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

TRV1 TRV2 ACRV GGRV ASRV JSRV10bp

150bp

100bp

50bp

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

Thank you for your Thank you for your attention!attention!