Neutralization properties of different genotypes and mutants of severe fever with thrombocytopenia syndrome virus / 中华微生物学和免疫学杂志

Objective:To analyze the neutralization properties of different genotypes and mutants of severe fever with thrombocytopenia syndrome virus (SFTSV).Methods:Pseudoviruses of SFTSV of different genotypes and mutants were constructed using VSVΔG-Fluc*G backbone. Neutralization assays were established based on the pseudoviruses. DNA vaccines for different SFTSV genotypes were prepared. Serum samples were collected from guinea pigs immunized with the DNA vaccines. Neutralizing antibodies in serum samples from immunized guinea pigs and naturally infected patients were detected using neutralization assays and analyzed.Results:The pseudoviruses of five genotypes and 43 mutants were successfully constructed and the neutralization assays based the pseudoviruses were successfully established after optimizing the reaction parameters. The dilution multiple corresponding to the inhibition rate of neutralizing antibody to half of the pseudovirus infection was taken as the titer of neutralizing antibody by the reduction in pseudovirus reporter gene. The neutralization antibody titers in naturally infected patients and immunized guinea pigs were respectively in the ranges of 1∶100-1∶43 000 and 1∶100-1∶2 500 when detected with the reference HB29 pseudovirus. The neutralization antibody titers ranged from 1∶100-1∶2 500 after immunization with different genotypes of DNA vaccines. No significant statistical difference in neutralization antibody titer was observed among different genotypes or mutant strains.Conclusions:The neutralization properties of different genotypes and mutants showed no significant change, which would be very useful for developing vaccines.

Development of a triplex real-time RT-PCR assay to detect Zika, Chikungunya and Mayaro viruses / 中华实验和临床病毒学杂志

Objective@#To establish a method for the simultaneous identification of Zika, Chikungunya and Mayaro viruses.@*Methods@#The complete genome sequences of Zika, Chikungunya and Mayaro virus were retrieved from Global Shared Database for comparative analysis, estimate its conservative region and determine the target gene location, specific primers and probes were designed, then a triplex real-time RT-PCR assay was developed. The specificity, sensitivity and repeatability of the assay were assessed by viral nucleic acid of Zika virus, Chikungunya virus a, in vitro transcriptional RNA of Mayaro virus, normal human serum and related virus simulation sample.@*Results@#The result showed that the established method could detect Zika virus, Chikungunya virus, as well as simulated Mayaro virus samples, the limit of detection (LOD) of Zika and Chikungunya virus was 16.22 Copy/PCR and 12.02 Copy/PCR, respectively, the LOD for simulated Mayaro virus RNA was 2.82 Copy/PCR, no significant difference was detected between the triplex and monoplex assays. No cross reaction was found in the detection of dengue virus, Hantavirus, severe fever with thrombocytopenia syndrome (SFTS) virus, yellow fever virus and influenza virus, and 100 healthy adults blood samples, the specificity of the method was 100%. The repeatability result showed that the standard deviation of all three detections were blow 0.5 and the coefficient of variation was less than 2% by selecting viral nucleic acids or transcribed RNA with high, medium and low concentration gradients.@*Conclusions@#A triplex real-time RT-PCR assay for detection of Zika, Chikungunya and Mayaro virus has been established with an acceptable specificity, sensitivity and repeatability.

Generation of human monoclonal antibody Fab fragment to Zika Virus by Phage Display Technology / 中华实验和临床病毒学杂志

Objective@#In this study, phage display technology was used to construct the human anti-Zika virus(ZIKV), phage antibody library and to obtain and express the monoclonal antibody. The aim was to master the preparation and expression of human phage antibody library screening method for highly specific antibodies.@*Methods@#The whole blood samples of Zika patients were collected and the lymphocytes were isolated. The RT-PCR method was used to amplify the antibody light chain and heavy chain Fab gene from lymphocyte Ig mRNA. The pComb3H system was used to construct the gene with genetic diversity Preparation of human anti-ZIKV phage antibody library. The purified antibody library was screened by using the purified ZIKV and the obtained ZIKV E protein antigen.@*Results@#The monoclonal antibody Fab fragment gene was successfully obtained for the ZIKV E protein antigen. The gene can be efficiently expressed in Escherichia coli.@*Conclusions@#According to the sequence analysis, this study showed that the monoclonal antibody was a new human genetically engineered antibody against ZIKV, which laid the foundation for the early diagnosis of ZIKV, and obtain a specific monoclonal antibody to ZIKV for human treatment of ZIKV infection.

Methods of packaging lentivirus library for CRISPR/cas9 -based screening / 中华实验和临床病毒学杂志

Objective@#To obtain the optimum of lentiviral library packaging based on CRISPR/cas9 (Clustered regularly interspaced short palindromic repeat sequences/CRISPR-associated protein 9).@*Methods@#Reverse transcription polymerase chain reaction (RT-PCR), immunofluorescence antibody (IFA) and enzyme linked immunosorbent assay (ELISA) were used to detect the lentivirus titers in condition of different ratio of packaging plasmids, different addition of lipofectamine 3000 reagent and different time points post-transfection. Then, high-throughput sequencing was performed to evaluate the representation and distribution of single guide (sg)RNAs in the library.@*Results@#The lentivirus titer was the highest when the molar ratio of psPAX2∶pMD2.0G∶Lentivirus library was 2∶1∶1, and the optimum addition of Lipofectamine 3000 reagent was 10 μl, while the result of ELISA were correspondent to that of RT-PCR. The IFA result showed that the lentivirus titer was the highest at 60 h post-transfecion. The coverage of sgRNAs in the lentivirus library packaged with the optimum we obtained was 99.3%, and the read counts of sgRNAs was observed in a normal distribution.@*Conclusions@#The optimal lentivirus library packaging was obtained, and this can provide basis for CRISPR/cas9-based screening.

Immortalization of spleen fibroblastic reticular cells infected by severe fever thrombocytopenia syndrome virus / 中华实验和临床病毒学杂志

Objective@#To isolate, purify and culture fibroblastic reticular cells (FRCs) of mouse in spleen, to develop a reliable and robust method to immortalize primary mouse FRCs, to filter stable FRCs cell lines, to prove that the clones can be infected by SFTSV in vitro.@*Methods@#After purifying FRCs by fluorescence activated cell sorting (FACS) from autoMACS-enriched stroma cells of mouse spleen, we infected FRCs by simian virus 40 large T antigen in vitro, screened the FRCs clones with puromycin, compared primary and immortalized FRCs by RNA sequencing(RNA-seq) technology, infected the clones with severe fever with thrombocytopenia syndrome virus (SFTSV) in vitro.@*Results@#We succeed in culturing purified FRCs from spleen, isolated four stable FRCs clones, two of which have a purity of 99%, survived for more than 50 passages, express the key FRCs marker podoplanin and do not express CD31 and CD45. Clone 01 lost the typical FRCs-like morphology, the rate of expansion of which is quite different from that of primary FRCs and Clone 02. Clone 02 can be infected with SFTSV, which has the same gene expression pattern and immunophenotype with primary FRCs.@*Conclusions@#The stable FRCs clone Clone 02 has FRCs-like morphology and express key FRCs surface markers podoplanin (GP38 or PDPN) and do not express endothelial cell markers CD31 and leukocyte common antigen CD45. The RNA expression profiles identified by RNA-seq are also characteristic of FRCs. Infected with SFTSV in vitro, Clone 02 will be a new platform to study SFTSV.

Development of a luminex-based multiplexed immunoassay for detection of IgG antibodies to the viral hemorrhagic fever epidemic in Africa / 中华实验和临床病毒学杂志

Objective@#To establish a multiplexed immunoassay for detection of IgG antibodies against viral hemorrhagic fever epidemic in Africa.@*Methods@#The recombinant antigens of hemorrhagic fever viruses (HFVs) epidemic in Africa were expressed and purified, and then coupled with the fluorescent microspheres. The coupling effects were evaluated by monoplexed detection of rabbit immune sera. Blood specimens were collected from people from Africa with fever, and multiplexed detection of IgG antibodies to HFVs was performed. Comparison of multiplexed assay and ELISA was performed by paired χ2 test using SPSS software.@*Results@#Both the purity and concentration of HFVs recombinant antigen met the standards for coupling and detection, and the antigens were successfully coupled with fluorescent microspheres. Seventy-eight sera samples of people from Africa with fever were multiplex detected. Among these, one was tested positive for LASV-specific IgG, one was tested positive for LUJV-specific IgG, 4 were tested positive for DENV-specific IgG and 6 tested positive for YFV-specific IgG. There was no statistically significant difference compared with ELISA, and the two method were highly correlated.@*Conclusions@#A multiplexed luminex-based immunoassay for detection of IgG antibodies to viral hemorrhagic fever epidemic in Africa was established, which laid the foundation for the differential diagnosis.

A fluorescent bead-based multiplex PCR assay for the simultaneous detection of seven viral diseases endemic in Africa / 中华实验和临床病毒学杂志

Objective@#To establish a fluorescent bead-based multiplex assay for the simultaneous detection of seven viral diseases endemic in Africa.@*Methods@#The genomic sequences of the viral pathogens causing Rift valley fever, Yellow fever, Marburg virus disease, Ebola virus disease, Lassa fever, Crimean-Congo hemorrhagic fever and Chikungunya fever were compared, PCR detection target fragments were selected, and amplification primers and hybrid probes were designed. The reference samples of related pathogens were prepared by chemical synthesis of DNA and in vitro transcription RNA. The sensitivity and stability of the detection method were evaluated. The specificity was evaluated by testing 30 samples of suspected dengue fever, and hantavirus diseases, and 32 healthy human blood samples.@*Results@#The fluorescent bead-based multiplex assay could specifically detect the corresponding pathogen, the detection limit was at a range of 102-105 copies/ μl, the specificity was 100%, and the intra-assay coefficient of variation was below 12%, and the inter-assay coefficient of variation was below 15%.@*Conclusions@#A fluorescent bead-based multiplex PCR assay for the simultaneous detection of seven viral diseases endemic in Africa was established, which may provide a new choice for the screening of suspected infectious diseases.

Distinguish vaccine strain and wild type strain of yellow fever virus imported to China using high-throughput sequencing technology / 中华实验和临床病毒学杂志

Objective@#To identify whether the three imported yellow fever cases in China in March 2016 were infections by wild type strain of yellow fever virus in Angola in 2016, vaccine-associated disease or co-infection of both.@*Methods@#Sequences of three yellow fever virus strains were obtained by high-throughput sequencing with IonTorrent PGM platform from blood or urine samples of three yellow fever cases, and their genomic characteristics were analyzed. Then the regions with relatively great difference between the wild type strain and 17D vaccine strain were identified, and then served as the reference sequences when mapping the reads obtained by high-throughput sequencing.@*Results@#Partial yellow fever virus genomes were obtained from three samples of yellow fever patients, among them a full length coding region sequence was gained in sample 2. Comparing the genome sequences, the three newly obtained strains of yellow fever virus were highly similar to strain CNYF01R / 2016 which was isolated from the first imported yellow fever case to China in 2016 and strain Angola 71 from Angola in 1971, and they all belonged to Angola genotype of yellow fever virus. In this study, we found five regions in yellow fever virus genomes with great diversity between the vaccine strain and the wild type strain. In these five regions, a number of short reads obtained by high-throughput sequencing of the three samples were mapped to the sequence of wild type virus, while no short reads matched the vaccine strain.@*Conclusions@#There were no viral nucleic acid of 17D vaccine strain in the blood or urine samples of these three cases of yellow fever. They are all infected by wild type strains of Angola in 2016.

Development of a double antibody sandwich ELISA for detection of Chikungunya virus antigen / 中华实验和临床病毒学杂志

Objective@#To establish a method for detection of chikungunya virus(CHIKV) antigen.@*Methods@#CHIKV virus like particle(VLP), that contains all structural proteins, was prepared by baculovirus expression system. Mice and rabbits were immunized with the VLP to develop antibodies against CHIKV. A double antibody sandwich ELISA was established for detection of CHIKV antigens. The concentrations of the antibodies used and the reaction conditions were optimized. The detection limit and repeatability of the ELISA was evaluated.@*Results@#The sensitivity and specificity was estimated by 10 mimicking CHIKV sera, 90 health person sera, 40 other virus infected sera. It was show that the specificity of DAS-ELISA was 100%, the detection limit was 10 TCID50, the coefficients of variation (CV) within plate was <5%, the CV of different plates was <10%.@*Conclusions@#The double antibody sandwich ELISA established in this study can be used to detect the CHIKV antigen in acute phase serum of patient and provide a method for detection of CHIKV.

Research of Human-mouse Chimeric Antibodies Against Ebola Virus Nucleoprotein / 病毒学报

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.

Inclusion Bodies are Formed in SFTSV-infected Human Macrophages / 病毒学报

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.

Establishment of Quality Control System of Nucleic Acid Detection for Ebola Virus in Sierra Leone-China Friendship Biological Safety Laboratory / 病毒学报

The quality control process throughout the Ebola virus nucleic acid detection in Sierra Leone-China Friendship Biological Safety Laboratory (SLE-CHN Biosafety Lab) was described in detail, in order to comprehensively display the scientific, rigorous, accurate and efficient practice in detection of Ebola virus of first batch detection team in SLE-CHN Biosafety Lab. Firstly, the key points of laboratory quality control system was described, including the managements and organizing, quality control documents and information management, instrument, reagents and supplies, assessment, facilities design and space allocation, laboratory maintenance and biosecurity. Secondly, the application of quality control methods in the whole process of the Ebola virus detection, including before the test, during the test and after the test, was analyzed. The excellent and professional laboratory staffs, the implementation of humanized management are the cornerstone of the success; High-level biological safety protection is the premise for effective quality control and completion of Ebola virus detection tasks. And professional logistics is prerequisite for launching the laboratory diagnosis of Ebola virus. The establishment and running of SLE-CHN Biosafety Lab has landmark significance for the friendship between Sierra Leone and China, and the lab becomes the most important base for Ebola virus laboratory testing in Sierra Leone.

Preparation and functional analysis of the monoclonal antibodies against severe fever with thrombocytopenia syndrome bunyavirus structural proteins / 病毒学报

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.

Development of human antibodies against the Gn protein of severe fever with thrombocytopenia syndrome virus / 病毒学报

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.

Purification and Preliminary Research on the Immunogenicity of Inactivated Severe Fever with Thrombocytopenia Syndrome Bunyavirus / 病毒学报

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.

New Method for Stable Expression of SFTS Virus-like Particles in CHO-K1 Cells / 病毒学报

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.

Infection of the Severe Fever with Thrombocytopenia Syndrome Virus in Balb/C Mice and Hamsters / 病毒学报

The severe fever with thrombocytopenia syndrome virus (SFTSV) is the causative pathogen of an emerging infectious disease severe fever with thrombocytopenia syndrome and a new member in the genus Phlebovirus of family Bunyaviridae. Immune responses and pathological lesions in SFTSV-infected Balb/C mice and hamsters were evaluated by inoculation of SFTSV at 105 TCID50 or 103 TCID50 per animal through four different routes of infection, including intravenous, intramuscular, intraperitoneal, and intracerebral injections. The vehicle control groups were also included. At different time points after the inoculation blood and plasma samples were collected. Blood cell counts, blood viral RNA copies, and plasma antibodies were detected by automatic blood cell counters, real-time PCR, and luminex assays, respectively. At two weeks post inoculation, the animals were sacrificed. Tissues including heart, liver, spleen, lung, kidney, intestine, muscle, and brain, were collected for pathological analyses. Results showed that the SFTSV could infect Balb/C mice and hamsters with SFTSV-specific immunoglobulin (Ig) M and IgG antibodies detected in plasma samples on day 7 post inoculation. The SFTSV-specific IgM levels peaked on day 7 post inoculation and then decreased, whereas the SFTSV-specific IgG levels started to increase on day 7 and then peaked on day 14 post inoculation. Pathological analyses indicated significant pathological lesions in liver and kidney tissues. In conclusion, SFTSV could can infect different strains of rodent animals and cause similar immunological and pathological responses.

Biological characteristics of dengue virus type 3 isolated from Yiwu, China / 中国人兽共患病学报

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

First confirmation of new bunyavirus-infected patients in Zhejiang province and molecular identification of the isolated virus / 中华微生物学和免疫学杂志

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.

Primary researches on human genetically engineered antibodies to HIV-1 / 中华微生物学和免疫学杂志

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.