ObjectiveTo determine the potential natural foci of new bunyavirus,and isolate and identify the new bunyavirus strain in sera from suspected new bunyavirus-infected patients.MethodsImmunofluorescence assay was used to detect the antigens of new bunyavirus in different tissue specimens of wild rodent animals in Tiantai area of Zhejiang province.Fluorescence quantitative real-time RT-PCR was applied to detect the viral nucleic acid in sera of suspected new bunyavirus-infected patients and the amplification products were analyzed by sequencing.The new bunyavirus in the pateints'sera was isolated using Vero cells.Using nucleocapsid protein encoding gene of new bunyavirus as the target gene,the isolated suspected new bunyavirus strain was identified by RT-PCR and sequencing of the amplification product.Moreover,sequence identity of the amplification product of nucleocapsid protein encoding gene of new bunyavirus was analyzed and compared.ResultsOf the 70 wild rodent animals,5.71% were positive in the immunofluorescence assay.The fluorescence quantitative real-time RT-PCR confirmed that two of the four detected patients'serum specimens were positive.One suspected strain of new bunyavirus was isolated from one pf the two positive patients'serum specimens.The results of RT-PCR and sequencing confirmed that the viral strain exactly belongs to new bunyavirus with 92.2% sequence identity to that of the new bunyavirus isolates in Hubei province but distinct with the new bunyavirus isolates from other areas in China.ConclusionThe presence of natural foci of new bunyavirus and new bunyavirus-infected patients in Zhejiang province are firstly confirmed by this study.There is a geographical diversity of the distribution of new bunyavirus in different groups.

Objective To pan and characterize anti-HIV-1 Fab by the phage antibody library technology. Methods Total RNA were extracted from lymphocytes which were isolated from peripheral blood collected from asymptomatic HIV-1 infected donors with high titer antibody against HIV-1. The genes of heavy chains Fd fragment and light chains of antibody were amplified by RT-PCR. The phagmids pComb3X cloned Fd and light chain genes were transformed into E. coli XL1-Blue by electroporation to construct phage Fab library. By three runs of "absorption-elution-neutralization-enrichment", the clones were induced by IPTG and characterized by ELISA. The positive clones were sequenced and analyzed the sequences. Subsequently, Fab antibodies of these positive clones were induced to expressed and purified, then the recombinant virus neutralization assay was performed. Results A phage Fab library was constructed with 8×106 members, and 11 positive clones were obtained by detecting IPTG-induced-expressing Fabs with ELISA. By analysis of the sequences, 10 light chain genes and 8 Fd genes were ensured to be obtained. Compared with the genes of anti-HIV-1 antibodies in HIV sequence database, the gene sequences we obtained were highly homologous to some patent genes of anti-HIV-1 gp120 antibodies in HIV sequence database( light chains with 60%-90% identity, Fd with 71%-85% identity); The CDRs of these positive clones were determined by comparing the positive clone genes with antibodies' genes in V base database, furthermore, CDRH3 of these positive clones has the length of 12-22 aa. Strand shift had little effect to improving affinity of our Fab clones. Fab antibodies were induced to express at the concentration of ＞ 10 mg/L. Three Fab antibodies neutralize HIV-1 virus to some extent. Conclusion The studies will provide the basis on further study on the anti-HIV-1 Fabs obtained successfully.

To explore a new method for stable expression of virus-like particles (VLPs) of the severe fever with thrombocytopenia syndrome (SFTS) virus, an expression plasmid for the membrane glycoprotein (GP) and nucleocapsid protein (NP) of the SFTS virus was constructed by fusion of the two proteins via a serine residue, and a yellow fluorescence protein (YFP) gene was introduced into the plasmid as a reporter. CHO-K1 cells were transfected with this plasmid, and stable cell lines constructed using the limited dilution method. Cellular colonies were hand-picked based on YFP with the help of fluorescence microscopy and expanded without selection pressure. Stability of cell lines was evaluated by monitoring of fluctuation of the intensity of YFP for 40 passages. VLP production was characterized using an indirect fluorescence assay, immunoblotting, and electronic microscopy. We showed that GP and NP fusion proteins could be assembled into VLPs in vivo, and that VLPs had similar morphologies to virus particles. Selected cell lines were stable for YFP expression: no significant fluctuation was detected in 40 passages. These data demonstrated the effectiveness of this new method for expression of structural proteins of the SFTS virus and screening for stable cell lines. Our results could provide new concepts for the production of biopharmaceuticals.
Animals ; Bunyaviridae Infections ; virology ; CHO Cells ; Cloning, Molecular ; methods ; Cricetinae ; Cricetulus ; Gene Expression ; Phlebovirus ; genetics ; metabolism ; Plasmids ; genetics ; metabolism ; Viral Proteins ; genetics ; metabolism ; Virion ; genetics ; metabolism ; Virus Assembly

To understand the immunogenicity of purified inactivated severe fever with thrombocytopenia syndrome bunyavirus (SFTSV), concentration by ultrafiltration as well as molecular-sieve chromatography (MSC) were used for purification of inactivated SFTSVs. Inactivated viruses in purified samples were analyzed and identified by western blotting and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the glycoprotein (GP) and nucleoprotein (NP) antigen titers of which were detected using a double-sandwich enzyme-linked immunosorbent assay (ELISA). Purified inactivated SFTSVs were enriched and observed by electron microscopy, and the total protein concentration detected using the bicinchoninic acid assay. Purified inactivated SFTSVs were applied to New Zealand rabbits via two immunization programs to evaluate immunogenicity and to compare the immune effect. After SFTSVs were inactivated and concentrated by ultrafiltration, MSC revealed two typical elution peaks. The sample of one peak was identified as inactivated virions, in which GP and NP were detected by SDS-PAGE, western blotting and ELISA. Main corponent of the other peak was NP. After concentration by ultrafiltration, purified inactivated SFTSVs with purity >90% and total protein concentration of 1. 1 mg/mL were obtained, and the typical electron microscopy of bunyavirus was observed. In the sera of animals immunized with purified inactivated SFTSVs, SFTSV-specific IgG antibody and neutralizing antibody were detected at high titers. However, antibody titers were affected by the immunization program. Effect of immunization on days 0, 14 and 28 was significantly better than that on days 0, 7 and 28. Our work revealed that cultivation of SFTSVs contained intact virus particles and large amounts of free NP. Using MSC, purified inactivated SFTSVs of high purity could be obtained. Purified inactivated SFTSVs induced high titers of neutralizing antibody and virus-specific IgG antibody showing satisfactory immunogenicity, which provides important clues for further study on a vaccine for the inactivated virus.
Animals ; Antibodies, Neutralizing ; immunology ; Antibodies, Viral ; immunology ; Bunyaviridae Infections ; immunology ; virology ; Humans ; Neutralization Tests ; Phlebovirus ; classification ; genetics ; immunology ; isolation & purification ; Rabbits

To obtain human antibodies against the Gn protein of Severe fever with thrombocytopenia syndrome virus (SFTSV) with phage display technology, this study aimed to screen anti-Gn protein antibodies from an anti-SFTSV Fab human phage display library. Antibody genes were identified by sequence analysis and the specificity of antibodies was confirmed by ELISA. The Fab antibody genes were cloned into the HL51-14 vector and expressed in a mammalian cell expression system. IgG antibodies were then purified by protein A affinity chromatography,and the results were further confirmed by ELISA,IFA,western blotting assays and micro-neutralization tests. The results showed that, after three rounds of panning, there were 390 human Fab antibodies against SFTSV particles, of which 364 were specific for nucleoprotein. Coated with the Gn protein, eight different Fab antibodies specific for Gn protein were obtained after the determination of the subtype and subclass of antibodies by gene sequencing; five of these antibodies were from the Lambda library and three were from the Kappa library. The eight IgG antibodies could specifically bind to Gn protein according to the ELISA, IFA and Western blotting assays. The micro-neutralization test showed that these eight antibodies had no neutralizing activity,but they could still provide a reference for research in human monoclonal antibodies against SFTSV.
Antibodies ; genetics ; immunology ; Bunyaviridae Infections ; genetics ; immunology ; virology ; Cell Line ; Cloning, Molecular ; Humans ; Immunoglobulin Fab Fragments ; genetics ; immunology ; Immunoglobulin G ; genetics ; immunology ; Neutralization Tests ; Phlebovirus ; genetics ; immunology ; Viral Proteins ; genetics ; immunology

To prepare monoclonal antibodies (mAbs) against structural proteins of severe fever with thrombocytopenia syndrome bunyavirus (SFTSV), BALB/c mice were immunized using purified inactivated SFTSV virions as the antigens. Subsequently, hybridoma cell lines that secreted monoclonal antibodies against nucleoprotein (NP) and glycoproteins (GP) were obtained using a hybridoma technique. The antigen specificities of prepared mAbs were examined by indirect immunofluorescence and immunoprecipitation assays. Functional analyses were then performed,including the detection of IFA antibody titers,the levels of neutralizing activity and antibody affinities. After cell fusion and cloning,13 hybridoma cell lines secreted mAbs specifically against SFTSV-GP and 7 hybridoma cell lines secreted mAbs specifically against SFTSV-NP. Immunofluorescence and immunoprecipitation assays showed that the mAbs had high levels of antigen specificity. Among the 13 anti-SFTSV-GP mAbs,6 recognized Gn,whereas the others reacted with Gc. IFA titers of most anti-SFTSV-GP mAbs were between 1,280 and 20,480, and four anti-SFTSV-Gn mAbs showed neutralizing activity. Seven of the obtained anti-SFTSV-NP mAbs reacted specifically with NP,of which the IFA titers ranged from 5,120 to 20,480 with no observed neutralizing activity. Furthermore, two anti-SFTSV-GP mAbs, 1C8 and 1G8, showed high levels of affinity via a non-competitive ELISA. Our study lays the foundation for the development of further diagnostic assays and basic research into SFTSV.
Animals ; Antibodies, Monoclonal ; immunology ; Antibodies, Viral ; immunology ; Antibody Specificity ; Bunyaviridae Infections ; immunology ; virology ; Female ; Humans ; Hybridomas ; immunology ; Mice ; Mice, Inbred BALB C ; Phlebovirus ; immunology ; Viral Structural Proteins ; immunology

The severe fever with thrombocytopenia syndrome virus (SFTSV) is a new member in the genus Phlebovirus of the family Bunyaviridae identified in China. The SFTSV is also the causative pathogen of an emerging infectious disease: severe fever with thrombocytopenia syndrome. Using immunofluorescent staining and confocal microscopy, the intracellular distribution of nucleocapsid protein (NP) in SFTSV-infected THP-1 cells was investigated with serial doses of SFTSV at different times after infection. Transmission electron microscopy was used to observe the ultrafine intracellular structure of SFTSV-infected THP-1 cells at different times after infection. SFTSV NP could form intracellular inclusion bodies in infected THP-1 cells. The association between NP-formed inclusion bodies and virus production was analyzed: the size of the inclusion body formed 3 days after infection was correlated with the viral load in supernatants collected 7 days after infection. These findings suggest that the inclusion bodies formed in SFTSV-infected THP-1 cells could be where the SFTSV uses host-cell proteins and intracellular organelles to produce new viral particles.
Cell Line ; China ; Humans ; Inclusion Bodies, Viral ; ultrastructure ; virology ; Macrophages ; ultrastructure ; virology ; Phlebotomus Fever ; virology ; Phlebovirus ; genetics ; physiology ; ultrastructure ; Thrombocytopenia ; virology

The Ebola virus is highly infectious and can result in death in ≤ 90% of infected subjects. Detection of the Ebola virus and diagnosis of infection are extremely important for epidemic control. Presently, Chinese laboratories detect the nucleic acids of the Ebola virus by real-time reverse transcription-polymerase chain reaction (RT-PCR). However, such detection takes a relatively long time and necessitates skilled personnel and expensive equipment. Enzyme-linked immunosorbent assay (ELISA) of serum is simple, easy to operate, and can be used to ascertain if a patient is infected with the Ebola virus as well as the degree of infection. Hence, ELISA can be used in epidemiological investigations and is a strong complement to detection of nucleic acids. Cases of Ebola hemorrhagic fever have not been documented in China, so quality-control material for positive serology is needed. Construction and expression of human-mouse chimeric antibodies against the nucleoprotein of the Ebola virus was carried out. Genes encoding variable heavy (VH) and variable light (VL) chains were extracted and amplified from murine hybridoma cells. Genes encoding the VH and VL chains of monoclonal antibodies were amplified by RT-PCR. According to sequence analyses, a primer was designed to amplify functional sequences relative to VH and VL chain. The eukaryotic expression vector HL51-14 carrying some human antibody heavy chain- and light chain-constant regions was used. IgG antibodies were obtained by transient transfection of 293T cells. Subsequently, immunological detection and immunological identification were identified by ELISA, immunofluorescence assay, and western blotting. These results showed that we constructed and purified two human- mouse chimeric antibodies.
Animals ; Antibodies, Monoclonal ; genetics ; immunology ; Cloning, Molecular ; Ebolavirus ; genetics ; immunology ; Hemorrhagic Fever, Ebola ; immunology ; virology ; Humans ; Immunoglobulin Heavy Chains ; genetics ; immunology ; Mice ; Nucleoproteins ; genetics ; immunology ; Viral Proteins ; genetics ; immunology

This study performed phylogenic analysis on a dengue strain isolated from an outbreak of dengue fever in 2009 at Yiwu City of Zhejiang Province,China,and further to analyze the immunogenicity of E protein of this viral isolate.Firstly,the viral genome was amplified by RT-PCR and phylogenetic trees were constructed by MEGA 4 based on both nucleotide and amino acid sequences of E and NS1 proteins.The phylogenetic analysis showed that the similarity of Yiwu strain with the Guangzhou GZ1D3 strain and the India GWL-25 strain was over 99％.Secondly,the expression plasmid of E protein was constructed and transfected into 293T cells.The secreted E protein were then purified by sucrose density gradient centrifugation and used to inoculate BALB/c mice.The humoral immunity was evaluated by ELISA and neutralizing antibody analysis.Resuits showed that the E protein of Yiwu strain could induce dengue specific IgG antibodies and neutralizing antibodies.Therefore,the study found that the Yiwu strain was classified into the subtype Ⅲ of dengue virus type 3 (DENV-3),and the E protein of this strain had strong immunogenicity

BACKGROUNDTo sequence, analyze and express the nucleotide sequences of S and M segments of hantavirus strain 84Fli.

METHODSS and M segments of hantavirus 84Fli strain were amplified by RT-PCR, the PCR products were cloned into plasmid pCR2.1-TOPOr. Three clones of each segment which have been sequenced were randomly selected. The coding region of S and M segments were amplified by PCR, and cloned into expressing vector pcDNA3.0. Transient expression of nucleocapsid protein, G1 and G2 glycoproteins in COS7 cells were performed by Lipofectin transfection. The expression of NP, G1 and G2 have been conformed by using immunofluorescence, Western blot and immuno-precipitation methods.

RESULTSThe full length S and M segments cDNA have been amplified and cloned. The S and M segments of hantavirus strain 84Fli contained 1 688 and 3 616 nucleotides respectively.

CONCLUSIONSDeduced amino acid sequences of NP and glycoproteins revealed high homologue to other Hantaan viruses. NP, G1 and G2 proteins of 84Fli can be transiently expressed in COS7 cells.

Animals ; COS Cells ; metabolism ; Cloning, Molecular ; Gene Expression ; Glycoproteins ; biosynthesis ; Hantavirus ; genetics ; metabolism ; Nucleocapsid Proteins ; biosynthesis ; Sequence Analysis ; Viral Proteins ; biosynthesis