1、Virology and Laboratory Diagnosis of Japanese Encephalitis Virus Infection乙脑病毒感染的病毒学实验室诊断,1,CHIK,SIN,DEN1-4,TBE (POW),SSH,EEE,WEE,VEE,MAY,DEN1-4,YF,BF,RR,SIN,MVE,DEN1-4,CHIK,SIN,YF,DEN1-4,TAH,SIN,TBE (POW),WN,(TAH,INK),LAC,SLE,POW,EEE,WEE,CTF,DEN1-4,EVE,EEE,VEE,WEE,MAY,DEN1-4,JE,(VEE),SLE,LAC,(VEE),
2、DEN1-4,Most common arboviruses World-wide 全球常见虫媒病毒分布,WN,WN,WN,WN,WN,SLE,SLE,2,uu,Flaviviruses in China 中国黄病毒 Japanese encephalitis virus (JEV) 乙型脑炎病毒 Dengue viruses (DEN) 登革热病毒 Tick Borne encephalitis virus (TBE) (rare, near Russian border) 蜱传脑炎病毒（TBE）（罕见，于与俄罗斯边境处） There are 10 members of the JEV se
3、rocomplex, the main human pathogens of which are JEV, West Nile (WNV), Murray Valley encephalitis virus (MVE) and St. Louis encephalitis virus (SLEV). Of these, only JEV is found in China 乙脑病毒有10个成员，对人类致病的主要有乙脑病毒、西尼罗河病毒（WNV）、默里谷脑炎病毒（MVE），和圣路易斯脑炎病毒（SLEV）。其中，中国只发现乙型脑炎病毒,3,Characteristics of JEV infect
4、ion and implications for laboratory diagnosis 乙脑病毒感染的特点和实验室诊断结果的解释,Diagnostic Detection Assays 诊断检测实验 Virus isolation detects virus 病毒分离检测病毒 Nucleotide amplification detection assays detect viral RNA 核苷酸扩增实验检测病毒RNA Serological assays detect antibodies 血清学实验检测抗体Enzyme-Linked ImmunoSorbent Assay antib
5、ody capture 酶联免疫吸附实验抗体捕捉IgM ELISA IgG ELISA Plaque reduction neutralization test (PRNT) 中和实验 Hemagglutination inhibition (HI) assay 血凝抑制实验 Complement fixation (CF) 补体结合实验,Theoretical Depiction of Human JE Viremia and Immune Response 人乙脑病毒血症和免疫反应理论曲线,DAYS POST ONSET 发病之后天数,1 2 3 4 5 6 7 8 9 10,-14 to
6、 -2 0,IgM,IgG,ELISA P/N (dependson test used) 根据使用测试方法,#pfu/ml,WN Viremia 病毒血症,250,CNS illness 中枢神经系统疾病 RNA difficult to detect in CSF at onset of encephalitis 在脑炎发病时难以在脑脊液中检测到RNA,2,20,Serologic Assays 血清学检测方法,Virus Assays 病毒检测方法,15,IgM detectable in serum & csf by onset (99%) 出现症状后，血清和脑脊液中IgM的检出率99
7、 6 exceptions serum of 800 cases, 1999-20021999-2002年，800个病例中6例血清学检测结果阴性 10 exceptions csf of 800 cases, 1999-200219992002年，800个病例中10例CSF结果阴性 IgG positive by day 7 post-onset发病7天后IgG检测结果阳性IgM persistence 1 YearIgM持续1年以上Secondary flavivirus Infections or previous flavivirus vaccinations are problemat
8、ic再次感染黄病毒或既往接种过黄病毒疫苗会造成其它问题,JE Human Serological Profile 乙脑血清学特点,Serological assays (IgM ELISA) more sensitive than virus isolation 血清学检测 （IgM ELISA）较病毒分离敏感,90% patients have IgM in CSF by 5 days after onset 发病5天后脑脊液中IgM的检出率90% Most patients have IgG by 10 days after onset大多数病人在发病10天后出现IgG Viral iso
9、lation and PCR on CSF are very insensitive assays for diagnosis of JE 通过脑脊液的病毒分离和PCR检测来诊断乙脑是不敏感的 Low levels of transient viremia 低水平、短暂的病毒血症 High levels of neutralizing antibodies 高水平的中和抗体 Thailand study 泰国的研究 0 of 34 non-fatal cases had JEV isolated from plasma or CSF 34例非死亡病例中没有病例在体液或脑脊液中分离出乙脑病毒 5
10、 (33%) of 15 fatal had JEV isolated from CSF 15例死亡病例中5例(33%)病例的CSF分离出乙脑病毒 7 (46%) of 15 fatal cases had JEV isolated from brain tissue 15例死亡病例中7例（46）病例的脑组织中分离出病毒标本,Specimen type 标本类型 Cerebral spinal fluid 脑脊液Serum血清,Clinical spectrum of JE disease 乙脑的临床特点,Death 死亡,Encephalitis 脑炎,Mild nonspecific il
11、lness 轻度非特异性疾病,Asymptomatic infection 没有症状的感染,1 hospitalized case to 300 people infected with JE, but with mild illness or no symptoms 300个感染乙脑的病例有1例住院，大多数为轻度非特异性和没有症状,-IgM in CSF and serum 在脑脊液和血清中可以检测到IgM-IgM in serum only 只在血清中检测到IgM- IgM in serum only 只在血清中检测到IgM,Long Term WN virus IgM activity
12、in human serum(including in JE vaccinees) 西尼罗病毒血清学IgM的持久性,NYCDOH-CDC collaborative study,N=28,N = 28,IgM +,CSF preferable diagnostic specimen due to: 脑脊液作为更理想的诊断标本是由于：JE IgM in serum from high proportion of inapparent and mild infections, which may not be cause of encephalitis 很大比例的没有症状或轻度症状的病例会检出乙脑
13、IgM， 但并没有出现脑炎的症状 Cross reactions in serological assays with other flaviviruses in serum which may not be cause of encephalitis 会与其它黄病毒引起交叉反应，而这些病毒又不引起脑炎IgM in serum from JE vaccine 血清中IgM可能是由接种乙脑疫苗引起 90% patients have IgM in CSF by 5 days after onset 发病5天后脑脊液中IgM的检出率90%,The Cross-Reactivity Problem
14、交叉反应问题,Human infections by flaviviruses elicit immune responses producing 人感染黄病毒产生免疫反应, virus species-specific antibodies病毒特异性抗体 flavivirus cross-reactive antibodies黄病毒交叉反应抗体,5,Flavivirus Virion Structure 黄病毒病毒体的结构,Virus particle 40-60 nm Single-stranded positive-sense RNA virus 病毒颗粒4060nm 单链正股RNA病毒
15、,4,E Glycoprotein Homodimer E糖蛋白同型二聚体 Top View 顶视图,Yellow residues are domain II of E glycoprotein; purple are conserved regions among the flaviviruses 黄色残基是E糖蛋白的结构域II；紫色收黄病毒的保守区域,6,Serologic cross-reactivity among the flaviviruses 黄病毒间血清交叉反应性 Both IgM and IgG antibodies exhibit significant cross-re
16、activity among the flaviviruses. For instance, anti-DEN IgM will react with JE virus antigen, even though DEN is not in the JE serocomplex. 黄病毒之间IgM和IgG抗体有交叉反应。例如抗-登革热病毒IgM与乙脑病毒抗原反应，尽管登革热病毒不是乙脑病毒血清群的。 IgM tends to be less cross-reactive than IgG This can be useful in diagnosis of unexpected agents (
17、eg when WNV entered the US it was first thought to be the endemic SLE) However, cross-reactivity can produce false-positives IgM通常比IgG交叉反应少些。 可以用于非预期病原的诊断（如，当西尼罗河病毒进入美国时，最先认为是地方圣路易斯病毒脑炎）。 然而，交叉反应可以产生假阳性。,7,Cross-reactivity continued交叉反应续 IgM to DEN and TBE (flaviviruses in China) will cross-react in
18、 a JEV IgM test. IgM对登革热病毒和蜱传脑炎病毒（中国的黄病毒）反应的，在乙脑病毒IgM检测中可以发生交叉反应。 TBE is rare in China, therefore false-positives due to TBE are unlikely. 蜱传脑炎在中国罕见，因此因蜱传脑炎病毒造成的假阳性可能性不大。 DEN does not cause encephalitis but does cause occasional encephalopathy (disturbances in mental status), and therefore a specime
19、n from a DEN patient could be submitted, and it is likely to test positive. 登革热病毒不引起脑炎，但是偶尔可以造成脑病（精神障碍），因此可能提供了登革热病人的病人标本，很可能测试结果阳性。,8,SHIPMENTS 运输,SERUM SPECIMENS: Acute and/or convalescent 血清标本：急性期和/或恢复期Sent on wet, or dry ice or ambient temperature在冰或干冰或常规环境运送 CEREBROSPINAL FLUID 脑脊液Sent on wet o
20、r dry ice or ambient temperature在冰或干冰或常规环境运送 Store specimens at 4 C if intended for immediate use; otherwise freeze at -20 C or below. 如果即可检测，在4 C条件下保存标本；否则在-20 C或更低温度冻存,9,Information needed for serological testing and interpretation 血清学检测和结果解说需要的资料,Dates of sample collection 样品采集日期 Evidence of clin
21、ical encephalitis 临床脑炎证据 Vaccination history 接种史 Date of onset of symptoms 症状出现日期,10,Safety安全性,Always wear latex or nitrile gloves, safety glasses and a lab coat when handling specimens and during all stages of the test. 处理样本和测试各个环节都要带乳胶和硅胶手套，安全眼镜和白大衣 Use of a Biosafety cabinet (BSC) Class 2 Type A2
22、 is recommended because acute specimens may contain infectious JE virus or other infectious agents. 由于急性期标本可能含传染性乙脑病毒或其它致病因子，建议用II类A2型生物安全柜 Class 2 is a ventilated cabinet that provides personnel, product and environmental protection. It has a limited fixed inward air flow access opening, a ventilat
23、ed downward HEPA filtered exhaust. II级生物安全柜有通风，可以提供人员、产品和环境保护。所带的内部空气接外界流量有限，使用了向下HEPA过滤通气装置。 Type 2 recirculates 70% of the air II型70的空气循环。 Do not use laminar flow hoods intended for cell culture! 细胞培养不要用层流罩。 If a BSC is unavailable, care should be taken especially when using the undiluted specimen
24、s, and during wash steps. Bleach should be added to the discard pan to inactivate viruses. 如果没有生物安全柜，操作要特别认真，特别是用未稀释的样品以及各洗涤步骤。在废物盘中加漂白粉灭活病毒。,11,Several kits are available to test for anti-JE IgM testing; the B B&C Beixi试剂盒将用于本项目,B&C (Beixi) EEB-IgM (48T) Cat No. IBCB350P,12,The kits use wells preco
25、ated with anti-human IgM antibody . 1. 本试剂盒利用抗人IgM抗体包被板。,The JEV IgM ELISA B&C Beixi kit works in the following way: B&C Beixi乙脑病毒IgMELISA检测试剂盒检测原理如下：,2. Diluted patient serum or CSF is added. If any IgM antibody is present it will be captured by the anti-IgM antibody. 2. 加稀释病人的血清或脑脊液。如果存在IgM抗体，就能被抗
26、IgM抗体捕获。,3. A mixture of JEV antigen ( ) and JEV-specific monoclonal antibody conjugated to horseradish peroxidase ( ) is added to the well. If the IgM captured in the last step has been made to JEV, the antigen-conjugate pair will attach to the IgM antibody. 3. 乙脑病毒抗原( )和标记辣根过氧化物酶乙脑病毒特异单克隆抗体( )的混合物
27、加入孔中。如果最后一步捕获的IgM是乙脑病毒的，抗原耦合将与IgM抗体结合。,4. A substrate is added which causes color to develop. The reaction is stopped and read at 450 nm 4. 加底物现色。终止反应在450nm波长条件下读数。,13,Test interpretation 检测结果解释,14,Use the blank well for blank. Read OD value设空白对照，读OD值P/N = positive/negativeP/N=阳性/阴性P/N 2.1 positive
28、P/N 2.1阳性OD sample/OD of negative control 样品的OD值/阴性对照的OD值 If the negative OD 0.05, use its OD value 如果阴性对照OD0.05，用该OD值,Results interpretation 结果的解释,Serum Used at 1:100 血清，稀释：1：100,Negative result:阴性结果 Interpreted as either NEG or IgM not yet present 解释为阴性和IgM未出现,Positive result: 阳性结果 IgM to JE or to
29、 vaccine if given 1 month prior to symptoms 乙脑IgM或出现症状前一个月内注射过疫苗,Action: Report as negative, or test convalescent specimen if available 措施：报告阴性结果，如果可能测定恢复期血清,Action: Report as positive; clinical correlation/vaccine history required 措施：报告阳性结果，需要临床相关/疫苗接种史,CSF (preferred specimen) Used at 1:10 脑脊液（最佳）
30、稀释： 1：10,Acute specimens 急性期标本 9 days after onset of symptoms 症状出现后 9天,Positive result: IgM to JE 阳性结果：乙脑病毒IgM阳性,16,Negative result:阴性结果 Interpreted as either NEG or IgM not yet present 解释为阴性和IgM未出现,Action: Report as negative, or test convalescent specimen if available 措施：报告阴性结果，如果可能测定恢复期血清,Action:
31、Report as positive; clinical correlation required 措施：报告阳性结果，需要临床相关资料,Negative result: Interpreted as NEG 阴性结果：解释为阴性,Action: Report as negative 措施：报告阴性结果,Convalescent specimens恢复期样本 9 days after onset of symptoms症状出现后 9天后,Positive result: IgM to JE 阳性结果：乙脑病毒IgM阳性,Note: The majority of specimens will
32、probably be single, acute CSF注：多数样本是单份急性期脑脊液,17,Serum Used at 1:100 血清，稀释：1：100,CSF (preferred specimen) Used at 1:10 脑脊液（最佳）稀释： 1：10,Positive result: 阳性结果 IgM to JE or to vaccine if given 1 month prior to symptoms 乙脑IgM和出现症状前一个月内可能注射过疫苗,Action: Report as positive; clinical correlation/vaccine histo
33、ry required 措施：报告阳性结果，需要临床相关/疫苗接种史,Action: Report as negative 措施：报告阴性结果,Negative result: Interpreted as NEG 阴性结果：解释为阴性,Action: Report as positive; clinical correlation required 措施：报告阳性结果，需要临床相关资料,Performance of test kit 试剂盒操作,18,Results of a limited analysis of B&C Beixi kit using serum samples: 用血清
34、样品测试B&Cbeixi试剂盒的初步分析结果,19,A more extensive assessment of the B&C (Beixi) kit was performed by a student of Dr. Liang. 学生梁博士对B&C (Beixi) 试剂盒进行了进一步评价 The following slide reports his data 以下的幻灯片报告他的数据,23,Analysis of B&C Beixi kit compared to IFA as a gold standard using 121 samples B&C Beixi试剂盒与间接荧光法作为
35、 金标准测定121份样品的比较分析,Agreement with IFA 与IFA一致的比例 False positive rate 假阳性率 False negative rate 假阴性率 Sensitivity 灵敏度 Specificity 特异度 Reproducibility 重复性 (297 samples) （297份样品）,B&C Beixi IgM capture 捕获 93.8% 13.33% 4.04% 95.96% 86.67% 97.31%,24,Test limitations 测试方法的局限性,21,Recommendations 建议If the B dilu
36、te as appropriate before use and use these negatives in your calculations for test samples (not for positive control). The kit negative isnt necessarily representative of your population, and it is unclear if it is serum or CSF. 除了说明书上的对照孔外，应对另外加一个对照孔。准备已知当地病人的阴性血清和阴性脑脊液库，分装并冻存；用前适当稀释，用这些阴性样品做计算测试样品（不用于阳性对照）。试剂盒的阴性对照不完全代表当地人群，不知是血清还是脑脊液。 If the kit positive control does not giv e a positive:negative (P/N) OD ratio of 5, consider rerunning the test. 如果试剂盒的阳性对照没有产生阳性/阴性（P/N) 光密度比值5， 考虑重新测试。,22,Thank you! 谢谢！,
