Characterization of the H7N3 Viruses

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Characterization of the H7N3 Viruses
Isolated During the BC Avian Influenza
Outbreak
John Pasick, DVM, MSc, PhD
Canadian Food Inspection Agency National Centre
for Foreign Animal Disease
Winnipeg, Manitoba
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NA
HA
PB1
PB2
PA
HA
NP
NA
M
NS
M1
M2

NS2
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Influenza A virus genome RNA segments
Segment Length (nts) Encoded
Polypeptide 1
2341 PB2 2
2341
PB1 3 2233
PA 4
1778 HA 5
1565
NP 6 1413
NA 7
1027 M1
315
M2 8
890 NS1
395
NS2
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Type A Influenza Virus Surface Antigens
Hemagglutinin (H) Subtypes
1 2 3 4 5 6 7 8
9 10 11 12 13 14 15 Human Equine Swine Avian
Neuraminadase (N) Subtypes
1 2 3 4 5 6 7
8 9 Human Equine Swine Avian

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Recent HPAI Outbreaks
2004 Canada (H7N3) 2004
South East Asia (H5N1) 2003 Netherland
s (H7N7) 2002 Chile (H7N3) 1999-2000 Italy
(H7N1) 1997 Italy (H5N2) 1997 Hong Kong
(H5N1) 1995 Pakistan (H7N3) 1994-1995 Mexico
(H5N2) 1983-1984 USA (H5N2)
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Avian Influenza Outbreak - Italy 1999-2000

• 4 epidemic waves
• LPAI HPAI
• 2 subsequent waves of LPAI
• HPAI resulted in 413 outbreaks in 4 months
  leading to the death or destruction of ? 14
  million birds
• 500 million euro

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70,000 birds/km2
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HPAI an infection of poultry caused either by
any influenza A virus which has an intravenous
pathogenicity index in 6-week-old chickens
greater than 1.2 or by any infection with
influenza A viruses of H5 or H7 subtype for which
nucleotide sequencing has demonstrated the
presence of multiple basic amino acids at the
cleavage site of the hemagglutinin
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Fresh infective allantoic fluid with HA titre gt
1/16 diluted 1/10 in PBS
Inoculate ten 4-6 week old SPF chickens with 0.1
ml of diluted virus intravenously
Birds are examined at 24 hour intervals for 10
days
Scoring 0 - Normal 1 and 2 -
3 - Dead
Sick Severely
Sick
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-4 -3 -2 -1 1
B-X-B-R/GLF
Nicholson, Webster, and Hay. Textbook of
Influenza (1998)
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Origins of HPAI

• HPAI of H5 and H7 subtypes appear to emerge from
  low pathogenic precursors only after introduction
  into domestic poultry
• HPAI viruses seem not to form a separate
  phylogenetic lineage or lineages in waterfowl

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Mechanisms

• Insertion of basic amino acids at the HA cleavage
  site possibly through the duplication of purine
  triplets resulting from a transcription fault of
  the polymerase complex
• Stepwise substitution of basic amino acids at the
  cleavage site
• Non-homologous recombination event resulting in
  the insertion of a foreign amino acid sequence at
  the cleavage site

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Non-Homologous Recombination

• Experimental systems
• DI particles resulting from intersegmental
  recombination
• Insertion of 28S rRNA sequence into the HA gene
  adaptation of H7N2 to growth in chicken embryo
  cells
• Insertion of 60 nucleotides from the NP gene into
  the HA gene adaptation of H7N7 virus to growth
  in chicken embryo cells
• Field conditions
• 30 nucleotide insert derived from the NP gene
  into the HA gene H7N3 virus from broiler
  breeder flock in Chile 2003

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H7N2 LPAI in Northeastern USA
1994 1995 P E N P K T R / G L F 1995 1998
P E N P K P R / G LF 1999 2001 P E K P K P
R / G LF 2002 P E K P K K R / GL F
LPAI by intravenous pathogenicity index Minimum
for HPAI B X B R / GLF
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Amino Acid Sequences of the HA Cleavage Site
of HPAI H7 Viruses
Ck/Victoria/85 P E I P K K R E K R /
G Ck/Victoria/92 P E I P K K K K R /
G Ck/Queensland/95 P E I P R K R K R /
G Ck/Pakistan/447/95 P E T P K R K R K R /
G Ck/N.S.W./220/97 R K R K R /
G Ty/Italy/4580/99 P E I P K G S R V
R R / G Ck/Chile/02 P E K P K T C S P
L S R C R E T R / G P E K P K T C S P
L S R C R K T R / G
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A/Ck/BC/514/2004 H7N3
PENPKTR / GLF PENPKQAYRKRMTR / GLF IVPI
2.96 Insert derived from the matrix gene
amino acid coordinates 238-244 Minimum required
for high pathogenicity R/K-X-R/K-R / GLF
IVPI 0

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H7N3 Viruses Isolated During the B.C. Avian
Influenza Outbreak
Amino Acid Sequence of Hemagglutinin of
Isolates IVPI
Cleavage Site
PENPKTRGLF 3
0.00 PENPKQAYQKRMTRGLF 24
2.17-2.96 PENPKQAYRKRMTRGLF
2 2.87-2.96 PENPRQAYRKRMTRGLF
1 ND PENPKQAYKKRMTRGLF
3
3.00 PENPKQAHQKRMTRGLF 1
2.95 PENPKQAYHKRMTRGLF 2
ND PENPKQSYQKRMTRGLF 1
2.93
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Summary

• A high path H7N3 influenza A virus appeared to
  emerge suddenly from a low path precursor
• The mechanism responsible for this emergence
  appears to involve a non-homologous
  recombination event
• This mechanism has been documented previously
  under experimental and natural conditions
• All previous reports have been associated with
  increased pathogenicity and all have involved
  viruses of the H7 sub-type
• This outbreak gives further support to the notion
  that all H5 and H7 influenza A infections in
  domestic poultry should be treated as potentially
  highly pathogenic

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Acknowledgements

• Dr. John Robinson
• Katherine Handel
• Deidre Ridd
• Helen Kehler
• Colleen Cottam-Birt
• Kevin Hills
• Margaret Krzyzelewski

• Dr. John Copps
• Stacey Halayko
• Shannon Toback
• Michelle French
• Marlee Ritchie
• Dr. Jim Neufeld
• Dr. Stephanie Czub
• Dr. Yohanes Bahrens