Antigenic subunits of Hantaan virus expressed by baculovirus and vaccinia virus recombinants.

Baculovirus and vaccinia virus vectors were used to express the small (S) and medium (M) genome segments of Hantaan virus. Expression of the complete S or M segments yielded proteins electrophoretically indistinguishable from Hantaan virus nucleocapsid protein or envelope glycoproteins (G1 and G2), and expression of portions of the M segment, encoding either G1 or G2 alone, similarly yielded proteins which closely resembled authentic Hantaan virus proteins. The expressed envelope proteins retained all antigenic sites defined by a panel of monoclonal antibodies to Hantaan virus G1 and G2 and elicited antibodies in animals which reacted with authentic viral proteins. A Hantaan virus infectivity challenge model in hamsters was used to assay induction of protective immunity by the recombinant-expressed proteins. Recombinants expressing both G1 and G2 induced higher titer antibody responses than those expressing only G1 or G2 and protected most animals from infection with Hantaan virus. Baculovirus recombinants expressing only nucleocapsid protein also appeared to protect some animals from challenge. Passively transferred neutralizing monoclonal antibodies similarly prevented infection, suggesting that an antibody response alone is sufficient for immunity to Hantaan virus.     

Hantaan virus infection causes an acute neurological disease that is fatal in adult laboratory mice

Hantaan virus, the etiological agent of Korean hemorrhagic fever, is transmitted to humans from persistently infected mice (Apodemus agrarius), which serve as the primary reservoir. Here we demonstrate that several strains of adult Mus musculus domesticus (C57BL/6, BALB/c, AKR/J, and SJL/J) were susceptible to Hantaan virus infection when infected intraperitoneally. First clinical signs were loss of weight, ruffled fur, and reduced activity, which were followed by neurological symptoms, such as paralyses and convulsions. Within 2 days of disease onset, the animals died of acute encephalitis. PCR analysis indicated a systemic infection with viral RNA present in all major organs. Immunohistochemical and in situ hybridization analyses of postmortem material detected viral antigen and RNA in the central nervous system (predominantly brain), liver, and spleen. In the central nervous system, viral antigen and RNA colocalized with perivascular infiltrations, the predominant pathological finding. To investigate the involvement of the interferon system in Hantaan virus pathogenesis, we infected alpha/beta interferon receptor knockout mice. These animals were more susceptible to Hantaan virus infection, indicating an important role of interferon-induced antiviral defense mechanisms in Hantaan virus pathogenesis. The present model may help to overcome shortcomings in the development of therapeutic and prophylactic measurements against hantavirus infections.     

Control of laboratory acquired hemorrhagic fever with renal syndrome (HFRS) in Japan

By the end of 1985, 126 human cases of laboratory acquired hemorrhagic fever with renal syndrome (HFRS) were recorded in Japan. Seroepidemiological studies revealed that laboratory rats exhibited high IFA titers against Hantaan or related viruses at locations where HFRS patients occurred. Laboratory researchers contracted HFRS more frequently than laboratory animal technicians or caretakers, although a laboratory animal caretaker died of the disease. Inhalation of HFRS-virus contaminated air in an animal facility is the likely cause of infection with this virus. Wound infection during animal experiments may be another important route of infection. Infection of laboratory rats can occur by transferring animals from contaminated to other animal facilities. Tissue fragments or cells of transplantable animal tumors are a potential source of spreading the HFRS virus. Eradication of HFRS virus from a contaminated animal facility can be achieved best by elimination of all animals in the room, especially when human HFRS is associated with an infected colony. In some cases, when IFA titers of the sera of the rats tested were low, infection apparently disappeared without instituting any particular control measures other than ordinary procedures for care and management of laboratory animals. HFRS viruses have not yet been eradicated from all animal facilities in Japan. Therefore, serological monitoring of laboratory rats continues.     

Study on the aerosol transmission of Hantaan virus

We studied some important aspects constituting aerosol transmission of Hantaan virus, including the possibility of viral aerosol generated by rodents, airborne stability, rodent’s susceptibility to aerosol challenge, and field air sampling for the virus. Our results showed that Hantaan virus aerosol could be generated through the activities of infected mice, and cause specific infection among the exposed animals. Several kinds of rodents such asApodemus agrarius, weaning mice and suckling mice were found to be rather sensitive to the aerosol challenge of Hantaan virus. The 50% of inhaled lethal dose (LD₅₀) of suckling mice is 0.73 (1.4–0.37) plaque-forming unit (pfu). Hantaan virus aerosol was relatively stable in the air at 18–20°C and 70–90% relative humidity. The biological decay rate of the viral aerosol was 4.1% per min during 90 min. We also successfully sampled and isolated Hantaan virus from the working field atmosphere. The data obtained in the study provided more solid evidence for Hantaan virus aerosol transmission among rodents and from rodents to human-beings.     

应用噬菌体展示随机肽库淘筛mAb 5H5识别的抗原表位

本文利用噬菌体随机9肽库探索汉滩病毒（HTNV）核衣壳蛋白（NP）B细胞抗原表位。以抗HTNV NP单克隆抗体(mAb）5H5作为筛选分子,生物淘洗噬菌体递呈的随机9肽库。阳性克隆经夹心ELISA、竞争ELISA鉴定后,随机挑取10个克隆,DNA测序,与HTNV76－118株S基因进行同源性分析。结果显示筛选到的噬菌体能特异地与5H5结合,这种结合可被天然抗原所抑制。10个克隆的氨基酸序列相同,均为VRDAEEQYE,与76－118株NP氨基端的aa25-33一致。证实了该线性表位是mAb 5H5识别的表位,噬菌体肽库有助于病毒抗原表位的确定。     

汉滩病毒感染诱导热休克蛋白70表达

为了解汉滩病毒感染后细胞的应激反应及ＨＳＰ７０的表达与病毒复制的关系,在汉滩病毒Ａ９株感染Ｖｅｒｏ－Ｅ６细胞后,用免疫组织化学及核酸分子原位杂交法,对细胞ＨＳＰ７０基因的表达进行了检测。结果表明,汉滩病毒感染细胞４ｈｙ后即可诱导Ｖｅｒｐ－Ｅ６细胞表达ＨＳＰ７０,表达可持续至感染后５ｄ且ＨＳＰ７０在细胞内的分布也有改变。提示汉滩病毒可直接诱导ＨＳＰ７０的高表达。     

Human Memory Cytotoxic T-Lymphocyte (CTL) Responses to Hantaan Virus Infection: Identification of Virus-Specific and Cross-Reactive CD8+ CTL Epitopes on Nucleocapsid Protein

Hantaan virus, the prototypic member of the Hantavirus genus, causes hemorrhagic fever with renal syndrome in humans. We examined the human memory T-lymphocyte responses of three donors who had previous laboratory-acquired infections with Hantaan virus. We demonstrated virus-specific responses in bulk cultures of peripheral blood mononuclear cells (PBMC) from all donors. Bulk T-cell responses were directed against either Hantaan virus nucleocapsid (N) or G1 protein, and these responses varied between donors. We established both CD4(+) and CD8(+) N-specific cell lines from two donors and CD4(+) G1-specific cell lines from a third donor. All CD8(+) cytotoxic T-lymphocyte (CTL) lines recognized one of two epitopes on the nucleocapsid protein: one epitope spanning amino acids 12 to 20 and the other spanning amino acids 421 to 429. The CTL lines specific for amino acids 12 to 20 were restricted by HLA B51, and those specific for amino acids 421 to 429 were restricted by HLA A1. The N-specific CTL lines isolated from these two donors included both Hantaan virus-specific CTLs and hantavirus cross-reactive CTLs. Responses to both epitopes are detectable in short-term bulk cultures of PBMC from one donor, and precursor frequency analysis confirms that CTLs specific for these epitopes are present at relatively high precursor frequencies in the peripheral T-cell pool. These data suggest that infection with Hantaan virus results in the generation of CTL to limited epitopes on the nucleocapsid protein and that infection also results in the generation of cross-reactive T-cell responses to distantly related hantaviruses which cause the distinct hantavirus pulmonary syndrome. This is the first demonstration of human T-lymphocyte responses to Hantaan virus.     

汉滩病毒S片段基因编码区在Vero-E6细胞中的表达

The coding region of S genome segment of Hantaan virus (76/118 strain) was inserted into the eukarytic expression plasmidpVR1012. The recombinant expression plasmid pVRS22 was constructed. Vero-E6 cells were transiently transfected in vitro with pVRS22 plasmid. The transient expression of Hantaan virus nucleocapsid proteins in Vero-E6 cells was detected by indirect immunofluorescence assay (IFA) with monoclonal antibody 5H5 against Hantaan virus.     

汉滩病毒S片段基因编码区在Vero—E6细胞中的表达

汉滩病毒属于布尼亚病毒科汉坦病毒属,主要引起人类肾综合征出血热［１］。我国是肾综合征出血热发生和流行的主要疫区,该病起病急、病死率高,流行范围广,因此寻找安全有效、低廉的疫苗是控制本病流行和发生的关键。基因免疫是近年来微生物学和免疫学研究的新热点［２...     

汉滩病毒感染血管内皮细胞后上调HLAI类分子的表达

目的:观察汉滩病毒感染血管内皮细胞(EVC304)前后,HLA-I类分子中HLA-A、HLA-B、HLA-Cw3、HLA-Cw5表达上的差异。方法:以汉滩病毒76-118株感染EVC304细胞作为实验组,以无任何刺激的EVC304细胞作为阴性对照组。6h后用RT-PCR的方法对HLA-A、HLA-B、HLA-Cw3、HLA-Cw5的mRNA进行反转录和基因片段的扩增,在mRNA水平上检测其表达差异。结果:在HTNV感染以后,EVC304细胞中HLA-A、HLA-B、HLA-Cw3、HLA-Cw5mRNA表达均呈升高现象,其中HLA-A、HLA-B和HLA-Cw5的mRNA表达与未感染细胞相比明显升高。结论:HTNV感染血管内皮细胞后,可以诱导HLA-A、HLA-B、HLA-Cw3、HLA-Cw5的表达升高,从而诱发机体特异性免疫应答,导致免疫损伤的发生。这或许是肾综合症出血热的发病机制之一。     

在大肠杆菌中分别表达汉滩病毒素膜糖蛋白G1和G2

利用PCR方法扩增了汉滩病毒76－118株囊膜糖蛋白G1和G2的编码区基因,并将PCR产物克隆到T-载体中,用限制性内切酶将G1和G2的编码区基因切下,并克隆到表达载体pBV220中构建G1和G2的表达质粒。诱导表达后在SDS－PAGE凝胶中未见表达产物带,表达的G1和G2能与部分抗G1和G2的单克隆抗体发生反应,但用Western－blot方法不能检测到表达产物。用表达的G1和G2免疫小白鼠能刺激小白鼠产生特异性抗汉摊病毒的抗体,间接免疫荧光抗体的滴度可分别达到1:160和1：320。     

汉滩病毒S片段基因编码区在Vero-E6细胞中的表达

The coding region of S genome segment of Hantaan virus (76/118 strain) was inserted into the eukarytic expression plasmidpVR1012. The recombinant expression plasmid pVRS₂₂ was constructed. Vero-E₆ cells were transiently transfected in vitro with pVRS₂₂ plasmid. The transient expression of Hantaan virus nucleocapsid proteins in Vero-E₆ cells was detected by indirect immunofluorescence assay (IFA) with monoclonal antibody 5H5 against Hantaan virus.     

The multimerization of hantavirus nucleocapsid protein depends on type-specific epitopes

Multimerization of the Hantaan virus nucleocapsid protein (NP) in Hantaan virus-infected Vero E6 cells was observed in a competitive enzyme-linked immunosorbent assay (ELISA). Recombinant and truncated NPs of Hantaan, Seoul, and Dobrava viruses lacking the N-terminal 49 amino acids were also detected as multimers. Although truncated NPs of Hantaan virus lacking the N-terminal 154 amino acids existed as a monomer, those of Seoul and Dobrava formed multimers. The multimerized truncated NP antigens of Seoul and Dobrava viruses could detect serotype-specific antibodies, whereas the monomeric truncated NP antigen of Hantaan virus lacking the N-terminal 154 amino acids could not, suggesting that a hantavirus serotype-specific epitope on the NP results in multimerization. The NP-NP interaction was also detected by using a yeast two-hybrid assay. Two regions, amino acids 100 to 125 (region 1) and amino acids 404 to 429 (region 2), were essential for the NP-NP interaction in yeast. The NP of Seoul virus in which the tryptophan at amino acid number 119 was replaced by alanine (W119A mutation) did not multimerize in the yeast two-hybrid assay, indicating that tryptophan 119 in region 1 is important for the NP-NP interaction in yeast. However, W119A mutants expressed in mammalian cells were detected as the multimer by using competitive ELISA. Similarly, the truncated NP of Seoul virus expressing amino acids 155 to 429 showed a homologous interaction in a competitive ELISA but not in the yeast two-hybrid assay, indicating that the C-terminal region is important for the multimerization detected by competitive ELISA. Combined, the results indicate that several steps and regions are involved in multimerization of hantavirus NP.     

汉坦病毒囊膜糖蛋白G1基因重组腺病毒载体的构建及其在Vero E6细胞中的表达

拟构建汉坦病毒Gl基因重组腺病毒载体并在VeroE6细胞中表达,为汉坦病毒基因疫苗的研究提供实验基础。PCR法从含汉坦病毒-76118株M基因的M56质粒扩增糖蛋白G1基因片段,利用穿梭质粒pShuttle,将其克隆入Adeno—X病毒DNA,获得重组腺病毒DNA,转染HEK293细胞,包装、扩增后得到汉坦病毒Gl基因重组腺病毒原种,感染VetoE6细胞,用IFA法和ELISA法检测表达产物。得到了含汉坦病毒G1基因的重组腺病毒,其滴度约为10^11pfu／ml,感染VeroE6细胞后检测到汉坦病毒糖蛋白G1的表达。     

Comparison of virulence between two strains of Rattus serotype hemorrhagic fever with renal syndrome (HFRS) virus in newborn rats

Two strains of hemorrhagic fever with renal syndrome (HFRS) virus from Rattus, SR-11 and KI-262, showed virtually identical antigenicity but differed from prototype strain Hantaan 76-118 (Apodemus origin) in a neutralization test. Wistar newborn rats inoculated intraperitoneally (i.p.) with SR-11, which was isolated from a laboratory rat associated with an outbreak of HFRS, developed clinical signs such as ataxia and limb paralysis and died at about 18 days after inoculation. The LD50 of SR-11 in 1-day-old rats was 10(1.2) focus-forming units (FFU). In contrast, the animals inoculated i.p. or intracerebrally with 10(4) FFU of KI-262, which was from a wild rat in a dumping-ground area--an enzootic focus where no human cases have been recorded--did not show any significant clinical signs. The susceptibility of rats to SR-11 fatal infection was age-dependent. Virus titers in brains, lungs, kidneys, and livers of the rats inoculated with SR-11 were significantly higher than those in the same organs of the animals infected with KI-262. Necrosis of neurons in the brain tissue occurred in the rats infected with SR-11, while it was mild in the animals infected with KI-262.     

Experimental Infection in Newborn Mice and Rats by Hemorrhagic Fever with Renal Syndrome (HFRS) Virus

Newborn mice and rats were inoculated intracerebrally (ic) or intraperitoneally (ip) with Hantaan virus (76–118 strain) or HFRS-related virus (B-1 strain). The mortality and the influence on the increase of body weight in newborn mice were higher in the groups infected with the 76–118 strain than in the groups infected with the B-1 strain, while the B-1 strain was more virulent in rats than the 76–118 strain. Virus isolation from rats inoculated with either strain was attempted 7 and 11 weeks after inoculation. Virus could be isolated from various organs of rats infected with the B-1 strain, while it was recovered from only the brain and lungs of rats infected with the 76–118 strain. Viral antigen was readily detected in various organs of rats infected with the B-1 strain, but the amount and distribution of antigens were less in rats infected with the 76–118 strain. Our results suggest that the virulence of HFRS-related virus is variable, depending on the species of infected animals as well as on the. virus strains. The virus also persists in the injected animals with high titers of antibodies for at least 11 weeks.     

汉滩病毒核蛋白与热休克蛋白GRP94、HSP27的相互作用

为研究汉滩病毒(Hantaan virus,HTNV)感染诱导乳鼠脑组织热休克蛋白GRP94、HSP27与病毒蛋白的相互关系,选出生2—3d的昆明乳鼠实验性感染汉滩病毒,取8d后发病乳鼠脑组织部分制石蜡切片,用免疫组化结合共聚焦显微镜检测组织中病毒抗原及GRP94、HSP27的表达,部分制匀浆液,用ELISA、免疫共沉淀方法分析病毒抗原和GRP94、HSP27的关系。结果示汉滩病毒感染诱导乳鼠脑组织神经细胞高表达GRP94且与细胞内病毒抗原有共定位关系,但未见HSP27诱导高表达;免疫共沉淀显示汉滩病毒核心抗原(HINV—NP)与GRP94、HSP27呈非共价复合物形式存在。该结果为进一步探讨HSPs在病毒感染复制中的作用以及抗病毒感染方面提供了有意义的实验资料。     

汉滩病毒诱导小鼠骨髓瘤SP2／0细胞凋亡的初步研究

为研究汉滩病毒对肿瘤细胞的诱导凋亡作用,以一定量病毒悬液感染体外培养的SP2／0细胞,接种后一定时间将细胞消化甩片行Gimsa染色观察凋亡细胞核的变化,制细胞悬液以流式细胞仪测细胞周期,并用免疫组化的方法检测凋亡分子Fas和FasL的表达。结果示经病毒诱导后细胞出现生长特性及形态学变化,Giemsa染色观察到典型凋亡细胞：流式细胞仪显示有凋亡峰出现;免疫组化检测出感染后SP2／0细胞中Fas和FasL表达明显升高。该结果表明汉滩病毒可诱导体外培养SP2／0细胞凋亡,其发生可能与凋亡分子Fas和FasL有关。