Characterization and Pathotyping of AIV and APMV-

1

Jan Pedersen, MicrobiologistNational 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 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

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

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 > 1.2

3. 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.

Low Pathogenic Notifiable AI (LPNAI)

All influenza A viruses of H5 and H7 subtype that are not HPNAI viruses

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

Intravenous Pathogenicity Index (IVPI)

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

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

Identification and Pathotyping of Newcastle Disease Virus

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

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 greateror

Multiple basic amino acids at the C-terminus of the F2 protein and phenylalanine at residue 117 (NH2 terminus of F1)

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

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

Intracerebral Pathogenicity Index (ICPI)

State of chicks after inoculation

Day

Total

Score Sum1 2 3 4 5 6 7 8

Normal 10 9 6 5 5 5 5 5 50 0 0

Sick 0 1 1 1 0 0 0 0 3 1 3

Dead 0 0 3 4 5 5 5 5 27 2 54

Total recordings 80 3 57

ICPI = Weighted Mean = 3 + 57 = 0.75 Number of Observations 80

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

Pathotyping AIV and APMV-1 by Nucleotide Sequencing

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

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

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)

LFNP HNMP 5'3'

N C

F1F2

Cleavage site

0 100 200 300 400 500

F2 F1

Cleavage site

Position 113 114 115 116 117

Avirulent R/K Q G R

Virulent R/K Q R/K R F

L

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)

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

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

Peaks are well defined and distinguishable

form background noise

Lower signal to noiseResults in occasional ambiguities

Peaks are less well definedand ambiguities increased

Unreliable sequencing

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

The amino acid sequence at the fusion gene cleavage site of avirulent APMV-1

SGGGKQGR / LIGAI

Basic amino acidsArginine (R) and Lysine (K)

Amino Acid Sequences at the Cleavage Site of the Fusion

Protein

LaSota L S G G G R Q G R / L I G A I

GB Texas NV S G G R R Q K R / F V G A I

Roakin M S G G R R Q K R / F I G A I

CK/CA/03 VV S G G R R Q K R / F V G A I

Strain Pathotype AA Sequence

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

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

Influenza A Virus Hemagglutinin

Theoretical minimum sequence for high pathogenicity

BASIC – ANY – BASIC – ARG / GLY

-4 -3 -2 -1 +1

HA1 / HA2

C-terminus of HA1

N-terminus of HA2

Basic amino acids

Arginine (R) and Lysine (K)

Pathogenic Strains Evolve from Nonpathogenic Lineages

LBM H7N2 LPAI: Cause for Concern1994 – 1995: P E N P K T R / G L F1995 – 1998: P E N P K P R / G L F1999 – 2001: P E K P K P R / G L F2002 : P E K P K K R / G L F2003 (33): P E K P K P R / G L F2004 (12): P E K P K K R / G L F2004 (178): P E K P K P R / G L F2004 (3): P E R P K P R / G L F

Minimum for HPAI = B X B R / G

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

Connecting Peptide Sequence of Selected H5 Viruses

A C A

G AArg Lys

HA1 / HA2

BASIC – ANY – BASIC – ARG / GLY

-4 -3 -2 -1 +1

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

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

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

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/13246-9/98

TK/NJ/9778-8/98

CK/PA/9801289/98

CK/NY/3572/98

CK/NJ/20621/99

CK/NY/30749-3/00

CK/FL/90348-4/01

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/260422-10/03

CK/DE/297267/04

CK/NY/292437/04

CK/NY/119055-7/01

CK/NJ/15827/99

CK/NY/34173-3/99

Avian/NY/73063-6/00

GuineaFowl/MA/148081-11/02

CK/NJ/294508-12/04

CK/NY/119256-7/01

Goose/NJ/8600-3/98

CK/NY/1398-6/99

CK/NY/13833-7/95

CK/NY/13142-5/94

Quail/PA/20304/98

CK/NY/12273-11/99

CK/NY/14714-9/99

Seal/Mass/1/80

Ck/Chile/176822/02

TK/Oregon/71

10 changes

Phylogenetic tree courtesy D. Suarez, SEPRL

Phylogenetic Analysis of

Ck/DE/04 H7N2 and other U.S. H7 Viruses

Hemagglutinin Gene Phylogeny

2003/04 LBM H7N2 viruses

and CT/03

H5 Phylogenetic Tree

Phylogenetic tree courtesy D. Suarez, SEPRL

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

H5N3H5N2

Chicken/QV4/Australia/66Chicken/Ulster/U.K./67

Chicken/LaSota/U.S./46Chicken/B1/U.S./47

Chicken/BeadetteC/U.S./45Chicken/TexasGB/U.S./48

Chicken/Australia-Victoria/32Chicken/Fontana-1083/CA/72

Pheasant/1208/CA/98Chicken/139/Kenya/90?

Pigeon/17498/U.S. (TX)/98Pigeon/Italy/00

Chicken/147/Korea/97Chicken/Italy/00

Game Chicken/24225/CA/98Chicken/3782-2/Mexico/96

Psittacine/28710/importU.S./93Chicken/3782-1/Mexico/96

Chicken/229808/CA/03236498/AZ/03232947/NV/03215149/CA/03

Gamefowl/223412/CA/02Gamefowl/211472/CA/02

Owl/220634/CA/02215151/CA/02

Pet bird/169302/CA/May,02215150/CA/02

Yellow Cheek/27345/U.S(TX)/96Chicken/141368/Mexico/00

Chicken/2/Mexico/00Chicken/1/Mexico/00Chicken/3073/Mexico/00

Chicken/4100/Mexico/99Chicken/290/Mexico/00

Chicken/6244/Mexico/98Chicken/5166//Mexico/98

Amazon parrot/36501/89Chicken/Honduras/00

Pigeon/44407/84Turkey/43804/92Cormorant/40068/92

Anhinga/44083/93Mixed species/Largo/71

1 change

8.2

6.8

8.2

7.84.2 1.9

3.1

2.8

4.9

0.2

7.9

0.7

3.4

7.0

1.0

0.6

1.86.8

4.40.9 5.1

6.96.0

15.712.6

11.9

3.9

0.4

0.6

3.0 1.14.9

1.1

1.4

0.8

0.9

0.6

0.6

1.6

1.4

1.61.1

1.01.0

0.51.4

0.5

0.7 3.49.6

5.03.9

4.60.1

2.96.1

Mex

ico-

Cal

ifor

nia

ND

V is

olat

es

California 2002/03 V-NDV Fusion

Gene Phylogeny

Chicken/U.S./BeaudetteC/52Chicken/U.S./Roakin/48

Chicken/U.S.(TX)/GB/48Chicken/U.S./B1./48

Turkey/U.S./VGGA/89Chicken/U.S./LaSota/46

Dove/Italy/2736/00Duck/Japan/D26/78

Chicken/Australia/QV4/66

Chicken/NorthernIreland/Ulster/64Chicken/Australia/AV/32Chicken/Italy/Milano/45

Chicken/Mexico/37821/96Chicken/Honduras/H15/00

Pigeon/U.S.(CA)/5658/75Psittacine/U.S(FL)/Largo/71

Anhinga/U.S.(FL)/44083/93Turkey/U.S.(ND)/430084/92

Chicken/Argentina/TL/71Cockatoo/Indonesia?/14698/90

Parakeet/Tanzania?/28710/93

Chicken/3286/Italy/00Pigeon/U.S.(GA)/21042/98

Pigeon/U.S.(TX)/17498/98Pigeon/Italy/1166/00

Pigeon/U.S(NY)/84Pigeon/U.S.(MD)/3981/84

Pigeon/Argentina/Capital/97

Pigeon/Argentina/Tigre/99Chicken/Korea/12a/89

Chicken/U.S./CA1083(Fontana)/71

10 changes

Pigeon-origin

ND

V Isolates

Matrix GenePhylogeny

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

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

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

Nucleotide sequence alignment of two avirulent APMV-1 isolates

Amino acid motif at cleavage site – SGGRQGR / GLFGAIand SGGKQGR / GLFGAI

NVSL, Ames, IowaOIE, FAO Reference Laboratory:Avian InfluenzaNewcastle disease

Thank You For Your Attention!