Genotyping of nucleocapsid protein gene of HCV in HIVHCV co-infected patients in Kunming in 2019 / 中国热带医学

@#Abstract: Objective　To investigate the distribution characteristics of HCV genotypes and subtypes in patients with HIV (human immunodeficiency virus, HIV)/HCV co-infection in Kunming based on the nucleocapsid protein gene sequence of HCV (hepatitis C virus). Methods　Serum was collected from HIV/HCV co-infected patients with household registration in 14 county-level cities, districts and counties under the jurisdiction of Kunming, who admitted to Yunnan Provincial Infectious Disease Hospital from March to August 2019. The viral RNA was extracted from the serum, reverse transcribed to synthesize cDNA, and the HCV nucleocapsid protein gene-specific primers were used for nested PCR amplification. The positive amplification products were sequenced, bioinformatics software such as DNAstar and MEGAX were used for sequence analysis. Results　A total of 64 samples from co-infected patients with clinical diagnosis of suspected HIV/HCV were collected and amplified by HCV nucleocapsid protein gene-specific primers, of which 17 samples were amplified positively. The results of sequence analysis showed that the sequences of 9 cases were located in the same evolutionary branch as the HCV 3b subtype sequence, and the nucleotide homology was 93.3%-95.2%; the sequences of 5 cases were located in the same evolutionary branch as the HCV 1b subtype sequence, and the nucleotide homology was 96.8%-97.6%; the sequence of one case and the subtype sequence of HCV 3a gene were located in the same evolutionary branch, and the nucleotide homology was 95.2%; the sequence of one case and HCV 6n gene subtype sequence were located in the same evolutionary branch, and the nucleotide homology was 97.9%; One case was located in the same evolutionary branch as the HCV 6u gene subtype sequence, and the nucleotide homology was 98.4%. Conclusions　HCV 1b, HCV 3a, HCV 3b, HCV 6n and HCV 6u genotypes or subtypes of HCV are prevalent in Kunming, and HCV 3b is the most prevalent genotype.

The nucleocapsid protein of rice stripe virus in cell nuclei of vector insect regulates viral replication

Rice stripe virus (RSV) transmitted by the small brown planthopper causes severe rice yield losses in Asian countries. Although viral nuclear entry promotes viral replication in host cells, whether this phenomenon occurs in vector cells remains unknown. Therefore, in this study, we systematically evaluated the presence and roles of RSV in the nuclei of vector insect cells. We observed that the nucleocapsid protein (NP) and viral genomic RNAs were partially transported into vector cell nuclei by utilizing the importin α nuclear transport system. When blocking NP nuclear localization, cytoplasmic RSV accumulation significantly increased. In the vector cell nuclei, NP bound the transcription factor YY1 and affected its positive regulation to FAIM. Subsequently, decreased FAIM expression triggered an antiviral caspase-dependent apoptotic reaction. Our results reveal that viral nuclear entry induces completely different immune effects in vector and host cells, providing new insights into the balance between viral load and the immunity pressure in vector insects.

Desenvolvimento de teste diagnóstico não invasivo capaz de detectar anticorpos anti-SARS-CoV-2 em urina / Development of a non-invasive diagnostic test capable of detecting anti-SARS-CoV-2 antibodies in urine

A plataforma de ELISA (ensaio de imunoabsorção por ligação enzimática) tem sido amplamente utilizada para detectar anticorpos anti-SARS-CoV-2 gerados após a exposição ao vírus ou à vacinação. A amostra comumente utilizada para a realização do teste é o soro. Até o momento, nenhum estudo havia investigado a urina do paciente como amostra para detectar anticorpos específicos para o vírus SARS-CoV-2. A urina é um espécime biológico que traz vantagens significativas inerentes ao tipo de amostra, que compreende coleta não invasiva, de fácil manuseio e armazenamento. Neste trabalho, propomos um ELISA indireto in house baseado no uso de urina e proteínas recombinantes do Nucleocapsídeo (N) ou da Spike (S) do vírus SARS-CoV-2. As proteínas recombinantes (r) de SARS-CoV-2, N e as subunidades da proteína S (S-Glic, S1-NGlic e RBD-NGlic), foram avaliadas usando um painel composto por aproximadamente 200 amostras de urina e de soro. A presença de anticorpos anti-SARS-CoV-2 na urina foi detectada com sensibilidade e especificidade similares ou superiores ao soro, nas quais foram obtidos valores de sensibilidade de 94,0%, 75,0%, 81,38% e 89,66%, e especificidade de 100%, 96,0%, 96,77% e 96,77%, frente às proteínas rSARS-CoV-2 N, S-Glic, S1-NGlic e RBDNGlic, respectivamente. Dessa forma, os dados apresentados sugerem que a urina poderia ser considerada como uma potencial amostra biológica para aplicação em plataformas de imunodiagnóstico para a infecção por SARS-CoV-2, trazendo benefícios tanto no contexto individual quanto populacional.

The Enzyme-linked immunosorbent assay (ELISA) method has been widely used to detect anti-SARS-CoV-2 antibodies generated after exposure to the virus or vaccination. The sample usually used to perform the test is the serum. Thus far, no study has investigated the urine of patients as biological sample to detect specific SARS-CoV-2 antibodies. Urine is a biological specimen with significant advantages inherent to the type of sample, which comprises non-invasive collection, easy handling and storage. In this work, we propose an in house urine-based indirect ELISA using recombinant proteins from Nucleocapsid (N) and Spike (S) of the SARSCoV-2 virus. SARS-CoV-2 recombinant N and S protein subunits (Gly-S, NonGly-S1 and NonGly-RBD) were evaluated in an ELISA platform with a panel composed about 200 urine and serum samples. The presence of anti-SARS-CoV-2 antibodies in urine was detected with similar or superior sensitivity and specificity to serum, in which sensitivity values of 94.0%, 75.0%, 81.38% and 89.66% were obtained, while specificity values were of 100.0%, 96.0%, 96.77% and 96.77%, respectively, against rSARS-CoV-2 N, S-Glic, S1-NGlic and RBD-NGlic proteins. In conclusion, the data presented suggest that urine could be considered as a potential biological sample for application in immunodiagnostic platforms for SARS-CoV-2 infection, with benefits to the individual and population context.

Development of monoclonal antibody based IgG and IgM ELISA for diagnosis of severe fever with thrombocytopenia syndrome virus infection

ABSTRACT Introduction: Severe fever with thrombocytopenia syndrome virus (SFTSV) is a newly emerged virus that poses a great threat to human health because of high fatality rate. Methods: To develop sensitive and specific sero-diagnostic systems for SFTSV infections, monoclonal antibodies (MAbs) against recombinant SFTSV nucleocapsid (rSFTSV-N) protein were developed by immunizing BALB/C mice with rSFTSV-N protein and fusing the spleen cells with SP2/0 myeloma cells. Three hybridoma cell lines secreting MAbs against rSFTSV-N were obtained. MAb based IgG sandwich enzyme linked immunosorbent assay (ELISA) and IgM capture ELISA systems were established by using the newly developed MAbs. One hundred fifteen clinical suspected SFTS patients serum samples were used to evaluate the newly established systems by comparing with the total antibody detecting sandwich ELISA system and indirect ELISA systems. Results: The MAbs based sandwich IgG ELISA was perfectly matched with that of the total antibody sandwich ELISA and the indirect IgG ELISA. IgM capture ELISA results perfectly matched with that of the total antibody sandwich ELISA while detecting eight additional positive samples missed by the indirect IgM ELISA. Conclusions: The MAbs against rSFTSV-N protein offer new tools for SFTSV studies and our newly developed MAb-based IgG and IgM capture ELISA systems would offer safe and useful tools for diagnosis of SFTS virus infections and epidemiological investigations.

Preparation of different fragments of SARS-CoV-2 N protein and its application in fluorescence chromatography / 生物工程学报

Different fragments of SARS-CoV-2 nucleocapsid (N) protein were expressed and purified, and a fluorescence immunochromatography method for detection of SARS-CoV-2 total antibody was established. The effect of different protein fragments on the performance of the method was evaluated. The N protein sequence was analyzed by bioinformatics technology, expressed in prokaryotic cell and purified by metal ion affinity chromatography column. Different N protein fragments were prepared for comparison. EDC reaction was used to label fluorescence microsphere on the synthesized antigen to construct sandwich fluorescence chromatography antibody detection assay, and the performance was systemically evaluated. Among the 4 prepared N protein fragments, the full-length N protein (N419) was selected as the optimized coating antigen, N412 with 0.5 mol/L NaCl was used as the optimal combination; deleting 91-120 amino acids from the N-terminal of N412 reduced non-specific signal by 87.5%. the linear range of detection was 0.312-80 U/L, the limit of detection was 0.165 U/L, and the accuracy was more than 95%. A fluorescence immunochromatographic detection method for analysis of SARS-CoV-2 total antibody was established by pairing N protein fragments. The detection result achieved 98% concordance with the commercially available Guangzhou Wanfu test strip, which is expected to be used as a supplementary approach for detection of SARS-CoV-2. The assay could also provide experimental reference for improving the performance of COVID-19 antibody detection reagents.

Prokaryotic expression, purification and antiserum preparation of severe acute respiratory syndrome coronavirus 2 nucleocapsid protein / 第二军医大学学报

Objective To express and purify the recombinant nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and prepare antiserum from immunized mice. Methods The prokaryotic plasmid pET28a-N containing SARS-CoV-2 N gene was transformed into Escherichia coli BL21 (DE3). The expression of recombinant SARS-CoV-2 N protein was induced by isopropyl-β-D-thiogalactopyranoside. The Ni-NTA affinity chromatography column was used to purify the recombinant SARS-CoV-2 N protein, and antiserum was obtained from the BALB/c mice immunized with recombinant SARS-CoV-2 N protein combined with manganese adjuvant through intramuscular and subcutaneous injections. The reactions of recombinant SARS-CoV-2 N protein with SARS-CoV-2 N monoclonal antibodies and severe acute respiratory syndrome coronavirus (SARS-CoV) N polyclonal antibodies were detected by Western blotting. The reaction of mouse antiserum with the recombinant SARS-CoV-2 N protein expressed in the cells transfected with eukaryotic expression plasmid was examined by indirect immunofluorescence assay. Results The recombinant SARS-CoV-2 N protein was successfully induced and expressed as a soluble protein with a molecular weight of about 55 000. High concentration of purified protein was obtained. The results of Western blotting showed that the recombinant SARS-CoV-2 N protein could be specifically recognized by the SARS-CoV-2 N monoclonal antibodies and the SARS-CoV N polyclonal antibodies. The prepared mouse antiserum could also correctly recognize the recombinant SARS-CoV-2 N protein expressed in mammalian cells by indirect immunofluorescence assay. Conclusion Recombinant SARS-CoV-2 N protein has been successfully expressed and purified from the prokaryotic expression system, and mouse antiserum has been prepared, which lays a foundation for establishing a rapid SARS-CoV-2 diagnostic tool and further studying the function of SARS-CoV-2 N protein..

Crystal structure of SARS-CoV-2 nucleocapsid protein RNA binding domain reveals potential unique drug targeting sites

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 Å 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 the -sheet core. Complemented by 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.

Natural versus recombinant viral antigens in SARS-COV-2 serology: challenges in optimizing laboratory diagnosis of COVID19

OBJECTIVES: COVID-19 is a public health emergency of international concern whose detection in recovered asymptomatic patients is dependent on accurate diagnosis as it enables the estimation of the susceptibility of the population to the infection. This demand has resulted in the development of several commercial assays employing recombinant proteins, but the results of these assays are not reliable as they do not involve comparison with natural viral antigens. We independently used the SARS-CoV-2 whole viral antigen (WVA) and recombinant nucleocapsid protein (rNP) to develop in-house ELISAs for IgG detection; the results of these ELISAs were then compared to obtain reliable results. METHODS: WVA and rNP ELISAs were performed on COVID-19 negative sera from patients before the pandemic in Brazil, and on RT-qPCR-positive or SARS-CoV-2-IgG against rNP and IgG against WVA-positive samples from recently infected patients in Sao Paulo, Brazil. RESULTS: Both ELISAs detected a large fraction of infected patients but exhibited certain drawbacks. Higher signals and lower numbers of false-negatives were observed in rNP ELISA; however, a higher fraction of false-positives was observed in control groups. A high number of false-negatives was observed with WVA ELISA. Correlating the results of rNP and WVA ELISAs resulted in improved performance for COVID-19 diagnosis. CONCLUSION: The choice of antigen is an important aspect in optimizing the laboratory diagnosis of COVID-19. The use of rNP ELISA for the detection of anti-SARS-CoV-2 IgG antibodies seems promising, but comparison of the results with those of WVA ELISA is crucial for accurate test development prior to commercialization. IgG serology using several assays, and with the spectral patterns of SARS-CoV-2, resulted in confusing information that must be clarified before the establishment of diagnostic serology criteria.

Construction and characterization of a recombinant vaccine encoding the nucleocapsid protein gene of avian infectious bronchitis virus / Construção e caracterização de uma vacina recombinante codificando o gene da proteina do nucleocapsideo do virus da bronquite infecciosa das galinhas

A vacinação é a forma mais utilizada para prevenir a bronquite infecciosa causada pelo vírus da bronquite infecciosa das galinhas (IBV). Contudo, as vacinas convencionais são incapazes de diferenciar aves infectadas de vacinadas. No presente trabalho foi construído, caracterizado, e avaliado como candidato vacinal, um adenovírus recombinante expressando o gene N do IBV. O gene N foi clonado em um adenovírus humano tipo 5 defectivo e transfectado para as células HEK-293A para gerar rAd5_N. Após o vetor ser obtido como esperado e a confirmação da expressão da proteína N em HEK-293ª, foi realizada inoculação pela via oculo-nasal na dose de 10 7 TCID 50 /0,1mL para imunização de galinhas livres de patógenos específicos (SPF). A resposta imunológica do Ad5_N e a proteção contra o desafio ao IBV foram avaliadas e comparadas com uma vacina viva comercial. Não foram detectados anticorpos anti-IBV em aves vacinadas com o Ad5_N. A vacina comercial induziu anticorpos detectáveis a partir do 7º dia pós-vacinal. Em aves vacinadas com o Ad5_N não houve aumento na expressão de IFNγ. Neste estudo, o rAd5_N obtido não conferiu proteção contra desafio com IBV-M41. Os resultados indicam a necessidade de avaliar adenovírus recombinantes expressando outros genes do IBV.(AU)

Antigenic and immunogenic properties of the canine distemper virus nucleocapsid protein expressed in Escherichia coli employing codon optimized synthetic gene / Propriedades antigênicas e imunogênicas da proteína do nucleocapsídeo do virus da cinomose canina expressa em Escherichia coli empregando gene sintético com codons otimizados

Despite common occurrence and importance of canine distemper disease the majority of tests currently available for diagnosis are hampered by either low sensitivity or specificity. In this study it was evaluated antigenic and immunogenic characteristics of a conserved region of nucleocapsid protein of canine distemper virus (rCDV NP) expressed in Escherichia coli employing a codon optimized synthetic gene. The expression of rCDVNP in Star strain (mean 300μg/mL, purified) was confirmed by SDS-PAGE and Western blot analysis by using His-Tag monoclonal antibodies. Western blot and ELISA, employing positive and negative control dog sera, demonstrated the rCDVNP antigenicity. The rCDVNP was inoculated in hens and immunoglobulin Y (IgY) was purified from the egg yolk. The mean yield of IgY was 28.55mg/mL. IgY reacted with the recombinant protein as demonstrated by Western blot and ELISA assays. In summary, our findings demonstrated that rCDVNP is antigenic since CDV positive dog sera recognized the protein in vitro. Additionally, the rCDVNP proved to be immunogenic in hens being possible to isolate a high concentration of specific IgY antibodies from the egg yolk. Taken together, these results indicate that the rCDVNP along with the specific IgY could be useful tools for development of the canine distemper immunodiagnostic assays.(AU)

Apesar da ocorrência comum e importância da cinomose canina, a maioria dos testes atualmente disponíveis para diagnóstico são prejudicados pela baixa sensibilidade ou especificidade. Neste estudo foram avaliadas características antigênicas e imunogênicas de uma região conservada da proteína do nucleocapsídeo do virus da cinomose canina (rCDV NP) expressa em Escherichia coli empregando um gene sintético e codons otimizados. A expressão na cepa Star (média de 300μg/mL, purificada) foi confirmada por SDS-PAGE e Western blot utilizando anticorpos monoclonais anti-His-Tag. A antigenicidade da rCDVNP foi demonstrada por western blot e ELISA empregando soros de cães positivos e negativos. A rCDVNP foi inoculada em galinhas e imunoglobulina Y (gY) foi obtida e purificada a partir da gema. A produção média de IgY foi 28.55mg/mL. Anticorpos IgY reagiram com a proteína recombinante, quando analisados por Western blot e ELISA. Em resumo, nossos achados demonstram que a rCDVNP produzida é antigênica, uma vez que os anticorpos de soro de cães positivos para CDV reconheceram a proteína in vitro. Além disso, a rCDVNP foi imunogênica em galinhas, sendo possível isolar anticorpos IgY específicos a partir da gema do ovo em altas concentrações. Tomados em conjunto, estes resultados indicam que a rCDVNP juntamente com a IgY específica podem ser ferramentas úteis para elaborar ensaios de imunodiagnóstico de cinomose canina.(AU)

Nucleocapsid protein from porcine epidemic diarrhea virus isolates can antagonize interferon-λ production by blocking the nuclear factor-κB nuclear translocation / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

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.

Recombinant expression of hantaan virus protein N with application of Western-blot in detecting anti-hantavirus antibody / 中华流行病学杂志

Objective S gene of hantavirus(HV) was expressed in insect cells by genetic engineering technology.The expression product of S gene was used as antigen to detect anti-HV specific antibody IgG in serum.Methods Gene encoding NP of the strain HV-Z10 was amplified by PCR and then its eukaryotic expression system rBAC-Z 10S-TN was constructed by using the routine genetic engineering method.SDS-PAGE was applied to measure the expression of rNP.Ion-exchange plus Ni-NTA-affinity chromatography was performed to purify the recombinant product.Indirect immuno-fluorescence assay (IFA) was used to determine the specific immune-reactivity of rNP.WB assay was established to detect the serum samples from 95 confirmed HFRS patients.Parameters related to the outcomes of detection were compared with the routine HV-IgG IFA method.Results rBAC-Z10S-TN was able to express rNP with high efficiency.The purified rNP only showed a single protein fragment in the gel after SDS-PAGE.HV IgG could efficiently recognize rNP and hybridize with the recombinant protein.97.67％ of the serum samples from the HFRS patients were positive confirmed by WB.Conclusions We successfully constructed a high efficient prokaryotic expression system of NP encoding gene from hantavirus strain HV-Z10.WB assay which was established in this study could be used as a new serological test for HFRS diagnosis,thanks to the simplicity,safety,sensitivity and specificity of this method.

Recombinant expression of hantaan virus protein N with application of Western-blot in detecting anti-hantavirus antibody / 中华流行病学杂志

Objective S gene of hantavirus(HV) was expressed in insect cells by genetic engineering technology.The expression product of S gene was used as antigen to detect anti-HV specific antibody IgG in serum.Methods Gene encoding NP of the strain HV-Z10 was amplified by PCR and then its eukaryotic expression system rBAC-Z 10S-TN was constructed by using the routine genetic engineering method.SDS-PAGE was applied to measure the expression of rNP.Ion-exchange plus Ni-NTA-affinity chromatography was performed to purify the recombinant product.Indirect immuno-fluorescence assay (IFA) was used to determine the specific immune-reactivity of rNP.WB assay was established to detect the serum samples from 95 confirmed HFRS patients.Parameters related to the outcomes of detection were compared with the routine HV-IgG IFA method.Results rBAC-Z10S-TN was able to express rNP with high efficiency.The purified rNP only showed a single protein fragment in the gel after SDS-PAGE.HV IgG could efficiently recognize rNP and hybridize with the recombinant protein.97.67％ of the serum samples from the HFRS patients were positive confirmed by WB.Conclusions We successfully constructed a high efficient prokaryotic expression system of NP encoding gene from hantavirus strain HV-Z10.WB assay which was established in this study could be used as a new serological test for HFRS diagnosis,thanks to the simplicity,safety,sensitivity and specificity of this method.

Expression of SARS-CoV nucleocapsid protein in Bac-to-Bac Baculovirus System and antigenic analysis / 中国免疫学杂志

Objective:To express SARS-CoV nucleocapsid protein in Bac-to-Bac Baculovirus Expression System and analyze the antigenicity of the recombinant protein.Methods: The SARS-CoV nucleocapsid gene was amplified by PCR.The PCR product digested with BamHⅠand SalⅠrestriction endonucleases was cloned into vector pFastBac HTC of Bac-to-Bac Baculovirus expression system.Recombinant plasmid was transformed DH 10Bac cells to obtain the recombinant Bacmid DNA.Recombinant Bacmid DNA was transferred into Sf9 cells which were inducted to express the recombinant protein in High Five cells.After purified by Ni affinity chroma-tography ,the antigenicity of the recombinant protein was analyzed by Western blot and ELISA.Results:Recombinant plasmid was con-structed successfully.The recombinant protein with the relative molecular mass of 48 kD was efficiently expressed in High Five cells and purified successfully by Ni affinity chromatography.Western blot and ELISA analysis showed that the recombinant protein could be spe -cifically recognized by the monoclonal antibody to SARS-CoV N protein and immune serum from rabbits ,respectively.The recombinant protein can specifically reacted with serum from SARS patients ,not with serum from healthy persons and patients infected with hCoV-229 E and hCoV-OC43.Conclusion: SARS-CoV nucleocapsid protein has been expressed successfully in the Bac-to-Bac Baculovirus Expression System ,and obtained good antigenicity.It is preliminary deemed that it can't reacted with serum from patients infected with hCoV-229E and hCoV-OC43.

The effect of nucleocapsid protein of severe acute respiratory syndrome-associated coronavirus on the expression of cyclooxygenase-2 / 中华传染病杂志

Objective To investigate the effect of nucleocapsid (N) protein of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) on the expression of cyclooxygenase-2 (COX-2). Methods 293T cells were co-transfected with reporter plasmid pCOX-2-Luc containing the luciferase gene under the control of COX-2 promoter and plasmids carrying individual genes of SARS-CoV, and luciferase activity was measured. Expression of COX-2 mRNA and protein was measured by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Results N protein of SARS-CoV enhanced COX-2 gene promoter activity, and upregulated COX-2 mRNA expression.COX-2 protein production in 293T cells was N protein concentration-dependent. Conclusion N protein of SARS-CoV could specifically activate COX-2 expression.

Serial Expression of the Truncated Fragments of the Nucleocapsid Protein of CCHFV and Identification of the Epitope Region / 中国病毒学·英文版

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(nueleoeapsid 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 polyclonai 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.

In Vivo Kinetics and Biodistribution of a Hantaan Virus DNA Vaccine after Intramuscular Injection in Mice / 中国病毒学·英文版

To study the kinetics in vivo of a Hantaan virus DNA vaccine, we constructed a fusion DNA vaccine,pEGFP/S, by cloning the S segment of Hantavirus into the vector, pEGFP-C1, which encodes Green fluorescent protein EGFP. In this report, we provide evidence that pEGFP/S was distributed and persistently expressed for more than 60 days in several organs after inoculation. Our findings suggest that the persistent immune responses induced by a Hantaan virus DNA vaccine are likely due to the plasmid pEGFP/S deposited in vivo, which acts as a booster immunization.

Production of specific antibodies against SARS-coronavirus nucleocapsid protein without cross reactivity with human coronaviruses 229E and OC43

Severe acute respiratory syndrome (SARS) is a life-threatening disease for which accurate diagnosis is essential. Although many tools have been developed for the diagnosis of SARS, false-positive reactions in negative sera may occur because of cross-reactivity with other coronaviruses. We have raised polyclonal and monoclonal antibodies (Abs) using a recombinant form of the SARS virus nucleocapsid protein. Cross-reactivity of these anti-SARS Abs against human coronavirus (HCoV) 229E and HCoV OC43 were determined by Western blotting. The Abs produced reacted with recombinant SARS virus nucleocapsid protein, but not with HCoV 229E or HCoV OC43.

Development and characterization of stable cell lines constitutively expressing the porcine reproductive and respiratory syndrome virus nucleocapsid protein

Despite global efforts to control porcine reproductive and respiratory syndrome virus (PRRSV) infection, the virus continues to cause economic problems in the swine industry worldwide. In this study, we attempted to generate and characterize a panel of stable BHK cell lines that constitutively express the nucleocapsid (N) protein of type 1 or type 2 PRRSV. The established BHK cell lines were found to react well with N-specific antibodies as well as the hyperimmune serum of pigs raised against each genotype of PRRSV. Taken together, the data implicate a potential usefulness for the newly generated stable cell lines as a diagnostic reagent for PRRSV serology.

Identification and Characterization of Nuclear Localization Signals within the Nucleocapsid Protein VP15 of White Spot Syndrome Virus / 中国病毒学·英文版

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. NLS 1 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 NLSI 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.