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Characterization and Pathotyping of AIV and APMV-1

Characterization and Pathotyping of AIV and APMV-1. Jan Pedersen, Microbiologist National Veterinary Services Laboratories, Veterinary Services, USDA, Animal Plant Health Inspection Service, Ames, IA. Identification of AIV. Type A Influenza can be identified by: AGID rRT-PCR – Matrix assay

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Characterization and Pathotyping of AIV and APMV-1

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  1. Characterization and Pathotyping of AIV and APMV-1 Jan Pedersen, Microbiologist National Veterinary Services Laboratories, Veterinary Services, USDA, Animal Plant Health Inspection Service, Ames, IA

  2. Identification of AIV Type A Influenza can be identified by: • AGID • rRT-PCR – Matrix assay • Antigen Detection assays Influenza virus can be subtyped by: • HI - 16 hemagglutinin subtypes • NI – 9 neuraminidase subtypes • Conventional RT-PCR or rRT-PCR N1 assay • rRT-PCR – H5 and H7 assays

  3. Avian InfluenzaFactors Influencing Pathogenicity • Polygenic • Associated with H5 and H7 subtypes • HA glycoprotein plays a dominate role • Presence of multiple basic amino acids at the cleavage site of the HA glycoprotein • Pathogenic strains evolve from nonpathogenic lineages

  4. Definition of Highly Pathogenic Notifiable AIV (HPNAI) • Any AIV that is lethal for 6-8 of eight 4-8 wk. old susceptible chickens within 10 days -Intravenous lethality test • Any AIV that has a IVPI in 4-8 wk. old chickens of > 1.2 • Any H5 or H7 subtype virus that does not meet the criteria in #1 & 2, but has an amino acid motif at the cleavage site of the hemagglutinin gene that is compatible with other HPAI viruses.

  5. Low Pathogenic Notifiable AI (LPNAI) • All influenza A viruses of H5 and H7 subtype that are not HPNAI viruses

  6. Notifiable AIV (NAI) • HPNAI – AIV or viral RNA detected in poultry or a poultry product • LPNAI - AIV or viral RNA of the H5 or H7 subtypes detected in poultry or a poultry product • Antibodies to H5 or H7 that are not a consequence of vaccination, nor indicative of a non-specific reaction

  7. Intravenous Pathogenicity Index (IVPI)

  8. Intravenous Pathogenicity Index (IVPI) • SPF or AIV antibody free commerical chickens • AIV isolate free of bacterial contamination is diluted 1:10 in sterile diluent (TBTB, PBS) • Ten 4-8 wk. old chickens are inoculated intravenouly with 0.2 ml of the diluted isolate • Number of normal, sick, morbid and dead chickens are recorded daily for 10 days

  9. Characterization of HPAI by IVPI • Any isolate with an IVPI > 1.2 is pathotyped as HPAI • Any isolate with an IVPI < 1.2 is pathotyped as LPAI

  10. Identification and Pathotyping of Newcastle Disease Virus

  11. Identification of APMV-1 • HI with APMV-1 monospecific antiserum • Cross neutralization with other PMV • rRT-PCR • Matrix assay identifies all APMV-1 • vNDV assay identifies virulent NDV & PPMV-1 • HI with monoclonal antisera • Identification of Pigeon paramyxovirus

  12. Definition of Newcastle disease (ND) as defined by OIE An infection in birds caused by a virus of avian paramyxovirus serotype 1 (APMV-1) that meets the following criteria • ICPI in day-old chicks of 0.7 or greater or • Multiple basic amino acids at the C-terminus of the F2 protein and phenylalanine at residue 117 (NH2 terminus of F1)

  13. Pathotyping Assays for ND • Intracerebral Pathogenicity Index (ICPI) • Differentiates vNDV from avirulent APMV-1 • ICPI for PPMV-1 are quite variable • Intracloacal Inoculation Pathogenicity Test • Differentiates clinicopathological forms (VVNDV, NVNDV, mesogenic) • Sequence Analysis • Nucleotide and amino acid sequencing of the F gene cleavage site • Phylogenetic Analysis

  14. Intracerebral Pathogenicity Index (ICPI) • Determined by inoculating 0.05 ml of a 1:10 dilution of infective, bacterial-free AAF in sterile isotonic saline (w/o antibiotics) in the brains of 10 one-day-old SPF chicks • Chicks are observed daily for 8 days and the number of normal, sick and dead chicks are recorded • ICPI is calculated by dividing the weighted mean by the number of observations

  15. Intracerebral Pathogenicity Index (ICPI) ICPI = Weighted Mean = 3 + 57 =0.75 Number of Observations 80

  16. Intracloacal Inoculation Pathogenicity Test • Determines virulence and tropism differentiating VVNDV from NVNDV • The cloaca of four 6-to-8-week-old SPF chickens are swabed with a 1:10 dilution of infective allantoic fluid • Birds are observed for 10 days • All dead birds are necrospied and the lesions are scored

  17. Pathotyping AIV and APMV-1 by Nucleotide Sequencing

  18. Nucleotide Sequencing & Analysis of AI HA Gene • Sequence analysis is conducted on all H5 and H7 viruses • Single tube one step RT-PCR for H5 • Conventional 2 step RT-PCR for H5 & H7 • Specific H5 and H7 PCR primers are selected for amplification of cDNA • Eurasian isolate • North American isolate

  19. Nucleotide Sequencing & Analysis of AI NA Gene • Conventional RT-PCR for the detection of N1 • WHO assay • 6.5 hrs. needed for results • rRT-PCR for the detection of N1 • WVDL assay • 4 hrs needed for results

  20. Nucleotide Sequencing & Analysis of F Gene for ND • Sequence analysis is conducted on the F gene • PCR is conducted to amplify a portion of the F gene including the cleavage site • 254 bp amplicon for analysis of cleavage site • 1000 bp amplicon for phylogenetic analysis (includes portions of the matrix and fusion genes)

  21. Position 113 114 115 116 117 Avirulent R/K Q G R Virulent R/K Q R/K R L F 3' 5' HN M F P L NP Cleavage site F2 F1 N C 0 100 200 300 400 500 Cleavage site F2 F1

  22. Automated Sequencing • Sanger sequencing with fluorescently labeled DNA fragments is the most frequently used technique • Tagged ddNTP’s are detected during electrophoresis with a four-color dye system • Sequence is automatically recorded to chromatograph which is interpreted by computer software into DNA sequence (ABI 3730 DNA analyzer)

  23. Sequencing PCR Amplicons Chromatograph from automated DNA sequencer of NDV F nucleotide sequence

  24. Chromatograph Analysis • The height of each of the 4 colored lines are indicative of fluorescence that corresponds to each of the 4 labeled dideoxynucleotides • Good sequence data • Well defined peaks • Peaks easily distinguished from background noise • Poor sequence data • Poorly defined peaks • Low signal-to-noise resulting in ambiguities

  25. Peaks are well defined and distinguishable form background noise Lower signal to noise Results in occasional ambiguities Peaks are less well defined and ambiguities increased Unreliable sequencing

  26. Sequence Analysis • Chromatograph is edited for quality of sequence and analyzed by commercial sequence analysis software • The nucleotide sequence is translated and the amino acid sequence at the cleavage site is identified • multiple basic amino acids ? • Fusion gene – NDV • Hemagglutinin gene - AIV

  27. The amino acid sequence at the fusion gene cleavage site of avirulent APMV-1 SGGGKQGR / LIGAI Basic amino acids Arginine (R) and Lysine (K)

  28. Amino Acid Sequences at the Cleavage Site of the Fusion Protein Strain Pathotype AA Sequence LaSota L S G G G R Q G R / L I G A I Roakin M S G G R R Q K R / F I G A I GB Texas NV S G G R R Q K R / F V G A I CK/CA/03 VV S G G R R Q K R / F V G A I PG/NY/01 PPMV-1 S G V R RKK R / F I G A I PG/NC/01 PPMV-1 S G E K R Q K R / F I G A I

  29. The amino acid at the fusion gene cleavage site of PPMV-1

  30. Influenza A Virus Hemagglutinin N-terminus of HA2 Theoretical minimum sequence for high pathogenicity HA1 / HA2 -4 -3 -2 -1 +1 BASIC – ANY – BASIC – ARG / GLY C-terminus of HA1 Basic amino acids Arginine (R) and Lysine (K)

  31. Pathogenic Strains Evolve from Nonpathogenic Lineages LBM H7N2 LPAI: Cause for Concern 1994 – 1995: P E N P K T R / G L F 1995 – 1998: P E N P K P R / G L F 1999 – 2001: P E K P K P R / G L F 2002 : P E K P K K R/ G L F 2003 (33): P E K P K P R / G L F 2004 (12): P E K P K K R/ G L F 2004 (178): P E K P K P R / G L F 2004 (3): P E R P K P R / G L F Minimum for HPAI = B X B R / G

  32. Connecting Peptide Sequence of Selected H5 Viruses A C A G A Arg Lys HA1 / HA2 -4 -3 -2 -1 +1 BASIC – ANY – BASIC – ARG / GLY

  33. Phylogenetic Analysis • Phylogenetic analysis is the study of evolutionary relationships Objective: to determine which data (sequences) are most closely related – how this family of viruses have evolved • Sequence phylogenetics – 2 sequences are the same at 95% of the nucleotide positions • Protein phylogenetics – 2 proteins are the same at 95% of the amino acid positions

  34. Phylogenetic Analysis Cont. • 2 sequences that are highly related (high % homology) will be located as neighboring branches – joined to a common branch • Analysis can be conducted with sequence (taxa) from a conserved gene (matrix) or non-conserved (HA/F) • An important tool for epidemiology • From what virus did the virus of interest most likely evolve • Is the virus of interest a new introduction of the disease, or an expansion of a previously introduced virus

  35. Chicken/U.S.(CA)/22908/03

  36. Phylogenetic Analysis of Ck/DE/04 H7N2 and other U.S. H7 Viruses Hemagglutinin Gene Phylogeny 2003/04 LBM H7N2 viruses and CT/03 Phylogenetic tree courtesy D. Suarez, SEPRL

  37. H5 Phylogenetic Tree TK/WI/68 Emu/Tx/39442/93 CK/NJ/17169/93 Chukkar/MN/14951-7/98 Env/NY/5626-2/98 UN/NY/101250-18/01 DK/NY/191255-59/02 DK/NY/186875/02 AV/NY/31588-3/00 DK/NY/44018-1/00 Avian/NY/31588-2/00 CK/TX/04 CK/TX/167280-4/02 TK/MN/10734/95 UN/NY/9899-6/01 DK/ME/151895-7A/02 TK/CA/D0208651-C/02 Pheasant/MD/4457/93 Ck/Hidalgo/26654-1368/94 Ck/Mexico/31381-1/94 Ck/Queretaro/14588-19/94 CK/Mich/28159-530/95 CK/Chiapas/15224/97 CK/FO-Guatemala/45511-3/00 CK/El Salvador/102711-2/01 CK/Mexico/37821-771/96 CK/Vera Cruz/232-6169/98 TK/TX/14802/82 Mallard/OH/345/88 Mallard/WI/428/75 Mallard/WI/944/82 Tk/Ontario/7732/66 Ck/PA/1/83 CK/PA/1370/83 CK/VA/40018/84 CK/OH/22911-10/86 CK/FL/22780-2/88 CK/FL/2507/89 H5 Eurasian 10 changes H5N2 H5N3 Phylogenetic tree courtesy D. Suarez, SEPRL

  38. Chicken/QV4/Australia/66 Chicken/Ulster/U.K./67 1.9 6.8 4.2 Chicken/LaSota/U.S./46 3.1 7.8 Chicken/B1/U.S./47 2.8 Chicken/BeadetteC/U.S./45 Chicken/TexasGB/U.S./48 7.9 Chicken/Australia-Victoria/32 7.0 Chicken/Fontana-1083/CA/72 6.8 8.2 Pheasant/1208/CA/98 0.2 3.4 1.8 5.1 0.9 Chicken/139/Kenya/90? 6.9 4.4 0.6 Pigeon/17498/U.S. (TX)/98 6.0 Pigeon/Italy/00 15.7 Chicken/147/Korea/97 0.7 1.0 12.6 Chicken/Italy/00 11.9 8.2 Game Chicken/24225/CA/98 1.1 Chicken/3782-2/Mexico/96 3.0 4.9 Psittacine/28710/importU.S./93 1.4 Chicken/3782-1/Mexico/96 Chicken/229808/CA/03 236498/AZ/03 0.6 232947/NV/03 215149/CA/03 4.9 1.4 Gamefowl/223412/CA/02 Gamefowl/211472/CA/02 Owl/220634/CA/02 1.6 215151/CA/02 California 2002/03 V-NDV Fusion Gene Phylogeny Mexico-California NDV isolates Pet bird/169302/CA/May,02 1.1 0.6 215150/CA/02 1.6 Yellow Cheek/27345/U.S(TX)/96 1.1 Chicken/141368/Mexico/00 0.4 0.6 Chicken/2/Mexico/00 Chicken/1/Mexico/00 0.9 Chicken/3073/Mexico/00 1.0 Chicken/4100/Mexico/99 1.0 Chicken/290/Mexico/00 0.5 0.8 Chicken/6244/Mexico/98 1.4 3.9 Chicken/5166//Mexico/98 3.4 0.7 Amazon parrot/36501/89 9.6 Chicken/Honduras/00 1 change 3.9 Pigeon/44407/84 0.5 5.0 Turkey/43804/92 0.1 4.6 Cormorant/40068/92 2.9 Anhinga/44083/93 6.1 Mixed species/Largo/71

  39. Chicken/U.S./BeaudetteC/52 Chicken/U.S./Roakin/48 Chicken/U.S.(TX)/GB/48 Chicken/U.S./B1./48 Turkey/U.S./VGGA/89 Chicken/U.S./LaSota/46 Dove/Italy/2736/00 Matrix Gene Phylogeny Duck/Japan/D26/78 Chicken/Australia/QV4/66 Chicken/NorthernIreland/Ulster/64 Chicken/Australia/AV/32 Chicken/Italy/Milano/45 Chicken/Mexico/37821/96 Chicken/Honduras/H15/00 Pigeon/U.S.(CA)/5658/75 Psittacine/U.S(FL)/Largo/71 Anhinga/U.S.(FL)/44083/93 Turkey/U.S.(ND)/430084/92 Chicken/Argentina/TL/71 Cockatoo/Indonesia?/14698/90 Parakeet/Tanzania?/28710/93 Chicken/3286/Italy/00 Pigeon/U.S.(GA)/21042/98 Pigeon/U.S.(TX)/17498/98 Pigeon-origin NDV Isolates Pigeon/Italy/1166/00 Pigeon/U.S(NY)/84 10 changes Pigeon/U.S.(MD)/3981/84 Pigeon/Argentina/Capital/97 Pigeon/Argentina/Tigre/99 Chicken/Korea/12a/89 Chicken/U.S./CA1083(Fontana)/71

  40. Alignment and BLAST Search Data • Process of comparing a new sequence with all other known sequences and determining homology (similarity) • Powerful tool to quickly determine relatedness • BLAST search should be conducted using a comprehensive and up-to-data repository • National Center for Biotechnology Information (NCBI) • GeneBank • Alignment is conducted using selected genomic sequence • Hemagglutinin – variable gene related to pathogenicity • Matrix – more conserved gene, not used for pathogenicity analysis

  41. Alignment and BLAST Search Analysis • Comparing an new sequence with all other known sequences • Can differentiate genotypes • LBM H7N2 viruses have a 24 nt deletion in the hemagglutinin gene

  42. Nucleotide sequence alignment of AI H7 viruses 24 nt deletion differentiates the two H7 genotypes

  43. Nucleotide sequence alignment of two avirulent APMV-1 isolates Amino acid motif at cleavage site – SGGRQGR / GLFGAI and SGGKQGR / GLFGAI

  44. NVSL, Ames, Iowa Thank You For Your Attention! OIE, FAO Reference Laboratory: Avian Influenza Newcastle disease

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