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.

Subcellular localization of severe fever with thrombocytopenia syndrome virus in macrophages / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the subcellular localization of severe fever with thrombocytopenia syndrome virus (SFTSV) in macrophages and understand the replication and assembly mechanism of SFTSV in host cells.</p><p><b>METHODS</b>Using two types of human macrophage cell lines THP-1 and U937, the study analyzed the intracellular colocalization of SFTSV with Golgi apparatus and endoplasmic reticulum by immunefluorescence staining and confocal microscopy.</p><p><b>RESULTS</b>SFTSV infected macrophage cell lines THP-1 and U937. Immunofluorescence staining showed that the SFTSV nuclear protein colocalized with Golgi apparatus and closely surrounded by endoplasmic reticulum in the perinuclear region.</p><p><b>CONCLUSION</b>The results suggested that Golgi complex and endoplasmic reticulum are probably the sites for formation and maturation of SFTSV viral particles.</p>

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>

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.

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>

Secreted expression of dengue virus type I envelope glycoprotein in 293T cells / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To expression prM/E gene of dengue virus type I in mammalia cells.</p><p><b>METHODS</b>The full-length prM/E gene of dengue virus type I strain GZ01/95 was amplified by RT-PCR, the signal peptide preceding the prM gene was added or the carboxyl-terminal 20% of DEN-1 E was replaced with the corresponding JE sequence in the meanwhile, and three of the constructions were cloned into the pcDNA5/FRT.Then they were transfected into 293T cells by lipofectamine respectively. The expression of recombinant proteins were identified by indirect immuno-fluorescence assay(IFA) as well as Western blot.</p><p><b>RESULTS</b>In the cytoplasm of 293T cells transfected with all the recombinant plasmids DNA, the expressed products for gene of dengue virus type I were confirmed by IFA. The secreted expression products for gene of dengue virus type I specific protein bands were confirmed by Western blot only existing in the cell supernatants transfected with the modified recombinant plasmids DNA.</p><p><b>CONCLUSION</b>The prM/E protein of dengue virus type 1 were expressed in 293T cells transfected with all the three recombinant plasmids DNA. The prM/E protein was obtained secretion after transfecting the modified recombinant plasmids adding a signal peptide preceding the prM gene or replacing the carboxyl-terminal 20% of E with the corresponding JE sequence.</p>

Identification and construction of replicon vectors of Japanese encephalitis virus (strain SA14-14-2) / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>In order to lay the groundwork for studying the novel vaccine Identified.</p><p><b>METHODS</b>(1) Two replicons were constructed. One's prM/E gene was deleted completely (Full AprM/E Replicon), the other's prM/E gene was deleted partially (213 bp of C terminal of E gene was reserved; Partial delta prM/E Replicon), and the deleted parts was replaced as the MCS. (2) Replicons RNA were which will use the JEV as the vector, replicon vectors of JEV was constructed and transfected into BHK-21 cell. After 24, 48, 72, 96 h, method of real-time PCR was used to identify Replicons' replication ability. (3) YFP gene was inserted into the MCS of those two replicons. Their RNA was transfected into BHK-21 cell. Expression of YFP was tested by the fluorescence microscopy and flow cytometer.</p><p><b>RESULTS</b>(1) After the two replicons RNA were transfected into BHK-21 cell, as time went by, the quantity of RNA increased. (2) After RNA of the replicons with YFP were transfected into BHK-21 cell, increasing trend of fluorescent signal and rate of YFP positive cell was observed and tested.</p><p><b>CONCLUSION</b>Full delta prM/E Replicon and Partial delta prM/E Replicon have the ability to duplicate itself and express the foreign protein.</p>

Construcion of a chimeric Japanese encephalits virus/dengue virus-2 / 病毒学报

The prM/E gene of DV2 was cloned into the JEV (SA14-14-2 strain) replicon vector which had been constructed previously, and the resulting recombinant plasmid was named pPartialdeltaprM/E. The constructed chimeric clone was linearized and then was transcripted into RNA in vitro. The produced RNA was transfected into BHK-21 cells. Five to seven days later, CPE could be observed on the transfected BHK-21cells, and then the supernatant containing the chimeric virus was collected. The Supernatant was inoculated to BHK-1 cells and C6/36 cells, respectively. CPE could be observed about 4 days post the infection of C6/36cell with the chimeric virus. The results from RT-PCR, IFA, Western blot showed that the virus contained the chimeric RNA and the envelop protein of DV2. However, the chimeric virus could not be passaged in BHK-21 cell. The successful construction of the infectious clone JE/DEN-2 laid the basis for the further research of the DV vaccine.

Fluorescent microbeads-based multiplex detection of IgM antibodies to pathogens caused viral hemorrhagic fever / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To develop and evaluate a multiplex detection of IgM antibodies to pathogens caused viral hemorrhagic fever.</p><p><b>METHODS</b>The nucleocapsid proteins (NP) of HTN, SEO, Puu MBV, Lassa, RFV and HPS viruses expressed in prokaryotic cells and purified were coupled to 7 different xMAP fluorescent microbeads. The assay was evaluated and optimized when screened against a panel of reference sera collected from HFRS patients, and compared to commonly used MacELISA Kits.</p><p><b>RESULTS</b>For detection of anti-NP antibodies, the sensitivity and specificity of the assay were comparable to a commonly used MacELISA kit, but it could detect different antigen specific antibodies in one reaction simultaneously.</p><p><b>CONCLUSION</b>A robust, rapid and multiplex assay based on NPs could be developed via Luminex xMAP platform for laboratory diagnosis of viral hemorrhagic fever and seroepidemiological investigation.</p>

Development and application of a two-step MacELISA for the early diagnosis of hemorrhagic fever with renal syndrome / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To develop and improve a MacELISA method for the early diagnosis of hemorrhagic fever with renal syndrome (HFRS) with simplified operation procedure.</p><p><b>METHODS</b>The nucleic proteins of hantavirus were labeled with horse raddish peroxidase (HRP) and used as detection antigens. A two-step MacELISA based HRP conjugated antigen was established and the detection sensitivity and specificity were compared with commonly used three-step MacELISA.</p><p><b>RESULTS</b>This method could be used to detect hantanvirus specific IgM with high sensitivity and specificity from human patient serum. There was not significant difference from commonly used three-step MacELISA and the sensitivity and specificity were 100%.</p><p><b>CONCLUSION</b>This method is simple, sensitive and rapid in operation, and therefore could be used for the early diagnosis of HFRS.</p>

Hantavirus mucosal vaccine through different mucosal with heat-labile enterotoxin B subunit as adjuvants / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To evaluation the effect of different mucosal vaccination pathway on hantavirus with the recombinant E. coli heat-labile enterotoxin B subunit (rLTB) as adjuvant.</p><p><b>METHODS</b>The rLTB was expressed and purified. Take the inactivated hantavirus strain 84Fli as vaccine, and immunized C57 BL/6 mice through intranasal, oral and vaginal respectively. Specific IgG and sectory IgA were detected by ELISA in serum, and vaginal washing samples respectively.</p><p><b>RESULTS</b>The rLTB was efficiently expressed under the induction of lactose, identified by western blotting and GM-1 binding experiment. The vaccination through intranasal, oral and vaginal, can induce IgG and sectory IgA response.</p><p><b>CONCLUSION</b>Inactivated hantavirus can produce mucosal immune response with rLTB as adjuvants through intranasal, oral and vaginal vaccination respectively.</p>

Recombinant envelope glycoprotein domain III of dengue virus inhibit virus infection / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To observe the ability of dengue virus recombinant envelope protein domain expressed in E. coli to inhibit virus infection and induce the neutralizing antibody.</p><p><b>METHODS</b>E III protein of Dengue virus serotypes 1-4 were expressed in E. coli BL21(DE3) then purified. Recombinant proteins were tested to inhibit DV2 from infecting BHK-21 cell. Rabbits were immunized with recombinant proteins to produce anti-E III serum. Antibody titers were determined by neutralizing assay.</p><p><b>RESULTS</b>The recombinant E III proteins of Dengue virus serotypes 1-4 were expressed in E. coli. They effectively protected BHK cells in culture against DV2 infection. All four type anti-E III sera can neutralize DV2 but their efficacies are different.</p><p><b>CONCLUSION</b>proteins of dengue virus expressed in E. coli can directly inhibit DV2 infection. Neutralizing antibodies were induced by E III proteins. Both E III protein of dengue virus and the neutralizing antibodies they induced are more efficient in inhibiting homologous dengue serotypes infection than heterologous serotypes.</p>