With the implementation of the C-strain vaccine,classical swine fever(CSF) has been under control in China,which is currently in a chronic atypical epidemic situation.African swine fever(ASF) emerged in China in 2018 ...With the implementation of the C-strain vaccine,classical swine fever(CSF) has been under control in China,which is currently in a chronic atypical epidemic situation.African swine fever(ASF) emerged in China in 2018 and spread quickly across the country.It is presently occurring sporadically due to the lack of commercial vaccines and farmers’ increased awareness of biosafety.Atypical porcine pestivirus(APPV) was first detected in Guangdong Province,China,in 2016,which mainly harms piglets and has a local epidemic situation in southern China.These three diseases have similar clinical symptoms in pig herds,which cause considerable losses to the pig industry.They are difficult to be distinguished only by clinical diagnosis.Therefore,developing an early and accurate simultaneous detection and differential diagnosis of the diseases induced by these viruses is essential.In this study,three pairs of specific primers and Taq-man probes were designed from highly conserved genomic regions of CSFV(5’ UTR),African swine fever virus(ASFV)(B646L),and APPV(5’ UTR),followed by the optimization of reaction conditions to establish a multiplex real-time PCR detection assay.The results showed that the method did not cross-react with other swine pathogens(porcine circovirus type 2(PCV2),porcine reproductive and respiratory syndrome virus(PRRSV),foot-and-mouth disease virus(FMDV),pseudorabies virus(PRV),porcine parvovirus(PPV),and bovine viral diarrhea virus BVDV).The sensitivity results showed that CSFV,ASFV,and APPV could be detected as low as 1 copy μL–1;the repeatability results showed that the intra-assay and interassay coefficient of variation of ASFV,CSFV,and APPV was less than 1%.Twenty-two virus samples were detected by the multiplex real-time PCR,compared with national standard diagnostic and patented method assay for CSF(GB/T 27540–2011),ASF(GB/T 18648–2020),and APPV(CN108611442A),respectively.The sensitivity of this triple real-time PCR for CSFV,ASFV,and APPV was almost the same,and the compliance results were the same(100%).A total of 451 clinical samples were detected,and the results showed that the positive rates of CSFV,ASFV,and APPV were 0.22% (1/451),1.3%(6/451),and 0%(0/451),respectively.This assay provides a valuale tool for rapid detection and accurate diagnosis of CSFV,ASFV,and APPV.展开更多
Reverse transcription polymerase chain reaction(RT-PCR) was used for the detection of classical swine fever virus(CSFV) in blood and tissue samples of field cases and experimentally inoculated pigs.The distribution of...Reverse transcription polymerase chain reaction(RT-PCR) was used for the detection of classical swine fever virus(CSFV) in blood and tissue samples of field cases and experimentally inoculated pigs.The distribution of CSFV in different organ samples showed some discrepancies in infected pigs.Four weaner pigs were inoculated with C-strain vaccine virus,then samples of spleen,tonsil,lung,mesenteric lymph node,kidney and brain were collected after slaughter and tested for E2 and NS5B genes using one-step RT-PCR and nested RT-PCR.Using the same method,12 field cases were simultaneously studied.A discrepancy of CSFV in different samples was found upon detecting the target gene.The most reliable diagnostic organs were spleen and tonsil,and the nested RT-PCR assay provided a highly sensitive and specific method with comparable performance to the one-step RT-PCR assay.展开更多
The NS5A non-structural protein of classical swine fever virus(CSFV)is a multifunctional protein involved in viral genomic replication,protein translation,assembly of infectious virus particles,and regulation of cellu...The NS5A non-structural protein of classical swine fever virus(CSFV)is a multifunctional protein involved in viral genomic replication,protein translation,assembly of infectious virus particles,and regulation of cellular signaling pathways.Previous report showed that NS5A inhibited nuclear factor kappa B(NF-κB)signaling induced by poly(I:C);however,the mechanism involved has not been elucidated.Here,we reported that NS5A directly interacted with NF-κB essential modulator(NEMO),a regulatory subunit of the IκB kinase(IKK)complex,to inhibit the NF-κB signaling pathway.Further investigations showed that the zinc finger domain of NEMO and the aa 126–250 segment of NS5A are essential for the interaction between NEMO and NS5A.Mechanistic analysis revealed that NS5A mediated the proteasomal degradation of NEMO.Ubiquitination assay showed that NS5A induced the K27-linked but not the K48-linked polyubiquitination of NEMO for proteasomal degradation.In addition,NS5A blocked the K63-linked polyubiquitination of NEMO,thus inhibiting IKK phosphorylation,IκBαdegradation,and NF-κB activation.These findings revealed a novel mechanism by which CSFV inhibits host innate immunity,which might guide the drug design against CSFV in the future.展开更多
Large-scale production of cell culture-based classical swine fever virus(CSFV)vaccine is hampered by the adverse reactions caused by contaminants from host cell and culture medium.Hence,we have developed an efficient ...Large-scale production of cell culture-based classical swine fever virus(CSFV)vaccine is hampered by the adverse reactions caused by contaminants from host cell and culture medium.Hence,we have developed an efficient method for purifying CSFV from cell-culture medium.Pure viral particles were obtained with two steps of tangential-flow filtration(TFF)and size-exclusion chromatography(SEC),and were compared with particles from ultracentrifugation by transmission electron microscopy(TEM),infectivity and recovery test,and real time fluorescent quantitative PCR(FQ-PCR).TFF concentrated the virus particles effectively with a retention rate of 98.5%,and 86.2%of viral particles were obtained from the ultrafiltration retentate through a Sepharose 4 F F column on a biological liquid chromatography system.CSFV purified by TFF-SEC or ultracentrifugation were both biologically active from 1.0×10–4.25 TCID50·mL^(–1) to 3.0×10^(–6.25) TCID50·mL^(–1),but the combination of TFF and SEC produced more pure virus particles than by ultracentrifugation alone.In addition,pure CSFV particles with the expected diameter of 40–60 nm were roughly spherical without any visible contamination.Mice immunized with CSFV purified by TFF-SEC produced higher antibody levels compared with immunization with ultracentrifugation-purified CSFV(P<0.05).The purification procedures in this study are reliable technically and feasible for purification of large volumes of viruses.展开更多
基金supported by the National Natural Science Foundation of China (31872484) to Zhang Qianyithe Non-profit Key Program of Veterinary Drug Industry from China Institute of Veterinary Drug Control (GY202011) to Xia Yingju。
文摘With the implementation of the C-strain vaccine,classical swine fever(CSF) has been under control in China,which is currently in a chronic atypical epidemic situation.African swine fever(ASF) emerged in China in 2018 and spread quickly across the country.It is presently occurring sporadically due to the lack of commercial vaccines and farmers’ increased awareness of biosafety.Atypical porcine pestivirus(APPV) was first detected in Guangdong Province,China,in 2016,which mainly harms piglets and has a local epidemic situation in southern China.These three diseases have similar clinical symptoms in pig herds,which cause considerable losses to the pig industry.They are difficult to be distinguished only by clinical diagnosis.Therefore,developing an early and accurate simultaneous detection and differential diagnosis of the diseases induced by these viruses is essential.In this study,three pairs of specific primers and Taq-man probes were designed from highly conserved genomic regions of CSFV(5’ UTR),African swine fever virus(ASFV)(B646L),and APPV(5’ UTR),followed by the optimization of reaction conditions to establish a multiplex real-time PCR detection assay.The results showed that the method did not cross-react with other swine pathogens(porcine circovirus type 2(PCV2),porcine reproductive and respiratory syndrome virus(PRRSV),foot-and-mouth disease virus(FMDV),pseudorabies virus(PRV),porcine parvovirus(PPV),and bovine viral diarrhea virus BVDV).The sensitivity results showed that CSFV,ASFV,and APPV could be detected as low as 1 copy μL–1;the repeatability results showed that the intra-assay and interassay coefficient of variation of ASFV,CSFV,and APPV was less than 1%.Twenty-two virus samples were detected by the multiplex real-time PCR,compared with national standard diagnostic and patented method assay for CSF(GB/T 27540–2011),ASF(GB/T 18648–2020),and APPV(CN108611442A),respectively.The sensitivity of this triple real-time PCR for CSFV,ASFV,and APPV was almost the same,and the compliance results were the same(100%).A total of 451 clinical samples were detected,and the results showed that the positive rates of CSFV,ASFV,and APPV were 0.22% (1/451),1.3%(6/451),and 0%(0/451),respectively.This assay provides a valuale tool for rapid detection and accurate diagnosis of CSFV,ASFV,and APPV.
基金supported by the grants from the National"863" Programme (2006AA10A2041)Development Programme of Science and Technology,Chengguan District of Gansu province (08-5-4)
文摘Reverse transcription polymerase chain reaction(RT-PCR) was used for the detection of classical swine fever virus(CSFV) in blood and tissue samples of field cases and experimentally inoculated pigs.The distribution of CSFV in different organ samples showed some discrepancies in infected pigs.Four weaner pigs were inoculated with C-strain vaccine virus,then samples of spleen,tonsil,lung,mesenteric lymph node,kidney and brain were collected after slaughter and tested for E2 and NS5B genes using one-step RT-PCR and nested RT-PCR.Using the same method,12 field cases were simultaneously studied.A discrepancy of CSFV in different samples was found upon detecting the target gene.The most reliable diagnostic organs were spleen and tonsil,and the nested RT-PCR assay provided a highly sensitive and specific method with comparable performance to the one-step RT-PCR assay.
基金the National Nature Science Foundation of China(Grant No.31972677)the Construction Project of Liaoning Provincial Key Laboratory,China(2022JH13/10200026).
文摘The NS5A non-structural protein of classical swine fever virus(CSFV)is a multifunctional protein involved in viral genomic replication,protein translation,assembly of infectious virus particles,and regulation of cellular signaling pathways.Previous report showed that NS5A inhibited nuclear factor kappa B(NF-κB)signaling induced by poly(I:C);however,the mechanism involved has not been elucidated.Here,we reported that NS5A directly interacted with NF-κB essential modulator(NEMO),a regulatory subunit of the IκB kinase(IKK)complex,to inhibit the NF-κB signaling pathway.Further investigations showed that the zinc finger domain of NEMO and the aa 126–250 segment of NS5A are essential for the interaction between NEMO and NS5A.Mechanistic analysis revealed that NS5A mediated the proteasomal degradation of NEMO.Ubiquitination assay showed that NS5A induced the K27-linked but not the K48-linked polyubiquitination of NEMO for proteasomal degradation.In addition,NS5A blocked the K63-linked polyubiquitination of NEMO,thus inhibiting IKK phosphorylation,IκBαdegradation,and NF-κB activation.These findings revealed a novel mechanism by which CSFV inhibits host innate immunity,which might guide the drug design against CSFV in the future.
基金the Special Fund for Agro-scientific Research in the Public Interest(201203039)China Agriculture Research System(CARS-36).
文摘Large-scale production of cell culture-based classical swine fever virus(CSFV)vaccine is hampered by the adverse reactions caused by contaminants from host cell and culture medium.Hence,we have developed an efficient method for purifying CSFV from cell-culture medium.Pure viral particles were obtained with two steps of tangential-flow filtration(TFF)and size-exclusion chromatography(SEC),and were compared with particles from ultracentrifugation by transmission electron microscopy(TEM),infectivity and recovery test,and real time fluorescent quantitative PCR(FQ-PCR).TFF concentrated the virus particles effectively with a retention rate of 98.5%,and 86.2%of viral particles were obtained from the ultrafiltration retentate through a Sepharose 4 F F column on a biological liquid chromatography system.CSFV purified by TFF-SEC or ultracentrifugation were both biologically active from 1.0×10–4.25 TCID50·mL^(–1) to 3.0×10^(–6.25) TCID50·mL^(–1),but the combination of TFF and SEC produced more pure virus particles than by ultracentrifugation alone.In addition,pure CSFV particles with the expected diameter of 40–60 nm were roughly spherical without any visible contamination.Mice immunized with CSFV purified by TFF-SEC produced higher antibody levels compared with immunization with ultracentrifugation-purified CSFV(P<0.05).The purification procedures in this study are reliable technically and feasible for purification of large volumes of viruses.
