发热伴血小板减少综合征布尼亚病毒微复制子的建立

发热伴血小板减少综合征布尼亚病毒(SFTSV)是我国新发现的一种布尼亚病毒,可引起人类严重发热伴血小板减少综合征。我们利用RNA聚合酶Ⅰ体系,分别构建SFTSV三个片段L、M、S微复制子,研究其非编码区调控功能。将报告基因绿色荧光蛋白(GFP)或荧光素酶(Luciferase)分别插入SFTSV三个片段5′和3′非编码区之间,所形成的嵌合cDNA反向插入含RNA聚合酶I的表达载体pHH21中,获得SFTSV微复制子重组质粒L-GFP-pHH21、M-GFP-pHH21、S-GFP-pHH21、L-Luc-pHH21、M-Luc-pHH21和S-Luc-pHH21,分别与成功表达SFTSV聚合酶蛋白(L蛋白)和结构蛋白(N蛋白)的质粒VR1012-L和VR-1012-NP共同转染293T细胞,24～48h后观察GFP表达情况或检测萤光素酶表达量。L、M、S片段GFP微复制子均可观察到特异性绿色荧光。荧光素酶定量结果显示其在不同节段非编码区中的表达量不同,提示SFTSV三个节段的非编码区启动微复制子转录和复制的强度不同。     

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。     

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。     

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。     

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...     

发热伴血小板减少综合征患者血清内抗体IgG中和活性及其影响因素分析

发热伴血小板减少综合征(SFTS)的病原体为发热伴血小板减少综合征布尼亚病毒,目前尚无特异性防治措施。本研究旨在明确SFTS患者不同时期血清内抗体的中和活性,为将来开发单克隆中和抗体药物和疫苗设计提供可靠的血源。7名患者15份血清收集后采用protein A亲和纯化抗体,SDS-PAGE分析抗体的纯度和分子量大小,中和实验评估抗体的中和能力,结果发现7个患者15份血清中4个恢复期患者血清内抗体具有广谱中和能力,其余3个患者未产生中和抗体,所有患者急性期均未产生IgG中和抗体,产生中和抗体的个体内抗体的中和能力随病毒消长而发生改变。因此,机体感染后部分康复者建立了特异性体液免疫力。此外,SFTS患者年龄亦影响中和抗体的产生,高龄并不预示着免疫力逐渐丧失,推测个体差异决定了免疫力强弱。具有广谱中和能力的血源将为开发治疗SFTS的候选抗体药物以及疫苗设计提供可能。     

Discovery of Severe Fever with Thrombocytopenia Syndrome Bunyavirus Strains Originating from Intragenic Recombination

This study analyzes available severe fever with thrombocytopenia syndrome virus (SFTSV) genomes and reports that a sublineage of lineage I bears a unique M segment recombined from two of three prevailing SFTSV lineages. Through recombination, the sublineage has acquired nearly complete G1 associated with protective epitopes from lineage III, suggesting that this recombination has the capacity to induce antigenic shift of the virus. Therefore, this study provides some valuable implications for the vaccine design of SFTSV.     

Risk Factors for Bunyavirus-Associated Severe Fever with Thrombocytopenia Syndrome,China

Background

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging disease that is caused by a novel bunyavirus, referred to as SFTS virus. During January 2011 to December 2011 we conducted a case-control study in Henan, Hubei and Shandong Provinces of China to determine the risk factors for SFTS.

Methods

Case-patients were identified in hospitals and reported to provincial Centers for Disease Control and Prevention while being notified electronically to the National Surveillance System. Controls were randomly selected from a pool of patients admitted to the same hospital ward within one week of the inclusion of the cases. They were matched by age (+/−5 years) and gender.

Results

A total of 422 patients participated in the study including 134 cases and 288 matched controls. The median age of the cases was 58.8 years, ranging from 47.6 to 70.1 years; 54.5% were male. No differences in demographics were observed between cases and controls; however, farmers were frequent and more common among cases (88.8%) than controls (58.7%). In multivariate analysis, the odds for SFTS was 2.4∼4.5 fold higher with patients who reported tick bites or presence of tick in the living area. Other independent risk factors included cat or cattle ownership and reported presence of weeds and shrubs in the working environment.

Conclusions

Our findings support the hypothesis that ticks are important vectors of SFTS virus. Further investigations are warranted to understand the detailed modes of transmission of SFTS virus while vector management, education on tick bites prevention and personal hygiene management should be implemented for high-risk groups in high incidence areas.     

Age Is a Critical Risk Factor for Severe Fever with Thrombocytopenia Syndrome

Background

Severe Fever with Thrombocytopenia Syndrome (SFTS) is an emerging infectious disease in East Asia. SFTS is a tick borne hemorrhagic fever caused by SFTSV, a new bunyavirus named after the syndrome. We investigated the epidemiology of SFTS in Laizhou County, Shandong Province, China.

Methods

We collected serum specimens of all patients who were clinically diagnosed as suspected SFTS cases in 2010 and 2011 in Laizhou County. The patients'' serum specimens were tested for SFTSV by real time fluorescence quantitative PCR (RT-qPCR). We collected 1,060 serum specimens from healthy human volunteers by random sampling in Laizhou County in 2011. Healthy persons'' serum specimens were tested for specific SFTSV IgG antibody by ELISA.

Results

71 SFTS cases were diagnosed in Laizhou County in 2010 and 2011, which resulted in the incidence rate of 4.1/100,000 annually. The patients ranged from 15 years old to 87 years old and the median age of the patients were 59 years old. The incidence rate of SFTS was significantly higher in patients over 40 years old and fatal cases only occurred in patients over 50 years old. 3.3% (35/1,060) of healthy people were positive to SFTSV IgG antibody. The SFTSV antibody positive rate was not significantly different among people at different age groups.

Conclusion

Our results revealed that seroprevalence of SFTSV in healthy people in Laizhou County was not significantly different among age groups, but SFTS patients were mainly elderly people, suggesting that age is the critical risk factor or determinant for SFTS morbidity and mortality.     

Seroprevalence of Severe Fever with Thrombocytopenia Syndrome Phlebovirus in Domesticated Deer in South Korea

Severe fever with thrombocytopenia syndrome phlebovirus(SFTSV) has a wide host range. Not only has it been found in humans, but also in many wild and domesticated animals. The infection of breeding deer on farms is a particularly worrisome public health concern due to the large amount of human contact and the diverse use of deer products, including raw blood. To investigate the prevalence of breeding domesticated deer, we examined the SFTSV infection rate on deer farms in South Korea from 2015 to 2017. Of the 215 collected blood samples, 0.9%(2/215) were found to be positive for viral RNA by PCR, and sequence analysis showed the highest homology with the KADGH human isolate. Both SFTSVspecific recombinant N and Gn protein-based ELISAs revealed that 14.0%(30/215) and 7.9%(17/215) of collected blood specimens were positive for SFTSV antibody. These results demonstrate that the breeding farm deer are exposed to SFTSV and could be a potential infection source for humans through direct contact or consumption of byproducts.     

Characterization of Severe Fever with Thrombocytopenia Syndrome in Rural Regions of Zhejiang,China

Severe fever with thrombocytopenia syndrome virus (SFTSV) infections have recently been found in rural regions of Zhejiang. A severe fever with thrombocytopenia syndrome (SFTS) surveillance and sero-epidemiological investigation was conducted in the districts with outbreaks. During the study period of 2011–2014, a total of 51 SFTSV infection cases were identified and the case fatality rate was 12% (6/51). Ninety two percent of the patients (47/51) were over 50 years of age, and 63% (32/51) of laboratory confirmed cases occurred from May to July. Nine percent (11/120) of the serum samples from local healthy people without symptoms were found to be positive for antibodies to the SFTS virus. SFTSV strains were isolated by culture using Vero, and the whole genomic sequences of two SFTSV strains (01 and Zhao) were sequenced and submitted to the GenBank. Homology analysis showed that the similarity of the target nucleocapsid gene from the SFTSV strains from different geographic areas was 94.2–100%. From the constructed phylogenetic tree, it was found that all the SFTSV strains diverged into two main clusters. Only the SFTSV strains from the Zhejiang (Daishan) region of China and the Yamaguchi, Miyazakj regions of Japan, were clustered into lineage II, consistent with both of these regions being isolated areas with similar geographic features. Two out of eight predicted linear B cell epitopes from the nucleocapsid protein showed mutations between the SFTSV strains of different clusters, but did not contribute to the binding ability of the specific SFTSV antibodies. This study confirmed that SFTSV has been circulating naturally and can cause a seasonal prevalence in Daishan, China. The results also suggest that the molecular characteristics of SFTSV are associated with the geographic region and all SFTSV strains can be divided into two genotypes.     

No Detection of Severe Fever with Thrombocytopenia Syndrome Virus from Ixodid Ticks Collected in Seoul

Larvae, nymphs, and adult stages of 3 species of ixodid ticks were collected by tick drag methods in Seoul during June-October 2013, and their infection status with severe fever with thrombocytopenia syndrome (SFTS) virus was examined using RT-PCR. During the period, 732 Haemaphysalis longicornis, 62 Haemaphysalis flava, and 2 Ixodes nipponensis specimens were collected. Among the specimens of H. longicornis, the number of female adults, male adults, nymphs, and larvae were 53, 11, 240, and 446, respectively. Ticks were grouped into 63 pools according to the collection site, species, and developmental stage, and assayed for SFTS virus. None of the pools of ticks were found to be positive for SFTS virus gene.     

Proteasome Inhibitor PS-341 Effectively Blocks Infection by the Severe Fever with Thrombocytopenia Syndrome Virus

Severe fever with thrombocytopenia syndrome(SFTS) is an emerging hemorrhagic fever disease caused by SFTSV, a newly discovered phlebovirus that is named after the disease. Currently, no effective vaccines or drugs are available for use against SFTSV infection, as our understanding of the viral pathogenesis is limited. Bortezomib(PS-341), a dipeptideboronic acid analog, is the first clinically approved proteasome inhibitor for use in humans. In this study, the antiviral efficacy of PS-341 against SFTSV infection was tested in human embryonic kidney HEK293 T(293 T) cells. We employed four different assays to analyze the antiviral ability of PS-341 and determined that PS-341 inhibited the proliferation of SFTSV in 293 T cells under various treatment conditions. Although PS-341 did not affect the virus absorption, PS-341 treatment within a non-toxic concentration range resulted in a significant reduction of progeny viral titers in infected cells.Dual-luciferase reporter assays and Western blot analysis revealed that PS-341 could reverse the SFTSV-encoded nonstructural protein(NS) mediated degradation of retinoic acid-inducible gene-1(RIG-I), thereby antagonizing the inhibitory effect of NSs on interferons and blocking virus replication. In addition, we observed that inhibition of apoptosis promotes virus replication. These results indicate that targeting of cellular interferon pathways and apoptosis during acute infection might serve as the bases of future therapeutics for the treatment of SFTSV infections.