All-trans retinoic acid alleviates transmissible gastroenteritis virus-induced intestinal inflammation and barrier dysfunction in weaned piglets

Background Transmissible gastroenteritis virus(TGEV)is one of the main pathogens causing severe diarrhea of pig-lets.The pathogenesis of TGEV is closely related to intestinal inflammation.All-trans retinoic acid(ATRA)is the main active metabolite of vitamin A,which has immunomodulatory and anti-inflammatory properties.However,it is unclear whether ATRA can alleviate TGEV-induced intestinal inflammation and barrier dysfunction in piglets.This study aimed to investigate the effects of ATRA on growth performance,diarrhea,intestinal inflammation and intesti-nal barrier integrity of TGEV-challenged piglets.Methods In a 19-d study,32 weaned piglets were randomly divided into 4 treatments:Control group(basal diet),TGEV group(basal diet+TGEV challenge),TGEV+ATRA5 group(basal diet+5 mg/d ATRA+TGEV challenge)and TGEV+ATRA15 group(basal diet+15 mg/d ATRA+TGEV challenge).On d 14,piglets were orally administered TGEV or the sterile medium.Results Feeding piglets with 5 and 15 mg/d ATRA alleviated the growth inhibition and diarrhea induced by TGEV(P<0.05).Feeding piglets with 5 and 15 mg/d ATRA also inhibited the increase of serum diamine oxidase(DAO)activ-ity and the decrease of occludin and claudin-1 protein levels in jejunal mucosa induced by TGEV,and maintained intestinal barrier integrity(P<0.05).Meanwhile,5 mg/d ATRA feeding increased the sucrase activity and the expres-sions of nutrient transporter related genes(GLUT2 and SLC7A1)in jejunal mucosa of TGEV-challenged piglets(P<0.05).Furthermore,5 mg/d ATRA feeding attenuated TGEV-induced intestinal inflammatory response by inhibit-ing the release of interleukin(IL)-1β,IL-8 and tumor necrosis factor-α(TNF-α),and promoting the secretion of IL-10 and secretory immunoglobulin A(sIgA)(P<0.05).Feeding 5 mg/d ATRA also down-regulated the expressions of Toll-like receptors and RIG-I like receptors signaling pathway related genes(TLR3,TLR4,RIG-I,MyD88,TRIF and MAVS)and the phosphorylation level of nuclear factor-κB-p65(NF-κB p65),and up-regulated the inhibitor kappa B alpha(IκBα)protein level in jejunal mucosa of TGEV-challenged piglets(P<0.05).Conclusions ATRA alleviated TGEV-induced intestinal barrier damage by inhibiting inflammatory response,thus improving the growth performance and inhibiting diarrhea of piglets.The mechanism was associated with the inhibi-tion of NF-κB signaling pathway mediated by TLR3,TLR4 and RIG-I.

Development of TaqMan-based Real-time PCR Assay for Detecting Transmissible Gastroenteritis Virus and Its Application in Vaccine Evaluation

[Objective] This study aimed to establish a TaqMan-based real-time PCR assay for detecting transmissible gastroenteritis virus （TGEV）. [Method] Primers and a probe were designed according to the conserved sequence of N gene in TGEV genome. After gradient dilution, the recombinant plasmid harboring the N gene was used as a standard for real-time PCR assay to establish the standard curve. [Re- sult] The results showed that the established real-time PCR assay exhibited a good linear relationship within the range of 102-10^10 copies/ul; the correlation coefficient was above 0.99 and the amplification efficiency ranged from 90% to 110%. The de- tection limit of real-time PCR assay for TGEV was 10 copies/μl, suggesting a high sensitivity; there was no cross reaction with other porcine viruses, indicating a good specificity; coefficients of variation within and among batches were lower than 3%, suggesting a good repeatability. The established real-time PCR method could be ap- plied in quantitative analysis and evaluation of the immune efficacy of TGEV vac- cines and detection of TGEV in clinical samples. [Conclusion] The TaqMan-based real-time PCR assay established in this study is highly sensitive and specific, which can provide technical means for the epidemiological survey of TGEV, development of TGEV vaccines and investigation of the pathogenesis of TGE.

Diagnosis and Treatment of Porcine Transmissible Gastroenteritis

Porcine transmissible gastroenteritis （TGE） caused by corona virus is an acute, highly contagious, and intestinal infectious disease. TGE is a common disease in intensive pig farms and prevalent throughout the world. Disease in affected pigs is characterized by diarrhea, vomiting and dehydration. In the present article, the pathogen, epidemiology, diagnosis and treatment were elaborated, so as to effectively prevent TGE and provide a reference for treatment.

Cloning and Homology Comparison of S Gene for Isolate TH-98 of Porcine Transmissible Gastroenteritis Virus

TH-98 isolate of transmissible gastroenteritis virus (TGEV) was propagated and harvested on swine testicle (ST) monolayer cell. Two pairs of primers were designed to amplify S gene by RT-PCR according to the published sequence of TGEV'S gene cDNA with Oligo version 4.1 and DNasis software. The products of PCR were named Sa and Sb, of 2.3 kb and 2.1 kb respectively. Sa was inserted in EcoR I and Kpn I sites after Sb was cloned in Kpn I and Pst I multiple cloning sites of the same pUC18 plasmid. The recombinant pUC-S plasmid was identified and analyzed by corresponding restriction endonuclease and nested PCR on the basis of the genetic sites of S gene and pUC18 plasmid, which was identified as S gene of TGEV. Recombinant pUC-S was sequenced and analyzed in comparison with the other strains. Gene sequence comparison indicated that TH-98 shared 99, 97, 98, 97 and 94% identities with Purdue-115(US), Miller(US), TO14(Japan), FS772(British), 96-1933(British), respectively, their deduced amino acid homology was 99, 97, 97, 96 and 93% correspondingly. In addition, the analysis report verified that pUC-S owned a complete open reading frame (ORF) including initiation codon, signal sequences, remaining sequences and termination codon as well. Therefore, the results affirmed that S gene of TGEV TH-98 was extremely conservative.

Studies on Mucosal Immunity Induced by Transmissible Gastroenteritis Virus Nucleocapsid Protein Recombinant Lactobacillus casei in Mice and Sow

Mucosal immunity plays an important role in protecting pigs against transmissible gastroenteritis virus （TGEV） infection. To elicit mucosal immune response against TGEV, we developed a surface antigen display system using the poly-γ- glutamate synthetase A （pgsA） protein of Bacillus subtilis as an anchoring matrix to express recombinant fusion proteins of pgsA and nucleocapsid protein of TGEV in Lactobacillus casei. Surface location of fusion protein was verified by ELISA and indirect immunofluorescence test. Oral and intranasal inoculations of pregnant sow and mice with recombinant L. casei resulted in high levels of serum immunoglobulin G （IgG） and secretory immunoglobulin A （slgA） against recombinant N protein as demonstrated by ELISA. More importantly, the level of specific slgA in colostrum significantly increased compared with that of IgG. The serum IgG levels of the piglets increased after suckling colostrum produced by sows was previously inoculated with recombinant L. casei. These results indicate that immunization with recombinant L. casei expressing TGEV N protein on its surface elicited high levels of specific slgA and circulating IgG against TGEV N protein.

Coronavirus transmissible gastroenteritis virus antagonizes the antiviral effect of the microRNA miR-27b via the IRE1 pathway

Although the functional parameters of micro RNAs(mi RNAs)have been explored to some extent,the roles of these molecules in coronavirus infection and the regulatory mechanism of mi RNAs in virus infection are still unclear.Transmissible gastroenteritis virus(TGEV)is an enteropathgenic coronavirus and causes high morbidity and mortality in suckling piglets.Here,we demonstrated that microRNA-27b-3p(miR-27b-3p)suppressed TGEV replication by directly targeting porcine suppressor of cytokine signaling 6(SOCS6),while TGEV infection downregulated miR-27b-3p expression in swine testicular(ST)cells and in piglets.Mechanistically,the decrease of miR-27b-3p expression during TGEV infection was mediated by the activated inositolrequiring enzyme 1(IRE1)pathway of the endoplasmic reticulum(ER)stress.Further studies showed that when ER stress was induced by TGEV,IRE1 acted as an RNase activated by autophosphorylation and unconventionally spliced m RNA encoding a potent transcription factor,X-box-binding protein 1(Xbp1s).Xbp1s inhibited the transcription of miR-27 and ultimately reduced the production of miR-27b-3p.Therefore,our findings indicate that TGEV inhibits the expression of an anti-coronavirus micro RNA through the IRE1 pathway and suggest a novel way in which coronavirus regulates the host cell response to infection.

Cloning and Identification of S Gene from Chinese Isolate TH-98 of Transmissible Gastroenteritis Virus

Chinese isolate of transmissible gastroenteritis virus(TGEV)was propagated and harvested in swine testicle(ST)cells.Two pairs of primers were designed according to the published sequence with Oligo 4.1 and DNasis softwares.The products of RT-PCR were named Sa and Sb,of 2.3kb and 2.1kb respectively.Sa was inserted in EcoR I and Kpn I sites after Sb was cloned in Kpn I and Pst I sites of the same pUC18 plasmid.The recombinant designated pUC-S was verified and analyzed by corresponding restriction endonuclease(RE)and nested PCR on the basis of genetic sites of S gene and physical map of pUC18 plasmid,which was identified as S gene from Chinese isolate of TGEV.

Effects of Different Alkaloids in Coptis chinensis on Inhibiting TGEV Proliferation

[Objectives]To study the effects of different alkaloids in Coptis chinensis on inhibiting the proliferation of Transmissible gastroenteritis virus(TGEV).[Methods]The components and content of the main alkaloids in the extract of C.chinensis were analyzed.The main alkaloids were selected as drugs to inhibit the proliferation of TGEV.The maximum non-toxic concentration of Columbamine,Jatrorrhizine,Epiberberine,Coptisine,Palmatine,and Berberine was screened.The protective rate of each drug on TGEV-infected ST cells was determined,and the transcriptional inhibitory effect of the drug on TGEV N gene was detected by fluorescent quantitative PCR.[Results]The extract of C.chinensis mainly contains 6 alkaloids:Columbamine,Jatrorrhizine,Epiberberine,Coptisine,Palmatine,and Berberine,accounting for 2.03%,8.88%,9.21%,15.07%,14.63%,and 50.18%,respectively.In the range of the safe concentration,Jatrorrhizine,Palmatine,and Coptisine had better protective effects on ST cells infected with TGEV;compared with the Columbamine group,the cell protection rate was significantly different(P<0.05);compared with the Berberine group,the difference was extremely significant(P<0.01).The Coptisine and Palmatine groups had significant inhibitory effects on the transcription of TGEV N gene,and the difference was extremely significant compared with the virus group(P<0.05).[Conclusions]Jatrorrhizine and Palmatine in C.chinensis are the main components to inhibit the proliferation of TGEV.

Detection of TGEV Antibody by Enzyme-Linked Immunosorbent Assay Using Recombinant Nucleocapsid Proteins

An enzyme linked immunosorbent assays (ELISA) based on recombinant nucleocapsid (N) protein generated in Escherichiacoli was evaluated for its sensitivity and specificity for diagnosis of transmissible gastroenteritis virus (TGEV) infection.The N gene encoding the N protein was cloned and expressed as a fusion protein with His tag protein in E. coli. Therecombinant N protein migrated at 42 kDa and reacted with His6 tag specific monoclonal antibody by immunoblotting.Recombinant N protein ELISA (rnELISA) demonstrated 97.5% specificity among 80 TGEV-free individuals, and 97.3%sensitivity ranging among 110 clinical samples with TGEV. Taken together, these results indicated that nucleocapsid maybe a useful antigen for the sera-diagnosis of TGEV and it was also suggested that the ELISA is a highly sensitive andspecific test for detecting antibodies against TGEV.

宿主细胞蛋白HSPA2与TGEV Nsp2的相互作用及其对病毒复制的影响

为了探究猪传染性胃肠炎病毒(Transmissible gastroenteritis virus,TGEV)非结构蛋白2(Nsp2)与宿主细胞热休克蛋白70-2(HSPA2)的相互作用,以及HSPA2表达对TGEV复制的影响,试验利用RT-PCR方法获得猪源HSPA2基因并构建其真核表达载体pCMV-Myc-HSPA2,通过Western-blot和间接免疫荧光试验检测真核表达载体pCMV-Myc-HSPA2在IPEC-J2细胞中的表达情况。利用免疫共沉淀(Co-IP)和共定位试验进一步验证TGEV Nsp2和宿主细胞蛋白HSPA2之间是否存在相互作用,利用基因过表达技术在IPEC-J2细胞中过表达HSPA2后接种TGEV,分别测定接种后36,48小时时病毒TCID50。结果表明:真核表达载体pCMV-Myc-HSPA2能够在IPEC-J2细胞中成功表达,HSPA2与Nsp2之间存在相互作用,并且HSPA2表达量升高后TGEV的TCID50略有升高。说明宿主细胞蛋白HSPA2与TGEV Nsp2之间存在相互作用,且HSPA2的表达对TGEV复制具有一定促进作用。