Detection of Porcine Epidemic Diarrhea Virus in Guangxi Province from 2011 to 2014 and Sequence Analysis of Its M Gene

Detection of pigs epidemic diarrhea virus （PEDV） was conducted on 331 piglets diarrhea fecal samples collected in Nanning, Yulin and other 12 areas of Guangxi Province from January of 2011 to April of 2014 by the method of reverse transcription-polymerase chain reaction （RT-PCR）. The results showed that the positive samples of PEDV were 210 and the positive rate was 63.44%. The clone and sequencing of M gene was carried out on 25 positive samples. PEDV reference strains were selected from GeneBank to conduct the sequence homology alignment analysis and the phylogenetic tree of M gene. The M gene homology and amino acid sequence identity between 25 isolated strains and 51 reference strains were 96.0% - 99.6% and 94.3% - 99.6%, respectively. The genetic variation anal- ysis of M gene showed that the genetic relationship of PEDV prevalent strains in Guangxi Province from 2013 to 2014 was close to that of the prevalent strains in Bei- jing, Anhui, Wuhan, Hebei and Guangdong from 2010 to 2013, and which were far from that of the Chinese early isolates CH/S （GenBank number： JN547228 ）, vaccine strain CV777 （GenBank number： AF353511 ） and Attenuated DR13 （GenBank number： JQ023162）. Indicating that the PEDV strains prevalent in Guan- gxi in recent years showed significant variation with the early isolates.

Construction and Immunogenicity of Recombinant Lactococcus lactis Expressing S1 Protein of Porcine Epidemic Diarrhea Virus(PEDV)

To evaluate the specific immune responses induced by recombinant Lactococcus lactis（L.lactis） which expresses porcine epidemic diarrhea virus（PEDV） S1 protein through oral administration,the spike gene fragment of PEDV was amplified from PEDV SDLY strain to construct p MG36 e-S1 recombinant plasmid.The recombinant plasmid was then electro-transferred into competent cells of L.lactis MG1363,to prepare the recombinant L.lactis expressing S1 protein of PEDV.The expression of target protein was identified by SDS-PAGE and Western-blot.New Zealand white rabbits were orally administered with the recombinant strain;the antibody titer in intestinal mucosa and serum was detected by neutralizing test;and the specific Ig G in serum was evaluated by indirect ELISA.The results showed that the recombinant L.lactis could effectively induce high level of Ig G in serum and high level of mucosal immune antibody.The recombinant L.lactis is qualified to be a potential oral vaccine because it could successfully stimulate both humoral and mucosal immune responses against PEDV.

Real-time Fluorescence Reverse-transcription Loop-mediated Isothermal Amplification for Detection of Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea,a highly contagious enteric infectious disease caused by the porcine epidemic diarrhea virus(PEDV)with symptoms of vomit,diarrhea,loss of appetite of suckling pig,has led to serious economic loss to the global swine industry.In this study,a real-time fluorescence reverse transcription loop-mediated isothermal amplification(RT-LAMP)assay was developed to detect PEDV RNA.The real-time fluorescence RT-LAMP assay was performed at62℃for 60 min,using a simple and portable device,the ESE-Quant Tube Scanner.The detection limit of RNA was 2.9×10^(6) copies/μl,10 times as sensitive as RT-PCR,and the detection was specific only to PEDV.Application of this method to clinical samples yielded a positivity rate of 93%,which was higher than that of RT-PCR.This technique saves time and is efficient,and is thus expected to be useful for the diagnosis of PEDV infection in the field.

Isolation and Identification of Porcine Epidemic Diarrhea Virus(PEDV) HLJ Strain with IPEC-J2 Cells and Phylogenetic Analysis of Its S Gene

Porcine epidemic diarrhea(PED)is caused by porcine epidemic diarrhea virus(PEDV),and is characterized by vomiting,diarrhea and dehydration of suckling pigs from 80% to 100% morbidity and 50% to 90% mortality,and resulted in tremendous economic losses to swine industry.The PEDV mainly infects small intestine of pigs,resulting in vacuolar degeneration and necrosis of mucosal epithelium.The IPEC-J2 is a pig intestine epithelial cell line,which is similar to the intestinal environment of piglets,can be used to isolate and identify the PEDV field isolates.In this study,it appeared the PEDV typical postmortem changes and histopathological lesion of degeneration and destruction of small intestine in infected piglets,and IHC identified that the PEDV distributed in the mucosa and submucosa of small intestine mostly.Furthermore,the PEDV HLJ strain was successfully isolated and characterized in the IPEC-J2 cells,and indicated that the IPEC-J2 cell line was sensitive to isolate and adapt the PEDV field strain,and could be utilized to multiply the PEDV rapidly.The S gene analysis indicated that the PEDV HLJ strain was the prevailed virus,belonged to Group 1 with attenuated virulent DR13,SC1402 and J-S2/2015 strains isolated in South Korea and China from 2014 to 2015.This study had important theoretical and practical significances on analyzing genetic variation of the PEDV,understanding the pathogenic characteristics of the virus and developing new vaccines for the PED.

Porcine NF-κB p65 Subunit:Molecular Characterization,Tissue Expression and Transcriptional Profile in Porcine Epidemic Diarrhea Virus-infected IPEC-J2 Cells

The p65 protein is a functional subunit of NF-κB family and exhibits a crucial role in host immune and inflammatory responses,apoptosis and tumor proliferation if improperly-regulated.Given its ubiquitous association with nearly all the animal cells and its pleotropic functions,the gene encoding NF-κB p65 subunit was cloned and sequenced from porcine kidney(PK-15)cells.The gene was 1662 bp in length,encoded a 553-amino acid protein and contained the prototypical NF-κB functional domains.Real-time quantitative RT-PCR and Western blot were used to characterize the transcription and expression levels of the p65 in different pig tissues.The results indicated that the p65 gene and protein were both broadly expressed in pig tissues,but most highly expressed in the intestine-associated lymph nodes and the lungs.To localize the recombinant protein in intestinal porcine epithelial cells(IPEC-J2),the gene was subcloned into the vector pEGFP(pEGFP-p65).Using fluorescence microscopy,the protein was found confined to the cytoplasm in normal cells;however,during porcine epidemic diarrhea virus(PEDV)infection,mRNA and protein expression were significantly up-regulated and the protein exhibited an overt tendency for nuclear translocalization consistent with a regulatory role in antiviral innate immunity.

Identification of niclosamide as a novel antiviral agent against porcine epidemic diarrhea virus infection by targeting viral internalization

Porcine epidemic diarrhea virus(PEDV),an enteropathogenic coronavirus,has catastrophic impacts on the global pig industry.However,there remain no effective drugs against PEDV infection.In this study,we utilized a recombinant PEDV expressing renilla luciferase(PEDV-Rluc)to screen potential anti-PEDV agents from an FDAapproved drug library in Vero cells.Four compounds were identified that significantly decreased luciferase activity of PEDV-Rluc.Among them,niclosamide was further characterized because it exhibited the most potent antiviral activity with the highest selectivity index.It can efficiently inhibit viral RNA synthesis,protein expression and viral progeny production of classical and variant PEDV strains in a dose-dependent manner.Time of addition assay showed that niclosamide exhibited potent anti-PEDV activity when added simultaneously with or after virus infection.Furthermore,niclosamide significantly inhibited the entry stage of PEDV infection by affecting viral internalization rather than viral attachment to cells.In addition,a combination with other small molecule inhibitors of endosomal acidification enhanced the anti-PEDV effect of niclosamide in vitro.Taken together,these findings suggested that niclosamide is a novel antiviral agent that might provide a basis for the development of novel drug therapies against PEDV and other related pathogenic coronavirus infections.

Nucleocapsid protein from porcine epidemic diarrhea virus isolates can antagonize interferon-λ production by blocking the nuclear factor-κB nuclear translocation

Porcine epidemic diarrhea virus（PEDV） is a highly infectious pathogen that can cause severe diseases in pigs and result in enormous economic losses in the worldwide swine industry. Previous studies revealed that PEDV exhibits an obvious capacity for modulating interferon（IFN） signaling or expression. The newly discovered type III IFN, which plays a crucial role in antiviral immunity, has strong antiviral activity against PEDV proliferation in IPEC-J2 cells. In this study, we aimed to investigate the effect of PEDV nucleocapsid（N） protein on type III IFN-λ. We found that the N proteins of ten PEDV strains isolated between 2013 and 2017 from different local farms shared high nucleotide identities, while the N protein of the CV777 vaccine strain formed a monophyletic branch in the phylogenetic tree. The N protein of the epidemic strain could antagonize type III IFN, but not type I or type II IFN expression induced by polyinosinic-polycytidylic acid（poly（I：C）） in IPEC-J2 cells. Subsequently, we demonstrated that the inhibition of poly（I：C）-induced IFN-λ3 production by PEDV N protein was dependent on the blocking of nuclear factor-κB（NF-κB） nuclear translocation. These findings might help increase understanding of the pathogenesis of PEDV and its mechanisms for evading the host immune response.

Isolation and oral immunogenicity assessment of porcine epidemic diarrhea virus NH-TA2020 strain:One of the predominant strains circulating in China from 2017 to 2021

Porcine epidemic diarrhea(PED)caused by porcine epidemic diarrhea virus(PEDV)is one of the most devastating diseases in the global pig industry due to its high mortality rate in piglets.Maternal vaccines can effectively enhance the gut-mammary gland-secretory IgA axis to boost lactogenic immunity and passive protection of nursing piglets against PEDV challenge.From 2017 to 2021,we collected 882 diarrhea samples from 303 farms in China to investigate the epidemiology of PEDV.The result showed that about 52.15%(158/303)of the farms were positive for PEDV with an overall detection rate of 63.95%(564/882)of the samples.The S1 fragments of S gene from 104 strains were sequenced for the phylogenetic analysis.A total of 71 PEDV strains(68.27%)sequenced in this study were clustered into the predominant G2c subgroup,while the newly-defined G2d strains(9.62%)were identified in three provinces of China.The NH-TA2020 strain of G2c subgroup was isolated and cultured,and its infection to piglets caused watery diarrhea within 24 h,indicating its strong pathogenicity.Oral administration of NH-TA2020 strain to pregnant gilts stimulated high levels of IgA antibody in colostrum.The piglets fed by the gilts above were challenged with NH-TA2020 strain or CH-HeB-RY-2020 strain from G2d subgroup,and the clinical symptoms and virus shedding were significantly reduced compared to the mock group.Our findings suggest that G2c subgroup is the predominant branch circulating in China from 2017 to 2021.Oral administration of NH-TA2020 enhances maternal IgA and lactogenic immune responses,which confer protection against the homologous and emerging G2d PEDV strains challenges in neonates.

Genome-wide transcriptome analysis of porcine epidemic diarrhea virus virulent or avirulent strain-infected porcine small intestinal epithelial cells

Porcine epidemic diarrhea virus(PEDV)is the main cause of diarrhea,vomiting,and mortality in pigs,which results in devastating economic loss to the pig industry around the globe.In recent years,the advent of RNAsequencing technologies has led to delineate host responses at late stages of PEDV infection;however,the comparative analysis of host responses to early-stage infection of virulent and avirulent PEDV strains is currently unknown.Here,using the BGI DNBSEQ RNA-sequencing,we performed global gene expression profiles of pig intestinal epithelial cells infected with virulent(GDS01)or avirulent(HX)PEDV strains for 3,6,and 12 h.It was observed that over half of all significantly dysregulated genes in both infection groups exhibited a down-regulated expression pattern.Functional enrichment analyses indicated that the differentially expressed genes(DEGs)in the GDS01 group were predominantly related to autophagy and apoptosis,whereas the genes showing the differential expression in the HX group were strongly enriched in immune responses/inflammation.Among the DEGs,the functional association of TLR3 and IFIT2 genes with the HX and GDS01 strains replication was experimentally validated by TLR3 inhibition and IFIT2 overexpression systems in cultured cells.TLR3 expression was found to inhibit HX strain,but not GDS01 strain,replication by enhancing the IFIT2 expression in infected cells.In conclusion,our study highlights similarities and differences in gene expression patterns and cellular processes/pathways altered at the early-stage infection of PEDV virulent and avirulent strains.These findings may provide a foundation for establishing novel therapies to control PEDV infection.

Significant Inhibition of Porcine Epidemic Diarrhea Virus In Vitro by Remdesivir,Its Parent Nucleoside and β-D-N^(4)-hydroxycytidine

Porcine epidemic diarrhea(PED)caused by porcine epidemic diarrhea virus(PEDV)is widespread in the world.In recent years,the increased virulence of the virus due to viral variations,has caused great economic losses to the pig industry in many countries.It is always worthy to find effective therapeutic methods for PED.As an important class of antivirals,nucleoside drugs which target viral polymerases have been applied in treating human viral infections for half a century.Herein,we evaluated the anti-PEDV potential of three broad-spectrum antiviral nucleoside analogs,remdesivir(RDV),its parent nucleoside(RDV-N)andβ-D-N^(4)-hydroxycytidine(NHC).Among them,RDV-N was the most active agent in Vero E6 cells with EC_(50) of 0.31μmol/L,and more potent than RDV(EC_(50)=0.74μmol/L)and NHC(EC_(50)=1.17μmol/L).The activity of RDV-N was further confirmed using an indirect immuno-fluorescence assay.Moreover,RDV-N exhibited a good safety profile in cells and in mice.The high sequence similarity of the polymerase functional domains of PEDV with other five porcine coronaviruses indicated a broader antiviral spectrum for the three compounds.Generally,RDV-N is a promising broad-spectrum antiviral nucleoside,and it would be worthy to make some structural modifications to increse its oral bioavailability.

Screening of Host Proteins Interacting with PorcineEpidemic Diarrhea Virus (PEDV) N Protein by YeastTwo-hybrid System

[Objective] The paper was to obtain host proteins interacting with porcine epidemic diarrhea virus （PEDV） N protein. [Method] The re-combinant vector pGBKT7-N of PEDV N gene was constructed and used as the bait plasmid to screen the proteins interacting with N protein ofPEDV from the cDNA library of porcine alveolar macrophage （PAM） by yeast two-hybrid method. [Result] There was no toxicity and self activationof bait protein in yeast hybridization system, and six proteins （FTH1, LGALS3, CORO1C, SNRPG, KRTAP5-3, ZNF598） interacting with N proteinwere indentified. It was confirmed that LGALS3 and SNRPG had specific interaction with N protein by return experiment and co-immunoprecipitation（CoIP） test. [Conclusion] The study lays a foundation for further studying the function of PEDV N protein and the pathogenic mechanism of PEDV.

Development of an accurate lateral flow immunoassay for PEDV detection in swine fecal samples with a filter pad design

Porcine epidemic diarrhea virus(PEDV),as the main causative pathogen of viral diarrhea in pigs,has been reported to result in high morbidity and mortality in neonatal piglets and cause significant economic losses to the swine industry.Rapid diagnosis methods are essential for preventing outbreaks and transmission of this disease.In this study,a paper-based lateral flow immunoassay for the rapid diagnosis of PEDV in swine fecal samples was developed using stable color-rich latex beads as the label.Under optimal conditions,the newly developed latex bead-based lateral flow immunoassay(LBs-LFIA)attained a limit of detection(LOD)as low as 10^(3.60) TCID_(50)/mL and no cross-reactivity with other related swine viruses.To solve swine feces impurity interference,by adding a filtration unit design of LFIA without an additional pretreatment procedure,the LBs-LFIA gave good agreement(92.59%)with RT-PCR results in the analysis of clinical swine fecal samples{n=108),which was more accurate than previously reported colloidal gold LFIA(74.07%)and fluorescent LFIA(86.67%).Moreover,LBs-LFIA showed sufficient accuracy(coefficient of variance[CV]<15%)and stable(room temperature storage life>56 days)performance for PEDV detection,which is promising for on-site analysis and user-driven testing in pig production system.

Inactivated Pseudomonas PE(△Ⅲ)exotoxin fused to neutralizing epitopes of PEDV S proteins produces a specific immune response in mice

Porcine epidemic diarrhea(PED)caused by the porcine epidemic diarrhea virus(PEDV),is a severe infectious and devastating swine disease that leads to serious economic losses in the swine industry worldwide.An increased number of PED cases caused by variant PEDV have been reported in many countries since 2010.S protein is the main immunogenic protein containing some B-cell epitopes that can induce neutralizing antibodies of PEDV.In this study,the construction,expression and purification of Pseudomonas aeruginosa exotoxin A(PE)without domain Ⅲ(PE△Ⅲ)as a vector was performed for the delivery of PEDV S-A or S-B.PE(△Ⅲ)PEDV S-A and PE(△Ⅲ)PEDV S-B recombinant proteins were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis.The immunogenicity of PEDV S-A and PEDV S-B subunit vaccines were evaluated in mice.The results showed that PEDV-S-B vaccine could not only induce specific humoral and Th1 type-dominant cellular immune responses,but also stimulate PEDV-specific mucosal immune responses in mice.PEDV-S-B subunit vaccine is a novel candidate mucosal vaccine against PEDV infection.

Calmodulin-like 5 promotes PEDV replication by regulating late-endosome synthesis and innate immune response

The infection caused by porcine epidemic diarrhea virus(PEDV)is associated with high mortality in piglets worldwide.Host factors involved in the efficient replication of PEDV,however,remain largely unknown.Our recent proteomic study in the virus-host interaction network revealed a significant increase in the accumulation of CALML5(EF-hand protein calmodulin-like 5)following PEDV infection.A further study unveiled a biphasic increase of CALML5 in 2 and 12 h after viral infection.Similar trends were observed in the intestines of piglets in the early and late stages of the PEDV challenge.Moreover,CALML5 depletion reduced PEDV mRNA and protein levels,leading to a one-order-of-magnitude decrease in virus titer.At the early stage of PEDV infection,CALML5 affected the endosomal trafficking pathway by regulating the expression of endosomal sorting complex related cellular proteins.CALML5 depletion also suppressed IFN-βand IL-6 production in the PEDV-infected cells,thereby indicating its involvement in negatively regulating the innate immune response.Our study reveals the biological function of CALML5 in the virology field and offers new insights into the PEDV-host cell interaction.

Next-generation sequencing and single-cell RT-PCR reveal a distinct variable gene usage of porcine antibody repertoire following PEDV vaccination

Porcine epidemic diarrhea virus(PEDV)is the most common diarrhea-causing pathogen in newborn piglets.The clarifications of the overall antibody repertoire and antigen-specific antibody repertoire are essential to provide important insights into the B-cell response and reshape new vaccines.Here,we applied next-generation sequencing(NGS)technology to investigate immunoglobulin(Ig)variable(V)gene segment usage of swine B-cells from peripheral blood lymphocytes(PBL)and mesenteric lymph node(MLN)cells following PEDV vaccination.We identified the transcripts of all functional Ig V-genes in antibody repertoire.IgHV1 S2,IgKV1-11,and IgLV3-4 were the most prevalent gene segments for heavy,kappa,and lambda chains,respectively,in PBL and MLN.Unlike previous studies,IgKV1,instead of IgKV2,and IgLV3,instead of IgLV8,were the prevalent Ig V-gene families for kappa and lambda light chains,respectively.We further examined the antibody repertoire of PEDV spike-specific B cells by single-cell RT-PCR.In contrast to the overall antibody repertoire,Ig V-gene segments of PEDV spike-specific B cells preferentially adopted IgHV1-4 and IgHV1-14 for heavy chain,IgKV1-11 for kappa chain,and IgLV3-3 for lambda chain.These results represent a comprehensive analysis to characterize the Ig V-gene segment usage in the overall and PEDV spike-specific antibody repertoire in PBL and MLN.

A Virulent PEDV Strain FJzz1 with Genomic Mutations and Deletions at the High Passage Level Was Attenuated in Piglets via Serial Passage In Vitro

Highly virulent porcine epidemic diarrhea virus(PEDV)strains re-emerged and circulated in China at the end of 2010,causing significant economic losses in the pork industry worldwide.To understand the genetic dynamics of PEDV during its passage in vitro,the PEDV G2 strain FJzzl was serially propagated in Vero cells for up to 200 passages.The susceptibility and adaptability of the FJzzl strain increased gradually as it was serially passaged in vitro.Sequence analysis revealed that amino acid(aa)changes were mainly concentrated in the S glycoprotein,which accounted for 72.22%-85.71%of all aa changes.A continuous aa deletion(^(55)I^(56)G^(57)E→^(55)K^(56)Δ^(57)Δ)occurred in the N-terminal domain of S1(Sl-NTD).To examine how the aa changes affected its virulence,FJzzl-F20 and FJzzl-F200 were selected to simultaneously evaluate their pathogenicity in suckling piglets.All the piglets in the FJzzl-F20-infected group showed typical diarrhea at 24 h postinfection,and the piglets died successively by 48 h postinfection.However,the clinical signs of the piglets in the FJzzl-F200-infected group were significantly weaker,and no deaths occurred.The FJzzl-F200-infected group also showed a lower level of fecal viral shedding and lower viral loads in the intestinal tissues,and no obvious histopathological lesions.TypeⅠandⅢinterferon were induced in the FJzzl-F200 infection group,together with pro-inflammatory cytokines,such as TNF-α,IL-1βand IL-8.These results indicate that the identified genetic changes may contribute to the attenuation of FJzzl strain,and the attenuated FJzzl-F200 may have the potential for developing PEDV live-attenuated vaccines.