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Genotype and Protectotype Characterization of IBV Variants in Israel

Genotype and Protectotype Characterization of IBV Variants in Israel. Rosie Meir, Ora Maharat and Yigal Farnushy Division of Avian and Aquatic Animal Diseases Kimron Veterinary Institute. IBV Classification. Serotyping – based on Virus Neutralization tests

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Genotype and Protectotype Characterization of IBV Variants in Israel

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  1. Genotype and Protectotype Characterization of IBV Variants in Israel Rosie Meir, Ora Maharat and Yigal Farnushy Division of Avian and Aquatic Animal Diseases Kimron Veterinary Institute

  2. IBV Classification Serotyping – based on Virus Neutralization tests Genotyping – based on the S1 gene sequence S1

  3. IBV Classification • Protectotyping – based on protection trials in vivo Protectotype ≠ Serotype/Genotype N protein

  4. S1 gene 5’ 3’ Primer Primer RT-PCR 5’ 3’ BstY I Hae III Xcm I Reverse Transcriptase-Polymerase Chain Reaction- Restriction Fragment Length Polymorphism (RT-PCR-RFLP)

  5. RT-PCR-RFLP Kwon et al. 1993; Jackwood et al. 1997 • Each serotype has a distinct RFLP pattern • Different RFLP patterns = different serotypes Serotyping was not performed in our laboratory RT-PCR-RFLP = rapid genotyping A new RFLP pattern S1 sequencing

  6. Israeli variants 1996-2007 • Variant 1 • Variant 2 • IS/720/99 • IS/885/00 • IS/1201/04 Massachusetts Relevantvaccine strains H120 4/91 Relevantvaccine strains H120 4/91

  7. RFLP patterns M 1 2 3 4 M 1 2 3 4 M 1 2 3 4 M 2 3 4 1720 1720 1720 Var II Var I Mass 1201 1. Uncut 2. BstY I 3. Hae III 4. XcM I 885 720

  8. Variant 1 genotype • First isolated in 1996 • Similar to 793B serotypes including the vaccine strains 4/91 and CR88 • Respiratory and Nephropathogenic M 1 2 3 4

  9. Variant 2genotype • First isolated in 1996 • Endemic - no similarity to published strains and variants • Respiratory and nephropathogenic M 1 2 3 4

  10. IS/720/99 genotype • Endemic • Also isolated in Egypt • Respiratory M 1 2 3

  11. IS/885/00 genotype • Endemic • Very similar to IS/720/99 • Nephropathogenic M 1 2 3 IS/720/99 = IS/885/00 (same genotypes)

  12. IS/1201/04 genotype • Similar to QXIBV • Respiratory M 1 2 3

  13. Variant prevalence 1996-2007

  14. Variant prevalence 2005-2007

  15. Comparison of S1 protein:Israeli IBV variants, H120 and 4/91 95% 98% 71-78% 92% 70-75%

  16. Israeli IBV Massachusettsgenotypes • Identity of 97-100% to H120 • Unknown role in IB outbreaks? • First identified in Israel in 1977 M 1 2 3 4

  17. Comparison of the S1 gene sequence of Mass-type isolates and H120 Difference range 0-2.7 % % 99.7-100

  18. Conclusions • Mass-type isolates are persistent in Israel • About 50% of sequenced isolates show 100% identity with H120 • S1 sequence difference of up to 3% from H120 - unknown significance • Not a major pathogen in severe IBV outbreaks

  19. Phylogenetic tree of world IB viruses Europe 793B serotype 4 Far east

  20. Comparison of Israeli and foreign IB viruses Europe Far East

  21. Protectotyping Efficacy of vaccines when challenged with a virulent virus (> 80% protection according to OIE)

  22. Efficacy of H120 (1999-2001)Tested with 1996-2000 isolates Efficient protection against Mass- types only

  23. Experimental Design Live vaccine eye drops At least 10 chicks /group Day old SPF chicks 40/47 days 42/49 days 14 days 35/42 days Bleeding Challenge Bleeding End of trial Bleeding Second vaccination (live or inactivated) Tracheal swabs Laboratory tests

  24. Laboratory Tests Sera Tracheal swabs 5 embryonated SPF eggs/bird ELISA ά IBV IgG (IDEXX) HI Mass Ag Aspiration of AF 2d PI Virus isolation: embryo mortality/lesions 8 day surveillance RT-PCR

  25. Resultsinterpretation Vaccine efficacy was determined by percentage of embryos negative for IBV (RT-PCR or virus isolation) ELISA/HI titers – indication of exposure/cross reaction

  26. Trial 1 – Efficacy of H120 Challenge viruses: • IS/1365/05 (Variant 1) • IS/1494/06 (Variant 2) • IS/1201/04 • M41 (Mass positive control)

  27. Vaccine efficacy results- trial 1

  28. Serology results– trial 1 ELISA HI Test (Ag – Mass)

  29. Summary – trial 1 • H120 afforded protection against the homologous virus and IS/1365/05 (Variant 1) • H120 did not protect against IS/1494/06 (Variant 2) and IS/1201 • Variant 2 results are in agreement with previous experiments (isolates from 1996)

  30. Trial 2 - Inactivated IS/1201 Vaccine Vaccination regimes: • H120 x2 • H120 + inactivated IS/1201 at 14 days • Only inactivated IS/1201 at 14 days Challenge virus: Live IS/1201

  31. Vaccine efficacy results-trial 2

  32. Serology results – trial 2

  33. Summary trial 2 • Inactivated vaccine did not improve protection against homologous live virus • Good humoral response was detected when chicks primed with H120 • Without priming – very low humoral response

  34. Trial 3 -Efficacy of 4/91 Vaccination regimes: • H120 at 1 and 14 days • 4/91 at 1 and 14 days • H120 at day 1 and 4/91 at 14 days Challenge viruses: • IS/1365/05 (Variant 1) • IS/1494/06 (Variant 2) • M41 (Mass positive control)

  35. Variant 1 challenge groups Group 4 4/91x2 Group 3 H120+4/91 Group 2 H120x2 Group 1 No vaccine X 3 challenge viruses = 12 groups

  36. Vaccine efficiency results – trial 3

  37. Serology results – trial 3 ELISA HI (Ag – Mass)

  38. Summary - trial 3 Vaccination with 4/91 alone or after H120 improved significantly the immunity against Variants 1 and 2

  39. Conclusions • Variant 1 = 4/91 genotype and protectotype • IS/1201 and Variant 2 ≠ H120 genotype and protectotype • Variant 2 ≠ 4/91 genotype = 4/91 protectotype

  40. Thank you!

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