A simple and rapid assay for the detection of Classical swine fever virus(CSFV)was established using reverse transcription loop-mediated isothermal amplification(RT-LAMP).This study describes the amplification of the ...A simple and rapid assay for the detection of Classical swine fever virus(CSFV)was established using reverse transcription loop-mediated isothermal amplification(RT-LAMP).This study describes the amplification of the genomic RNA of CSFV under isothermal conditions(63℃)within one hour,using a set of six primers(two outer primers,two inner primers and two loop primers).This RT-LAMP assay showed 100-fold higher sensitivity than the standard RT-PCR method and identified eighteen additional positive cases that were negative when tested by RT-PCR.This RT-LAMP was able to detect all the 13 strains of CSFV but not the BVDV.PRRSV.SIV. PRV-PCV,thus showed a good specificity.Products amplified by RT-LAMP can be visualized by agarose gel electrophoresis and in addition,either as a white precipitate at the bottom of the tube after a pulse spin or as a color change when dyed with SYBR Green I which are visible to the naked eye.Because RT-LAMP is low-cost and produces rapid results,it has the potential to be an excellent tool for CSFV surveillance in the field,especially in developing countries.展开更多
In this study,a synthesized quadruple antigenic epitope gene region of the classical swine fever virus (CSFV)E2 glycoprotein was expressed in E.coli to a obtain target protein.This target protein was used as a coating...In this study,a synthesized quadruple antigenic epitope gene region of the classical swine fever virus (CSFV)E2 glycoprotein was expressed in E.coli to a obtain target protein.This target protein was used as a coating antigen to establish an indirect ELISA for specifically detecting anti-CSFV antibodies in serum samples from pigs.The P/N cut-off value of this assay was 1.92 by receiver operating characteristic curve(ROC)analysis based on 30 negative sera and 80 positive samples.The test gave 97.5%sensitivity and 96.7%specificity compared with the indirect hemagglutination(IHA)test.The inter-assay and intra-assay coefficients of variation (CVs)for 16 sera were both≤6.8%.No cross-reactivity between the coating antigen and anti-bovine viral diarrhoea virus(BVDV)antibodies was observed.展开更多
Full genomic sequence of a newly isolated persistent infection strain of classical swine fever virus was firstly determined. It was demonstrated by sequence analyses that nucleotides homologies of this strain compared...Full genomic sequence of a newly isolated persistent infection strain of classical swine fever virus was firstly determined. It was demonstrated by sequence analyses that nucleotides homologies of this strain compared with virulent Shimen and vaccine HCLV were 89.7%and 87.7%, and homologies of amino acids were 94.8%and 93.3%, respectively. The sequencing results primarily suggest a tighter relationship between this persistent infection strain and virulent Shimen strain than vaccine HCLV strain.展开更多
Classical swine fever virus (CSFV) is the causative agent of Classical swine fever which is a highly contagious disease affecting swine and resulting in severe economic losses. In this study, we developed reverse tran...Classical swine fever virus (CSFV) is the causative agent of Classical swine fever which is a highly contagious disease affecting swine and resulting in severe economic losses. In this study, we developed reverse transcription loopmediated isothermal amplification (RT-LAMP) assay targeting the 5’UTR gene for the detection of CSFV. This amplification method can be obtained in 1 h under isothermal conditions (65°C) employing a set of six specific primers mixtures. Amplification product was visualized by using hydroxynaphthol blue (HNB) dye and agarose gel electrophoresis. The sensitivity was 100 copy numbers. No cross-reactivity related to Japanese encephalitis virus (JEV) and porcine reproductive and respiratory syndrome virus (PRRSV) was demonstrated. The results demonstrated that the RT-LAMP assay is a useful tool for the rapid and sensitive for CSFV detection in swine.展开更多
A multi-epitope-vaccine MEVABc consisting of two linear neutralizing determinants （BCI： aa693-716; A6： aa844-865） located on antigenic unit B/C and unit A of glycoprotein E2 was prepared to evaluate whether a comb...A multi-epitope-vaccine MEVABc consisting of two linear neutralizing determinants （BCI： aa693-716; A6： aa844-865） located on antigenic unit B/C and unit A of glycoprotein E2 was prepared to evaluate whether a combination strategy is effective in the design of peptide vaccines. After immunization, pig sera collected every one to two weeks were evaluated by enzyme linked immunosorbent assay. C-straininduced anti-sera and hyper-immune sera cannot recognize overlapping peptides that cover the E2 N-terminus, while MEVAgC is able to elicit high levels of peptide-specific antibody response. When compared with previously studied peptide vaccines PV-BC1 and PV-A6, the same dose of either component in the MEMABc increases the BC1- or A6-specific antibodies （to 1/3-1/2 of the levels of the separate vaccines）. However, the synergy between the antibodies may make MEVAgc much more potent. Moreover, anti-C-strain immunity pre-existing in pigs does not disturb the sequent MEVABc vaccination. Thus, MEVABc can be administrated to pigs which already possess anti-classical swine fever virus immunity. MEVAgC is a promising candidate marker vaccine.展开更多
Ems is a highly glycosylated envelope protein of classical swine fever virus （CSFV） with RNase ac- tivity. Ems can induce neutralizing antibodies and provide immune protection against CSFV infection. In this study, ...Ems is a highly glycosylated envelope protein of classical swine fever virus （CSFV） with RNase ac- tivity. Ems can induce neutralizing antibodies and provide immune protection against CSFV infection. In this study, the RNase domain of the Ems was produced in Eschenchia coil. Its reactivity with CSFV-positive sera and its ability to induce antibodies and to provide protective immunity were then investigated. The serological tests showed that the prokaryotically expressed RNase domain of the Ems retained its antigenicity and in- duced high titers of humoral responses. However, only partial protection and a limited amount of neutralizing antibodies were demonstrated by an in vitro neutralization test and an immunization/challenge test. The re- sults suggest that other essential factors rather than simply enhancing the immunogenicity of Ems should be taken into consideration when Er＂s is enrolled as one of the components of a candidate vaccine.展开更多
Previous investigations demonstrated that the envelope glycoprotein E2 (gp55) of classical swine fever virus (CSFV) is the most immunogenic protein. Interestingly, recombinant protein E2 that contains only one stru...Previous investigations demonstrated that the envelope glycoprotein E2 (gp55) of classical swine fever virus (CSFV) is the most immunogenic protein. Interestingly, recombinant protein E2 that contains only one structural antigenic unit (unit B/C or A) could protect pigs from a lethal challenge of CSFV. Based on these findings, we designed and prepared five overlapping synthetic peptides that covered the sequence unit B/C (aa 693777) of Shimen E2 and conjugated individual peptides with bovine serum albumin (BSA). After the vaccination, the specificity of the rabbit sera was analyzed in the enzyme linked immunosorbent assay (ELISA) and the fast protein liquid chromatography (FPLC). The results show that each of the five candidate peptide vaccines can successfully induce a high titer of specific antibodies in New Zealand White Rabbits (n=3). Subsequently, the five candidate peptide vaccines were applied in combination for immunization of pigs (n=10) and induced specific and strong humoral responses against all of the five designed peptides in pigs. Our studies indicate that the candidate multi peptide vaccine would prove an excellent marker vaccine against CSFV and provide a model for developing effective synthetic peptide vaccines to stop viral epidemics in humans and animals.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
Full E2 gene fragment of Classical swine fever virus (CSFV)shimen strain was amplified by RT-PCR method, then a specific fraction -the A, D antigenic domains- , which encodes protein with good antigenicity and suitabl...Full E2 gene fragment of Classical swine fever virus (CSFV)shimen strain was amplified by RT-PCR method, then a specific fraction -the A, D antigenic domains- , which encodes protein with good antigenicity and suitable for being expressed in E. coli with high level, was amplified respectively by 2 different pair of primers. Then the 2 amplified fragments were tandemly linked and inserted into prokaryotic expressing vector pET-32a to obtain the plasmid pET-2e. The SDS-PAGE assay showed that although the proteins were present in the form of inclusion body, the linked proteins were expressed from plasmid pET-2e as expected, and can be expressed with high level when induced with IPTG.Western-blotting showed good antigenicity of the target protein.展开更多
E2 is an envelope glycoprotein of Classical swine fever virus (CSFV) and contains sequential neutralizing epitopes to induce virus-neutralizing antibodies and mount protective immunity in the natural host. In this stu...E2 is an envelope glycoprotein of Classical swine fever virus (CSFV) and contains sequential neutralizing epitopes to induce virus-neutralizing antibodies and mount protective immunity in the natural host. In this study, four antigen domains (ABCD) of the E2 gene was cloned from CSFV Shimen strain into the retroviral vector pBABE puro and expressed in eukaryotic cell (PK15) by an retroviral gene expression system, and the activity of recombinant E2 protein to induce immune responses was evaluated in rabbits. The results indicated that recombinant E2 protein can be recognized by fluorescence antibodies of CSFV and CSFV positive serum (Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China) using Western blot, indirect immunofluorescence antibody test (IFAT) and ELISA, Furthermore, anti-CSFV specific antibodies and lymphocyte proliferation were elicited and increased by recombinant protein after vaccination. In the challenge test, all of rabbits vaccinated with recombinant protein and Chinese vaccine strain (C-strain) were fully protected from a rabbit spleen virus challenge. These results indicated that a retroviral-based epitope-vaccine carrying the major antigen domains of E2 is able to induce high level of epitope-specific antibodies and exhibits similar protective capability with that induced by the C-strain, and encourages further work towards the development of a vaccine against CSFV infection.展开更多
【目的】全面了解中国猪群中猪瘟病毒(CSFV)的流行感染情况,为后期CSFV的流行病学调查提供证据。【方法】本研究在中国知网、万方、维普、PubMed、Web of Science和Science Direct国内外6种主要数据库中检索中国猪群CSFV流行情况的相关...【目的】全面了解中国猪群中猪瘟病毒(CSFV)的流行感染情况,为后期CSFV的流行病学调查提供证据。【方法】本研究在中国知网、万方、维普、PubMed、Web of Science和Science Direct国内外6种主要数据库中检索中国猪群CSFV流行情况的相关文献,检索时间范围为2005年1月1日-2023年1月1日。通过文献筛选、文献质量评分和CSFV流行率的数据提取,对文献中提取的数据利用StataMP17软件进行Meta分析和系统评价,分析CSFV的整体流行率,利用Meta回归分析和亚组分析CSFV整体流行率的异质性来源和影响因素。【结果】从6种主要数据库中共筛选出55篇CSFV流行情况的相关文献纳入本次Meta分析及系统评价,其中18篇文献被纳为高质量文献,37篇文献纳为中等质量文献。Meta分析结果表明,2005-2023年中国猪群中CSFV的整体流行率为12%(95%CI:0.09~0.15);Meta回归分析和亚组分析结果表明,文献的异质性来源不包括采样时间和猪种类(P>0.05),而影响CSFV流行率的因素包括诊断方法、季节变化、气候因素、海拔高度、纬度因素、经度因素及地区分布(P<0.05)。【结论】本研究从数据上确定了中国猪群中CSFV整体流行情况,其整体流行率不高,流行趋势为地区性的散在流行,以慢性和非典型毒株流行为主。本研究结果对掌握猪群CSFV流行的全局状况和有效防控及猪瘟(CSF)净化有一定的参考意义。展开更多
基金The National Science and Technology supporting plan of the Eleventh Five-year(2006BAD06A18 and 2006BAD06A03)Beijing Natural Science Foundation(5072041)
文摘A simple and rapid assay for the detection of Classical swine fever virus(CSFV)was established using reverse transcription loop-mediated isothermal amplification(RT-LAMP).This study describes the amplification of the genomic RNA of CSFV under isothermal conditions(63℃)within one hour,using a set of six primers(two outer primers,two inner primers and two loop primers).This RT-LAMP assay showed 100-fold higher sensitivity than the standard RT-PCR method and identified eighteen additional positive cases that were negative when tested by RT-PCR.This RT-LAMP was able to detect all the 13 strains of CSFV but not the BVDV.PRRSV.SIV. PRV-PCV,thus showed a good specificity.Products amplified by RT-LAMP can be visualized by agarose gel electrophoresis and in addition,either as a white precipitate at the bottom of the tube after a pulse spin or as a color change when dyed with SYBR Green I which are visible to the naked eye.Because RT-LAMP is low-cost and produces rapid results,it has the potential to be an excellent tool for CSFV surveillance in the field,especially in developing countries.
文摘In this study,a synthesized quadruple antigenic epitope gene region of the classical swine fever virus (CSFV)E2 glycoprotein was expressed in E.coli to a obtain target protein.This target protein was used as a coating antigen to establish an indirect ELISA for specifically detecting anti-CSFV antibodies in serum samples from pigs.The P/N cut-off value of this assay was 1.92 by receiver operating characteristic curve(ROC)analysis based on 30 negative sera and 80 positive samples.The test gave 97.5%sensitivity and 96.7%specificity compared with the indirect hemagglutination(IHA)test.The inter-assay and intra-assay coefficients of variation (CVs)for 16 sera were both≤6.8%.No cross-reactivity between the coating antigen and anti-bovine viral diarrhoea virus(BVDV)antibodies was observed.
基金Supported by National Basic Research Developmental Project ( G19990 1190 0 ) . Gen Bank NO.:AF40 7339
文摘Full genomic sequence of a newly isolated persistent infection strain of classical swine fever virus was firstly determined. It was demonstrated by sequence analyses that nucleotides homologies of this strain compared with virulent Shimen and vaccine HCLV were 89.7%and 87.7%, and homologies of amino acids were 94.8%and 93.3%, respectively. The sequencing results primarily suggest a tighter relationship between this persistent infection strain and virulent Shimen strain than vaccine HCLV strain.
文摘Classical swine fever virus (CSFV) is the causative agent of Classical swine fever which is a highly contagious disease affecting swine and resulting in severe economic losses. In this study, we developed reverse transcription loopmediated isothermal amplification (RT-LAMP) assay targeting the 5’UTR gene for the detection of CSFV. This amplification method can be obtained in 1 h under isothermal conditions (65°C) employing a set of six specific primers mixtures. Amplification product was visualized by using hydroxynaphthol blue (HNB) dye and agarose gel electrophoresis. The sensitivity was 100 copy numbers. No cross-reactivity related to Japanese encephalitis virus (JEV) and porcine reproductive and respiratory syndrome virus (PRRSV) was demonstrated. The results demonstrated that the RT-LAMP assay is a useful tool for the rapid and sensitive for CSFV detection in swine.
基金the National Natural Science Foundation of China (No. 30221003)
文摘A multi-epitope-vaccine MEVABc consisting of two linear neutralizing determinants （BCI： aa693-716; A6： aa844-865） located on antigenic unit B/C and unit A of glycoprotein E2 was prepared to evaluate whether a combination strategy is effective in the design of peptide vaccines. After immunization, pig sera collected every one to two weeks were evaluated by enzyme linked immunosorbent assay. C-straininduced anti-sera and hyper-immune sera cannot recognize overlapping peptides that cover the E2 N-terminus, while MEVAgC is able to elicit high levels of peptide-specific antibody response. When compared with previously studied peptide vaccines PV-BC1 and PV-A6, the same dose of either component in the MEMABc increases the BC1- or A6-specific antibodies （to 1/3-1/2 of the levels of the separate vaccines）. However, the synergy between the antibodies may make MEVAgc much more potent. Moreover, anti-C-strain immunity pre-existing in pigs does not disturb the sequent MEVABc vaccination. Thus, MEVABc can be administrated to pigs which already possess anti-classical swine fever virus immunity. MEVAgC is a promising candidate marker vaccine.
基金Supported by the National Natural Science Foundation of China (No. 30221003) and the Beijing Feikai Biotech Ltd
文摘Ems is a highly glycosylated envelope protein of classical swine fever virus （CSFV） with RNase ac- tivity. Ems can induce neutralizing antibodies and provide immune protection against CSFV infection. In this study, the RNase domain of the Ems was produced in Eschenchia coil. Its reactivity with CSFV-positive sera and its ability to induce antibodies and to provide protective immunity were then investigated. The serological tests showed that the prokaryotically expressed RNase domain of the Ems retained its antigenicity and in- duced high titers of humoral responses. However, only partial protection and a limited amount of neutralizing antibodies were demonstrated by an in vitro neutralization test and an immunization/challenge test. The re- sults suggest that other essential factors rather than simply enhancing the immunogenicity of Ems should be taken into consideration when Er＂s is enrolled as one of the components of a candidate vaccine.
基金Supported by the National Key Basic Research Specific Funds(No.G19990 75 60 7) the National Science Foundation for Outstanding Young Scientists of China (No.3 0 0 2 5 0 3 8)
文摘Previous investigations demonstrated that the envelope glycoprotein E2 (gp55) of classical swine fever virus (CSFV) is the most immunogenic protein. Interestingly, recombinant protein E2 that contains only one structural antigenic unit (unit B/C or A) could protect pigs from a lethal challenge of CSFV. Based on these findings, we designed and prepared five overlapping synthetic peptides that covered the sequence unit B/C (aa 693777) of Shimen E2 and conjugated individual peptides with bovine serum albumin (BSA). After the vaccination, the specificity of the rabbit sera was analyzed in the enzyme linked immunosorbent assay (ELISA) and the fast protein liquid chromatography (FPLC). The results show that each of the five candidate peptide vaccines can successfully induce a high titer of specific antibodies in New Zealand White Rabbits (n=3). Subsequently, the five candidate peptide vaccines were applied in combination for immunization of pigs (n=10) and induced specific and strong humoral responses against all of the five designed peptides in pigs. Our studies indicate that the candidate multi peptide vaccine would prove an excellent marker vaccine against CSFV and provide a model for developing effective synthetic peptide vaccines to stop viral epidemics in humans and animals.
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.
文摘Full E2 gene fragment of Classical swine fever virus (CSFV)shimen strain was amplified by RT-PCR method, then a specific fraction -the A, D antigenic domains- , which encodes protein with good antigenicity and suitable for being expressed in E. coli with high level, was amplified respectively by 2 different pair of primers. Then the 2 amplified fragments were tandemly linked and inserted into prokaryotic expressing vector pET-32a to obtain the plasmid pET-2e. The SDS-PAGE assay showed that although the proteins were present in the form of inclusion body, the linked proteins were expressed from plasmid pET-2e as expected, and can be expressed with high level when induced with IPTG.Western-blotting showed good antigenicity of the target protein.
基金The National "973" (2005CB523201)Key Technology R&D Programme (2006BAD06A03)
文摘E2 is an envelope glycoprotein of Classical swine fever virus (CSFV) and contains sequential neutralizing epitopes to induce virus-neutralizing antibodies and mount protective immunity in the natural host. In this study, four antigen domains (ABCD) of the E2 gene was cloned from CSFV Shimen strain into the retroviral vector pBABE puro and expressed in eukaryotic cell (PK15) by an retroviral gene expression system, and the activity of recombinant E2 protein to induce immune responses was evaluated in rabbits. The results indicated that recombinant E2 protein can be recognized by fluorescence antibodies of CSFV and CSFV positive serum (Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China) using Western blot, indirect immunofluorescence antibody test (IFAT) and ELISA, Furthermore, anti-CSFV specific antibodies and lymphocyte proliferation were elicited and increased by recombinant protein after vaccination. In the challenge test, all of rabbits vaccinated with recombinant protein and Chinese vaccine strain (C-strain) were fully protected from a rabbit spleen virus challenge. These results indicated that a retroviral-based epitope-vaccine carrying the major antigen domains of E2 is able to induce high level of epitope-specific antibodies and exhibits similar protective capability with that induced by the C-strain, and encourages further work towards the development of a vaccine against CSFV infection.
文摘【目的】全面了解中国猪群中猪瘟病毒(CSFV)的流行感染情况,为后期CSFV的流行病学调查提供证据。【方法】本研究在中国知网、万方、维普、PubMed、Web of Science和Science Direct国内外6种主要数据库中检索中国猪群CSFV流行情况的相关文献,检索时间范围为2005年1月1日-2023年1月1日。通过文献筛选、文献质量评分和CSFV流行率的数据提取,对文献中提取的数据利用StataMP17软件进行Meta分析和系统评价,分析CSFV的整体流行率,利用Meta回归分析和亚组分析CSFV整体流行率的异质性来源和影响因素。【结果】从6种主要数据库中共筛选出55篇CSFV流行情况的相关文献纳入本次Meta分析及系统评价,其中18篇文献被纳为高质量文献,37篇文献纳为中等质量文献。Meta分析结果表明,2005-2023年中国猪群中CSFV的整体流行率为12%(95%CI:0.09~0.15);Meta回归分析和亚组分析结果表明,文献的异质性来源不包括采样时间和猪种类(P>0.05),而影响CSFV流行率的因素包括诊断方法、季节变化、气候因素、海拔高度、纬度因素、经度因素及地区分布(P<0.05)。【结论】本研究从数据上确定了中国猪群中CSFV整体流行情况,其整体流行率不高,流行趋势为地区性的散在流行,以慢性和非典型毒株流行为主。本研究结果对掌握猪群CSFV流行的全局状况和有效防控及猪瘟(CSF)净化有一定的参考意义。
