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.

In situ hybridization detection of persistent infection and viral RNA distribution in laboratory rats with hemorrhagic fever with renal syndrome virus

Digoxigenin-labelled Hantaan specific cDNA probes were used in the present study for in situ hybridization detection of hemorrhagic fever with renal syndrome virus(HFRSV) persistent infection and viral RNA distribution in naturally infected laboratory rat

Hemorrhagic fever with renal syndrome virus infection in livers studied by in situ hybridization and immunohistochemistry

To clarify whether hemorrhagic fever with renal syndrome (HFRS) virus is cytopathic or not for the liver, HFRS virus RNA in liver tissue from 19 patients with HFRS and the livers of naturally infected rats with virus were examined by in situ hybridization

Tracing in vivo of the vascular and cellular membranous permeabilities in the experimentally infected suckling mice with Chen strain hemorrhagic fever with renal syndrome virus

the vascular and cellular membranous permeabilities in the experimentally infected suckling BALB/c mice with Chen strain hemorrhagic fever with renal syndrome virus (HFRSV) were studied by employing horseradish peroxidase (HRP), colloidal lanthanum and colloidal gold labelled antibodies as tracers and applying the tracers to the mice in vivo via tail veins, and the tissues were observed under light mcroscope and electron microscope. The vascular and cellular membranous permeabilities in the infected increased as the tracers appeared in the perivascular and interstitial tissues as well as in the cytoplasms of some parenchymal cells of the organs ,while the permeabilities remained normal in the control. The rasults suggest that in the infected mice, it might be the virus infection that was mainly responsible for the abnormal permeabilities.

Tracing of the viral antigens by gold-labelled antibodies in the experimentally infected suckling mice with Chen strain hemorrhagic fever with renal syndrome virus and ultrastructural observation

Our previous works were done on the abnormal permeability of vasculature and cellular membrane after hemorrhagic fever with renal syndrome virus (HFRSV) infection. In this study, the internalization of viral antigens in the experimentally infected suckling BALB/c mice with Chen strain HFRSV were further studied by the application of the colloidal gold-labelled polyclonal and monoclonal antibodies against HFRSV (GLAb) to the infected animals via tail veins. The normal mice with GLAb and the infected mice with colloidal gold-labelled indifferent antibodies (GLIg) were also employed as experimental controls. At certain time intervals after the injection,the animals were sacrificed and tissues were observed under light and electron microscopes. In the normal mice,the conjugates were confined to the vasculature and reliculo-endothelial system and localized in the lysosomes of phagocytes. In the infected animals,the GLIg conjugates could be used to demonstrate the abnormal permeability,but could not show the antigen localization,while the GLAb could be internalized into the cytoplasm of the parenchymal cells and localized in the free ribosome,Golgi apparatus,granules within vesicles,and inclusion body-like structures Compared to the infected animals with GLIg,obvious tissue structure alternations under LM were observed in the infected mice with GLAb. The ultrastructural changes of destruction and abnormal structures frequently occurred in the cells of the infected animals. The typical virion,immature virion and inclusion body could be found but only in a few cells. The results indicated that the free ribosomes,Golgi apparatus and vesices may be related to viral infection and positive viral antigen could not represent the virus structure only. It is suggested that the antibodies produced in the bodies after HFRSV infection can bind not only with the extracel lular viral antigens to form immunocomplexes to induce tissue lesions but also the cytoplasmic viral antigens of the infected cells through the damaged cellular membranes.

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.

CLINICAL SIGNIFICANCE OF DETECTING SERUM ANTIBODIES TO NUCLEOPROTEIN AND GLYCOPROTEIN G_2 OF HANTAAN VIRUS IN PATIENTS WITH HEMORRHAGIC FEVER WITH RENAL SYNDROME

Antibody blocking enzyme linked immunosorbent assays, respectively detecting antibodies to Hantaan virus nucleoprotein (NPAb) and glycoprotein GZ (G,Ab), were developed using monoclonal antibody L133, L13r3, LV48A and LVZB28B NPAb and GZAb in 291 serum samples from 65 patientswith kemorrkagic fever with renal syudrome (HFRS) were detfrmlned by these methods. The positive rates or NPAb were 90N on day 2-3 and 100 % on day 8-9 arter onset of disease, respectively.NPAb titers Increased during fever period and reached Peak levels during kypotensive and oliguric periods of HFRS. It was suggested that NPAb might be an important component Involved in the immunopathogenlc lin'alrmeut of HFRS and the detection of NPAb might be useful for the early diagnosis or HFRS. The I,osltlve rates and titers of GZAh were very low during the rirst three periods,namely rever, hypoteuslve and ollgurlc periods, and reached high levels during the convalescent period. GRAb titers were negatively related to the I,rotelnurla levels during the course of HFRS. It wasIndicated that GZAb might be the main component or neutralizing autlhodles to Hantaan virus Infection and the efrlclent production or GZAb was a good marker ror predicting the recovery and betterprognosis 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.

Diffuse alveolar hemorrhage complicating dengue haemorrhagic fever in a 15-year-old boy:A case report

Rationale:Dengue fever is a viral infection that is spread through the bites of infected female Aedes mosquitos.It can cause life threatening complications,including dengue haemorrhagic fever(DHF)and dengue shock syndrome.Patient concerns:A 15-year-old male presented with fever and petechiae and later developed hemoptysis.Diagnosis:Dengue fever with DHF with diffuse alveolar hemorrhage.Interventions:Invasive ventilation with high positive end expiratory pressure,multiple transfusions of packed red blood cells,fresh frozen plasma,single donor platelets and inotropic support Outcomes:The patient was stabilized and discharged on minimal supplemental oxygen.Lessons:Diffuse alveolar hemorrhage,although very rare,should be considered in a patient with dengue who presents with hemoptysis.The treatment is directed at providing respiratory and circulatory support,and preventing the progression of microcirculation damage.

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.

HLA-E-restricted Hantaan virus-specific CD8^(+)T cell responses enhance the control of infection in hemorrhagic fever with renal syndrome

Infection with the Hantaan virus(HTNV)may result in severe hemorrhagic fever with renal syndrome(HFRS).The functions of HLA-E-restricted CD8^(+)T lymphocytes in virus control and vaccine development have recently received increased attention.The purpose of this research is to discover HLA-E-restricted CD8^(+)T cell epitopes on HTNV as well as the features of these epitope-specific CD8^(+)T cells in HFRS patients.To anticipate HLA-Erestricted HTNV epitopes,the NetMHCpan servers were utilized.The K562/HLA-E cell binding test and the enzyme-linked immunospot assay were used to confirm epitope binding to HLA-E.The number and features of HLA-E-restricted epitope-specific CD8^(+)T lymphocytes in HFRS patients were investigated using tetramer staining,intracellular cytokine labeling,proliferation,and cytotoxicity assays.Six HTNV-derived HLA-Erestricted CD8^(+)T cell epitopes were found in this study.In mild/moderate HFRS patients,the frequency of HLA-E-restricted epitope-specific CD8^(+)T cells was greater than in severe/critical patients.CD38+HLA-DR+HLA-E-restricted CD8^(+)T cells were identified.Meanwhile,CD45RA^(+)CCR7^(-)effector memory-re-expressing CD45RA T cells with early and intermediate maturation and differentiation characteristics predominated.Notably,CD8^(+)T cells from milder HFRS patients produced more interferon-γ,interleukin-2,and granzyme B,had a stronger proliferative potential,and were inversely linked with the amount of plasma HTNV virus load.Furthermore,HLA-E-restricted epitope-specific CD8^(+)T cells demonstrated improved cytotoxic activity in vitro during the acute stage of HFRS.Taken together,the findings demonstrate the protective effects of HLA-E-restricted CD8^(+)T cells during HTNV infection,suggesting that HLA-E-targeted vaccines against HTNV might be developed for HLA-diverse populations.

Research Progress on the Pathogenesis of Hemorrhagic Fever with Renal Syndrome

Hemorrhagic fever with renal syndrome(HFRS)is an acute natural focus epidemic disease characterized by fever,shock,hemorrhage and kidney injury caused by hantavirus infection.Hantavirus mainly infects human vascular endothelial cells,and induces extensive damage to small blood vessels and capillaries.Increased vascular permeability is the pathological basis for clinical manifestations of HFRS.Although domestic and foreign scholars have carried out many studies on the hantavirus pathogenesis,such as the immune pathological response induced by hantavirus,host genetics and apoptosis,thrombocytopenia,coagulation and fibrinolysis dysfunction,and the vascular endothelial damage,the pathogenesis of HFRS has not been fully elucidated and there is no effective drug yet.In-depth discussion of the molecular mechanism of HFRS and finding effective therapeutic drugs are still the research hotspots on the field of hantavirus/HFRS.This review will elaborate the research progress on the pathogenesis of HFRS in recent years.

Distribution and appearance of insoluble granular antigen（inclusionbody） of Hantaan virus infection of human，rat and mouse

Objective: The study aimed at looking into the expression and distribution of Hantaan virus antigens in human, rat and mouse tissues. Methods: With 15 kinds of monoclonal antibodies(MAbs) and 1 polyclonal antibody, the repeated PAP or the repeated PAP plus ABC immunohistochemical methods and immunoelectron microscopic techniques were used. Results: Two kinds of viral antigens of hemorrhagic fever with renal syndrome, the soluble and the insoluble(inclusion), were found in human, rat and mouse tissues with G2 , NP and hemagglutinin MAbs and 1 polyclonal antibody, etc. The insoluble granular inclusion body like antigen was mainly found in the epithelial cells of the 20 autopsy cases and the puncture bone marrow cells. Cells with inclusion body sometimes showed cytolytic or eosinophilic necrosis or proliferative changes. Under the immunoelectron microscope the granular antigen was either fibrous solid or vesicular a structure at the side of nucleus near Golgi complex. Conclusion: The granular antigen of Hantaan virus was indeed a special form of antigen, which was of great importance to the inducrion of the cellular injury and pathogenesis of the disease.

河南省肾综合征出血热不同类型疫区监测比较研究

目的 :研究河南省不同类型肾综合征出血热 (HFRS)疫区的人间疫情、宿主动物监测及影响因素 ,为控制该病提供依据。方法 :应用描述流行病学、血清流行病学、分子生物学及统计学方法进行研究。结果 :( 1) 1990至 2 0 0 0年 ,河南省HFRS呈稳定下降趋势 ,其中姬鼠型疫区一直呈下降趋势 ,家鼠型有一定的周期波动 ,混合型走势较为平稳。( 2 )我省姬鼠型及家鼠型疫区分别在冬季和春季各有一发病高峰 ,而混合型疫区一年有春、冬两个季节高峰。 ( 3)家鼠型疫区居民区以褐家鼠为优势鼠种 ( 5 8.0 7% ) ,黄胸鼠、小家鼠次之。野外以黑线姬鼠 ( 33.6 7% )和大仓鼠 ( 4 2 .5 % )为优势种。姬鼠型疫区居民区以褐家鼠 ( 4 6 .12 % )、小家鼠 ( 4 2 .92 % )为优势种 ;野外以黑线姬鼠占绝对优势 ( 83.0 4% ) ,大仓鼠为次要鼠种 ( 11.49% )。混合型疫区居民区褐家鼠为优势鼠种 ( 73.8% ) ,黄胸鼠为次要鼠种 ( 17.83% ) ;野外以黑线姬鼠为优势种 ( 4 9.6 6 % ) ,大仓鼠次之 ( 36 .43% )。 ( 4 )家鼠型疫区居民区褐家鼠带病毒率较高 ,其次为黄胸鼠、小家鼠 ,野外为褐家鼠、黑线姬鼠。姬鼠型疫区居民区以黑线姬鼠带病毒率最高 ,褐家鼠及小家鼠次之 ,野外仅有黑线姬鼠带病毒。混合型疫区居民区带病毒鼠包括褐家鼠、黄胸鼠及小?

肾综合征出血热与鼠的监测和防控

肾综合征出血热(HFRS)的主要储存宿主是黑线姬鼠(姬鼠型)和褐家鼠(家鼠型)。在疫区,鼠密度与发病率密切相关,因此,监测鼠密度对预测疫情具有重要意义。防控的主要方法是防鼠和灭鼠。灭鼠应使用灭鼠药,大面积同时进行。只有当鼠密度明显下降后,才能有效控制HFRS流行。必要时,可接种HFRS疫苗。

浙西地区肾综合征出血热监测研究

目的掌握肾综合征出血热(HFRS)流行规律与疫源地情况,为控制HFRS的流行提供依据。方法运用描述流行病和疫源地调查方法,对人间疫情和主要宿主动物进行分析,采集标本送实验室采用间接免疫荧光法测抗体,直接免疫荧光法测抗原。结果1965年后的近40年来,衢州共发生HFRS18422例,年均发病率为21.87/10万,死亡271例,病死率1.47%。发病季节随年代发生明显变化,20世纪80年代以前,冬春峰发病数之比为7.22∶1,2000年以后为1.18∶1,农民发病占82.41%,男女之比为2.20∶1,25～54岁组发病占78.26%。开化县和衢江区是我市最主要HFRS疫区。2002～2004年室外鼠密度为3.64%,室内为4.20%。褐家鼠和黑线姬鼠分别为室内和野外优势鼠种,带病毒鼠指数分别为0.026和0.034。3年的带病毒鼠指数分别是0.048、0.036和0.032,与发病率变化存在相关性。结论自1965年报告首发病例后40年来,发病率曲线呈单峰型,表明HFRS存在散发、流行、逐步静息的自然规律性。我市仍然是一个以姬鼠型为主的混合型疫区,近年来疫情的下降,不仅在于易感人群接触病原体机会的减少,也在于自然界主要宿主动物的带病毒鼠指数的下降。HFRS的防制工作仍应引起高度重视。

用PCR技术检测革螨、恙螨体内EHFV的研究

本次研究拟用PCR技术直接检测鼠巢、鼠体中革螨和恙螨体内EHFV-RNA。结果从5份革螨和恙螨标本中有4份检出EHFV-RNA,分别为格氏血厉螨、厩真厉螨、巴氏厉螨和小盾纤恙螨。表明用PCR技术可直接从革螨、恙螨体内检测出EHFV-RNA.为流行病学调查提供了一种方法。

肾综合征出血热病毒对人血管内皮细胞感染性的研究

目的 研究肾综合征出血热 (HFRS)病毒在血管内皮细胞 (HEC)内的增殖 ,及病毒对HEC产生几种细胞因子的影响。方法 采用微量免疫酶斑法检测HFRS病毒A6 9株感染HEC后病毒繁殖动态变化 ,间接免疫荧光法检测HEC内HFRS病毒抗原。ELISA法检测HFRS病毒感染上清液中TNF -α、IL - 6和IFN - β的含量。 结果 病毒感染后 1d即可在培养上清中用微量免疫酶斑法测出病毒 ,病毒滴度 5d达高峰 ,以后下降。在一定范围内病毒产量随病毒感染复数 (multiplicityofinfection ,M0I)的增加而增高。间接免疫荧光法证明感染的HEC胞浆及胞膜上携带肾综合征出血热病毒抗原。电镜和光镜下 ,感染细胞未见明显的形态和结构改变。病毒对HEC产生TNF -α无影响 ;产生IL - 6和IFN - β明显增高。 结论 HFRS病毒可在原始靶细胞HEC内增殖。HFRS病毒感染后所出现的微血管损害可能不是病毒直接损害所致 ,而很可能与间接因素有关。

肾综合征出血热病毒对人外周血单个核细胞产生细胞因子的影响

目的研究肾综合征出血热(HFRS)病毒对人外周血单个核细胞(PMBC)产生细胞因子的影响。方法用HFRS病毒A69株及脂多糖(LPS)单独或联合作用于PMBC,于作用后12、24、48、72、96h分别收集培养上清液,用ELISA法检测上清液中细胞因子TNF-α、IL-6和IFN-γ的含量,实验数据用方差分析处理。结果病毒感染和LPS能使PBMC分泌TNF-α、IL-6和IFN-γ能力增强。感染后12h,TNF-α和IFN-γ开始升高,于感染后24h达高峰,然后开始下降;而IL-6水平于感染后72h达高峰,然后开始下降。HFRS病毒与LPS联合作用显著提高病毒诱导PBMC分泌TNF-α、IL-6和IFN-γ的能力。结论HFRS病毒能提高PBMC分泌TNF-α、IL-6和IFN-γ的能力,LPS可增强病毒诱导PBMC分泌细胞因子的能力,在HFRS病毒感染过程中合并细菌感染可能调节细胞因子介导的疾病进程,细胞因子在HFRS病毒的致病与免疫过程中可能起重要作用。

江苏省肾综合征出血热灭活疫苗免疫效果现场考核初报

对东海、赣榆、灌云东辛农场３个ＨＦＲＳ高发地区分别进行４８７１人份Ⅰ型疫苗、４５７１人份Ⅱ型疫苗和６６５８人份Ⅲ型疫苗的人群接种，并进行了１年期观察。结果：该两型苗的副反应均较低，安全性好，Ⅰ型疫苗全程免后２周荧光抗体阳转率为８４．４％，中和抗体阳转率为３７．０％；Ⅱ型疫苗荧光抗体阳转率为４８．８％，中和抗体阳转率为９４．５８％；Ⅰ型疫苗１年期内保护率为５６．０％，Ⅱ型疫苗为９３．０％。