2013 International Symposium on Ranaviruses By Rachel Marschang

1. Ranaviruses in snakes,Ranaviruses in snakes,
lizards, and chelonianslizards, and chelonians
Rachel E. Marschang1
, Anke C. Stöhr1
, Silvia Blahak2
,
Maria Filomena Alcobia da Silva3,4
, Antonio Pedro Alves
de Matos4,5
, Tibor Papp6
1
Fachgebiet für Umwelt- und Tierhygiene, University of Hohenheim, Germany
2
Chemisches und Veterinäruntersuchungsamt OWL, Detmold, Germany
3
University of Lisbon, Faculty of Sciences, Department of Plant Biology, Lisbon, Portugal
4
CESAM – Centre for Environmental and Marine Studies, Aveiro University, Aveiro, Portugal
5
Anatomic Pathology Department, Curry Cabral Hospital, Lisbon, Portugal
6
Institute for Veterinary Medical Research, Centre for Agricultural Research, Hungarian Academy
of Sciences, Budapest, Hungary

2. • Screening of reptiles for ranavirusesScreening of reptiles for ranaviruses
– Conventional PCR targeting a portion of the MCPConventional 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

3. • 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

4. • 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 different64 chelonians, 14 species (5 positive from 4 different
owners)owners)
• 20132013
– 52 chelonians, 8 species (3 positive from 3 different52 chelonians, 8 species (3 positive from 3 different
owners)owners)
Diagnosis: chelonians

5. • 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

6. • 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

7. • 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 different114 animals, 22 species, 5 positive from 5 different
ownersowners
Diagnosis: positives

8. • Species in which ranaviruses have been detectedSpecies 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,, VaranusVaranus
macraeimacraei,, Anolis carolinensisAnolis carolinensis,, Iguana iguanaIguana iguana,, PogonaPogona
vitticepsvitticeps (n=2),(n=2), Lacerta agilisLacerta agilis
– Snakes: 2Snakes: 2 Python regiusPython regius, 2, 2 Python molurusPython molurus + 1+ 1 EunectesEunectes
spp.spp.
Diagnosis: positives

9. Comparison
Virus No. Host Country Year
Short case
history/clinical
signs
Ref.
Testudo
hermanni
ranavirus
CH8/96
Hermann’s
tortoise
Testudo
hermanni
Switz. 1996
Stomatitis, death of
several tortoises.
Marschang
et al., 1999
Testudo
ranavirus 1
TRV1
(882/96)
Egyptian
tortoise
Testudo
kleinmanni
Germany 1996
Rhinitis, stomatitis,
hepatitis. 2nd animal in
collection survived.
Blahak and
Uhlenbrok,
2010
Testudo
ranavirus 2
TRV 2
(5187/97)
Hermann’s
tortoise
Testudo
hermanni
Germany 2007 Stomatitis, emaciation.
Blahak and
Uhlenbrok,
2010
TRV 2
(CU60/09)
Marginated
tortoise
Testudo
marginata
Germany 2009
Stomatitis, necrosis in
the trachea.
Blahak and
Uhlenbrok,
2010

10. Virus No. Host Country Year Case history Ref.
German
Gecko
ranavirus
GGRV
(2000/99)
Leaf-tailed
gecko
Uroplatus
fimbriatus
Germany 2001
Granulomatous lesions on
the tongue, hepatitis
Marschang
et al., 2005
Lacerta
monticola
ranavirus
LMRV
Iberian
mountain lizard
Lacerta
monticola
Portugal
2003/
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)
2008
2011
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

11. Comparison
Virus No. Host Country Year
Short 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 ranavirusTotal: 10 different reptilian ranavirus
isolatesisolates

12. • 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 etet 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 regionLength of microsatellite region (Mavian et al., 2012)(Mavian et al., 2012)
Comparison

13. 0 .0 0 5
S S T IV ( C h in a )
E S V (G e r )
B IV ( A u s t r a lia )
R m a x - D K
A C R V (U S A - > G e r )
P B R V (A s ia -> G e r )
C M T V ( S p a in + N e t h .)
J S R V (C h in a -> U S A - > G e r )
T R V 2 (G e r )
E H N V (A u s t r a lia )
T F V (C h in 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 itz e r la n d )
Z P R V 2 (E u r o p e )
R E V ( Ita 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 ia - > G e r )
F V 3 (U S A )
S E R V
P P IV ( F in n l)
N C R V ( Ir a q - > G e r )
R g r y l (C h in 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 /1
8 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
Concatenated
tree 3223 bp
Red=full length vIF-2a
Green=truncated vIF2a
Black=no data available

14. 100bp50bpTRV1TRV2TRV2ASRV GGRV PBRV ACRV JSRV FV3 LMO DGRV CH8/96 ZPRV1 ZPRV2 NK
Microsatellite PCR

15. TRV1 TRV2 ACRV GGRV ASRV JSRV10bp
150bp
100bp
50bp

16. 0 .0 0 5
S S T IV (C h in a )
E S V (G e r )
B IV (A u s tr a lia )
R m a x - D K
A C R V (U S A -> G e r )
P B R V (A s ia -> G e r )
C M T V (S p a in + N e th .)
J S R V ( C h in a -> U S A -> G e r )
T R V 2 (G e r )
E H N V (A u s tr a lia )
T F V (C h in 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 itz e r la n d )
Z P R V 2 (E u r o p e )
R E V (Ita 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 ia -> G e r )
F V 3 ( U S A )
S E R V
P P IV ( F in n l)
N C R V ( Ir a q -> G e r )
R g r y l (C h in 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 /1
8 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
Russian tort, 2012, D
Retic. python, 2012, D
Retic. python, 2012, D
Anaconda, 2012, D
Bearded dragon, 2012, D
Hermann‘s tort, 2012, D
Ball python, 2013, D
Bearded dragon, 2013, D
Red-eared slider, 2013, D
Hermann‘s tort, 2013, UK

17. 0 .0 0 5
S S T IV (C h in a )
E S V (G e r )
B IV (A u s tr a lia )
R m a x - D K
A C R V (U S A -> G e r )
P B R V (A s ia -> G e r )
C M T V (S p a in + N e th .)
J S R V ( C h in a -> U S A -> G e r )
T R V 2 (G e r )
E H N V (A u s tr a lia )
T F V (C h in 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 itz e r la n d )
Z P R V 2 (E u r o p e )
R E V (Ita 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 ia -> G e r )
F V 3 ( U S A )
S E R V
P P IV ( F in n l)
N C R V ( Ir a q -> G e r )
R g r y l (C h in 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 /1
8 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
Leopard tort, 2012, D
Hermann‘s tort, 2012, D
Ball python, 2012, D
Monitor lizard, 2012, D
Hermann‘s tort, 2012, I
Iguana, 2012, D

18. 0 .0 0 5
S S T IV (C h in a )
E S V (G e r )
B IV (A u s tr a lia )
R m a x - D K
A C R V (U S A -> G e r )
P B R V (A s ia -> G e r )
C M T V (S p a in + N e th .)
J S R V ( C h in a -> U S A -> G e r )
T R V 2 (G e r )
E H N V (A u s tr a lia )
T F V (C h in 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 itz e r la n d )
Z P R V 2 (E u r o p e )
R E V (Ita 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 ia -> G e r )
F V 3 ( U S A )
S E R V
P P IV ( F in n l)
N C R V ( Ir a q -> G e r )
R g r y l (C h in 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 /1
8 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
Sand lizard, 2012, UK

19. 0 .0 0 5
S S T IV (C h in a )
E S V (G e r )
B IV (A u s tr a lia )
R m a x - D K
A C R V (U S A -> G e r )
P B R V (A s ia -> G e r )
C M T V (S p a in + N e th .)
J S R V ( C h in a -> U S A -> G e r )
T R V 2 (G e r )
E H N V (A u s tr a lia )
T F V (C h in 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 itz e r la n d )
Z P R V 2 (E u r o p e )
R E V (Ita 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 ia -> G e r )
F V 3 ( U S A )
S E R V
P P IV ( F in n l)
N C R V ( Ir a q -> G e r )
R g r y l (C h in 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 /1
8 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
Europ. pond turtle, 2012, D

20. • Ranaviruses found in reptiles areRanaviruses 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

21. • American Association of ZooAmerican Association of Zoo
VeterinariansVeterinarians
Helge Behncke, Waldkirch, Jutta Wiechert, Mainz, TonyHelge Behncke, Waldkirch, Jutta Wiechert, Mainz, Tony
Sainesbury, London, Ross Martin, Leeds, SabineSainesbury, London, Ross Martin, Leeds, Sabine
Öfner, Munich, Kim Heckers, Bad Kissingen, JürgenÖfner, Munich, Kim Heckers, Bad Kissingen, Jürgen
Seybold, Baiersbronn, Karina Mathes, HannoverSeybold, Baiersbronn, Karina Mathes, Hannover
Acknowledgements

22. Thank you for yourThank you for your
attention!attention!