University of Tennessee 1Center for Wildlife Health2CVM Department of BDS

Debra L. Miller1,2 and Matthew J. Gray1

A Pathogen Lurking in the Darkness: Global Emergence of Ranavirus

in Ectothermic Vertebrates

M. Niemiller

26 September 2014, 12:00 PM University of Georgia

Outline

I. Ranavirus Characteristics and Die-offs

II.II. Pathology of RanavirusesPathology of Ranaviruses

III.III. Learning about Ranavirus (FV3) EcologyLearning about Ranavirus (FV3) Ecology

IV. Can Ranaviruses Contribute to Declines?

Ranavirus Characteristics•dsDNA, 150-280K bp

•120-300 nm in diameter (3x smaller than bacteria)

•Icosahedral Shape (20)

Family: Iridoviridae

Virion

Chinchar et al. (2011)

Iridovirus, Chloriridovirus, Ranavirus, Megalocytivirus, and Lymphocystivirus Genera:

Invertebrates Ectothermic Vertebrates

Paracrystalline Array

Species (6)Ambystoma tigrinum virus (ATV)

Bohle iridovirus (BIV)

Frog virus 3 (FV3)

ICTV (2012)

BalseiroUne

Epizootic haematopoietic necrosis virusEuropean catfish virus

Santee-Cooper Ranavirus

History of Ranavirus Die-offsFirst Isolated: •Dr. Allan Granoff

•Rana pipiens (1962)

First Large-scale Die-offs:•Dr. Andrew Cunningham

•Rana temporaria (1992)

•St. Jude Hospital

•Institute of Zoology, ZSL

First North American Die-offs:•Dr. Jim Collins and students

•Arizona State University

•Ambystoma tigrinum stebbinsi (1985, 1997)

A. Duffus

Acta Herpetologica

6 Continents: 1965, 1992Duffus et al. (2015)

Global Distribution of Ranavirus Cases: Amphibians

All Latitudes, All Elevations18 Families: Alytidae, Ranidae, Hylidae, Bufonidae, Centrolenidae, Craugastoridae, Dendrobatidae,

Discoglossidae, Leptodactylidae, Pipidae, Myobatrachidae, Rhacophoridae, Scaphiopodidae, Ambystomatidae, Salamandridae, Hynobiidae, Cryptobranchidae>100 Species

Global Distribution of Ranavirus Cases: Reptiles

12 Families: Agamidae, Anguidae, Boidae, Dactyloidae, Emyididae, Gekkonidae, Iguanidae, Lacertidae, Pythonidae, Testudinidae, Trionychidae, Varanidae

>30 Species

4 Continents: 1982, 1990s

Most FV3-like Ranaviruses: Captivity

Duffus et al. (2015)

Global Distribution of Ranavirus Cases: Fishes

22 Families:>50 Species

4 Continents: 1991

Most non-FV3-like Ranaviruses

Duffus et al. (2015)

Acipenseridae, Anguillidae, Centrarchidae, Channidae, Cobitoidae, Cyprinidae, Eleotridae, Esocidae, Gadidae, Gasterosteidae, Ictaluridae, Labridae, Latidae, Lutjanidae, Moronidae, Percidae, Poeciliidae, Salmonidae, Sciaenidae, Scophthalmidae, Serranidae, Siluridae

>90% of Reports Since

2010

Maine 2013 Die-off

1000 carcasses/m2

>200,000 deadqPCR Confirmed

6/14/13

Wheelwright et al. (2014)

<24 hrs

6/15/13

Ranaviral Disease in Eastern Box Turtles

13 February 2012

26 of 31 Box Turtles Die

from Ranaviral

Disease

Larval anurans and salamanders

dead too

Farnsworth and Seigel, Towson U.

2008 – 2011

North Branch Stream Valley

State Park

How Does Ranavirus Infect A Host?

Brunner et al. (2004), Harp & Petranka (2006), Brunner et al. (2007), Hoverman et al. (2010), Robert et al. (2011)

Routes of Transmission

Indirect Transmission

Skin, Gills, Intestines

(epithelial cells)

(3 hrs viral transcription)

Water or

Sediment

Ingestion

Incidental, Necrophagy, Cannibalism,

Predation

(Mortality 2X Faster)

Direct Contact

One Second Skin Contact

Ranaviral DiseaseRanaviral Disease

Gross changesGross changes

Lesions can look similar across classes (amphibian, reptile, fish)Lesions can look similar across classes (amphibian, reptile, fish)– Hemorrhage, swelling and necrosis (tissue death) are common gross Hemorrhage, swelling and necrosis (tissue death) are common gross

changeschanges

W. Sutton N. Haislip T. Waltzak

Amphibians: larvae

Photo: J. ChaneyBoreal Toad

Photo: Nathan HaislipBullfrog

affected

unaffectedPhoto: N. HaislipBullfrog

Photo: N. HaislipWood frog

Amphibians: metamorphs

Photo: Jordan ChaneyBoreal Toad

Amphibian: adultsAmphibian: adults

Midwife toad (Alytes obstetricans)Photos: Amanda Duffus

ulceration

Eastern spotted newt (Notophthalmus viridescens)Photo: Betsie B. Rothermel

Hemorrhage

Friable spleen

Hemorrhage

Gopher frogPhotos: B Sutton and R Hardman

Amphibian: adultsAmphibian: adultsEurope: 2 presentationsEurope: 2 presentations

Common frog (Rana temporaria)Photos: Amanda Duffus

ulceration

hemorrhage

Brazil : 2 Brazil : 2 presentations: presentations:

tadpoles vs tadpoles vs adultsadults

(Mazzoni, in prep)(Mazzoni, in prep)

Edema and Hemorrhages

Neurologic: vestibular syndrome

Hemorrhage and necrosis

Photos: R. Mazzoni

Is this Is this ranaviral ranaviral disease in disease in hellbendershellbenders?? Ozark hellbender (K. Irwin)

Ozark hellbender (K. Irwin)

It is in Chinese Giant Salamanders! (Y. Geng)

How about this?How about this?

Cryptobranchus alleganiensis alleganiensisPhoto: Dale McGinnity and Sherri Reinsch

And what role do And what role do ectoparasites (leeches) play?ectoparasites (leeches) play?

Photos: B Sutton and R Hardman

Bullfrog (~10%; 0% FV3)

Cope’s Gray tree frog (~70% RI; ~40% FV3)

Wood frog (~ 100% for both)

Varies by host-susceptibil i ty & virus isolateVaries by host-susceptibil i ty & virus isolate(mortality: RI [ranaculture isolate] vs FV3)(mortality: RI [ranaculture isolate] vs FV3)

Are isolates from captive (culture) facil i t ies more virulent?

Necrotic (white ) areas inside the mouth (circles and arrows). Upper photo with mouth opened. Lower photo with mandible removed

Photo: Mark RuderPhoto: Mark Ruder

Photo: Mark RuderPhoto: Mark Ruder

Photo: Mark RuderPhoto: Mark Ruder

Reptiles(most often chelonian reports)Similar reports in snakes

and l izards(including being off feed, weight

loss, dermatitis)

Eastern Box Turtle (West Virginia)(Terrapene carolina carolina) Eastern Box Turtle (Kentucky)

(Terrapene carolina carolina)

Blue arrows = lef t lungYellow arrows = necrosis

LiverIntest ine

Stomach

Ocular discharge

Ranavirus-NegativeHerpesevirus-NegativeMycoplasma-POSITIVE (M. agassizzi)

Ranavirus-POSITIVEHerpesvirus-NegativeMycoplasma-Negative

Determining etiologyDetermining etiology

Oral mucosal necrosisPhoto: Mark RuderPhoto: Mark Ruder

Lung necrosis

Photo: Mark RuderPhoto: Mark Ruder

Eastern Box Turtle (Virginia)(Terrapene carolina carolina)

Eastern Box Turtle (Kentucky)(Terrapene carolina carolina)

Fish

Photo: Emilie TravisPhotos: Tom Waltzek

Photo: Ted Henry

HistologyHistology

HistologyHistology Lesions can look similar across classes (amphibian, reptile, fish)Lesions can look similar across classes (amphibian, reptile, fish)

– Cellular necrosis of the hematopoietic tissue, vascular endothelium and Cellular necrosis of the hematopoietic tissue, vascular endothelium and epithelial cells and intracytoplasmic inclusion bodies are common epithelial cells and intracytoplasmic inclusion bodies are common microscopic lesionsmicroscopic lesions

Endothel ium

Endothel ial necrosis

Spleen Necrosis

AmphibiansAmphibians

Vestibular hemorrhage and necrosis(Mazzoni, in prep)

FV3 (originally isolated from frog in the wild)

Box turt le isolate(Die-off in wild)

Pall id sturgeon isolate(From culture facil i ty)

Ranaculture isolate(From culture faci l i ty)

Wood Frog Spleen

Repti lesRepti les

Pallid isolate; bath exposure

Pallid isolate; bath exposure

Skeletal muscle (neck): degeneration

Liver: endothelial necrosis, vacuolar degeneration

Trachea: serosal fibrinous necrosisThymus: fibrinous necrosisLung: occasional endothelial necrosis

Lung

Thymus

Trachea

Intestine: endothelial necrosis and hemorrhage

IntestineLu

men

Mucosa: severe epithelial necrosis

unaffected mucosa

vasculitis

Myofibers disrupted

Ulcerated crust on oral mucosa

Disrupted muscle fibers

Photos: Mark RuderPhotos: Mark Ruder

FishFish

Photo: Emilie Travis

Hematopoietic necrosis; tubular epithelial necrosis

Endothelium

Endothelial necrosis

Photo: Tom Waltzek

Visualizing the virusVisualizing the virus

Concurrent Concurrent

InfectionsInfections

Concurrent Concurrent infectionsinfections

URTIURTI

Gopher Tortoise (Gopherus polyphemus)Photo: Jess Gonynor McGuire

Eastern Box Turtle(Terrapene carolina carolina)

Blue arrows = lef t lungYellow arrows = necrosis

LiverIntest ine

Stomach

Ocular discharge

Ranavirus-NegativeHerpesevirus-NegativeMycoplasma-POSITIVE (M. agassizzi)

Ranavirus-POSITIVEHerpesvirus-NegativeMycoplasma-Negative

Determining etiologyDetermining etiology

Oral mucosal necrosisPhoto: Mark RuderPhoto: Mark Ruder

Lung necrosis

Photo: Mark RuderPhoto: Mark Ruder

Eastern Box Turtle (Virginia)(Terrapene carolina carolina)

Eastern Box Turtle (Kentucky)(Terrapene carolina carolina)

46(Y. Geng)

- Antivirals?

- Temperature?

- Vaccine development (Asia)- Much focus on fish

ranaviruses (Japan)- DNA and Live vaccines- Oral delivery

- In China, an inactivated vaccine is being studied for use in Chinese Giant Salamanders.

What about Treatment?What about Treatment?

UT CWH ResearchFV3-like Ranaviruses

0.360.4

0.3

0.15

0

0.1

0.2

0.3

0.4

0.5

Bullfrog Green Frog

FV

3 P

reva

len

ce

Access

Non-access

Cattle Land Use

A

A

A

B

n =104 tadpoles n =80 tadpoles

P =0.78 P =0.02

3.9X More

Likely!!!

Ranavirus in TNCattle Land Use

Life History and PhylogenyAmphibians

P = 0.354

• Fast larval development time*• Low aquatic index • Breeding habitat (ephemoral)

Hoverman et al. (2011); Brenes (2013)

All Three Isolates

No Phylogenetic Signal

35 spp

Physiological Trade-off Hypothesis

Susceptibility: 0 – 100%

1/3 of Species: >75%

Mortality

Single-species FV3-like ChallengesChelonians

Terrapene carolina, T. ornata, Elseya latisternum, Emydura krefftii , Trachemys scripta

Water bath exposure sufficient for transmission with some species.

Greatest infection and morbidity with IP injection or oral inoculation.

Ariel (1997), Johnson et al. (2007), Allender et al. (2013)

Control Turtle Fish Amph

Graptemys pseudogeographica kohni

Brenes et al (2014a)

Eastern River Cooter – no infection

Apalone ferox

Single-species FV3-like ChallengesFishes

Control Turtle Fish Amph Control Turtle Fish Amph

No Transmission: Nile tilapia, bluegill and fathead minnow

Brenes et al. (2014a)

Journal of Aquatic Animal Health 26:118-126

Gambusia affinis

Ictalurus punctatus

Reservoirs or Amplification Hosts?FV3-like Ranaviruses

Low Mortality(Subclinical)

Low Mortality(Subclinical)

Low – High Mortality(Subclinical & Clinical)

Reservoir Reservoir or Amplification

Reservoir

Suitable Hosts

Can Interclass Transmission Occur?

Bandin & Dopazo (2011)

Experiment

• Direct exposure– Exposed to 103 PFU/mL

– 3 days

• 12-L containers divided in half by a 2000 µm plastic mesh

• Different species in each side of the container

Amphibian: Hyla chrysocelis ; Turtle: Trachemys scripta elegans; Fish: Gambusia affinis

Turtle and Fish Results

• All classes tested can transmit the virus

• Turtles infected tadpoles– 50% mortality

• Fish infected tadpoles– 10% mortality

50%

10%

Brenes et al. (2014b)

Amphibian Results

• Amphibians transmitted to turtles but not fish

• No mortality of turtles or fish exposed to infected tadpoles

• Suggests that turtles and fish may be reservoirs of FV3-like ranavirus

• Amphibians may be amplifying species

Brenes et al. (2014b)

Community Level TransmissionBrenes, Gray, & Miller (unpubl. data)

Does Exposure Order or Composition Matter?

Inoculated in Lab103 PFU/mL FV3Exposure Order

Appalachian: Wood frog, chorus frog, spotted salamander

Coastal Plains: Gopher frog, chorus, southern toad

45% of initially exposed tadpoles.

Nearly 0 transmission of ranavirus & no mortality.

Cope’s gray treefrog 100% of initially exposed

tadpoles became infected.

Transmission to and mortality of >80% of unexposed tadpoles.

Wood Frogs

Superspreading and Amplification Superspreading and Amplification of Ranavirusof Ranavirus

Reilly, Gray, Miller (unpubl. data)

Temperature Dependency

FV3-like ranaviruses appears to become pathogenic at 12 C (54 F) but infection in vivo can occur at lower temperatures.

Survival: Brand, Reilly, Chaney et al. (unpubl. data)

FV3 In Vitro Replication Stops at 12 C (Chinchar 2002)

10oC RC Isolate

L. sylvaticus

Are Ranaviruses Capable of Causing

Local Extirpations and Species Declines?

0

50

100

150

200

250

1960

1963

1966

1969

1972

1975

1978

1981

1984

1987

1990

1993

1996

Nu

mb

er

of

Po

pu

lati

on

s

Collins & Crump (2009)

Muths et al. (2006)

Evidence of Local ExtinctionDr. Amber TeacherSoutheastern England

Animal Conservation

13:514-522

1996/97 and 2008

Ranavirus (+) populations

81% Median Reduction

Larger PopulationsGreatest

Proportional Declines

A. Teacher

A. Teacher

Teacher et al. 2010

81%

Rana temporaria

Female Population SizeEarl and Gray (2014)

Closed Population

Time to ExtinctionEarl and Gray (2014)

Closed Population

25 yearsExposed Every Year = 5 years

300 years

Evidence of Rare Species EffectsSutton et al. (accepted)

Endangered Dusky Gopher FrogDiseases of Aquatic

Organisms

n = 18 /trt

ADULTS

Water bath (103 PFU/mL)

Lithobates sevosus

Evidence of Rare Species EffectsChaney et al. (in progress)

Endangered Dusky Gopher Frog

Ranavirus and Small/Isolated Populations

Pearman and Garner (2005)

Isolated Populations had Faster Mortality and Less Genetic Diversity

Isolated Populations

Rana latastei

Factors Contributing to Emergence

Other Possible Stressors: Pesticide Mixtures, Nitrogenous Waste, Endocrine Disruptors, Acidification, Climate Change, Heavy Metals

Pathogen Pollution:

Anthropogenic introduction of novel strains to naïve populations

(Cunningham et al. 2003)

•Fishing Bait •Ranaculture Facilities

•Biological Supply Companies•International Food & Pet Trade

•Contaminated FomitesPicco et al. (2007) Schloegel et al. (2009)

Anthropogenic Stressors:

1) Herbicide (Atrazine)

Forson & Storfer (2006); Gray et al. (2007); Greer et al. (2008); Kerby et al. (2011)

ATV SusceptibilityA. tigrinum

2) Cattle Land Use: Prevalence Green Frogs and Tiger Salamanders

Insecticide (Carbaryl)

Should we be concerned?

•Ranavirus are Multi-species Pathogens •Amphibians with fast-developing larvae most susceptible

•Isolated populations (rare species) greatest threat•Interclass Transmission can occur •Community Composition matters

•Transmission is efficient – Multiple Routes •Environmental Persistence is long (1 – 3 mo)

•Anthropogenic Stressors and Pathogen Pollution contribute to Ranavirus Emergence

Ranaviruses represent a significant threat to the global biodiveristy of ectothermic vertebrates

What can we do?

•Establish surveillance programs (broad then focus on hotspots, >40% infection)

•Identify mechanisms of emergence(natural, stressors, novel strains)

•Identify and implement intervention strategies(break host-pathogen cycle, reduce stressors,

biosecurity precautions)

Gray and Miller (2013)

World Organization for Animal Health

Chytridiomycosis Ranaviral disease

2008

OIE Aquatic Code International Transport of

Animals

•Bleach >4%•EtOH >70%•Virkon >1%

•Nolvasan >0.75%

$75/ bottle

Notifiable Diseases

Schloegel et al. (2010)

Certification for Shipment

Disinfection: Johnson et al. (2003), Bryan et al. (2009), Gold et al. (2013)

Global Ranavirus Consortiumhttp://fwf.ag.utk.edu/mgray/ranavirus/ranavirus.htm

The goal of the GRC is to facilitate communication and collaboration among scientists and veterinarians conducting research on ranaviruses and diagnosing

cases of ranaviral disease

GRC@LISTSERV.UTK.EDU

SymposiaDiscussion Groups

WebsiteReporting System

Outreach ResourcesSpringer eBook

Third International Symposium on Ranaviruses

2015 Florida Marine Health Conference

30 May – 1 June 2015 (3 days): Gainesville, FL

Dr. Tom Waltzek

Invited Talks and Poster SessionDirected Topic Discussions

WorkshopsField Trips

Presentation Contributors:

A. Balseiro, SERIDAM. Brand, University of TennesseeR. Brenes, Carroll University J. Chaney, University of TennesseeA. Duffus, Gordon CollegeR. Goodman, Hampden-Sydney CollegeR. Hardman, Laboklin GmbH & Co KGR. Hill, University of TennesseeJ. Hoverman, Purdue UniversityA. Kouba, Memphis ZooJ. Lankton, USGS NWHCR. Mazzoni, Universidade Federal de GoiásD. McGinnity, Nashville ZooP. Reilly, University of TennesseeM. Ruder, USDAS. Schlosshan, UT Histology (CVM)B. Sutton, Tennessee State UniversityT. Waltzek, University of FloridaB. Wilkes, UT Virology (CVM)

Unpublished Data or Gratis Service

Questions??

dmille42@utk.edumgray11@utk.edu

Photo: N. Wheelwright