Characterization and Pathotyping of AIV and APMV-1 Jan

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 q r. RT-PCR – Matrix assay q Antigen Detection assays q Influenza virus can be subtyped by: HI - 16 hemagglutinin subtypes q NI – 9 neuraminidase subtypes q Conventional RT-PCR or r. RT-PCR N 1 assay q r. RT-PCR – H 5 and H 7 assays q

Avian Influenza Factors Influencing Pathogenicity q Polygenic q Associated with H 5 and H 7 subtypes q HA glycoprotein plays a dominate role q Presence of multiple basic amino acids at the cleavage site of the HA glycoprotein q Pathogenic strains evolve from nonpathogenic lineages

Definition of Highly Pathogenic Notifiable AIV (HPNAI) 1. 2. 3. 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 H 5 or H 7 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.

Low Pathogenic Notifiable AI (LPNAI) q All influenza A viruses of H 5 and H 7 subtype that are not HPNAI viruses

Notifiable AIV (NAI) q HPNAI – AIV or viral RNA detected in poultry or a poultry product q LPNAI - AIV or viral RNA of the H 5 or H 7 subtypes detected in poultry or a poultry product q Antibodies to H 5 or H 7 that are not a consequence of vaccination, nor indicative of a non-specific reaction

Intravenous Pathogenicity Index (IVPI)

Intravenous Pathogenicity Index (IVPI) q SPF or AIV antibody free commerical chickens q AIV isolate free of bacterial contamination is diluted 1: 10 in sterile diluent (TBTB, PBS) q Ten 4 -8 wk. old chickens are inoculated intravenouly with 0. 2 ml of the diluted isolate q Number of normal, sick, morbid and dead chickens are recorded daily for 10 days

Characterization of HPAI by IVPI q Any isolate with an IVPI > 1. 2 is pathotyped as HPAI q Any isolate with an IVPI < 1. 2 is pathotyped as LPAI

Identification and Pathotyping of Newcastle Disease Virus

Identification of APMV-1 q HI with APMV-1 monospecific antiserum Ø q r. RT-PCR Ø Ø q Cross neutralization with other PMV Matrix assay identifies all APMV-1 v. NDV assay identifies virulent NDV & PPMV-1 HI with monoclonal antisera Ø Identification of Pigeon paramyxovirus

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 q ICPI in day-old chicks of 0. 7 or greater or q Multiple basic amino acids at the C-terminus of the F 2 protein and phenylalanine at residue 117 (NH 2 terminus of F 1)

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

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 q Chicks are observed daily for 8 days and the number of normal, sick and dead chicks are recorded q ICPI is calculated by dividing the weighted mean by the number of observations q

Intracerebral Pathogenicity Index (ICPI) State of chicks after inoculation Normal Day 1 2 3 4 5 6 7 8 10 9 6 5 5 Total Score Sum 5 50 0 0 Sick 0 1 1 1 0 0 3 1 3 Dead 0 0 3 4 5 5 27 2 54 80 3 57 Total recordings ICPI = Weighted Mean = 3 + 57 = 0. 75 Number of Observations 80

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

Pathotyping AIV and APMV-1 by Nucleotide Sequencing

Nucleotide Sequencing & Analysis of AI HA Gene Sequence analysis is conducted on all H 5 and H 7 viruses q Single tube one step RT-PCR for H 5 q Conventional 2 step RT-PCR for H 5 & H 7 q Specific H 5 and H 7 PCR primers are selected for amplification of c. DNA q Eurasian isolate q North American isolate q

Nucleotide Sequencing & Analysis of AI NA Gene q Conventional RT-PCR for the detection of N 1 Ø Ø q WHO assay 6. 5 hrs. needed for results r. RT-PCR for the detection of N 1 Ø Ø WVDL assay 4 hrs needed for results

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

3' P NP M F HN 5' L Cleavage site F 2 F 1 N 0 C 100 200 300 400 Cleavage site F 2 F 1 Position 113 114 115 116 117 Avirulent R/K Q G R L Virulent R/K Q R/K R F 500

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

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

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

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

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

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

Amino Acid Sequences at the Cleavage Site of the Fusion Protein Strain Pathotype AA Sequence La. Sota L SGGGRQGR/LIGAI Roakin M SGGRRQKR/FIGAI GB Texas NV SGGRRQKR/FVGAI CK/CA/03 VV SGGRRQKR/FVGAI PG/NY/01 PPMV-1 SGVRRKKR/FIGAI PG/NC/01 PPMV-1 SGEKRQKR/FIGAI

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

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

Pathogenic Strains Evolve from Nonpathogenic Lineages LBM H 7 N 2 LPAI: Cause for Concern 1994 – 1995: 1995 – 1998: 1999 – 2001: 2002 : 2003 (33): 2004 (12): 2004 (178): 2004 (3): PENPKTR/GLF PENPKPR/GLF PEKPKKR/GLF PEKPKPR/GLF PERPKPR/GLF Minimum for HPAI = B X B R / G

Connecting Peptide Sequence of Selected H 5 Viruses Amino Acid Sequence Virus Virulence CK/Penn/83 Virulent Pro Gln Lys Lys Arg/Gly Ck/Scott/59 Virulent Pro Gln Arg 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 -4 -3 HA 1/HA 2 HA 1 / HA 2 -2 -1 +1 BASIC – ANY – BASIC – ARG / GLY ACA G A Arg Lys

Phylogenetic Analysis q 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 Ø Sequence phylogenetics – 2 sequences are the same at 95% of the nucleotide positions Ø Protein phylogenetics – 2 proteins are the same at 95% of the amino acid positions

Phylogenetic Analysis Cont. q 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) q An important tool for epidemiology q Ø Ø 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

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

Phylogenetic Analysis of Ck/DE/04 H 7 N 2 and other U. S. H 7 Viruses Hemagglutinin Gene Phylogeny 2003/04 LBM H 7 N 2 viruses and CT/03 Phylogenetic tree courtesy D. Suarez, SEPRL

H 5 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 H 5 N 2 H 5 N 3 UN/NY/9899 -6/01 DK/ME/151895 -7 A/02 TK/CA/D 0208651 -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 H 5 Eurasian 10 changes Phylogenetic tree courtesy D. Suarez, SEPRL

7. 8 8. 2 0. 7 8. 2 4. 9 California 2002/03 V-NDV Fusion Gene Phylogeny 1 change 4. 2 1. 9 3. 1 Chicken/La. Sota/U. S. /46 Chicken/B 1/U. S. /47 2. 8 Chicken/Beadette. C/U. S. /45 Chicken/Texas. GB/U. S. /48 7. 9 7. 0 Chicken/Australia-Victoria/32 6. 8 Chicken/Fontana-1083/CA/72 Pheasant/1208/CA/98 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 1. 0 12. 6 Chicken/Italy/00 11. 9 Game Chicken/24225/CA/98 3. 0 1. 1 4. 9 Chicken/3782 -2/Mexico/96 Psittacine/28710/import. U. S. /93 1. 4 Chicken/3782 -1/Mexico/96 Chicken/229808/CA/03 236498/AZ/03 0. 6 232947/NV/03 215149/CA/03 1. 4 Gamefowl/223412/CA/02 Gamefowl/211472/CA/02 Owl/220634/CA/02 1. 6 215151/CA/02 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. 8 1. 40. 5 Chicken/6244/Mexico/98 3. 9 Chicken/5166//Mexico/98 0. 7 3. 4 Amazon parrot/36501/89 9. 6 Chicken/Honduras/00 3. 9 Pigeon/44407/84 0. 5 5. 0 0. 1 Turkey/43804/92 4. 6 Cormorant/40068/92 2. 9 Anhinga/44083/93 6. 1 Mixed species/Largo/71 Mexico-California NDV isolates 6. 8 Chicken/QV 4/Australia/66 Chicken/Ulster/U. K. /67

Chicken/U. S. /Beaudette. C/52 Chicken/U. S. /Roakin/48 Chicken/U. S. (TX)/GB/48 Chicken/U. S. /B 1. /48 Turkey/U. S. /VGGA/89 Chicken/U. S. /La. Sota/46 Dove/Italy/2736/00 Duck/Japan/D 26/78 Chicken/Australia/QV 4/66 Chicken/Northern. Ireland/Ulster/64 Chicken/Australia/AV/32 Chicken/Italy/Milano/45 Chicken/Mexico/37821/96 Chicken/Honduras/H 15/00 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/12 a/89 Chicken/U. S. /CA 1083(Fontana)/71 Matrix Gene Phylogeny Pigeon-origin NDV Isolates 10 changes

Alignment and BLAST Search Data q q q 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 Ø Ø q National Center for Biotechnology Information (NCBI) Gene. Bank Alignment is conducted using selected genomic sequence q q Hemagglutinin – variable gene related to pathogenicity Matrix – more conserved gene, not used for pathogenicity analysis

Alignment and BLAST Search Analysis q Comparing an new sequence with all other known sequences q Can differentiate genotypes Ø LBM H 7 N 2 viruses have a 24 nt deletion in the hemagglutinin gene

Nucleotide sequence alignment of AI H 7 viruses 24 nt deletion differentiates the two H 7 genotypes

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

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