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 soft...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.展开更多
The complete genome sequence of transmissible Gastroenteritis virus (TGEV) strain TS, previously isolated from Gansu province, was cloned and compared with published sequence data from other TGEV strains. Phylogenetic...The complete genome sequence of transmissible Gastroenteritis virus (TGEV) strain TS, previously isolated from Gansu province, was cloned and compared with published sequence data from other TGEV strains. Phylogenetic tree analysis based on the amino acid and nucleotide sequences of the S gene showed that the TGEV strains were divided into 3 clusters. TGEV TS showed a close evolutionary relationship to the American Miller cluster but had a 5' non-translated region (NTR) sequence closely related to the American Purdue cluster. Continued culture in different cell types indicated that TGEV TS virulence could be attenuated after fifty passages in Porcine kidney (PK-15) cells, and that the Porcine kidney cell line IB-RS-2 (IBRS) was not suitable for culture of the TGEV strain TS.展开更多
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...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.展开更多
[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...[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.展开更多
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)...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.展开更多
文摘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.
基金National Basic Research Program(2004CCA00500)National High-tech Development Research Program of China (2006AA02Z440)
文摘The complete genome sequence of transmissible Gastroenteritis virus (TGEV) strain TS, previously isolated from Gansu province, was cloned and compared with published sequence data from other TGEV strains. Phylogenetic tree analysis based on the amino acid and nucleotide sequences of the S gene showed that the TGEV strains were divided into 3 clusters. TGEV TS showed a close evolutionary relationship to the American Miller cluster but had a 5' non-translated region (NTR) sequence closely related to the American Purdue cluster. Continued culture in different cell types indicated that TGEV TS virulence could be attenuated after fifty passages in Porcine kidney (PK-15) cells, and that the Porcine kidney cell line IB-RS-2 (IBRS) was not suitable for culture of the TGEV strain TS.
文摘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.
基金Supported by General Project of Chongqing Natural Science Foundation(cstc2020jcyj-msxmX0757)Pilot Project of National Center of Technology Innovation for Pigs(NCTIP-XD/B12)Regional Innovation Cooperation in Sichuan Province(2022YFQ0024)。
文摘[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.
基金The present study was supported by Sichuan Science and Technology Program(2021ZDZX0009)the Sichuan Pig Innovation Team of National Modern Agricultural Industry Technology System of China(scsztd-2021-08-11)the Sichuan Natural Science Foundation of China(2023NSFSC1141).
文摘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.
