[ Objective] The aim of this study was to investigate the construction and identification of siRNA expression vector targeting nucleocapsid protein N gone of PRRSV. [Method] Three siRNA oligonucleotides targeting nucl...[ Objective] The aim of this study was to investigate the construction and identification of siRNA expression vector targeting nucleocapsid protein N gone of PRRSV. [Method] Three siRNA oligonucleotides targeting nucleocapsid protein N gone sequence of PRRSV were designed or synthesized, and then inserted into CMV promoter downstream to clone into pSilencer 4,1 -CMV eukaryotic expression vector. The recombinant expression vector was identified by enzyme digestion and DNA sequencing. [ Result] The results showed that the siRNA interference recombinant plasmid vector pSilencer-N targeting nucleocapsid protein gone expression had been successfully constructed. [ Conclusion] This study lays a foundation for studies on the controlling PRRSV by RNA interference technique .展开更多
Porcine epidemic diarrhea virus（PEDV） is a highly infectious pathogen that can cause severe diseases in pigs and result in enormous economic losses in the worldwide swine industry. Previous studies revealed that PED...Porcine epidemic diarrhea virus（PEDV） is a highly infectious pathogen that can cause severe diseases in pigs and result in enormous economic losses in the worldwide swine industry. Previous studies revealed that PEDV exhibits an obvious capacity for modulating interferon（IFN） signaling or expression. The newly discovered type III IFN, which plays a crucial role in antiviral immunity, has strong antiviral activity against PEDV proliferation in IPEC-J2 cells. In this study, we aimed to investigate the effect of PEDV nucleocapsid（N） protein on type III IFN-λ. We found that the N proteins of ten PEDV strains isolated between 2013 and 2017 from different local farms shared high nucleotide identities, while the N protein of the CV777 vaccine strain formed a monophyletic branch in the phylogenetic tree. The N protein of the epidemic strain could antagonize type III IFN, but not type I or type II IFN expression induced by polyinosinic-polycytidylic acid（poly（I：C）） in IPEC-J2 cells. Subsequently, we demonstrated that the inhibition of poly（I：C）-induced IFN-λ3 production by PEDV N protein was dependent on the blocking of nuclear factor-κB（NF-κB） nuclear translocation. These findings might help increase understanding of the pathogenesis of PEDV and its mechanisms for evading the host immune response.展开更多
The Crimean-congo hemorrhagic fever virus(CCHFV)is a geographically widespread fatal pathogen. Identification of the epitope regions of the virus is important for the diagnosis and epidemiological studies of CCHFV inf...The Crimean-congo hemorrhagic fever virus(CCHFV)is a geographically widespread fatal pathogen. Identification of the epitope regions of the virus is important for the diagnosis and epidemiological studies of CCHFV infections.In this study,expression vectors carrying series truncated fragments of the NP(nucleocapsid protein)gene from the S fragment of CCHFV strain YL04057 were constructed.The recombinant proteins were expressed in E.coli and purified for detection.The antigenic of the truncated fragments of NP was detected with a polyclonal serum(rabbit)and 2 monoclonal(mAbs)(14B7 and 43E5)against CCHFV by Western-blot analyses. The results showed that the three expressed constructs,which all contained the region 235AA to 305AA could be detected by mAbs polyclonal serum.The results suggest that region 235-305 aa of NP is a highly antigenic region and is highly conserved in the NP protein.展开更多
Porcine epidemic diarrhea (PED), a devastating enteric disease in pigs, is caused by PEDvirus (PEDV)(1)Reduced severity of clinical diseases was reported to associate with neutralizing antibody titers in colostrum. Ho...Porcine epidemic diarrhea (PED), a devastating enteric disease in pigs, is caused by PEDvirus (PEDV)(1)Reduced severity of clinical diseases was reported to associate with neutralizing antibody titers in colostrum. However, viral neutralization assay(VN) is laborious and not suitable for routine diagnosis. Spike protein plays an important role in stimulating neutralizing antibody that might be suitable for PEDV diagnosis.展开更多
The nucleocapsid protein(N) is a major structural protein of coronaviruses. The N protein of bat SARS-like coronavirus(SL-CoV) has a high similarity with that of SARS-CoV. In this study,the SL-CoV N protein was expres...The nucleocapsid protein(N) is a major structural protein of coronaviruses. The N protein of bat SARS-like coronavirus(SL-CoV) has a high similarity with that of SARS-CoV. In this study,the SL-CoV N protein was expressed in Escherichia coli,purified and used as antigen. An Indirect Enzyme-Linked Immunosorbent Assay(indirect ELISA) was developed for detection of SARS-or SL-CoV infections in bat populations. The detection of 573 bat sera with this indirect ELISA demonstrated that SL-CoVs consistently circulate in Rhinilophus species,further supporting the proposal that bats are natural reservoirs of SL-CoVs. This method uses 1-2 μl of serum sample and can be used for preliminary screening of infections by SARS-or SL-CoV with a small amount of serum sample.展开更多
Baculoviruses are insect-specific viruses with a circular double-stranded DNA genome ranging in size from 80-180 kb （Lu et al., 2012）. Two distinct types of viri- ons have been identified during the infectious cycle...Baculoviruses are insect-specific viruses with a circular double-stranded DNA genome ranging in size from 80-180 kb （Lu et al., 2012）. Two distinct types of viri- ons have been identified during the infectious cycle of baculoviruses, namely budded virions （BVs） and occlu- sion-derived virions （ODVs）. BVs mediate infection from cell to cell, while ODVs initiate oral infection in the insect midgut （Braunagel and Summers, 2007）.展开更多
In order to establish the eukaryotic cell lines for inducible control of SARS-CoV nucleocapsid gene expression.The recombinant plasmid of pTRE-Tight-SARS-N was constructed by using the plasmid p8S as the PCR template ...In order to establish the eukaryotic cell lines for inducible control of SARS-CoV nucleocapsid gene expression.The recombinant plasmid of pTRE-Tight-SARS-N was constructed by using the plasmid p8S as the PCR template which contains a cDNA clone covering the nucleocapsid gene of SARS-CoV HKU-39449. Restriction enzymes digestion and sequence analysis indicated the recombinant plasmid of pTRE-Tight-SARS-N contained the nucleocapsid gene with the optimized nucleotide sequence which will improve the translation efficiency. Positive cell clones were selected by cotransfecting pTRE-Tight-SARS-N with the linear marker pPUR to BHK-21 Tet-on cells in the presence of puromycin. A set of double-stable eukaryotic cell lines (BHK-Tet-SARS-N) with inducible control of the SARS-CoV neucleocapsid gene expression was identified by using SDS-PAGE and Western-blot analysis. The expression of SARS-CoV nucleocapsid protein was tightly regulated by the varying concentration of doxcycline in the constructed double-stable cell line. The constructed BHK-Tet-SARS-N cell strains will facilitate the rescue of SARS-CoV in vitro and the further reverse genetic research of SARS-CoV.展开更多
Autographa californica multiple nucleopolyhedrovirus(Ac MNPV)orf13(ac13)is a conserved gene in all sequenced alphabaculoviruses.However,its function in the viral life cycle remains unknown.In this study,we found that ...Autographa californica multiple nucleopolyhedrovirus(Ac MNPV)orf13(ac13)is a conserved gene in all sequenced alphabaculoviruses.However,its function in the viral life cycle remains unknown.In this study,we found that ac13 was a late gene and that the encoded protein,bearing a putative nuclear localization signal motif,colocalized with the nuclear lamina.Deletion of ac13 did not affect viral genome replication,nucleocapsid assembly or occlusion body(OB)formation,but reduced virion budding from infected cells by approximately 400-fold compared with the wild-type virus.Deletion of ac13 substantially impaired the egress of nucleocapsids from the nucleus to the cytoplasm,while the OB morphogenesis was unaffected.Taken together,our results indicated that ac13 was required for efficient nuclear egress of nucleocapsids during virion budding,but was dispensable for OB formation.展开更多
Objective: To express the 26 kD fragment of Hantaan virus nucleocapsid protein that contains the major antigenic epitopes in insect cells, and make a preliminary analysis of its immunological characteristics. Methods:...Objective: To express the 26 kD fragment of Hantaan virus nucleocapsid protein that contains the major antigenic epitopes in insect cells, and make a preliminary analysis of its immunological characteristics. Methods: The recombinant baculovirus bac-S0.7 with the 700 bp fragment of S gene 5' terminal of Hantaan virus was constructed, and the antigenicity of the expression product was tested. Mice were injected with Sf9 cells infected by the recombinant baculovirus. The humoral and cellular immunological effects were identified by indirect immunofluorescence assay, micro-cell culture neutralization test and T lymphocytes stimulation test. Results: Immunized by bac-S0.7 infecting insect cells, specific antibody with the highest titer of 1∶1 600 was observed. The stimulation indexes of splenocytes of immunized mice to nucleocapsid protein of Hantaan virus was higher than the negative control. Conclusion: The expression product of S0.7 gene fragment in insect cells is immunogenic.展开更多
Avian influenza, subtype H5N1, causes occasional but serious infections in humans and efforts to produce vaccines against this strain continue. Current influenza vaccines are prophylactic and utilize the two major ant...Avian influenza, subtype H5N1, causes occasional but serious infections in humans and efforts to produce vaccines against this strain continue. Current influenza vaccines are prophylactic and utilize the two major antigens, hemagglutinin and neuraminidase. Nucleocapsid protein (NP) is an attractive alternative antigen because it is highly conserved across all influenza strains, has been shown to increase the rate of viral clearance, and potential therapeutic vaccines would elicit cytotoxic T lymphocyte responses in an infected person. The NP antigen from H5N1 was characterized using a variety of physico-chemical methods to gain insights into both the biological and physical properties of the antigen which are important from a regulatory viewpoint when considering therapeutic vaccines. Results obtained to date show that NP is relatively unstable and indicate that the conformation of the H5N1 NP antigen is highly dependent upon purification procedure, buffer conditions, pH and the presence or absence of RNA. These factors will need to be clearly defined and taken into consideration when manufacturing and regulating NP vaccine preparations.展开更多
Dear Editor,Severe fever with thrombocytopenia syndrome virus(SFTSV)is a newly identified viral pathogen of the genus Phlebovirus in the family Bunyaviridae(Sun et al.,2012).SFTSV was first identified from patient ser...Dear Editor,Severe fever with thrombocytopenia syndrome virus(SFTSV)is a newly identified viral pathogen of the genus Phlebovirus in the family Bunyaviridae(Sun et al.,2012).SFTSV was first identified from patient serum samples in China(Li et al.,2013;Ning et al.,2015).SFTSV can cause a severe hemorrhagic fever-like disease with a reported case fatality rate ranging from 2.5%展开更多
The nucleocapsid protein VP15 of white spot syndrome virus (WSSV) is a basic DNA-binding protein. Three canonical bipartite nuclear localization signals (NLSs), called NLS1 (aa 11-27), NLS2 (aa 33-49) and NLS3 (44-60)...The nucleocapsid protein VP15 of white spot syndrome virus (WSSV) is a basic DNA-binding protein. Three canonical bipartite nuclear localization signals (NLSs), called NLS1 (aa 11-27), NLS2 (aa 33-49) and NLS3 (44-60), have been detected in this protein, using the ScanProsite computer program. To determine the nuclear localization sequence of VP15, the full-length open reading frame, or the sequence of one of the three NLSs, was fused to the green fluorescent protein (GFP) gene, and transiently expressed in insect Sf9 cells. Transfection with full-length VP15 resulted in GFP fluorescence being distributed exclusively in the nucleus. NLS1 alone could also direct GFP to the nucleus, but less efficiently. Neither of the other two NLSs (NLS2 and 3) was functional when expressed alone, but exhibited similar activity to NLS1 when they were expressed as a fusion peptide. Furthermore, a mutated VP15, in which the two basic amino acids (11RR12) of NLS1 were changed to two alanines (11AA12), caused GFP to be localized only in the cytoplasm of Sf9 cells. These results demonstrated that VP15, as a nuclear localization protein, needs cooperation between its three NLSs, and that the two residues (11RR12) of NLS1 play a key role in transporting the protein to the nucleus.展开更多
In order to elucidate the molecular and immunological mechanisms as well as the pathogenesis of hemorrhagic fever with renal syndrome (HFRS), the CD8 + cytotoxic T lymphocytes (CTL) clone was established directly from...In order to elucidate the molecular and immunological mechanisms as well as the pathogenesis of hemorrhagic fever with renal syndrome (HFRS), the CD8 + cytotoxic T lymphocytes (CTL) clone was established directly from peripheral blood mononuclear cells (PBMC) of patients with HFRS. The activities of CTL were detected as usual with EBV-transformed lymphoblastoid cell line (BLCL) as target cells. The results showed that the CTL clone could recognized and killed the target cells with specificity of nucleocapsid protein of Hantaan virus (HTNVNP) with the cytotoxicity percentages of 50.2%, 25.4% and 39.0% respectively. These results demonstrated that the antigenic epitopes of HTNVNP mainly located on the C-terminal of the viral nucleocapsid protein.展开更多
The nucleocapsid protein(NP)plays a crucial role in SARS-CoV-2 replication and is the most abundant structural protein with a long half-life.Despite its vital role in severe acute respiratory syndrome coronavirus 2(SA...The nucleocapsid protein(NP)plays a crucial role in SARS-CoV-2 replication and is the most abundant structural protein with a long half-life.Despite its vital role in severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)assembly and host inflammatory response,it remains an unexplored target for drug development.In this study,we identified a small-molecule compound(ciclopirox)that promotes NP degradation using an FDA-approved library and a drug-screening cell model.Ciclopirox significantly inhibited SARS-CoV-2 replication both in vitro and in vivo by inducing NP degradation.Ciclopirox induced abnormal NP aggregation through indirect interaction,leading to the formation of condensates with higher viscosity and lower mobility.These condensates were subsequently degraded via the autophagy-lysosomal pathway,ultimately resulting in a shortened NP half-life and reduced NP expression.Our results suggest that NP is a potential drug target,and that ciclopirox holds substantial promise for further development to combat SARS-CoV-2 replication.展开更多
冠状病毒能够有效逃避宿主天然免疫应答,这是其感染过程中的显著特征。天然免疫信号通路的激活依赖多个启动抗病毒基因表达的转录因子进入细胞核,而这一过程需要宿主核质运输系统的协助,因此,该系统成为冠状病毒拮抗宿主免疫反应的关键...冠状病毒能够有效逃避宿主天然免疫应答,这是其感染过程中的显著特征。天然免疫信号通路的激活依赖多个启动抗病毒基因表达的转录因子进入细胞核,而这一过程需要宿主核质运输系统的协助,因此,该系统成为冠状病毒拮抗宿主免疫反应的关键靶点。近期,中国农业科学院上海兽医研究所廖瑛研究员团队在PLoS Pathogens上发表了题为“Coronavirus nucleocapsid protein enhances the binding of p-PKCαto RACK1:Implications for inhibition of nucleocytoplasmic trafficking and suppression of the innate immune response”的研究论文。该研究以禽冠状病毒——传染性支气管炎病毒(IBV)为模型,系统探讨了冠状病毒干扰宿主核质运输系统的保守机制。研究发现,IBV感染能够动态抑制多种转录因子入核,进而抑制关键抗病毒基因的转录。进一步研究表明,核孔复合体(NPC)的重要组成部分FG-Nups在感染过程中从核膜剥离并弥散至胞质。IBV的核衣壳蛋白(N蛋白)被鉴定为导致这一现象的关键病毒蛋白。研究揭示,N蛋白通过与支架蛋白RACK1相互作用,促使活化的蛋白激酶PKCα(p-PKCα)锚定至RACK1,并将RACK1-PKCα复合物重新定位至胞质。这一过程促进了PKCα介导的NUP62磷酸化及其解聚,从而阻碍抗病毒基因的表达并增强病毒复制能力。更重要的是,这一机制在多种冠状病毒的N蛋白中具有高度保守性。展开更多
BACKGROUND In the initial stages of the coronavirus disease 2019(COVID-19)pandemic,healthcare workers(HCWs)who were immunologically naive to COVID-19,were exposed to a highly transmissible virus.AIM To compare infecti...BACKGROUND In the initial stages of the coronavirus disease 2019(COVID-19)pandemic,healthcare workers(HCWs)who were immunologically naive to COVID-19,were exposed to a highly transmissible virus.AIM To compare infection risk among HCWs in high-risk(HR)and low-risk(LR)areas.METHODS Data on reverse transcriptase-polymerase chain reaction confirmed clinical infection and samples for nucleocapsid,and spike protein antibodies were collected at five time-points(T1 to T5)from HCWs in the emergency department and intensive care unit(HR group)and pre-clinical and para-clinical areas(LR).For the sero-study,only participants who provided at least one baseline sample and one during the second wave(T4 or T5)were analysed.Since CovishieldTM elicits only spike protein antibodies,subclinical infection was diagnosed if asymptomatic unvaccinated and CovishieldTM vaccinated individuals tested positive for nucleocapsid antibody.RESULTS Overall,by T5,clinical infection rate was similar in the HR(120/366,32.8%)and LR(22/82,26.8%)groups(P=0.17).However,before vaccination(T3),more HCWs in the HR group developed COVID-19 infection(21.9%vs 8.8%,P=0.046).In the sero-study group,clinical infection occurred in 31.5%(45/143)and 23.7%(14/59)in the HR and LR groups respectively(P=0.23).Spike antibody was detected in 140/143(97.9%)and 56/59(94.9%)and nucleocapsid antibody was positive in 95/143(66.4%)and 35/59(59.3%)in the HR and LR groups respectively(P=0.34).Subclinical infection rate(HR 34.9%,LR 35.6%,P=0.37)and hospitalization rate were similar.There was no mortality.CONCLUSION Before vaccination,HCWs in HR areas had a higher risk of infection.Seroprevalence studies suggest that subclinical infection was not uncommon.展开更多
Porcine deltacoronavirus(PDCoV)is an emerging swine enteropathogenic coronavirus that can cause acute diarrhea and vomiting in newborn piglets and poses a potential risk for cross-species transmission.It is necessary ...Porcine deltacoronavirus(PDCoV)is an emerging swine enteropathogenic coronavirus that can cause acute diarrhea and vomiting in newborn piglets and poses a potential risk for cross-species transmission.It is necessary to develop an effective serological diagnostic tool for the surveillance of PDCoV infection and vaccine immunity effects.In this study,we developed a monoclonal antibody-based competitive ELISA(cELISA)that selected the purified recombinant PDCoV nucleocapsid(N)protein as the coating antigen to detect PDCoV antibodies.To evaluate the diagnostic performance of the cELISA,122 swine serum samples(39 positive and 83 negative)were tested and the results were compared with an indirect immunofluorescence assay(IFA)as the reference method.By receiver operating characteristic(ROC)curve analysis,the optimum cutoff value of percent inhibition(PI)was determined to be 26.8%,which showed excellent diagnostic performance,with an area under the curve(AUC)of 0.9919,a diagnostic sensitivity of 97.44%and a diagnostic specificity of 96.34%.Furthermore,there was good agreement between the cELISA and virus neutralization test(VNT)for the detection of PDCoV antibodies,with a coincidence rate of 92.7%,and theκanalysis showed almost perfect agreement(κ=0.851).Overall,the established cELISA showed good diagnostic performance,including sensitivity,specificity and repeatability,and can be used for diagnostic assistance,evaluating the response to vaccination and assessing swine herd immunity.展开更多
The nucleocapsid protein (N protein) has been found to be an antigenic protein in a number of coronaviruses. Whether the N protein in severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is antigenic re...The nucleocapsid protein (N protein) has been found to be an antigenic protein in a number of coronaviruses. Whether the N protein in severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is antigenic remains to be elucidated. Using Western blot and Enzyme-linked Immunosorbent Assay (ELISA), the recombinant N proteins and the synthesized peptides derived from the N protein were screened in sera from SARS patients. All patient sera in this study displayed strong positive immunoreactivities against the recombinant N proteins, whereas normal sera gave negative immunoresponses to these proteins, indicating that the N protein of SARS-CoV is an antigenic protein. Furthermore, the epitope sites in the N protein were determined by competition experiments, in which the recombinant proteins or the synthesized peptides competed against the SARS-CoV proteins to bind to the antibodies raised in SARS sera. One epitope site located at the C-terminus was confirmed as the most antigenic region in this protein. A detailed screening of peptide with ELISA demonstrated that the amino sequence from Codons 371 to 407 was the epitope site at the C-terminus of the N protein. Understanding of the epitope sites could be very significant for developing an effective diagnostic approach to SARS.展开更多
基金Supported by Based on Cuttingedge technology and research Project of Henan Province(072300430060)The focus of Scientific andTechnological Project of Henan Province(072102130023)Colleges and Universities of Henan Province in Support of TechnologicalInnovation Plan~~
文摘[ Objective] The aim of this study was to investigate the construction and identification of siRNA expression vector targeting nucleocapsid protein N gone of PRRSV. [Method] Three siRNA oligonucleotides targeting nucleocapsid protein N gone sequence of PRRSV were designed or synthesized, and then inserted into CMV promoter downstream to clone into pSilencer 4,1 -CMV eukaryotic expression vector. The recombinant expression vector was identified by enzyme digestion and DNA sequencing. [ Result] The results showed that the siRNA interference recombinant plasmid vector pSilencer-N targeting nucleocapsid protein gone expression had been successfully constructed. [ Conclusion] This study lays a foundation for studies on the controlling PRRSV by RNA interference technique .
基金Project supported by the National Natural Science Foundation of China(No.31702250)the Key Research and Development Project Funds of Zhejiang Provincial Science and Technology Department(Nos.2015C02044 and 2018C02028)+2 种基金the Agricultural Technology Extension Funds of Zhejiang Universitythe Dabei Agricultural Discipline Development and Talent Training Fund(No.2017ZDNT004)the Three Rural and Six Party Funds,China
文摘Porcine epidemic diarrhea virus（PEDV） is a highly infectious pathogen that can cause severe diseases in pigs and result in enormous economic losses in the worldwide swine industry. Previous studies revealed that PEDV exhibits an obvious capacity for modulating interferon（IFN） signaling or expression. The newly discovered type III IFN, which plays a crucial role in antiviral immunity, has strong antiviral activity against PEDV proliferation in IPEC-J2 cells. In this study, we aimed to investigate the effect of PEDV nucleocapsid（N） protein on type III IFN-λ. We found that the N proteins of ten PEDV strains isolated between 2013 and 2017 from different local farms shared high nucleotide identities, while the N protein of the CV777 vaccine strain formed a monophyletic branch in the phylogenetic tree. The N protein of the epidemic strain could antagonize type III IFN, but not type I or type II IFN expression induced by polyinosinic-polycytidylic acid（poly（I：C）） in IPEC-J2 cells. Subsequently, we demonstrated that the inhibition of poly（I：C）-induced IFN-λ3 production by PEDV N protein was dependent on the blocking of nuclear factor-κB（NF-κB） nuclear translocation. These findings might help increase understanding of the pathogenesis of PEDV and its mechanisms for evading the host immune response.
基金National Key Technologies R&D Program of China during the 10th Five-Year Plan Period(2003BA712A08-03)The Knowledge Innovation Program of the Chinese Academy of Sciences(KSCX2-YW-N-065)+1 种基金The Foundation scientific and technological project from MOST(2007FY210700)The NSFC Grant(30860255)
文摘The Crimean-congo hemorrhagic fever virus(CCHFV)is a geographically widespread fatal pathogen. Identification of the epitope regions of the virus is important for the diagnosis and epidemiological studies of CCHFV infections.In this study,expression vectors carrying series truncated fragments of the NP(nucleocapsid protein)gene from the S fragment of CCHFV strain YL04057 were constructed.The recombinant proteins were expressed in E.coli and purified for detection.The antigenic of the truncated fragments of NP was detected with a polyclonal serum(rabbit)and 2 monoclonal(mAbs)(14B7 and 43E5)against CCHFV by Western-blot analyses. The results showed that the three expressed constructs,which all contained the region 235AA to 305AA could be detected by mAbs polyclonal serum.The results suggest that region 235-305 aa of NP is a highly antigenic region and is highly conserved in the NP protein.
文摘Porcine epidemic diarrhea (PED), a devastating enteric disease in pigs, is caused by PEDvirus (PEDV)(1)Reduced severity of clinical diseases was reported to associate with neutralizing antibody titers in colostrum. However, viral neutralization assay(VN) is laborious and not suitable for routine diagnosis. Spike protein plays an important role in stimulating neutralizing antibody that might be suitable for PEDV diagnosis.
基金The State Key Program for Basic Research Grant (2005CB523004) The Knowledge InnovationProgram Key Project (KSCX1-YW-R-07).
文摘The nucleocapsid protein(N) is a major structural protein of coronaviruses. The N protein of bat SARS-like coronavirus(SL-CoV) has a high similarity with that of SARS-CoV. In this study,the SL-CoV N protein was expressed in Escherichia coli,purified and used as antigen. An Indirect Enzyme-Linked Immunosorbent Assay(indirect ELISA) was developed for detection of SARS-or SL-CoV infections in bat populations. The detection of 573 bat sera with this indirect ELISA demonstrated that SL-CoVs consistently circulate in Rhinilophus species,further supporting the proposal that bats are natural reservoirs of SL-CoVs. This method uses 1-2 μl of serum sample and can be used for preliminary screening of infections by SARS-or SL-CoV with a small amount of serum sample.
基金supported by the grants from the National Science Foundation of China(No.31570153,31130058,31321001 and 31400142)the Strategic Priority Research Program of the Chinese Academy of Sciences(Grant No.XDB11030400)the Core Facility and Technical Support of Wuhan Institute of Virology for technical assistance
文摘Baculoviruses are insect-specific viruses with a circular double-stranded DNA genome ranging in size from 80-180 kb （Lu et al., 2012）. Two distinct types of viri- ons have been identified during the infectious cycle of baculoviruses, namely budded virions （BVs） and occlu- sion-derived virions （ODVs）. BVs mediate infection from cell to cell, while ODVs initiate oral infection in the insect midgut （Braunagel and Summers, 2007）.
基金This work was supported by the European Commission (SARS-DTV ) SP22-CT-2004–511064)the State Key Laboratory of Pathogen and Biosecunity SKLPBS0918
文摘In order to establish the eukaryotic cell lines for inducible control of SARS-CoV nucleocapsid gene expression.The recombinant plasmid of pTRE-Tight-SARS-N was constructed by using the plasmid p8S as the PCR template which contains a cDNA clone covering the nucleocapsid gene of SARS-CoV HKU-39449. Restriction enzymes digestion and sequence analysis indicated the recombinant plasmid of pTRE-Tight-SARS-N contained the nucleocapsid gene with the optimized nucleotide sequence which will improve the translation efficiency. Positive cell clones were selected by cotransfecting pTRE-Tight-SARS-N with the linear marker pPUR to BHK-21 Tet-on cells in the presence of puromycin. A set of double-stable eukaryotic cell lines (BHK-Tet-SARS-N) with inducible control of the SARS-CoV neucleocapsid gene expression was identified by using SDS-PAGE and Western-blot analysis. The expression of SARS-CoV nucleocapsid protein was tightly regulated by the varying concentration of doxcycline in the constructed double-stable cell line. The constructed BHK-Tet-SARS-N cell strains will facilitate the rescue of SARS-CoV in vitro and the further reverse genetic research of SARS-CoV.
基金supported by the National Key Research and Development Program of China(2017YFD0201206)the WIV “One-Three-Five”strategic program(Y602111SA1 to XS)。
文摘Autographa californica multiple nucleopolyhedrovirus(Ac MNPV)orf13(ac13)is a conserved gene in all sequenced alphabaculoviruses.However,its function in the viral life cycle remains unknown.In this study,we found that ac13 was a late gene and that the encoded protein,bearing a putative nuclear localization signal motif,colocalized with the nuclear lamina.Deletion of ac13 did not affect viral genome replication,nucleocapsid assembly or occlusion body(OB)formation,but reduced virion budding from infected cells by approximately 400-fold compared with the wild-type virus.Deletion of ac13 substantially impaired the egress of nucleocapsids from the nucleus to the cytoplasm,while the OB morphogenesis was unaffected.Taken together,our results indicated that ac13 was required for efficient nuclear egress of nucleocapsids during virion budding,but was dispensable for OB formation.
基金National Natural Science Foundation of China (No.30070686)Chinese Educational Deputy Fund for skeleton teachers
文摘Objective: To express the 26 kD fragment of Hantaan virus nucleocapsid protein that contains the major antigenic epitopes in insect cells, and make a preliminary analysis of its immunological characteristics. Methods: The recombinant baculovirus bac-S0.7 with the 700 bp fragment of S gene 5' terminal of Hantaan virus was constructed, and the antigenicity of the expression product was tested. Mice were injected with Sf9 cells infected by the recombinant baculovirus. The humoral and cellular immunological effects were identified by indirect immunofluorescence assay, micro-cell culture neutralization test and T lymphocytes stimulation test. Results: Immunized by bac-S0.7 infecting insect cells, specific antibody with the highest titer of 1∶1 600 was observed. The stimulation indexes of splenocytes of immunized mice to nucleocapsid protein of Hantaan virus was higher than the negative control. Conclusion: The expression product of S0.7 gene fragment in insect cells is immunogenic.
文摘Avian influenza, subtype H5N1, causes occasional but serious infections in humans and efforts to produce vaccines against this strain continue. Current influenza vaccines are prophylactic and utilize the two major antigens, hemagglutinin and neuraminidase. Nucleocapsid protein (NP) is an attractive alternative antigen because it is highly conserved across all influenza strains, has been shown to increase the rate of viral clearance, and potential therapeutic vaccines would elicit cytotoxic T lymphocyte responses in an infected person. The NP antigen from H5N1 was characterized using a variety of physico-chemical methods to gain insights into both the biological and physical properties of the antigen which are important from a regulatory viewpoint when considering therapeutic vaccines. Results obtained to date show that NP is relatively unstable and indicate that the conformation of the H5N1 NP antigen is highly dependent upon purification procedure, buffer conditions, pH and the presence or absence of RNA. These factors will need to be clearly defined and taken into consideration when manufacturing and regulating NP vaccine preparations.
基金supported by the grants from the National Science Foundation of China(No.81460303)the Ministry of Science and Technology of China(2013FY113500)Funded by the Open Research Fund Program of the State Key Laboratory of Virology of China(No.2015IOV003)
文摘Dear Editor,Severe fever with thrombocytopenia syndrome virus(SFTSV)is a newly identified viral pathogen of the genus Phlebovirus in the family Bunyaviridae(Sun et al.,2012).SFTSV was first identified from patient serum samples in China(Li et al.,2013;Ning et al.,2015).SFTSV can cause a severe hemorrhagic fever-like disease with a reported case fatality rate ranging from 2.5%
基金State Key Program for Basic ResearchGrants (2006CB101801)the Chinese Academy ofSciences (KSCX2-SW-302).
文摘The nucleocapsid protein VP15 of white spot syndrome virus (WSSV) is a basic DNA-binding protein. Three canonical bipartite nuclear localization signals (NLSs), called NLS1 (aa 11-27), NLS2 (aa 33-49) and NLS3 (44-60), have been detected in this protein, using the ScanProsite computer program. To determine the nuclear localization sequence of VP15, the full-length open reading frame, or the sequence of one of the three NLSs, was fused to the green fluorescent protein (GFP) gene, and transiently expressed in insect Sf9 cells. Transfection with full-length VP15 resulted in GFP fluorescence being distributed exclusively in the nucleus. NLS1 alone could also direct GFP to the nucleus, but less efficiently. Neither of the other two NLSs (NLS2 and 3) was functional when expressed alone, but exhibited similar activity to NLS1 when they were expressed as a fusion peptide. Furthermore, a mutated VP15, in which the two basic amino acids (11RR12) of NLS1 were changed to two alanines (11AA12), caused GFP to be localized only in the cytoplasm of Sf9 cells. These results demonstrated that VP15, as a nuclear localization protein, needs cooperation between its three NLSs, and that the two residues (11RR12) of NLS1 play a key role in transporting the protein to the nucleus.
文摘In order to elucidate the molecular and immunological mechanisms as well as the pathogenesis of hemorrhagic fever with renal syndrome (HFRS), the CD8 + cytotoxic T lymphocytes (CTL) clone was established directly from peripheral blood mononuclear cells (PBMC) of patients with HFRS. The activities of CTL were detected as usual with EBV-transformed lymphoblastoid cell line (BLCL) as target cells. The results showed that the CTL clone could recognized and killed the target cells with specificity of nucleocapsid protein of Hantaan virus (HTNVNP) with the cytotoxicity percentages of 50.2%, 25.4% and 39.0% respectively. These results demonstrated that the antigenic epitopes of HTNVNP mainly located on the C-terminal of the viral nucleocapsid protein.
基金supported by grants from Shenzhen Science and Technology Program(Grant No.JCYJ20220530163206015,China)National Key Research and Development Program of China(Grant No.2021YFA0910900)+4 种基金Shenzhen Science and Technology Program(Grant No.JCYJ20220818103017036,China)the National Science Fund for Distinguished Young Scholars(Grant No.82025022,China)Guangdong Basic and Applied Basic Research Foundation(Grant No.2023A1515110033,China)Guangdong Science and Technology Plan Project,construction of high-level biosafety laboratories(Grant No.2021B1212030010,China)Guangdong Basic and Applied Basic Research Foundation(Grant No.2023A1515110033,China).
文摘The nucleocapsid protein(NP)plays a crucial role in SARS-CoV-2 replication and is the most abundant structural protein with a long half-life.Despite its vital role in severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)assembly and host inflammatory response,it remains an unexplored target for drug development.In this study,we identified a small-molecule compound(ciclopirox)that promotes NP degradation using an FDA-approved library and a drug-screening cell model.Ciclopirox significantly inhibited SARS-CoV-2 replication both in vitro and in vivo by inducing NP degradation.Ciclopirox induced abnormal NP aggregation through indirect interaction,leading to the formation of condensates with higher viscosity and lower mobility.These condensates were subsequently degraded via the autophagy-lysosomal pathway,ultimately resulting in a shortened NP half-life and reduced NP expression.Our results suggest that NP is a potential drug target,and that ciclopirox holds substantial promise for further development to combat SARS-CoV-2 replication.
文摘冠状病毒能够有效逃避宿主天然免疫应答,这是其感染过程中的显著特征。天然免疫信号通路的激活依赖多个启动抗病毒基因表达的转录因子进入细胞核,而这一过程需要宿主核质运输系统的协助,因此,该系统成为冠状病毒拮抗宿主免疫反应的关键靶点。近期,中国农业科学院上海兽医研究所廖瑛研究员团队在PLoS Pathogens上发表了题为“Coronavirus nucleocapsid protein enhances the binding of p-PKCαto RACK1:Implications for inhibition of nucleocytoplasmic trafficking and suppression of the innate immune response”的研究论文。该研究以禽冠状病毒——传染性支气管炎病毒(IBV)为模型,系统探讨了冠状病毒干扰宿主核质运输系统的保守机制。研究发现,IBV感染能够动态抑制多种转录因子入核,进而抑制关键抗病毒基因的转录。进一步研究表明,核孔复合体(NPC)的重要组成部分FG-Nups在感染过程中从核膜剥离并弥散至胞质。IBV的核衣壳蛋白(N蛋白)被鉴定为导致这一现象的关键病毒蛋白。研究揭示,N蛋白通过与支架蛋白RACK1相互作用,促使活化的蛋白激酶PKCα(p-PKCα)锚定至RACK1,并将RACK1-PKCα复合物重新定位至胞质。这一过程促进了PKCα介导的NUP62磷酸化及其解聚,从而阻碍抗病毒基因的表达并增强病毒复制能力。更重要的是,这一机制在多种冠状病毒的N蛋白中具有高度保守性。
基金Supported by Internal Institutional Research Fund.
文摘BACKGROUND In the initial stages of the coronavirus disease 2019(COVID-19)pandemic,healthcare workers(HCWs)who were immunologically naive to COVID-19,were exposed to a highly transmissible virus.AIM To compare infection risk among HCWs in high-risk(HR)and low-risk(LR)areas.METHODS Data on reverse transcriptase-polymerase chain reaction confirmed clinical infection and samples for nucleocapsid,and spike protein antibodies were collected at five time-points(T1 to T5)from HCWs in the emergency department and intensive care unit(HR group)and pre-clinical and para-clinical areas(LR).For the sero-study,only participants who provided at least one baseline sample and one during the second wave(T4 or T5)were analysed.Since CovishieldTM elicits only spike protein antibodies,subclinical infection was diagnosed if asymptomatic unvaccinated and CovishieldTM vaccinated individuals tested positive for nucleocapsid antibody.RESULTS Overall,by T5,clinical infection rate was similar in the HR(120/366,32.8%)and LR(22/82,26.8%)groups(P=0.17).However,before vaccination(T3),more HCWs in the HR group developed COVID-19 infection(21.9%vs 8.8%,P=0.046).In the sero-study group,clinical infection occurred in 31.5%(45/143)and 23.7%(14/59)in the HR and LR groups respectively(P=0.23).Spike antibody was detected in 140/143(97.9%)and 56/59(94.9%)and nucleocapsid antibody was positive in 95/143(66.4%)and 35/59(59.3%)in the HR and LR groups respectively(P=0.34).Subclinical infection rate(HR 34.9%,LR 35.6%,P=0.37)and hospitalization rate were similar.There was no mortality.CONCLUSION Before vaccination,HCWs in HR areas had a higher risk of infection.Seroprevalence studies suggest that subclinical infection was not uncommon.
基金supported by the National Key Research and Development Program(2023YFD1800501)the National Natural Science Foundation of China(32373030,32202787)+5 种基金the S&T Program of Hebei(21322401D)the Jiangsu Province Natural Sciences Foundation(BK20221432,BK20210158)the Jiangsu Agricultural Science and Technology Innovation Fund(CX(22)3028)the Special Project of Northern Jiangsu(SZ-LYG202109)the Open Fund of Shaoxing Academy of Biomedicine of Zhejiang Sci-Tech University(SXAB202215)the Open Fund of Key Laboratory for Prevention and Control of Avian Influenza and Other Major Poultry Diseases,Ministry of Agriculture and Rural Affairs(YDWS202213).
文摘Porcine deltacoronavirus(PDCoV)is an emerging swine enteropathogenic coronavirus that can cause acute diarrhea and vomiting in newborn piglets and poses a potential risk for cross-species transmission.It is necessary to develop an effective serological diagnostic tool for the surveillance of PDCoV infection and vaccine immunity effects.In this study,we developed a monoclonal antibody-based competitive ELISA(cELISA)that selected the purified recombinant PDCoV nucleocapsid(N)protein as the coating antigen to detect PDCoV antibodies.To evaluate the diagnostic performance of the cELISA,122 swine serum samples(39 positive and 83 negative)were tested and the results were compared with an indirect immunofluorescence assay(IFA)as the reference method.By receiver operating characteristic(ROC)curve analysis,the optimum cutoff value of percent inhibition(PI)was determined to be 26.8%,which showed excellent diagnostic performance,with an area under the curve(AUC)of 0.9919,a diagnostic sensitivity of 97.44%and a diagnostic specificity of 96.34%.Furthermore,there was good agreement between the cELISA and virus neutralization test(VNT)for the detection of PDCoV antibodies,with a coincidence rate of 92.7%,and theκanalysis showed almost perfect agreement(κ=0.851).Overall,the established cELISA showed good diagnostic performance,including sensitivity,specificity and repeatability,and can be used for diagnostic assistance,evaluating the response to vaccination and assessing swine herd immunity.
文摘The nucleocapsid protein (N protein) has been found to be an antigenic protein in a number of coronaviruses. Whether the N protein in severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is antigenic remains to be elucidated. Using Western blot and Enzyme-linked Immunosorbent Assay (ELISA), the recombinant N proteins and the synthesized peptides derived from the N protein were screened in sera from SARS patients. All patient sera in this study displayed strong positive immunoreactivities against the recombinant N proteins, whereas normal sera gave negative immunoresponses to these proteins, indicating that the N protein of SARS-CoV is an antigenic protein. Furthermore, the epitope sites in the N protein were determined by competition experiments, in which the recombinant proteins or the synthesized peptides competed against the SARS-CoV proteins to bind to the antibodies raised in SARS sera. One epitope site located at the C-terminus was confirmed as the most antigenic region in this protein. A detailed screening of peptide with ELISA demonstrated that the amino sequence from Codons 371 to 407 was the epitope site at the C-terminus of the N protein. Understanding of the epitope sites could be very significant for developing an effective diagnostic approach to SARS.
