1、 中國人禽流感（H5N1）病例的 實驗室診斷及結(jié)果分析 2007年流感培訓 中國疾病預(yù)防控制中心 病毒病預(yù)防控制所 國家流感中心Country20032004200520062007TotalcasesdeathscasesdeathscasesdeathscasesdeathscasesdeathscasesdeathsAzerbaijan000000850085Cambodia000044221177China110085138322516Djibouti000000100010Egypt00000018102053815Indonesia000020135545282410382Iraq

2、000000320032Lao000000002222Nigeria000000001111Thailand0017125233002517Turkey00000012400124Total4446329843115795735320193全球人禽流感（H5N1）病例數(shù)Total number of cases includes number of deaths.WHO reports only laboratory-confirmed cases.16 August 2007呼吸道標本的采集血清標本的采集組織標本的采集消化道標本的采集第一部分 檢測標本的采集人禽流感病例呼吸道標本的采集盡量采

3、集深部呼吸道標本肺穿刺液深部吸取液血清標本的采集采集急性期和恢復(fù)期血清；急性期血清應(yīng)該在病人發(fā)病后立即采集，一般不要超過7天，如果沒有采集到7天內(nèi)血清，在能夠采集的時候立即采集；恢復(fù)期血清在病人發(fā)病后2-3周采集，如果病人死亡，在死亡前應(yīng)采集一份血清；病人恢復(fù)后應(yīng)定期采集血清；采集血清時不能少于5ml；最好采集全血和血清標本。消化道標本的采集糞便標本特別是有腹瀉癥狀的糞便標本PBS 或者細胞培養(yǎng)液 加入以下抗菌素和0.5%BSA -氨芐青霉素 (2 x 106 IU/litre)- 鏈霉素 (200 mg/litre) - 多粘菌素 B (2 x 106 IU/litre) - 慶大霉素 (2

4、50 mg/litre) - 制霉菌素 (0.5 x 106 IU/litre) - 鹽酸氧氟沙星 (60 mg/litre), and - sulfamethoxazole (0.2 g/litre) 標本的保存和運輸用于病毒分離的標本應(yīng)保存于4oC,并且立即送往實驗室進行接種，如果在48小時內(nèi)不能進行接種，應(yīng)該保存于-70oC；標本應(yīng)避免反復(fù)的凍融（降低分離率）；血清標本可以在4oC保存7天，否則應(yīng)保存于-20oC；標本的保存和運輸應(yīng)遵守國家生物安全的相關(guān)規(guī)定。任何成功的流感大流行的準備都依賴于對新出現(xiàn)毒株的正確的診斷。對于可能是A型流感病毒的每個樣品最初的實驗室檢測應(yīng)該快速并且能夠排除其

5、它常見的呼吸道病毒感染。最理想檢測方法是24小時內(nèi)能出結(jié)果。第二部分：H5N1檢測方法的建立符合以下標準之一的病例可以診斷為疑似或可能的H5N1病例H5N1 病毒分離陽性。 兩種不同的引物針對不同的片段檢測H5病毒的PCR結(jié)果為 陽性。微量中和實驗恢復(fù)血清(發(fā)病后2-4周)比急性期血清(發(fā)病后7日內(nèi)) H亞型抗體滴度升高4倍或以上， 且恢復(fù)期的血清的微量中和抗體滴度在1:80以上。單份恢復(fù)期血清(癥狀出現(xiàn)后的14天或以后)的微量抗體滴度在1:80或 以上,這以陽性結(jié)果采用不同的血清學方法可以驗證,如馬血球?qū)嶒災(zāi)种茖嶒灴贵w滴度在1:160 以上或 H5特異的 westerblot 檢測結(jié)果陽

6、性。確認人禽流感病例(notify WHO) 核酸檢測抗體檢測病毒分離抗原檢測HIMNSRH細胞分離雞胚分離PCRMDDNASBAIFAELISA膠體金診斷快速；特異度高；靈敏度低；診斷快速；特異度高；靈敏度高；得不到病毒；特異度高；無法進行早期診斷；金標準；病毒可用于其它研究；需要較嚴格的實驗條件；H5N1病毒的檢測方法 2005年以來中國人禽流感病例的實驗室診斷結(jié)果（續(xù)） Methods Cases RT-PCRReal-time PCRMDDHIMNELISA Virus IsolationSichuan case1+NDNDND+case2+NDNDND+case3+NDNDND+Sh

7、anghaicase1+NDNDND+Guangdongcase1+NDNDND+case2+NDNDND+Hubeicase1+NDNDND+Zhejiangcase1+NDNDND+Liaoning case1-+-Xinjiangcase1-NDNDND+RT-PCR技術(shù)的建立Real-time PCR 靈敏度檢測臨床標本的Real-time PCR檢測Luminex xMAP technologyMDD 對呼吸道病毒的診斷MDD對流感病毒亞型的鑒別1號為氣管吸取物標本一臨床病例的MDD檢測結(jié)果NASBA H5N1 標本的檢測結(jié)果VirusesH5 NASBA results N1NAS

8、BA resultsH1N1NegativeNegativeH3N2NegativeNegativeFlu BNegativeNegativeANHUI/1/2005/H5N1PositivePositiveGUANGDONG/1/2005/H5N1PositivePositiveXINJIANG/1/2006/H5N1PositivePositivesamplesH5 NASBA results N1 NASBA resultsH5 Real-time RT-PCR resultsH5N1(100TCID50)PositivePositivePositiveH5N1(10TCID50)Pos

9、itivePositivePositiveH5N1(1TCID50)PositivePositivePositiveH5N1(10-1TCID50)PositivePositivePositiveH5N1(10-2TCID50)NegativeNegativeNegativeH5N1(10-3TCID50)NegativeNegativeNegativeH5N1(10HA)PositivePositivePositiveH5N1(1HA)PositivePositivePositiveH5N1(10-1HA)PositivePositivePositiveH5N1(10-2HA)Negativ

10、ePositiveNegativeH5N1(10-3HA)NegativeNegativeNegative NASBA 與Real-time RT-PCR敏感性的比較血清學檢測方法 HI( HRBC)ELISASRHMNHI assayRBCChickenTurkeyGuinea pigHuman (o)Horse Conc0.5%0.5%0.75%0.75%1%Plate shapeVVUUUIncubation temperature RTRTRTRT4oCIncubation time30 min30 min60min60 min60 min不同種屬 紅細胞HI實驗的比較RBCSerum

11、 (USCDC)Serum (CNIC)ChickenTurkeyHorse16016016032051205120H5 HI titer using different red blood cellHRCCRCH5（ positive ，HS)16020H5（ positive ，USCDC)1280640H5（negative，Human)1010H5（ negative ，Human)1010H5（ negative ，Chicken)1010H5（ negative ，Chicken)10108day101017day 32016022day320160Antigen: A/chick

12、en/hunanxiangtan-he/21/2005(H5N1)(provided by HARBIN Veterinary Research Institute )Serum:HS: Chicken serum from HARBIN Veterinary Research Institute USCDC: goat serum from USCDCHI(HRBC) assay使用U型板4 代替室溫孵育。每次病毒的滴度要回滴馬血球要新鮮采集，一周內(nèi)使用。SRH原理示意圖SRH 實驗的優(yōu)化最適的抗原濃度是常規(guī)血凝板滴定 的1000 HAI。1% agarose。雞血球更適合于SRH實驗中的溶

13、血圈的產(chǎn)生。SRH技術(shù)的特點簡便、可靠、重復(fù)性好、特異性強、敏感性高，不受血清中非特異性抑制素影響。血清用量少, 并不必進行稀釋。可對大量血清標本進行測定。一般實驗室均可開展，特別適用于我國基層地區(qū)。單擴溶血的特異性對H5N1患者血清的測定微量中和實驗 (MN）的特點微量中和實驗 是診斷人感染禽流感的最好的血清雪方法。 敏感性高。特異性強( strain selection) 。每次實驗要設(shè)病毒和細胞對照，病毒要回滴A/CK/LNHS-JHL/23/05A/Anhui/1/2005A/CK/Hunanxiangtan-he/21/05Case 1Acute serum202020Case 1C

14、onvalescence serum2080160Case 2Acute serum202020Case 2Convalescence serum1604040Case 3Acute serum202020Case 3Convalescence serum208080Positive control1280640640MN assay of confirmed human cases 第三部分: 檢測結(jié)果和分析antigenserumA/CK/HN/21/2005(H5N1)A/CK/LN/23/2005(H5N1)A/AH/1/2005(H5N1)A/AH/2/2005(H5N1)A/FJ/

15、1/2005(H5N1)A/GX/1/2005(H5N1)A/JX/1/2005(H5N1)NIBRGA/CK/HN/21/2005(H5N1)121.412112.8A/CK/LN/23/2005(H5N1)12.85.785.72.82A/AH/1/2005(H5N1)11.41112.8A/AH/2/2005(H5N1)12.81.414A/FJ/1/2005(H5N1)111.42A/GX/1/2005(H5N1)115.7A/JX/1/2005(H5N1)18NIBRG1The antigenic ratio of Human H5N1 isolates by HI assayAnt

16、igen Serum A/CK/HN/21/2005(H5N1)A/CK/LN/23/2005(H5N1)A/AH/1/2005(H5N1)A/AH/2/2005(H5N1)A/FJ/1/2005(H5N1)A/GX/1/2005(H5N1)A/JX/1/2005(H5N1)NIBRGA/CK/HN/21/2005(H5N1)121.41.41222A/CK/LN/23/2005(H5N1)12.8222.848A/AH/1/2005(H5N1)111.411.42.8A/AH/2/2005(H5N1)11.412.85.7A/FJ/1/2005(H5N1)1125.7A/GX/1/2005(

17、H5N1)115.7A/JX/1/2005(H5N1)14NIBRG1The antigenic ratio of Human H5N1 isolates by MN assayReference ferret serumA/Vietnam/1203/2004A/Indonesia/5/2005A/Turkey/15/2005A/Anhui/1/2005A/Anhui/2/2005A/Guangxi/1/2005A/Fujian/1/2005A/Sichuan/1/2006A/Jiangxi/1/2005VN/1203IND/5TK/15AH/1AH/232020104040206404016

18、080803201280802040320101280640201605320320208056403201080516016040160101280640806404025601280Antigenic analysis of human H5N1isolates by HI assay Antigenic analysis of human H5N1isolates by HI assay using chicken serumAntigen Reference chicken serumAH/1AH/2XJ/1VN/1194A/Anhui/1/20053201608080A/Anhui/

19、2/2005 640 64016080A/Xinjiang/1/2006401016020A/Vietnam/194/2004-PR8RG404020320South China North China VietNam Antigenic assay of human H5N1 isolates By MN assay using confirmed human case serumAntigen Confirmed casescase1*case2#A/Anhui/1/200516040A/Xinjiang/1/200640320case1 is a survived confirmed h

20、uman H5N1 case from Anhui province(southern china), the serum was collected 4 weeks after the illness onset.#Case 2 is a survived confirmed human H5N1 case from Liaoning province(northern china), the serum was collected 4 weeks after the illness onset.Human H5N1 confirmed cases in mainland of china

21、since Nov,2005death(15)survival(9)China CDCClade 1Clade2.1Clade2.3 Clade 2.2HA Phylogenetic tree generated by the neighbor-joining method using the Mega3.1 program and the boottrap value 1000, the result showing the relationships of HA genes of representative influenza A H5N1 virus from different re

22、gions. The red dots denote viruses isolated from humans in China; green dots denote viruses isolated from Viet Nam and were recommended as vaccine strains by the WHO. NAPB2PB1NPNSPAMA/JX/1/2005A/Ck/GXNN/6/2004Clade IA/VN/1194/2004Clade 2.1A/BHG/5/2005A/XJ/1/2006Clade II 2.2A/IND/5/2005Clade II 2.3A/

23、AH/1/2005A/AH/2/2005A/SC/1/2006A/SC/2/2006A/SC/3/2006A/HB/1/2006A/ZJ/1/2006Virus isolatesHA(226-228)a 2-3specific A/AH/1/05QSGA/AH/2/05RSG?A/GX/1/05QSGA/JX/1/05QSGA/FJ/1/05QSGA/SC/1/06QSGA/SC/2/06QSGA/HN/1/06QSGA/AH/1/06QSGA/ZJ/1/06QSGA/GD/1/06QSGA/SH/1/06QSGA/HB/1/06QSGA/SC/3/06QSGA/GD/2/06QSGA/XJ/

24、1/06QSGA/FJ/1/2007QSGA/AH/1/2007QSGReceptor Specificity (HA 226-228)Virus isolatesM2(25-42)Sensitive A/AH/1/05PLVVAASIIGILHLILWILA/AH/2/05PLVVAASIIGILHLILWILA/GX/1/05PLVVAASIIGILHLILWILA/JX/1/05PLVVAASIIGILHLILWILA/FJ/1/05PLVVAASIIGILHLILWILA/SC/1/06PLVVAASIIGILHLILWILA/SC/2/06PLVVAASIIGILHLILWILA/H

25、N/1/06PLVVAASIIGILHLILWILA/AH/1/06PLVVAASIIGILHLILWILA/ZJ/1/06PLVVAASIIGILHLILWILA/GD/1/06PLVVAASIIGILHLILWILA/SH/1/06PLVVAASIIGILHLILWILA/HB/1/06PLVVAASIIGILHLILWILA/SC/3/06PLVVAASIIGILHLILWILA/GD/2/06PLVVAASIIGILHLILWILA/XJ/1/06PLVVAASIIGILHLILWILA/FJ/1/2007PLVVAANIIGILHLILWILXA/AH/1/2007PLVVAASII

26、GILHLILWILDrug surveillance (M2 inhibitor drugs)Drug surveillance ( NA inhibitor drugs)Virus isolatesNA(274)Sensitive(H)Resistant(Y)A/AH/1/05LNAPNYHYEEA/AH/2/05LNAPNYHYEEA/GX/1/05LNAPNYHYEEA/JX/1/05LDAPNYHYEEA/FJ/1/05LDAPNYHYEEA/SC/1/2006LNAPNYHYEEA/SC/2/2006LNAPNYHYEEA/HN/1/2006LNAPNYHYEEA/AH/3/06L

27、DAPNYHYEEA/ZJ/1/06LNAPNYHYEEA/GD/1/06LDAPNYHYEEA/SH/1/06LDAPNYHYEEA/HB/1/06LNAPNYHYEEA/SC/3/2006LNAPNYHYEEA/GD/2/06LNAPNYHYEEA/XJ/1/06LDAPNYHYEEA/FJ/1/07LNAPNYHYEEA/AH/1/07LDAPNYHYEEVirus isolatesHA connecting peptideHPAIVA/AH/1/05LRERRRKRGA/AH/2/05LRERRRKRGA/GX/1/05LRERRRKRGA/JX/1/05LRERRRKRGA/FJ/1

28、/05LRERRRKRGA/SC/1/2006LRERRRKRGA/SC/2/2006LRERRRKRGA/HN/1/2006LRERRRKRGA/AH/3/06LRERRRKRGA/ZJ/1/06LRERRRKRGA/GD/1/06LRERRRKRGA/SH/1/06LRERRRKRGA/SC/3/06LRERRRKRGA/HB/1/06LRERRRKRGA/GD /2/06LRERRRKRGA/FJ/1/07LRERRRKRGA/AH/1/07LRERRRKRGA/XJ/1/06QGERRRKKRGHA connecting peptideWHO recommended candidate

29、 H5N1 vaccine viruses for potential use as pre-pandemic vaccines A/Hongkong/213/03 A/Vietnam/1194/04 A/Vietnam/1203/04 A/Indonesia/5/2005 A/BHG/QH/1A/2005 A/WPS/Mongolia/244/2005 A/Turkey/Turkey/1/2005A/AH/1/2005 printable version Availability of new recombinant H5N1 vaccine virus21 December 2006The

30、 WHO Global Influenza Programme is continuing to monitor the antigenic and genetic evolution of circulating H5N1 viruses and their human isolates. In August 2006, WHO reported1 that analysis of newly isolated viruses collected from both animals and humans indicated that the H5 haemagglutinin (HA) ge

31、nes had become genetically distinguishable from the H5N1 viruses that had previously been selected for vaccine development. Furthermore, there was also evidence of antigenic variation among the recent viruses. Since then, WHO Collaborating Centres (WHO CCs) and H5 Reference Laboratories have been de

32、veloping several new recombinant H5N1 vaccine viruses that are representative of this newly discovered genetic sub-group of clade 2 viruses.The WHO CC in the Centers for Disease Control and Prevention (CDC), Atlanta USA and the China Centers for Disease Control, Beijing China have together developed

33、 a new recombinant H5N1 virus from A/Anhui/1/2005 selected from clade 2, sub-clade 3. The recombinant vaccine virus is available for distribution, under a Material Transfer Agreement (MTA). The sequences of haemagglutinin (HA) and neuraminidase (NA) of the new H5N1 recombinant vaccine virus can be f

34、ound on the public web site of Los Alamos National Laboratory database together with all other WHO selected and developed influenza vaccine viruses for both seasonal and H5N1 influenza.Institutions, companies and others interested in pandemic vaccine development, who wish to receive this recombinant

35、 vaccine virus should contact either the WHO Global Influenza Programme at or WHO CC CDC at the address below:WHO Collaborating Center for Surveillance, Epidemiology and Control of Influenza, Centers for Disease Control and Prevention, 1600 Clifton Road, Mail stop G16, Atlanta, GA 30333, USA (fax: +1 404 639 23 34; e-mail: ).Review of the needs and the actual development of H5N1 vaccine viruse