-localize in the cytoplasm.Moreover,PEDV-S1 and Siglec15 can interact with each other as shown by co-immunoprecipitation.Therefore,Siglec15 is likely a receptor of PEDV.Treatment of wild-type or Siglec15-overexpressing 293 T cells with humanized Siglec15 monoclonal antibody can significantly inhibited the proliferation of PEDV.It is further shown that PEDV uses Siglec15 as the receptor to mediate its infection of target cells.We infected Siglec15 humanized mice(which we made previously)and wild-type mice with PEDV through the nose and mouth.Strikingly,PEDV RNA(N gene)and N protein could be detected in the lung,spleen and small intestine of these mice.The abundance of PEDV m RNA was positively correlated with the expression of Siglec15,and the abundance of PEDV m RNA in Siglec15 humanized mice was higher than that in wild-type mice.HE staining showed that 72 h after PEDV infection,the small intestinal epithelial cells of Siglec15 humanized mice were severely shed,and the lungs were edematous and infiltrated by a large number of immune cells,indicating that PEDV proliferated in the lungs and small intestines and caused tissue damage,although no diarrhea symptoms were observed.The detection of PEDV in wild-type mice is a unexpected finding,as it is currently believed that PEDV only infects pigs.In addition,PEDV can proliferate in porcine,human and mouse peripheral blood mononuclear cells(PBMC),therefore,we suggest that PEDV has a risk of infecting humans and mice.Whether PEDV is transmitable between humans,mice and pigs in the natural environment remains to be more explored.In order to search for other host-dependent factors of PEDV,genome-wide CRISPR-Cas9 knockout library of 293 T and 293 A cells were constructed.Through next-generation sequencing,it was found that the coverage of sg RNA in the genome-wide knockout libraries of 293 T and 293 A cells was 95.35% and 97.82%,respectively.The 293 T and 293 A cell genome-wide knockout libraries were used for 3rounds of screening.After next-generation sequencing,we found 13 common sg RNAs among 3 rounds of screens.The targets of these sg RNAs are LRP12,ELTD1,OR52M1,PDZD2,MGEA5,SPAG8,EBLN1,GNG7,C14ORF159,PRKCQ,METTL17,TPBGL and TMCO5 A.We performed further demonstration.Knockout of LRP12 and OR52M1 could significantly reduce the proliferation of PEDV in 293 T cells.Knockout of ELTD1 also significantly reduced PEDV proliferation in 293 A cells.These data suggest that LRP12,ELTD1 and OR52M1 are likely to be PEDV host-dependent factors,but how they affect the proliferation of PEDV remains to be further explored.In this study,a new PEDV CHFJFQ strain was successfully isolated.PEDV CHFJFQ is a virulent strain that causes diarrhea,vomiting,dehydration and death in pigs.It will provide a reference for PEDV clinical diagnosis,prevention and control and vaccine development.We found that porcine CD13 and human CD13,ACE2,DPP4 and CEACAM1 are not receptors for PEDV.The effect of sialic acid-binding protein on the proliferation of PEDV was investigated for the first time,and it shows that Siglec15 of human and pig is one of the receptors that mediate PEDV infection of target cells.It was proposed for the first time that PEDV may have a risk of cross-species transmission among pigs,humans and mice.For the first time,the genome-wide CRISPR-Cas9 knockout library was used to screen PEDV host-dependent factors,and several potential PEDV host-dependent factors were obtained,and verified including LRP12,ELTD1 and OR52M1,but how do these host-dependent factors affect PEDV infection remain to be uncovered.