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.

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.

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.

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.

Evaluation of purified recombinant spike fragments forassessment of the presence of serum neutralizing antibodiesagainst a variant strain of porcine epidemic diarrhea virus

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.

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.

Coexistence of multiple genotypes of porcine epidemic diarrhea virus with novel mutant S genes in the Hubei Province of Chinain 2016

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.

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.

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.

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.

二乙烯亚胺在猪流行性腹泻病毒PEDV-WN株灭活疫苗中的应用

[目的]通过甲醛和二乙烯亚胺对猪流行性腹泻病毒PEDV-WN株的灭活效果比较,获得一种适合猪流行性腹泻病毒疫苗灭活的生产工艺。[方法]将二乙烯亚胺接种小白鼠观察二乙烯亚胺的毒性。分别使用甲醛和二乙烯亚胺,在室温条件下灭活PEDV-WN株病毒液8、12、16、20和24 h,取灭活后的病毒液接种生长良好的单层Vero细胞,进行灭活效果的检验;同时,取灭活检验合格的病毒液进行无菌检验。灭活合格的病毒液乳化配苗,进行安全性和效力试验。[结果]接种二乙烯亚胺的小白鼠28 d均健活,状态良好;甲醛体积分数为0.3%,灭活16 h可使病毒液完全灭活;二乙烯亚胺浓度为0.010 mol/L,灭活12 h可使病毒液完全灭活;灭活合格的病毒液无菌;将不同方法灭活后的病毒液乳化配苗,接种3~5日龄的仔猪,安全性较好,未观察到不良反应,然后用PEDV-WN毒株攻毒,二乙烯亚胺灭活制备的疫苗比甲醛灭活制备的疫苗保护率高。[结论]在猪流行性腹泻病毒PEDV-WN株病毒液中加入浓度为0.010 mol/L的二乙烯亚胺,在室温灭活12 h,可使病毒完全灭活,且制备的疫苗安全性好,保护率高。

永生化猪视网膜色素上皮细胞的建立及初步应用

【目的】建立永生化猪视网膜色素上皮细胞系,为病毒学研究提供一种新的细胞材料。【方法】合成猪腺病毒3型E1基因,将其克隆至真核表达质粒pCI-neo中,构建E1基因真核表达质粒pCI-neo-E1,进行PCR和测序鉴定。提取pCI-neo-E1质粒,转染原代猪视网膜色素上皮细胞,用新霉素G418筛选猪永生化视网膜色素上皮细胞系(RPECs)。检测RPECs的角蛋白18和19,通过细胞计数测定其生长曲线,流式细胞仪检测细胞周期,并对其进行染色体核型分析。分别将猪流行性腹泻病毒(PEDV)CV77毒株、猪圆环病毒2型(PCV2)DBN-SX07株和猪伪狂犬病毒(PRV)Bartha-K61株接种RPECs,同时将PEDV CV77毒株接种绿猴肾细胞(Vero细胞),将PCV2 DBN-SX07株接种猪肾细胞(PK15细胞),将PRV Bartha-K61株接种仓鼠肾成纤维细胞(BHK细胞),观察细胞病变效应(CPE),并采用间接免疫荧光法测定病毒的滴度。【结果】PCR和测序结果显示,真核表达质粒pCI-neo-E1构建成功。将pCI-neo-E1转染原代猪视网膜色素上皮细胞,经G418筛选得到永生化细胞株,该细胞株表达角蛋白18和19,传代50代仍保持上皮样细胞形态,增殖活性良好,细胞周期及染色体核型特征与正常的二倍体细胞一致。PEDV CV77毒株感染猪RPECs 24 h后,CPE明显,滴度为10^(5.25)TCID_(50)/mL,低于其在Vero细胞中的滴度(10^(8.125) TCID_(50)/mL)。PCV2 DBN-SX07毒株感染猪RPECs后可产生明显CPE,病毒滴度为10^(6.125)TCID_(50)/mL,略高于其在PK15细胞上的滴度(106.0 TCID_(50)/mL)。PRV Bartha-K61毒株感染猪RPECs后,CPE明显,病毒滴度为10^(8.75)TCID_(50)/mL,略低于其在BHK上的滴度(108.875 TCID_(50)/mL)。【结论】成功构建了永生化猪视网膜细胞系,可用于猪源病毒的培养,尤其是培养PCV2可产生明显CPE,易于观察,为后续开展疫苗工艺的提升提供了一种新的细胞材料。

PEDV乳酸菌工程菌株对小鼠的免疫效果研究

本研究以引起猪流行性腹泻的猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)为研究目标,利用含有PEDV S基因的植物乳杆菌活载体菌株LP1522-PEDS口服免疫小鼠,应用ELISA试验检测免疫小鼠粪便中SIgA含量、血清中IgG、IL-4、IL-2和IFN-γ含量,评价乳酸菌重组菌株LP1522-PEDS对小鼠的免疫效果。结果表明,首次免疫后,商品灭活疫苗组和工程菌株免疫组粪便中的SIgA、血清中的IgG、IL-4、IL-2和IFN-γ含量较对照组显著升高(P<0.05)。在免疫42~56 d,灭活疫苗组和重组菌株组抗体水平均达到最大值,抗体和免疫因子水平依次为:商品灭活疫苗组>LP1522-PEDS组>空载组≈对照组。随后商品灭活疫苗组、LP1522-PEDS组抗体和免疫因子水平逐渐下降。免疫70 d,商品灭活疫苗组抗体水平降幅度相对较小,工程菌株免疫组降幅较大。结果表明,小鼠口服携带PEDV S基因的基因工程植物乳杆菌LP1522-PEDS后能够诱导机体产生PEDV免疫应答。

猪lncRNA LOC102157897表达、定位及其对PEDV复制的调控作用

猪流行性腹泻(porcine epidemic diarrhea,PED)是由猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)引起的高度传染性疾病,前期基于组学测序筛选出1个关键lncRNA(LOC102157897),有关其表达定位以及对PEDV复制的调控作用目前尚不清楚。首先在组织和细胞水平上测定PEDV感染前后LOC102157897的表达水平;其次利用lncLocator软件预测和核质分离试验分析其细胞定位情况,并验证其表达水平与PEDV复制的关系;最后利用转录组测序筛选其下游靶基因及信号通路。结果表明:PEDV感染可极显著提高猪空肠组织和IPEC-J2细胞中LOC102157897的表达水平;LOC102157897主要定位于细胞核中,这可能与其生物学功能有关;成功构建了LOC102157897干扰细胞系,且干扰后IPEC-J2细胞中PEDV复制水平呈极显著下降。表达水平下调有利于提高宿主细胞对PEDV感染的抵抗能力。转录组测序分析显示,LOC102157897干扰前后存在151个差异表达基因,主要参与病毒蛋白互作、趋化因子信号、白细胞受体互作、白细胞介素17信号等免疫通路,并根据功能注释和差异程度,筛选出2个组蛋白基因H2AC18和H3C14。综上,这一研究确定了1个PEDV抗性相关lncRNA分子LOC102157897,并初步探究了其功能和下游调控机制,为揭示lncRNA在PEDV感染宿主过程中的重要作用以及为今后制定PEDV的抗病育种工作策略和筛选分子标记物奠定基础。

猪流行性腹泻病毒微芯片荧光定量RT-qPCR检测方法的建立

为了建立简便快速的猪流行性腹泻病毒(PEDV)荧光定量RT-qPCR检测方法,根据GenBank数据库公布的猪流行性腹泻病毒囊膜蛋白E基因,设计特异性引物和探针结合微芯片技术,建立微芯片荧光定量RT-qPCR检测方法。该方法除对PEDV检测为阳性外,对与其同源性较近的致病病毒的检测均为阴性;最低检测限为1 copies/μL,常规荧光RT-qPCR检测限为10 copies/μL。批内、批间重复性试验表明,Ct值变异系数均在2%以内,可对PEDV临床样本进行检测。建立的检测方法特异性好、敏感性高、检测时间短且重复性好,适用于临床检测。

CD44通过影响猪流行性腹泻病毒复制调节钠氢交换体3活性

本研究旨在研究CD44(cluster of differentiation 44)对感染猪流行性腹泻病毒(porcine epidemic diarrhea virus, PEDV)的猪小肠上皮细胞(IPEC-J2)中钠氢交换体3(NHE3)表达及膜转移的影响。以IPEC-J2为细胞模型,采用RT-qPCR和Western blot检测感染PEDV后不同时间点IPEC-J2细胞中NHE3和PEDV N表达量变化;转染质粒调控IPEC-J2中CD44表达后,采用TCID50和Western blot检测PEDV感染后不同时间点PEDV复制水平和NHE3蛋白表达变化,采用火焰原子吸收法检测IPEC-J2细胞内外Na^(+)浓度变化。转录组数据和细胞试验结果显示,与对照组相比,PEDV感染后IPEC-J2细胞中CD44蛋白表达水平和mRNA表达量均呈上调趋势,24~48 h内上升显著(P<0.05),而PEDV N蛋白表达水平在12~48 h内则呈显著下降趋势(P<0.05)。此外,CD44重组质粒转染试验结果显示,与PEDV感染组相比,过表达CD44后感染PEDV组细胞中病毒滴度和PEDV N蛋白表达水平显著降低(P<0.05),而干扰CD44后感染PEDV组细胞中病毒滴度和PEDV N蛋白表达水平则显著上升(P<0.05)。以上结果表明,高表达CD44具有抑制PEDV复制的作用,干扰CD44后PEDV复制增多。同时,为研究PEDV感染情况下,CD44是否参与了IPEC-J2细胞中NHE3表达的调节,采用Western blot和火焰原子吸收法检测了调节CD44后膜NHE3蛋白的表达水平和细胞内外Na^(+)浓度。结果表明,过表达CD44显著促进了膜NHE3蛋白的表达和活性(P<0.05),细胞内外Na^(+)浓度逐渐恢复正常水平。相反,干扰CD44显著降低了膜NHE3蛋白的表达和活性(P<0.05),细胞内外Na^(+)浓度呈现失衡状态。结果提示,CD44可能是缓解PEDV引发仔猪腹泻的潜在治疗靶点,它通过抑制IPEC-J2细胞中PEDV的复制来增加转移至质膜上的NHE3数量,从而维持细胞内外Na^(+)运转平衡。

猪流行性腹泻病毒变异毒株一步法RT-PCR鉴别检测方法的建立及临床应用

为了建立一种快速鉴别检测猪流行性腹泻病毒(PEDV)变异毒株的病原学方法和掌握洛阳市PEDV变异毒株的流行规律,试验根据GenBank中PEDV变异毒株的特异性序列设计引物,通过优化退火温度、模板添加量、引物添加量建立PEDV变异毒株一步法RT-PCR鉴别检测方法,并分析了该方法的特异性、敏感性和重复性;同时应用该方法检测采集自洛阳市的251份临床样品,并对不同地区、不同年份、不同养殖模式的检测结果进行比较分析。结果表明:优化后的退火温度为51℃,模板添加量为5μL,引物添加量为0.5μL;该方法对PEDV变异毒株能够扩增出550 bp特异性条带,而检测的PEDV经典毒株、猪传染性胃肠炎病毒(TGEV)、猪轮状病毒(RV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪瘟病毒(CSFV)、猪细小病毒(PPV)、猪伪狂犬病病毒(PRV)、猪圆环病毒2型(PCV-2)均为阴性;对PEDV变异毒株RNA的最低检测限达到0.74 ng,对3份PEDV变异毒株阳性病料和3份PEDV变异毒株阴性病料重复检测3次的结果完全一致;检测采集于洛阳市251份临床样品的平均阳性率为29.48%,其中不同地区阳性率介于13.33%~44.44%之间,2018—2022年阳性率介于28.30%~31.58%之间,散养户和规模化猪场的阳性率分别为38.24%和19.13%。说明试验建立的PEDV变异毒株一步法RT-PCR鉴别检测方法特异、敏感、稳定、准确,洛阳市PEDV变异毒株的流行特点为个别地区较严重的情况、近几年流行率基本持平、散养户流行情况较规模化猪场严重。