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...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.展开更多
[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 plas...[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.展开更多
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 resul...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.展开更多
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 me...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.展开更多
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 ...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.展开更多
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...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.展开更多
Since 2010, variant strains of porcine epidemic diarrhea virus(PEDV) have caused disasters in the pork industry. The spike(S) protein, as the major immunity-eliciting antigen, has previously been used for serological ...Since 2010, variant strains of porcine epidemic diarrhea virus(PEDV) have caused disasters in the pork industry. The spike(S) protein, as the major immunity-eliciting antigen, has previously been used for serological testing and has been found to correlate significantly with the results of the serum neutralization(SN) test. However, further evaluation of this method is needed as new epidemic strains of PEDV emerge. Hence, the main objective of this study was to assess sow sera and determine the correlation between enzyme-linked immunosorbent assay(ELISA) results(involving a newly isolated GDS01 virus-based ELISA and ELISAs based on seven recombinant fragments comprising overlapping S1 and partial S2 sequences) and SN titers. Furthermore, we determined the reliability of the ELISAs based on receiver operating characteristics(ROC) curve analyses. For the most promising ELISA, i.e., the SP4 ELISA, the correlation coefficient(r) and the area under curve(AUC) were determined to be 0.6113 and 0.8538, respectively. In addition, we analyzed the homology of the SP4 sequences obtained from different strains(including vaccine strains) and found that various strains showed a high degree of homology in this region. Thus, we conclude that SP4 is a promising serological testing protein for use in the field.展开更多
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 effective...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.展开更多
The emergence of highly virulent porcine epidemic diarrhea virus(PEDV) variants in China caused huge economic losses in 2010. Since then, large-scale sporadic outbreaks of PED caused by PEDV variants have occasionally...The emergence of highly virulent porcine epidemic diarrhea virus(PEDV) variants in China caused huge economic losses in 2010. Since then, large-scale sporadic outbreaks of PED caused by PEDV variants have occasionally occurred in China. However, the molecular diversity and epidemiology of PEDV in different provinces has not been completely understood. To determine the molecular diversity of PEDV in the Hubei Province of China, we collected 172 PED samples from 34 farms across the province in 2016 and performed reverse transcription polymerase chain reaction(RTPCR)by targeting the nucleocapsid(N) gene. Seventy-four samples were found to be PEDVpositive.We further characterized the complete spike(S) glycoprotein genes from the positive samples and found 21 different S genes with amino acid mutations. The PEDV isolates here presented most of the genotypes which were found previously in field isolates in East and SouthEast Asia, North America, and Europe. Besides the typical Genotypes Ⅰ and Ⅱ, the INDEX groups were also found. Importantly, 58 new amino acids mutant sites in the S genes, including 44 sites in S1 and 14 sites in S2, were first described. Our results revealed that the S genes of PEDV showed variation and that diverse genotypes of PEDV coexisted and were responsible for the PED outbreaks in Hubei in 2016. This work highlighted the complexity of the epidemiology of PEDV and emphasized the need for reassessing the efficacy of classic PEDV vaccines against emerging variant strains and developing new vaccines to facilitate the prevention and control of PEDV in fields.展开更多
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 PED...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.展开更多
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 RNAsequen...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.展开更多
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 ...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.展开更多
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 reco...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.展开更多
基金Supported by Priority Academic Talent Team Cultivation Program of Shandong Colleges and Universities and Agricultural Industry Research System of Shandong Province(SDAIT-06-022-08)People’s Livelihood Science and Technology Program of Qingdao City(16-6-2-42-nsh)
文摘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.
基金Supported by National Natural Science Foundation of China(31201915,31502071)Key Project of Science and Technology Promoting Agriculture in Shanghai City[HNKGZ(2013)No.3-6,No.5-5]
文摘[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.
基金Supported by the National Natural Science Foundation of China(31201911 31372438)
文摘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.
基金Supported by Scientific Research Project of Guangxi Bureau of Animal Husbandry&Veterinary Medicine(12049031)Systemic Research Subject of Guangxi Key Laboratory of Animal Vaccines and New Technology(12-071-28-A-5)Guangxi Basal Research Specific Fund(14-2)
文摘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.
基金Supported by Science and Technology Research Project of Universities in Hebei Province,China(QN2014220)
文摘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.
基金Supported by the National Natural Science Foundation of China(31370140,31372438)。
文摘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.
基金supported by the National Key Research and Development Program(2016YFD0500101)
文摘Since 2010, variant strains of porcine epidemic diarrhea virus(PEDV) have caused disasters in the pork industry. The spike(S) protein, as the major immunity-eliciting antigen, has previously been used for serological testing and has been found to correlate significantly with the results of the serum neutralization(SN) test. However, further evaluation of this method is needed as new epidemic strains of PEDV emerge. Hence, the main objective of this study was to assess sow sera and determine the correlation between enzyme-linked immunosorbent assay(ELISA) results(involving a newly isolated GDS01 virus-based ELISA and ELISAs based on seven recombinant fragments comprising overlapping S1 and partial S2 sequences) and SN titers. Furthermore, we determined the reliability of the ELISAs based on receiver operating characteristics(ROC) curve analyses. For the most promising ELISA, i.e., the SP4 ELISA, the correlation coefficient(r) and the area under curve(AUC) were determined to be 0.6113 and 0.8538, respectively. In addition, we analyzed the homology of the SP4 sequences obtained from different strains(including vaccine strains) and found that various strains showed a high degree of homology in this region. Thus, we conclude that SP4 is a promising serological testing protein for use in the field.
基金supported by the National Key R&D Program of China(2021ZD0113803)the"Pioneer"and"Leading Goose"R&D Program of Zhejiang (2022C02031)the National Natural Science Foundation of China (No. 31701424)
文摘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.
基金funded by the National Natural Science Foundation of China(31470260 and 81401672)the"Fundamental Research Funds for the Central Universities"(531107040975)
文摘The emergence of highly virulent porcine epidemic diarrhea virus(PEDV) variants in China caused huge economic losses in 2010. Since then, large-scale sporadic outbreaks of PED caused by PEDV variants have occasionally occurred in China. However, the molecular diversity and epidemiology of PEDV in different provinces has not been completely understood. To determine the molecular diversity of PEDV in the Hubei Province of China, we collected 172 PED samples from 34 farms across the province in 2016 and performed reverse transcription polymerase chain reaction(RTPCR)by targeting the nucleocapsid(N) gene. Seventy-four samples were found to be PEDVpositive.We further characterized the complete spike(S) glycoprotein genes from the positive samples and found 21 different S genes with amino acid mutations. The PEDV isolates here presented most of the genotypes which were found previously in field isolates in East and SouthEast Asia, North America, and Europe. Besides the typical Genotypes Ⅰ and Ⅱ, the INDEX groups were also found. Importantly, 58 new amino acids mutant sites in the S genes, including 44 sites in S1 and 14 sites in S2, were first described. Our results revealed that the S genes of PEDV showed variation and that diverse genotypes of PEDV coexisted and were responsible for the PED outbreaks in Hubei in 2016. This work highlighted the complexity of the epidemiology of PEDV and emphasized the need for reassessing the efficacy of classic PEDV vaccines against emerging variant strains and developing new vaccines to facilitate the prevention and control of PEDV in fields.
基金Project supported by the National Natural Science Foundation of China(No.31702250)the Key Research and Development Project Funds of Zhejiang Provincial Science and Technology Department(Nos.2015C02044 and 2018C02028)+2 种基金the Agricultural Technology Extension Funds of Zhejiang Universitythe Dabei Agricultural Discipline Development and Talent Training Fund(No.2017ZDNT004)the Three Rural and Six Party Funds,China
文摘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.
基金funded by National Key Research and Development Program of China(No.2016YFD0500101)China Postdoctoral Science Foundation(No.2020M672974)
文摘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.
基金supported by the following funding sources,a grant from the Shanghai Science and Technology Committee in China(Number:19430750100)the National Science&Technology Major Project"Key New Drug Creation and Manufacturing Program",China(Number:2018ZX09711002)+1 种基金the Shandong Key Provincial Research and Development Program(2019GNC106044)the Agricultural Scientific and Technological Innovation Project of Shandong Academy of Agricultural Sciences(CXGC2016B14)。
文摘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.
基金supported by the National Natural Science Foundation of China(Grant No.31602033 and 32172839)the China Scholarship Council under grant 201908410129.
文摘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.