2013年河南省新布尼亚病毒的分离与S片段基因特征分析

目的 分离2013年河南省新布尼亚病毒分离株,并了解S片段序列特征。方法 采集2013年的发热伴血小板减少综合征急性期血清,用Vero细胞分离新布尼亚病毒,对分离到的毒株采用RT-PCR法扩增病毒S片段基因,并进行同源性分析,构建系统进化树。结果 从采集的64份标本中分离到新布尼亚病毒22株, 新分离的毒株的S片基因序列比对发现,同源性在94.6%～100%之间,其双义编码的两个蛋白质,NSs蛋白的氨基酸的同源性在95.90%～100%之间,N蛋白的氨基酸的同源性在99.50%～100%之间。系统进化树分析发现, 22株病毒株分属于A、B和E三个基因型,其中A型18株,B和E型各2株。结论 2013年河南省新布尼亚病毒以A基因型为主要流行型别。     

新型布尼亚病毒感染研究进展

<正>新型布尼亚病毒即发热伴血小板减少综合征布尼亚病毒(sever fever with thrombocytopnia syndrome virus,SFTSV)是近年来被分离鉴定出的新病毒。该病毒感染人体引起的发热伴血小板减少综合征(sever fever with thrombocytopnia syndrome,SFTS)以发热、血小板和白细胞减少为主要临床表现,多数病例有乏力,消化道症状,肌肉酸痛和淋巴结肿大等临床症状。病情危重者可     

1株汉坦病毒的分离及其S基因片段的克隆和序列分析

目的对浙江省肾综合征出血热(Hemorrhagic Fever with Renal Syndrome,HFRS)疫区-龙泉,新分离到的毒株的S基因片段进行克隆并进行序列测定及分析,以确定毒株的型别和基因变异程度,为进一步研究病毒进化和变异提供了有利条件。方法采用直接免疫荧光检测疫区鼠肺HV抗原。将HV抗原阳性的鼠肺标本接种Vero-E6细胞,分离病毒。参考GenBank发表的汉坦病毒核蛋白基因序列,设计合成一对引物,提取分离株细胞培养物的总RNA,应用逆转录聚合酶链反应(RT-PCR)方法扩增新毒株S片段基因,并克隆入载体,纯化后进行核苷酸序列测定及分析。结果成功分离到的新毒株S片段的全基因序列共1 700个核苷酸,只有一个开放读码框架,共编码429个氨基酸。新毒株与HTN型毒株比较,核苷酸同源率为85.7%~91.9%。与SEO型毒株比较,核苷酸同源率为71.2%~75%,表明该毒株为HTN型毒株。结论浙江HFRS疫区新分离汉坦病毒株可以定型为HTN型毒株,可能为新的亚型。     

聚合酶链反应一步法检测汉坦病毒RNA

肾综合征出血热病毒（ＨＦＲＳＶ）属布尼亚病毒科汉坦病毒属（ＨＶ）,长期以来人们希望从组织和其它标本中直接检测出病原体,尽管用动物和细胞培养分离病毒是最为可靠的,但费时、费力、费钱。而常用的ＩＦＡ和ＥＬＩＳＡ法对少量抗原的直接检测不够灵敏。聚合酶链反应...     

黑龙江省肾综合征出血热病毒基因序列变化特点

目的研究黑龙江省肾综合征出血热病毒基因序列特点,以期寻找近年来黑龙江省肾综合征出血热临床特点变化的原因。方法收集鼠肺标本110例及2005—2015年就诊于哈尔滨医科大学附属第一医院及齐齐哈尔市第七医院临床诊断为轻型和不典型肾综合征出血热的患者早期血标本121例,及部分典型出血热患者外周血标本100例,提取病毒核酸,进行序列扩增,电泳分析及基因测序,所得序列分型并与既往毒株基因序列进行比较,进行系统进化及同源性分析,结合核苷酸及氨基酸位点的变化,探讨肾综合征出血热临床特点变化原因。结果轻型及不典型肾综合征出血热基因序列扩增成功26例,其中HTN型14例,SEO型12例。典型肾综合征出血热标本扩增成功22例,其中HTN型19例,SEO型3例;黑龙江省人源与鼠源汉坦病毒与76-118株均存在变异,非典型病例标本、典型病例标本、鼠源汉坦病毒与标准株核苷酸同源性分别为74.4%～89.2%、87.4%～90.3%、88.1%～88.5%;人源及鼠源汉坦病毒同源性为79.7～99.1%,其中人源的Hljh38、Hljh39与其他毒株差异较大,与既往Amur病毒株H5、H8205同源性高,可达94.9%～97.6%,为同一亚型。推导的氨基酸与标准毒株比较部分位点存在变异,但未见明显的变异规律。结论黑龙江省肾综合征出血热临床特征较之前发生变化的原因与病毒型别的变化相关;同时也存在汉坦病毒变异,氨基酸位点变化,但具体变异规律及与临床表现的相关性有待进一步验证。。     

CODEHOP RT-PCR方法在汉坦病毒基因型别鉴定和分子溯源中的应用

分析CODEHOP RT-PCR方法对于汉坦病毒(HV)基因型别鉴定和分子溯源中的应用效果。方法 选取实验室保存的汉滩型病毒阳性鼠肺样品DX0901和汉城型病毒阳性鼠肺样品DX1101,用CODEHOP RT-PCR扩增HV病毒L基因片段并测序,进行Blast同源性比对和系统发生分析。结果 两份样品均获得478 bp大小的RT-PCR扩增产物,汉滩病毒DX0901与汉滩病毒株Nc167同源性最为接近,遗传距离为0.142。汉城病毒DX1101与汉城病毒株ZT10,ZT71,Z37同源性最为接近,遗传距离为0.049。结论 CODEHOP RT-PCR方法可以有效的用于对汉坦病毒进行基因型别鉴定和分子溯源。     

一种汉滩病毒和汉城病毒双重实时荧光定量RT-PCR检测方法的建立

目的 建立一种汉滩病毒(Hantaan virus,HTNV)和汉城病毒(Seoul virus,SEOV)双重实时定量荧光RT-PCR检测方法。方法 根据HTNV和SEOV S基因设计引物和探针、优化反应条件,建立2种汉坦病毒的双重实时荧光定量RT-PCR方法。以甲型流感病毒、登革热病毒、新布尼亚病毒、寨卡病毒、新冠病毒阳性核酸为模板验证方法的特异性,将建立的方法与巢氏RT-PCR比较,确定方法对临床样本的适用性。结果 建立了一种HTNV和SEOV 双重实时定量荧光RT-PCR检测方法,该方法对2种型别病毒的最低检测限均为10 copies/μL,不同浓度标准品Ct值批内和批间差异均小于2%。与登革热病毒、新布尼亚病毒、寨卡病毒、新型冠状病毒、甲型流感病毒均无交叉反应。对10份肾综合征出血热急性期血清样本进行检测,结果9份为HTNV、1份为SEOV,与巢式RT-PCR方法结果一致。结论 建立的双重实时荧光定量RT-PCR方法可以快速对HTNV和SEOV 进行分型和定量检测,适用于肾综合征出血热临床早期诊断。     

2007年浙江丽水地区汉坦病毒的分离与型别鉴定

目的对2007年从浙江省丽水地区肾综合征出血热(hemorrhagic fever with renal syndrome,HFRS)疫区鼠肺分离的汉坦病毒进行型别鉴定和分子生物学特性研究。方法将汉坦病毒抗原阳性的鼠肺标本接种Vero-E6细胞,分离汉坦病毒。提取病毒总RNA,应用逆转录聚合酶链反应(RT-PCR)扩增病毒S基因全片段,克隆入质粒载体,进行核苷酸序列测定及分析,应用DNA STAR软件分析比较,确定病毒型别。结果从疫区7份阳性鼠肺中分离到2株汉坦病毒,经汉坦病毒单克隆直接荧光血清检查和基因序列测定,结果为汉滩型(HTN)病毒,2株病毒与国际标准株76-118(HTN型)和80-39(SEO型)S片段核苷酸的同源性分别为84.7%、84.6%和64.2%、64.0%。结论浙江省丽水地区可能存在以HTN型病毒为主的肾综合征出血热自然疫源地。     

河南省部分区域肾综合征出血热患者汉坦病毒的分子特征

目的 分析河南省部分区域肾综合征出血热患者汉坦病毒的分子特征。方法 采集2017-2018年就诊于河南省郑州市第六人民医院临床诊断为肾综合征出血热患者血标本32例,采用半巢式PCR筛选汉坦病毒阳性样本,然后再对阳性样本中病毒S片段和M部分片段基因进行扩增和测序,并对得到的S片段基因序列进行组装。所得序列与已分离到的国内其他省份相对应的基因序列进行同源分析并构建系统发生树。结果 收集的32份血标本中,16份血标本扩增出目的片段,序列分析表明均为汉滩病毒(HTNV)。系统发生分析显示16株病毒株S片段和M部分片段聚类分支关系基本一致。其中HN2018/138病毒株与其余15株S片段和M部分片段核苷酸序列的同源性分别为91.4%~92.1%和83.2%~84.4%,差异较大,其余15株亲缘关系较近。结论 本研究所统计的河南省部分区域内肾综合征出血热患者以HTNV为主导基因型,且其表现出明显的地域聚集现象,并存在基因差异较大的变异株或省外输入病毒株的可能性。     

新型布尼亚病毒感染17例临床观察

"发热伴血小板减少综合征"是以发热、血小板减少和多脏器功能损害为主要临床表现的一类疾病,其病因尚不确切,根据我国疾病预防控制中心研究表明,人粒细胞无形体、新型布尼亚病毒可能是该综合征的致病原,是经蜱传播的一种自然疫源性疾病[1].2006年,我国安徽省报道了首例人粒细胞无形体病[2],此后在其他省份陆续有散发病例报道.     

Seroprevalence and Risk Factors for Severe Fever with Thrombocytopenia Syndrome Virus Infection in Jiangsu Province,China, 2011

Severe fever with thrombocytopenia syndrome (SFTS), which is caused by a novel bunyavirus, is an emerging infectious disease in China. In 2011, this new virus was designated as severe fever with thrombocytopenia syndrome virus (SFTSV). The aim of the present study was to determine the seroprevalence and risk factors of SFTSV infection. The investigation was conducted among the general population in Jiangsu Province, China in 2011. A total of 2,510 serum samples were collected. Testing by enzyme-linked immunosorbent assay was conducted to determine the seroprevalence of SFTSV infection. Result showed that the overall seroprevalence of SFTSV infection was 0.44% (11 of 2,510) in seven counties in Jiangsu Province. Multiple variable logistic regression analysis showed that raising goats, farming, and grazing were risk factors for SFTSV infection. Raising goats, farming, and grazing might be important risk factors for virus exposure, and appropriate health education could be useful in preventing infections.     

In Silico Structure-Based Design of Antiviral Peptides Targeting the Severe Fever with Thrombocytopenia Syndrome Virus Glycoprotein Gn

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne bunyavirus in Asia that causes severe disease. Despite its clinical importance, treatment options for SFTSV infection remains limited. The SFTSV glycoprotein Gn plays a major role in mediating virus entry into host cells and is therefore a potential antiviral target. In this study, we employed an in silico structure-based strategy to design novel cyclic antiviral peptides that target the SFTSV glycoprotein Gn. Among the cyclic peptides, HKU-P1 potently neutralizes the SFTSV virion. Combinatorial treatment with HKU-P1 and the broad-spectrum viral RNA-dependent RNA polymerase inhibitor favipiravir exhibited synergistic antiviral effects in vitro. The in silico peptide design platform in this study may facilitate the generation of novel antiviral peptides for other emerging viruses.     

大连市“蜱咬”患者新布尼亚病毒(SFTSV)的检测分析

目的 通过对大连市一起“蜱咬”患者流行病学调查及病原体检测,探讨新布尼亚病毒(Severe fever with thrombocytopenia syndrome bunyavirus, SFTSV)在辽宁省南部沿海地区的流行范围。方法 采用ELISA方法对“蜱咬”患者急性期血清样本及同居住地的26份非患者血清样本进行SFTSV的IgM、IgG抗体检测;采用实时荧光定量RT-PCR方法检测“蜱咬”患者血液样本SFTSV核酸;采用VERO-E6细胞对“蜱咬”患者血液样本进行病毒分离并鉴定。结果 “蜱咬”患者急性期血清样本SFTSV的IgM抗体阳性、IgG抗体阴性;26份非患者血清样本中4份SFTSV的IgG抗体阳性;“蜱咬”患者血液样本SFTSV核酸阳性;从“蜱咬”患者血液样本和体表叮咬的蜱虫中各分离出1株病毒,经SFTSV的M片段全基因测序引物测序鉴定为SFTSV。结论 “蜱咬”患者血液分离株与蜱虫分离株均为SFTSV,同源性达99.7%,该病毒是导致患者发病的重要原因。本次分离到的2株病毒与我省东部(丹东、抚顺),北部(铁岭)山区分离株遗传距离相对较远。SFTSV在正常人群中存在隐性感染者。     

Person-to-person transmission of severe fever with thrombocytopenia syndrome virus

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by a newly discovered bunyavirus, SFTS virus (SFTSV), and causes high fatality (12% on average and as high as 30%). The objective of this study was to determine whether SFTSV could be transmitted from person to person. We analyzed sera of 13 patients from two clusters of unknown infectious diseases that occurred between September and November of 2006 in Anhui Province of China for SFTSV antibody by indirect immunofluorescence assay and for SFTSV RNA by RT-PCR. We found that all patients (n=14) had typical clinical symptoms of SFTS including fever, thrombocytopenia, and leukopenia and all secondary patients in both clusters got sick at 6-13 days after contacting or exposing to blood of index patients. We demonstrated that all patients in cluster 1 including the index patient and nine secondary patients and all three secondary patients in cluster 2 had seroconversion or fourfold increases in antibody titer to SFTSV and/or by RT-PCR amplification of SFTSV RNA from the acute serum. The index patient in cluster 2 was not analyzed because of lack of serum. No person who contacted the index patient during the same period, but were not exposed to the index patient blood, had got illness. We concluded that SFTSV can be transmitted from person to person through contacting patient's blood.     

M Segment-Based Minigenome System of Severe Fever with Thrombocytopenia Syndrome Virus as a Tool for Antiviral Drug Screening

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne bunyavirus that causes severe disease in humans with case fatality rates of approximately 30%. There are few treatment options for SFTSV infection. SFTSV RNA synthesis is conducted using a virus-encoded complex with RNA-dependent RNA polymerase activity that is required for viral propagation. This complex and its activities are, therefore, potential antiviral targets. A library of small molecule compounds was processed using a high-throughput screening (HTS) based on an SFTSV minigenome assay (MGA) in a 96-well microplate format to identify potential lead inhibitors of SFTSV RNA synthesis. The assay confirmed inhibitory activities of previously reported SFTSV inhibitors, favipiravir and ribavirin. A small-scale screening using MGA identified four candidate inhibitors that inhibited SFTSV minigenome activity by more than 80% while exhibiting less than 20% cell cytotoxicity with selectivity index (SI) values of more than 100. These included mycophenolate mofetil, methotrexate, clofarabine, and bleomycin. Overall, these data demonstrate that the SFTSV MGA is useful for anti-SFTSV drug development research.     

汉坦病毒感染致血小板减少的机制

汉坦病毒主要感染血管内皮细胞,引起两种人类疾病,即肾综合征出血热和汉坦病毒心肺综合征,它们的共同特点是毛细血管广泛损伤,通透性升高。汉坦病毒感染者血小板数量严重减少,不仅与“外周破坏增加”有关,而且还可能与“血小板生成和功能障碍”有关。尽管汉坦病毒感染引起血小板减少的机制尚不完全明确,但近年也取得了一些重要进展。本文主要围绕血小板减少相关的两种机制展开讨论,旨在阐明汉坦病毒感染发病机制,为治疗肾综合征出血热的药物设计提供指导。     

发热伴血小板减少综合征流行病学研究进展

发热伴血小板减少综合征（Severe fever with thrombocytopenia syndrome,SFTS）是近几年来新发现的一种以发热伴血小板减少为主要临床表现的传染性疾病,新布尼亚病毒是引起该病的病原体.蜱叮咬是主要的传播途径,可通过人-人接触传播.该病发病急,病情进展快,部分患者可因多脏器功能衰竭而死亡,病死率约10％.本文对该病病原学、病例分布特点及其传染源、传播途径和易感人群三个流行环节方面的最新研究进展作一综述.     

Hemorrhagic fever with renal syndrome in the Dolenjska region of Slovenia--a 10-year survey.

This report describes the first investigation of clinical findings for a larger series of patients with hemorrhagic fever with renal syndrome (HFRS) who were infected with Dobrava virus. From 1985 to 1995, 38 patients with serologically confirmed HFRS were hospitalized at the regional hospital in Novo mesto in the Dolenjska region of Slovenia. On the basis of results of serological examination, 24 patients had Dobrava virus infection, and 14 patients had Puumala virus infection. Complete clinical data were available for 31 patients. Eleven patients underwent hemodialysis for treatment of acute oliguric or anuric renal failure. Four patients, all infected by Dobrava virus, had signs of shock and severe bleeding. Three severely ill Dobrava virus-infected patients died of hemorrhagic complications. We have demonstrated that Dobrava and Puumala viruses coexist in a single region of endemicity and are capable of causing HFRS with significant differences in severity.     

肾综合征出血热感染者尿液RT-PCR法检测

目的应用逆转录聚合酶链反应法(RT-PCR)对临床诊断及拟诊断为肾综合症出血热(HFRS)病例尿样标本进行检测,为临床诊断及科研提供一种新的检测手段。方法对临床诊断和拟诊断为HFRS病例者125名(男91例,女34例)和30名健康者的尿液样本,用RT-RCR法进行检测,同时设置标准病毒株进行对照。结果临床诊断为HFRS病例的125人,其中103人尿样标本为阳性,阳性率为82.4%;标准病毒株为阳性;30名健康者尿样标本为阴性。结论RT-PCR法具有方法简便、灵敏度高、特异度好,而且快速的特点,适用于临床对HFRS的检测及科研应用。     

Evidence that maternal dengue antibodies are important in the development of dengue hemorrhagic fever in infants

To establish the role of maternal dengue-specific antibodies in the development of dengue hemorrhagic fever and dengue shock syndrome caused by dengue 2 virus in infants, we examined sera from mothers of infants and toddlers with dengue hemorrhagic fever or dengue shock syndrome and mothers of infants with pyrexia of unknown origin. The mean titers of hemagglutination inhibition, neutralization, and infection-enhancing activities against dengue 2 virus were not statistically different among the three groups. However, among infants who developed dengue hemorrhagic fever/dengue shock syndrome there was a strong correlation between the mothers' dengue 2 neutralizing titers and infant age at the time of onset of severe illness, where no such correlation was found among the other two groups. Furthermore, the actual age at which dengue hemorrhagic fever/dengue shock syndrome occurred in each infant correlated with the age at which maximum enhancing activity for dengue 2 infection in mononuclear phagocytes was predicted. This critical time for the occurrence of dengue hemorrhagic fever/dengue shock syndrome was observed to be approximately 2 months after the time calculated for maternal dengue 2 neutralizing antibodies to degrade below a protective level. In addition, sera of mothers of infants with dengue hemorrhagic fever/dengue shock syndrome enhanced dengue 2 virus infection to a slightly greater degree than did sera from mothers of infants with pyrexia of unknown origin and toddlers with dengue hemorrhagic fever/dengue shock syndrome. These data are consistent with the hypothesis that maternal dengue antibodies play a dual role by first protecting and later increasing the risk of development of dengue hemorrhagic fever/dengue shock syndrome in infants who become infected by dengue 2 virus.