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    VIRUSOLOGIE

    CursProf. Dr. Simona Ruta

    Lucrari Practice:

    Asist. Univ. dr. Camelia SultanaAsist. Univ. dr. Loredana ManolescuAsist. Univ. Dr. Cornel PopescuPreparator Dr. Aura Temereanca

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    VARIOLA (smallpox)

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    Variolizare1% vs. 25% mortalitate

    Imunitate pe viata

    Marea Britanie: sfarsit ani 1700

    China 1950

    Pakistan/Afghanistan/Ethiopia-1970

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    Cowpox-Vaccinia

    Jenner, 1796

    1800- Vaccinare obligatorie copiiMarea Britanie 1930-ultimul caz indigen in Marea

    Britanie 1940-ultimul caz indigen in StateleUnite

    1958- programul OMS de eradicareOctombrie 1977: Ultimul caz din lume(Somalia)1980- eradicarea variolei

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    Dimensiunea virusurilor Exceptie- Virus gigant 650nm (780nm cufibrilele de pe suprafata) denumit Mimivirus

    (de la Mimicking Microbe) ce infecteazaamoebae, nou patogen uman asociat

    pneumoniilor?

    Mimivirus a fost izolat prima

    data in 2003, din apa din

    turnurile de racire in cursul

    unei epidemii de pneumonie

    din Anglia

    In 2008 a fost identificat un

    virofag ce infecteaza

    Mimivirus, denumit sputnick

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    1892- Dimitri Ivanovski -virusul mozaicului tutunului-Martinus Willem Beijerinckcontagium vivum fluidum

    1898- Loeffler si Frosch- virusul febrei aftoase.

    1909-Landsteiner si Popper-virusul poliomielitic

    1949 - Enders, Weller si Robins introduc culturile de celule ca substrat pentru propagareavirusurilor

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    (a) Adenovirus.

    (b) Rotavirus.

    (c) Influenza virus (courtesy of George Leser).

    (d) Vesicular stomatitis virus.(e) Tobacco mosaic virus.

    (f) Alfalfa mosaic virus.

    (g) T4 bacteriophage.

    (h) M13 bacteriophage.

    Microscopia electronic

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    James Batcheller Sumner John Howard Northrop Wendell Meredith Stanley

    1/2 of the prize 1/4 of the prize 1/4 of the prize

    USA USA USA

    Cornell UniversityIthaca, NY, USA

    Rockefeller Institute for

    Medical ResearchPrinceton, NJ, USA

    Rockefeller Institute for

    Medical ResearchPrinceton, NJ, USA

    b. 1887d. 1955

    b. 1891d. 1987

    b. 1904d. 1971

    Tobacco mosaic disease virus can be crystallized in the same way as proteins and enzymes

    Premiul Nobel pentru chimie 1946

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    SUMARSTRUCTURAVIRUSURILOR.

    Virusurile nu contin dect o singur specie de acid nucleic: ADN (dezoxiribovirusuri) sau ARN(ribovirusuri). Acidul nucleic constituie genomul, depozitarul infectivittii virale.

    Genomul viral este protejat de un nvelis proteic numit capsid. Capsida rezult dinasamblarea unor subunitti numite capsomere care se asociaz potrivit regulilor de simetrieicosaedric sau helicoidal. Adesea genomul si capsida alctuiesc un ansamblu numit

    nucleocapsid. Capsida este nconjurat la unele virusuri de un al doilea nvelis numit anvelop sau peplos.

    Anvelopa este dublu codificat: de genomul viral si de genomul celulei gazd. Aceasta

    nseamn c n cursul eliberrii virionilor din celulele infectate ei includ n anvelop structuri

    codificate de genomul viral si structuri proprii membranei celulei infectate

    Genomul viral codific proteine structurale care alctuiesc nvelisurile genomului (capsida sianvelopa)responsabile de antigenicitatea virala si proteine reglatorii sau cu functieenzimatic, importante in cursul replicarii virale. Numrul proteinelor codificate de genomul

    virusurilor este foarte diferit: de la numai dou la virusul hepatitei delta la cteva sute la

    herpesvirusuri sau poxvirusuri.

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    STRUCTURA VIRUSURILOR

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    SIMETRIE ICOSAEDRICA

    SIMETRIE HELICOIDALA

    ASAMBLAREA CAPSOMERELOR

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    VIRUSURI ADN

    Mimivirus -ADNds liniar 1,2Mb-cel mai mare genom

    viral descris pana in prezent, mai mare ca genomul

    unor bacterii

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    VIRUSURI ARNSS CU POLARITATE POZITIVA

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    VIRUSURI ARNss CU POLARITATE NEGATIVA

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    Genomul Familii representative

    ARN ss (+) Picorna-, Calici-, Astro-,Flavi-, Toga-viridae

    ARN ss (-) Orthomixo-, Paramixo-, Arena-, Rhabdo-, Bunya-viridae

    ARN ss RT Retro-viridae

    ARN ds REO-viridae: (Orbivirus, rotavirus, reovirus)

    ADNss Parvo-viridae

    ADNds Papova-, Adeno-, Herpes-, Pox-viridae

    ADNds RT Hepadna-viridae

    PRINCIPALELE FAMILII VIRALE PATOGENE PENTRU OM

    RT- cu reverstranscriptaza, ce sintetizeaza ADN pe matrita ARN, inversand fluxul normal ainformatiei genetice.

    Pentru detalii privind clasificarea consultati Index Virum: www.life.anu.edu.au/viruses

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    DETALII ULTRASTRUCTURALEALE VIRUSURILOR INMICROSCOPIE ELECTRONICA

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    Vaccinia virus. (A) Electron micrograph of purified virus, negative stain (Westwood et al.,1964). (B) Electron micrograph of a purified virion, whole mount, negative stain (Wilton et

    al., 1995). (C) Freeze etch electron micrograph of a purified virion (Nermut, 1973).(D) Deep

    etch electron micrograph of a purified virion (Heuser, 2005).(E) Atomic force micrograph of a

    purified virion (Malkin et al., 2003). (F) Cryoelectron micrograph of a whole mount

    preparation of purified virions (Griffiths et al., 2001).

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    PARAZITISM STICT INTRACELULAR

    Mimivirus infectand Acanthamoeba polyphaga.

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    SUMAR

    Virusurile se deosebesc de bacterii prin:

    parazitism exclusiv intracelular datorat dependentei de sursele de energie

    ale celulei (virusurile nu au mitocondrii) si de mecanismele de sintez

    proteic (virusurile nu au ribozomi) ale celulelor parazitate;-->mecanism de

    nmultire diferit de microbi -numit replicare viral si cultivare numai pe

    substraturi celulare vii

    talia mic, de 100 - 1000 ori mai mic dect a microbilor; virusurile se

    msoar n nanometrii (10-9 metri)--ultrafiltrabilitate si vizualizare in

    microscopie electronica.

    N.B. Singura exceptie par a fi virusurile din familia virusurilor largi

    nucleocitoplasmatice ADN (poxviridae, iridoviridae, asfarviridae,

    phycodnaviridae si mimiviridae).

    organizare simpl dictat de codificarea informatiei genetice de o specie

    unic de acid nucleic: ARN la ribovirusuri si ADN la dezoxiribovirusuri

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    http://www.virology.net/Big_Virology/BVHomePage.htmlDepartamentul de Microbiologie si Imunologie al Universittii deMedicin din Tulane, New Orleans, SUA

    Fotografii de microscopie electronica: Institutul Robert Koch din Berlin -

    http://www.rki.de/infekt/enivd/emqm/fs_pict3.htm- Universitatea din Cape Town - I spy with my little eye

    http://www.uct.ac.za/depts/mmi/stannard/linda.html,

    Preparate colorate cu efecte citopatice determinate devirusuri n culturi celulare:http://www.uct.ac.za/depts/stannard/cpe.html

    Please advise your students:Don't watch TV - listen to medical podcasts !http://freemedicalpodcasts.com

    http://www.rki.de/INFEKT/ENIVD/EMQM/fs_pict3.htm-http://www.rki.de/INFEKT/ENIVD/EMQM/fs_pict3.htm-http://www.uct.ac.za/depts/mmi/stannard/linda.htmlhttp://www.uct.ac.za/depts/mmi/stannard/linda.htmlhttp://www.uct.ac.za/depts/mmi/stannard/linda.htmlhttp://www.uct.ac.za/depts/mmi/stannard/linda.htmlhttp://www.uct.ac.za/depts/stannard/cpe.htmlhttp://www.uct.ac.za/depts/stannard/cpe.htmlhttp://freemedicalpodcasts.com/http://freemedicalpodcasts.com/http://freemedicalpodcasts.com/http://www.uct.ac.za/depts/stannard/cpe.htmlhttp://www.uct.ac.za/depts/mmi/stannard/linda.htmlhttp://www.rki.de/INFEKT/ENIVD/EMQM/fs_pict3.htm-http://www.rki.de/INFEKT/ENIVD/EMQM/fs_pict3.htm-
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    IZOLAREA VIRUSURILOR IN

    CULTURI CELULARE

    EFECTE CITOPATICE

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    DIRECIILE DIAGNOSTICULUI

    VIRUSOLOGIC

    1. Izolarea viral

    2. Demonstrarea prezeneivirusului / componentelor sale

    3. Diagnosticul serologic

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    Izolarea virusurilor

    3 sisteme de izolare

    Culturi de celule

    Ou de gin embrionat

    Animalul de laborator

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    AVANTAJE / DEZAVANTAJE ALESISTEMELOR DE IZOLARE VIRALA

    AVANTAJE DEZAVANTAJE

    ANIMAL DE LAB Infectie naturala Cost / etica

    O.G.E. Cost Susceptibilitate

    limitata

    CULTURI DECELULE

    Sensibilitate buna/ disponibilitate

    Susceptibilitatevariabila

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    Mediu nutritiv solutie salina tamponata - Earle, Hanks

    substane nutritive ( SFV, lizate proteice)

    vitamine

    antibiotice i antifungice

    Condiii necesare pentru pstrarea n via a celulelor din cultura celular

    Condiii aseptice

    pH adecvat Temperatur constant

    atmosfer cu 10% CO2

    Componentele culturilor de celule: Celule dispersate+ mediu nutritiv

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    CLASIFICAREA

    CULTURILORDE CELULE

    Culturi de celule dispersate Culturi primare

    Tulpini celulare

    Linii celulare- CELULE TRANSFORMATE

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    CELULE TRANSFORMATE1. conin gene implicate n imortalizarea n vitro (cultivare nr.infinit pasaje) modificari cromozomiale => cariotip

    aneuploid;2. se multiplic haotic, cerine nutritive foarte sczute (semultiplic la concentraii foarte sczute de SFV), glicolizaanaerob = sursa principala de energie.

    3. membran celular modificat n compoziie (absena

    fibronectinei => scaderea adeziunii intercelulare4. nu au inhibitie de contact, se dispun dezordonat diferitde esutul din care deriv

    5. inoculare la animale => TUMORI

    6. modificarea morfologiei (rotunde, refrigente)

    7. au antigene de suprafa diferite de N codif. de virus/celule gazda=> neoantigene

    8. aglutineaz uor cu lectinele (antigene vegetale)

    9. modificarea mesageri secunzi (AMPc)

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    Echipament de laborator

    Hote cu flux laminar pentru obtinerea, sipropagarea culturilor de celule si izolarea

    virala

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    TIPURI DE EFECTE CITOPATICE

    SINCITIAL: HIV, v rujeolos, VRS

    PICNOTIC LITIC

    enterovirusuri v. poliomielitice; v. Coxsackie; ECHO

    INCLUZIONAR

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    EFECT CITOPATIC SINCITIAL

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    EFECT CITOPATIC INCLUZIONAR

    Intracitoplasmatic acidofil: v rabic, v vaccinal

    Intranuclear -acidofil: papovavirusuri

    - bazofil: adenovirusuri

    Mixt: v herpetice, v rujeolos

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    ECP picnotic litic

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    INCLUZIICITOPLASMATICE

    (RABIE)patognomonic in SNC - CORPII BABESNEGRI in cornul lui Ammonsi neuroniipiramidali din cerebel

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    INCLUZII INTRANUCLEARE(BOALA CUINCLUZII CMV)

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    INCLUZIIINTRANUCLEARE

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    Diagnostic de laborator

    in neuroviroze

    1. SUSPICIUNEA CLINICA

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    Definitie de caz meningita virala

    Febra 38.5 C asociata cu minim un simptom dintre:

    Cefalee severa i persistenta;

    Redoare de ceafa

    Durere de ceafa asociata cu minim 2 din urmatoarele:

    Fotofobie

    Greata

    Varsaturi

    Faringita exudativa

    Dureri abdominale

    La sugari si copiii mici semnele principale sunt febra 38.5 C asociata cuiritabilitate si pulsarea fontanelei

    1. SUSPICIUNEA CLINICA

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    Algoritm de diagnostic virusologic

    ETIOLOGIE

    POSIBILA

    Produs

    Patologic

    IZOLARE Dg. DIRECT

    =detectiedirecta

    Dg. INDIRECT

    =serologie

    80%-90%

    Enterovirusuri

    Polio

    Cocsackie A si B

    E.C.H.O.

    10 20% alte

    virusuri:VZV; V.URLIAN; HSV; CMV; V.PARAGRIPALE

    Materii

    Fecale*SNF

    LCR**

    De electie

    Culturi

    celulareAnimal delaborator

    NU DA

    Este important de notat ca enterovirusurile se excreta saptamani la rand, dar sansele de izolare scad. Se recomanda

    recoltarea a 2 - 3 probe de la pacient la interval de 24-48 ore

    ** Sansa de izolare din acest produs creste cnd participarea meningiana este evidenta. Acest lucru se traduce prinpleiocitoza (cresterea numarului de celule limfo-monocitare in LCR).

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    IZOLAREA ENTEROVIRUSURILOR IN CULTURI DE CELULE ECPpicnotic litic

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    RSN:IDENTIFICAREA VIRUSURILOR=VIRUS NEUTRALIZARE

    TITRAREA ANTICORPILOR= REACTIA DE SERONEUTRALIZARE

    VIRUSNEUTRALIZAREVIRUS NECUNOSCUT

    ANTICORPI CUNOSCUTI=SER IMUN DE REFERINTA

    SERONEUTRALIZARETITRU DE ANTICORPI NECUNOSCUTVIRUS CUNOSCUT = TULPINA DE REFERINTA

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    ETAPE RSN

    1. TITRAREA INFECTIVITATII VIRALE

    2. BLOCAREA INFECTIVITATII VIRALE

    3. DEMONSTRAREA NEUTRALIZARII

    INFECTIVITATII VIRALE

    1. TITRAREA INFECTIVITATII VIRALE

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    1. TITRAREA INFECTIVITATII VIRALE metoda plajelor

    Dilutii seriale

    ale virusului

    Insamantare pe celulesusceptibiledupa adsorbtie, 1h la 37,se acopera cu agar

    Restrictia raspandirii virusului

    determina liza focalizata amonostratului celular vizibilasub forma plajelor, PFU

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    Metoda plajelor

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    2. BLOCAREA INFECTIVITATII VIRALE

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    3. DEMONSTRAREA NEUTRALIZARII INFECTIVITATII VIRALE

    = Repetarea etapei 1 cu amestecurileantigen-anticorp

    Id tifi t i il d t d T bl d h

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    Identificarea enterovirusurilor dupa metoda Tabla de sah

    Lim - Benyesh Melnick

    ENCEFALITE Algoritm de diagnostic virusologic

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    ENCEFALITE Algoritm de diagnostic virusologic

    SINDROM ETIOLOGIE

    POSIBILA

    Prod.

    Patologic

    IZOLARE Dg.DIRECT

    =detect.directa

    Dg.INDIRECT

    =serologie

    ENCEFALITA

    ACUTAMENINGO-

    ENCEFALITA

    ENCEFALO-MIELITA

    HERPES

    VIRUSURI:HSV1

    HSV2

    VZV,EBV,CMV

    Lichidvezicular

    Biopsiecerebrala

    LCR

    Culturicelulare

    Animal delaborator

    PCR

    IF

    NU

    V. RABIC

    ARBOVIRUSURITOGAVIRUSURI

    FLAVIVIRUSURI

    BUNYAVIRUSURI

    SALIVA,AMPRENTEDERMICE,CORNEENE,

    BIOPSIECEREBRALA

    LCR

    SANGE

    Culturicelulare

    Animal delaborator

    PCRIF

    De electie pt.ARBO

    VIRUSURI

    POSTINFECTIOASE:

    V. RUJEOLOS

    V. URLIAN

    V. GRIPALE

    VVZ

    SALIVA,

    BIOPSIECEREBRALA

    LCR

    Culturicelulare

    PCR

    IF

    NU

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    NEUROVIROZE

    POLIOMIELITA

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    POLIOMIELITA

    (gr- polios/myelos)

    Infectie subclinica (90 - 95%)

    Infectie abortiva (4 - 8%)

    Meningita polio non-paralitica (1- 2%)Invazie SNC prin :1. Transport axonal retrograd

    2. Traversare BHE

    3. Transport prin macrofage infectate

    Poliovirus- receptor CD155 gp TM din super familia Ig stabilire

    jonctiuni intercelulare la nivel celule epiteliale; posibil implicat in

    RIU intestin subtire

    6 saptamani, intermitent, > imunocompromisi

    POLIOMIELITA

    http://upload.wikimedia.org/wikipedia/commons/1/1d/Poliovirus_binding_receptor_1DGI.png
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    POLIOMIELITA

    1 in 200-1000cazuri in infectia cu virusul salbatic la copii

    1 in 75 cazuri in infectia cu virusul salbatic la adulti

    Boala majora (0.1 0.5%)-polio

    spinala sau bulbara -paralizii acute flasceprin leziuni de neuron motor periferic

    Sindromul Post-polio (15-30 ani postpolio- slabiciune musculara, dureri, astenie)

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    Polio bulbaraRata mortalitatii

    25% copii1530% adulti

    1909 poliomielita poate fi tansmis la

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    1911-Suedia -epidemie (4000 cazuri cu

    zeci de decese). Izolarea virusului pe explant de tesut

    histopatologia bolii experimentale

    demonstrarea ultrafiltrabilitatii agentuluietiologic

    existenta infectiilor inaparente si

    seroconversia.- investigarea a 6 familiicare au avut contact cu cazuri de polio

    Constantin Levaditi (1874 1953)lInstitutul Pasteur, Paris, 1946

    1909 - poliomielita poate fi tansmis lamaimutele superioare prin filtrat demduv spinal (Landsteiner,K., and Levaditi C. Latransmission de la paralysie infantile aux singes. C. R. Soc.Biol. 1909, 592-594).

    1949- cultivarea virus polio pe culturi de celule non-

    neuronale- Enders, Robbins, Weller- 1954 Nobel

    VACCINURI VIRALE

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    VACCINURI VIRALE

    Virusuri antigenic inrudite cu agentul etiologic al virozeiumane, dar nevirulente pentru om (vaccinia)

    Tulpini virale natural atenuate( Max Theiler)

    Atenuare in laborator sub selectia presori fizici, chimici,biologici

    Virusul neatenuat in doze mici sau asociat cu anticorpispecifici/administrat pe cai de inoculare diferite

    VACCINURI INACTIVATE

    VACCINURI VII ATENUATE

    VACCINURI ANTIPOLIO

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    VACCINVIRUS

    Determinantiantigenici

    deteminantvirulenta

    VACCINURI ANTIPOLIOVaccin antipolio inactivat -SALK1952-

    National Foundation for Infantile Paralysis -1938

    Franklin D. Roosevelt - Al 32-lea presedinte SUA 19331945

    Incident Cutter 120,000 doze - 40,000 polio abortiva, 56 polio paralitica , 5 decese

    VACCIN ANTIPOLIO VIU ATENUAT SABIN 1962

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    VACCIN ANTIPOLIO VIU ATENUAT- SABIN 1962

    Tulpini atenuate prin pasaje seriale la temperaturi suboptimale

    -Baza moleculara a atenuarii distincta pentru fiecare serotip-Numar mic de mutatii disting tulpina atenuata Sabin deserotipul natural virulent (11 substitutii nucleotidice pt tip 3, 8 pt tip 2 si celputin 56 pt tip 1)

    -Mutatii esentiale in IRES 5 NT (nucleotide apropiate pt toateserotipurile-480 tip1; 481 tip 2, 472 tip 3)- destabilizarea structurii secundare

    a ARN si VP1

    IRES

    Element esential in replicare si initiere traducere

    http://upload.wikimedia.org/wikipedia/commons/6/65/Poliovirus_genome.pnghttp://upload.wikimedia.org/wikipedia/commons/6/65/Poliovirus_genome.pnghttp://upload.wikimedia.org/wikipedia/commons/6/65/Poliovirus_genome.png
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    AVANTAJELE VACCINURILOR VII

    Induc raspuns imun global (celular, umoral si local)

    Raspuns imun impotriva tuturor antigenelor(inactivarea poate altera antigenicitatea)

    Imunogene consecutiv administarii pe cale naturala

    (pret scazut, acceptabilitate mare)

    Imunogene consecutiv administrarii unice, imunitatedurabila

    Capacitate de a disemina de la vaccinati la contactiiacestora- duc la eliminarea virusului din colectivitate

    DEZAVANTAJELE VACCINURILOR VII

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    DEZAVANTAJELE VACCINURILOR VII

    Reversie la virulenta-poliomielita paralitica asociata vaccinrii - PPAV

    1 in 2-3 milioane - polio paralitic asociat vaccinarii

    (Risc ~ 8-10 cazuri/an in SUA- din 2000 exclusiv VPI)

    Instabilitate la transport sau stocaj, in absenta lantului de frig

    Interferare de catre virusuri cu care impart acelasi habitat

    Reactii severe la imunosupresati

    Reactogene-grad de atenuare al tulpinii virale

    Contaminarea vaccinului cu agenti criptici din substrat cu potential patogen sau

    oncogen?

    1988 - 350 000 cazuri in 125 tari endemice

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    1988 350 000 cazuri in 125 tari endemice

    2006 1874 cazuri in 4 tari

    1988- OMS - Campania de eradicarea a poliomieliteivaccin trivalent oral Sabin

    1994- Eradicare poliomielita in emisfera vestica-(1991 ultimul caz in Peru; 1979 ultimul caz in SUA)2000- eradicare in 36 tari din Pacificul de vest ( inclusiv China and Australia)

    2002 eradicare in Europa

    Polio endemica in :

    - Nord Nigeria -epidemie 2003 27, 379 cazuri de PFA intre 2001-2007-reinfectia > 20 tari anterior polio free

    - Nord India, granita PakistanAfghanistan (acoperire vaccinala insuficienta si eficienta neadecvata a

    vaccin trivalent)

    Strategie eradicare polio:1. Mentinere nivel crescut de imunizare de rutina

    2. Zile nationale de Imunizare- vaccinare tuturor copiilor

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    ROMANIA

    Calendarul de vaccinari valabil de la 1 decembrie 2008

    Varsta recomandata Vaccin Comentarii

    Primele 24 de ore

    4-7 zile

    HepB

    BCG

    In maternitate

    2 luni DTPaIPV, HepB Simultan

    4 luni DTPaIPV Simultan

    6 luni DTPaIPV, HepB Simultan

    12 luni DTPa IPV, RRO Simultan

    4 ani** DTPa

    7 ani (in cls. I) RRO Campanii scolare

    9 ani (in cls. a III-a) VPO Campanii scolare

    14 ani (in cls. a VIII-a) dT, Rubeolic *** Campanii scolare

    Enterovirusuri:Polioviruses 1 2 3

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    Polioviruses 1, 2, 3Coxsackie A1-24/ B1-6ECHO - 33 (Enteric cytopatogenic human orphans)Enteroviruses 68-71

    CARACTERE GENERALE ALE ENTEROVIRUSURILOR

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    CARACTERE GENERALE ALE ENTEROVIRUSURILOR

    Sunt virusuri cu genom ARNss, cu polaritate pozitiv, de talie mica (~30 nm) si neanvelopate.Lipsa anvelopei explic rezistenta picornavirusurilor la dezinfectante, la pH-ul acid al suculuigastric, la srurile biliare etc.

    Afectiuni produse de unele enterovirusuri. Virusul Boala frecvent determinat:

    Virusurile Cox A Herpangina, diarei minore de vara, meningite aseptice

    Virusurile Cox B Mialgie epidemica sau pleurodinia Bornholm,moarte subit a nou-nscutilor, meningite aseptice,miocardite, pancreatite, hepatite, etc.

    Virusurile ECHO Meningite aseptice, guturai, conjunctivite, exanteme

    Enterovirusurile 68-71 Encefalite, mieloradiculonevrite.

    Virusurile ECHO produc uneori epidemii extinse de meningite aseptice. Astfel, n vara anului1999 o epidemie cu virusul ECHO 33 de peste 5000 cazuri a afectat judetele din Moldovaextinzndu-se ulterior si n restul trii. Cazurile s-au datorat consumului apei infectate sauscldatului n ape contaminate si au prevalat la grupa de vrst 5-15 ani.

    Impactul economic al meningitelor virale (meningite aseptice sau cu lichid cefalorahidian clar)este considerabil fiind estimat la cheltuieli n jur de 5000 $ per caz (costuri directe) si 8 zile deinactivitate (costuri indirecte). n 90% din cazuri aceste meningite sunt de etiologieenteroviral. Cheltuielile directe sunt mai mari pentru cazurile pediatrice si deriva dinadministrarea empirica a antibioticelor si ngrijirea cazurilor cu deshidratare severa n serviciilede reanimare.

    Virusul rabic Familia

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    Virusul rabic-FamiliaRhabdoviridae, genul Lyssavirus

    Rabia- zoonoz major anualcirca 60 000 decese

    -Incubatie (10 zile- 6 luni-2

    ani!?)

    - Prodrom

    - Sindrom neurologic acut

    - Coma- Deces

    Virusul rabic

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    Virusul rabic

    Virusul rabic (lyssavirus) face parte din familia Rhabdoviridaevirusuri ARNss cu polaritatenegativn form de glont.

    Virusul rabic salbatic este numit "de strada" si are o incubatie variabila; pasaje repetate laaceiasi specie stabilizeaza si scurteaza incubatia, tulpinile fiind etichetate ca "virus rabicfix".

    Leziunile citopatice sunt exclusiv intraneuronale, citoplasmatice si au aspectul unor incluziigranulare numite corpii Babes Negri.

    Tulpinile de virus fix dau incluzii mici. Corpii Babes Negri predomina n neuronii din cornulAmmon din hipocamp si n celulele Purkinje din cerebel. Aceste leziuni corespund situsurilorreplicarii virale.

    In celulele glandelor salivare virusul se replica si se elibereaza prin nmugurire la nivelulmembranei celulare ceea ce reprezinta o adaptare eficienta pentru propagarea infectiei la onoua gazda. n afara neuronilor si celulelor glandelor salivare antigenul rabic mai poate fievidentiat n cornee, piele, mucoasa bucala - elemente care au valoare diagnostica.

    Patogenia infectiei este conditionat de diseminarea septinevritic centripet a virusului de la

    nivelul plcutelor neuromusculare periferice ctre SNC. Din SNCvirusul se propag centrifugpe calea filetelor senzitive (septinevritic) si se secret n saliv.

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    Incluzii i-citoplasmice Negri Antigene rabice

    Neuroni fara incluzii

    1885 -Pasteur -atenuare virus rabic prin pasaje repetate intra-

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    cerebral la iepure(L.Pasteur: Methode pour prevenir la rage morsure. C.R. Acad Sci,101, 765)

    1887- Babes si Puscariu -metoda romneasc de vaccinare

    antirabic cu virus fix -atenuat prin pasaje la cobai si princldur. (V.Babes: Ann. Inst. Pasteur 1889, 7, 384).

    1894- Babes recomand utilizarea serurilor de la animaleleimunizate cu virus fix pentru tratamentul rabiei dup musctur(V. Babes: Romnia Medical, 1894, p. 467-469).

    Vaccinarea la om- vaccin inactivat preparat pe culturi decelule diploide umane (vaccinoterapia Pasteur)

    Administrarea de IgG in jurul plagii muscate (seroprofilaxia

    Babes). Vaccinarea animalelor domestice(obligatorie) si salbatice-

    vaccinuri vii atenuate- cu mutatii si deletii n proteina G- uzveterinar

    http://americanhistory si edu/polio/virusvaccine/index htm

    http://americanhistory.si.edu/polio/virusvaccine/index.htmhttp://americanhistory.si.edu/polio/virusvaccine/index.htm
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    http://americanhistory.si.edu/polio/virusvaccine/index.htm

    http://www.astdhpphe.org/infect/polio.html-polio facts- FAQ

    http://www.nlm.nih.gov/medlineplus/polioandpostpoliosyndro

    me.html http://w3.whosea.org/EN/Section1226/Section1228.htm-

    vaccin antipolio http://www.post-polio.org/

    Cello J, Paul AV, Wimmer E: Chemical synthesis of poliovirus cDNA: generation ofinfectious virus in the absence of natural template. Science 2002, 297:1016-1018.http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=12114528- sintetizare virus polio in laborator

    http://americanhistory.si.edu/polio/virusvaccine/index.htmhttp://www.astdhpphe.org/infect/polio.html-http://www.nlm.nih.gov/medlineplus/polioandpostpoliosyndrome.htmlhttp://www.nlm.nih.gov/medlineplus/polioandpostpoliosyndrome.htmlhttp://w3.whosea.org/EN/Section1226/Section1228.htm-http://www.post-polio.org/http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=12114528-http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=12114528-http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=12114528-http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=12114528-http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=12114528-http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=12114528-http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=12114528-http://www.post-polio.org/http://www.post-polio.org/http://www.post-polio.org/http://w3.whosea.org/EN/Section1226/Section1228.htm-http://w3.whosea.org/EN/Section1226/Section1228.htm-http://www.nlm.nih.gov/medlineplus/polioandpostpoliosyndrome.htmlhttp://www.nlm.nih.gov/medlineplus/polioandpostpoliosyndrome.htmlhttp://www.astdhpphe.org/infect/polio.html-http://www.astdhpphe.org/infect/polio.html-http://americanhistory.si.edu/polio/virusvaccine/index.htm
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    ORTHOMYXOVIRIDAE

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    Matrice

    Nucleocapsida helicoidala(ARNss -, segmentat)

    HA - hemaglutinina

    transcriptaza

    anvelopa

    NA - neuraminidaza

    tip A, B, C : NP, Msubtipuri: HA si NA16 tipuri de HA si 9 de NAtip/subtip/ loc, an izolare/nr. pasaje-A/H5N1/Guangdong/1996/1

    HEMAGLUTININA

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    Virusul gripal tip A de laspecia/organul

    Receptorul culegatura de tip

    Uman, trahee uman SA-2-6-Gal beta

    Aviar (trahee sau epiteliu

    intestinal), ecvin

    SA-2-3-Gal beta

    Porcin, trahee porcin;

    prepelit

    SA-2-3-Gal beta si

    SA-2-6-Gal beta

    REASORTANTI

    principaliideterminanti

    antigenici

    Endocitoza mediata de receptorschimbari

    conformationale cu expunerea peptid de

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    conformationale cu expunerea peptid de

    fuziune N-terminal HA2- -complex

    ribonucleoproteic transportat in nucleu

    Proteina M- rol de canal ionic

    INHIBITORII DECAPSIDARII- AMANTADINA SI

    RIMANTADINA

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    INHIBITORII DE NEURAMINIDAZA-

    O SELTAMIVIR -75 mg x 2/ziZANAMIVIR -10 mg (2 inhalatii de 5 mg/zi)PERAMIVIR-injectabil- aprobat in regim de urgentade FDA pe 23 Octombrie 2009, pt utilizare in cazurisevere, spitalizate in care ceilalti inhibitori de NA suntneutilizabili (rezistenta la Oseltamivir , imposibilitatea

    administrarii Zanamivir)

    NS1 inhibitor al sintezei de IFN

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    V. Gripale A: VARIABILITATE DRIFT ANTIGENIC-mutatii punctiforme- EPIDEMII

    VARIABILITATEA VIRUSURILOR GRIPALE A

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    SHIFT ANTIGENIC- PANDEMII

    1. reasortarea genic ntre tulpini gripale de la specii diferite

    2. transferul direct al unor tulpini gripale de la animal la om- modificriconformationale in structura hemaglutininei.

    3. Re-emergenta unui virus ce a determinat epidemii cu ani inainte

    Psri acvatice slbatice (rate,gste, pescrusi)-rezervorulnatural al virusurilor gripale A

    La psrile domestice gripa poate evolua n dou forme,-minor (LPAI) sau asimptomatic, - afecteaz productia ou in cresctorii- majora (HPAI), foarte sever, nalt contagioas, cu mortalitate 50-100%. (aminoacizi bazici suplimentari la

    nivelul situs de clivare HA)

    Pandemii gripale

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    Pandemii gripale

    1918-H1N1gripa spaniola -20-50 milioane decese Virus aviar

    1957-H2N2 gripa asiatica-1-2 milioane decese

    reasortant

    1968- H3N2 gripa Hong Kong -700,000 decese-reasortant

    1977- H1N1gripa ruseasca-re-emergenta

    A/H1N1/ 2009 pandemic

    414 000 i fi t d

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    Influenza A H1N1/20096 Minigene de la tulpinile

    porcine Nord America(reasortant 1998

    suin/aviar/uman) si2 de la tulpinile porcine izolate

    in Europa/Asia

    Aceste tulpini au coexistatpentru mai mult de 10 ani -

    Reasortarea actuala probabilla porc- gazda ramane totusinecunoscuta (supraveghereinsuficienta; infectie

    asimptomatica la porc,

    reasortare recenta?)

    414,000 cazuri confirmate delaborator

    cca 5000 decese raportate la OMS

    Primul val pandemic (H1N1) 2009

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    80

    Primul val pandemic (H1N1) 2009

    12% din forta de muncaVarf rata de absenteism

    0.1% - 0.2% (pana la 0.35%)Rata de fatalitate

    2% din cazuri clinic aparenteRata de spitalizare

    15% din cazuri clinic aparenteRata Complicatii

    6.5% (4.5% - 8%) per saptamanaVarf rata de atac

    30%Rata de atac

    Courtesy of Department of Health, UK, http://www.dh.gov.uk/en/Publicationsandstatistics/Publications/PublicationsPolicyAndGuidance/DH_102892

    Data valabile in Iulie 2009 pt UK

    45 decese asociate cu cazuri confirmate delaborator de H1N1/pandemic 2009 in 13 tari in

    i d 21 27 O t b i

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    53 State Membre in regiunea Europeana OMS

    WHO/Europe influenza surveillance(EuroFlu.org)

    perioada 21- 27 Octombrie.Numar total de decese confirmate din Aprilie

    2009 in 27 tari EU si 4 EFTA 317.Ukraina a raportat 12 decese cauzate depneumonii regiunea Ternopol alte 6 decese in

    regiuni invecinate

    SHIFT ANTIGENIC- Modificri conformationale n structurahemaglutininei pentru traversarea barierei de specie

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    2. transferul direct al unor tulpinigripale de la animal la om

    hemaglutininei pentru traversarea barierei de specie Toate virusurile gripale umane au singur reziduu bazic de arginin la nivelul sitului de clivare

    a hemaglutininei.

    Achizitia unor aminoacizi bazici suplimentari la acest nivel, duc la o conformatie

    asemntoare virusurilor aviare HPAI (situri de clivare multiple) si permit si altor proteaze nafara tripsinei s cliveze hemaglutinina, ceea ce duce la alterarea tropismului tisular (cuposibilitatea afectrii tesutului cerebral, miocardic si vaselor de snge) si la crestereavirulentei.

    O serie de mutatii punctiforme afecteaza legarea la receptori si cresc legarea de rezidurisialozidice specifice mamiferelor

    V.GRIPAL 1918

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    Caracteristica Tulpina H1N1 A/New

    Caledonia/20/99

    A/New Caledonia/20/99 cu HA si N A

    de la tulpina pandemic 1918

    LD50/Soarece >106 PFU

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    Decembrie 2003- cazuri letale gripa H5N1 lafeline mari Thailanda, continua in octombrie2004 -147/441 tigrii 2004--H5N1- Coreea deSud, Vietnam, Cambodia, Laos, Japonia,

    Thailanda, Indonezia, China, Hong Kong

    2005-iulie-august Rusia , Kazakstan , Mongolia

    octombrie- decembrie-Turcia, Romania),Croatia , Kuwait, Ukraina

    China, Indonezia, Vietnam, Cambodia,Turcia (cazuri umane)

    2006- Iraq, Nigeria, Azerbaijan, Egipt, India,Franta (februarie-I data in UE), Niger,Albania - pasari domesticeBulgaria, Greece, Italy, Slovenia ,

    Iran, Austria, Germania, Franta,

    Ungaria, Slovacia, Bosnia-Herzegovina, Georgia, Suedia, Elvetia ,Serbia-Muntenegru, Polonia-pasarisalbaticelebede, rate

    2008-2009 China, Indonezia, VietNam, Egipt,Thailanda, Iraq, Turcia-cazuri umane

    Transferul tulpinilor nalt patogene H5N1 dinAsia ctre Estul Europei este atribuit ruteipsrilor deasupra Eurasiei continentale saude-a lungul litoralului Mrii Arctice.

    Aprilie 2005- extinderea spectrului de gazda la pasarisalbatice - 6,345 pasari din specii diferite mor in citeva

    saptamini in regiunea marilor lacuri (Qinghai Lake) din Chinacentrala- se extind in n Siberia, Caucaz, Balcani.

    2009- 442 cazuri umane/262 fatale

    Conditii necesare izbucnirii unor pandemii

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    Conditii necesare izbucnirii unor pandemii

    noutatea antigenica a virusului

    lipsa imunittii n populatia uman

    facilitatea transmiterii interumane anoilor tulpini.

    VACCIN INACTIVAT CU VIRUS INTREGSAU SUBUNITAR (1931-izolare in OGE

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    SAU SUBUNITAR (1931-izolare in OGE,1940- vaccin inactivat)

    - Virus izolat in OGE, lichid alantoidian imbogatit,particule virale centrifugate in gradient de densitate

    pentru concentrare si purificare, inactivare cuformaldehida sau -propiolactona +/_ tratare cudetergent si purificare HA si NA- vaccin trivalent

    - Petrescu si colab., 1980 - NIVGRIP- administrareaintranazal a vaccinului inactivat preparat cu virusul

    ntregprioritate romneasc.

    VACCIN VIU ATENUAT CU TULPINIRECI

    Tulpina master atenuata, adaptata pentru cresterela 250 C (A /H2N2 /Ann Arbor/6/60 si B/AnnArbor/1/66) in care se insera genele pentru HA/ NA-

    mutatii stabile in genele complexului polimerazicPA, PB1, and PB2, propagate n pasaje seriate latemperaturi din ce n ce mai sczute pe celule renalede pui de gin SPF (specific pathogen free)

    Fiecare doz de vaccin continehemaglutinina corespunztoare a 10bilioane de virioni n cazul cultivriivirusului pe OGE (15 ug per 0.5 ml) .

    Cantitatea de neuraminidaz nu este

    standardizat -protein labil n cursulprocesului de preparare si stocare avaccinului.

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    PB2PB1

    PAHANANP

    MNS

    PB2PB1PA

    HANANP

    MNS

    PB2PB1PA

    HA

    NANPMNS

    Tulpina matca -PR8 (H1N1A/PR/8/34)atenuata,nonpatogena, incapabila de

    replicare la om

    Tulpina antigenic noua

    X

    Reasortantvirus cu 6 gene PR8, dar HA siNA tulpinii circulante

    Vaccinuri Influenza A (H1N1) 2009 monovalente aprobate in US, 6 Octombrie 2009

    V i P d t P tg Hg/0.5

    G tNo.

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    Vaccin Producator Prezentareg g

    mLGrup varsta

    doze

    Inactivat Sanofi Pasteur SA

    0.25 mL siringa 0 6--35 luni 2

    0.5 mL siringa 0 36 luni 1 or 2

    5.0 mL fiola multidoza 25.0 6 luni 1 or 2

    InactivatNovartis Vaccines

    Diagnostics Ltd

    5.0 mL fiola multidoza 25.0 4 ani 1 or 2

    0.5 mL siringa

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    EUROPEANA Celvapan(Baxter AG);- virion ntreg, A/California/07/2009 (H1N1) inactivat,

    cultivat pe celule Vero 7,5 micrograme HA per doz de 0,5 ml

    . Focetria(Novartis Vaccines and Diagnostics S.r.l)- Antigene de suprafaale virusului gripal (hemaglutinin i neuraminidaz) din tulpina:

    A/California/7/2009 (H1N1) v (X-179A) cultivata n OGE 7,5 micrograme HAper doz de 0,5 ml -adjuvant (MF59C.1)

    Pandemrix(GlaxoSmithKline Biologicals S.A)- Virus gripal fragmentat,inactivat, coninnd HA din tulpin similar virusului A/California/7/2009

    (H1N1) (X-179A) cultivata n OGEechivalent cu: 3,75 micrograme** per doz de 0,5 ml- adjuvant AS03

    GRUPE DE RISC

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    Persoane cu risc nalt de complicatii

    copii < 2 ani (rate de spitalizare de 2.3 ori > ca cei 2- 4 ani, acesrtia la randul

    lor sanse de 20% mai frecvente complicatii)

    adulti > 65 anifemeile gravide n trimestrele II si III de sarcin, 2 saptamani postpartum

    inclusiv dupa pierdere sarcina

    pacienti institutionalizati

    adulti si copii cu boli cardiace (cu exceptia HTA) si pulmonare cronice

    (inclusiv astm), boli metabolice (inclusiv diabet zaharat), disfunctii hepatice,

    renale, hematologice , imunosupresie (inclusiv cei HIV pozitivi)

    Obezitate (BMI 30-35), mai ales obezitate morbida (BMI )>40 n

    Persoane care pot transmite infectia gripal celor cu risc de complicatii

    personalul medico-sanitar

    angajatii institutiilor psihiatrice si cei ce ngrijesc bolnavi cronici

    membrii familiilor persoanelor la risc

    Persoane la care vaccinarea poate fi recomandat

    persoane care cltoresc n zone cu epidemii de grip

    persoane implicate n servicii comunitare esentiale

    copiii din gradinite si scoli, studentii, militarii

    UP TO DATE

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    http://ec.europa.eu/health/ph_threats/com/Influenza/h1n1_en.htm

    http://ec.europa.eu/health-eu/newsletter/influenza/1/newsletter_en.htm

    http://www.emea.europa.eu/influenza/vaccines/home.htm http://www.euro.who.int/surveillance

    http://www.cdc.gov/flu/weekly

    http://www.ecdc.eu.int/

    http://www.oie.int Organizatia Mondiala Pentru Sanatatea Animala

    www.virology.ro- INSTITUTUL DE VIRUSOLOGIE St. S. Nicolau date despre debutul pandemiei

    www.who.int/csr/disease/avian-influenza

    http://www.efsa.eu.int/science/- The European Food Safety Authority

    http://europa.eu.int/comm/food/animal/diseases/controlmeasures/avian/

    avain_influenza_22092005.pdf

    Taubenberger et al in Nature ( VOL. 437 October 6th 2005 )http://www.nature.com/news/2005/051003/full/437794a.html http://www.nature.com/news/2005/051003/full/nature04230.html

    Tumpey et al in Science (2005; 310: 77-80).

    http://ec.europa.eu/health/ph_threats/com/Influenza/h1n1_en.htmhttp://ec.europa.eu/health-eu/newsletter/influenza/1/newsletter_en.htmhttp://www.emea.europa.eu/influenza/vaccines/home.htmhttp://www.euro.who.int/surveillancehttp://www.euro.who.int/surveillancehttp://www.cdc.gov/flu/weeklyhttp://www.ecdc.eu.int/http://www.oie.int/http://www.who.int/csr/disease/avian-influenzahttp://www.who.int/csr/disease/avian-influenzahttp://www.who.int/csr/disease/avian-influenzahttp://www.who.int/csr/disease/avian-influenzahttp://www.efsa.eu.int/science/-http://europa.eu.int/comm/food/animal/diseases/controlmeasures/avian/avain_influenza_22092005.pdfhttp://europa.eu.int/comm/food/animal/diseases/controlmeasures/avian/avain_influenza_22092005.pdfhttp://www.nature.com/news/2005/051003/full/437794a.htmlhttp://www.nature.com/news/2005/051003/full/nature04230.htmlhttp://www.nature.com/news/2005/051003/full/nature04230.htmlhttp://www.nature.com/news/2005/051003/full/437794a.htmlhttp://europa.eu.int/comm/food/animal/diseases/controlmeasures/avian/avain_influenza_22092005.pdfhttp://europa.eu.int/comm/food/animal/diseases/controlmeasures/avian/avain_influenza_22092005.pdfhttp://www.efsa.eu.int/science/-http://www.efsa.eu.int/science/-http://www.who.int/csr/disease/avian-influenzahttp://www.who.int/csr/disease/avian-influenzahttp://www.who.int/csr/disease/avian-influenzahttp://www.who.int/csr/disease/avian-influenzahttp://www.who.int/csr/disease/avian-influenzahttp://www.who.int/csr/disease/avian-influenzahttp://www.who.int/csr/disease/avian-influenzahttp://www.who.int/csr/disease/avian-influenzahttp://www.who.int/csr/disease/avian-influenzahttp://www.who.int/csr/disease/avian-influenzahttp://www.oie.int/http://www.ecdc.eu.int/http://www.cdc.gov/flu/weeklyhttp://www.euro.who.int/surveillancehttp://www.euro.who.int/surveillancehttp://www.emea.europa.eu/influenza/vaccines/home.htmhttp://ec.europa.eu/health-eu/newsletter/influenza/1/newsletter_en.htmhttp://ec.europa.eu/health-eu/newsletter/influenza/1/newsletter_en.htmhttp://ec.europa.eu/health-eu/newsletter/influenza/1/newsletter_en.htmhttp://ec.europa.eu/health/ph_threats/com/Influenza/h1n1_en.htm
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    HERPESVIRIDAEh i

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    gr. herpein

    Centers for Disease Control and Prevention's PublicHealth Image Library (PHIL), identification nr #1878.

    Proteine virale deja sintetizate:-inhiba sinteza proteinelor celulare,- inhiba mecanismele de aparare celulara-stimuleaza expresia genelor virale.

    GENOMUL HERPESVIRUSURILOR

    http://en.wikipedia.org/wiki/Centers_for_Disease_Control_and_Preventionhttp://phil.cdc.gov/phil/home.asphttp://phil.cdc.gov/phil/home.asphttp://phil.cdc.gov/phil/home.asphttp://phil.cdc.gov/phil/home.asphttp://en.wikipedia.org/wiki/Centers_for_Disease_Control_and_Preventionhttp://upload.wikimedia.org/wikipedia/commons/1/16/Varicella_(Chickenpox)_Virus_PHIL_1878_lores.jpg
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    Genomul este ADNds, liniar, asamblat ca un toroid care circularizeaz cu usurint.Fiecare spir ADN contine dou componente L (long) si S (short) mrginite desecvente repetate inverse. Componentele L si S pot face rocada producnd formelineare izomere care explic ntre altele latenta

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    HERPESVIRIDAE

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    ALPHAHERPESVIRINAE- Neuronii senzitivi din rdcinaposterioar a nervilor spinali si nucleii senzitivi ai nervilor cranieni

    HERPES SIMPLEX 1 (HSV1)

    HERPES SIMPLEX 2 (HSV2)

    VARICELO-ZOSTERIAN (VZV)

    BETAHERPESVIRINAE-Limfocitul T, monocite, organe solide

    (plamni, rinichi) +/- epiteliul glandelor salivare CITOMEGALIC (CMV)

    HUMAN HERPESVIRUS 6 (HHV6)

    HUMAN HERPESVIRUS 7 (HHV7)

    GAMAHERPESVIRINAE-Limfocitul B, endoteliu capilar EPSTEIN BARR (EBV)

    HUMAN HERPESVIRUS 8 (HHV8)-KAPOSI ASSOCIATED

    gp C=receptori-

    glicosaminoglicani

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    glicosaminoglicani

    gpD =co-receptori

    (nectine, HVEM-herpes

    virus entry mediator, 3OS

    heparansulfat -formamodificata a heparan sulfat sub

    influenta 3-O sulfotranferaza

    enzima prezenta in creier)

    -TEGUMENT-

    -VHS virion host shut-offprotein (de decuplare asintezei proteinelor celulare)

    -factor de transcripie virional

    (VP16 sau alfa TIF -alfa trans-

    inducing factor)-proteina "DNA-binding"Oct-1 n celulele nouinfectate - activarespecifica a unor promotorivirali

    ADN-ul viral ---ARN polimeraza II celulara----sinteza ARNm pt 3 specii de proteine:

    alfa (IE)si beta (E) (reglatorii, enzimatice, timpurii) si gama (L) (structurale, tardive).

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    alfa (IE)si beta (E) (reglatorii, enzimatice, timpurii) si gama (L) (structurale, tardive).

    Sinteza ADN- Rolling circleconcatemeri clivati in

    monomeri in cursul asamblarii nucleocapsidei

    Deficiente in transportul-factor de transcripie virional (VP16/ alfa TIF-trans-inducing factor)

    -proteina "DNA-binding" Oct-1 n celulele nou infectate - activare

    LATENTA

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    moleculelor alfa-TIF dincitoplasma catre nucleulneuronilor -blocheaza

    infectia HSV productiva

    Fixarea unor proteinecelulare cu actiunerepresoare, la nivelulsecventelor din ADN-ul viral

    care controleaza expresiagenelor .

    Reducerea sau absentaexpresiei proteinelor celularenecesare activarii genelor .

    Prezenta de proteine careinhiba activitatea alfa-TIF.

    proteina DNA binding Oct 1 n celulele nou infectate activare

    specifica a unor promotori virali

    Expresia diferentiata a proteinelor celulare implicate in reglarea expresiei genelor virale

    reprezinta factorul major care hotareste daca un neuron infectat va evolua catre o infectie

    latenta sau productiva.

    LATENTA

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    - Dou gene IE(ICP 4 si ICP 27) conditioneaz propagarea HSV 1 numai n celule

    permisiv, fiind transactivatori ai genelor asociate cu infectia litica.

    - O singura zona a genomului viral este functionala in timpul latentei- situata in

    secventele repetitive ale genomului si codifica un numar inca necunoscut de molecule

    de ARN, denumite LATs (Latency Associated Transcript) , care nu vor fi traduse in

    proteine

    Expresia LAT in neuroni inhiba expresia unor gene litice (ICP4 si timidin kinaza),

    prin modificari ale histonelor ce convertesc portiuni din ADN viral in forme

    neproductive

    Elemente din jurul genei pt ICP4 leaga o proteina - NRSF- factor de silentiere

    neuronala restrictiva (element represor al transcriptieiREST Repressor Element

    Silencing Transcription Factor)- duc la blocarea unor gene imediat timpuriistabilirea latentei.

    Forme truncate ale NRSF sunt produse in neuroni si controleaza selectiv expresia

    unor canale pentru neurotransmitatori

    LAT interfera cu mecanismele naturale ale mortii celulare

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    GINGIVOSTOMATITA HERPETICA HERPES FACIAL

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    HERPES GENITAL

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    HERPESNEONATAL

    Episoade recurenteadulti imunocompetenti:

    F -20% (8-80%); M-15% (5-50%)

    Excretie virala asimptomatica(EVA)-75% din recurente

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    1988 -PREMIUL NOBEL PENTRU MEDICINA

    NOI PRINCIPII TERAPEUTICE

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    PO4PO4PO4

    ACYCLOVIRAnalog nucleozidic purinic aciclic , derivat din guanin- activ informa trifosforilata

    NOI PRINCIPII TERAPEUTICE

    Sir James W. Black, Gertrude B. Elion , George H. Hitchings

    S i h l i it l i t i l i

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    Supresia herpesului genital prin terapia cu acyclovir:

    Numarul mediu de episoade de herpes genital per

    persoana per an

    VARICELA

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    VARICELA (LEZIUNI PROGRESIVE, IN VALURI- MACULE, VEZICULE, CRUSTE)-vs RUJEOLA(ERUPTIE MACULO-PAPULARA)

    RUJEOLA (ERUPTIE MACULO-PAPULARA CUFEBRA, RINOREE, CONJUNCTIVITA, DIAREE)

    VACCIN VIU ATENUANT ANTI VARICELOSadministrare la 19-35 luni

    VACCIN VIU ATENUANT MMRmeasles,mumps, rubella(rujeola, oreion, rubeola)

    administrare la 12 luni

    Reactivarea virusului varicelo- zosterian latent = zona zoster- eruptie herpetica pe

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    traseul unui nerv

    BETAHERPESVIRINAECITOMEGALIC (CMV)

    HUMAN HERPESVIRUS 6 (HHV6)

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    HUMAN HERPESVIRUS 6 (HHV6)

    HUMAN HERPESVIRUS 7 (HHV7)

    Transmiterea materno-fetal a CMV majoritar la primoinfectia in sarcina:

    Prenatal-teratogen determin malformatii congenitale severe (microcefalie, retard mental,chorioretinit, anomalii osoase);

    Perinatal - determin boala cu incluzii citomegalice a nou nscutului (hepatosplenomegalie,

    icter, anemie hemolitic);

    Postnatalinfectie asimptomatic sau asemntoare mononucleozei

    Citomegalie si incluziiintranucleare tipice in ochi de

    bufnita

    INFECTIA CU CMV

    http://upload.wikimedia.org/wikipedia/commons/b/b1/CMV_placentitis2_mini.jpg
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    La imunosupresati infectii primare si reactivari simptomatice:

    CMV POST-TRANSFUZII: mononucleosis-like

    CMV +SIDA: RETINITE/ encefalopatii CMV POST- TRANSPLANT :

    PNEUMONIEletalitate ridicata>85%

    Hepatite, colite, encefalite

    GANCICLOVIRCytovene

    CIDOFOVIR- RETINITA CMV

    FOSCARNET

    TRATAMENT

    MONONUCLEOZA INFECTIOASA

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    EPSTEIN BARR (EBV)- GAMAHERPESVIRINAE

    LATENTA- EXPRESIA PROTEINELOR

    VIRALE TIMPURII:

    FACTORI DE CRESTERE AUTOCRINI PT.

    LIMFOCITELE B

    RECEPTORI AI FACTORILOR DE

    CRESTERE

    ACTIVATORI AI UNOR ONCOGENE

    CELULARE

    HUMAN HERPES VIRUS 8 (HHV8)- SARCOMUL KAPOSI- cancerl l d t li l

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    celule endoteliale

    MAI MULTE INFORMATII

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    MAI MULTE INFORMATII

    http://www.ashastd.org/- American Social HealthAssociation. http://www.gotherpes.com/ http://www.famvir.com/ si http://www.herpeshelp.com/

    GlaxoSmithKline si Novartis.

    http://www.ihmf.org/ International Herpes ManagementForum. http://www.herpesdiagnosis.com/

    http://www.focusdx.com/focus/splash/splash.asp http://www.webmd.com/

    http://www.herpeshomepage.com/

    http://www.ihmf.org/http://www.ihmf.org/http://www.herpesdiagnosis.com/http://www.ihmf.org/http://www.herpesdiagnosis.com/http://www.westoverheights.com/http://www.herpeshomepage.com/http://www.herpeshomepage.com/http://www.herpeshomepage.com/http://www.herpeshomepage.com/http://www.webmd.com/http://www.westoverheights.com/http://www.herpesdiagnosis.com/http://www.herpesdiagnosis.com/http://www.ihmf.org/http://www.ihmf.org/http://www.gotherpes.com/
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    DIAGNOSTIC DE LABORATOR INHEPATITE DE ETIOLOGIE VIRALA

    ALGORITM DE DIAGNOSTIC IN HEPATITE

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    ALGORITM DE DIAGNOSTIC IN HEPATITE

    1. Tablou clinic

    2. Teste biochimice Markerii necrozei hepatocitare (transaminaze, LDH)

    Markeri colestaza (FAL, GGT, bilirubina)

    Markeri ai incapacitatii de sinteza la nivel hepatic (proteine serice, colesterol,trigliceride)

    3. Date epidemiologice: cale transmitere, contactinfectant, per. incubatie, vaccinuri

    4. Diagnostic etiologic = detectia markerilor virali: antigene virale (ag)

    anticorpi antivirali (anti)

    acid nucleic viral

    Hepatita B

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    Hepatita B

    Hepatita acuta

    Hepatita cronica persistenta

    Hepatita cronica agresivaCiroza hepatica

    Carcinom hepato-celular

    ALGORITM

    Sisteme antigenice VHB

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    Sisteme antigenice VHB

    Ag HBs Ac antiHBS

    Ag HBc Ac antiHBc

    Ag HBe Ac antiHBe

    Infectia acuta cu VHB

    evolutie spre vindecare

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    SimptomeAgHBeADN/VHB

    anti-HBe

    anti-HBc totali

    anti-HBc IgM anti-HBsAgHBs

    0 4 8 12 16 20 24 28 32 36 52 100

    evolutie spre vindecare

    Saptamini post expunere

    Infectia cronica cu VHB

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    IgM anti-HBc

    Total anti-HBc

    HBsAg

    Acute

    (6 months)HBeAg

    Chronic

    (Years)anti-HBe

    0 4 8 12 16 20 24 28 32 36 52 Years

    Weeks after Exposure

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    ELISA de detectie a Ag viraleELISA de detectie a Ac antivirali

    Tehnica ELISA sandwich

    1. Incubare ser pacient 1h la 37C

    ELISA de detectie a Ag virale

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    1. Incubare ser pacient 1h la 37 C2. Spalare3. Incubare CONJUGAT 30min la 37C

    4. Spalare5. Incubare SUBSTRAT 20 min la t.camerei6. Citire la spectrofotometru => D.O., MN, MP, C.O.

    Ag HBs

    Anti HBs

    Anti HBs cuplat cuo enzima =CONJUGAT

    SUBSTRAT+

    EVOLUTIA MARKERI DE INFECTIE VHA

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    0 1 2 3 4 5 6 7 8 9 10 11 12 13

    saptamana

    simptomatologie

    ALT

    IgM

    IgG

    VHA in MFInfectie

    Viremie

    Diagnostic hepatita acuta A = detectie Ac anti VHA IgM (ELISA de captura)

    ELISA de captura

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    p

    Anti-IgM

    IgM

    Ag marcatenzimatic =conjugat

    ETAPE

    1. Incubare ser pacient 1h la 37C

    2. Spalare

    3. Incubare conjugat 30 min la 37C

    4. Spalare

    5. Incubare substrat 20 min la t. camerei

    6. Citire la spectrofotometru

    SUBSTRAT

    +

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    HEPATITE VIRALE

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    AInfectioasa

    Serica

    HEPATITA

    VIRALA

    Transmitere

    Enterica

    TransmitereParenterala

    E

    NANB

    B D

    C

    Virusuri hepatitice F,GCoxackie, EBV, CMV, arbovirusuri

    VIRUSUL HEPATITEI A PICORNAviridae; gen HEPARNA-viridae

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    VHA este rezistent la- cldur,- pH acid,- alcool-ether.Inactivare- fierberela > 85 oC, cel putin 3minute

    CONCENTRATIA VHA IN DIFERITE FLUIDE

    CORPORALE

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    Soursa: Viral Hepatitis and Liver Disease 1984;9-22

    J Infect Dis 1989;160:887-890

    Materii fecale

    Ser

    Saliva

    100 102 104 106 108 1010

    TITRUL/ml

    Digestiv

    Contact personal direct

    Sange (rar postransfuzional,

    IDU)(e.g., injection drug use, r

    Incubatie: medie 25 zile (limite 15-50 ) Icter:

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    0 1 2 3 4 5 6 7 8 9 10 11 12 13

    saptamana

    simptomatologie

    ALT

    IgM

    IgG

    VHA in MFInfectie

    Viremie

    y

    Complicatii: hepatita fulminanta; colestaza(rare)- vindecarecompleta regula in hepatita A

    In Romnia, peste 80% dincopiii sub 10 ani au trecutprin infectie

    profilaxie specific: vaccin inactivat

    profilaxie nespecific: igien personal sau gamaglobuline la contacti

    Virusul Hepatitei E- Calicivirus1983-IEM n preparat provenit de la unvoluntar care a fost infectat cu filtrat

    Incubatie: Medie 40 zile (15-60)

    Severitate creste cu varsta

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    voluntar care a fost infectat cu filtratde materii fecale de la un pacient cuhepatit enteric nonA nonB.

    Ulterior, virusul transmis la maimutecynomolgus- clonat, genomulsecventiat.

    Retrospectiv, probe arhivate prelevaten cursul unor epidemii hidrice -circulatia VHE din anii 50.

    Epidemii hidrice extinse n subcontinentul indian si n America Central

    Rata de atac: 1-15%; adulti (3 30%); copii (0.2-10%).Transmitere interpersonala minima

    Nu exista vaccin- eficienta Ig din sangele donatorilor din zone ne-

    endemice?

    Severitate creste cu varsta

    Rata fatalitatii : 1%-3%;

    femei gravide 15-25%- avorturi sau nasteri premature: 1230%- moartea ftului in utero: 25%;-decesul mamei prin hepatit fulminant: 25%

    Hepatita Eo zoonoz ?

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    Un nou virus desemnat swine VHE a fost identificat la

    porci Porcii peste 3 luni din unele cresctorii prezint anticorpi,

    desi nu au semne clinice. Porcii

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    GENOMUL VHB- ADNpartial ds,

    partial circularizat

    Genomul VHB este ADN partial dublu catenar cu o

    spir lung (L, negativ) si una scurt (S, pozitiv).

    Pe spira lung se gsesc genele ce codific

    proteinele VHB:

    Antigenul de suprafa AgHBs mediaz atasarea la

    receptorii celulari si este implicat n

    virusneutralizare- Codificat de gena S- cu trei

    componente: preS1+preS2+S;Antigenul de centru AgHBc (gena c) care prin

    clivare produce AgHBe (marker de infectivitate);

    Reverstranscriptaza , ADN polimeraza, si

    proteinkinaza VHB(gena P-pol)

    Factori transactivatori ai unor gene celulare (gena

    x, implicat n cancerizare).

    Genomul ADN partial ds, partialcircular cu dimensiuni mici (3200nucleotide) este foarte eficient

    organizat. Prezenta orf suprapusepermite codificarea a 50% proteine

    n plus, n raport cu ceea ce ar fiteoretic posibil.

    CICLUL REPLICATIV VHBDup completarea secventei spirei scurte, pe matrita ADN ds se sintetizeaz o copie ADN circular relaxat (DNAccc - Dclosed covalent circular) care este transferat n nucleu n form episomal, neintegrat cromozomial. Pe acest ADN sesintetizeaz un ARN relaxat numit ARN pregenomic Acesta serveste fie ca ARNm pentru sinteza proteinelor virale fie c

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    Adapted from Lai CL, et al. J Med Virol. 2000;61:367-373.

    Virion VHB infectios

    ADN partial ds

    Completarea spireiscurte de catre ADN

    polimeraza virala

    Particule subvirale

    Hepatocyte

    mRNA

    Citoplasma

    NucleuPrecore/core

    HBeAg

    ER

    HBcAg

    HBsAg

    cccDNA

    ADNss-

    ARN mpregenomic incapsidat

    cccDNA, covalently closed

    circular DNA

    sintetizeaz un ARN relaxat, numit ARN pregenomic. Acesta serveste fie ca ARNm pentru sinteza proteinelor virale, fie cmatrit pentru reverstranscriptaza care va sintetiza genomul progen.

    Caracteristic pentru VHB este sinteza

    disproporionat ntre particulele virale complete

    (particulele Dane) cu genom ( 108

    ) si cele vide (1014)

    .

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    In serul bolnavilor se gsesc diferite forme ale VHB: particulele omogene, sferice de 42 nm

    care reprezint virionul complet (numite particule Dane) si particule neomogene, sferice saufilamentoase, (cu diametrul de 22 nm) constituind structuri lipsite de ADN si de infectivitate.

    Concentratia VHB in fluide

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    INALT Moderat Scazut/

    nedetectabil

    Sange Lichid spermatic Urina

    Ser Secretie vaginala Materii fecale

    Exudat rani Saliva Gl. sudoripare

    Lacrimi

    Lapte matern

    corporale

    CAI DE TRANSMITERE VHBParenteral Sexual

    Perinatal

    Contact

    100100

    Evolutia hepatitei B in functie de varsta

    Incubatie: medie 75 zile (45-180)

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    Simptomatologie

    Cronicizare

    Varsta la achizitia infectiei

    Infectia cronica (%) Infectia Simptomatica (%)

    nastere 1-6 luni 7-12 luni 1-4 ani Copii mari si adulti

    0

    20

    40

    60

    8080

    60

    40

    20

    0

    ( )

    Icter:

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    Unul din urmtoarele patru criterii pentru diagnostic:

    persistenta AgHBs >6 luni,

    valori ale ncrcrii virale >104

    copii/ml ADN/VHB, transaminaze persistent sau intermitent crescute,

    scor Knodell >4 pentru activitatea necroinflamatorie pe biopsia hepatic.

    Immuno-

    toleranta

    hepatita B

    cronicaAgHBe+

    Purtator inactiv

    Ag HBs

    hepatita B cronica

    AgHBe-(Mutante Precore )

    AgHBs + + + +

    AgHBe + +

    Anti-HBe + +

    ALT Normal Normal

    ADN VHB> 20,000 IU/mL

    (> 105copii/mL)

    > 20,000 IU/mL(> 105

    copies/mL)

    < 200 IU/mL(< 103

    copies/mL)

    > 2000 IU/mL(> 104 copies/mL*)

    HistologieNormal/moder

    Activa Normal Activa

    Fazele infectiei cronice cuVHB

    eliminarea unor subseturi delimfocite Tc sensibilizate (prinantigene care induc tolerant)blocarea functiilor LTc si APC prinsinteza exagerat de IL10

    Pierderea efectului tolerogenprecipit liza imun indus ahepatocitelor infectateGenerarea limfocitelor Tcitotoxice specifice infectieiVHB it li i i

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    HBeAg+ HBeAg-/anti-HBe+ (precore/core promoter variants)

    ALT

    HBV DNA

    Normal/mild

    CHInflamatie Moderate/severe CHNormal/mild CH

    Cirrhosis

    Immune

    Tolerance

    Immune

    Clearance

    Low Replicative

    Phase

    Reactivation

    Phase

    Cirrhosis

    < 2000 IU/mL

    > 2000 IU/mL

    Inactive cirrhosis

    2 x 108

    -2 x 1011 IU/mL

    Slide courtesy of A. S. F. Lok, MD.

    200,000 - 2 x 109 IU/mL

    Inactive-carrier state HBeAg-chronic hepatitis

    HBeAg+chronic hepatitis

    viremia nalt nu se asociaz cutransaminaze crescute

    lipsa citopatogenittii VHB.

    VHBcitoliza- mecanism imunmediat

    Hepatita B Cronica activa, cu AgHBe negativ la pacienti cu mutante precore/core

    Variantele precore previn sinteza AgHBe

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    p p g

    - sunt determinate de mutatia nonsens G1896A inlturun semnal indispensabil ncapsidrii

    Mutantele core sintetizeaz numai un produs truncat

    care nu este detectat de trusele ELISA pentru

    AgHBe/antiHBe. Cele mai frecvente mutatii sunt: A1762Tsi G1764A. Mutatiile duble reduc si productia viral cupeste 20%.

    Variantele core promoterprevin initierea traducerii

    Sunt determinate de mutatii punctiforme la codonul start

    ATG. In majoritatea cazurilor, devin prevalente nc

    nainte de seroconversia anti HBe

    6-15% -remisiuni spontane definitive (rata anual 0,5%)

    Prognosticul mai rezervat dect n hepatitele cronice AgHBepozitive att n ceea ce priveste probabilitatea cirozei ct si acarcinomului hepatic primitiv (CHP).

    Evolutia Hepatitei B400 milioane infectii

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    1. Hepatita cronica persistentaasimptomatica

    2. Hepatita cronica Activasimptomatica

    3. Ciroza hepatica

    4. Carcinom Hepatocelular

    cronice

    500 000- 1 milion

    decese/an

    10% HIV/ HBV

    coinfectii

    Prevalentahepatitei B cronice

    2 miliarde de infectii curente sau vindecat4 milioane infectii acute/an300-400 milioane infectii cronice

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    HBsAg Prevalence

    > 8% - High

    2-8% - Intermediate

    < 2% - Low

    Numarul imigrantilor pe fiecare continent intre 1996-2006

    ~ 2 million Asians

    ~ 400,000

    South Americans

    ~ 350,000Africans

    ~ 930, 000

    Europeans

    Centers for Disease Control. Hepatitis B fact sheet. Available at: http://www.cdc.gov/hepatitis. 2008. Mahoney FJ. Clin Microbiol Hepatitis B statistics1. WHO. Hepatitis B. 2002. 2. Maynard JE, et al. In: Viral Hepatitis and Liver Disease. New York: Alan R. Liss, Inc. 1988. 3. CDC. Epidemiology &prevention of vaccine-preventable diseases. The Pink Book. 8th ed. 4. CDC. MMWR. 2001;50:RR-11

    300 400 milioane infectii cronice25% decedeaza datorita cirozei sau CHP - 1 milion dedecese /an

    60-80% din cauzele de CHP

    OMS global-VHB a 10-a cauza demortalitateVHB este al doilea agent carcinogen dupatutunVHB este de 100 ori mai contagios ca HIV

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    ncetinirea progresiei fibrozei ctre ciroz, prevenirea insuficientei hepatice, prevenirea

    aparitiei CHP

    TRATAMENTUL CU ANTIVIRALE N HEPATITA B CRONIC

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    aparitiei CHP

    Hepatit cronic AgHBs +

    Transaminaze peste 2xLSN Trateaz

    AgHBe Prezent Trateaz

    ADN VHB >1000 copii Trateaz

    Fibroz Prezent Trateaz

    Formele replicative ale genomului viral rmn n nucleul hepatocitelor infectate sub

    forma ADN ccc, - recderi la ntreruperea terapiei sau n conditii de imunosupresie.

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    DESCOPERIREA VHC 1989- Clonare molecular- Choo Q-L., Science244, 359-62

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    Adsorbtiacomplex de receptori: CD 81, receptorul scavenger B I (SR-B I); moleculelede adeziune DC-SIGN Si LSIGN, claudina 1 si receptorii pentru LDL- Susceptibilitatea

    VIRUSUL HEPATITIC C- FLAVIVIRIDAE-

    HEPACIVIRUS

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    Traducere IRES mediata -

    procesarea unui precursorpoliproteic larg clivat ulterior depeptidaze semnal SP si proteaze

    virale in 10 proteine virale .

    , p p p

    crescut a vrstnicilor cu leziuni de ateroscleroz

    Replicarea VHC - n zone

    alterate ale endoteliuluicitoplasmic - alctuiesc

    reteaua /nodul replicativ

    (membranous web);morfologic determin incluzii

    intracitoplasmice cu aspect

    spongios.

    Prelucrarea posttraducerencomplexul replicativ -

    proteinele nonstructurale +

    proteinele celulei gazd.

    Eliberarea la nivelul polului apical al hepatocituluistructur cu bogat continut LDL- celulele depletate deinsulele membranare lipidice (rafturi) sunt slabproductive n particule virale infectante

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    Rata replicarii: 10 trilioane virioni/zi; Titruri plasmatice > 10 milioane copii ARN

    VHC/mlrata mutatiilor 105 per nucleotid per ciclu; la fiecare sit din genom produc 105 mutatii

    tif

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    punctiforme

    HETEROGENITATE VIRALA

    6 genotipuri majore -20-48% diferente

    Cvasispecii-SECVENTA CONSENS- 2-10%-diferente escape mutants

    proteaza HCV NS3/4A cliveaza moleculeadaptator ce intervin in caile desemnalizarea dependente de TLR si RIG I

    RISC REZIDUAL AL TRANSFUZIEI:

    Virus fereeastra serologica risc rezidual

    HTLV 51 il (32 72) 1 56/1 ili d

    Ecranarea antigenicitatii virale

    de lipoproteine plasmatice

    determina seroconversia

    t di t 4 i 10 t i

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    Virus si test Riscrezidualprin triajserologic

    Riscrezidualprin testareacizinucleici

    Reducereariscului

    CostPer test(USD)

    VHB -

    ADN/PCR

    15.83 9.12 42.4% 30

    VHCARN/Rt- PCR

    9.7 2.72 72.0% 200

    HIV - Ag p24

    2.03

    1.48 27.3% 5

    HIV - ADN/PCR 1.48 27.3% 50

    HIV

    1.01 50.0% 200

    Reducerea riscului rezidual asociat transfuziilor

    HTLV: 51 zile (32-72) 1,56/1 milion dedonatii

    HIV: 22 zile (6-38) 2,03/1 milion de

    donatii

    HCV: 82 zile ( 54-192) 9.7/1 milion dedonatii

    HBV: 75 zile (37-87) 15.8/1 milion de

    donatii

    tardiva, ntre 4 i 10 sptmni

    post-infectie. Aceast

    fereastr serologic mentineriscul transmiterii VHC prin

    transfuzii.

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    PATOGENIE- CLINICA

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    Incubatie medie 6-7 saptamani (2-

    26 )

    Icter 85%

    Hepatita cronica activa 10%-60%

    Ciroza 6 luni; Inflamatie/necroza

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    moderata/fibroza scazuta

    Factori predictivi pentru RSV:

    Genotip 2/3

    VL baseline

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    Rspuns tranzitoriu(dispariia ARN VHC la sfritul tratamentului, cu recderi ulterioare)- 10-25% din pacieni -retratarea acestorpacieni poate furniza uneori RSV, dar la doze mai mari i pe termen mai lung de administrare amedicamentelor.

    Absena rspunsului la tratament -30% din pacieni -chiar dac se nregistreaz scderea nivelului ncrcriivirale sub tratament nu se produce niciodatdispariia virusului din organism- recderea este regul.

    Denumire Criterii

    VEVR

    Rspuns virusologic foarte rapid

    Scadere >1.4 log10 HCV ARN la 24 ore ; recul viral dupa 7

    zile?

    RVR

    Rspuns virusologic rapid

    HCV ARN nedetectabil la 4 saptamani ; predictor RSV

    EVR

    Rspuns virusologic timpuriu

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    Imunomodulator Creste rata de mutatii

    VHC (viral fitness)

    Mutageneza letala aVHC

    Scade frecventareculului viral

    Accentueaza apoptozacelulelor infectate

    Antiviral

    Imunomodulator- stimuleaz activitatea citolitic a limfocitelor CD8

    - contribuie la limitarea accesului intralobular al

    limfocitelor implicate n rspunsul inflamator.

    cresterea markerilor de activare a macrofagelor

    (exemplu, CD 69);

    cresterea activittii celulelor NK si armareaperforinelor;

    adugarea ribavirinei creste rspunsul

    antiinflamator prin scderea citokinelor IL-2 si IL-

    12 si cresterea IL-10.

    PEG-IFN alpha-2a (PEGASYS;Roche)-180 godat pe sptmn.

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    VIRUSUL HEPATITEIDELTA = virus

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    HBsAg

    RNA HDV

    antigen

    DEFECTIV

    1977 Mario Rizzetto

    Coinfectie VHB - VHDBOALA ACUTA SEVERARISC SCAZUT DE CRONICIZARE

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    Ac anti-

    HBs

    Simptome

    ALTcrescute

    Ac anti-VHDtotali

    IgM anti-HDV+ IgM antiHBc

    HDV RNA

    AgHBs;ADN VHB

    Timp dupa

    Titru

    RISC SCAZUT DE CRONICIZAREPROFILAXIA HEPATITEI B

    SUPRAINFECTIA VHB - VHDBOALA ACUTA ?RISC CRESCUT DE CRONICIZARE INFORME SEVERE

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    Icter

    Simptome

    ALTAc Totali anti-HDV + anti HBc

    IgG

    IgM anti-HDV

    ARN VHD

    AgHBs

    Timp dupa

    Titru

    FORME SEVERETRATAMENT VHB/REDUCERE RISC

    TRANSMITERE VHD

    Asociatia American pentru studiul bolilor hepatice

    http://www.aasld.org/netFORUMAASLD/eweb/docs/update

    http://www.aasld.org/netFORUMAASLD/eweb/docs/updatehttp://www.aasld.org/netFORUMAASLD/eweb/docs/update
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    Centrul pentru Controlul si Profilaxia Bolilor CDCAtlanta

    http://www.cdc.gov

    Clinical Care Options for Hepatitis

    http://www.clinicalcareoptions.com/hepatitis.aspx

    Medscape Hepatitis Resource Center

    http://www.medscape.com/resource/hbv

    Projects In Knowledge, Advanced Certificate Program in the Management

    of Chronic Hepatitishttp://www.projectsinknowledge.com/1712

    Viral Hepatitis Serology A-E: CDC-sponsored

    http://www.cdc.gov/ncidod/diseases/hepatitis/serology/

    http://www.cdc.gov/ncidod/diseases/hepatitis/slideset/index.htmhttp://www.clinicalcareoptions.com/hepatitis.aspxhttp://www.medscape.com/resource/hbvhttp://www.projectsinknowledge.com/1712http://www.cdc.gov/ncidod/diseases/hepatitis/serology/http://www.cdc.gov/ncidod/diseases/hepatitis/serology/http://www.projectsinknowledge.com/1712http://www.medscape.com/resource/hbvhttp://www.clinicalcareoptions.com/hepatitis.aspxhttp://www.cdc.gov/ncidod/diseases/hepatitis/slideset/index.htm
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    Hepatologie 2009www.HepatologyTextbook.comVersiunea in limba romana-

    http://www.fb4d.com hepatologie 2009

    Versiunea in limba romana.

    Traducerea a fost asigurata de

    colectivul Disciplinei de Virusologie,

    UMF Carol Davila

    Institutul de Virusologie St. S. Nicolau

    285, sos. Mihai Bravu, Bucuresti RomaniaTel/fax +40213242590

    http://www.fb4d.com/http://www.fb4d.com/
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    DIAGNOSTIC SEROLOGIC

    1. DIAGNOSTIC SEROLOGIC IN INFECTIA HIV/SIDA

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    TESTE DE TRIAJ- sensibilitate inaltaELISA de detectie a Ac antiviraliTESTE RAPIDE

    TESTE DE CONFIRMARE- specificitate inalta

    WESTERN BLOTRADIOIMUNOPRECIPITAREA (RIPA)IMUNOFLUORESCENTA INDIRECTA

    2. DETECTIA DIRECTA A VIRUSULUI SAU A

    PRODUSELOR VIRALEIZOLARE VIRALA

    DETECTIA ANTIGENEMIEI p24 ELISA de detectie a Ag virale

    DETECTIA ACIDULUI NUCLEIC VIRAL

    ELISA de detectie a anticorpilor antivirali

    TEHNICA INDIRECTA

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    ETAPE

    1. Incubare ser pacient 1h la 37C

    2. Spalare

    3. Incubare conjugat (Ac anti IgG uman x enzima)- 1h la 37C

    4. Spalare

    5. Incubare substrat 20 30 min la temp camerei

    6. Citire la spectrofotometru DO probe, martori, cut off, zona gri

    ELISA de capturadetectia de Ac de clasa IgM

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    Ac Anti-IgM

    IgM

    Ag marcatenzimatic =

    conjugat

    ETAPE

    1. Incubare ser pacient 1h la 37C

    2. Spalare

    3. Incubare conjugat 30 min la 37C

    4. Spalare

    5. Incubare substrat 20 min la t. camerei

    6. Citire la spectrofotometru

    TESTE DE CONFIRMARE

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    Tehnica

    WESTERN BLOT

    1. SDS-PAGE

    2. BLOT (transfer)

    3. DETECTIA ANTICORPILOR

    WESTERN BLOT detectia Ac serici

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    Substrate

    Stop

    color

    Antiviral antibody

    Western blotin infectia HIV

    gp160

    gp120

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    gp41

    p66

    p31

    p24

    p17

    p10/11

    p7/9

    p51

    p55

    Criteriul minim de

    pozitivitate

    2 env

    +

    1 gag

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    Test imunoblot pt

    confirmarea

    infectiei VHC

    RIBA

    (Recombinant

    Imunoblotting Assay)

    DIAGNOSTIC SEROLOGIC IN INFECTIA HIV

    ELISA

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    POZITIV IN DUPLICAT

    WESTERN BLOT

    NEGATIV=

    ABSENTAINFECTIEI HIV

    ! Ferestraserologicarisc rezidual al

    transfuziei = 2/1 milion

    NEGATIV

    INDETERMINAT

    - repeta la 6-8 saptamini- alt test de confirmare

    POZITIV=

    INFECTIE HIV

    Ag p24- 1.48/1 milionADN proviral1.48/1 milionARN HIV 1.01/1 milion

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    HIV/SIDA

    173

    Human Immunodeficiency Virus

    Acquired immunodeficiency syndrome

    Sindrome dimmunodeficience aquise

    rem u o e pen ru e c na sau z o og e

    2008

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    174

    Franoise Barr-

    SinoussiLuc Montagnier

    1/4 of the prize 1/4 of the prize

    Regulation of

    Retroviral Infections

    Unit, Institut

    Pasteur Paris,

    France

    World Foundation for AIDS

    Research and Prevention

    ,Paris, France

    b. 1947 b. 1932

    1981- primele cazuri AIDS (sindrom de imunodeficienta umana dobandita ) raportate inSUA la homosexuali tineri, anterior sanatosi

    1981 83 d i AIDS b l i l l (h i h l)

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    175

    1981-83- grupe de risc, AIDS- boala transmisa parenteral, sexual (hetero si homosexual),

    materno-fetal1983 - se stabileste etiologia virala a bolii prin izolarea virusului - tulpina numita LAV(Lymphadenopathy Associated Virus) - Luc Montaigner, Institutul Pasteur din Paris / tulpinanumita HTLV III (Human T-cell Leukemia Virus III) - Robert Gallo, Bethesda, SUA/tulpinaARV AIDS associated retrovirus (J. Levy, San Francisco, SUA)

    1985cele 3 tulpini sunt demonstrate a apartine aceluiasi virus redenumit HIV (HumanImunodeficiency Virus), Familia RETROVIRIDAE, GEN LENTIVIRINAE

    1985 - sunt livrate primele truse de diagnostic imunoenzimatic

    1986 - se descopera HIV2 (Africa de Vest)

    1998secventierea prin Rt-PCR celei mai vechi probe de plasma HIV1 pozitivacunoscuta -recoltata n 1959, de la un barbat din Congo.

    Argumente SIV-HIV:

    Similitudini in organizarea

    genomului

    Inrudire filogenetica

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    176

    Inrudire filogenetica

    Prevalenta in gazda

    naturala

    Areal geografic

    Cai plauzibile de

    transmitere

    1992 - 1993 - variantele genomice ale HIV1-M sunt ncadrate n 8 clade (A-H), Media distanteiintercladale este de aproximativ 27%, izolatele din aceeasi clada nu se deosebesc decit prin

    11% din nucleotide

    Camerun; Gabon,Guineea Ecuatoriala

    HIV-1 NCamerun

    1999 - se descopera rezervorul natural

    al HIV1SIV cpz- provenit de la o

    subspecie din Africa Centrala, Pan

    troglodites troglodites

    2006- identificare v. la cimpanzei care

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    177

    3 transferuri distincte ale

    SIV cpz de la aceeasi

    specie:

    Pan troglodites trogloditesGao.F et al, Nature, 1999, 397, 436-41Weiss RA,

    Wrangham RW, Nature, 1999, 397, 385-6.

    Keele, BF, 2006 Science www. sciencexpress.org

    p

    traiesc in salbaticie in sud Camerun

    Gp160-precursor

    Gp120-

    GENOMUL HIV:1.Gene structurale:env (anvelopa-gp120, gp41)gag :antigene de grup: (capsida, matricea, nucleocapsida;)pol: cx polimerazic-(reverstranscriptaza, integraza, proteaza)

    l ii f

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    178

    superficiala

    Gp41-transmembranara

    P66-Rt

    P32-proteaza

    P24- capsida

    P17- matricea

    p10/11-integraza

    p7/9- nucleocapsida

    P55-Rt

    Western

    2.Gene reglatorii(rev, tat, nef)3. Gene accesorii (de maturare)

    CELULE TINTA IN INFECTIA HIV- celule CD4 pozitive :

    limfocitele T helper

    macrofage

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    macrofage

    celule foliculare dendritice (CFD)

    179

    CELULE TINTA IN INFECTIA HIVCelule murine transfectate cu CD4 nu sunt infectate

    cofactor in celulele umane

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    180

    Co-Receptori?

    CD8+ Cells

    MIP-1 alpha MIP-1 beta RANTESChemokine

    Blocare infectie HIV -Co-Receptori=Receptori pentru chemokine

    CCR5 Macrofage CXCR4

    limfocite

    celule Th de memorie din tesuturi nonlimfoide- celule Th nave si de memorie

    Tropism HIV

    Tulpini R5

    M-tropeTulpini

    Tulpini X4

    T-trope

    Asociate cu progresia

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    Macrofage

    CD4+ CCR5+

    Linii celulare TCD4+ CCR5+/CXCR4+

    Limfocite TCD4+ CXCR4+

    M-trope

    Asociate cu transmiterea

    HIV

    R5/X4

    Dual-trope

    Asociate cu progresia

    bolii

    Forma modificata a CCR5- Deletie 32 bp

    Alela prezenta in forma homozigota- pacienti expusi dar neinfectati HIV

    Heterozigota- supravietuitori de lunga durata (non-progresori, lent progresori)

    Polimorfism populational

    In stadiile timpurii ale infectiei, tulpini:

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    182

    p p

    Macrofagotrope

    Nonsincitizante

    rata replicativa scazuta

    In stadiile tardive ale infectiei, tulpini:

    limfotrope

    sincitizante

    rata replicativa inalta

    Ciclul Replicativ viral: faza de eclipsa

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    183

    CRESTEREA LOGARITMICA

    NUCLEU

    In fiecare zi circa 10 miliarde de virioni

    Pol II rata mare de erori 1:2,000-10,000

    vp

    r

    Rata mare de erori a Rt-azei:

    3x10-5 mutatii/ nucleotid/ ciclu

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    184

    INTEGRAZA

    LTR

    ARN polimeraza ADN

    dependenta -POLII

    CITOPLASMA

    genom HIV 9749 nucleotidesFIECARE virion progen poate avea cel putin o

    mutatie!

    r

    Tat, rev

    replicativ

    Rata mare de recombinari in

    cursul reverstranscrierii

    ROLUL PROTEINELOR REGLATORII SI ACCESORII

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    NEF interactioneaza cu proteine celulare implicate insemnalizarea celulara permitand supravietuirea pe

    termen lung a LTh infectate si la promovarea

    apoptozei LT neinfectate

    VIF Interactioneaza cu citozin-

    deaminaza celulara

    APOBEC3G

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    186

    AADDNN pprroovviirraalleverstranscriptaza

    virala

    AARRNN HHIIVV

    PROTEINE

    ADN

    celular

    integraza

    COFACTORI

    LTR

    HIV

    Proteine celulare implicate in

    replicarea HIV:

    -TRIM5 (TRIpartite Motif) laprimate intervine in degradarea cx de

    reverstranscriere in proteazomi

    -APOBEC3G citozin deaminaza

    converteste citozina in uracil in curs

    MATURARE- ASAMBLARE-

    ELIBERARE

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    187

    Maturarea-Clivarea precursorilor polipeptidici sub

    influenta proteazei viraleEliberare prin

    inmugurire

    TINTE ALE MEDICAMENTELOR ANTIRETROVIRALE

    Inhibitori de RT

    NRTI

    NNRTI

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    188

    NNRTI

    INHIBITORI DE

    INTEGRAZA

    INHIBITORI DE

    PROTEAZA

    INHIBITORI

    ATASARII DE

    CORECEPTORI

    SI INHIBITORII

    INTERNALIZARII

    3 CLASE MAJORE DE INHIBITORI AI FAZEI DE ECLIPSA

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    Inhibitori CD4??-neaprobati pt

    utilizare- in curs de

    testare

    Inhibitori ai

    corecptorilor CCR5

    Inhibitorii Fuziunii

    NativeTrimer

    CD4 BindingENV binding siteexposure

    CoR Binding Fusionpeptide insertion?

    6-Helix BundleFormation Membranefusion

    Maraviroc (R5)

    Vicriviroc (R5)Fuzeon (Enfuvirtide)TNX-355

    gp120

    Variableloops

    gp41

    CD4

    INHIBITORII FUZIUNII

    FUZEON (ENFIVURTIDE)

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    Source: www.trimeris.com

    PRINCIPALELE CLASE DE ANTIRETOVIRALE FOLOSITE IN PRACTICA

    De prima linie:

    Analogii nucleozidici inhibitori ai reverstranscriptazei virale (NRTI) - compusi apartinndclasei dideoxinucleozidelor substante chimice formate dintr-o baza azotat si un rest glucidic a crui

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    clasei dideoxinucleozidelor, substante chimice formate dintr-o baza azotat si un rest glucidic a crui

    grupare hidroxil din pozitia 3' a fost nlocuit cu un alt substituent care nu permite participarea

    acestei structuri la legturi fosfodisterice. In celulele umane, analogii nucleozidici sunt fosforilati de

    enzimele celulare Mecanismele care stau la baza acestei activitti sunt:

    - blocarea elongatiei lantului ADN rezultat prin retrotranscriere, datorit imposibilittii pozitiei 3' a

    dideoxinucleozidelor de a participa la legturi fosfodiesterice cu alte nucleozide;

    - competitia cu nucleozidele fiziologice la nivelul situsurilor de legare ale reverstranscriptazei.

    Inhibitorii nonnucleozidici ai reverstranscriptazei virale (NNRTI)sunt activi direct, fr anecesita fosforilare intracelular-blocheaz direct activitatea enzimei, fixndu-se la nivelul unui "buzunar"hidrofob, situat foarte aproape de situl catalitic al Rt

    Inhibitorii proteazei virale (PI)-blocheaza clivarea precursorului gag-pol n:- proteinestructurale gag(p24, p17, p7) si pol- enzime virale (RT-aza, intrgraza si proteaza p32 nssi).Sunt medicamente active ca atare-blocheaz maturarea si inhib infectivitatea virionilor

    progeni att n celulele infectate activ ct si n cele infectate cronic, penetrarea lorintracelular fiind bun (inclusiv n macrofage si celule foliculare dendritice - CFD).

    De rezerva- Inhibitorii fuziunii (Fuzeon), antagonistii coreceptorilor CCR5 (Maraviroc,

    vicriviroc) si inhibitorii integrazei (Raltegravir)

    191

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    Global estimates for adults and children, 2008

    Situatia globala a epidemiei HIV la 1 decembrie 2009 (date

    UNAIDS)

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    December 2009

    People living with HIV 33.4 million [31.1 35.8million]

    New HIV infections in 2008 2.7 million [ 2.4 3.0million]

    Deaths due to AIDS in 2008 2.0 million [1.7 2.4 million]

    Adults and children estimated to be living with HIV, 2008

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    December 2009

    Total: 33.4 million (31.1 35.8 million)

    Western &Central Europe

    850 000[710 000 970 000]

    Middle East & North Africa

    310 000[250 000 380 000]

    Sub-Saharan Africa

    22.4 million[20.8 24.1 million]

    Eastern Europe& Central Asia

    1.5 million[1.4 1.7 million]

    South & South-East Asia

    3.8 million[3.4 4.3 million]

    Oceania

    59 000[51 000 68 000]

    North America

    1.4 million[1.2 1.6 million]

    Latin America

    2.0 million[1.8 2.2 million]

    East Asia

    850 000[700 000 1.0 million]

    Caribbean

    240 000[220 000 260 000]

    Estimated number of adults and children

    newly infec