发热伴血小板减少综合征布尼亚病毒研究进展

发热伴血小板减少综合征布尼亚病毒是一种新型布尼亚病毒,其研究结果在《新英格兰医学杂志》上发表之后,引起了国内外学者的高度重视.从病原学、流行病学特征、临床表现、实验室诊断和治疗等方面重点介绍新布尼亚病毒引起的发热伴血小板减少综合征.     

家养动物体表蜱中发热伴血小板减少综合征布尼亚病毒分离及鉴定

为了解发热伴血小板减少综合征布尼亚病毒(SFTSV)的传播机制,采集了山东疫区家养牛、羊和狗等动物体表蜱,分类鉴定后,通过Real-time PCR筛查、病毒分离培养和基因组序列分析等方法分离鉴定蜱中的病毒。所采集的蜱,以长角血蜱为主,占91.4%。其中3头SFTSV核酸检测阳性,阳性率为2.14%,并在其中一份羊体表蜱标本中分离到SFTSV病毒,命名为SDLZTick12。序列分析显示与我国在不同省份患者标本中分离的病毒全基因序列具有高度同源性,且病毒的抗原性和生长特性与人源病毒相同。本研究首次在山东疫区蜱中分离到新型布尼亚病毒,并与人源病毒进行了系统比较研究,提示蜱可能为该新病原体的传播媒介,对疾病的防控具有重要的指导意义。     

发热伴血小板减少综合征布尼亚病毒规模化生产培养工艺的建立

目的通过对发热伴血小板减少综合征布尼亚病毒（简称“新布尼亚病毒”）进行规模化生产培养工艺研究,为新布尼亚病毒规模化生产提供有力支持。方法采用细胞工厂培养Vero细胞,待其长成致密单层后,取工作种子批新布尼亚病毒毒种接种细胞,采用连续收获或细胞病变充分时收获培养液上清的方法收获病毒,并以病毒滴度、抗原含量作为评价指标选择基础培养基、培养基pH、人血白蛋白添加浓度、接种细胞日龄、接种病毒MOI以及病毒培养温度。结果按0．01-0．001MOI接种3-4日龄Vero细胞,病毒培养液选择含0．3％人血白蛋白pH7．6-7．8的DMEM溶液,35℃培养7d收获,病毒收获液病毒滴度7．87LgCCID50／mL、抗原含量170．1μ/mL。结论初步建立了新布尼亚病毒规模化生产培养工艺,为后续工业化生产提供了数据支持。     

发热伴血小板减少综合征布尼亚病毒结构和非结构蛋白表达研究

发热伴血小板减少综合征布尼亚病毒(SFTSV)是我国2010年新发现的新布尼亚病毒,可导致人类严重发热伴血小板减少综合征。SFTS新布尼亚病毒全基因组已解析,但病毒分子生物学结构蛋白特征及功能尚需更多研究。本文通过蔗糖密度梯度离心确定发热伴血小板减少综合征布尼亚病毒(HB29株)病毒颗粒的沉降密度及超离纯化条件,得出该病毒颗粒在蔗糖中的沉降密度为1.135g/mL。利用PCR方法扩增SFTSV病毒株HB29株病毒RNA聚合酶(RdRp)、糖蛋白前体蛋白(M)、包膜糖蛋白(Gn)、包膜糖蛋白(Gc)、核蛋白(NP)及非结构蛋白(NSs)的编码区基因片段,分别克隆入真核表达载体pcDNA5/FRT或VR1012,在293T细胞上获得上述基因表达。通过SDS-PAGE分析纯化病毒颗粒和重组蛋白,并通过免疫印迹(Western blotting)和间接免疫荧光(IFA)确定蛋白活性和分子量。本研究结果将有利于对新布尼亚病毒分子生物学特征的认识,为后期研究提供基础。     

发热伴血小板减少综合征病毒微基因组模型的构建与应用

发热伴血小板减少综合征病毒(Severe fever with thrombocytopenia syndrome bunyavirus, SFTSV)是一种新发蜱传布尼亚病毒,人群普遍易感,病死率高,目前没有针对SFTSV感染的特异性疫苗和治疗药物。为开发针对它的研究方法和工具,本研究用反向遗传学方法构建了基于SFTSV S片段非编码区的微基因组模型,并利用该模型验证了广谱RNA病毒RdRp抑制剂,法匹拉韦(Favipiravir,T-705)的活性。在表达报告基因Gluc的微基因组模型中,T-705表现出显著的抑制活性(IC₅₀=3.17μmol/L),且具有良好的剂量相关性(r²=0.95),证明了该微基因组模型用于SFTSV RdRp抑制剂筛选的可行性。     

人源抗发热伴血小板减少综合征布尼亚病毒-Gn蛋白重组抗体的研究

为研制人源抗发热伴血小板减少综合征布尼亚病毒(Severe fever with thrombocytopenia syndrome virus,SFTSV)Gn蛋白重组抗体,本研究利用噬菌体表面展示技术,以SFTSV全病毒颗粒和重组表达SFTSV-Gn蛋白为抗原,从人源抗SFTSV Fab噬菌体抗体库中筛选抗SFTSV-Gn蛋白的重组Fab抗体,通过ELISA对Fab抗体的结合特异性进行检测。将Fab抗体基因克隆入哺乳动物细胞表达载体HL51-14,瞬时转染293T细胞获得分泌表达的IgG抗体。通过ELISA、IFA和Western-blotting检测IgG抗体的结合特异性。采用亲和层析纯化IgG抗体,并用微量中和试验检测IgG抗体的中和活性。结果表明经过三轮富集筛选,以SFTSV病毒颗粒为抗原筛选出364株针对SFTS病毒核蛋白Fab抗体,没有筛选出特异性结合Gn蛋白的阳性克隆,而通过Gn蛋白筛选得到8株特异结合Gn蛋白的Fab抗体,其中5株来自Lambda库,3株来自Kappa库。ELISA、IFA和Western-blotting检测证实这8株IgG抗体均能特异性结合Gn蛋白。微量中和试验显示8株新筛抗体没有中和活性,但仍可为后续SFTSV人源单克隆抗体的研究提供借鉴和参考。     

无血清细胞培养在发热伴血小板减少综合征布尼亚病毒生长中的应用

目的建立无血清培养基培养Vero细胞制备发热伴血小板减少综合征布尼亚病毒(severe fever with thrombocytopenia syndrome bunyavirus,SFTSV)的工艺。方法分别采用含10%牛血清的MEM(10%MEM培养基)和无血清M2培养基(SF-M2培养基)在方瓶中培养Vero细胞制备SFTSV,比较无血清与含血清培养基培养Vero细胞制备SFTSV在病毒滴度及病毒繁殖曲线之间的差异。在生物反应器里用无血清培养的方式进行工艺放大,收获病毒原液并进行检定。结果无血清培养的Vero细胞能够满足SFTSV培养需求,与含血清细胞培养相比,单位细胞病毒产量没有降低,达到30~60个活病毒/细胞。可以实现在生物反应器的工艺放大,病毒高峰时病毒滴度均在7.0lg PFU/m L以上。结论无血清细胞培养可以应用于SFTSV的培养,有利于降低疫苗生产过程中的纯化难度,提高疫苗安全性。     

发热伴血小板减少综合征患者血清中核蛋白抗体的动态变化

目的:观察新发传染病发热伴血小板减少综合征(SFTS)的病原体新型布尼亚病毒(SFTSV)核蛋白(NP)IgM和IgG型抗体在SFTS患者外周血中的变化规律,为疾病的早期诊断和发病机制的认识提供依据.方法:用ELISA方法检测28例SFTS患者不同病程阶段血清中NP特异性IgM、IgG抗体.结果:①28例SFTS患者中,IgM阳性检出率为89.3 ％(25/28),IgG阳性检出率为85.7 ％(24/28).②IgM和IgG均在发病5天后开始出现,随着病程延长血清中抗体水平逐渐上升,其峰值出现在发病2周左右.③死亡组患者的抗体检出时间迟于痊愈组患者,且抗体水平低下.结论:①在SFTSV感染早期,SFTS患者血清中NP特异性抗体IgG和IgM的变化趋势一致,NP特异性抗体IgG和IgM一样是SFTS早期诊断的重要指标.②因疾病严重而死亡的患者,抗体出现延迟、抗体水平低下可能与患者细胞免疫系统严重受损及多脏器功能障碍有关,致使机体体液免疫应答减退或应答无能.③抗体出现延迟且抗体水平低下可能是病情严重患者预后不良的预测指标.     

新布尼亚病毒的流行病学及临床特征

发热伴血小板减少综合征布尼亚病毒(Severe fever with thrombocytopenia syndrome bunyavirus,SFTSV)是引起人类的一种病死率较高的新发传染病——发热伴血小板减少综合征(Severe fever with thrombocytopenia syndrome,SFTS)的病原体。本文从SFTSV的病原学、流行病学及临床特征等作一综述。     

发热伴血小板减少综合征病毒在不同环境条件下的稳定性研究

为评估发热伴血小板减少综合征病毒(Severe fever with thrombocytopenia syndrome virus, SFTSV)在环境中的稳定性,本研究比较分析了实验室制备的SFTSV在不同介质表面的稳定性以及温度、自然通风干燥和常用酒精消毒剂等因素对其生存能力的影响。不同时间点收获的病毒样本,通过接种于Vero细胞中传代培养、空斑试验和实时荧光定量RT-PCR等方法测定病毒的感染性、滴度和复制培养过程中的动态变化。结果显示,在室温(约25℃)自然通风干燥条件下,不同介质表面的SFTSV感染性快速下降,在硬币、塑料等介质表面的病毒感染性可维持24 h,但病毒感染性滴度24 h内显著下降分别约10^4.46倍和10^4.6倍,在无纺布和纸张表面的病毒感染性滴度在6 h内就下降分别约10^3.82倍和10^4.12倍。如果将SFTSV置于密闭湿润环境中,病毒感染性可维持长时间的稳定性,24 h病毒感染性滴度下降不明显,1周内下降约10^1.49倍,在3周时仍可通过细胞培养...     

Entry of severe fever with thrombocytopenia syndrome virus

Severe fever with thrombocytopenia syndrome virus(SFTSV) is a globe-shaped virus covered by a dense icosahedral array of glycoproteins Gn/Gc that mediate the attachment of the virus to host cells and the fusion of viral and cellular membranes. Several membrane factors are involved in virus entry, including C-type lectins and nonmuscle myosin heavy chain ⅡA. The post-fusion crystal structure of the Gc protein suggests that it is a class Ⅱ membrane fusion protein, similar to the E/E1 protein of flaviviruses and alphaviruses. The virus particles are internalized into host cell endosomes through the clathrin-dependent pathway, where the low pH activates the fusion of the virus with the cell membrane. With information from studies on other bunyaviruses, herein we will review our knowledge of the entry process of SFTSV.     

Isolation,characterization, and phylogenic analysis of three new severe fever with thrombocytopenia syndrome bunyavirus strains derived from Hubei Province,China

Hubei Province is a major epidemic area of severe fever with thrombocytopenia syndrome bunyavirus(SFTSV) in China. However, to date, a few SFTSV strains have been isolated from Hubei Province, preventing effective studies of epidemic outbreaks. Here, we report three confirmed patients(2015–2016) with typical symptoms of severe fever with thrombocytopenia syndrome disease(SFTS) who were farmers resident in different regions in Hubei Province. Three new SFTSV strains were isolated from the serum samples of each patient. Characterization of viral growth properties showed that there were no significant differences in virus production. All strains were completely sequenced, and phylogenetic analysis showed that unlike the other strains from Hubei province, which belonged to the SFTSV C3 genotype, one of the three strains belonged to the SFTSV C2 genotype. These results suggested that multiple SFTSV genotypes have been circulating in Hubei Province, providing insights into SFTSV evolution and improving our understanding of SFTSV prevalence in Hubei Province.     

A new luciferase immunoprecipitation system assay provided serological evidence for missed diagnosis of severe fever with thrombocytopenia syndrome

Severe fever with thrombocytopenia syndrome (SFTS), caused by SFTS virus (SFTSV) infection, was first reported in 2010 in China with an initial fatality of up to 30%. The laboratory confirmation of SFTSV infection in terms of detection of viral RNA or antibody levels is critical for SFTS diagnosis and therapy. In this study, a new luciferase immunoprecipitation system (LIPS) assay based on pREN2 plasmid expressing SFTSV NP gene and tagged with Renilla luciferase (Rluc), was established and used to investigate the levels of antibody responses to SFTSV. Totally 464 serum samples from febrile patients were collected in the hospital of Shaoxing City in Zhejiang Province in 2019. The results showed that 82 of the 464 patients (17.7%) had antibody response to SFTSV, which were further supported by immunofluorescence assays (IFAs). Further, qRT-PCR and microneutralization tests showed that among the 82 positive cases, 15 patients had viremia, 10 patients had neutralizing antibody, and one had both (totally 26 patient). However, none of these patients were diagnosed as SFTS in the hospital probably because of their mild symptoms or subclinical manifestations. All the results indicated that at least the 26 patients having viremia or neutralizing antibody were the missed diagnosis of SFTS cases. The findings suggested the occurrence of SFTS and the SFTS incidence were higher than the reported level in Shaoxing in 2019, and that LIPS may provide an alternative strategy to confirm SFTSV infection in the laboratory.     

Current status of severe fever with thrombocytopenia syndrome in China

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by SFTS virus (SFTSV). SFTSV is associated with a high mortality rate and has been reported in China, South Korea and Japan. SFTSV undergoes rapid changes owing to evolution, gene mutations, and reassortment between different strains of SFTSV. In this review, we summarize the recent cases and general properties of SFTS, focusing on the epidemiology, genetic diversity, clinical features, and diagnostics of SFTSV in China. From 2010 to October 2016, SFTS cases were reported in 23 provinces of China, with increased numbers yearly. Infection and death cases are mainly found in central China, where the Haemaphysalis longicornis ticks are spread. The national average mortality rate of SFTS infection was 5.3%, with higher risk to elder people. The main epidemic period was from May to July, with a peak in May. Thus, SFTS reminds a significant public health problem, and development of prophylactic vaccines and effective antiviral drugs will be highly needed.

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Severe fever with thrombocytopenia syndrome and its pathogen SFTSV

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging hemorrhagic fever in East Asia with case fatality up to 50%. SFTS is caused by SFTSV, a tick borne bunyavirus. In endemic area in China 1%–3% population was infected with SFTSV, but age is critical risk factor for hospitalization and death of SFTS patients.     

发热伴血小板减少综合征病毒中和抗体酶联免疫吸附试验方法的建立及应用

本文旨在对发热伴血小板减少综合征(severe fever with thrombocytopenia syndrome,SFTS)患者中和抗体进行定性和效价评估,建立中和抗体酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)。用96孔微量培养板培养非洲绿猴肾细胞(Vero-E6)并接种发热伴血小板减少综合征病毒(severe fever with thrombocytopenia syndrome virus,SFTSV),以抗核衣壳蛋白(nucleocapsid protein,NP)单克隆抗体为一抗,使用间接ELISA检测SFTSV NP,根据光密度(optical density,OD)判断阳性孔数,采用ReedMuench方法计算病毒半数组织培养感染剂量(50%tissue culture infective dose,TCID_(50)),以反映SFTSV在Vero-E6细胞中的复制水平。ELISA检测中和抗体作用后的病毒残余量,可间接反映中和抗体的作用效果并进行定量。应用以上建立的微量中和-ELISA对10例SFTS患者的双份血清进行中和抗体效价测定,8例患者恢复期血清效价较急性期增高4倍以上,7份患者恢复期血清效价达1∶1 280,急性期血清效价最高为1∶640。结果提示,本研究建立的ELISA操作简便,结果判定客观,所需时间短,可用于临床血清抗体诊断,也可用于血清流行病学调查和疫苗效果临床评价等。     

Establishment of a Reverse Genetic System of Severe Fever with Thrombocytopenia Syndrome Virus Based on a C4 Strain

Virologica Sinica - Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne bunyavirus that causes hemorrhagic fever-like disease (SFTS) in humans with a case fatality...