北京市褐家鼠中汉坦病毒的分离和初步鉴定

肾综合征出血热(hemorrhagic fever with renal syndrome，HFRS)是由鼠类传播的自然疫源性疾病。现已证实我国主要存在汉滩型(HTN)和汉城型(SEO)汉坦病毒。近年来，北京市HFRS发病出现不断上升趋势，流行形势比较严峻。近年的流行病学研究证实北京地区存在鼠间SEO型汉坦病毒感染。为了解近期北京地区HFRS流行毒株的型别和特点，从病原学角度阐明疫情原因，我们在对北京地区宿主动物进行汉坦病毒分子流行病学调查研究的基础上，进行了病毒分离和初步鉴定。     

烟台地区肾综合征出血热患者血清抗体检测和汉坦病毒序列测定

目的 了解烟台地区肾综合征出血热(HFRS)患者血清中IgG、IgM抗体水平，确定引起该地区HFRS流行的汉坦病毒的型别分布。方法 收集临床HFRS急性期和恢复期患者血清；用EMSA检测IgG、IgM抗体；用交叉空斑减少中和试验检测中和抗体；采用Trizol法提取患者血清中HFRS病毒RNA，用套式PCR产物做TA试验，测定核苷酸序列。结果 HFRS患者血清IgM阳性率为82．2％(88／107)，IgG阳性率为85．7％(66／77)。该地区两城市38份HFRS患者血清中，有32份属家鼠型病毒(SE0)感染，另6份未能定型；另一城市16份HFRS患者血清中，有15份属姬鼠型病毒感染，1份未定型。该地区HFRS病毒与SE0的同源性达90％以上。结论 引起烟台地区HFRS流行的汉坦病毒，属于以SE0为主的混合型病毒。     

西安地区汉坦病毒L及S部分片段的编码序列分析

应用巢氏PCR法从陕西西安地区间接免疫荧光抗原初检为阳性的黑线姬鼠标本中扩增汉坦病毒的L和S片段部分序列。序列分析显示：不同标本来源的汉坦病毒均为汉滩型病毒,彼此之间L和S部分片段序列同源性较高,与贵州地区优势汉滩型病毒S2／L4群毒株同源性最高,分别为95．0％～97．0％和97．7％～99．3％,但与该地区已报道的汉坦病毒同源性较低。系统发育树显示：所有来自西安的病毒株处在同一个进化分支上,但可分成两个较小的分支。证实目前检测到的西安地区流行的汉坦病毒株与贵州地区汉滩型病毒株亲缘关系最近,而与当地既往流行毒株的差异较大,究竟是西安地区汉坦病毒流行株发生了变化还是当地同时流行多种毒株还有待于进一步研究证实。     

家鼠型肾综合征出血热病毒单克隆抗体的制备与鉴定

以家鼠型HFRS病毒R22株免疫BALB/c小鼠,取免疫鼠脾细胞与小鼠骨髓瘤细胞系Sp2/0-Ag14融合,获得了23株能稳定分泌HFRS病毒特异性McAb的杂交瘤细胞系。以18株不同型别的HFRS病毒株进行分析,结果表明上述McAb中有些为HFRS病毒组特异性的,有些为家鼠型特异性的。有2株McAb对家鼠型HFRS病毒株具有较高的中和活性,对HFRS病毒R22株感染乳鼠也有明显的保护作用。     

汉坦病毒与肾综合征出血热

汉坦病毒在世界上分布广泛,肾综合征出血热是由汉坦病毒属的汉滩型、汉城型、Puumala型和Dobrave型病毒引起的流行性疾病,分别由携带有不同型病毒的鼠类感染人.临床症状分五个时期.本文主要对我国流行的汉坦病毒及其引发的肾综合征出血热做一综述.     

螨类传播汉坦病毒的研究进展

肾综合征出血热 (hemorrhagicfeverwithrenalsyndrome,HFRS)在我国多称流行性出血热 (epidemichemorrhagicfever,EHF) ,是一种疫区分布广、发病率及病死率较高的自然疫源性疾病 ,其病原属布尼亚病毒科 (Bunyaviridae)、汉坦病毒属(Hantavirus,HV)。在中国流行的有由黑线姬鼠携带传播的汉滩病毒 (Hantaanvirus,HTNV)和由褐家鼠携带传播的汉城病毒 (Seoulvirus ,SEOV)。HFRS病原体的多型性及传播途径的多样化构成HFRS复杂多变的流行特征。查明HFRS多样化传播途径的构成及不同型HFRS在不同环境条件下的主要传播途径 ,对有针对性…     

单克隆抗体ELISA间接夹心法检测家鼠型HFRS疫区人和动物血清中特异性抗体

用McAb-ELISA间接夹心法和IFAT或酶标SPA染色法平行检测山西地区(家鼠型HFRS疫区)的人及动物血清中HFRS病毒特异性IgM和/或IgG抗体。结果ELISA检测174份HFRS病人血清的阳性率及抗体滴度均明显高于IFAT。检测295份无明确HFRS病史的健康人血清,ELISA的阳性率也高于IFAT。检测215份鼠类血清,102份兔血清及108份猪血清,ELISA的阳性检出率与IFAT或酶标SPA染色法基本相同。用阻断试验等证明本ELISA检出的确是HFRS病毒特异性抗体。对ELISA的某些试验条件作了讨论,并认为,McAb-ELISA在帮助临床早期诊断及血清流行病学调查等方面均有实用价值。     

汉滩病毒感染成龄鼠模型用于特异性单克隆抗体保护作用的研究

目的建立汉滩病毒感染成龄鼠的模型,以进行mAb的保护实验.方法将7～8wkBalb/c小鼠于感染病毒前1d及感染后1d,2d和4d,分别经腹腔注射环磷酰胺65mg/kg体重,总剂量为260mg/kg体重,观察其发病及死亡情况,并检测其体内不同脏器中汉滩病毒抗原的分布及滴度.用mAb对上述感染小鼠进行保护实验.结果经环磷酰胺处理的成龄鼠,由汉滩病毒隐性感染变为急性致死性感染(死亡率达100%).其发病症状与汉滩病毒感染的乳鼠类似,平均发病与死亡时间较感染乳鼠晚2～3d,且较为规律和集中.汉滩病毒抗原在感染的成龄鼠和乳鼠体内的分布及滴度基本相同.用mAb对感染的成龄鼠和乳鼠分别进行保护实验,结果完全一致.结论经环磷酰胺处理的成龄鼠,可作为汉滩病毒和HFRS研究用的动物模型.     

深圳市HFRS疫源地鼠携带汉坦病毒与人感染汉坦病毒的基因特征对应性研究

目的 了解深圳市肾综合征出血热(HFRS)疫源地鼠携带汉坦病毒与人感染汉坦病毒二者的病原学特征及联系,为HFRS的防控提供科学依据.方法 收集疑似HFRS病人急性期血清标本,并在相同区域开展笼日法捕鼠,无菌取鼠肺.分别采用ELISA和直接免疫荧光法筛选阳性标本,选择代表性血清标本以及鼠肺标本进行逆转录-套式聚合酶链反应(RT-nested PCR)扩增部分M和S片段及核苷酸序列测定,与国内外的汉坦病毒毒株一起进行同源性比对和系统进化树分析.结果 472份鼠肺经直接免疫荧光检测共获得阳性鼠肺47份,带毒率为9.96%.对60份IgM抗体阳性的血液标本进行RT-nested PCR扩增,检出12份阳性,阳性率为20%.从代表性的8份鼠肺和8份病人血清中提取的标本二者之间M片段核苷酸序列的同源性高达95%以上,S片段之间的同源性高达96.5%以上.其推导的氨基酸序列同源性分别为98.0%～100%、98.4%～100%.深圳市HFRS疫源地鼠携带的汉坦病毒和人感染汉坦病毒均为汉城型S2亚型.结论 深圳市流行的汉坦病毒为SEO型S2亚型.无论是同一地区的鼠和人之间,还是不同地区的鼠和人之间,汉坦病毒都是具有较高的同源性.     

病毒载量检测鉴别诊断HIV早期感染

目的 研究病毒载量检测在鉴别诊断HIV早期感染中的应用.方法 对13份HIV抗体检测结果高度提示为早期感染的样本进行病毒载量检测,并对这些个体进行随访和抗体检测以证实其感染状况.结果 13份样本中,有12份病毒载量阳性,随访确定1例HIV抗体阳性婴幼儿感染者,11例窗口期感染者;1例HIV抗体呈阳性的婴幼儿,病毒载量阴性,随访证实未感染.病毒载量检测结果与最终的感染状况相符.结论 通过病毒载量检测能够有效鉴别诊断早期感染中的婴幼儿感染(18个月以内抗体呈阳性)和窗口期感染者.病毒载量检测可以作为HIV感染早期不确定样本的诊断依据.     

Puumala and Dobrava viruses cause hemorrhagic fever with renal syndrome in Bosnia-Herzegovina: Evidence of highly cross-neutralizing antibody responses in early patient sera

Hantavirus infection was diagnosed serologically by μ-capture IgM and IgG ELISAs in hemorrhagic fever with renal syndrome (HFRS) patients admitted to Tuzla Hospital, Bosnia-Herzegovina. The results indicated that more than one hantavirus caused the outbreak. To address the question of which hantavirus serotypes were involved, sequentially drawn sera were analyzed by focus reduction neutralization test (FRNT) for antibodies against Puumala, Hantaan, Dobrava, and Seoul hantaviruses. The data revealed that acute- or early convalescent-phase sera, even when drawn as late as 3 weeks after the onset of disease, could not be used for typing of the causative hantavirus; a significant number of these samples showed similar reactivity of neutralizing antibodies to several different hantavirus serotypes. Moreover, although several acute-phase sera showed the highest FRNT titer to Hantaan virus, convalescent sera from these patients in all cases showed high specificity for Puumala or Dobrava viruses. This phenomenon, interpreted as a cross-neutralizing primary antibody response, makes several earlier reports concerning causative agents of HFRS questionable. Serological examination of small rodents trapped in the endemic area identified Puumala- and Dobrava-like virus infections. RT-PCR and sequencing of rodent lung samples identified Dobrava virus in one yellow-necked field mouse (Apodemus flavicollis). Cross-FRNT data, using polyclonal rabbit antibodies, clearly confirmed Dobrava virus as a unique hantavirus serotype. In conclusion, the results revealed that both Puumala- and Dobrava-like viruses caused HFRS in Bosnia-Herzegovina, whereas no signs of Hantaan or Seoul virus involvement were found. J. Med. Virol. 53:51–59, 1997. © 1997 Wiley-Liss, Inc.     

Hantaviruses in Estonia

Human serum samples collected from healthy individuals in 14 counties were screened by ELISA in order to investigate the presence of hantavirus infections in Estonia. Out of 1,234 serum samples, 124 were found positive for hantavirus-specific IgG and were subsequently serotyped by a focus reduction neutralization test. A total of 112 samples neutralized at least one of the examined hantaviruses-Puumala (PUUV), Saaremaa (SAAV), Dobrava (DOBV), Hantaan, and Seoul viruses-and thereby, the focus reduction neutralization test confirmed the overall hantavirus seroprevalence rate in Estonia to be 9.1%. Most of the sera showed a specific reaction (at least 4-fold higher endpoint titer) of neutralizing antibodies to PUUV (5.1%), while 3.4% showed a SAAV- or SAAV/DOBV-specific reaction. The fact that seven sera (0.6%) could not be serotyped may indicate the presence of an unknown hantavirus serotype. Hantavirus infections were confirmed in 13 of 14 investigated counties, with highly varying seroprevalence rates (1.0-28.4%). The sex ratio was 1.8:1.0 (M:F), and the antibody prevalence peaked in the age group 45-54 years. A total of 513 rodents of seven species trapped in seven counties were examined for the presence of hantavirus antigen, in order to study the distribution of hantavirus natural carriers. Two species, Clethrionomys glareolus and Apodemus agrarius, were found positive for hantaviral antigen in 13.7% and 4.5% of the investigated rodents, respectively. Analyses of viral sequences recovered from infected C. glareolus tissue samples showed that the infecting virus belonged to the PUUV genotype, confirming that PUUV circulates in mainland Estonia. The Estonian PUUV strains were placed in the closest proximity to Russian PUUV strains in phylogenetic trees, suggesting a common evolutionary history. Together with earlier data on SAAV in A. agrarius, the results revealed that two hantaviruses, PUUV and SAAV, are common in Estonia and that the incidence of human infection is high in both cases.     

Generation of an HFRS patient-derived neutralizing recombinant antibody to Hantaan virus G1 protein and definition of the neutralizing domain

Hantaan virus (HTNV) in the Hantavirus genus, family Bunyaviridae, is the major cause of severe hemorrhagic fever with renal syndrome (HFRS). We prepared a combinatorial phage display library of human Fabs to HTNV from RNA extracted from the blood lymphocytes of a convalescent HFRS patient. We selected two G1 glycoprotein-specific clones and one nucleocapsid protein (N)-specific clone from the Fab library for further studies. The human Fab antibodies were converted to IgG form in baculovirus/insect cells system by using cassette vectors that we developed earlier. Characterization of the recombinant antibodies revealed that the two G1-specific IgGs, could bind to and neutralize HTNV but not Seoul virus (SEOV). The N-specific IgG did not neutralize either HTNV or SEOV. Sequence analysis revealed that the two G1-specific clones differed by only one predicted amino acid in their complementarity determining regions, CDR3. Epitope mapping studies were carried out with one of the two G1-specific clones and synthetic peptides representing portions of HTNV G1. Results indicated that the recombinant antibody recognizes the core amino acid sequence LTKTLVIGQ, which is found near the C-terminus of HTNV G1. These results are the first to define a neutralizing epitope on the G1 protein of HTNV using an antibody derived from an HFRS patient.     

肾综合征出血热患者肝分离株的生物学特性研究

从20例3～12病日的肾综合征出血热肝活检组织中分离到5株汉坦病毒。其中一株L20在Vero—E_6细胞传代稳定,滴度高,抗原分析结果显示,L20株与HTN型的交叉反应很小,可确定不是HTN型,与SEO型虽有一定交叉反应但又明显不同,与PUU型亦有一定的交叉反应。进一步用PCR扩增分析,结果L20株可被汉坦病毒属特异性引物所扩增,但不能被HTN和SEO型特异性引物所扩增。这些结果表明L20株的抗原性似有特殊。     

汉坦病毒浙江分离株ZT71株的全基因核苷酸序列测定及分析

目的 为了获得浙江省汉坦病毒基因组更为详尽的资料,研究汉坦病毒的进化状况及变异程度,为疫苗病毒株的选择使用提供科学依据.方法 设计特异性引物,RT-PCR分段扩增ZT71株全长L、M和S片段,PCR产物纯化后克隆于T载体并进行序列测定.然后进行遗传进化分析.结果 汉坦病毒ZT71株L、M及S基因分别由6530、3651和1753个核苷酸组成,分别编码2151、1133和429个氨基酸.基因比较分析表明,ZT71株与SEO型汉坦病毒比较其L片段核苷酸同源性为95.5%～99.7%,氨基酸同源性为99.0%～99.3%;M片段核苷酸同源性为84.1%～99.6%,氨基酸同源性为95.3%～99.2%;S片段核苷酸同源性为88.7%～99.5%,氨基酸同源性为96.5%～99.1%;而与其他汉坦病毒同源性则较低.结论 测定了ZT71株的全基因核苷酸序列,其L、M和S片段具有和其他汉坦病毒L、M、S片段相似的核苷酸一级结构,但与SEO型更为接近,属于SEO型病毒.     

Immune responses to inactivated vaccine in people naturally infected with hantaviruses

An inactivated Hantaan virus vaccine for hemorrhagic fever with renal syndrome (HFRS) was given by injection to 15 people who were naturally infected with either Hantaan or Seoul viruses. Immunofluorescent antibody (IFA), reversed passive hemagglutination inhibition (RPHI), hemagglutination inhibition (HI), and neutralization antibody (NA) assays were used to measure the antibody titers of the vaccinated people before and after three doses of vaccine. The results indicated that IFA and RPHI antibody titers were boosted significantly (P < 0.05) after the vaccination. Either Hantaan or Seoul virus could induce two-way cross-reactive neutralization antibody responses in humans. After HTNV vaccine immunization, the NA titers of people with natural infection increased significantly (P < 0.05) to both Hantaan and Seoul viruses, while the relative dominance between these two type responses was still similar to that of natural infection. It is worthwhile to studying the procedure further to inoculate two different virus vaccines for improving the cross-protective effect. © 1996 Wiley-Liss, Inc.     

Identification of three novel CTL epitopes within nucleocapsid protein of Hantaan virus

Hantaan virus (HTNV) is a member of the Hantavirus genus that causes human hemorrhagic fever with renal syndrome (HFRS) in humans. The CTL response seems to play a key role in control of viral infection, but only a few HTNV epitopes recognized by the CTLs have been reported. Herein, we screened a panel of overlapping peptides covering the HTNV nucleocapsid protein by ELISPOT assays for those that can elicit IFN-γ production in vitro. Three novel CD8(+) CTL epitopes, N197-205 (RYRTAVCGL), N245-253 (KLLPDTAAV), and N258-266 (GPATNRDYL), were defined on the nucleocapsid protein and were found to be restricted by various HLA alleles including A11, A24, and B7. The epitopes were highly conserved among the reported HTNV strains and other hantanviruses, including Dobrava-Belgrade virus and Seoul virus, supporting their potential use in vaccine designs.     

Identification of Hantaan virus-related structures in kidneys of cadavers with haemorrhagic fever with renal syndrome

Summary The etiologic agent of haemorrhagic fever with renal syndrome (HFRS), Hantaan virus, was first isolated in 1976. Since then numerous Hantaan-like viruses have been isolated and five serotypes of Hantavirus have been recognized. Serological studies indicate that these viruses are globally distributed, with each serotype occurring in specific areas. Hantaan virus has been intensively studied antigenically, biochemically, and genetically. However there is still a paucity of information on the pathogenesis of Hantaan virus in the human host.In this paper, we report the detection by thin section immune electron microscopy of the occurrence of numerous dense precipitates, typical inclusion bodies, a surface antigen layer, as well as Hantaan virion-like structures in the kidneys of patients that died during the acute phase of HFRS. These findings may shed some light on understanding the pathogenesis of HFRS in target organs most affected by the disease, such as the kidneys.