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

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

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

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

Influenza as a public health threat

• Influenza Viruses are the respiratory viruses of greatest public health importance, particularly Influenza A

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

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

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.

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

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

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

Influenza Diagnostic Table

Procedure Influenza

Types Detected

Acceptable Specimens

Time for Results

Rapid result

available

Viral culture A and B NP swab2, throat swab, nasal wash, bronchial wash, nasal aspirate, sputum

3-10 days 3

No

Immunofluorescence DFA An tibody Staining

A and B NP swab2, nasal wash, bronchial wash, nasal aspirate, sputum

2-4 hours

No

RT-PCR5 A and B NP swab2, throat swab, nasal wash, bronchial wash, nasal aspirate, sputum

1-2 days No

Serology A and B paired acute and convalescent serum samples6

>2 weeks

No

Enzyme Immuno Assay (EIA)

A and B NP swab2 , throat swab, nasal wash, bronchial wash

2 hours No

Rapid Diagnost ic Tests

Directigen Flu A7 (Becton-Dickinson)

A NP wash and aspirate <30 minutes

Yes

Directigen Flu A+B7,9 (Becton-Dickinson)

A and B NP swab2,aspirate, wash; lower nasal swab; throat swab; bronchioalveolar lavage

<30 minutes

Yes

Directigen EZ Flu A+B7,9 (Becton-Dickinson)

A and B NP swab2, aspirate, wash; lower nasal swab; throat swab; bronchioalveolar lavage

<30 minutes

Yes

FLU OIA4,7 (Biostar)

A and B NP swab2 , throat swab, nasal aspirate, sputum

<30 minutes

Yes

FLU OIA A/B 7, 9 (Biostar)

A and B NP swab2 , throat swab, nasal aspirate,

<30 minutes

Yes

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FluAlert1000SAS FluAlert-046230 (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$250.00

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QDL-20183Quidel 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$449.00

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INV-416-110BINAX 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$175.00

PRUEBAS RÁPIDAS

ImmunofluorescenceImmunofluorescence

• Direct– Ab to tissue Ag is labeled with fluorochrome

Ag

FluorochromeLabeled Ab

Tissue Section

ImmunofluorescenceImmunofluorescence

• Indirect– Ab to tissue Ag is

unlabeled– Fluorochrome-labeled

anti-Ig is used to detect binding of the first Ab.

Ag

FluorochromeLabeled Anti-Ig

Tissue Section

UnlabeledAb

• Qualitative to Semi-Quantitative

Figure A-17

PARTICIPANTES HA Y HI

Flu virus

AGLUTINACIÓN NO AGLUTINA

AnticuerpoEritrocitos

Hemaglutinación (HA)Flu virus

No virus

AGLUTINA NO AGLUTINA

Hemagutinación para virus

Diluciones del Flu virus No virus

AGLUTINAN NO AGLUTINAN

Inhibición de la hemaglutinación (HI)

Flu virus

Virus esbloqueado por Ac

AGLUTINAN NO AGLUTINAN

HI para virus

Antivirus específicoS

Ac H5N1 Ac H3N2

HI NEGATIVA hay aglutinación de eritrocitos

HI POSITIVA hay inhibición por el Ac H3N2

Figure A-7

ELISA DE ELISA DE CAPTURA DE CAPTURA DE ANTÍGENO ANTÍGENO

Técnicas de amplificación Técnicas de amplificación

de Ac. Nucleicosde Ac. Nucleicos

• 1-PCR1-PCR

• 2-LCR2-LCR

• 3-NASBA, TMA3-NASBA, TMA

• 4-Amplificación de señal4-Amplificación de señal

• 5-Boomerang 5-Boomerang

PCRPCR

• 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

PCRPCR• Amplifica en forma exponencial el DNA del Amplifica en forma exponencial el DNA del

microorganismo. Esto le confiere un enorme microorganismo. Esto le confiere un enorme potencial para detectar pequeñas cantidades del potencial para detectar pequeñas cantidades del material inicialmaterial inicial

• Tipos de PCRTipos de PCR– MúltipleMúltiple– RT-PCRRT-PCR– Anidada (nested-PCR)Anidada (nested-PCR)– En tiempo realEn tiempo real– PCR-RFLPPCR-RFLP– PCR-ELISAPCR-ELISA

PCR

PCR

TermocicladorTermociclador

Detección del producto de PCRDetección del producto de PCR

Enzima

SustratoSustrato

CromógenoCromógeno ColorColor

DETECCIÓN COLORIMÉTRICA DE RT-PCR-DETECCIÓN COLORIMÉTRICA DE RT-PCR-ELISA PARA ENTEROVIRUSELISA PARA ENTEROVIRUS

Amplificado con biotina

Sonda de captura

Estreptavidina

Sistema automatizadoSistema automatizado

PCRPCR

• VARIEDADESVARIEDADES– PCR simplePCR simple– RT-PCRRT-PCR– RAPD´sRAPD´s– PCR semicuantitativaPCR semicuantitativa– PCR competitivaPCR competitiva– Nested PCRNested PCR– PCR múltiplePCR múltiple– Otras: real time PCR: Otras: real time PCR: Higuchi et al.Higuchi et al.

RT-PCR