Post on 14-Jul-2015
Ranaviruses in snakes, Ranaviruses in snakes, lizards, and chelonians lizards, and chelonians
Rachel E. Marschang1, Anke C. Stöhr1, Silvia Blahak2, Maria Filomena Alcobia da Silva3,4, Antonio Pedro Alves
de Matos4,5, Tibor Papp6
1Fachgebiet für Umwelt- und Tierhygiene, University of Hohenheim, Germany2Chemisches und Veterinäruntersuchungsamt OWL, Detmold, Germany
3University of Lisbon, Faculty of Sciences, Department of Plant Biology, Lisbon, Portugal4CESAM – Centre for Environmental and Marine Studies, Aveiro University, Aveiro, Portugal
5Anatomic Pathology Department, Curry Cabral Hospital, Lisbon, Portugal6Institute for Veterinary Medical Research, Centre for Agricultural Research, Hungarian Academy
of Sciences, Budapest, Hungary
• Screening of reptiles for ranavirusesScreening of reptiles for ranaviruses– Conventional PCR targeting a portion of the MCP Conventional PCR targeting a portion of the MCP
genegene– Virus isolation in cell culture (TH-1, IgH2, VH2)Virus isolation in cell culture (TH-1, IgH2, VH2)
Diagnosis
• 2010-2013:2010-2013:– Screening of samples from:Screening of samples from:
• 834 reptiles, 82 species834 reptiles, 82 species• Chelonians: 232 animals, at least 24 speciesChelonians: 232 animals, at least 24 species• Lizards: 423 animals, at least 35 speciesLizards: 423 animals, at least 35 species• Snakes: 179 animals, at least 23 speciesSnakes: 179 animals, at least 23 species
Diagnosis
• 2010:2010:– 85 chelonians, 13 species (all negative)85 chelonians, 13 species (all negative)
• 20112011– 31 chelonians, 9 species (all negative)31 chelonians, 9 species (all negative)
• 20122012– 64 chelonians, 14 species (5 positive from 4 different 64 chelonians, 14 species (5 positive from 4 different
owners)owners)
• 20132013– 52 chelonians, 8 species (3 positive from 3 different 52 chelonians, 8 species (3 positive from 3 different
owners)owners)
Diagnosis: chelonians
• 2010:2010:– 141 lizards, 19 species (all negative)141 lizards, 19 species (all negative)
• 20112011– 190 lizards, 18 species (1 group positive)190 lizards, 18 species (1 group positive)
• 20122012– 60 lizards, 13 species (6 positive)60 lizards, 13 species (6 positive)
• 20132013– 32 lizards, 7 species (1 positive)32 lizards, 7 species (1 positive)
Diagnosis: lizards
• 2010:2010:– 4 snakes, 3 species (all negative)4 snakes, 3 species (all negative)
• 20112011– 68 snakes, 17 species (all negative)68 snakes, 17 species (all negative)
• 20122012– 77 snakes, 13 species (4 positive from 2 different owners)77 snakes, 13 species (4 positive from 2 different owners)
• 20132013– 30 snakes, 7 species (1 positive)30 snakes, 7 species (1 positive)
Diagnosis: snakes
• 2010:2010:– 230 animals, 35 species, all negative230 animals, 35 species, all negative
• 20112011– 289 animals, 44 species, positive in one group of lizards289 animals, 44 species, positive in one group of lizards
• 20122012– 201 animals, 40 species, 15 positive from 12 owners201 animals, 40 species, 15 positive from 12 owners
• 20132013– 114 animals, 22 species, 5 positive from 5 different 114 animals, 22 species, 5 positive from 5 different
ownersowners
Diagnosis: positives
• Species in which ranaviruses have been detected Species in which ranaviruses have been detected (2010-2013):(2010-2013):– Chelonians: Chelonians: Stigmochelys pardalisStigmochelys pardalis, , T. graecaT. graeca, , T. T.
hermannihermanni (n=2), (n=2), T. horsfieldiiT. horsfieldii, , Emys orbicularisEmys orbicularis
– Lizards: Lizards: Japalura splendidaJapalura splendida, , Dopasia gracilisDopasia gracilis, , Varanus Varanus macraeimacraei, , Anolis carolinensisAnolis carolinensis, , Iguana iguanaIguana iguana, , Pogona Pogona vitticeps vitticeps (n=2), (n=2), Lacerta agilisLacerta agilis
– Snakes: 2 Snakes: 2 Python regiusPython regius, 2 , 2 Python molurus Python molurus + 1 + 1 EunectesEunectes spp.spp.
Diagnosis: positives
Comparison
Virus No. Host Country YearShort case
history/clinical signs
Ref.
Testudo hermanni ranavirus
CH8/96
Hermann’s tortoiseTestudo
hermanni
Switz. 1996 Stomatitis, death of several tortoises.
Marschang et al., 1999
Testudo ranavirus 1
TRV1
(882/96)
Egyptian tortoiseTestudo
kleinmanni
Germany 1996Rhinitis, stomatitis,
hepatitis. 2nd animal in collection survived.
Blahak and Uhlenbrok, 2010
Testudo ranavirus 2
TRV 2
(5187/97)
Hermann’s tortoiseTestudo
hermanni
Germany 2007 Stomatitis, emaciation.Blahak and Uhlenbrok, 2010
TRV 2
(CU60/09)
Marginated tortoiseTestudo
marginata
Germany 2009Stomatitis, necrosis in
the trachea.
Blahak and Uhlenbrok, 2010
Virus No. Host Country Year Case history Ref.
German Gecko
ranavirus
GGRV
(2000/99)
Leaf-tailed gecko
Uroplatus fimbriatus
Germany 2001Granulomatous lesions on
the tongue, hepatitisMarschang et al., 2005
Lacerta monticolaranavirus
LMRV
Iberian mountain lizard
Lacerta monticola
Portugal2003/2004
Wild-caught, co-inf. with erythrocytic necrosis virus
Alves de Matos et al., 2011
Japalura splendida ranavirus
JSRV
Green striped tree dragon
Japalura splendida
Germany (imported from Asia
via Florida)
2011
Skin lesions, systemic haemorrhages, liver
necrosis, large number of animals died.
AdV / IIV in the same group
Behncke et al., 2013
Anolis sagrei
ranavirus
ASRV
Brown anole Anolis sagrei
Germany (imported
from Florida)
20082011
RV found repeatedly in different imported groups
during 3 years. Low to high mortality, apathy, skin
lesions. Coinfection with reovirus in one animal.
Stöhr et al., submitted for publication
Dopasia gracilis
ranavirus
DGRV
Asian glass lizard
Dopasia gracilis
Germany (imported from Asia)
2012
Illegally imported animals, a number of animals died.
Skin lesions. IIV in the same animal.
Stöhr et al., submitted for publication
Anolis carolinensis
ranavirus
ACRV
Green anole Anolis
carolinensis
Germany (imported
from Florida)
2012
Several animals in poor body condition separated, high mortality, skin lesions.
AdV and IIV in the same animal.
Stöhr et al., submitted for publication
Comparison
Virus No. Host Country YearShort case
history/clinical signs
Ref.
Python brongersmai
ranavirus
PBRV
Red blood python
Python
brongersmai
Germany(imported
from Indonesia)
2007
100 animals imported, 30 % developed severe
diphteroid stomatitis and hepatitis. An unknown
number of snakes died.
Blahak, unpub.
• Total: 10 different reptilian ranavirus Total: 10 different reptilian ranavirus isolatesisolates
• Based on:Based on:– Sequence comparison of partial genes:Sequence comparison of partial genes:
• MCP (1402 bp), DNA polymerase(560 bp), RNR-MCP (1402 bp), DNA polymerase(560 bp), RNR-αα (806 bp), RNR-(806 bp), RNR-ββ (646 bp) (646 bp) (Mao et al., 1997; Marschang (Mao et al., 1997; Marschang et al., 1999; Ariel et al., 2010; Hyatt et al., 2000; Holopainen et et al., 1999; Ariel et al., 2010; Hyatt et al., 2000; Holopainen et al., 2009)al., 2009)
– Length and sequences of vIF-Length and sequences of vIF-22αα (247 or 1050 (247 or 1050 bp) bp) (Essbauer et al., 2001; Chinchar, personal comm.)(Essbauer et al., 2001; Chinchar, personal comm.)
– Length of microsatellite region Length of microsatellite region (Mavian et al., 2012)(Mavian et al., 2012)
Comparison
C o n c a t - D N A d i s t t r e e ( R N R - a l p h a , R N R - b e t a , D N A p o l , M C P g e n e = 3 2 2 3 b p )
0 . 0 0 5
S S T I V ( C h i n a )
E S V ( G e r )
B I V ( A u s t r a l i a )
R m a x - D K
A C R V ( U S A - > G e r )
P B R V ( A s i a - > G e r )
C M T V ( S p a i n + N e t h . )
J S R V ( C h i n a - > U S A - > G e r )
T R V 2 ( G e r )
E H N V ( A u s t r a l i a )
T F V ( C h i n a )
G G R V ( 2 0 0 0 / 9 9 ) ( G e r )
T R V 1 ( G e r )
L M R V ( P o r t )
E C V ( F r a n c e )
A S R V ( U S A - > G e r )
C H 8 / 9 6 ( S w i t z e r l a n d )
Z P R V 2 ( E u r o p e ) R E V ( I t a l )
Z P R V 1 ( G e r )
C o d V ( D K ) A T S V ( U S A )
D G R V ( A s i a - > G e r )
F V 3 ( U S A )
S E R V
P P I V ( F i n n l ) N C R V ( I r a q - > G e r )
R g r y l ( C h i n a )
1 0 0 / 1 0 0 / 1
6 9 / 5 7 / 0 . 9 4
1 0 0 / 1 0 0 / 1
1 0 0 / 1 0 0 / 1
8 2 / 6 4 / 0 . 9 7
6 2 / 4 8 / 0 . 9
1 0 0 / 8 4 / 1
8 3 / 7 2 / 0 . 9 7
1 0 0 / 1 0 0 / 1
1 0 0 / 8 6 / 1 7 8 / 5 9 / 0 . 7 4
1 0 0 / 1 0 0 / 1
1 0 0 / 1 0 0 / 1
1 0 0 / 1 0 0 / 18 7 / 6 8 / 0 . 8
6 8 / 5 9 / 0 . 8 1
1 0 0 / 1 0 0 / 1
9 8 / 9 3 / 1
8 5 / n . a . / 0 . 9 9
1 0 0 / 9 9 / 1
7 4 / 7 3 / 0 . 9 6
N u m b e r s a t t h e n o d e s o f t h e t r e e i n d i c a t e b o o t s t r a p v a l u e s o f 1 0 0 r e p l i c a t e s i n D N A d i s t - F i t c h
a n d m a x i m u m l i k e l i h o o d c a l c u l a t i o n s a n d M r B a y e s p o s t e r i o r p r o b a b i l i t i e s .
A l l c a l c u l a t e d t r e e s s h o w e d s i m i l a r t o p o l o g i e s .
= f u l l l e n g h t v I F - 2 a l p h a - g e n e
= t r u n c a t e d v I F - 2 a l p h a - g e n e
B l a c k = n o d a t a a v a i l a b l e
R e d
G r e e n
Concatenated tree 3223 bpRed=full length vIF-2aGreen=truncated vIF2aBlack=no data available
100bp 50bp TRV1TRV2TRV2ASRV GGRV PBRV ACRV JSRV FV3 LMO DGRV CH8/96 ZPRV1 ZPRV2 NK
Microsatellite PCR
C o n c a t - D N A d i s t t r e e ( R N R - a l p h a , R N R - b e t a , D N A p o l , M C P g e n e = 3 2 2 3 b p )
0 . 0 0 5
S S T I V ( C h i n a )
E S V ( G e r )
B I V ( A u s t r a l i a )
R m a x - D K
A C R V ( U S A - > G e r )
P B R V ( A s i a - > G e r )
C M T V ( S p a i n + N e t h . )
J S R V ( C h i n a - > U S A - > G e r )
T R V 2 ( G e r )
E H N V ( A u s t r a l i a )
T F V ( C h i n a )
G G R V ( 2 0 0 0 / 9 9 ) ( G e r )
T R V 1 ( G e r )
L M R V ( P o r t )
E C V ( F r a n c e )
A S R V ( U S A - > G e r )
C H 8 / 9 6 ( S w i t z e r l a n d )
Z P R V 2 ( E u r o p e ) R E V ( I t a l )
Z P R V 1 ( G e r )
C o d V ( D K ) A T S V ( U S A )
D G R V ( A s i a - > G e r )
F V 3 ( U S A )
S E R V
P P I V ( F i n n l ) N C R V ( I r a q - > G e r )
R g r y l ( C h i n a )
1 0 0 / 1 0 0 / 1
6 9 / 5 7 / 0 . 9 4
1 0 0 / 1 0 0 / 1
1 0 0 / 1 0 0 / 1
8 2 / 6 4 / 0 . 9 7
6 2 / 4 8 / 0 . 9
1 0 0 / 8 4 / 1
8 3 / 7 2 / 0 . 9 7
1 0 0 / 1 0 0 / 1
1 0 0 / 8 6 / 1 7 8 / 5 9 / 0 . 7 4
1 0 0 / 1 0 0 / 1
1 0 0 / 1 0 0 / 1
1 0 0 / 1 0 0 / 18 7 / 6 8 / 0 . 8
6 8 / 5 9 / 0 . 8 1
1 0 0 / 1 0 0 / 1
9 8 / 9 3 / 1
8 5 / n . a . / 0 . 9 9
1 0 0 / 9 9 / 1
7 4 / 7 3 / 0 . 9 6
N u m b e r s a t t h e n o d e s o f t h e t r e e i n d i c a t e b o o t s t r a p v a l u e s o f 1 0 0 r e p l i c a t e s i n D N A d i s t - F i t c h
a n d m a x i m u m l i k e l i h o o d c a l c u l a t i o n s a n d M r B a y e s p o s t e r i o r p r o b a b i l i t i e s .
A l l c a l c u l a t e d t r e e s s h o w e d s i m i l a r t o p o l o g i e s .
= f u l l l e n g h t v I F - 2 a l p h a - g e n e
= t r u n c a t e d v I F - 2 a l p h a - g e n e
B l a c k = n o d a t a a v a i l a b l e
R e d
G r e e n
Russian tort, 2012, DRetic. python, 2012, DRetic. python, 2012, DAnaconda, 2012, DBearded dragon, 2012, DHermann‘s tort, 2012, DBall python, 2013, DBearded dragon, 2013, DRed-eared slider, 2013, DHermann‘s tort, 2013, UK
C o n c a t - D N A d i s t t r e e ( R N R - a l p h a , R N R - b e t a , D N A p o l , M C P g e n e = 3 2 2 3 b p )
0 . 0 0 5
S S T I V ( C h i n a )
E S V ( G e r )
B I V ( A u s t r a l i a )
R m a x - D K
A C R V ( U S A - > G e r )
P B R V ( A s i a - > G e r )
C M T V ( S p a i n + N e t h . )
J S R V ( C h i n a - > U S A - > G e r )
T R V 2 ( G e r )
E H N V ( A u s t r a l i a )
T F V ( C h i n a )
G G R V ( 2 0 0 0 / 9 9 ) ( G e r )
T R V 1 ( G e r )
L M R V ( P o r t )
E C V ( F r a n c e )
A S R V ( U S A - > G e r )
C H 8 / 9 6 ( S w i t z e r l a n d )
Z P R V 2 ( E u r o p e ) R E V ( I t a l )
Z P R V 1 ( G e r )
C o d V ( D K ) A T S V ( U S A )
D G R V ( A s i a - > G e r )
F V 3 ( U S A )
S E R V
P P I V ( F i n n l ) N C R V ( I r a q - > G e r )
R g r y l ( C h i n a )
1 0 0 / 1 0 0 / 1
6 9 / 5 7 / 0 . 9 4
1 0 0 / 1 0 0 / 1
1 0 0 / 1 0 0 / 1
8 2 / 6 4 / 0 . 9 7
6 2 / 4 8 / 0 . 9
1 0 0 / 8 4 / 1
8 3 / 7 2 / 0 . 9 7
1 0 0 / 1 0 0 / 1
1 0 0 / 8 6 / 1 7 8 / 5 9 / 0 . 7 4
1 0 0 / 1 0 0 / 1
1 0 0 / 1 0 0 / 1
1 0 0 / 1 0 0 / 18 7 / 6 8 / 0 . 8
6 8 / 5 9 / 0 . 8 1
1 0 0 / 1 0 0 / 1
9 8 / 9 3 / 1
8 5 / n . a . / 0 . 9 9
1 0 0 / 9 9 / 1
7 4 / 7 3 / 0 . 9 6
N u m b e r s a t t h e n o d e s o f t h e t r e e i n d i c a t e b o o t s t r a p v a l u e s o f 1 0 0 r e p l i c a t e s i n D N A d i s t - F i t c h
a n d m a x i m u m l i k e l i h o o d c a l c u l a t i o n s a n d M r B a y e s p o s t e r i o r p r o b a b i l i t i e s .
A l l c a l c u l a t e d t r e e s s h o w e d s i m i l a r t o p o l o g i e s .
= f u l l l e n g h t v I F - 2 a l p h a - g e n e
= t r u n c a t e d v I F - 2 a l p h a - g e n e
B l a c k = n o d a t a a v a i l a b l e
R e d
G r e e n
Leopard tort, 2012, DHermann‘s tort, 2012, DBall python, 2012, DMonitor lizard, 2012, DHermann‘s tort, 2012, IIguana, 2012, D
C o n c a t - D N A d i s t t r e e ( R N R - a l p h a , R N R - b e t a , D N A p o l , M C P g e n e = 3 2 2 3 b p )
0 . 0 0 5
S S T I V ( C h i n a )
E S V ( G e r )
B I V ( A u s t r a l i a )
R m a x - D K
A C R V ( U S A - > G e r )
P B R V ( A s i a - > G e r )
C M T V ( S p a i n + N e t h . )
J S R V ( C h i n a - > U S A - > G e r )
T R V 2 ( G e r )
E H N V ( A u s t r a l i a )
T F V ( C h i n a )
G G R V ( 2 0 0 0 / 9 9 ) ( G e r )
T R V 1 ( G e r )
L M R V ( P o r t )
E C V ( F r a n c e )
A S R V ( U S A - > G e r )
C H 8 / 9 6 ( S w i t z e r l a n d )
Z P R V 2 ( E u r o p e ) R E V ( I t a l )
Z P R V 1 ( G e r )
C o d V ( D K ) A T S V ( U S A )
D G R V ( A s i a - > G e r )
F V 3 ( U S A )
S E R V
P P I V ( F i n n l ) N C R V ( I r a q - > G e r )
R g r y l ( C h i n a )
1 0 0 / 1 0 0 / 1
6 9 / 5 7 / 0 . 9 4
1 0 0 / 1 0 0 / 1
1 0 0 / 1 0 0 / 1
8 2 / 6 4 / 0 . 9 7
6 2 / 4 8 / 0 . 9
1 0 0 / 8 4 / 1
8 3 / 7 2 / 0 . 9 7
1 0 0 / 1 0 0 / 1
1 0 0 / 8 6 / 1 7 8 / 5 9 / 0 . 7 4
1 0 0 / 1 0 0 / 1
1 0 0 / 1 0 0 / 1
1 0 0 / 1 0 0 / 18 7 / 6 8 / 0 . 8
6 8 / 5 9 / 0 . 8 1
1 0 0 / 1 0 0 / 1
9 8 / 9 3 / 1
8 5 / n . a . / 0 . 9 9
1 0 0 / 9 9 / 1
7 4 / 7 3 / 0 . 9 6
N u m b e r s a t t h e n o d e s o f t h e t r e e i n d i c a t e b o o t s t r a p v a l u e s o f 1 0 0 r e p l i c a t e s i n D N A d i s t - F i t c h
a n d m a x i m u m l i k e l i h o o d c a l c u l a t i o n s a n d M r B a y e s p o s t e r i o r p r o b a b i l i t i e s .
A l l c a l c u l a t e d t r e e s s h o w e d s i m i l a r t o p o l o g i e s .
= f u l l l e n g h t v I F - 2 a l p h a - g e n e
= t r u n c a t e d v I F - 2 a l p h a - g e n e
B l a c k = n o d a t a a v a i l a b l e
R e d
G r e e n
Sand lizard, 2012, UK
C o n c a t - D N A d i s t t r e e ( R N R - a l p h a , R N R - b e t a , D N A p o l , M C P g e n e = 3 2 2 3 b p )
0 . 0 0 5
S S T I V ( C h i n a )
E S V ( G e r )
B I V ( A u s t r a l i a )
R m a x - D K
A C R V ( U S A - > G e r )
P B R V ( A s i a - > G e r )
C M T V ( S p a i n + N e t h . )
J S R V ( C h i n a - > U S A - > G e r )
T R V 2 ( G e r )
E H N V ( A u s t r a l i a )
T F V ( C h i n a )
G G R V ( 2 0 0 0 / 9 9 ) ( G e r )
T R V 1 ( G e r )
L M R V ( P o r t )
E C V ( F r a n c e )
A S R V ( U S A - > G e r )
C H 8 / 9 6 ( S w i t z e r l a n d )
Z P R V 2 ( E u r o p e ) R E V ( I t a l )
Z P R V 1 ( G e r )
C o d V ( D K ) A T S V ( U S A )
D G R V ( A s i a - > G e r )
F V 3 ( U S A )
S E R V
P P I V ( F i n n l ) N C R V ( I r a q - > G e r )
R g r y l ( C h i n a )
1 0 0 / 1 0 0 / 1
6 9 / 5 7 / 0 . 9 4
1 0 0 / 1 0 0 / 1
1 0 0 / 1 0 0 / 1
8 2 / 6 4 / 0 . 9 7
6 2 / 4 8 / 0 . 9
1 0 0 / 8 4 / 1
8 3 / 7 2 / 0 . 9 7
1 0 0 / 1 0 0 / 1
1 0 0 / 8 6 / 1 7 8 / 5 9 / 0 . 7 4
1 0 0 / 1 0 0 / 1
1 0 0 / 1 0 0 / 1
1 0 0 / 1 0 0 / 18 7 / 6 8 / 0 . 8
6 8 / 5 9 / 0 . 8 1
1 0 0 / 1 0 0 / 1
9 8 / 9 3 / 1
8 5 / n . a . / 0 . 9 9
1 0 0 / 9 9 / 1
7 4 / 7 3 / 0 . 9 6
N u m b e r s a t t h e n o d e s o f t h e t r e e i n d i c a t e b o o t s t r a p v a l u e s o f 1 0 0 r e p l i c a t e s i n D N A d i s t - F i t c h
a n d m a x i m u m l i k e l i h o o d c a l c u l a t i o n s a n d M r B a y e s p o s t e r i o r p r o b a b i l i t i e s .
A l l c a l c u l a t e d t r e e s s h o w e d s i m i l a r t o p o l o g i e s .
= f u l l l e n g h t v I F - 2 a l p h a - g e n e
= t r u n c a t e d v I F - 2 a l p h a - g e n e
B l a c k = n o d a t a a v a i l a b l e
R e d
G r e e n
Europ. pond turtle, 2012, D
• Ranaviruses found in reptiles are Ranaviruses found in reptiles are genetically variablegenetically variable
• More common than we realizedMore common than we realized• Outbreaks?Outbreaks?• Pet trade?Pet trade?• Pathogenicity?Pathogenicity?
Summary
• American Association of Zoo American Association of Zoo VeterinariansVeterinarians
Helge Behncke, Waldkirch, Jutta Wiechert, Mainz, Tony Helge Behncke, Waldkirch, Jutta Wiechert, Mainz, Tony Sainesbury, London, Ross Martin, Leeds, Sabine Sainesbury, London, Ross Martin, Leeds, Sabine Öfner, Munich, Kim Heckers, Bad Kissingen, Jürgen Öfner, Munich, Kim Heckers, Bad Kissingen, Jürgen Seybold, Baiersbronn, Karina Mathes, HannoverSeybold, Baiersbronn, Karina Mathes, Hannover
Acknowledgements