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Influenza Virus review Family Orthomyxoviridae: three types –Influenza A –Influenza B –Influenza C (not considered of critical importance) –Segmented (8)

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Presentation on theme: "Influenza Virus review Family Orthomyxoviridae: three types –Influenza A –Influenza B –Influenza C (not considered of critical importance) –Segmented (8)"— Presentation transcript:

1 Influenza Virus review Family Orthomyxoviridae: three types –Influenza A –Influenza B –Influenza C (not considered of critical importance) –Segmented (8) ssRNA genome with lipid envelop

2 Influenza A Further classified by Hemagglutinin (H) and Neuraminidase (N) sub-types Current circulating strains are H1N1 and H3N2 Human subtypes include H1N1, H3N2, H1N2, and H2N2 Avian subtypes include H1 to H15 and N1 to N9 Bird  human H5N1, H9N2, H7N7, H7N2, H7N3

3 Influenza B Produces less serious disease than does Influenza type A Not categorized as by H or N type as Influenza A is First isolated in 1949 Not known to be responsible for epidemics Influenza C

4 Influenza as a public health threat Influenza Viruses are the respiratory viruses of greatest public health importance, particularly Influenza A

5 Epidemiology, Prevention, and Control of Influenza; esp. Influenza A Why is Influenza A such a public health threat? Antigenic drift (variation within the HN sub- type) or Antigenic shift (variation between different HN sub-types) makes large portions of the population immunologically naïve on a regular basis Influenza epidemics can be characterized as inter-pandemic or pandemic

6 Global surveillance –Avian influenza: H5N1: HIGHLY PATHOGENIC Present in Asia since 1996 Extent/distribution not firmly established Threat level is high In 1997 there were 18 confirmed cases of H5N1 with 6 deaths Other avian strains being watched include H7N7 and H9N2

7 Pandemic Influenza –Recipe for a human pandemic Emergence of a novel sub-type of influenza to which the population is immunologically naïve Replication in humans  disease Efficient human-to-human transmission Note: H5N1 has meet all criteria except the third one.

8 Pandemic planning –An influenza pandemic will be unlike other public health emergencies or common disasters. Inevitable Will arrive with very little warning Locally explosive epidemics Widespread, not focused like a bio-terrorism event Will put an extraordinary strain on human and material resources Effect will be relatively prolonged –weeks to months

9 Laboratory issues Laboratory safety Tissue culture techniques Rapid test kits HA/HI sub-typing Immuno-fluorescent testing Real time PCR analysis –Molecular typing and sub-typing

10 Previous testing algorithm –Inoculation of specimens into cell culture; one diploid, one Hep-2, one Viromed Rhmk and two Diagnostic Hybrid Rhmk –In the absence of CPE, “blind” Hemadsorption (HAD) at days 7 and 14. –In the absence of CPE “blind” passage of Hep2 with “blind” FA for RSV at day 14 for all patients ≤5 years old –Identification of Influenza isolates: Immuno-fluorescent testing to identify type (A or B) followed by Hemagglutination Inhibition (HI) testing to identify sub-type

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13 FluAlert1000 SAS FluAlert (15 tests) The SAS Influenza A and B tests are visual and rapid assays for presumptive qualitative detection of Influenza A and B antigens from nasal washes and aspirates, respectively. This test is FDA Cleared and CLIAwaived. Use CPT CODE: 87804QW $ QDL Quidel QuickVue Influenza A/B-(25 tests) The QuickVue ® Influenza A+B test (dipstick format)allows for the rapid, qualitative detection of influenza type A and type B antigens directly from nasal swab, nasopharyngeal swab,nasal wash and/or nasal aspirate specimens. FDA Cleared and CLIAwaived $ INV BINAX NOW Influenza A/B (10 per kit) BINAX NOW Influenza A & B is an in vitro immunochromatographic assay to aid in the rapid differential diagnosis of influenza type A and B viral infections. 15-minute test for Influenza Types A& B w/ excellent sensitivity. FDA Cleared & CLIAwaived $ PRUEBAS RÁPIDAS

14 Immunofluorescence Direct – Ab to tissue Ag is labeled with fluorochrome Ag Fluorochrome Labeled Ab Tissue Section

15 Immunofluorescence Indirect –Ab to tissue Ag is unlabeled –Fluorochrome-labeled anti-Ig is used to detect binding of the first Ab. Ag Fluorochrome Labeled Anti-Ig Tissue Section Unlabeled Ab Qualitative to Semi- Quantitative

16 Figure A-17

17 PARTICIPANTES HA Y HI Flu virus AGLUTINACIÓN NO AGLUTINA Anticuerpo Eritrocitos

18 Hemaglutinación (HA) Flu virus No virus AGLUTINANO AGLUTINA

19 Hemagutinación para virus Diluciones del Flu virusNo virus AGLUTINAN NO AGLUTINAN

20 Inhibición de la hemaglutinación (HI) Flu virus Virus es bloqueado por Ac AGLUTINAN NO AGLUTINAN

21 HI para virus Antivirus espec í ficoS Ac H5N1 Ac H3N2 HI NEGATIVA hay aglutinación de eritrocitos HI POSITIVA hay inhibición por el Ac H3N2

22 Figure A-7 ELISA DE CAPTURA DE ANTÍGENO

23 Técnicas de amplificación de Ac. Nucleicos 1-PCR1-PCR 2-LCR2-LCR 3-NASBA, TMA3-NASBA, TMA 4-Amplificación de señal4-Amplificación de señal 5-Boomerang5-Boomerang

24 PCR 1-Qué es?1-Qué es? 2-En qué consiste?2-En qué consiste? 3-Cómo?3-Cómo? 4-Quién?4-Quién? 5-Ventajas5-Ventajas 6-Desventajas6-Desventajas

25 PCR Amplifica en forma exponencial el DNA del microorganismo. Esto le confiere un enorme potencial para detectar pequeñas cantidades del material inicialAmplifica en forma exponencial el DNA del microorganismo. Esto le confiere un enorme potencial para detectar pequeñas cantidades del material inicial Tipos de PCRTipos de PCR –Múltiple –RT-PCR –Anidada (nested-PCR) –En tiempo real –PCR-RFLP –PCR-ELISA

26 PCR

27 Termociclador

28 Detección del producto de PCR

29 Enzima SustratoCromógeno Color DETECCIÓN COLORIMÉTRICA DE RT-PCR- ELISA PARA ENTEROVIRUS Amplificado con biotina Sonda de captura Estreptavidina

30 Sistema automatizado

31 PCR VARIEDADESVARIEDADES –PCR simple –RT-PCR –RAPD´s –PCR semicuantitativa –PCR competitiva –Nested PCR –PCR múltiple –Otras: real time PCR: Higuchi et al.

32 RT-PCR


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