Avian influenza diagnostic further analysis

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Avian influenza diagnostic further analysis
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Laboratory analyses procedure
Mix
RNA extraction
Sample sorting
Discard Swabs

• Influenza A Q RT-PCR M gene
• Primers M25, M-124 and the probe M25 (Spackman
  et al, 2002)
• Brilliant QRT-PCR one-step Master mix kit
  (Stratagene)

Positive samples
Positive samples

• Influenza H5 and H7 Q RT-PCR
• primers and probes recommended by the reference
  laboratory VLA (Weybridge, UK)
• one-step RT-PCR kit (Qiagen)

RT-PCR on HA cleavage site
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Hemagglutinine and HPAI
Probe H5PRO
H5-Kha-1
H5-Kha-3
H5LH1
H5RH1
HA-875F
HA-1114R
J3
B2a
Site de clivage
HA Gene
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Sequencing
H5Kha
Sequencing of the PCR product
Alignment with other haemaglutinin sequences
using bio informatic software or a blast search
on genebank
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HA1-HA2 cleavage site on avian isolates
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(No Transcript)
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Isolation in embryonated chicken eggs

• Inoculation of sample in allantoic cavity of SPF
  embryo chicken eggs of 9 to 11 days
• 5 eggs per sample
• About 200 µl of sample
• Confirmation of isolation after 2 to 7 days of
  incubation at 37C
• Mortality
• Detection of haemagglutinating activity of
  allantoic fluid

1/4096
1/2
Serial dilutions of allantoic fluid
HA
HA-
1h

AIV and NDV free chicken red blood cells 1
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Isolation in embryonated chicken eggs

• Confirmation of the presence of Influenza A virus
  by Agar gel immunodiffusion test
• Determination of the subtype by inhibition
  haemagglutination assay with specific antiserums

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Detection of H5 or H7 subtypes

• High variation of haemagglutinin gene

Necessary to verify the sequences of the
circulating strains to design new primers so that
false negative can be avoided
ex design of H5 real-time RT-PCR primers by
Spackman et al. (2002)
Observation of differences on HA gene between
North American Strain and Eurasian strains
Modification of H5 primers used in Europe and
Africa (Slomka et al. 2007)