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Characterization of the H7N3 Viruses Isolated During the BC

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. NA. HA. PB1. PB2.

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Characterization of the H7N3 Viruses Isolated During the BC

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  1. 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

  2. NA HA PB1 PB2 PA HA NP NA M NS M1 M2 NS2

  3. Influenza A virus genome RNA segments SegmentLength (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

  4. 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

  5. Recent HPAI Outbreaks 2004 Canada (H7N3) 2004 South East Asia (H5N1) 2003 Netherlands (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)

  6. 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

  7. 70,000 birds/km2

  8. 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”

  9. Fresh infective allantoic fluid with HA titre > 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

  10. -4 -3 -2 -1 +1 B-X-B-R/GLF Nicholson, Webster, and Hay. Textbook of Influenza (1998)

  11. 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

  12. 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

  13. 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

  14. H7N2 LPAI in Northeastern USA 1994 – 1995: P E N P K T R / G L F 1995 – 1998: P E N P KPR / G LF 1999 – 2001: P E K P KPR / 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

  15. Amino Acid Sequences of the HA Cleavage Site of HPAI H7 Viruses Ck/Victoria/85P E I P K K R E K R / G Ck/Victoria/92P E I P K K K K R / G Ck/Queensland/95P E I P R K R K R / G Ck/Pakistan/447/95P E T P K R K R K R / G Ck/N.S.W./220/97R K R K R / G Ty/Italy/4580/99P E I P K G S R V R R / G Ck/Chile/02 P E K P K T C S P L SRCRE TR / G P E K P K T C S P L SRCR KTR / G

  16. 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

  17. H7N3 Viruses Isolated During the B.C. Avian Influenza Outbreak Amino Acid Sequence of Hemagglutinin # of Isolates IVPI Cleavage Site PENPKTR*GLF 3 0.00 PENPKQAYQKRMTR*GLF 24 2.17-2.96 PENPKQAYRKRMTR*GLF 2 2.87-2.96 PENPRQAYRKRMTR*GLF 1 ND PENPKQAYKKRMTR*GLF 3 3.00 PENPKQAHQKRMTR*GLF 1 2.95 PENPKQAYHKRMTR*GLF 2 ND PENPKQSYQKRMTR*GLF 1 2.93

  18. 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

  19. 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 Acknowledgements

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