Thomas B. Waltzek MS, DVM, PhDAssistant Professor

UF College of Veterinary Medicine Department of Infectious Diseases and Pathology

Repeated Detection of Frog Virus 3 During Repeated Detection of Frog Virus 3 During Aquaculture Health SurveysAquaculture Health Surveys

Decline of Pallid Sturgeon within the Missouri River Basin (MRB)Decline of Pallid Sturgeon within the Missouri River Basin (MRB)

• Overfishing & damming MRB led to PS decline

• 1990 listed as federally endangered • Breeding program in 6 hatcheries

Pallid Sturgeon Restoration EffortPallid Sturgeon Restoration Effort• Endangered adults are captured annually & transported to 1/6

hatcheries

• Adults spawned & then returned to the wild

• Progeny reared at 6 hatcheries for restocking 3 management zones within the MRB

Blind Pony State Fish Hatchery (BPSFH)

2009 Blind Pony State Fish Hatchery Epizootic2009 Blind Pony State Fish Hatchery Epizootic• July – Sept. YOY PS experienced heavy losses (80-100% over 3 months)

• Mortality (550/d) greatest at higher temps (15.5 – 25.5 °C)

• Dying fish displayed external and internal hemorrhagic lesions

• Histology revealed necrosis of hematopoietic (K, S) organs et al…

Cell debris within tubuleRenal tubular

necrosis

• Mixed pop. of bact. cultured but mort. continued despite repeated antib. Tx

• Replicating agent observed on all cell lines (FHM, sturgeon lines)

• Negative staining EM revealed icosahedral particles (~ 150 nm)

BPSFH PS Epizootic – Bacteriology, Cell Culture, EMBPSFH PS Epizootic – Bacteriology, Cell Culture, EM

PS Isolate Genomic Characterization PS Isolate Genomic Characterization

•PS isolate genome sequenced (Illumina MiSeq)> PSRV co-linear to FV3, 99% ID ( ~ 105 kbp)

•PS represents a new fish host for Frog Virus 3 (FV3) = interclass host shift!

PSRV Lethal to Pallid Sturgeon - Koch’s Postulates FulfilledPSRV Lethal to Pallid Sturgeon - Koch’s Postulates Fulfilled

• 90% mortality in low & high titer exposures

• High virus titers recov. from 36/36 morts, 2/4 virus exposed survivors, 0/5 controls

Percent survival of individuals (n = 20/treatment) exposed to PSRV isolate. Bath exposure was 103 PFU/ml (low) or 104 PFU/ml (high); experiment duration = 21 d.

Host Range of Frog Virus 3 like AgentsHost Range of Frog Virus 3 like Agents

Decrea sin

g Su

sceptib

ility to FV

3D

ecrea sing S

uscep

tibility to F

V3

Percent survival of individuals (n = 20/treatment) exposed to one of two ranavirus isolates. Bath exposure was 103 PFU/ml; experiment duration = 21 d.

PSRV (FV3) Lethal to AmphibiansPSRV (FV3) Lethal to Amphibians

PSRV (FV3) Lethal to ReptilesPSRV (FV3) Lethal to Reptiles

Red-eared slider

Percent survival of individuals (n = 20/treatment) exposed to PRSV (FV3). Bath exposure was 103 PFU/ml; experiment duration = 28 d.

Ranavirus Ranavirus Tree of Life Tree of Life

0.05

CGSV

EHNV

GIV

SGIV

PPIV

ATV

PSRV

SERV

BIV

REV

FV3

ESV

WSRV

TFV

RMAX

ECV

CIV

STIV

Rana Esculenta Virus

> 99 % NA Seq ID

Frog Virus 3 Isolates

> 99% NA Seq IDBayesian Analysis: Full DNA Polymerase & Major Capsid Genes

• Lowest host specificity observed among dsDNA viruses?

• Implications for control/eradication efforts & thus regulatory implications

‒ Only amphibian RVs reportable?

• Conservation concern‒ Endangered species‒ Ecosystem-level effects?

SignificanceSignificance

• Ranaviruses are a global threat to both cultured & feral populations of poikilothermic vertebrates (fish, amphibians, & reptiles)

• RVs are especially concerning emerging pathogens given their high virulence & low host specificity (interclass host shifts/reservoirs)

• These epizootics are especially concerning given the federally endangered status of pallid sturgeon

• The importance of FV3 in aquaculture and the role of aquaculture in disseminating FV3 is unclear

• Explore the impact of FV3 on sturgeon aquaculture– White Sturgeon RV (1998 CA epizootic) – 2001 Pallid Sturgeon RV (2001 MO Hatchery Epizootic)– Russian/Lake Sturgeon RV (2004 GA Hatchery Epizootic)– Pallid Sturgeon RV (2009 MO Hatchery Epizootic)

• Determine the role aquaculture plays in FV3 dissemination– Northern Pike RV (VHSV Surveillance)– Fathead Minnow RV (VHSV Surveillance)– Walleye RV (VHSV Surveillance)

Future DirectionsFuture Directions

Future DirectionsFuture Directions

• vIF-2α truncated in less virulent FV3 strains

• PSRV has full length vIF-2α

• More thorough SNP and positive selection analyses needed

• Genomic comparisons across a wider range of isolates of known virulence needed…

• Molecular determinants of FV3 virulence• Comparative genomics using isolates of differing virulence

UF Aquatic Animal Health Program/CVM* Dr. Charles Courtney* Dr. Ruth Francis-Floyd* Dr. Mike Walsh* Dr. Iske Larkin* Dr. Nicole Stacy* Dr. Denise Petty* Dr. Galaxia Cortes-Hinojosa* Heather Maness

UF Aquatic Pathobiology Laboratory* Dr. Andy Kane* Dr. James Wellehan* Dr. Brian Stacy* Dr. Don Behringer* Linda Archer

UF Tropical Aquaculture Laboratory* Dr. Roy Yanong* Dr. Kathleen Hartman* Craig Watson* Debbie Pouder

AcknowledgmentsAcknowledgments

UF Global Pathogens Laboratory* Dr. Gregory Gray* Dr. Gary Heil* Dr. John Lednicky* Dr. Kalina Atanasova* Dr. Whitney Krueger* John Burks* Robin Derby* John Friary* Clint McDaniel* Ben Anderson

Fish & Wildlife Research Institute* Dr. Jan Landsberg* Theresa Cody* Micah Bakenhaster* Yasunari Kiryu* Clark Gray* Maki Jenkins

Thanks for your Thanks for your attention! Questions?attention! Questions?