醫(yī)學(xué)微生物學(xué)課件：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(冠狀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（禽類-豬-人類）1918H1N1(Spanish flu)1968

7、H3N2(Hong Kong flu)1957H2N2(Asian flu)直接適應(yīng)：突變（禽類）重排（禽類-人類）重排（禽類-人類）1977H1N1(Russian flu)H1N1毒株再現(xiàn)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形態(tài)與結(jié)構(gòu)核衣殼：螺旋對(duì)稱型， 分節(jié)段的RNA是變異的基礎(chǔ) 核糖核蛋白（RNP) NPMP：型特異性，劃分型的依據(jù)。包膜-MP：堅(jiān)韌， 保持病毒形狀和完整性， 型特異性。刺突：HA+NA，亞型的主要依據(jù)。23 Receptor宿主細(xì)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現(xiàn)象HAHA1+HA2 病毒方具有感染性HA1-病毒吸附蛋白，受體（唾液酸）結(jié)合HA2-膜融合活性誘導(dǎo)中和抗體抗原改變劃分甲型流感v.亞型的主要依據(jù)血凝素的功能 28Structure & function of NATetramer on virus particleFunctions at end of the viral life cyclehydrolyze cellular neuraminide (神經(jīng)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)型別type 宿主名 host of origin分離地點(diǎn)location病毒株序號(hào)Isolate number分離年代 year亞型號(hào)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 （抗原轉(zhuǎn)變）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)變異幅度小(基因點(diǎn)突變，HAaa變異率1%,量變)已有中和抗體不能完全保護(hù)中小流行 抗原轉(zhuǎn)變 (antigenic shift)變異幅度大(基因重組，質(zhì)變, HAaa變異率20-50%,)新的亞型抗體無(wú)保護(hù)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變異發(fā)生de可能機(jī)制1. 基因的重組（Recombination）2. 病毒的進(jìn)化3. 動(dòng)物來(lái)源的病毒跨種屬傳播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簡(jiǎn)稱，是由甲型流感病毒引起的禽類傳染性疾病，容易在鳥類（尤其是雞）之間引起流行，過(guò)去在民間稱作雞瘟（Fowl plague)。 禽類感染后死亡率很高。Basic imformation58History of avian flu1878年由Perroncito首次報(bào)道于意大利。1901年證實(shí)其病原為“濾過(guò)性”病原體。1955年證實(shí)其

26、為甲型流感病毒的一員。 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響！59時(shí)間地點(diǎn)波及范圍型別1983-1984美國(guó)1700多萬(wàn)只雞H5N21978美國(guó)140多萬(wàn)只火雞1997香港7000只雞死亡H5N12002.3香港捕殺86萬(wàn)只雞H5N12003.12 2004.1日本山口死亡6000只雛雞3萬(wàn)只雞感染H5N12004.1韓國(guó)9000只雞死亡H5N12004.1越南河內(nèi)100萬(wàn)家禽死亡捕殺200萬(wàn)只雞H5N12004.1臺(tái)灣捕殺3.5萬(wàn)只雞H5N2禽流感流行情況60流行情況流感病毒感染具有物種特異性，僅在罕見(jiàn)情況下超越范圍感染其它物種。1997年，香港發(fā)生H5N1型人禽流感

27、 （18人感染，6人死亡），在世界范圍內(nèi)引起了廣泛關(guān)注。 首次發(fā)現(xiàn)直接由禽類傳染人類的流感病毒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亞毒株H7亞毒株高致病性禽流感因在禽類中傳播快、病死率高，被世界動(dòng)物衛(wèi)生組織列為A類動(dòng)物疫病，我國(guó)列為一類動(dòng)物疫病。 2003年報(bào)告有1例獸醫(yī)感染H7N7死亡。2013年中國(guó)有多例H7N9。H5N1型不斷進(jìn)化，其寄生的動(dòng)物范圍會(huì)不斷擴(kuò)大，可感染虎、家貓等哺乳動(dòng)物，家鴨攜帶并排出病毒的比例增加，尤其是在豬體內(nèi)更常被檢出。低致病性非致病性高致病性禽流感病毒可以直接感染人類。1997

28、年， 香港地區(qū)，高致病性禽流感病毒H5N1型導(dǎo)致了18人感染，6人死亡，首次證實(shí)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 （怎樣認(rèn)識(shí)？）. # 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在浙江叢岺島重

29、組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傳染源 主要為患禽流感或攜帶病毒的雞、鴨、鵝等禽類。目前尚無(wú)人與人之間傳播的確切證據(jù)（可能性？）。71傳播途徑 經(jīng)呼吸道傳播，或密切接觸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齡均可被感染，但在H5N1感染病例中，13歲以下兒童所

31、占比例較高。從事家禽養(yǎng)殖業(yè)者及其同地居住的家屬、在發(fā)病前1周內(nèi)到過(guò)家禽飼養(yǎng)、銷售及宰殺等場(chǎng)所者、接觸禽流感病毒感染材料的實(shí)驗(yàn)室工作人員、與禽流感患者有密切接觸的人員為高危人群。73Avian flu pathogenesis H5N1亞型病毒感染者早期急性起病。類似普通感冒，重癥者進(jìn)展迅速，可出現(xiàn)急性肺損傷、急性呼吸窘迫綜合征（ARDS）、多臟器功能衰竭等多種并發(fā)癥。并可繼發(fā)細(xì)菌感染。H9N2和H7N7亞型癥狀輕微74禽流感防治管理傳染源，加強(qiáng)禽類疾病的監(jiān)測(cè)，受感染動(dòng)物立即銷毀切斷傳播途徑，監(jiān)測(cè)密切接觸禽類人員75禽流感防治抗病毒藥物： Rimantadine， Amantadine， Zan

32、amivir， Oseltamivir（達(dá)菲） 疫苗： 禽用 人用？76怎樣認(rèn)識(shí)1997 Hongkong avian flu 的發(fā)生？禽流感病毒受體：唾液酸-2, 3Gal 分布特點(diǎn)：下呼吸道 （呼吸細(xì)支氣管和肺泡）人流感病毒受體：唾液酸-2, 6Gal 分布特點(diǎn)： 上呼吸道 （氣管、支氣管） 隨著支氣管分級(jí)漸低分布減少，肺泡最少. 77怎樣認(rèn)識(shí)1997 Hongkong avian flu 的發(fā)生？ 可能與個(gè)體間上呼吸道禽流感病毒受體唾液酸-2,3Gal受體表達(dá)差異有關(guān). H5N1病毒進(jìn)入下呼吸道.H6N1 human Infection in Taiwan 201379甲型H1N1流感（

33、Swine flu ） 80關(guān)于命名 疫情暴發(fā)早期世界各地稱謂不一： 德國(guó)：“Schweinegrippe” 法國(guó)：“Ia Grippe A” 荷蘭：“Mexican Flu” 墨西哥衛(wèi)生部： “Ia epidemia” “北美流感” “加利弗尼亞流感” 豬流感病毒 豬源A型H1N1流感病毒（ swine-origin influenza A(H1N1) virus ）目前WHO稱之為流感大流行H1N1 2009(Pandemic H1N1 2009)我國(guó)稱之為甲型H1N1流感病毒（A/H1N1 influenza virus）8150個(gè)病例：37例平民和13例軍人37例平民：6例發(fā)生在夏天

34、29例發(fā)生在9-3月 2例不詳6例死亡：4例死于病毒性肺炎， 1例有細(xì)菌混合感染 1例有其它器官的損傷61%有豬的暴露史年齡中位數(shù)為22.5歲13例軍人病例（1976）：沒(méi)有暴露史1例死亡年齡中位數(shù)18歲這起疫情涉及230名士兵豬流感病毒A（H1N1）感染人病例82NEJM 360;25 , 200983致病性大多數(shù)病人表現(xiàn)為流感樣癥狀可發(fā)生肺炎等并發(fā)癥。少數(shù)病例病情進(jìn)展迅速，出現(xiàn)呼吸衰竭、多臟器功能不全或衰竭。84流行病學(xué)傳染源：主要為甲型H1N1流感病人；豬體內(nèi)已發(fā)現(xiàn)甲型H1N1流感病毒，但動(dòng)物是否為傳染源？ 傳播途徑：主經(jīng)呼吸道傳播，亦可通過(guò)接觸傳播易感人群：普遍易感85實(shí)驗(yàn)室檢查(1)

35、病毒核酸檢測(cè)： RT-PCR real-time PCR (2)病毒分離： 呼吸道標(biāo)本/ 肺組織 (3)血清學(xué)檢查86防治抗病毒藥物：初步藥敏試驗(yàn)提示，此甲型H1N1流感病毒對(duì)奧司他韋(oseltamivir)和扎那米韋(zanamivir)敏感，對(duì)金剛烷胺和金剛乙胺耐藥關(guān)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) 不分節(jié)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