白纹伊蚊经实验感染后涎腺中基孔肯雅病毒和乙型脑炎病毒抗原检测

用基孔肯雅和乙型脑炎病毒经口感染白纹伊蚊，采用免疫荧光试验对雌蚊涎腺作病毒抗原检查，结果于感染后第８￣１４天基孔肯雅的阳性为５０％￣９１．６７％，乙型脑炎为７０％￣８０％。试验认为，白纹伊蚊在感染后第６￣８天即可传播基孔肯雅和乙型脑炎病毒。     

我国乙型脑炎病毒优势基因型转换及其风险和对策

乙型脑炎是由乙型脑炎病毒引起的人畜共患病,在自然界中主要是通过蚊虫叮咬进行传播,能够引起人的中枢神经系统损伤和猪的繁殖障碍等疾病,对公共卫生安全造成了严重威胁。 由于乙型脑炎病毒在水禽宿主中的适应性差异,导致了病毒优势基因型由 GⅢ型逐渐向 GⅠ型转变。 乙型脑炎病毒优势基因型的转换和流行范围的扩大给疾病防控带来了新挑战,本文对我国乙型脑炎病毒优势基因型转换及其风险和对策进行了综述。     

流行性乙型脑炎患儿25例临床分析

流行性乙型脑炎是由乙型脑炎病毒引起的中枢神经系统急性传染病,病毒主要侵犯大脑,又称大脑炎。2005年6月～2008年8月,我院收治流行性乙型脑炎患儿25例。现将其发病特点分析如下。     

食蚊鱼和双硫磷对三带喙库蚊的综合防制

三带喙库蚊是东南亚乙型脑炎和西尼罗病毒的重要传播媒介。印度许多地区曾发生乙型脑炎的暴发流行,从三带咏库蚊中已分离出乙型脑炎的病毒,证明是主要媒介。这种库蚊在德里     

3批乙型脑炎减毒活疫苗病毒全基因序列测定和分析

目的对3批乙型脑炎减毒活疫苗病毒进行全基因测序,并与疫苗原始种子序列进行比较。方法随机选取某厂家生产的3批乙脑减毒活疫苗,提取病毒全基因组RNA,逆转录为cDNA后分段PCR扩增并测序。序列拼接后利用MegAlign软件与GenBank中乙脑减毒活疫苗原始种子全基因序列进行同源性比较。结果 3批乙型脑炎减毒活疫苗病毒基因组全长10 977个核苷酸,与乙脑减毒活疫苗原始种子序列同源性为100%。结论三批次乙型脑炎减毒活疫苗遗传特性高度稳定。     

基因Ⅲ型乙型脑炎病毒Real-time PCR检测方法的建立及应用

目的建立针对云南地区流行的基因Ⅲ型乙型脑炎病毒株Real-time PCR检测方法。方法根据云南地区分离株乙型脑炎病毒株(Yunnan0901)基因组核苷酸序列设计特异性引物,构建pMD18-T-JEV-E质粒,以此为模板进行SYBR GreenⅠReal-time PCR扩增并制作标准曲线,摸索最佳反应体系条件,建立乙型脑炎病毒荧光定量PCR检测方法,并与普通RT-PCR方法相比较。结果建立的SYBR GreenⅠReal-time PCR标准曲线呈现良好的重复性和特异性,与模板浓度呈现良好的线性关系。与RT-PCR方法相比,SYBR GreenⅠReal-time PCR的灵敏度高10倍,而且不与猪瘟病毒、猪蓝耳病毒、猪圆环病毒、基孔肯雅病毒、辛德毕斯病毒核酸发生非特异性扩增,具有良好的特异性。用该方法检测云南部分地区的蚊虫样品,其中库蚊、按蚊乙型脑炎病毒阳性率分别为17.4%和30.7%,且主要为基因型Ⅲ型。结论本实验建立的检测基因Ⅲ型乙型脑炎病毒的SYBR GreenⅠReal-time PCR方法具有特异、敏感、快速、定量、重复性好等特点,可应用于乙型脑炎疫情的监测。     

基因Ⅲ型乙型脑炎病毒Real-time PCR检测方法的建立及应用北大核心CSCD

目的建立针对云南地区流行的基因Ⅲ型乙型脑炎病毒株Real-time PCR检测方法。方法根据云南地区分离株乙型脑炎病毒株(Yunnan0901)基因组核苷酸序列设计特异性引物,构建pMD18-T-JEV-E质粒,以此为模板进行SYBR GreenⅠReal-time PCR扩增并制作标准曲线,摸索最佳反应体系条件,建立乙型脑炎病毒荧光定量PCR检测方法,并与普通RT-PCR方法相比较。结果建立的SYBR GreenⅠReal-time PCR标准曲线呈现良好的重复性和特异性,与模板浓度呈现良好的线性关系。与RT-PCR方法相比,SYBR GreenⅠReal-time PCR的灵敏度高10倍,而且不与猪瘟病毒、猪蓝耳病毒、猪圆环病毒、基孔肯雅病毒、辛德毕斯病毒核酸发生非特异性扩增,具有良好的特异性。用该方法检测云南部分地区的蚊虫样品,其中库蚊、按蚊乙型脑炎病毒阳性率分别为17.4%和30.7%,且主要为基因型Ⅲ型。结论本实验建立的检测基因Ⅲ型乙型脑炎病毒的SYBR GreenⅠReal-time PCR方法具有特异、敏感、快速、定量、重复性好等特点,可应用于乙型脑炎疫情的监测。     

云南省洱源县流行性乙型脑炎暴发流行的病原分离与鉴定

１９９１年７￣１０月,云南省洱源县发生流行性乙型脑炎暴发流行,采用滤过性试验,免疫荧光试验,交互ＨＩ试验,交互ＣＦ试验和中和试验方法,对从该县流行期采集的病人血清分离的２株病毒和从三带喙库蚁分离到的２株病毒进行鉴定,证实４株病毒均为流行性乙型脑炎病毒,从病原学证实为流行性乙型脑炎暴发流行,同时还查明了三带喙库蚊是本次乙型脑炎流行的主要传播媒介。     

培养猪肝细胞中空纤维型生物反应器的制备

目的 探讨培养猪肝细胞中空纤维型生物反应器的制备方法。方法 将两步法分离的新生实验小型猪肝细胞接种于中空纤维器内,用培养液循环式人工毛细管培养系统进行培养,观察生物反应器的蛋白分泌、利多卡因转换能力;检测培养猪肝细胞的乳酸脱氢酶(LDH)漏出量及细胞形态和活力。结果在反应器制备后第2、4、6天可检测出猪肝细胞分泌的白蛋白,生物反应器对利多卡因的转换率为89.6％～96.1％。培养结束时LDH漏出量由(23.7±4.6)U/L升高至(127.8±17.4)U/L(F=39.582,P<0.01),细胞活力由95.8％±0.3%降低至83.8％±4.7％(t=5.135,P<0.01)。结论 可用人工毛细管培养系统制备出1周内性能较好的培养猪肝细胞中空纤维型生物反应器。     

多重PCR结合核酸侵入反应及纳米金显色技术检测病毒性脑炎脑膜炎病原体

目的建立一种病毒性脑炎脑膜炎病原体多重PCR结合核酸侵入反应及纳米金显色的检测方法。方法针对几种重要的脑炎脑膜炎病毒(东方马脑炎病毒、西方马脑炎病毒、流行性乙型脑炎病毒、西尼罗病毒和尼帕病毒)保守区基因设计引物,进行多重PCR反应、核酸侵入反应及纳米金显色反应,对多种脑炎脑膜炎病毒同时进行检测,以森林脑炎病毒、圣路易脑炎病毒、基孔肯雅病毒和登革病毒4种病原核酸评价其检测特异性,以体外转录的病毒RNA或扩增的PCR片段评价其检测敏感性,并对流行性乙型脑炎患者临床标本进行检测。结果成功建立了一种脑炎脑膜炎病毒多重PCR结合核酸侵入反应及纳米金显色的检测技术。建立的检测方法可特异的检测目的病原体,且与森林脑炎病毒、圣路易脑炎病毒、基孔肯雅病毒和登革病毒无交叉反应。该方法对不同靶标的检测灵敏度均为103拷贝/μL,临床标本检测结果均为阳性。结论建立的脑炎脑膜炎病毒多重PCR结合核酸侵入反应及纳米金显色技术的检测方法,具有较高的检测特异性及灵敏度,检测通量高,肉眼即可观察结果,在传染病病原体检测方面具有广阔的应用前景。     

Immunological analysis of Japanese encephalitis virus using anti-Kamiyama monoclonal antibodies

Fifteen hybridomas were produced by fusing P3X63Ag8.653 mouse myeloma cells with spleen cells from BALB/c mice immunized with Japanese encephalitis (JE) virus Kamiyama strain. Antigenic analysis of twenty-five strains of JE virus was carried out by hemagglutination inhibition (HI) test with anti-Kamiyama monoclonal antibodies (KAMIMAs). Twenty-one JE virus strains were isolated from various parts of Japan, and four foreign countries. These strains had been isolated from different host between 1935 and 1979. According to the HI test against the five species-specific monoclonal antibodies, the twenty-five JE virus strains were classified serologically as follows: Group A: Kamiyama, Sekiya, Mochizuki, Nishizono, Sasazaki, Mie 44-1, Fukuoka 7101, Fukuoka 7202, Fukuoka 7309, Fukuoka 7311, Fukuoka 7452, Fukuka 7463, Fukuoka 7506, Kumamoto 80679, Chang Mai and JaGAr 02 strains. Group B: Nakayama-RFVL and Nakayama-Yoken strains. Group C: Nakayama-Yakken, Kalinina, G-1 late, JaGAr 01, Beijing 1 and 691004 strains. Group D: Muar strain. These results mostly corresponded with the serological classification by anti-Nakayama-RFVL monoclonal antibodies. Analysis of the biological activities of KAMIMAs revealed that there were no correlations among HI titer, ELISA titer and neutralization titer. Neutralizing and some non-neutralizing monoclonal antibodies protected mice infected with lethal doses of JE virus Kamiyama strain. SDS-PAGE and immunoblotting analysis revealed that three antibodies reacted with a 52.0 kD band under non-reducing conditions and with a 53.0 kD band under reducing conditions, five antibodies reacted with a 52.0 kD band only under non-reducing conditions, and that seven antibodies reacted with a 14.5 kD band under both non-reducing and reducing conditions.     

Mac ELISA测定乙脑患者血清和脑脊液中特异性IgM的评价

用抗体捕捉ELISA法(Mac ELISA)检测临床疑似流行性乙型脑炎(下称乙脑)病人脑脊液88份,阳性51份,阳性率57.95％;血清91份,阳性56份,阳性率为61.53％,两者无显著性差异(X~2=0.238 P>0.05);包括取自同一患者的脑脊液和血清各72份,阳性率分别为61.11％和68.05％;而分析其中14例IgG呈4倍以上增高,乙脑患者脑脊液IgM阳性13例,而血清IgM阳性却只有8例,提示在疫区流行季节内仅仅血清学IgM阳性有时可能为乙脑隐性感染的同时还伴其它有关病毒感染。     

Studies on the inhibitory activities of human serum lipoproteins for Japanese encephalitis virus.

Human serum lipoproteins were purified by ultracentrifuging and their concentrations adjusted as required to be within the normal male/female serum range for all assays. The activities in inhibition of hemagglutination (HAI) for Japanese encephalitis virus were--low density lipoprotein (LDL) greater than very low density lipoprotein (VLDL) greater than high density lipoprotein (HDL). Heating (56 degrees C/30 minutes) caused the LDL titer to fall and freeze-thawing (20 degrees C/room temperature) the VLDL titer to rise slightly, possibly as a result of alteration in lipoprotein structure. The additon of lipoprotein depleted serum appeared to dampen these effects and there was no nett change in titer when it was added to a lipoprotein mixture. Similarly, unfractionated normal serum showed no significant change in titer after these treatments. The lipoproteins lacked significant virus neutralizing (VN) activity and this remained so in spite of fluctuations in HAI titer after heating and freeze-thawing.     

Rapid diagnosis of viral infections in the central nervous system

Rapid diagnosis of viral infections in the central nervous system has become increasingly important. Antiviral treatment, prevention of spread of disease and differentiation from infections caused by agents sensitive to antibiotics may be the important consequences of a virus specific diagnosis gained early in the disease. The diagnosis can be obtained by detection of virus or viral antigen in the human specimen: herpes simplex virus by electron microscopy, immunofluorescence or immunosorbent assays in brain biopsies; rabies virus by immunofluorescence in corneal cells or skin and mucous membranes. The presence of measles or influenza antigens in nasopharyngeal secretions, shown by immunofluorescence or enzyme immunoassays, may diagnose an encephalitis caused by either of these viruses. Where suitable material is not available the detection of virus-specific IgM in a single serum specimen may be used for diagnosis. Mumps specific IgM activity is detected by enzyme-linked immunosorbent assay (ELISA) or indirect immunofluorescence techniques; tick-borne encephalitis (TBE) specific IgM by immunosorbent assays or by reduction of hemagglutination-inhibition (HI) titer by 2-mercaptoethanol treatment of serum. Reports have been given on the detection of IgM activity by ELISA also in other arboviral infections such as Japanese and LaCrosse encephalitis. The demonstration of an intrathecal production of virus-specific immunoglobulins may reveal the type of virus causing the infection in the central nervous system.     

Proposal for Japanese encephalitis surveillance using captured invasive mongooses under an eradication project on Okinawa Island, Japan

A project to eradicate invasive small Asian mongooses (Herpestes javanicus) is underway to conserve the unique ecosystem of Okinawa Island, Japan. In the present study, we tried to elucidate whether the mongoose is a host of Japanese encephalitis virus (JEV) and to evaluate the reliability of surveillance of Japanese encephalitis (JE) using this species. Culex tritaeniorhynchus, the main vector mosquito of JEV, feeds on the mongoose. Eighty-five (35.4%) of 240 wild small Asian mongooses captured between 2001 and 2005 had neutralizing antibodies against more than one of four JEV strains. Prevalence rates of JEV antibodies tended to increase with body weight and length of the animals. One of three sentinel mongooses showed a temporal change in antibody titer. These results indicate that the small Asian mongooses on Okinawa Island are sensitive to JEV. From the antibody titers and the locations of capture, the JEV active area was clarified. We propose that surveillance of JE using mongooses captured under the eradication program is reliable.     

MDCK悬浮细胞制备及流感病毒敏感性研究

目的 建立无血清悬浮培养MDCK细胞的方法,分析其传代、线性放大过程中的稳定性及增殖流感病毒的敏感性。方法 运用逐步降血清悬浮驯化法将贴壁培养型MDCK细胞驯化为无血清全悬浮培养型MDCK细胞;进一步分析其生长动力学特性、连续传代稳定性,在5L生物反应器中扩大培养;接种流感和禽流感病毒,测定不同时间HA效价、CCID₅₀滴度,分析其病毒敏感性。结果 成功将ATCC引进的贴壁培养型MDCK细胞驯化为无血清全悬浮培养型MDCK细胞(命名为MDCK-XF02细胞)并冻存;不同接种密度培养MDCK-XF02细胞最大增殖密度均达13.0×10⁶ cells/mL以上,且差异无统计学意义(P>0.05);连续传代培养过程中细胞形态和生长状况稳定,比生长速率差异无统计学意义(P>0.05);三个代次的MDCK-XF02细胞以2.0×10⁶ cells/mL接种于5 L生物反应器,细胞生长状态良好,倍增时间小于21 h,在批培养中细胞浓度高达(14.57±0.47)×10⁶ cells/mL,比生长速率分别为(0.027±0.012)h^-1、(0.028±0.013)h^-1、(0.027±0.013)h^-1(P>0.05);接种甲型流感病毒H1N1和H3N2,HA效价达到(6~7)log₂HA/25 μL、CCID₅₀为(4.35~4.68)lgCCID₅₀/mL;接种乙型流感病毒B/P和BX-35增殖效果更好,HA效价达到(9~10)log₂HA/25 μL,CCID₅₀为(6.38~7.31)lgCCID₅₀/mL。接种重组禽流感病毒H5亚型Re-5、Re-6、Re-10均能很好的增殖,HA效价达到(7~9)log₂HA/25 μL、CCID₅₀为(6.21~6.96)lgCCID₅₀/mL。结论 本研究获得一株具有自主知识产权的流感/禽流感病毒敏感的无血清悬浮培养型MDCK-XF02细胞株,实现了生物反应器高密度放大培养,为我国开展流感疫苗研究和生产提供细胞基质,也为其他动物细胞悬浮驯化提供参考。     

Three Japanese encephalitis cases in Okinawa, Japan, 1991

Since 1974, no Japanese encephalitis (JE) case had been reported on Okinawa island in either Okinawan people or US servicemen. In 1991, three US marines stationed on Okinawa island developed encephalitis symptoms. Neutralization (N) test and IgM-capture ELISA were carried out on the serial samples of serum and cerebrospinal fluid (CSF) taken from the patients. In each patient N test on both serum and CSF samples gave a significant rise in JE antibody titer in the comparison between the acute and convalescent phases, indicating that all the cases were infected with JE virus. The IgM-capture ELISA also showed a significant rise of antibody titer of the serum and CSF samples in the convalescent phase in patients 2 and 3, while in patient 1 a significant rise in IgM antibody was observed in the serum sample, but not in the CSF sample. None of the patients had been administered JE vaccine. This report underscores the importance of JE vaccination.     

Arbovirus infections in reptiles: studies on the presence of Japanese encephalitis virus antibody in the plasma of the turtle, Trionyx sinensis.

This study was undertaken to examine further the natural infection of poikilothermic animals e.g. turtles, to Japanese encephalitis (JE) virus. Plasma samples from 75 soft-shelled fresh water turtles (Trionyx sinensis Wiegman) from China were examined in virus neutralization (VN) and hemagglutination inhibition (HAI) tests for the presence of specific antibody. The total incidence of antibody detected by either test to a titer of 10 or greater was 89% while 77% and 60% were positive by VN and HAI tests, respectively. Forty-one per cent were jointly positive by both tests. Mean HAI and VN titers were similar and showed no obvious differences between spring/summer and autumn/winter seasons. The HAI reactivity was associated with a 7S component for both seasons. The significance of this inhibition in the serology of poikilothermic hosts and the possible behaviour of T. sinensis in the natural history of JE virus is briefly considered.     

澜沧江流域健康人群血清乙型脑炎病毒抗体调查

采用血凝抑制试验检测澜沧江上游的维西县、中游的凤庆县和景东县及下游的勐腊县健康人血清乙型脑炎病毒抗体阳性率分别为7.50%（18/240）、20.97%（65/310）、24.46%（57/233）和23.84%（77/323）。表明该地区健康人群中普遍存在流行性乙型脑炎病毒隐性感染。