IntroductionThe Crimean-Congo hemorrhagic fever virus (CCHFV), the most geographically widespread tick-borne virus, is endemic in Africa, Eastern Europe and Asia, with infection resulting in mortality in up to 30% of cases. Currently, there are no approved vaccines or effective therapies available for CCHF. The CCHFV should only be manipulated in the BSL-4 laboratory, which has severely hampered basic seroprevalence studies. MethodsIn the present study, two antibody detection methods in the forms of an enzyme-linked immunosorbent assay (ELISA) and a surrogate virus neutralization test (sPVNT) were developed using a recombinant glycoprotein (rGP) and a vesicular stomatitis virus (VSV)-based virus bearing the CCHFV recombinant glycoprotein (rVSV/CCHFV) in a biosafety level 2 (BSL-2) laboratory, respectively. ResultsThe rGP-based ELISA and rVSV/CCHFV-based sVNT were established by using the anti-CCHFV pre-GC mAb 11E7, known as a broadly cross-reactive, potently neutralizing antibody, and their applications as diagnostic antigens were validated for the specific detection of CCHFV IgG and neutralizing antibodies in experimental animals. In two tests, mAb clone 11E7 (diluted at 1:163840 or 512) still displayed positive binding and neutralization, and the presence of antibodies (IgG and neutralizing) against the rGP and rVSV/CCHFV was also determined in the sera from the experimental animals. Both mAb 11E7 and animal sera showed a high reactivity to both antigens, indicating that bacterially expressed rGP and rVSV/CCHFV have good immunoreactivity. Apart from establishing two serological testing methods, their results also demonstrated an imperfect correlation between IgG and neutralizing antibodies. DiscussionWithin this limited number of samples, the rGP and rVSV/CCHFV could be safe and convenient tools with significant potential for research on specific antibodies and serological samples.

Marburg virus (MARV) is one of the principal members of the filovirus family, which can cause fatal hemorrhagic fever in humans. There are currently no prophylactic and therapeutic drugs on the market, and the high pathogenicity and infectivity of MARV make its research highly dependent on biosafety level 4 conditions, severely hindering the development of vaccines and therapies. Therefore, the development of medicines, such as MARV serological diagnosis, vaccines, and therapeutic antibody drugs, urgently needs a safe, convenient, and biosafety level 2 detection method to measure the neutralizing activity of MARV antibodies. To this end, we report a neutralization assay relying on a Rabies virus (RABV) reverse genetic operating system. We constructed infectious clones carrying the eGFP reporter gene and the full length of the original unmodified MARV GP gene. Based on the critical parameters of phylogenetic analysis, recombinant viruses targeting representative strains in the two major MARV lineages were successfully rescued. These pseudoviruses are safe in mice, and their inability to infect cells after being neutralized by antibodies can be visualized under a fluorescence microscope. We tested the system using the neutralizing antibody MR191. MR191 can significantly block the infection of BSR cells with pseudovirus. We compared it with the traditional lentivirus-type pseudovirus system to verify the system’s credibility and obtained the same results as reported in the literature. In general, we have established a safe and visualized method for evaluating the neutralizing activity of MARV antibodies. Compared with traditional methods, it has the advantages of convenient operation, short cycle, and low cost. It is a candidate method that can replace actual viruses for a neutralization assay.

The Crimean Congo Hemorrhagic Fever Virus (CCHFV) is a tick-borne bunyavirus of the Narovirus genus, which is the causative agent of Crimean Congo Hemorrhagic Fever (CCHF). CCHF is endemic in Africa, the Middle East, Eastern Europe and Asia, with a high case-fatality rate of up to 50% in humans. Currently, there are no approved vaccines or effective therapies available for CCHF. The GEM-PA is a safe, versatile and effective carrier system, which offers a cost-efficient, high-throughput platform for recovery and purification of subunit proteins for vaccines. In the present study, based on a GEM-PA surface display system, a GEM-PA based vaccine expressing three subunit vaccine candidates (G-GP, including G-eGN, G-eGC and G-NAb) of CCHFV was developed, displaying the ectodomains of the structural glycoproteins eGN, eGC and NAb, respectively. According to the immunological assays including indirect-ELISA, a micro-neutralization test of pseudo-virus and ELISpot, 5 μg GPBLP3 combined with Montanide ISA 201VG plus Poly (I:C) adjuvant (A-G-GP-5 μg) elicited GP-specific humoral and cellular immunity in BALB/c mice after three vaccinations via subcutaneous injection (s.c.). The consistent data between IgG subtype and cytokine detection, ELISpot and cytokine detection indicated balanced Th1 and Th2 responses, of which G-eGN vaccines could elicit a stronger T-cell response post-vaccination, respectively. Moreover, all three vaccine candidates elicited high TNF-α, IL-6, and IL-10 cytokine levels in the supernatant of stimulated splenocytes in vitro. However, the neutralizing antibody (nAb) was only detected in A-G-eGC and A-G-eGC vaccination groups with the highest neutralizing titer of 128, suggesting that G-eGC could elicit a stronger humoral immune response. In conclusion, the GEM-PA surface display system could provide an efficient and convenient purification method for CCHFV subunit antigens, and the G-GP subunit vaccine candidates will be promising against CCHFV infections with excellent immunogenicity.

为了构建携带eGFP标签并表达马尔堡病毒(Marburg virus,MARV)囊膜糖蛋白(GP)的复制型水泡性口炎病毒(Vesicular stomatitis virus,VSV)假型病毒,通过将VSV感染性克隆全长质粒中的G基因替换成马尔堡病毒的GP基因,与4个辅助质粒p3.1-VSV-N、p3.1-VSV-P、p3.1-VSV-L、p3.1-VSV-G共转染BSR细胞,拯救成功的重组VSV病毒传5代后,进行Western-blot、间接免疫荧光试验、RT-PCR鉴定和生长动力学曲线检测.显微镜检测结果显示,3个重组全长质粒分别与4个辅助质粒共转染BSR细胞培养48h后均能使细胞发生合胞体病变并伴有绿色荧光蛋白表达;Western-blot、间接免疫荧光试验鉴定结果显示,3株马尔堡病毒的囊膜糖蛋白在重组VSV病毒中正确表达;生长动力学曲线显示,3株重组VSV病毒的毒力相较于母本VSV病毒均降低,但病毒滴度依旧能达到1×106～1×107 TCID50 mL-1.结论:本研究成功构建3株携带eGFP标签表达MARV GP的复制型VSV假型病毒,为后续MARV疫苗与抗体的效果评价等奠定了基础.

Since its first emergence in 2012, cases of infection with Middle East respiratory syndrome coronavirus (MERS-CoV) have continued to occur. At the end of January 2020, 2519 laboratory confirmed cases with a case-fatality rate of 34.3% have been reported. Approximately 84% of human cases have been reported in the tropical region of Saudi Arabia. The emergence of MERS-CoV has highlighted need for a rapid and accurate assay to triage patients with a suspected infection in a timely manner because of the lack of an approved vaccine or an effective treatment for MERS-CoV to prevent and control potential outbreaks. In this study, we present two rapid and visual nucleic acid assays that target the MERS-CoV UpE and N genes as a panel that combines reverse transcription recombinase polymerase amplification with a closed vertical flow visualization strip (RT-RPA-VF). This test panel was designed to improve the diagnostic accuracy through dual-target screening after referencing laboratory testing guidance for MERS-CoV. The limit of detection was 1.2×101 copies/μl viral RNA for the UpE assay and 1.2 copies/μl viral RNA for the N assay, with almost consistent with the sensitivity of the RT-qPCR assays. The two assays exhibited no cross-reactivity with multiple CoVs, including the bat severe acute respiratory syndrome related coronavirus (SARSr-CoV), the bat coronavirus HKU4, and the human coronaviruses 229E, OC43, HKU1 and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Furthermore, the panel does not require sophisticated equipment and provides rapid detection within 30 min. This panel displays good sensitivity and specificity and may be useful to rapidly detect MERS-CoV early during an outbreak and for disease surveillance.

目的 原核表达并纯化克里米亚-刚果出血热病毒(Crimean-Congo hemorrhagic fever virus,CCHFV)M基因编码的包膜糖蛋白片段Gn1和Gc1,以两段目的蛋白为包被抗原分别建立检测CCHFV抗体的间接ELISA方法.方法 PCR扩增CCHFV M基因的抗原保守区胞外域Gn1和Gc1基因片段,分别克隆至pET-30a(+)载体,转化大肠埃希菌BL21 (DE3),并对不同诱导条件(温度、IPTG浓度、时间)进行优化,使用His-Ni柱纯化目的蛋白,进行SDS-PAGE和Western blot鉴定,分别以纯化的Gn1和Gc1蛋白作为包被抗原建立抗体ELISA检测方法.结果 表达并纯化了CCHFV糖蛋白片段Gn1、Gc1,分子质量分别为35 ku和23 ku;Western blot检测两种重组蛋白均能被抗Gn1,Gc1抗体识别.以纯化蛋白为抗原建立的间接ELISA方法检测已知阳性血清CCHFV抗体滴度为1∶10240,检测RVFV、WNV、JEV感染者血清均阴性,变异系数＜8％.结论 表达纯化的Gn1和Gc1蛋白纯度均在90％以上,两种蛋白均表现出良好的免疫原性,以两种蛋白为包被抗原建立的抗体间接ELISA检测方法,该方法具有良好的特异性和敏感性.重组蛋白的制备为克里米亚刚果出血热疫苗免疫效果评价、疫病监测和新型疫苗研发奠定了基础.

Rift Valley fever (RVF) is an acute, febrile zoonotic disease that is caused by the RVF virus (RVFV) and is spread by arthropod vectors. Virus-like particle (VLP) vaccines, which have the advantages of strong immunogenicity and safety, play an important role in the prevention of this disease. VLPs for RVFV were successfully prepared by our research group using a baculovirus-insect cell expression system. To study the immunogenicity of these RVFV VLPs, a correct 3rd or 4th generation recombinant baculovirus, rBac-N-G, was identified and used to infect Sf9 cells, which were cultured in suspension at a large scale. Subsequently, cell debris was removed by centrifugation, and the VLPs were concentrated by ultracentrifugation and purified using a sucrose gradient, after which they were used to immunize BALB/c mice by intramuscular injection. The results showed that the RVFV VLPs prepared by our research group could effectively induce mice to produce RVFV neutralizing antibodies and that the prepared VLPs could stimulate mouse spleen cells to produce high levels of the cytokines IL-4 and IFN-γ. Moreover, the proportion of lymphocytes producing IL-4 and IFN-γ in the spleen of mice immunized with RVFV VLPs was significantly increased. Therefore, the RVFV VLPs prepared in this study had strong immunogenicity and could effectively activate humoral and cellular immunity in mice. This study lays a solid foundation for the development of RVFV VLP vaccine candidates and promotes the healthy development of animal husbandry and human public health.

Radiation biodosimetry is an important part of radiation medicine. Some new discoveries and progresses have been made in radiation biodosimetry studies in recent years. Cytogenetic method represented by chromosome aberration analysis as the golden standard of radiation biodosimeter is being transformed to automate analysis, and a number of international, regional and national laboratory networks of radiation biodosimetry are being established. As a widely acceptable molecular marker of DNA damage,γ-H2AX has made rapid progress in radiation dose estimation. Based on the expressions of protein and genes, further advancements have been made in the studies of metabolites and miRNAs. At the same time, with the development of proteomics technology, there are some breakthroughs in the study of using molecular expression profiling to evaluate radiation dose. The research progresses of radiation biodosimetry is reviewed in this paper.

BACKGROUND:Marburg virus (MARV) causes severe haemorrhagic fever in humans and nonhuman primates and has a high mortality rate. However, effective drugs or licensed vaccines are not currently available to control the outbreak and spread of this disease.METHODS:In this study, we generated MARV virus-like particles (VLPs) by co-expressing the glycoprotein (GP) and matrix protein (VP40) using the baculovirus expression system. MARV VLPs and three adjuvants, Poria cocos polysaccharide (PCP-II), poly(I:C) and aluminium hydroxide, were evaluated after intramuscular vaccination in mice.RESULTS:Murine studies demonstrated that vaccination with the MARV VLPs induce neutralizing antibodies and cellar immune responses. MARV VLPs and the PCP-II adjuvant group resulted in high titres of MARV-specific antibodies, activated relatively higher numbers of B cells and T cells in peripheral blood mononuclear cells (PBMCs), and induced greater cytokine secretion from splenocytes than the other adjuvants.CONCLUSION:MARV VLPs with the PCP-II adjuvant may constitute an effective vaccination and PCP-II should be further investigated as a novel adjuvant.