Transcript Influenza: The Virus

Influenza
The Virus
Family Medicine Forum
28 October 2009
Dr. Kevin Fonseca
Clinical Virologist & Influenza Program Lead
Alberta Provincial Laboratory
[email protected]
Topics
• Biology of Influenza
• Seasonal Influenza & “Pandemic
H1N1”
• Laboratory Testing
• Surveillance
Agents Causing Influenza-Like
Illness (ILI)
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Influenza A & B
Rhinovirus (30%)
Coronaviruses (>10%)
Parainfluenza group (5%)
Enteroviruses (?)
Respiratory Syncytial Virus (5%)
Human Metapneumovirus (3-5%)
Bacterial agents (legionella, mycoplasma &
S.pneumo)
• (Bocavirus & polyomaviruses)
Influenza Viruses
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ss enveloped RNA virus
Segmented genome
Orthomyxovirus
3 types – influenza A, B & C
Prototypic lineages of Influenza A
(based on Nucleoprotein gene)
Avian
Ancient
Equine
Recent
Equine
Gull
Likely avian
ancestor
Swine
Two main lineages of Influenza A
•Eurasian
•North American
Human
The Influenza viruses
Influenza A
Influenza B
Influenza C
Genome
8 genes
8 genes
7 genes
Variants
Subtypes
Lineages
?
Reassortment &
Recombination
Reassortment &
Recombination
?
Hosts
Avian, human,
animals
Human, seals (dogs)
Human ? Only
Disease
Pandemics &
outbreaks
Outbreaks
Sporadic
Clinical
Mild to severe
“mild to less severe”
“mild”
Genetic
expansion
Structure & Function
Segment
1
2
3
4
5
6
7
8
Size
2341
2341
2233
1778
1565
1413
1027
890
Peptide(s)
PB2
PB1
PA
HA
NP
NA
M1
M2
NS1
NS2
Function
RNA polymerase subunit
RNA polymerase subunit
RNA polymerase
Haemagglutinin
Nucleoprotein
Neuraminidase: release of virus
Matrix protein: major component of virion
Integral membrane protein - ion channel
Anti-interferon protein/virulence factor.
“Virulence factor”
Subtypes of Influenza A
H1 - swine, human
H2 - human
H3 - human
H4 - avian
H5* - avian, (human)
H6 - avian
H7* - equine,avian, (human)
H8 - avian
H9 - avian, (human)
H10 – H16 - avian
N1
N2
N3
N4
N5
N6
N7
N8
N9
- human
- human
- avian
- avian
- avian
- avian
- equine
- equine
- avian
Characterization of Influenza
Strains
Pandemic Influenza (H1N1) A/California/07/2009
A/Fujian/411/02-like [H3N2]
Type Geographical Isolate Year of HA component
Origin Number Isolation
NA component
Structure of Haemagglutinin
Diagrammatic Representation of influenza A haemagglutinin
Avian influenza
strains
Preferentially bind to
N-acetyneuraminic
acid-ά2,3-galactose
Human influenza
strains
Preferentially bind to
N-acetyneuraminic
acid-ά2,6-galactose
Distribution of Receptors in the Respiratory Tree
2,6
2,3
How do Pandemics Arise ?
Possible
Mechanisms
for the
Emergence
of Potential
Pandemic
strains
Emergence of pandemic influenza viruses.
G Neumann et al. Nature 000, 1-9 (2009) doi:10.1038/nature08157
Pandemics of Influenza A
Year
Subtype
Deaths
x106
Origin
1889
H2N2
6
Europe
1898
H3N2
0.5
Europe
1918 Spanish flu
H1N1
20-40
Europe
1957 Asian flu
H2N2
4
Asia
1968 Hong Kong
flu
1977 Russian flu
H3N2
2
Asia
H1N1
?
Asia /lab
2009 Swine-origin H1N1
3200 or so ?Mexico
JS Oxford: Rev Med Virol:2000:10;119-33
Smith et al; Nature 2009
Shinde et al NEJM 2009
Testing for Influenzavirus
Sample Type
Upper respiratory
infection
• Nasopharyngeal swab
• Nasopharyngeal wash
• Auger suction or
nasopharyngeal aspirate
• Throat swab
• [Nasal swab]
• ((Sputum))
• ((Saliva))
Lower respiratory tract
infection
• Endotracheal suctions
• Bronchoalveolar lavage (BAL)
• Lung tissue
Sample type
Sensitivity
Nasopharyngeal swab
77-100
Nasopharyngeal
aspirate/wash
79-97
Auger suctions
Throat swabs
No data
91
BAL
No data
Endotracheal
secretions
No data
Detection of Influenzavirus
• Antigen Assays
– Point of Care
– Direct Fluorescent Antigen
• Molecular Based (RT-PCR)
– End point (gel-based)
– Real time (single or duplex)
– Array based
• Serology
– Microneutralization
– Hemagglutination inhibition (HAI)
• Culture
– Shell vial
– Tube culture
Antigen Tests for Influenza
Rapid Influenza Tests
•Rapid TAT (15-20 mins)
•Cannot tell subtype
•Variable sensitivity
•Restricted sample type
Direct Fluorescent Antigen
•More labour intensive
•TAT 2-3 hrs
•Cannot tell subtype
•Restricted sample type
•Can assess sample quality
•Sensitivity about 50% for H1N1
Culture of Influenzaviruses
Disadvantages
• Takes 3-10 days
• May require level 3 facility
• Poorly sensitive (approx <30%
relative to PCR)
• Requires various cell lines
• Not all subtypes grow well
easily
• Sample has to be fresh for
virus to be viable
• Expertise & tissue culture
facilities required
• Permits for importation of cell
lines
Advantages
• Need for seed strains for the
vaccine
• Required for characterization
(is the virus changing ?)
• Required for antiviral
susceptibility
• Required for research
Serological Testing
Hemagglutination inhibition assay (HAI)
• Cross reactive between similar subtypes
• Highly labour intensive
• For optimal interpretation requires acute &
convalescent bloods
• No absolute titre for immunity
Microneutralization Assay
• May require level 3 facility
• Good for H5, H7 subtypes
• Require paired bloods (min 3 weeks apart)
• Current data shows that cross-neutralization to
related subtypes can occur
• Different strains may give different results
• (Individuals >70 yrs appear to have neutralizing
antibody to Pandemic influenza)
• No data to definitively link this finding to immunity
Assays for Detection & Identification of
Influenza A
• CDC M PCR
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–
–
–
Detects Matrix (M) gene of influenza A
Detects both seasonal & Pandemic influenza
TAT 24 hrs
Highly sensitive
• Respiratory Viral Panel (Luminex assay)
– Detects influenza A & B and other respiratory
viruses
– Detect & discriminates seasonal subtypes (H1
or H3)
– TAT 36 to 48 hrs
• Confirmatory Assays
– Pandemic influenza confirmatory RT-PCR
– Seasonal influenza subtyping RT-PCR
Molecular Testing
Advantages:
• Highly sensitive
• Rapid TAT
• Can subtype directly from
sample
• High through put
• Virus does not have to be
viable
• Variety of sample types can be
used
• Quantification a possibility
• Variety of various viruses can
be detected from the same
sample
• Currently recommended
technique to detect influenza
Disadvantages
• Only works if you know what to
look for
• Mutations in crucial regions
can give false negative results
• Awareness of pitfalls
• Specialized facilities required
(clean air, no contamination,
etc)
• Specialized supplies required
• High staff expertise required
• Cost
• Testing only available in
specialized centres
• Can result in restricted testing
Current
Algorithm
Influenza Like Illness
DFA T esting Criteria:
<1 year
Hospit al Inpat ient
In ER pending admit
Respiratory outbreaks
Influenza A M olecular
T esting
(CDC M gene)
12 hours
24 hours
Report
Result
T arrant
(Surveillance)
Report
Negative
Result
Report
P osit ive
Result
Molecular Respirat ory
Viral P anel
Confirmation of
Subtype
48 Hrs
24 hours
Report
Result
Report
Seasonal or
P andemic
Distribution of Respiratory Specimens by Week
March 2009 to Present
11 June
25 Apr
14 Apr
Data from DIAL
Surveillance
Distribution of Specimens & Viruses by Month
Data from DIAL
Distribution of Viruses by Period
Others
9%
Others
5%
Flu A
10%
Flu A
39%
Rhino/Ent
49%
Flu B
3%
27/April to 30/July 09
Others
1%
Rhino/Ent
85%
31/July to 1/Oct 09
Rhino/Ent
30%
Flu A
69%
1/Oct to 26/Oct 09
Antiviral Sensitivity (Dr. Y. Li, NML)
How to Take an NP swab
Collection of a nasopharyngeal swab (NP)
1. Assemble all supplies such as gloves, mask, pen, appropriate collection kit containing ProvLab
requisition, nasopharyngeal flock swab and transport medium
2. Check expiry date of transport medium
3. Perform hand hygiene by washing hands with soap and water or using alcohol hand rub
4. Put on gloves and mask (and eye protection if required, or if splashing is anticipated)
5. Have the patient sit in a chair or lie on a bed – elevate the head of the bed so that their head can be
tilted back (see diagram)
6. Remove any mucous from the patient’s nose, with a tissue or cotton tipped swab prior to collecting the
NP swab
7. How deep is the NP swab inserted into the nasopharynx ?
Measure the distance from the corner of the nose to the
front of the ear and insert the shaft ONLY half this length.
In adults, this distance is usually about 4 cm, (finest
thickness of this swab shaft). In children this distance is
less.
8. Tilt the patient’s head back slightly (about 70o ) to straighten
the passage from the front of the nose to the nasopharynx
to make insertion of the swab easier
9. Gently insert the swab along the medial part of the septum,
along the base of the nose, until it reaches the posterior
nares – gentle rotation of the swab may be helpful. (If
resistance is encountered on one side, try the other nostril,
as the patient may have a deviated septum)
10. Rotate the swab several times to dislodge the columnar epithelial cells, and then remove the swab.
Note – insertion of the swab usually induces a cough
11. Put the NP swab into the transport medium and break it at the score mark on the shaft so that it does
not protrude above the rim of the container. Failure to do so will result in the transport medium leaking
and the sample being discarded.
12. Ensure that the lid of the container is screwed on tight.
13. Remove and discard gloves. Perform hand hygiene by washing hands with soap and water or using
alcohol hand rub
14. Remove and discard face mask, and repeat hand hygiene if hands become contaminated
15. Follow the labeling and transport instructions given in the collection kit insert.
1.