Influenza & Avian Flu

INFLUENZA VIRUS II MBBS

Common Properties myxo- mucous tropic orthomyxovirus = influenza • (-) polarity ssRNA • enveloped viruses • encapsidates RNA polymerase • causes worldwide disease • responsible for > 10% work/school absence • acute infections and diseases • known since 1500’s

Orthomyxovirus: Classification Three types • Influenza A - highly variable - infects humans, birds, pigs, horses, seals - most prevalent human virus - cause of most human epidemics • Influenza B - infects only animals • Influenza C - infects human and swine - antigenic stable, only mild illness

Structure of Virus     5. 6.

Spherical, 80-120nm in diameter but pleomorphism is common Nucleocapsid – helical symmetry Genome – 8 segmented RNA (ribonucleoprotein - RNP) Envelope – Inner membrane protein layer – “M” protein, 2 components: M1 & M2 Outer lipid layer – 2 types of spikes: Hemagglutinin(HA) & Neuraminidase(NA).

Antigenic Structure  •

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Influenza virus Ags – 2 types: Internal Ags – Type specific Ags. Includes: - Ribonucleoprotein (RNP) Ag - M protein Ag Surface/ External Ags – Strain specific Ags Composed of 2 virus coded proteins : - HA & NA

Internal Antigens  Ribonucleoprotein

- found free in infected tissues - Used to classify into types A, B & C - Stable, does not exhibit any significant antigenic variation  M protein

- type specific

Surface Antigens - Hemagglutinin  Glycoprotein – made up of 2 polypeptides

HA1 & HA2.  Responsible for hemagglutination & hemadsorption  Adsorbs to mucoprotein receptor on RBCs as well as on epithelial cells  Capable of great variation  16 HA subtypes, named H1 to H16 have been identified.

Surface Antigens - Neuraminidase  Glycoprotein enzyme  Destroys sialic acid (neuraminic acid)

receptors on cell surface during exit  Promotes the release & spread of progeny virions  Also helps in the penetration of the mucus layers in the respiratory tract.  Antigenic variation  9 subtypes, N1 to N9.

Properties of Influenza virus  Hemagglutination  Hemadsorption  Antigenic variation – Antigenic drift

- Antigenic shift

Hemagglutination  When mixed with a suspension of fowl

erythrocytes, the virus is adsorbed onto the mucoprotein receptors on the cell surface – clumping of cells occurs – known as Hemagglutination (brought about by HA)  After some time, the virus detaches itself from

the cell surface by the action of Neuraminidase (NA) enzyme – reversal of hemagglutination – known as Elution.

Hemagglutination  Virus particles which have eluted from RBCs

can still agglutinate fresh RBCs.  RBCs that have been acted on by the virus are not susceptible to agglutination by the same strain – due to destruction of specific cell receptors by initial contact with the virus.  Occurs with in a wide range of temp. 0°C to 37°C  Can agglutinate RBCs of different species Type A & B – fowl, human, guinea pig & some other species Type C – fowl only, at 4°C

Application of Hemagglutination  Detection & titration of the influenza virus in

egg & other culture fluids.  Hemagglutination titre – highest

dilution of virus suspension that produces agglutination of a fixed quantity of RBCs.

 Can also be used for the titration of

inactivated influenza virus.

Hemagglutination Inhibition  A convenient method for the detection

and quantitation of antibody to the virus  Disadvantage – certain substances in

serum can also cause non specific inhibition of HA

Hemadsorption  A technique employed for the

identification of growth of influenza virus in cell cultures.

 The plasma membrane of tissue culture

cells in which the viruses are multiplying contain HA, hence RBCs are adsorbed onto the surface of such cells.

Antigenic Variation  Unique feature

of influenza virus  Important in the epidemiology of disease  Highest variability in Influenza type A, none in type C.  Both surface/ external Ags, HA & NA, undergo independent antigenic variations  Two types – Antigenic drift - Antigenic shift

Antigenic drift  Gradual sequential change in antigenic

structure occurring at regular intervals.  HA and NA accumulate mutations  New Ags, though different from the previous Ags, are yet related to them. So the pre existing Abs will provide only partial protection  Occurs due to mutation & selection  Results in sporadic outbreaks & limited epidemics

Antigenic Shift  An abrupt, drastic, discontinuous variation in

antigenic structure  ‘New’ NA or HA proteins - Results in a new virus strain antigenically unrelated to the predecessor strain  Pre-existing Abs do not protect.  New variants can spread widely in population causing major epidemics or pandemics.

Pathogenesis 

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Portal of entry - airborne droplets - direct contact with aerosols (105 to 106 virions/ droplet) Person to person transmission - sneezing - hands on contaminated surfaces Incubation period – 18 to 72 hrs Movement to target organ inside host - Initial replication in nasal passage cells - Spread to URT aided by NA activity - No viremia involved

Pathogenesis  Neuraminidase – dilution of mucus film

lining respiratory epithelium – exposure of cell surface receptors  Entry of virus in to the epithelial cells via HA – virus adsorption.  Chief target cells – Ciliated cells, destroyed nearly after 3 days  Cell death occurs due to - direct effects of virus on the cell - effects of Interferons - action of cytotoxic T cells

Pathogenesis  2. 3. 4. 5.

All this results in: Decreased ciliary clearance Exposure of basal cells in trachea & bronchi Increased risk of bacterial invasion However, viremia is rare

NORMAL TRACHEAL MUCOSA

3 DAYS POST-INFECTION

7 DAYS POST-INFECTION

Recovery from disease  2. 3.

Interferons: Reduce virus production Symptoms of influenza – fever, myalgia, fatigue, malaise

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Cell mediated immunity – viral clearance

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Repair of respiratory epithelium

Interferon

Time course of virus production will vary from virus to virus

Interferon

Interferon

antiviral state

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Interferon

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Interferon

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BACK TO INFLUENZA

Protection / Immunity  Main source – humoral immunity  IgA & IgG

- protects against re-infection - IgG less efficient but lasts longer  Abs to HA - can neutralize the virus (blocks binding) - prevents initiation of infection  Abs to NA - slows the spread of virus * usually develops short term immunity due to frequent antigenic variations.

Symptoms  Fever  Headache  Myalgia  Dry Cough  Rhinitis  Ocular symptoms - photophobia

Clinical Findings  SEVERITY Very Young Elderly Immuno-compromised Heart OR Lung disease

Pulmonary Complications  Croup (Acute laryngotracheobronchitis) in

young children – cough, stridor, difficulty breathing  Primary influenza virus pneumonia  Secondary bacterial infections - S. pneumoniae - S. aureus - H. influenzae

Non-pulmonary Complications  Myositis (rare, > in children, > with type B)  Cardiac complications  Recent studies

report encephalopathy

 2002/2003 season studies of patients

younger than 21 yrs in Michigan - 8 cases (2 deaths)

 Liver and CNS  Reye’s syndrome

 Peripheral nervous system  Guillian-Barré syndrome

Diagnosis 

Provisional diagnosis – clinical picture + knowledge of influenza outbreak

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Laboratory Diagnosis: Demonstration of virus Ag Isolation of the virus Serology

4. 5. 6.

Laboratory Diagnosis 1. Demonstration of virus Ag 

Immunofluorescence – for virus Ag on the surface of the nasopharyngeal cells, rapid diagnosis

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RT-PCR: for viral RNA, highly sensitive

2. Isolation of Virus    •

Best during the first 2-3 days of illness Specimen – throat gargles, should be treated with antibiotics to destroy bacteria Methods of isolation Amniotic cavity of 11 –13 days old chick embryos: incubate at 35°C for 3 days, test the amniotic & allantoic fluid for hemagglutination at RT & 4°C. - identification & typing of isolate by CFT with antisera to types A, B & C - subtyping by HI test.

2. Isolation of Virus  •

Methods of isolation Tissue culture - primary monkey kidney cell culture, other continuous cell cultures like baboon kidney - incubate at 33°C in roller drums - growth is detected & identified by hemadsorption with human O group, fowl or guinea pig erythrocytes - rapid results by demonstration of viral Ag in infected cell cultures by IF.

Serology Examination of paired sera to demonstrate rise in titre of Abs. • CFT with RNP Ags of types A, B & C as Abs are formed only during infection 2. HI - convenient & sensitive test - highest dilution of serum that inhibits hemagglutination is its HI titre. • Radial Immunodiffusion – useful screening test 

- detection of Abs to RNP Ag, HA & NA.

Prophylaxis  Yearly vaccine - formulated with the types

& strains of influenza predicted to be a major problem.  Current vaccine – multivalent , has 2 strains of type A & 1 of type B. - inactivated virus, grown in chick embryo - short lived protection  New vaccines - HA & NA subunit - “cold adapted” live attenuated virus (Flu-mist)

Who should receive vaccine?  Health care workers  High risk individuals

- elderly >65 years - children 6-59 months - people with underlying diseases  Women in 2nd or 3rd trimester of pregnancy during influenza season  Persons who are clinically or sub clinically infected and can transmit to high risk individuals.  Travellers & general population who wish to be vaccinated.

Treatment   3. 4. 

Antibiotics – to control 2 bacterial infections Antiviral drugs can be given after exposure Rimantadine / Amantadine – Type A Zanamivir (Relenza)/ Oseltamivir (Tamiflu) – Type A & B Other treatment – rest, liquid, anti febrile agents (no aspirin to ages 6 mths to 18 years)

Influenza A Pandemics

where do “new” HA and NA come from?  16 types HA 

9 types NA  all circulate in birds

 pigs  avian and human

Where do “new” HA and NA come from?

Where do “new” HA and NA come from - can ‘new’ bird flu directly infect humans? Bird flu H5N1?

Avian Influenza  Avian influenza is an infectious disease of birds

caused by type A strains of the influenza virus.

 Wide spectrum

of symptoms in birds - ranging from mild illness to a highly contagious and rapidly fatal disease resulting in severe epidemics. The latter is known as “highly pathogenic avian influenza”. This form is characterized by sudden onset, severe illness, and rapid death, with a mortality that can approach 100%.

Avian Influenza  To date, all outbreaks of the highly pathogenic

form have been caused by influenza A viruses of subtypes H5 and H7.

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Avian influenza viruses do not normally infect species other than birds and pigs. The first documented infection of humans with an avian influenza virus occurred in Hong Kong in 1997, when the H5N1 strain caused severe respiratory disease in 18 humans, of whom 6 died.

Avian Influenza  An outbreak

of highly pathogenic H7N7 avian influenza, which began in the Netherlands in February 2003, caused the death of one veterinarian two months later, and mild illness in 83 other humans.

 The most recent cause for alarm occurred in

January 2004, when laboratory tests confirmed the presence of H5N1 avian influenza virus in human cases of severe respiratory disease in the northern part of Viet Nam.

Avian Influenza  Based on historical patterns, influenza

pandemics can be expected to occur, on average, three to four times each century when new virus subtypes emerge and are readily transmitted from person to person.

 In the 20th century, the great influenza

pandemic of 1918–1919, which caused an estimated 40 to 50 million deaths worldwide, was followed by pandemics in 1957–1958 and 1968–1969.

why do we not have influenza B pandemics?  so far no shifts

have been recorded  no animal

reservoir known

H5N1 – in birds  Avian H5N1 has spread to humans  So far human cases in Asia and Africa  256 cases (12-1-03 through 10-16-06)  151 (59%) fatal

 Have been a few instances where may have

spread human-to-human  So far no sustained spread in humans AND  Surveillance continues

SURVEILLANCE

CDC/Katherine Lord