VSITA, an Improved Approach of Target Amplification in the Identification of Viral Pathogens / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>Unbiased next generation sequencing (NGS) is susceptible to interference from host or environmental sequences. Consequently, background depletion and virome enrichment techniques are usually needed for clinical samples where viral load is much lower than background sequences.</p><p><b>METHODS</b>A viral Sequence Independent Targeted Amplification (VSITA) approach using a set of non-ribosomal and virus-enriched octamers (V8) was developed and compared with traditionally used random hexamers (N6). Forty-five archived clinical samples of different types were used in parallel to compare the V8 and N6 enrichment performance of viral sequences and removal performance of ribosomal sequences in the step of reverse transcription followed by quantitative PCR (qPCR). Ten sera samples from patients with fever of unknown origin and 10 feces samples from patients with diarrhea of unknown origin were used in comparison of V8 and N6 enrichment performance following NGS analysis.</p><p><b>RESULTS</b>A minimum 30 hexamers matching to viral reference sequences (sense and antisense) were selected from a dataset of random 4,096 (46) hexamers (N6). Two random nucleotides were added to the 5' end of the selected hexamers, and 480 (30 × 42) octamers (V8) were obtained. In general, VSITA approach showed higher enrichment of virus-targeted cDNA and enhanced ability to remove unwanted ribosomal sequences in the majorities of 45 predefined clinical samples. Moreover, VSITA combined with NGS enabled to detect not only more viruses but also achieve more viral reads hit and higher viral genome coverage in 20 clinical samples with diarrhea or fever of unknown origin.</p><p><b>CONCLUSION</b>The VSITA approach designed in this study is demonstrated to possess higher sensitivity and broader genome coverage than traditionally used random hexamers in the NGS-based identification of viral pathogens directly from clinical samples.</p>

Efficacy of mild hypothermia for the treatment of patients with cardiac arrest / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Therapeutic hypothermia has been recommended for the treatment of cardiac arrest patients who remain comatose after the return of spontaneous circulation. The aim of this study was to evaluate the effectiveness and safety of mild hypothermia on patients with cardiac arrest by conducting a meta-analysis.</p><p><b>METHODS</b>The relevant trials were searched in Cochrane Library, PubMed, Web of Science, Embase, CNKI and Wan Fang Data from the date of their establishment to October 2014. Thereafter, the studies retrieved were screened based on predefined inclusion and exclusion criteria. Data were extracted, and the quality of the included studies was evaluated. A meta-analysis was conducted using the Cochrane Collaboration Review Manager 5.2 software.</p><p><b>RESULTS</b>Six randomized controlled trials involving 531 cases were included, among which 273 cases were assigned to the treatment group and the other 258 cases to the control group. The meta-analysis indicated that mild hypothermia therapy after cardiac arrest produced significant differences in survival rate (relative risk [RR] =1.23, 95% confidence interval [CI]: 1.02-1.48, P = 0.03) and neurological function (RR = 1.33, 95% CI: 1.08-1.65, P = 0.007) after 6 months compared with normothermia therapy. However, no significant differences were observed in the survival to the hospital discharge (RR = 1.35, 95% CI: 0.87-2.10, P = 0.18), favorable neurological outcome at hospital discharge (RR = 1.53, 95% CI: 0.95-2.45, P = 0.08) and adverse events.</p><p><b>CONCLUSIONS</b>The meta-analysis demonstrated that mild hypothermia can improve the survival rate and neurological function of patients with cardiac arrest after 6 months. On the other hand, regarding the survival to hospital discharge, favorable neurological outcome at hospital discharge, and adverse events, our meta-analysis produced nonsignificant results.</p>

Study on serological cross-reactivity of six pathogenic phleboviruses / 病毒学报

This article aimed to study the antigenicity of nucleocapsid proteins (NPs) in six pathogenic phleboviruses and to provide theoretical evidence for the development of serological diagnostic reagents. NPs of six pathogenic phleboviruses were expressed and purified using a prokaryotic expression system and rabbits were immunized with individual recombinant NPs. Cross-reactions among NPs and rabbit sera were determined by both indirect ELISA and Western blotting analyses, and the sera titer was determined by indirect ELISA. Furthermore, sera from SFTS patients were also detected by each recombinant NP as a coating antigen using indirect ELISA. The cross-reactions and the sera titer were subsequently determined. Both the concentration and purity of recombinant NPs of six pathogenic phleboviruses met the standards for immunization and detection. The results of indirect ELISA and Western blotting showed that each anti-phlebovirus NP rabbit immune serum had potential serological cross-reactivity with the other five virus NP antigens. Furthermore, the sera from SFTS patients also had cross-reactivity with the other five NP antigens to a certain extent. Our preliminary study evaluated the antigenicity and immune reactivity of six pathogenic phleboviruses NPs and laid the foundation for the development of diagnostic reagents.

Virus-like particle-based immunoglobulin M capture enzyme-linked immunosorbent assay for the detection of IgM antibodies against Chikungunya virus / 病毒学报

To establish a MacELISA method for the detection of IgM antibodies against Chikungunya virus (CHIKV), we prepared virus like particle (VLP) antigens of CHIKV using the whole structural protein C-E3-E2-6K-E1 encoding gene with a baculovirus expression system in Sf9 insect cells. The VLPs were purified and used to immunize Kunming mice. Then, polyclonal antibodies were purified from the samples of ascites with a protein G HiTrap SP column and labeled with horseradish peroxidase. A MacELISA method for the detection of IgM antibodies against CHIKV was assembled with goat anti-human IgM antibody, VLP antigens and an enzyme-labeled polyclonal antibody. The results were evaluated with a serum panel containing serum samples from laboratory-confirmed CHIK, HFRS patients, healthy donors, and commercially available CHIKV IgM as a quality control. It was shown that the MacELISA had a specificity of 99% (99/100), the coefficients of variation (CoV) within a plate were <10%, and the CoV of different ELISA plates in terms of the plate variation coefficient was <15%. A comparative analysis was performed to compare the current method against a commercial CHIKV IgM antibody detection kit for IIFA-IgM. The detection limit of MacELISA was significantly lower than that of the IIFA-IgM commercial kit (P< 0.0001). Here, we demonstrate that the VLP-based MacELISA is a promising tool for the early diagnosis and epidemiological investigation of CHIKV infection, with a high level of sensitivity and specificity for the detection of IgM antibodies against CHIKV.

Study on adjuvant effect of oral recombinant subunit vaccine formulated with chitosan against human enterovirus 71 / 病毒学报

To evaluate the adjuvant effect of recombinant enterovirus 71 (EV71) subunit vaccine formulated with chitosan, rabbits were orally immunized with recombinant VP1 (rVP1) or rVP1 mixed with chitosan adjuvant. Levels of virus-specific IgG and IgA antibodies in sera, mucosal wash buffer (intestine, nasal cavity, and lung), and feces were determined by indirect enzyme-linked immunosorbent assay (ELISA). The titers of neutralizing antibodies against EV71 were determined using cytopathic effect-based neutralizing assay, and levels of cytokines (IFN-gamma and IL-4) secreted from in vitro-cultured rabbit splenic lymphocytes under antigen stimulation were also determined by ELISA. Results showed that immunization with rVP1 alone could only induce low levels of serum IgG and mucosal IgA, while rVP1 combined with chitosan adjuvant were able to induce significantly higher levels of antibodies, rVP1 can only induce neutralizing antibodies when used in combination with chitosan. Levels of IFN-gamma and IL-4 in the group immunized with rVP1 plus chitosan were significantly higher than those in the group immunized with rVP1 only or those in the control groups. Our study lays the foundation for development of oral VP1 vaccine against EV71 infection.

Severe fever with thrombocytopenia syndrome virus nucleoprotein specifically binds to 60kD SSA/Ro protein in host cells / 病毒学报

This study aims to investigate whether the nucleoprotein (NP) of severe fever with thrombocytopenia syndrome virus (SFTSV) can impact the cellular immunity of host cells. Gene segments that encode the NP and non-structural protein (NSs) of SFTSV were inserted into eukaryotic expression vector VR1012. Host proteins that interact with NP and affect immunity were identified with co-immunoprecipitation (IP), SDS-PAGE, mass spectrometry (MS), and Western blot. Co-localization of NP and the identified host proteins was confirmed by confocal microscopy. A 60kD SSA/Ro, a protein related to immunity, interacted with NP, as found by IP and MS. Confocal microscopy showed that NP and SSA/Ro were co-localized in cytoplasm. These results indicated that SFTSV NP may specifically bind to 60kD SSA/Ro and cause a series of immune responses and clinical symptoms.

Investigation of RNA viral genome amplification by multiple displacement amplification technique / 病毒学报

In order to facilitate the detection of newly emerging or rare viral infectious diseases, a negative-strand RNA virus-severe fever with thrombocytopenia syndrome bunyavirus, and a positive-strand RNA virus-dengue virus, were used to investigate RNA viral genome unspecific amplification by multiple displacement amplification technique from clinical samples. Series of 10-fold diluted purified viral RNA were utilized as analog samples with different pathogen loads, after a series of reactions were sequentially processed, single-strand cDNA, double-strand cDNA, double-strand cDNA treated with ligation without or with supplemental RNA were generated, then a Phi29 DNA polymerase depended isothermal amplification was employed, and finally the target gene copies were detected by real time PCR assays to evaluate the amplification efficiencies of various methods. The results showed that multiple displacement amplification effects of single-strand or double-strand cDNA templates were limited, while the fold increases of double-strand cDNA templates treated with ligation could be up to 6 X 10(3), even 2 X 10(5) when supplemental RNA existed, and better results were obtained when viral RNA loads were lower. A RNA viral genome amplification system using multiple displacement amplification technique was established in this study and effective amplification of RNA viral genome with low load was achieved, which could provide a tool to synthesize adequate viral genome for multiplex pathogens detection.

A double antibody sandwich ELISA based assay for titration of severe fever with thrombocytopenia syndrome virus / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To develop an assay for titration of severe fever with thrombocytopenia syndrome virus (SFTSV) based on double antibody sandwich ELISA.</p><p><b>METHODS</b>A double antibody sandwich ELISA was developed for detection of SFTSV based on SFTSV nucleocapsid (N) protein specific poly- and monoclonal antibodies, procedures were optimized and evaluated. This ELISA based titration assay was compared with fluorescence assasy and plaque assay based titration method.</p><p><b>RESULTS</b>The results suggested that the titers obtained by ELISA based method are consistent with those obtained by IFA based method (R = 0.999) and the plaque assay titration method (R = 0.949).</p><p><b>CONCLUSION</b>The novel ELISA based titration method with high sensitivity and specificity is easy to manage and perform, and can overcome the subjectivity associated with result determination of the fluorescence assay and plaque assay based methods. The novel ELISA based titration method can also be applied to high throughput detection.</p>

Epidemic situation and surveillance on SFTS in China, 2011-2012 / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To analyze the data of surveillance on severe fever with thrombocytopenia syndrome (SFTS), from 2011 to 2012 in China.</p><p><b>METHODS</b>Descriptive methods were conducted to analyze the surveillance data from 2011 to 2012 which were collected from the internet-based National Notifiable Disease Reporting System.</p><p><b>RESULTS</b>From 2011 to 2012, a total of 1229 SFTS cases and 107 deaths were reported in China with the average annual incidence rate of 0. 046/100 000 and case fatality rate of 8.7%. Compared to 2011, morbidity of 2012 has increased by 23.5% and mortality has decreased by 32%. 16 provinces reported SFTS cases. More cases occurred in spring and summer seasons,with the peak in May to July, during this period, 69% of the total cases were reported. The ages of the patients ranged from 1 to 85 years, 44.2% of total case was 55 to 70 years old, there were no differences in sex. Of all the cases 86. 8% was farmer.</p><p><b>CONCLUSION</b>Severe fever with thrombocytopenia syndrome in widely distributed in China, especially in the central and eastern regions, the incidence has obvious seasonal. Surveillance and immigration quarantine should be strengthened.</p>

Laboratory diagnosis of viral hemorrhagic fevers / 病毒学报

Viral hemorrhagic fevers (VHFs) refer to a group of acute infections with high case fatality rates that are caused by four distinct families of RNA viruses belonging to the families Bunyaviridae, Flaviviridae, Filoviridae and Arenaviridae, the main clinical symptoms of these diseases are accompanied by fever and bleeding. For the reason that these infections have similar primary clinical symptoms, it is difficult to diagnose and distinguish them; rapid and reliable laboratory diagnostic tests are required in suspected cases for epidemiological investigation and controlling the spread of VHFs. This review addresses the laboratory diagnostics of VHFs, covering etiological classification and different diagnostic techniques, such as virus isolation, nucleic acid detection, as well as antigen and antibody assays. Prospects for novel diagnostic tools are also discussed.

Hepatitis A virus mimotope mapping by phage display peptide library / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>A 12 mer phage display peptide library was used to identify hepatitis A virus mimotopes of antigenic determinants, to provide the feasibility of virus epitope mapping by using this approach.</p><p><b>METHODS</b>Using purified anti-hepatitis A virus monoclonal antibody as affinity selective molecule, phage display peptide library was biopanned and positive clones were selected by ELISA, competition assay and DNA sequencing.</p><p><b>RESULTS</b>10 ELISA positive clones were chosen for DNA sequencing, and the displayed peptide sequences were deduced. 9 of them showed identical nucleotide sequence, and similarity in their amino acid sequence with VP1 of HAV HM175 was found, but no sequence homology was found between the other phage clone and the capsid proteins of HAV. Those peptides may behave as mimotopes of HAV.</p><p><b>CONCLUSION</b>The mimotope of HAV was selected by using phage display peptide library screening. The results provide the potential of this method to search for the mimotopes of the virus.</p>

Survival analysis of patients with spontaneous rupture of hepatocellular carcinoma / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To explore the prognostic factors influencing overall survival (OS) in patients with spontaneous rupture of hepatocellular carcinoma (HCC-SR).</p><p><b>METHODS</b>The medical records of 44 patients with HCC-SR treated in our department from January 1, 2005 to April 1 2011 were retrospectively reviewed. The clinical and prognostic data of 19 HCC-SR patients who received curative hepatectomy were compared with data of 137 HCC patients with no SR who were managed by curative hepatectomy during the same period. Type of HCC-SR was defined according to previously established criteria. The clinicopathological data were evaluated for possible associations with OS of HCC-SR by univariate analysis with the Kaplan-Meier method followed by multivariate analysis with the Cox proportional hazard model.</p><p><b>RESULTS</b>While some clinical features differed between the HCC-SR patients and non-HCC-SR patients, the postoperative prognosis was comparable between the two groups: (1) The 1-, 2-, 3- and 5-year postoperative cumulative recurrence rates were 78.9% (15/19), 89.5% (17/19), 94.7% (18/19) and 94.7% (18/19) in the HCC-SR group but 43.1% (59/137), 54.0% (74/137), 59.1% (81/137) and 66.4% (91/137) in the non-HCC-SR group respectively, and the differences reached statistical significance (P = 0.006, 0.003, 0.002, and 0.014); (2) The 1-, 2-, 3- and 5-year postoperative disease-free survival rates were 10.5% (2/19), 5.3% (1/19), 5.3% (1/19) and 5.3% (1/19) in the HCC-SR group but 40.1% (55/137), 21.2% (29/137), 12.4% (17/137) and 4.4% (6/137) in the non-HCC-SR group respectively, and only the 1-year disease-free survival rate was significantly different (P = 0.032); (3) The 1-, 2-, 3- and 5-year postoperative OS rates were 42.1% (8/19), 10.5% (2/19), 5.3% (1/19) and 5.3% (1/19) in the HCC-SR group but 59.1% (81/137), 32.8% (45/137), 19.0% (26/137) and 6.6% (9/137) in the non-HCC-SR group, and none of the differences reached statistical significance (P = 1.972, 0.061, 0.200, 1.000). Multivariate analysis identified that severity of concomitant liver cirrhosis, levels of alpha fetoprotein (AFP), choice of treatment modality, and type of HCC-SR acted as factors influencing OS.</p><p><b>CONCLUSIONS</b>Patients with HCC-SR receiving curative hepatectomy have higher postoperative recurrence rates than their non-HCC-SR counterparts, but the two groups have similar postoperative OS rates. OS is influenced by severity of concomitant liver cirrhosis, level of AFP, choice of treatment modality, and type of HCC-SR.</p>

Establishment of minireplicon system for severe fever with thrombocytopenia syndrome bunyavirus / 病毒学报

Severe fever with thrombocytopenia syndrome bunyavirus is a newly emerging virus in China, enveloped with a tripartite, single-stranded RNA genome of negative polarity. The regulatory elements for viral transcription and replication, as well as encapsidation and packaging signals, are thought to be located within these noncoding regions (NCRs). The terminal nucleotides are genus specific and highly conserved. The function of the remaining nucleotides of the NCRs is still not well understood. In this study, we developed the plasmid-driven RNA polymerase I minireplicon system for SFTSV firstly, using reporter genes GFP and luciferase. The function of the noncoding regions of the three Bunyaviridae RNA segments (L, M, S) in transcription was analyzed. Reporter genes are successfully expressed in SFTSV minireplicon system. Our results suggest that the NCRs of SFTSV from all three segments contain the necessary signals to initiate transcription. Quantitative detection of the luciferase expression level shows that promoter activity in the three segments is different.

Isolation, identification and characterization of SFTS bunyavirus from ticks collected on the surface of domestic animals / 病毒学报

To understand the maintenance and transmission of SFTS virus, the potential vector ticks were collected from sheep, cattle and dogs in the endemic areas of SFTSV in Shandong Province. Among the collected ticks, the dominant species was H. longicornis ticks. Real-time PCR for RNA detection, virus isolation and characterization, genomic sequencing, phylogenetic and antigenic analysis were performed in this investigation. The results showed that the SFTS viral RNA was detected in 2.14% H. longicornis, and a SFTS virus was isolated from one of viral RNA positive ticks collected from sheep. Whole genome analysis of the SFTSV isolates with 11 human-origin SFTS virus revealed a highly pairwise similarity, and the growth curve analysis showed nearly identical in virus yield and the dynamic of virus reproduction compared to human derived viral isolates. Immunofluorescence and neutralization test showed identical serological reaction character of the two different origin viral strains. In this study, the characters of a SFTSV isolate was firstly described, which suggested that the tick species H. longicornis acting important vector role in the transmission of SFTS virus.

Secreted expression of dengue virus type 2 envelope glycoprotein in eukaryotic cells / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To secreted express envelope glycoprotein (E) of dengue virus type 2 extracellularly.</p><p><b>METHODS</b>The entire prM/E gene was amplified by RT-PCR. An optimized signal sequence gene from Japanese encephalits virus (JEV, SA14-14-2 strain) was introduced using fusion PCR. The impact of E protein transmembrane and cytoplasmatic domains was compared by amplifying prM and E with full length of E gene, with 20% truncation of the E gene at 3' terminus and one chimeric gene, which was generated by replacing the 3' terminal 20% region of E gene with the corresponding sequence of JEV (SA14-14-2 strain). The PCR segments were inserted into the NheI and NotI sites of pcDNA5/FRT vector or into the NheI and XhoI sites of pAcUW51-M. Then they were transfected into 293T cells or Sf9 cells respectively. The expression and secretion of E protein were detected by immunofluorescence assay (IFA) and Western Blot.</p><p><b>RESULTS</b>After transected into 293T cells or Sf9 cells, all constructs expressed E protein intracellularly indentified by IFA while only two plasmids could secret detectable E protein into tissue culture using Western Blot analysis.</p><p><b>CONCLUSION</b>Signal peptide as well as the transmembrane and cytoplasmatic domains is crucial for the secretion of dengue E protein.</p>

Generation of recombinant human antibodies for EV71 virus / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To obtain recombinant human anti-EV71 antibodies from a EV71-associated hand-foot-and-mouth disease patient-derived antibody phage library.</p><p><b>METHODS</b>A combinatorial human scFv library to enterovirus 71 (EV71) virus was constructed using antibody genes harvested from the blood of EV71 virus patients. The library was panned and selected by using purified VP1 protein of EV71 virus with phage display. After that the specific antibody was converted to full human IgG antibody with recombinant baculovirus/insect cell system.</p><p><b>RESULTS</b>One unique human scFv antibody specific for EV71 virus VP1 protein was obtained by ELISA, IFA and analysis of the antibody DNA sequence. The specific anti-VP1 human scFv antibody was converted to full human IgG antibody with recombinant baculovirus/insect cell system. The full human IgG antibody was tested in vitro for EV71 virus neutralization, resulting in no neutralizing activity with EV71 A type and EV71 C4 subtype.</p><p><b>CONCLUSION</b>The obtained human anti-EV71 antibodies without neutralizing activity laid the foundation for diagnosis of human EV71-associated hand-foot-and-mouth disease.</p>

Expression of structural and non-structural proteins of severe fever with thrombocytopenia syndrome bunyavirus / 病毒学报

Severe fever with thrombocytopenia syndrome bunyavirus (SFTSV) is a novel phlebovirus, causing a life-threatening illness associated with the symptoms of severe fever and thrombocytopenia syndrome. The sequence and structure of the genome have already been illustrated in previous study. However, the characteristics and function of the structure and non-structure proteins is still unclear. In this study, we identified the density of the purified SFTSV virions as 1.135 g/mL in sucrose solution. Using RT-PCR method, we amplified the full coding sequence of RNA dependent RNA polymerase(RdRp), glycoprotein precursor (M), glycoprotein n (Gn), glycoprotein c (Gc), nuclear protein (NP) and non structural protein (NSs) of SFTSV (strain HB29). Respectively inserted the target genes into eukaryotic expression vector pcDNA5/FRT or VR1012, the target protein in 293T cell were successfully expressed. By analyzing the SFTSV virions in SDS-PAGE and using recombinant viral proteins with SFTS patients sera in Western blotting and Immunofluorescent assay, the molecule weight of structure and non-structure proteins of SFTSV were defined. The study provides the first step to understand the molecular characteristics of SFTSV.

The development of neutralizing human antibodies against rabies virus / 病毒学报

A combinatorial human Fab library to the rabies virus was constructed using antibody genes derived from the blood of vaccinated donors. The library were panned and selected on purified rabies virus particles of aG or CTN strain with phage display. Eleven unique human Fab antibodies specific for the rabies virus glycoprotein were obtained by ELISA, IFA and DNA sequences analysis of these antibodies. Among these Fab antibodies, five human Fab antibodies were converted to full-length human IgG antibodies with recombinant baculovirus system. The five full-length human IgG antibodies were tested in vitro for rabies virus neutralization, resulting in all specificities to neutralize the virus. The obtained human anti-rabies antibodies lay the basis for the production of cocktail of anti-rabies monoclonal antibody with chinese intellectual property.

Development of pseudoviral competitive internal controls for RT-PCR detection of dengue virus / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>Development of pseudoviral competitive internal controls for RT-PCR laboratory detection of dengue virus.</p><p><b>METHODS</b>The internal controls target gene were obtained by insertion of a 180 bp non-related DNA fragment into RT-PCR detection target of dengue virus between the forward and reverse PCR primer binding regions. A yellow florescence protein reporter gene was induced at downstream of internal controls target gene via internal ribosome entry site gene. HEK 293T cells were transfected with plasmid containing this whole cassette and lentiviral packaging support plasmid. Pseudoviral particle was recovered from the supernatant and analyzed quantitatively and qualitatively in simulated samples at the same tube under different experimental conditions.</p><p><b>RESULTS</b>The established pseudoviral competitive internal controls can be used in the RT-PCR detection of different serotype dengue virus and the whole detection process can be monitored. The obtained fragment is easy to be differentiated in agarose electrophoresis.</p><p><b>CONCLUSION</b>The pseudoviral competitive internal controls could be used for the quality control of the laboratory diagnosis process, simple to prepare, stable for storage, easy to be transformed into internal controls for other RNA virus.</p>

Expression and function analysis of dengue virus type 1 to 4 envelope domain III recombinant fusion protein / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To observe the ability of dengue virus type 1-4 envelope domain III fusion protein to inhibit virus infection and analyze the neutralizing ability of polyclonal antibodies against rE III.</p><p><b>METHODS</b>After being connected by linker peptide, E III protein of Dengue virus serotypes 1-4 were expressed in E coli BL21 (DE3) then purified. Fusion proteins were verified by Western Blot and ELISA. Rabbits were immunized with fusion proteins to produce anti-rE III serum. The activity of anti-rE III serum were detected through indirect immunofluorescence assay test. Inhibition of dengue virus type 1 to 4 infection in BHK-21 cells by rE III fusion protein were tested. Neutralizing activity of anti-rE III serum was analyzed.</p><p><b>RESULTS</b>Dengue virus type 1 to 4 envelope domain III recombinant fusion protein was expressed in E coli BL21 and purified successfully. Then rE III fusion protein and anti-rE III serum were analyzed respectively and rE III fusion protein can effectively inhibit dengue virus type 1 to 4 from infecting BHK cells. The anti-rE III serums can neutralize dengue virus type 1 to 4 but with different neutralizing titer.</p><p><b>CONCLUSION</b>Dengue virus type 1-4 envelope domain III fusion protein can directly inhibit DV infection. Antibodies induced by rE III fusion proteins can neutralize dengue virus type 1-4.</p>