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.

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>

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.

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>