医学微生物学课件：呼吸道病毒

1、1 呼吸道病毒 Respiratory Viruses2OverviewTo gain access to human body via respiratory tract and cause local (season cold) and /or systemic infection (SARS). More than 90% of acute respiratory infection are caused by the respiratory viruses .3Key points in this chapter1. Biological properties: morphology

2、and structure as well as cultivation properties of influenza V. 2. Variation of influenza V antigenic shift and antigenic drift3. Pathogenesis of other respiratory viruses (measle v, mumps v & rubella v)4Classification Orthomyxoviridae(正粘v) : influenza v. A, B and C2. Paramyxoviridae(副粘v) Measles v.

3、 and Mumps v. Para influenza ( 副流感v) v. RSV (respiratory syncytial v.)3. Others: Rhinovirus(鼻v)* Rubella v. Coronavirus(冠状v.) : SARS v. 5Human main respiratory viruses and caused diseases Viruses Diseases Influenza v. Influenza Measles v. Measles Mumps v. Mumps Rhino v. Season cold Rubella v. Rubell

4、a (Congential rubella syndrome) 6 Sec. 1 Influenza viruses (Orthomyxoviridae)7Biological propertiesof OrthomyxoviridaeMedium-sized, 80120nm enveloped viruses exhibiting helical symmetry. Particles are either round or filamentous, with surface projections that contain hemagglutinin or neuraminidase a

5、ctivity. The genome is linear, segmented, negative sense, single-stranded RNA.8OrthomyxoviridaeEnvelopedShape: either round or filamentousSurface projections (spikes): hemagglutinin(HA) or neuraminidase (NA)Core: helical symmetryGenome: linear, segmented, -ssRNA. 910Brief history- Influenza pandemic

6、sDeaths:11The big pandemic of 1918An Emergency Hospital for Influenza Patients 121918 FLU-An serious threat to human health Flu in recent years131997, H5N1 in Hong Kong 2009, H1N1 in Mexico2013, H7N9 in mainland ChinaSpread from animal to human流感大流行2009H1N1(pdm09)重排（禽类-猪-人类）1918H1N1(Spanish flu)1968

7、H3N2(Hong Kong flu)1957H2N2(Asian flu)直接适应：突变（禽类）重排（禽类-人类）重排（禽类-人类）1977H1N1(Russian flu)H1N1毒株再现Watanabe, Y., et al., Trends Microbiol，201215 1. Morphology and structure 2. Nomenclature and variation 3. Multiplication 4. Cultivation properties 5. Resistance 一. Biological properties16I. Biological ch

8、aracteristics# Filamentous: ( newly isolated viruses)Enveloped Round Influenza virus under EM18I. Morphology and structure linear, -ssRNA : 8 or 7 segments Core: nucleoproteins (NP) （nucleocapsid） RNP : ribonucleoprotein= RNA + NP helical symmetry 8 RNA segments for A & B; 7 RNA segments for C(2) Ma

9、trix proteins: M1 and M2 NP & M protein: type-specific. (3) Envelope: HA-binding NAreleaaseHA & NA protein: subtype-specific19Segments:0.9-2.4 kb20Structure of Influenza virus21Summary of structure & compositionType A, B, C : NP M protein Sub-types: HA(H1H15) NA (N1N9) proteinThree parts: core, matr

10、ix proteins and envelopeGenome: segmented -ssRNA22形态与结构核衣壳：螺旋对称型， 分节段的RNA是变异的基础 核糖核蛋白（RNP) NPMP：型特异性，划分型的依据。包膜-MP：坚韧， 保持病毒形状和完整性， 型特异性。刺突：HA+NA，亚型的主要依据。23 Receptor宿主细胞Structure of HA Trimer on virus particleRBS: Receptor Binding SiteReceptor: sialic acid (acetylneuraminc acid)HemagglutininHemaggluti

11、nin24SSSSSScell enzymesacid pH HA0 HA1 + HA2protease*Disulfide bond25Function of HA agglutinate RBC (erythrocytes)(named) the cleavage is necessary for virus infection HA1: binds to receptor on cell HA2: necessary for the viral envelope to fuse with the cell membrane Induce neutralizing Ab antigenic

12、 variation subtype the virus 26流感病毒吸附RBC27凝集RBC，HI现象HAHA1+HA2 病毒方具有感染性HA1-病毒吸附蛋白，受体（唾液酸）结合HA2-膜融合活性诱导中和抗体抗原改变划分甲型流感v.亚型的主要依据血凝素的功能 28Structure & function of NATetramer on virus particleFunctions at end of the viral life cyclehydrolyze cellular neuraminide (神经氨酸) damage viral receptors on cell membra

13、ne ficilitates viral buddingAntigenic variation subtype the virus Antibodies to NA: useful for diagnosis29 NA30A/equine/Saskatoon/1/90(H3N8)型别type 宿主名 host of origin分离地点location病毒株序号Isolate number分离年代 year亚型号Serotype of HA and NANomenclatureWorld Health Organization. A revision of the system of nome

14、nclature for influenza viruses.(1980) 31 Antigenic variation Minor antigenic changes (quantitative changes) resulting from mutation of the HA and/or NA genes are called antigenic drift.HA and NA accumulate point mutationsResult in changes of amino acid and antigenicityimmune response only protects p

15、artially.Limited epidemics.ANTIGENIC DRIFT (抗原漂移)32Three key points in the driftPoint mutations in the gene of HA or NAAntigenicity of the proteins have a little change -pre-existing immune responseMay cause a limited epidemics. 33ANTIGENIC SHIFT （抗原转变）Major antigenic changes in HA and/or NA (qualit

16、ative changes)“New” HA or NA proteins new subtypesPre-existing antibodies do not protectAn epidemic/pandemic may occur Antigenic variation34Three key points in the shiftMajor mutation in the genes (genetic reassortment)Major antigenic changes on HA or NA new subtypespre-existing antibodies do not pr

17、otect epidemic/pandemic35 抗原漂移 (antigenic drift)变异幅度小(基因点突变，HAaa变异率1%,量变)已有中和抗体不能完全保护中小流行 抗原转变 (antigenic shift)变异幅度大(基因重组，质变, HAaa变异率20-50%,)新的亚型抗体无保护作用大流行36Where do “new” HA and NA come from?Genetic reassortment: in doubly infected cells (the virus from human and avian).Based on segmented genome.

18、Type A is easy to change. Types B and C do not display shift because few related viruses exist in animal.37变异发生de可能机制1. 基因的重组（Recombination）2. 病毒的进化3. 动物来源的病毒跨种属传播38Life cycle of the virus39The steps for replication of influenza virusesAttachment and binding with receptorEndocytosis and uncoatingRNA

19、 entry into the nucleus and biosynthesis (* the most of RNA virus multiplicate at the cytoplasm but influ.v performs this process at the nucleus.)Assembly, maturation and budding Natural Cycle of the Influenza virusesLiu et al., 2005, Science 41 Where do “new” HA and NA come from?Pigs as Mixing Vess

20、els for the Creation of New Pandemic Influenza A Viruses42Life cycle of the virusDuring the replication, intermingling of genome segments from different parents in co-infected cells is responsible for the reassortment of the viruses. 43NEJM 360;25 , 200944Cultivation properties Cultivation in embryo

21、nated egg Give a higher positive rate for primary isolation. 2. Cell culture (MDCK cells) Animal inoculation (ferret)45embryonated egg-culture amniotic cavity is often used 46Virus Culture Cell(MDCK) cultureNormal Inoculated47Sensitive to heat, dry, UV, ether and lactic acid. (easy to be inactivated

22、) Resistance: weak 流 感 病 毒48二. Pathogenesis & Immunity49TransmissionDroplets By sneezing and coughingDirect contact 1-3 days incubation50SymptomsFeverHeadacheSore throat (咽喉痛)CoughRhinitis(鼻炎)Nasal discharge51Pathogenesis & ImmunityMultiplication in local siteViraemia is rarely observedSystemic symp

23、toms: Cytokines from host cellsMay have secondary bacterial infectionimmunity to the same subtype (anti-HA): long (no cross-reaction among subtypes)52NORMAL TRACHEAL MUCOSA3 DAYS POST-INFECTION7 DAYS POST-INFECTIONLycke and Norrby Textbook of Medical Virology 1983Normal cilia53三. Laboratory Diagnosi

24、sIsolation and identification of virusRapid diagnosis: PCRSerology: paired acute and convalescent sera (ELISA)* During epidemics or pandemics of influenza54Samples: Nasal washing Gargles Throat swabs2. Timing for sample collection: acute phase (within 3 days)3. Methods: Embryonated egg Cell culture5

25、5四. Prevention and TreatmentVaccine development Cut off transmissionDrugs and Chinese medicine56禽 流 感（avian influenza ）57 禽流感是禽流行性感冒的简称，是由甲型流感病毒引起的禽类传染性疾病，容易在鸟类（尤其是鸡）之间引起流行，过去在民间称作鸡瘟（Fowl plague)。 禽类感染后死亡率很高。Basic imformation58History of avian flu1878年由Perroncito首次报道于意大利。1901年证实其病原为“滤过性”病原体。1955年证实其

26、为甲型流感病毒的一员。 禽流感病毒一直在世界各地家禽中普遍存在，并造成不同程度的影响！59时间地点波及范围型别1983-1984美国1700多万只鸡H5N21978美国140多万只火鸡1997香港7000只鸡死亡H5N12002.3香港捕杀86万只鸡H5N12003.12 2004.1日本山口死亡6000只雏鸡3万只鸡感染H5N12004.1韩国9000只鸡死亡H5N12004.1越南河内100万家禽死亡捕杀200万只鸡H5N12004.1台湾捕杀3.5万只鸡H5N2禽流感流行情况60流行情况流感病毒感染具有物种特异性，仅在罕见情况下超越范围感染其它物种。1997年，香港发生H5N1型人禽流感

27、 （18人感染，6人死亡），在世界范围内引起了广泛关注。 首次发现直接由禽类传染人类的流感病毒61高致病性禽流感病毒（highly pathogenic avian influenza, HPAI）禽流感病毒高致病性H5亚毒株H7亚毒株高致病性禽流感因在禽类中传播快、病死率高，被世界动物卫生组织列为A类动物疫病，我国列为一类动物疫病。 2003年报告有1例兽医感染H7N7死亡。2013年中国有多例H7N9。H5N1型不断进化，其寄生的动物范围会不断扩大，可感染虎、家猫等哺乳动物，家鸭携带并排出病毒的比例增加，尤其是在猪体内更常被检出。低致病性非致病性高致病性禽流感病毒可以直接感染人类。1997

28、年， 香港地区，高致病性禽流感病毒H5N1型导致了18人感染，6人死亡，首次证实高致病性禽流感可以危及人的生命。62Where do “new” HA and NA come from?Direct spread of animal viruses to human （怎样认识？）. # In 1997, in Hong Kong, infection of human by avain influenza A virus occurred. # During the outbreak, 19 persons were infected and 6 persons died.63在浙江丛岺岛重

29、组Basic InformationStarting February, 2013Confirmed March, 2013 134 Cases/45 Deaths12 provinces/municipalitiesMost Recent Cases: June/JulySpatial distribution of 131 confirmed H7N9 casesNatural Cycle of the Influenza virusesLiu et al., 2005, Science Molecular basis of the “host jump”Science, 2013Scie

30、nce, 2013Journal of Virology, 2013Flu receptor binding2,62,3 & 2,62,62,62,62,32,32,32,3 HA 226LH1N1H5N1H7N9Binding model of HAs from human and avian influenza viruses70传染源 主要为患禽流感或携带病毒的鸡、鸭、鹅等禽类。目前尚无人与人之间传播的确切证据（可能性？）。71传播途径 经呼吸道传播，或密切接触感染的家禽分泌物和排泄物、受病毒污染的物品和水等。72易感及高危人群任何年龄均可被感染，但在H5N1感染病例中，13岁以下儿童所

31、占比例较高。从事家禽养殖业者及其同地居住的家属、在发病前1周内到过家禽饲养、销售及宰杀等场所者、接触禽流感病毒感染材料的实验室工作人员、与禽流感患者有密切接触的人员为高危人群。73Avian flu pathogenesis H5N1亚型病毒感染者早期急性起病。类似普通感冒，重症者进展迅速，可出现急性肺损伤、急性呼吸窘迫综合征（ARDS）、多脏器功能衰竭等多种并发症。并可继发细菌感染。H9N2和H7N7亚型症状轻微74禽流感防治管理传染源，加强禽类疾病的监测，受感染动物立即销毁切断传播途径，监测密切接触禽类人员75禽流感防治抗病毒药物： Rimantadine， Amantadine， Zan

32、amivir， Oseltamivir（达菲） 疫苗： 禽用 人用？76怎样认识1997 Hongkong avian flu 的发生？禽流感病毒受体：唾液酸-2, 3Gal 分布特点：下呼吸道 （呼吸细支气管和肺泡）人流感病毒受体：唾液酸-2, 6Gal 分布特点： 上呼吸道 （气管、支气管） 随着支气管分级渐低分布减少，肺泡最少. 77怎样认识1997 Hongkong avian flu 的发生？ 可能与个体间上呼吸道禽流感病毒受体唾液酸-2,3Gal受体表达差异有关. H5N1病毒进入下呼吸道.H6N1 human Infection in Taiwan 201379甲型H1N1流感（

33、Swine flu ） 80关于命名 疫情暴发早期世界各地称谓不一： 德国：“Schweinegrippe” 法国：“Ia Grippe A” 荷兰：“Mexican Flu” 墨西哥卫生部： “Ia epidemia” “北美流感” “加利弗尼亚流感” 猪流感病毒 猪源A型H1N1流感病毒（ swine-origin influenza A(H1N1) virus ）目前WHO称之为流感大流行H1N1 2009(Pandemic H1N1 2009)我国称之为甲型H1N1流感病毒（A/H1N1 influenza virus）8150个病例：37例平民和13例军人37例平民：6例发生在夏天

34、29例发生在9-3月 2例不详6例死亡：4例死于病毒性肺炎， 1例有细菌混合感染 1例有其它器官的损伤61%有猪的暴露史年龄中位数为22.5岁13例军人病例（1976）：没有暴露史1例死亡年龄中位数18岁这起疫情涉及230名士兵猪流感病毒A（H1N1）感染人病例82NEJM 360;25 , 200983致病性大多数病人表现为流感样症状可发生肺炎等并发症。少数病例病情进展迅速，出现呼吸衰竭、多脏器功能不全或衰竭。84流行病学传染源：主要为甲型H1N1流感病人；猪体内已发现甲型H1N1流感病毒，但动物是否为传染源？ 传播途径：主经呼吸道传播，亦可通过接触传播易感人群：普遍易感85实验室检查(1)

35、病毒核酸检测： RT-PCR real-time PCR (2)病毒分离： 呼吸道标本/ 肺组织 (3)血清学检查86防治抗病毒药物：初步药敏试验提示，此甲型H1N1流感病毒对奥司他韦(oseltamivir)和扎那米韦(zanamivir)敏感，对金刚烷胺和金刚乙胺耐药关于疫苗。 87Sec. 2 Paramyxovirus (副粘v) Similar to but larger (150300nm) than orthomyxoviruses. Particles are pleomorphic. The genome is linear, single-stranded, nonsegm

36、ented, negative-sense RNA.88 Difference with Orthomyxoviruses 1. Non-segmented genomeGenetic reassortment rareRate of antigenic change: low2. Spikes: F(fusion) + H/HN/G3. Replication & transcription of RNA: in the cytoplasm of host cell89Measles v. (Incidence of measles has been obviously reduced by

37、 using live virus vaccine, but this disease is still a leading cause of death of young children in some developing countries. )Mumps v.RSV:Parainfluenza v.Paramyxoviruses90一、 Measles v-morphology1. enveloped & round 120-250nm, MP2. spikes： HA HL(hemolysin)3. genome：-ssRNA nucleocapsid: helical symme

38、tryM proteinhelical nucleocapsid (RNA plusNP protein) 不分节段HN/H/G glycoprotein SPIKESpolymerase(2 proteins) lipid bilayer membrane F glycoprotein SPIKESParamyxoviruses150-300nm92Biologic properties Cultivation: grown in human kindey cells typical CPE (cytopathic effects): multinucleated giant cell wi

39、th inclusion body antigen: only one antigenic type Resistance：weak93MEASLES GIANT CELL PNEUMONIAMurray et al. Medical Microbiology 94Pathogenesis and immunity Sourse : acute patients/carriers Transmssion route: dropletpathogenesis：entrylocal lymph node 1st viremiaspread to the RES (reticulo-endothel

40、ial system) 2nd viremia disseminate systemic symptoms 95Pathogenesis and immunityPrognosis: Recovery completely Secondary bacterial pneumonia(15% serious) Otitis media Subacute sclerosing panencephalitis（SSPE): rare and late. (incidence: 1:300,000 to 1:1000,000)Lifelong immunity96Diagnosis, preventi

41、on and treatmentDiagnosis： on clinical ground Kopliks spots: pathognomonic (usually absent ) typical CPE: multinucleated giant cell with inclusion body Prevention：attenuated live measles virus vaccineTreatment: no specific treatment 97Measles - Kopliks spotsMurray et al. Medical Microbiology 98Measles - rashCDC - B.RiceMurray et al. Medical Microbiology 99MumpsNonsuppurative enlargement of parotid gland 100Mumps VBiological properties: similar to other paramyxoviruses such as measles v101Pathogenesis Transmission: by droplet Cause mu