[ 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 (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
In order to investigate immunogenicity in the induction of humoral and cellular immune responses, severe acute respiratory syndrome associated coronavirus （SARS-CoV）-N gene recombinant replication-defective adenovir...In order to investigate immunogenicity in the induction of humoral and cellular immune responses, severe acute respiratory syndrome associated coronavirus （SARS-CoV）-N gene recombinant replication-defective adenoviral vector, rAd-N, was generated and immunized BALB/c mice in a pcDNA3.1-N prime-rAd-N boost regimen. After humoral and cellular immune response detection, different levels of SARS-CoV N protein specific antibodies and interferon-γ （IFN-γ） secretion are shown compared to controls. The humoral immune response was induced more effectively by the DNA priming and recombinant adenovirus boosting regimen. There is a significant difference between heterogeneous and homologous vaccinations. The heterogeneous combinations were all higher than those of the homologous combinations in the induction of anti-N antibody response. Among the three heterogeneous combinations, pcDNA3.1-N/pcDNA3.1-N/pcDNA3.1-N/rAd-N induced the strongest antibody response. In the induction of IFN-γ production, the homologous combination of rAd-N/rAd-N/rAd-N/rAd-N was significantly stronger than that of pcDNA3.1-N/pcDNA3.1-N/pcDNA3.1-N/pcDNA3.1-N, but was relatively weaker than the heterogeneous combination of pcDAN3.1-N/pcDAN3.1-N/pcDAN3.1-N/rAd-N. This combination was a most efficient immunization regimen in induction of SARS-CoV-N-specific （IFN-γ） secretion just as the antibody response. These results suggest that DNA immunization followed by recombinant adenovirns boosting could be used as a potential SARS-CoV vaccine.展开更多
The outbreak of coronavirus disease(COVID-19)caused by SARS-CoV-2 virus continually lead to worldwide human infections and deaths.Currently,there is no specific viral protein-targeted therapeutics.Viral nucleocapsid p...The outbreak of coronavirus disease(COVID-19)caused by SARS-CoV-2 virus continually lead to worldwide human infections and deaths.Currently,there is no specific viral protein-targeted therapeutics.Viral nucleocapsid protein is a potential antiviral drug target,serving multiple critical functions during the viral life cycle.However,the structural information of SARS-CoV-2 nucleocapsid protein remains unclear.Herein,we have determined the 2.7 A crystal structure of the N-terminal RNA binding domain of SARS-CoV-2 nucleocapsid protein.Although the overall structure is similar as other reported coronavirus nucleocapsid protein N-terminal domain,the surface electrostatic potential characteristics between them are distinct.Further comparison with mild virus type HCoV-OC43 equivalent domain demonstrates a unique potential RNA binding pocket alongside theβ-sheet core.Complemented by in vitro binding studies,our data provide several atomic resolution features of SARS-CoV-2 nucleocapsid protein N-terminal domain,guiding the design of novel antiviral agents specific targeting to SARS-CoV-2.展开更多
Ebola virus （EBOV） is a key member of Filoviridae family and causes severe human infectious diseases with high morbidity and mortality. As a typical negative-sense single-stranded RNA （-ssRNA） viruses, EBOV posses...Ebola virus （EBOV） is a key member of Filoviridae family and causes severe human infectious diseases with high morbidity and mortality. As a typical negative-sense single-stranded RNA （-ssRNA） viruses, EBOV possess a nucleocapsid protein （NP） to facilitate genomic RNA encapsidation to form viral ribonucleoprotein complex （RNP） together with genome RNA and polymerase, which plays the most essential role in virus proliferation cycle. However, the mechanism of EBOV RNP formation remains unclear. In this work, we solved the high resolution structure of core domain of EBOV NP. The polypeptide of EBOV NP core domain （NPcore） pos- sesses an N-lobe and C-lobe to clamp a RNA binding groove, presenting similarities with the structures of the other reported viral NPs encoded by the members from Mononegavirales order. Most strikingly, a hydrophobic pocket at the surface of the C-lobe is occupied by an a- helix of EBOV NPcore itself, which is highly conserved among filoviridae family. Combined with other bio- chemical and biophysical evidences, our results provides great potential for understanding the mechanism underlying EBOV RNP formation via the mobility of EBOV NP element and enables the development of antiviral therapies targeting EBOV RNP formation.展开更多
A key to tackling the coronavirus disease 2019(COVID-19)pandemic is to understand how severe acute respiratory syndrome coronavirus 2(SARS-Co V-2)manages to outsmart host antiviral defense mechanisms.Stress granules(S...A key to tackling the coronavirus disease 2019(COVID-19)pandemic is to understand how severe acute respiratory syndrome coronavirus 2(SARS-Co V-2)manages to outsmart host antiviral defense mechanisms.Stress granules(SGs),which are assembled during viral infection and function to sequester host and viral m RNAs and proteins,are part of the antiviral responses.Here,we show that the SARS-Co V-2 nucleocapsid(N)protein,an RNA binding protein essential for viral production,interacted with RasGTPase-activating protein SH3-domain-binding protein(G3 BP)and disrupted SG assembly,both of which require intrinsically disordered region1(IDR1)in N protein.The N protein partitioned into SGs through liquid-liquid phase separation with G3 BP,and blocked the interaction of G3 BP1 with other SG-related proteins.Moreover,the N protein domains important for phase separation with G3 BP and SG disassembly were required for SARS-Co V-2 viral production.We propose that N protein-mediated SG disassembly is crucial for SARS-Co V-2 production.展开更多
In order to develop clinical diagnostic tools for rapid detection of SARS-CoV (severe acute respiratory syndrome-associated coronavirus) and to identify candidate proteins for vaccine development, the C-terminal porti...In order to develop clinical diagnostic tools for rapid detection of SARS-CoV (severe acute respiratory syndrome-associated coronavirus) and to identify candidate proteins for vaccine development, the C-terminal portion of the nucleocapsid (NC) gene was amplified using RT-PCR from the SARS-CoV genome, cloned into a yeast expression vector (pEGH), and expressed as a glutathione S-transferase (GST) and Hisx6 double-tagged fusion protein under the control of an inducible promoter. Western analysis on the purified protein confirmed the expression and purification of the NC fusion proteins from yeast. To determine its antigenicity, the fusion protein was challenged with serum samples from SARS patients and normal controls. The NC fusion protein demonstrated high antigenicity with high specificity, and therefore, it should have great potential in designing clinical diagnostic tools and provide useful information for vaccine development.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.
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.
文摘In order to investigate immunogenicity in the induction of humoral and cellular immune responses, severe acute respiratory syndrome associated coronavirus （SARS-CoV）-N gene recombinant replication-defective adenoviral vector, rAd-N, was generated and immunized BALB/c mice in a pcDNA3.1-N prime-rAd-N boost regimen. After humoral and cellular immune response detection, different levels of SARS-CoV N protein specific antibodies and interferon-γ （IFN-γ） secretion are shown compared to controls. The humoral immune response was induced more effectively by the DNA priming and recombinant adenovirus boosting regimen. There is a significant difference between heterogeneous and homologous vaccinations. The heterogeneous combinations were all higher than those of the homologous combinations in the induction of anti-N antibody response. Among the three heterogeneous combinations, pcDNA3.1-N/pcDNA3.1-N/pcDNA3.1-N/rAd-N induced the strongest antibody response. In the induction of IFN-γ production, the homologous combination of rAd-N/rAd-N/rAd-N/rAd-N was significantly stronger than that of pcDNA3.1-N/pcDNA3.1-N/pcDNA3.1-N/pcDNA3.1-N, but was relatively weaker than the heterogeneous combination of pcDAN3.1-N/pcDAN3.1-N/pcDAN3.1-N/rAd-N. This combination was a most efficient immunization regimen in induction of SARS-CoV-N-specific （IFN-γ） secretion just as the antibody response. These results suggest that DNA immunization followed by recombinant adenovirns boosting could be used as a potential SARS-CoV vaccine.
基金supported by National Natural Science Foundation of China(31770801)Special Fund for Scientific and Technological Innovation Strategy of Guangdong Province of China(2018B030306029 and 2017A030313145)+2 种基金National Natural Science Foundation of China(81430041,81620108017)National Key Basic Research Program,China(SQ2018YFC090075)National Natural Science Foundation of China(81870019)
文摘The outbreak of coronavirus disease(COVID-19)caused by SARS-CoV-2 virus continually lead to worldwide human infections and deaths.Currently,there is no specific viral protein-targeted therapeutics.Viral nucleocapsid protein is a potential antiviral drug target,serving multiple critical functions during the viral life cycle.However,the structural information of SARS-CoV-2 nucleocapsid protein remains unclear.Herein,we have determined the 2.7 A crystal structure of the N-terminal RNA binding domain of SARS-CoV-2 nucleocapsid protein.Although the overall structure is similar as other reported coronavirus nucleocapsid protein N-terminal domain,the surface electrostatic potential characteristics between them are distinct.Further comparison with mild virus type HCoV-OC43 equivalent domain demonstrates a unique potential RNA binding pocket alongside theβ-sheet core.Complemented by in vitro binding studies,our data provide several atomic resolution features of SARS-CoV-2 nucleocapsid protein N-terminal domain,guiding the design of novel antiviral agents specific targeting to SARS-CoV-2.
文摘Ebola virus （EBOV） is a key member of Filoviridae family and causes severe human infectious diseases with high morbidity and mortality. As a typical negative-sense single-stranded RNA （-ssRNA） viruses, EBOV possess a nucleocapsid protein （NP） to facilitate genomic RNA encapsidation to form viral ribonucleoprotein complex （RNP） together with genome RNA and polymerase, which plays the most essential role in virus proliferation cycle. However, the mechanism of EBOV RNP formation remains unclear. In this work, we solved the high resolution structure of core domain of EBOV NP. The polypeptide of EBOV NP core domain （NPcore） pos- sesses an N-lobe and C-lobe to clamp a RNA binding groove, presenting similarities with the structures of the other reported viral NPs encoded by the members from Mononegavirales order. Most strikingly, a hydrophobic pocket at the surface of the C-lobe is occupied by an a- helix of EBOV NPcore itself, which is highly conserved among filoviridae family. Combined with other bio- chemical and biophysical evidences, our results provides great potential for understanding the mechanism underlying EBOV RNP formation via the mobility of EBOV NP element and enables the development of antiviral therapies targeting EBOV RNP formation.
基金supported by the National Natural Science Foundation of China(81830004,31970755,and 31970173)the Local Grant(608285568031)。
文摘A key to tackling the coronavirus disease 2019(COVID-19)pandemic is to understand how severe acute respiratory syndrome coronavirus 2(SARS-Co V-2)manages to outsmart host antiviral defense mechanisms.Stress granules(SGs),which are assembled during viral infection and function to sequester host and viral m RNAs and proteins,are part of the antiviral responses.Here,we show that the SARS-Co V-2 nucleocapsid(N)protein,an RNA binding protein essential for viral production,interacted with RasGTPase-activating protein SH3-domain-binding protein(G3 BP)and disrupted SG assembly,both of which require intrinsically disordered region1(IDR1)in N protein.The N protein partitioned into SGs through liquid-liquid phase separation with G3 BP,and blocked the interaction of G3 BP1 with other SG-related proteins.Moreover,the N protein domains important for phase separation with G3 BP and SG disassembly were required for SARS-Co V-2 viral production.We propose that N protein-mediated SG disassembly is crucial for SARS-Co V-2 production.
文摘In order to develop clinical diagnostic tools for rapid detection of SARS-CoV (severe acute respiratory syndrome-associated coronavirus) and to identify candidate proteins for vaccine development, the C-terminal portion of the nucleocapsid (NC) gene was amplified using RT-PCR from the SARS-CoV genome, cloned into a yeast expression vector (pEGH), and expressed as a glutathione S-transferase (GST) and Hisx6 double-tagged fusion protein under the control of an inducible promoter. Western analysis on the purified protein confirmed the expression and purification of the NC fusion proteins from yeast. To determine its antigenicity, the fusion protein was challenged with serum samples from SARS patients and normal controls. The NC fusion protein demonstrated high antigenicity with high specificity, and therefore, it should have great potential in designing clinical diagnostic tools and provide useful information for vaccine development.
