Characterization and Pathotyping of AIV and APMV-1

Size: px

Start display at page:

Download "Characterization and Pathotyping of AIV and APMV-1"

Vivian Patterson
5 years ago
Views:

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 Influenza Factors 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) 1. Any AIV that is lethal for 6-8 of eight 4-8 wk. old susceptible chickens within 10 days -Intravenous lethality test 2. Any AIV that has a IVPI in 4-8 wk. old chickens of > 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 Highly Pathogenic Avian Influenza 1. Any influenza that kills 6, 7, or 8 of 8 chickens 2. Any influenza virus that is not an H5 or H7 that kills 1 to 5 chickens and grows in cell culture in the absence of trypsin 3. Any H5 or H7 subtype that does not meet the criteria in item, but has an amino acid sequence at the cleavage site of the hemagglutinin that is compatible with other HPAI viruses Nucleotide and amino acid sequencing of the HA gene Phylogenetic analysis

8 Intravenous Pathogenicity Index (IVPI)

9 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

11 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

12 Identification and Pathotyping of Newcastle Disease Virus

13 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

14 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)

15 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

17 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

18 Intracerebral Pathogenicity Index (ICPI) State of chicks after inoculation Day Total Score Sum Normal Sick Dead Total recordings ICPI = Weighted Mean = = 0.75 Number of Observations 80

19 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

20 Scoring of Lesions for the Intracloacal Inoculation Pathogenicity Test +4 = edema of head and neck, hemorrhage in trachea, hemorrhage and necurosis/ulceration throughout the gastrointestinal tracts = lesions in repiratory and intestinal tracts but in decreasing severity Viruses are viscerotropic (VVNDV) if one bird shows +4 lesions or at least two birds show +2 or +3 lesions NVNDV virus causes death in birds which display neurological signs

21 Pathotyping AIV and APMV-1 by Nucleotide Sequencing

22 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

23 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

24 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)

25 3' NP P M F HN L 5' Cleavage site N F2 F1 C Cleavage site F2 F1 Position Avirulent R/K Q G R L Virulent R/K Q R/K R F

Automated Sequencing Sanger sequencing with fluorescently labeled DNA fragments is the most frequently used technique Tagged ddntp s are detected during

26 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)

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

28 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

29 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

30 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

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

32 Amino Acid Sequences at the Cleavage Site of the Fusion Protein Strain Pathotype AA Sequence LaSota Roakin L S G G G R Q G R / L I G A I 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 R K K R / F I G A I PG/NC/01 PPMV-1 S G E K R Q K R / F I G A I

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

34 Amino Acid Sequence at the Fusion Gene Cleavage Site Isolate U.S./B1/48 TX GB/48 Pigeon/Italy/00 Turkey/ND/92 Mexico/1/00 TX/248306/03 CA/211472/02 Dove/CA/9547/03 Chick/CA/634/03 F2 F1/117 Gly Gly Gly Arg Gln Gly Arg / Leu Iso Gly Gly Gly Arg Arg Gln Lys Arg /Phe Iso Gly Gly Gly Arg Arg Gln Lys Arg /Phe Iso Gly Arg Gly Arg Arg Gln Lys Arg /Phe Val Gly Gly Gly Arg Arg Gln Lys Arg /Phe Val Gly Gly Gly Arg Arg Gln Lys Arg /Phe Val Gly Gly Gly Arg Arg Gln Lys Arg /Phe Val Gly Gly Gly Arg Arg Gln Arg Arg /Phe Val Gly Gly Gly Arg Arg Gln Arg Arg /Phe Val Gly Pathotype L M PPMV-1 NV V V V V V

35 Influenza A Virus Hemagglutinin Theoretical minimum sequence for high pathogenicity HA1 / HA BASIC ANY BASIC ARG / GLY N-terminus of HA2 C-terminus of HA1 Basic amino acids Arginine (R) and Lysine (K)

37 Pathogenic Strains Evolve from Nonpathogenic Lineages LBM H7N2 LPAI: Cause for Concern : P E N P K T R / G L F : P E N P K P R / G L F : 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

38 Connecting Peptide Sequence of Selected H5 Viruses Amino Acid Sequence Virus Virulence HA1/HA2 CK/Penn/83 Virulent Pro Gln Lys Lys Lys Arg/Gly Ck/Scott/59 Virulent Pro Gln Arg Lys Lys Arg/Gly Dk/Penn/84 Avirulent Pro Gln Arg Glu Thr Arg/Gly Ck/Penn/93 Avirulent Pro Gln Arg Lys Thr Arg/Gly HA1 / HA BASIC ANY BASIC ARG / GLY A C A G A Arg Lys

39 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

40 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

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

42 Phylogenetic Analysis of Ck/DE/04 H7N2 and other U.S. H7 Viruses Hemagglutinin Gene Phylogeny 10 changes TK/NY/4450-5/94 CK/NY/3112-1/95 CK/NY/8030-2/96 CK/NY/3202-7/96 CK/NY/1387-8/98 GuineaFowl/NJ/ /98 TK/NJ/9778-8/98 CK/PA/ /98 CK/NY/3572/98 CK/NJ/20621/99 CK/NY/ /00 CK/FL/ /01 TK/VA/66/02 TK/NC/11165/02 CK/PA/143586/01 CK/VA/32/02 Avian/NY/244451/03 CK/CT/260413/03 Avian/NY/ /03 CK/DE/297267/04 CK/NY/292437/04 CK/NY/ /01 CK/NJ/15827/99 CK/NY/ /99 Avian/NY/ /00 GuineaFowl/MA/ /02 CK/NJ/ /04 CK/NY/ /01 Goose/NJ/8600-3/98 CK/NY/1398-6/99 CK/NY/ /95 CK/NY/ /94 Quail/PA/20304/98 CK/NY/ /99 CK/NY/ /99 Seal/Mass/1/80 Ck/Chile/176822/02 TK/Oregon/ /04 LBM H7N2 viruses and CT/03 Phylogenetic tree courtesy D. Suarez, SEPRL

43 H5 Phylogenetic Tree 10 changes TK/WI/68 Emu/Tx/39442/93 CK/NJ/17169/93 Chukkar/MN/ /98 Env/NY/5626-2/98 UN/NY/ /01 DK/NY/ /02 DK/NY/186875/02 AV/NY/ /00 DK/NY/ /00 Avian/NY/ /00 CK/TX/04 H5N2 CK/TX/ /02 H5N3 TK/MN/10734/95 UN/NY/9899-6/01 DK/ME/ A/02 TK/CA/D C/02 Pheasant/MD/4457/93 Ck/Hidalgo/ /94 Ck/Mexico/ /94 Ck/Queretaro/ /94 CK/Mich/ /95 CK/Chiapas/15224/97 CK/FO-Guatemala/ /00 CK/El Salvador/ /01 CK/Mexico/ /96 CK/Vera Cruz/ /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/ /86 CK/FL/ /88 CK/FL/2507/89 H5 Eurasian Phylogenetic tree courtesy D. Suarez, SEPRL

44 Chicken/QV4/Australia/66 Chicken/Ulster/U.K./67 1 change 4.9 California 2002/03 V-NDV Fusion Gene Phylogeny Chicken/LaSota/U.S./46 Chicken/B1/U.S./47 Chicken/BeadetteC/U.S./45 Chicken/TexasGB/U.S./ Chicken/Australia-Victoria/ Chicken/Fontana-1083/CA/72 Pheasant/1208/CA/ Game Chicken/24225/CA/ Chicken/3782-2/Mexico/96 Psittacine/28710/importU.S./93 Chicken/3782-1/Mexico/96 Chicken/229808/CA/ /AZ/ /NV/ /CA/03 Gamefowl/223412/CA/02 Gamefowl/211472/CA/02 Owl/220634/CA/ /CA/02 Pet bird/169302/ca/may, /CA/02 Yellow Cheek/27345/U.S(TX)/ Chicken/141368/Mexico/00 Chicken/2/Mexico/00 Chicken/1/Mexico/00 Chicken/3073/Mexico/ Chicken/4100/Mexico/99 Chicken/290/Mexico/00 Chicken/6244/Mexico/ Chicken/5166//Mexico/ Amazon parrot/36501/ Chicken/Honduras/00 Pigeon/44407/ Turkey/43804/ Cormorant/40068/ Anhinga/44083/93 Mixed species/largo/71 Chicken/139/Kenya/90? Pigeon/17498/U.S. (TX)/98 Pigeon/Italy/00 Chicken/147/Korea/97 Chicken/Italy/00 Mexico-California NDV isolates

45 10 changes CK/Jalisco/ /94 CK/Jalisco/ /95 CK/Hidalgo/ /95 CK/Queretaro/ /94 CK/Queretaro/ /95 CK/Queretaro/ /96 CK/GTO/ /95 CK/Mich/ /95 CK/Hidalgo/ /95 Mexico/VACCINE/95 CK/Queretaro/ /94 CK/Morelos/ /95 CK/Hidalgo/ /94 CK/Mexico/ /94 CK/Mexico/ /94 CK/Mexico/ /94 CK/Mexico/ /94 CK/Mexico/ /94 CK/Mexico/ /94 CK/Mexico/ /94 CK/VeraCruz/ /95 CK/Mexico/ /96 CK/Chiapas/15406/97 CK/Chiapas/15408/97 CK/Vera Cruz/ /98 CK/Puebla/ /98 CK/Morelos/FO22189/98 CK/Morelos/ /98 CK/Jalisco/ /98 CK/AG/ /98 CK/Puebla/ /95 CK/Chiapas/15224/97 CK/Chiapas/15405/97 CK/TAB/ /98 CK/FO/22066/98 CK/FO-Guatemala/ /00 CK/FO-Guatemala/ /00 Parrot/CA/04 (H5N2) CK/FO-Guatemala/ /00 CK/FO-Guatemala/ /00 CK/El Salvador/ /01 CK/El Salvador/ /01 CK/FO-Guatemala/ /00 CK/FO-GUATEMALA/194573/02 CK/Guatemala/ /03 CK/Guatemala/ /03 CK/Puebla/ /94 CK/Puebla/ /94 CK/Puebla/ /94 CK/Puebla/ /94 TK/CA/D /02 Ck/PA/13609/93 Ck/PA/1/83 TK/WI/68 Eurasian H5 H5 tree for Parrot/CA/04 Mexican-lineage H5N2 viruses

46 10 changes 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 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/Italy/1166/00 Pigeon/U.S(NY)/84 Pigeon/U.S.(MD)/3981/84 Pigeon/Argentina/Capital/97 Pigeon/Argentina/Tigre/99 Chicken/Korea/12a/89 Chicken/U.S./CA1083(Fontana)/71 Matrix Gene Phylogeny Pigeon-origin NDV Isolates

47 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

48 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

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

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

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

Introduction to Avian Influenza

Introduction to Avian Influenza David L. Suarez D.V.M., Ph.D. Research Leader Exotic and Emerging Avian Viral Disease Research Unit Agricultural Research Service United States Department of Agriculture