1.Genome sequence and bioinformatics analysis of an ovine-derived Salmonella enterica strain
Yao WANG ; Longbin KANG ; Renjie WU ; Qiuyong CHEN ; Longbai WANG ; Dingping BAI ; Lunjiang ZHOU ; Yongliang CHE
Chinese Journal of Veterinary Science 2025;45(10):2187-2195
To elucidate the genomic characteristics of Salmonella strains derived from sheep,this study employed various methods,including bacterial isolation and identification,biochemical identi-fication,pathogenicity test,whole-genome sequencing,and BLAST comparison,along with the screening of integrative conjugative elements(ICE)using ICEfinder and EasyFig for comparative analysis,as well as plasmid comparisons utilizing PlasmidBrig.The results revealed the isolation of a Gram-negative,non-spore-forming bacillus from nasal swabs of diseased sheep,which formed gray-white,smooth-surfaced,and neatly edged circular colonies on TSA sheep blood agar.On XLT-4 agar medium,it produced smooth-surfaced,white,circular colonies.The bacterium was identified as Salmonella enterica through 16S rRNA sequencing and biochemical identification.This bacteri-um induces hemorrhaging in the intestines of guinea pigs,resulting in their demise within a 48-hour period.The pathogen exhibits high virulence.Whole-genome alignment demonstrated a high degree of homology with Salmonella enterica subsp.diarizonae serotype 61:k:1,5,(7).ICE screen-ing and comparative analysis indicated the presence of a novel ICE in this strain,characterized by a core structural framework that includes an integrative shear module,a mobilizable processing mod-ule,a conjugative pair formation module,and a regulatory module.Notably,ICE from different spe-cies containing the same integrase exhibited identical inverted repeat sequences and insertion sites at tRNAPhe.Plasmid homology comparisons revealed that plasmid sequences from different strains of Salmonella enterica subsp.diarizonae serotype 61:k:1,5,(7)also showed high homology;however,the homology with plasmid sequences from other Salmonella and Escherichia coli strains was only 50%.These findings indicate that the isolated strain is Salmonella enterica subsp.diarizonae serotype 61:k:1,5,(7)and contains a novel ICE as well as a plasmid.This study fur-ther enriches the molecular epidemiology of Salmonella and provides a theoretical basis for the prevention and control of infections caused by this pathogen.
2.Establishment of Vero cell line overexpressing pAPN gene and its effect on porcine epidemic diarrhea virus replication
Qiuyong CHEN ; Zhihua SUN ; Rujing CHEN ; Xuemin WU ; Renjie WU ; Jinli QIU ; Bing HE ; Yutao LIU ; Longbai WANG ; Lunjiang ZHOU
Chinese Journal of Veterinary Science 2025;45(2):181-186
pAPN is a zinc-dependent metalloprotease,mediating the fusion between virus and host cell,and playing a role as the receptor of coronavirus.To explore the effect of pAPN on PEDV rep-lication,the full-length pAPN gene was amplified from the porcine small intestinal by PCR,and was cloned into the lentiviral vector via the homologous site digested with BamH Ⅰ and Not Ⅰ to obtain the recombinant lentiviral vector PLVX-pAPN-mCMV-ZsGreen1-puro.The recombinant lentiviral vector and helper plasmids pLP1,pLP2,pLP-VSVG were co-transfected into 293T cells for lentiviral packaging.Vero cells were infected with the packaged lentivirus and the pAPN gene overexpressing cells were screened by puromycin.The stable expression of Vero-pAPN monoclonal cell line was screened by a limited dilution method,and the effect of the cell line on the replication of PEDV was determined by qPCR for N mRNA transcription level,Western blot for N protein level,and TCID50.The results showed that the packaged lentivirus could infect Vero cells,and the monoclonal cell line Vero-pAPN(2C5)could stably expressed pAPN.The Vero-pAPN cell line can promote the replication of PEDV,the N gene mRNA transcription level was significantly different at 12-48 h(P<0.05),the N protein expression level increased,and the TCID50 was significantly different at 24 and 48 h(P<0.05).In conclusion,the Vero-pAPN cell line was constructed in this study and it can significantly promote the replication of PEDV,which provides a candidate cell line for PEDV vaccine production and isolation.
3.Genome sequence and bioinformatics analysis of an ovine-derived Salmonella enterica strain
Yao WANG ; Longbin KANG ; Renjie WU ; Qiuyong CHEN ; Longbai WANG ; Dingping BAI ; Lunjiang ZHOU ; Yongliang CHE
Chinese Journal of Veterinary Science 2025;45(10):2187-2195
To elucidate the genomic characteristics of Salmonella strains derived from sheep,this study employed various methods,including bacterial isolation and identification,biochemical identi-fication,pathogenicity test,whole-genome sequencing,and BLAST comparison,along with the screening of integrative conjugative elements(ICE)using ICEfinder and EasyFig for comparative analysis,as well as plasmid comparisons utilizing PlasmidBrig.The results revealed the isolation of a Gram-negative,non-spore-forming bacillus from nasal swabs of diseased sheep,which formed gray-white,smooth-surfaced,and neatly edged circular colonies on TSA sheep blood agar.On XLT-4 agar medium,it produced smooth-surfaced,white,circular colonies.The bacterium was identified as Salmonella enterica through 16S rRNA sequencing and biochemical identification.This bacteri-um induces hemorrhaging in the intestines of guinea pigs,resulting in their demise within a 48-hour period.The pathogen exhibits high virulence.Whole-genome alignment demonstrated a high degree of homology with Salmonella enterica subsp.diarizonae serotype 61:k:1,5,(7).ICE screen-ing and comparative analysis indicated the presence of a novel ICE in this strain,characterized by a core structural framework that includes an integrative shear module,a mobilizable processing mod-ule,a conjugative pair formation module,and a regulatory module.Notably,ICE from different spe-cies containing the same integrase exhibited identical inverted repeat sequences and insertion sites at tRNAPhe.Plasmid homology comparisons revealed that plasmid sequences from different strains of Salmonella enterica subsp.diarizonae serotype 61:k:1,5,(7)also showed high homology;however,the homology with plasmid sequences from other Salmonella and Escherichia coli strains was only 50%.These findings indicate that the isolated strain is Salmonella enterica subsp.diarizonae serotype 61:k:1,5,(7)and contains a novel ICE as well as a plasmid.This study fur-ther enriches the molecular epidemiology of Salmonella and provides a theoretical basis for the prevention and control of infections caused by this pathogen.
4.Establishment of Vero cell line overexpressing pAPN gene and its effect on porcine epidemic diarrhea virus replication
Qiuyong CHEN ; Zhihua SUN ; Rujing CHEN ; Xuemin WU ; Renjie WU ; Jinli QIU ; Bing HE ; Yutao LIU ; Longbai WANG ; Lunjiang ZHOU
Chinese Journal of Veterinary Science 2025;45(2):181-186
pAPN is a zinc-dependent metalloprotease,mediating the fusion between virus and host cell,and playing a role as the receptor of coronavirus.To explore the effect of pAPN on PEDV rep-lication,the full-length pAPN gene was amplified from the porcine small intestinal by PCR,and was cloned into the lentiviral vector via the homologous site digested with BamH Ⅰ and Not Ⅰ to obtain the recombinant lentiviral vector PLVX-pAPN-mCMV-ZsGreen1-puro.The recombinant lentiviral vector and helper plasmids pLP1,pLP2,pLP-VSVG were co-transfected into 293T cells for lentiviral packaging.Vero cells were infected with the packaged lentivirus and the pAPN gene overexpressing cells were screened by puromycin.The stable expression of Vero-pAPN monoclonal cell line was screened by a limited dilution method,and the effect of the cell line on the replication of PEDV was determined by qPCR for N mRNA transcription level,Western blot for N protein level,and TCID50.The results showed that the packaged lentivirus could infect Vero cells,and the monoclonal cell line Vero-pAPN(2C5)could stably expressed pAPN.The Vero-pAPN cell line can promote the replication of PEDV,the N gene mRNA transcription level was significantly different at 12-48 h(P<0.05),the N protein expression level increased,and the TCID50 was significantly different at 24 and 48 h(P<0.05).In conclusion,the Vero-pAPN cell line was constructed in this study and it can significantly promote the replication of PEDV,which provides a candidate cell line for PEDV vaccine production and isolation.
5.Therapeutic value of endoscopic resection of tumors originated from gastric fundus muscularis propria
Qiuyong XU ; Junjie CHEN ; Yadong LAI ; Hui PU ; Qiying LIU ; Shuhui LIN ; Xuefen CHEN ; Xiangnong XU
Chinese Journal of Digestive Endoscopy 2015;(3):175-179
Objective To investigate the safety and effectiveness of endoscopic resection of tumors originated from gastric fundus muscularis propria.Methods Data of 53 patients with tumors originated from gastric fundus muscularis propria detected by endoscopic ultrasonograpy,treated by endoscopic resection and followed up at our hospital between January 2012 and June 2014 were reviewed.The postoperative pathology and complications were retrospectively analyzed to evaluate the therapeutic effect and safety.Results The procedure was successfully performed on all patients and all lesions were removed in one procedure.The lesion size ranged from 0.5 to 4.5 cm and the operation time was 25-155 min[mean(46.7 ±18.2)min].Mild bleeding (5 ~150 ml)occurred in all cases,which was successfully managed by argon plasma coagulation,hot biopsy probe or endoclip.Perforation occurred in 8 patients(8 /53),seven of whom were closed with titanium clips and titanium clips combined with nylon cord.Laparoscopic intervention was applied to 1 case because of severe perforation.Gastrointestinal decompression,acid suppression with proton pump inhibitors and antibiotics were performed on all cases.No severe hemorrhage occurred.The average length of hospitalization was (5.3 ± 1.4)days(3-14 d).Pathology confirmed 46 cases of gastrointestinal stromal tumors and 7 cases of leiomyoma. The patients were followed up for 3 to 27 months,and no tumor residue or recurrence was observed. Conclusion Endoscopic resection is a method not only to get the accurate pathologic diagnosis but also to meet principle of the local resection for stomach.It is safe,effective and worthy of recommendation.

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