Transcript PRRSV - MSD Animal Health Nederland

PRRSV in Europe: origin, evolution
and diversification
Tomasz Stadejek
National Veterinary Research institute
Pulawy, Poland
NVRI, Pulawy
Acknowledgments
Zygmunt Pejsak – Head of DSD, NVRI
Barbara Frącek
Katarzyna Chabros
Ewelina Skrzypiec
Katarzyna Podgórska
Martin B. Oleksiewicz, Denmark
Jonas S. Krabbe, Denmark
Torben Storgaard, Denmark
Anette Bøtner, Denmark
Roald Forsberg, Denmark
Arunas Stankevicius, Lithuania
Alexei Scherbakov, Russia
Marian Porowski, Poland
Iwona Stankiewicz, Poland
Denis Potapchuk, Belarus
PoRRSCon, FP7
EuroPRRSnet, COST
National grants, MNiSW
PRRS
• North America
– USA – 1987 (Keffaber, 1989)
– Mystery swine disease
• Asia
– Japan – 1989 (Shimizu et al., 1994)
– Heko-heko
• Europe
– Germany – 1990 (Ahl, 1991)
– Blue ear disease
PRRSV
•
•
•
•
The Netherlands: Lelystad virus (Wensvoort et al. 1991)
USA: VR-2332 (Collins et al. 1992)
Enveloped ss RNA virus
Arteriviridae family
– Arterivirus genus
• Replication in macrophages
• Prolonged viremia
• Persistent infection
• SHFV: 100% mortality within a week
• LDV: asymptomatic infection with extremely high viremia
Non-structural proteins
Structural proteins
GP2 & 2b GP4
1a
2
1b
4
3
GP3
M
6
5
GP5
7
N
Two viruses – one disease
•
Only 55-70% nucleotide identity in
different genes
•
European genotype (EU-PRRSV)
– emerged in Europe
– Mostly in Europe but also in
America and Asia
•
American genotype (NA-PRRSV)
– emerged in North America
– Mostly in America and Asia but
also in Europe
•
New terminology:
– type 1 = EU
– type 2 = NA
Early evidence for PRRSV emergence
•
North America
– 1979 Canada (Carman et al., 1995) (antibodies)
– 1985 USA (Zimmermann et al., 1997) (antibodies)
•
Asia
– 1985 South Korea (Shin et al., 1993)
– 1986 far eastern part of the former Soviet Union (Grebennikova et al.,
2005)
– 1987 Japan (Yoshii et al., 2005), Taiwan (Shin et al., 1993), Philippines
(Thanawongnuwech et al., 2003)
•
Europe
– 1987 former East Germany (Ohlinger et al., 2000) (antibodies)
European strains grouped in clades:
1. French, Dutch and Belgian
2. Spanish
Italian PRRSV - most closely
related to the original outbreak of
the disease in Europe
1996: EU-PRRSV genetic diversity lower than NA-PRRSV
Three major groups of European type
PRRSV isolates:
1. Lelystad-like clade: Western Europe
including Italy, Spain and Denmark
2. Purely Danish clade
3. A group of highly diverse Italian-like
strains from Italy, Spain, and Denmark.
2002: EU-PRRSV genetic diversity same as in NA-PRRSV
• EU-PRRSV diversity in Central Europe (PL, CZ, HU) like in
Western Europe
• EU-PRRSV diversity in Lithuania is extremelly high
• ORF7 in Lithiuanian PRRSV was 378 nucleotides, intermediate
between prototypical EU-type (387 nucleotides) and NA-type
(372 nucleotides) ORF7 lengths.
• Lithuanian strains represent strains that are most closely related
to the common ancestor of all porcine arteriviruses.
14 0
Le lystad
Au S-Lit huani a
vr 2332
150
160
GGACA GGCCAA ---AA AGAAAA AGCCT G
..... ..T... TAA.. ....G. ....C .
...A. .AAA.. TAAG. ...... .C..G .
370
380
390
CTACATCCGCCAGTCAGGGTGCAAGTTAA
.C.....T......T..
.AT..C..T.AGCATGA
2002: EU-PRRSV genetic more diverse than NA-PRRSV
• Four genetic subtypes of EUPRRSV
• Exceptional diversity of ORF7:
375, 378, 387 (prototype), 393
nucleotides long
1 40
Lel ystad
AuS-Lith uani a
ZaD-Bela rus
BoR-Bela rus
vr2 332
150
16 0
GGAC AGGCC---AAAAA GAAA AAGC CTG
.... ...T.AAT..... ...G .... .C.
.... ....G---..... .... .... ...
.... ....TCAC....G .... .... ...
...A ..AAAAAT..G.. .... ..C. .G.
370
38 0
390
CTAC ATCCG CCAG TCAG GGTG CAAG TTAA
.C.. ...T. .... .T..
.C.. .C.T. .T.. .T..
.CG. .C..A ATGA .TT. ..C. ...C .C.. TAG
.AT. .C..T .AGC ATGA
Arctic Ocean
NO
SE
FI
Baltic Sea
PL K LT LV
BY
Pacific Ocean
EE
NW +
*+
+
+
Moscow
**
N
FE
+
+
#
V-V
UA * CBE +*+ +
+ +
**
* * +,**++
*
C
+
*
*
V
+
NC
+
KZ
AZ
IR
•
•
Only EU genotype
Majority clustered in previously
defined subtypes
– Subtype 1: 24 sequences from
18 farms
– Subtype 2: 24 sequences from
19 farms
– Subtype 3: 1 sequence
TM
28
104
107,108 42
106
24
44
20
4
38
97
17
22
18
21
57
9
87
27
46
933
686
60
61
48
47
93
72
71
89
88
43 91
90
92
Belarus
81
843
75
35
77
55
65
Russia
85
Subtype 2
343
56
66
86
533
816
JP
Subtype 3
19
8
52
37,109
45
Russian subtype 1 strains
are phylogenetically
different from subtype 1
strains from other countries
54
13
15
6
10
Sea of Japan
KP
CN
UZ
49
25
26
40
MN
41
95
CN
Aral s.
Subtype 1
39
* *
Caspian
sea
AM
ES
WS
U
*
TR GA
Sea of Okhotsk
x
36
67
68
79
7
53
64
83
94
14
84
82
69
62
78
76
16
5
74
50
70
63
12
73
51
80
58
59
Lithuania
Western/Central Europe, North America, Asia
ORF5 identity down to
69.5% (Clustal V)
Subtype 3
21
29
30
40
18 9
22
7
10
11
32
ORF7 identity down to
75.4% (Clustal V)
87
19
28
99
96
24
27
34
39
23 97
Subtype 2
59
42
63
988
80
327
41
109
55
37
100
3
781
743
94
35
108 107
106
2
25
98
103
53
50
1
102
69
36
26 105
44
25
26
40
33
56
44
54
71
89
90
43
92
61
16
83
97
Belarus
Lithuania
Western Europe, Asia, North America
18
57
9
87
27
46
933
686
60
61
816
48
47
93
72
71
89
88
43 91
90
92
81
85
Subtype 2
843
75
343
35
77
56
66
86
533
55
68
79
7
53
64
83
94
14
84
82
69
62
78
76
36
67
16
5
74
50
70
63
12
73
51
80
58
Russia
17
22
21
65
93
20
37,109
45
95
Subtype 1
Subtype 3
19
4
38
45
12
13
8
54 52
28
104
107,108 42
106
24
101
38
49
15
6
10
Subtype 1
66 (Subtype 1)
5
31
41
95
15 13
14
59
62 Type 1 and 68 Type 2 ORF 5 sequences were aligned in Clustal X. The
phylogenetic tree was drawn using the neighbor-joining method. Sequences
were selected to represent overall diversity. By Juan Abrahante.
EU‐PRRSV
NA‐PRRSV
The genetic diversity range in NA-PRRSV
is comparable to the genetic diversity of a
single subtype within the EU-PRRSV
genotype
0.03
EU-PRRSV strains on farms over time
SOZ’06
KH‘05
KH’04
41
95
49
107,108 42
106
25
26
40
24
IL‘97
44
54
28
104
4
38
97
37,109
17
22
VL’06
18
21
57
9
87
933
48
686
60
61
71
89
88
43
85
Subtype 2
843
72
75
343
35
91
90
92
77
56
45
55
65
66
81
TM’00
NV’96
NV’04
68
79
53
64
83
7
94
16
NV’06
NK’04
14
NK’99
84
82
69
62
78
76
36
67
5
74
50
70
63
12
73
51
80
58
59
SOZ’06
KH ‘05
TM’05
VL’04
86
533
816
47
93
NV: 19962006
20
27
IL‘99
GB‘97
Subtype 3
19
8
52
46
GB‘00
15
6
10
Subtype 1
39
SOZ’04
13
MB‘04
MB‘05
VL’01
EU-PRRSV emergence
Za d-1 BY’04
Subtype 3
Yuz-34 BY’04
A very sharp geographical demarcation of
highly diverse EU genotype PRRSV is
observed along the eastern Polish border
(established only in 1945)
Obu- 1 BY’05
Soz-6 BY’04
Vos-49 BY’04
Subtype 4
Bel-42 BY’04
Za p-36-40 BY’04
Okt-35 BY’04
Sid LT’00
Dzi-62 PL’05
982
361-4 DK’94
Sok-4 PL’04
Vas-2 BY’05
836
1000
2029/97 I’97
+ Sid
+Au s
Aus LT’00
Prz-6 6-70 PL’05
Up a-13 PL’05
Sno-4 BY ’04
Ch e-46 PL’05
Pyrsvac-187 E vac
Amervac PRRS E vac
Lelystad NL’91
Porc ilis P RRS NL vac
28639/98 DK’9 8
Subt ype 1
Lithuania
Bor-41 BY’04
L56/2/91 E’91
Subtype 2
2567/96 I’96
Upa*
* Sma
Prz *
* Ch e
Kon
Bie
*
Gro Kwi
*
Sok
*
*
*
* Lek Poland
*
*
Rak
Krz
Gra
Po l
*
* Zbr
Observed diversity pattern supports
hypothesis of PRRSV emergence
in the former Soviet Union
Bor
+
Sn o
+
Dzi
Ancestral populations are
expected to be more
diverse than descendant
populations
+Vas
# Obu
x Okt
100 km
*
Zad
#
Vos
#
Belarus
*
+/# Bel
# Zap
Yuz
#
Soz
#
* Jed
* Nie
•
•
Kemerovo: Siberia, Far East, 1961
North Caucasian: Caukasus, Central
Asia, 1955
• Semirechensk, arid regions of Central
Asia, 1978
• Forest Mountain: Caucasus
Germany reunification
could merge two
completely different pig
populations
Geographic distribution
of genotype 1 PRRSV
subtypes in Europe
Multiple
subtypes
Subtype 1
?
?
?
Porcilis PR RS
Lel ystad
U pa-13 PL 2 005
Amervac Hipra
Pyrsvac Syva
361-4_15 DK 1994
Subtype
28639_ 98_12 DK 1998
Che-46 PL 2005
Prz-66-70 PL 2 005
Sok-4 PL 2004
2029_97_ 6 IT 1997
2567_96_9 IT 1996
Yuz-3 4 BY 2004
Zad -1 BY 2004
Obu-1 BY 2005
Vos-49 BY 2 004
Subtype
Za p-36-40 BY 2004
Bel -42 BY 2004
Soz-6 BY 20 04
Aus LT 20 00
Sno-4 BY 2 004
Sid LT 2000
Subtype
24717 D 1999
Bo r-41 BY 20 04
20
15
10
5
Nucleo tide Substitutions (x10 0)
3
2
Subtype 4
Okt-35 BY 2004
20.5
1
West and Central
Europe
Belarus
Belarus, Lithuania
and Germany!
Belarus, Latvia
0
Phylogenetic tree of ORF5 amino acid sequences including German sequence 24717
from Pesch et al., 2005
Can wild boars transmit
East European strains
westward?
+ Sid
+Au s
Lithuania
Upa*
* Sma
Stankevicius et al., Barcelona 2011
Prz *
* Ch e
Kon
*
Bor
+
Sn o
+
Dzi
Bie
+Vas
# Obu
x Okt
Gro Kwi
*
Sok
*
*
*
* Lek Poland
*
*
Rak
Krz
Gra
Po l
*
* Zbr
100 km
*
Zad
#
Vos
#
Belarus
*
+/# Bel
# Zap
Yuz
#
Soz
#
* Jed
* Nie
EU-PRRSV west of Lithuania, Belarus, Ukraine
„Original”
PRRSV
outbreak in
Europe
Prof. Frederic Leung, EuroPRRS2010, Warsaw, Poland
PRRSV in the Netherlands
•
Wellenberg et al., EAVLD 2010
•
•
•
Dutch PRRSV strains (n= 284; 20052009)
No sign of introduction of EastEuropean PRRSV strains
Identity between field strains and DVstrain decreased from 90% to 86%
Amervac PRRS vac E
Amervac PRRS vac E_ORF5
10-1646-78 Hrus
Pyrsvac-183 vac E
11-01098-10.1
11-01098-10.2
11-01098-16
S473
10-3407-41 Vian
09-NUMER-golina(110416)_1
09-NUMER-golina(110416)_2
Che PL 2005
10-16410-4
10-16410-8
Prz PL 2005
Upa PL 2005
SZ2009-01885-6-NL
11-3143
11-01104-14.3
Porcilis full-1
Porcilis full-1
VA2011-01722-NL
11-01104-16.1
11-01104-19.2
VA2011-01230-NL
10-1267-3 Rzep
Lelystad ORF5-1
Lelystad ORF5-1
10-5458-1 Sier
VA2011-01239-2-NL
10-6346-Wilc
VA2008-10003 seq 8-1-NL
VA2011-00883 1-4-NL
SZ2010-04736-1-NL
SZ2011-01253
VA2011-01874-4-NL
VA2011-01874-1-NL-DIRT
11-946-6-10 PK
11-946-26-30 PK
11-946-PL2 PK
11-495-5 PK
SZ2011-01172-1
VA2008-10003 seq 8-4-NL
VA2008-10003 seq 8-6-NL
VA2008-10003 seq 8-7-NL
10-15824-2
10-17940-21.4
10-17940-7.1
10-03191-3 Gali
10-03191-5 Gali
10-03191-4 Gali
VA2011-01238 9-12-NL
10-17451-14.1
10-17451-17.4
10-17940-5.4
10-17453-7.4
11-1111-10.3
10-17451-8.4
2567_96_9-IT-1
28639_98_12-DK-1
994-2005-NL
9191_1 2005 NL
361-4_15-DK-1
L56_2_91-1
VA2011-1470 1-4-NL
Sok PL 2004
09-NUMER-Kar_ORF5
10-6346-Seko
2029_97_6-IT-1
Yuz BY 2004
Zad-1 orf5 trimm ed
Obu-1 orf5 trimmed
Zap BY 2004
Vos-49 orf5 trimm ed
Bel-42 orf5 trim med
Soz-6 orf5 trimm ed
Soz F2A_48_trimm ed
Soz F2_21_trimm ed
Soz F3_19_trimm ed
Okt BY 2004
PK BY 2002
Sid LT 2000
VR-2005 Chuvashia RU
KH1-2005 Khabarovsk RU
NV-2006 Novosibirsk RU
SHV-2006 Kostrom a RU
TM-2005 Tatarstan RU
Aus LT 2000
GK-2005 Yaroslavl RU
IL-1997 Nizhny Vovgorod RU
MB-2005 Mordovia RU
ZV-2004 Tver RU
Sno BY 2004
Bor BY 2004
KH2-2005 Khabarovsk RU
NB-2006 Novgorod RU
VL-2006 Vladimir RU
IN-2005 Krasnodar RU
BT-2006 Vologda RU
PMP-2005 Penza RU
ND-1998 Vologda RU
ND-2006 Vologda RU
KZ-2004 Moscow RU
VSH-2005 Voronez RU
BLG-2006 Belgorod RU
RS-2005 Bashkiria RU
BK-2006 Belgorod RU
Subtype 1
Subtypes 2, 3, 4 etc…
vr2332~1
VA2009-02578-5-NL
VA2009-04185-1-NL_DIRT
37.0
35
30
25
20
15
Nucleotide Substitution per 100 residues
10
5
0
The Netherlands
Poland
Amervac PRRS vac E
Amervac PRRS vac E _ORF5
10-1646-78 Hrus
Pyr svac- 183 v ac E
11-01098-10.1
11-01098-10.2
11- 01098 -16
S473
10- 3407- 41 Vian
09- NUMER-golina(110416)_1
09- NUMER-golina(110416)_2
Che PL 2005
10-16410-4
10-16410-8
Prz PL 2005
Upa PL 2005
SZ2 009-01885-6-NL
11- 3143
11- 01104-14.3
Por cilis full -1
Por cilis full -1
VA2011-01722-NL
11- 01104 -16.1
11- 01104 -19.2
VA2 011-0 1230- NL
10-1267-3 Rzep
Lelystad ORF5-1
Lelystad ORF5-1
10- 5458-1 Sier
VA2011-01239-2-NL
10-6346-Wilc
VA2008-1 0003 seq 8 -1-NL
VA2011-00883 1-4-NL
SZ2010-0 4736- 1-NL
SZ2011-01253
VA2011-0 1874- 4-NL
VA2 011-01874-1-NL-DIRT
11- 946-6 -10 P K
11- 946-2 6-30 PK
11- 946-P L2 PK
11- 495-5 PK
SZ2011-01172-1
VA2 008-10003 seq 8-4-NL
VA2 008-10003 seq 8-6-NL
VA2 008-10003 seq 8-7-NL
10-15824-2
10- 17940 -21.4
10- 17940 -7.1
10-03191-3 Gali
10-03191-5 Gali
10-03191 -4 Ga li
VA2 011-0 1238 9-12-NL
10-17451 -14.1
10-17451 -17.4
10-17940 -5.4
10- 17453-7.4
11-1111- 10.3
10-17451 -8.4
2567_96_9-IT- 1
28639_98_12-D K-1
994-2005-NL
9191_1 2005 NL
361-4_15-DK-1
L56 _2_91 -1
VA2011-1470 1-4-NL
Sok PL 2004
09-NUMER-Kar_ORF5
10-6346-Seko
202 9_97_6-IT-1
Genetic diversity, subtypes,
lineages, clusters etc.:
does it matter?
• Vaccination with US type
vaccine does not protect
lungs against challenge with
Lelystad virus
• Vaccination with EU type
vaccine provides incomplete
but significant protection of
lungs against challenge with
Lelystad
Genetic diversity between
EU-type and US-type
PRRSV affects the efficacy
of immunization
• All vaccinated pigs remained
free of virus after Lelystad-like
challenge (98% identity of
vaccine and challenge virus
in ORF5)
• All vaccinated pigs had virus in
BAL and serum after Italian-like
challenge (84% identity of
vaccine and challenge virus
in ORF5)
Genetic diversity within
EU-type PRRSV may
affect the efficacy of the
current EU-type vaccines
ORF5
sequence
shouldofnot
be
The
most
dissimilarsimilarity
ORF5 sequences
PRRSVEU can
lessefficacy
than 70%prediction
identity!!!
used
for share
vaccine
Better vaccine, safer, more efficacious?
•
Killed?
– No scientific proof that they work…
•
MLV based on „recent” strains?
– PRRSV is fast evolving agent
– We will be chasing PRRSV diversity forever!
– Present day MLV’s are as distant (genetically and antigenically) from recent strains,
as any two of them!
Zad-1 BY’04
Subtype 3
Yuz-34 BY’04
Obu-1 BY’05
Vos-49 BY’04
Bel-42 BY’04
Soz-6 BY’04
Zap-36-40 BY’04
Subtype 4
Okt-35 BY’04
Sid LT’00
Dzi-62 PL’05
982
361-4 DK’94
Sok-4 PL’04
Vas-2 BY’05
836
1000
2029/97 I’97
Aus LT’00
Prz-66-70 PL’05
Upa-13 PL’05
Sno-4 BY’04
Che-46 PL’05
Pyrsvac-187 E vac
Amervac PRRS E vac
Lelystad NL’91
Porcilis PRRS NL vac
28639/98 DK’98
Subtype 1
L56/2/91 E’91
Bor-41 BY’04
Subtype 2
2567/96 I’96
And if the vaccine does not work..?
Did pigs get the vaccine antigen at all?
Virulence?
• EU-PRRSV is thought to be less pathogenic
than NA-PRRSV
– All studies (but one) performed with relatively similar
strains from one genetic subtype
Belarusian PRRSV isolate
Vietnamese PHFD PRRSV isolate
Diagnosis
• ORF7 is a target for PCR
• Capsid protein (encoded by ORF7) induced antobodies
are detected in commercial ELISAs
• EU-PRRSV nucleotide identity in ORF7 can be as low as
75%
Za d-1 BY’04
Subtype 3
Yuz-34 BY’04
Obu- 1 BY’05
Soz-6 BY’04
Vos-49 BY’04
Bel-42 BY’04
Za p-36-40 BY’04
Subtype 4
Okt-35 BY’04
Sid LT’00
Dzi-62 PL’05
982
361-4 DK’94
Sok-4 PL’04
Vas-2 BY’05
836
1000
2029/97 I’97
Aus LT’00
Prz-6 6-70 PL’05
Up a-13 PL’05
Sno-4 BY ’04
Ch e-46 PL’05
Pyrsvac-187 E vac
Amervac PRRS E vac
Lelystad NL’91
Porc ilis P RRS NL vac
28639/98 DK’9 8
Subt ype 1
L56/2/91 E’91
Bor-41 BY’04
Subtype 2
2567/96 I’96
Very high aa diversity of N protein in East European subtypes affects important
antigenic regions of the ORF7 protein
Sera from infected pigs:
6 with virulent genotype 1 subtype 3 Lena
(124 aa)
10 with Lelystad (128 aa)
7 with genotype 2 US5
N protein AA identities between Lena ,
Lelystad and US5 strains (Clustaw W)
Lelystad Lena
***
90.4
***
US5
63.6
65.3
***
Lelystad
Lena
US5
Despite >90% AA identity between N protein of Lena and Lelystad the antigenic cross reaction was similar to that between Lelystad and US5.
Sensisivity of PRRSV antibody detection in 9 farms expressed as % of positive results.
G. 1/1: genotype 1, subtype 1 (N: 128 aa); g. 1/2: genotype 1, subtype 2 (N: 125 aa); g.
1/3: genotype 1, subtype 3 (N: 124 aa); g. 1/atyp.: genotype 1, atypical (N: 130 aa); g. 2:
genotype 2.
The differences in sensitivity are not related to antigenic
diversity
Sensitivity and specificity of 10 ELISA tests detecting PRRSV-specific antibodies. Sensitivity
was expressed as a percentage of positive results obtained in 169 sera samples collected in
11 farms affected with PRRS. Specificity was expressed as a percentage of negative results
obtained in 140 samples collected in 8 farms free from PRRS.
Sows!
Farm
in
house
IDEXX
2XR
10/15740
(22)
0%
4,5%
0%
13,6%
10/15738
(36)
0%
Nd
0%
76,9%
0%
singletons
7,8%
(13)
IDEXX
ING
ING
ING
X3
Universal Europa America
ING
DR
ING
CIVTEST CIVTEST
Korea
Compac
E
A
9,1%
90,9%
9,1%
0%
4,5%
63,6%
22,2%
0%
0%
19,4
%
0%
2,8%
13,9%
0%
0%
15,4%
15,4%
0%
84,6% 38,5%
45,5
%
61,1
%
0%
Courtesy of Dr. Bernd
Hoffmann, FLI,
Germany
Monoclonals for typing?
Strain
EU subtype
SDOW17
(EU+NA)
VO17 (NA)
WB4 (EU)
231/Sza
3
415/And
nd
6695/Bie
2
8380/Okt
4
Bor
atypical
+
+
+
+
+
+
+
+
+
-
Anette Botner, unpublished
NA-PRRSV
NA-PRRSV strains in Europe
Courtesy of Dr. Ivan Psikal, Brno,
Czech Republic
Take home messages
•
Diversity of EU-PRRSV is extremely high in comparison to NAPRRSV
• The spread of East European variants to the Western Europe is
a matter of time
• Sensitivity of current RT-PCR assays to divergent EU-PRRSV is not
known: We may not have the complete picture of the diverse strains
distribution!
• Further exploration of the PRRSV genetic diversity is necessary to
understand the virus’ evolution and monitor distribution of different
clusters and lineages
• Serology works well but differences in sensitivity to detect antibodies
against some variants do exist
Thank you for your attention!
Questions?