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
 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 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
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 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


rRT-PCR



Cross neutralization with other PMV
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 (APMV1) 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)
Pathotyping Assays
for ND

Intracerebral Pathogenicity Index (ICPI)



Intracloacal Inoculation Pathogenicity Test


Differentiates vNDV from avirulent APMV-1
ICPI for PPMV-1 are quite variable
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
Normal
Day
1
2
3
4
5
6
7
8
10 9
6
5
5
5
5
Total
Score
Sum
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
80
3
57
Total recordings
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)
3'
P
NP
M
F
HN
5'
L
Cleavage site
F2
F1
N
0
C
100
200
300
400
Cleavage site
F2
F1
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
 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 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
 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 acids
Arginine (R) and Lysine (K)
Amino Acid Sequences at the
Cleavage Site of the Fusion Protein
Strain
Pathotype
AA Sequence
LaSota
L
SGGG RQGR /LIGAI
Roakin
M
SGG RRQKR/FIGAI
GB Texas
NV
SGG RR QKR/FVGAI
CK/CA/03
VV
SGG RR QKR/FVGAI
PG/NY/01
PPMV-1
SGVRR KKR /FIGAI
PG/NC/01
PPMV-1
S G E K R Q KR / FI GAI
The amino acid at the
fusion gene cleavage site of PPMV-1
Influenza A Virus Hemagglutinin
Theoretical minimum sequence
for high pathogenicity
HA1 / HA2
-4
-3
-2
-1
+1
BASIC – ANY – BASIC – ARG / GLY
N-terminus of HA2
C-terminus of HA1
Basic amino acids
Arginine (R) and Lysine (K)
Pathogenic Strains Evolve from
Nonpathogenic Lineages
LBM H7N2 LPAI: Cause for Concern
1994
1995
1999
2002
2003
2004
2004
2004
– 1995:
– 1998:
– 2001:
:
(33):
(12):
(178):
(3):
PENPKTR/GLF
PENPKPR/GLF
PEKPKPR/GLF
PEKPKKR/GLF
PEKPKPR/GLF
PEKPKKR/GLF
PEKPKPR/GLF
PERPKPR/GLF
Minimum for HPAI = B X B R / G
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
ACA
HA1 / HA2
-4
-3
-2
-1
+1
BASIC – ANY – BASIC – ARG / GLY
G A
Arg
Lys
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
Phylogenetic Analysis of
Ck/DE/04 H7N2 and
other U.S. H7 Viruses
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
Hemagglutinin Gene
Phylogeny
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
2003/04 LBM
H7N2 viruses
and CT/03
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
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
H5N2
H5N3
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
Phylogenetic tree
courtesy D. Suarez,
SEPRL
7.8
8.2
0.2
0.7
8.2
4.9
California 2002/03
V-NDV Fusion
Gene Phylogeny
1 change
4.2
1.9
3.1
Chicken/LaSota/U.S./46
Chicken/B1/U.S./47
2.8
Chicken/BeadetteC/U.S./45
Chicken/TexasGB/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/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
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 1.40.5Chicken/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/QV4/Australia/66
Chicken/Ulster/U.K./67
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
10 changes
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 / GLFGAI
and SGGKQGR / GLFGAI
Thank You For Your
Attention!
NVSL, Ames, Iowa
OIE, FAO Reference Laboratory:
Avian Influenza
Newcastle disease