Study and Application of Monoclonal Antibodies against Virus of Haemorrhagic Fever with Renal Syndrome

This paper summarizes the study and application of monoclonal antibodies (McAbs)against haemorrhagic fever with renal syndrome (HFRS) virus in our department over the past sever-al years. The following six points are discussed: (1) establishment and characterization of thehybridoma cell lines secreting McAbs against HFRS virus and hemagglutinin of the virus: (2) theantigenic analysis of HFRS viruses in China by McAbs: (3) purification and application of theMcAbs: (4) purification and characterization of HFRS virus 50K stnactural protein by McAba-affini-ty chromatography and the McAbs possessing different characteristics: (5) detection of HFRS virusantigen in peripheral blood lymphocytes from HFRS patients by the McAb-IFAT; and (6)development of McAb-ELISA indirect sandwich methods, and detection of HFRS virus antigen andIgM, IgG and/or HI antibodies in human and animals. The results of the studies show that theMcAbs can be used for early diagnosis, epidemiological investigation, preparation of vaccine andimmunotherapy of HFRS.

Hemorrhagic Fever with Renal Syndrome Associated with Acute Pancreatitis

Hemorrhagic fever with renal syndrome (HFRS) is a systemic infectious disease caused by Hantaviruses and characterized by fevers, bleeding tendencies, gastrointestinal symptoms and renal failure. It encompasses a broad spectrum of clinical presentations, ranging from unapparent or mild illnesses to fulminant hemorrhagic processes. Among the various complications of HFRS, acute pancreatitis is a rare find. In this report, based on clinical data, laboratory and radiologic examination findings, we describe a clinical case, with HFRS from Dobrava virus, associated with acute pancreatitis. The patient was successfully treated by supportive management. Clinicians should be alert to the possibility of HFRS when examining patients with epidemiological data and symptoms of acute pancreatitis.

DETERMINATION OF URINE TUMOR NECROSIS FACTOR, IL-6, IL-8 AND SERUM IL-6 IN PATIENTS WITH HEMORRHAGIC FEVERS WITH RENAL SYNDROME

Objective To explore the roles of cytokines in the pathogenesis of hemorrhagic fever with renal syndrome(HFRS). Methods Double-antibody sandwich ELISA was used to determine serum interleukin (IL)-6, urine tumor necrosis factor (TNF), IL-6 and IL-8 levels in 56 patients with HFRS. Results Serum IL-6, urine TNF, IL-6 and IL-8 concentrations in HFRS patients were significantly higher than those in control group, respectively (P<0.001). The concentrations increased at fever stage, then continued to increase during hypotension stage and peaked at oliguria stage. The concentrations of serum IL-6, urine TNF, IL-6 and IL-8 increased in accord with the severity of the disease and differed greatly among different types of the disease. Serum IL-6 had remarkable relationships with serum specific antibodies. It was positively related to serum β_2-microglobulin (β_2-MG), blood ureanitrogen (BUN) and creatinine (Cr). Significant positive relationships were also found both between urine IL-6 and TNF, and between IL-6 and IL-8 (r=0.5768, P<0.05; r=0.3760, P<0.01). Conclusion TNF, IL-6 and IL-8 activated during the course of the disease. IL-6 is associated with the immunopathological lesions caused by the hyperfunction of humoral immune response. IL-6, IL-8 and TNF are involved in the renal immune impairment. Determining them might, in certain extent, be used in predicting the prognosis and outcome of patients with HFRS.

Study on the Cytotoxic T Lymphocytes Clone Specific for the Nucleocapsid Protein of Hantaan Virus from Peripheral Blood in Patients with Hemorrhagic Fever with Renal Syndrome

In order to elucidate the molecular and immunological mechanisms as well as the pathogenesis of hemorrhagic fever with renal syndrome (HFRS), the CD8 + cytotoxic T lymphocytes (CTL) clone was established directly from peripheral blood mononuclear cells (PBMC) of patients with HFRS. The activities of CTL were detected as usual with EBV-transformed lymphoblastoid cell line (BLCL) as target cells. The results showed that the CTL clone could recognized and killed the target cells with specificity of nucleocapsid protein of Hantaan virus (HTNVNP) with the cytotoxicity percentages of 50.2%, 25.4% and 39.0% respectively. These results demonstrated that the antigenic epitopes of HTNVNP mainly located on the C-terminal of the viral nucleocapsid protein.

Detection of Hantavirus gene from peripheral blood of patients with HFRS in Heilongjiang province and the epidemiological significance

The aim of this study is to further understand the genotype of Hantavirus （HV） from peripheral blood of patients with hemorrhagic fever with renal syndrome （HFRS） and the epidemiological significance of this disease in Heilongjiang province in recent years. Thirty-one serum samples of clinically diagnosed patients with HFRS were examined by RT-PCR to decide the genetic subtype. On the basis of infection season, the serum samples were divided into two groups： winter （Nov, 2003--Feb, 2004）, spring and summer （April, 200d--Sep, 2004）. Further analysis was performed in combination with clinical symptoms. It was found that among the total 31 samples, 22 were sero-positive. Among 14 serum samples in winter, 8 were sero-positive, of which 5 cases were of type Ⅰ （Hantaan virus, HTNV） and 3 of type Ⅱ （Seoul virus, SEOV）. Among 17 samples in spring and summer, 14 were sero-positive, of which 5 cases were of type Ⅰ and 9 of type Ⅱ . So it concludes that both of the two types of Hantavirus exist in Heilongjiang. The type Ⅰ is the main pathogen of HFRS in winter, and typeⅡ is the main in spring and summer.

STUDY ON AEROSOL TRANSMISSION ROUTES OF HEMORRHAGIC FEVER WITH RENAL SYNDROME (HFRS)

The present paper reviews our studies on transmission routes and prophylactic measures of Hemorrhagic Fever with Renal Syndrome (HFRS), including aerosol inhalation, skin injury/mucosa, insect vectors, peroral infection and vertical transmission. The results show that HFRS is transmitted by Hantaan virus via multi-routes. One of them, the aerosol transmission, is perhaps the main route transmitting HFRS from mouse to human beings. The injury and mu-cosa is the main transmission route from mouse to mouse and also an important agent from mouse to human beings. The peroral infection may occur in very serious pollution of foods. The insect vectors may play an important role in the focus of HFRS and in the transmission of HFRS from mouse to human. The epidemiological significance of vertical transmission is yet uncertain. According to the results, four proposals should be emphasized including killing insects and mice, long lasting and combined prophylactics, intensive studies on prophylactic measures on viral aerosols and the blocking of transmission of HFRS from mouse to human via aerosols.

Immunization effect of purified bivalent vaccine to haemorrhagic fever with renal syndrome manufactured from primary cultured hamster kidney cells

Haemorrhagic fever with renal syndrome (HFRS) is a worldwide epidemic plaguing over thirty nations. This disease has spread across 26 provinces, cities and autonomous regions of China with an annual onsets number of more than a hundred thousand and a mortality rate of 5% to 15%, accounting for over 80% of cases in the world, and threatening the safety and health of Chinese people. 1 Analysis of serum samples over recent years indicates that the plagued areas are expanding. Instead of a single type Ⅰ or Ⅱ strain, each area now has a combination with one type predominant. 2 These demographic changes revealed a shortcoming of the monovalent vaccine in use, urging China to develop a purified bivalent vaccine based on monovalent one. This research on clinical observation and immunization effects led to a purified bivalent vaccine manufactured by Changchun Institute of Biological Products, China from primary cultured hamster kidney cells.

HFRS病毒Z_(37)株在绿猴肾传代细胞的病变作用和传代适应

〔目的〕研究肾综合征出血热 (HFRS)病毒Z3 7疫苗株在绿猴肾传代细胞 (vero ,vero -E6)上的病变和传代适应增殖情况 ,为应用vero细胞研制出血热疫苗提供科学依据。〔方法〕将肾综合征出血热疫苗株Z3 7适应在绿猴肾传代细胞 (vero ,vero-E6)上 ,对细胞进行细胞病变 (CPE)的动态观察 ,并于不同的时间取样 ,测定病毒滴度 ,比较静置培养、转瓶培养滴度的差异情况。〔结果〕Z3 7株不使vero -E6细胞产生病变 ,用HEPES诱导 ,3d可见明显病变现象。Z3 7株不使vero细胞产生病变 ,用HEPES诱导病变现象未获成功 ,Z3 7株在vero细胞中可高滴度繁殖 ,滴度达到 10 7/ml,转瓶培养病毒滴度略高于静置培养。

用三维趋势面模型分析预测HFRS的时空动态趋势

目的 探讨三维趋势面模型技术在ＨＦＲＳ时－空动态分析预测中的应用价值。方法 在二维趋势面模型基础，通过加入时间变量ｔ，构造三维趋势面模型。结果 获得三维趋势面模型及其建模方法。结论 该模型不仅可分析ＨＦＲＳ的空间趋势。

浙江省肾综合征出血热(HFRS)-Ⅰ型鼠脑纯化疫苗安全性、血清学、防病效果和免疫策略研究

目的 现场观察、考核兰州生物制品研究所生产的HFRS -Ⅰ型鼠脑纯化疫苗的安全性、血清免疫学与防病效果 ,并确定其免疫程序和免疫策略。方法 试验组和对照组按随机整群分组法进行分组 ;采用 0、7、2 8d及免后 1年加强 1针的免疫程序 ;分别采集免前、全程接种后 2周、加强前、加强后两周、加强后 1、2、3、4、5年的部分接种者全血和微量耳垂血 ,分别测定其中和抗体和IFA抗体 ,观察疫苗的血清免疫学效果和现场流行病学防病效果。结果 在 1995年 8月～ 2 0 0 1年 12月的 6年时间里从疫苗开始接种到加强后 5年 ,观察了HFRS -Ⅰ型鼠脑灭活纯化疫苗的安全性、血清免疫学效果和流行病学防病效果等。纵观结果 :证明该疫苗除了因疫苗中残留蔗糖导致较重较普遍的局部反应外 ,未发现其他严重的副反应 ,证明疫苗有较好安全性。从血清免疫效果看 ,86例全程接种后两周的免疫者血清 ,IFA抗体阳转率达 10 0 % ,中和抗体阳转率为4 4 4 4 % (8/ 18)。 1年后 ,IFA抗体和中和抗体阳性率分别下降到 2 8 5 7%和 14 80 %。但加强后 2周的血清IFA抗体和中和抗体阳性率分别反弹至 83 33%和 5 5 5 6 % ,其抗体几何平均滴度也随之回升 ,但不十分明显。此外 ,在加强后 2年IFA和中和抗体阳性率再次下降到较低水平 。

用硫酸鱼精蛋白从HFRSV感染材料中提取病毒蛋白及鉴定

本文采用硫酸鱼精蛋白沉淀方法，从感染肾综合征出血热病毒（HFRSV）的鼠脑和细胞中提取病毒蛋白，结果表明：鼠脑先后用鱼精蛋白1mg／ml和2mg／ml两次沉淀HFRSV蛋白时，细胞先后用鱼精蛋白4mg／ml和2mg／ml两次沉淀HFRSV蛋白时，效果均较好。SDS－PAGE电泳显示：提取的病毒蛋白分子量为67kD、55kD、45kD。Western－Blotting及ELISA表明：提取的病毒蛋白为病毒的特异性蛋白。血凝试验表明：从鼠脑及细胞中提取的病毒蛋白血凝效价分别为1：1024、1：4096。液闪计数可观察到[3H]-氨基葡萄糖只结合入了67KD和55kD两种蛋白。

HFRS混合型疫区人群感染特征

于1987年4月和l988年3月,在安徽某县进行了HFRS隐性感染调查,发现混合型疫源地的隐性感染率为4.82%,介于野鼠型和家鼠型病源地之间,同时发现,HFRS隐性感染男、女间无性别差异;各年龄组均有隐性感染,但以20～50岁组为高;经过一个流行年度后,隐性感染似有上升趋势,且发现发病率的高低与该地人群隐性感染率高低相一致;家中有患者的正常人群隐性感染率高于家中无患者的人群。

保定地区HFRS流行高峰年中1株家鼠型病毒株的分离鉴定及其核苷酸序列分析

目的 探讨保定地区 HFRS流行高峰的原因。方法 对 HFRS病人急性期血清用 RT- PCR方法进行病毒基因分型 ,从 1例家鼠型 (SEO型 )汉坦病毒感染的病人血清中分离病毒 ,并进行核苷酸序列测定。结果 31例病人中 ,2例为姬鼠型(HTN型 ) ,2 4例为家鼠型 (SEO型 ) ,1例 SEO型病毒经分离鉴定测序后与 SEOU L、R2 2 株进行比较 ,M基因核苷酸同源性分别为 :95 .15 %、93.94% ;氨基酸同源性均为 97.2 7%。结论 所分离 SEO型汉坦病毒为 1996年流行高峰年的主要流行株 ,未发现病毒基因片段的重排 ,流行不是由病毒基因重排引起。不同地区 SEO型病毒分子结构差异较小 。

含HTNV S基因重组痘苗病毒在HFRS患者B淋巴母细胞系中的表达

采用Ficoll密度梯度离心法(淋巴细胞分离液)分离肾综合征出血热(HFRS)患者外周血单个核细胞(PBMC),并用EB病毒(EBV)感染B淋巴细胞,建立永生化的B淋巴母细胞系(B-LCL)。然后,用含汉滩病毒(Hantaan virus,HTNV)S基因的重组痘苗病毒感染B-LCL,应用间接免疫荧光检测核衣壳蛋白的表达。结果表明,B淋巴细胞经EBV感染4周左右,可形成永生化B-LCL。成功转化后的B-LCL,体积增大,且增殖的淋巴细胞积聚成团。汉滩病毒S基因在B-LCL中能有效表达核衣壳蛋白。含S基因的重组痘苗病毒感染的B-LCL可用作HTNV核衣壳蛋白特异性CTL活性研究的靶细胞。

鼠源抗HFRSV衣壳蛋白McAb F3株单链抗体基因克隆构建及表达

为了获得鼠源抗HFRSV衣壳蛋白McAbF3 株轻链和重链可变区基因 ,由连接肽体外连接获得单链抗体基因 ,在大肠杆菌中表达。从鼠源抗HFRSV衣壳蛋白McAbF3株细胞中分离总RNA ,以oligo(dT) 18为引物逆转录成cDNA ,通过PCR扩增出抗体的轻链 (VL)和重链可变区 (VH)基因 ,由连接肽体外连接获得单链抗体 (ScFv)基因。将此单链抗体 (ScFv)基因插入原核表达载体PET2 8a ,转化大肠杆菌BL2 1(DE3) ,经IPTG诱导后表达。结果构建的鼠源抗HFRSV衣壳蛋白McAbF3株单链抗体 (ScFv)基因 ,全长 741bp ,在大肠杆菌中获得了表达。结论为表达产物经SDS—PAGE及Western印迹分析 ,此单链抗体分子相对分子量为 2 9,0 0 0 ,与兔抗 鼠IgG产生特异反应。

HFRS患者特异性IgA、IgE抗体及其免疫复合物测定

为进一步研究ＨＦＲＳ免疫损伤机制，用ＥＬＩＳＡ法同步测定了１０８例不同临床型、不同病日、病期ＨＦＲＳ患者血清中特异性ＩｇＡ、ＩｇＥ抗体以及ＨＦＲＳ病毒特异性ＩｇＡ、ＩｇＥ型ＣＩＣ的水平及检出率。发现ＨＦＲＳＩｇＡ型抗体在轻型病例高于中、重型病例；ＨＦＲＳＩｇＥ型抗体及ＩｇＥ型ＣＩＣ在重型病例高于中、轻型病例。上述差异在病程早期（发热、休克少尿期，或是３～８病日）尤为突出。ＩｇＡ型ＣＩＣ则未见到上述差异。

野鼠和家鼠型HFRS所致残余损害的探讨

本文报道天台HFRS监测点研究野鼠型、家鼠型病毒对患者所致的残余损害。结果表明:天台县HFRS不仅发病串较高,且病后较长时间内机体的机能和功能不能完全恢复,尤以野鼠型患者更为严重。主要表现为肾脏损害综合征和神经衰竭综合征。提示在HFRS防治中,对病后残余损害的体检和随访工作不可忽视。

大规模人群中接种HFRS疫苗预防HFRS的流行病学效果评价

[目的]分析评价莒南县1995～2005 年大规模接种肾综合征出血热疫苗的流行病学效果.[方法]采用实验流行病学研究方法,通过设立接种人群和对照人群观察队列,对比分析接种组与对照组人群的发病密度、疫苗保护率、效果指数等实验流行病指标.[结果]接种组人群的发病密度明显低于对照组人群的发病密度（P＜0.01）,保护率为89.36%（95% CI 为81.42～97.31）,效果指数为9.40（95% CI：4.45～19.85）.[结论]大规模接种肾综合征出血热疫苗的流行病学效果良好.

Lack of association between integrin αvβ3 gene polymorphisms and hemorrhagic fever with renal syndrome in Han Chinese from Hubei, China

Hantaviruses belong to the family Bunyaviridae and cause hemorrhagic fever with renal syndrome （HFRS） in humans. 133 integrins, including αvβ3 and αⅡbβ3 integrins, act as receptors on endothelial cells and play key roles in cellular entry during the pathogenesis of hantaviruses. Previous study demonstrated that the polymorphisms of integrin αⅡbβ3 are associated with susceptibility to hantavirus infection and the disease severity of HFRS in Shaanxi Province of China, rather than in Finland. However, the polymorphisms of integrin av133 in patients with HFRS was incompletely understood. Here, we aimed to investigate the associations between polymorphisms in human integrin αvβ3 and HFRS in Han Chinese individuals. Ninety patients with HFRS and 101 healthy controls were enrolled in this study. Analysis of five single nucleotide polymorphism （SNP） sites （rs3768777 and rs3738919 on ITGAV; rs13306487, rs5921, and rs5918 on ITGB3） was performed by TaqMan SNP genotyping assays and bi-directional PCR allele-specific amplification method. No significant differences were observed between the HFRS group and controls regarding the genotype and allele frequency distributions of any of the five SNP sites, and no associations were found between ITGAV polymorphisms/genotypes and disease severity. In conclusion, our results implied that these five SNPs in the integrin αvβ3 gene were not associated with HFRS susceptibility or severity in Han Chinese individuals in Hubei Province.

Epidemic Prediction of Hemorrhagic Fever with Renal Syndrome Using Grey Multivariable Model

In this paper,we set up a grey multivtriable model to predict the HFRS morbidity.Forecasting test results show that this new model is of general use in the prediction Of the disease.It is particularly appropriate for HFRS investigations and controls.