Thomas B. Waltzek MS, DVM, PhD
Assistant Professor
UF College of Veterinary Medicine
Department of Infectious Diseases and...
Decline of Pallid Sturgeon within the Missouri River Basin (MRB)Decline of Pallid Sturgeon within the Missouri River Basin...
Pallid Sturgeon Restoration EffortPallid Sturgeon Restoration Effort
• Endangered adults are captured annually & transport...
2009 Blind Pony State Fish Hatchery Epizootic2009 Blind Pony State Fish Hatchery Epizootic
• July – Sept. YOY PS experienc...
• Mixed pop. of bact. cultured but mort. continued despite repeated antib. Tx
• Replicating agent observed on all cell lin...
PS Isolate Genomic CharacterizationPS Isolate Genomic Characterization
•PS isolate genome sequenced (Illumina MiSeq)
> PSR...
PSRV Lethal to Pallid Sturgeon - Koch’s Postulates FulfilledPSRV Lethal to Pallid Sturgeon - Koch’s Postulates Fulfilled
•...
Host Range of Frog Virus 3 like AgentsHost Range of Frog Virus 3 like Agents
DecreasingSusceptibilitytoFV3DecreasingSusceptibilitytoFV3
Percent survival of individuals (n = 20/treatment) exposed to o...
PSRV (FV3) Lethal to ReptilesPSRV (FV3) Lethal to Reptiles
Red-eared slider
Percent survival of individuals (n = 20/treatm...
RanavirusRanavirus
Tree of LifeTree of Life
CGSV
EHNV
PPIV
ATV
PSRV
SERV
BIV
REV
FV3
ESV
WSRV
TFV
RMAX
ECV
CIV
STIV
Rana E...
SignificanceSignificance
• Ranaviruses are a global threat to both cultured & feral
populations of poikilothermic vertebra...
• Explore the impact of FV3 on sturgeon aquaculture
– White Sturgeon RV (1998 CA epizootic)
– 2001 Pallid Sturgeon RV (200...
Future DirectionsFuture Directions
• vIF-2α truncated in less virulent
FV3 strains
• PSRV has full length vIF-2α
• More th...
UF Aquatic Animal Health
Program/CVM
* Dr. Charles Courtney
* Dr. Ruth Francis-Floyd
* Dr. Mike Walsh
* Dr. Iske Larkin
* ...
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Repeated detection of frog virus 3 during aquaculture health surveys

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2013 International Symposium on Ranaviruses
by Thomas Waltzek

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  • The genomic DNA of KHV is found in the core of the virus and is protected by a capsid with a shape described as an icosadeltahedron or 20 juxtaposed triangles. The capsid is built from 162 structural units known as capsomers as shown in blue for human herpesvirus 8 and evident in the negative stain of KHV. This capsid structure and size is highly conserved among the herpesviruses found in a wide range of hosts. The tegument lies between the capsid and the envelope and is composed of proteins essential to virus replication or that influence host cell protein synthesis. The envelope is obtained from host membranes by “budding” as previously mentioned. The envelope contains lipid of host origin and numerous virus-coded glycoproteins that are critical to virus attachment and penetration of the host cell wall. Viral glycoproteins serve as key antigens recognized by the host’s immune response and are essential to immunity to virus infection. The loss of envelope functions render the virus noninfectious and are one reason the virus is labile to drying, detergents, lipid solvents, low pH, freezing and other environmental insults. Now let’s turn to genome characteristics of KHV….
  • The genomic DNA of KHV is found in the core of the virus and is protected by a capsid with a shape described as an icosadeltahedron or 20 juxtaposed triangles. The capsid is built from 162 structural units known as capsomers as shown in blue for human herpesvirus 8 and evident in the negative stain of KHV. This capsid structure and size is highly conserved among the herpesviruses found in a wide range of hosts. The tegument lies between the capsid and the envelope and is composed of proteins essential to virus replication or that influence host cell protein synthesis. The envelope is obtained from host membranes by “budding” as previously mentioned. The envelope contains lipid of host origin and numerous virus-coded glycoproteins that are critical to virus attachment and penetration of the host cell wall. Viral glycoproteins serve as key antigens recognized by the host’s immune response and are essential to immunity to virus infection. The loss of envelope functions render the virus noninfectious and are one reason the virus is labile to drying, detergents, lipid solvents, low pH, freezing and other environmental insults. Now let’s turn to genome characteristics of KHV….
  • Repeated detection of frog virus 3 during aquaculture health surveys

    1. 1. Thomas B. Waltzek MS, DVM, PhD Assistant Professor UF College of Veterinary Medicine Department of Infectious Diseases and Pathology Repeated Detection of Frog Virus 3 DuringRepeated Detection of Frog Virus 3 During Aquaculture Health SurveysAquaculture Health Surveys
    2. 2. 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
    3. 3. 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
    4. 4. 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 tubule Renal tubular necrosis
    5. 5. • 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
    6. 6. PS Isolate Genomic CharacterizationPS 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!
    7. 7. 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.
    8. 8. Host Range of Frog Virus 3 like AgentsHost Range of Frog Virus 3 like Agents
    9. 9. DecreasingSusceptibilitytoFV3DecreasingSusceptibilitytoFV3 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
    10. 10. 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.
    11. 11. RanavirusRanavirus Tree of LifeTree of Life CGSV EHNV 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?
    12. 12. 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
    13. 13. • 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
    14. 14. 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
    15. 15. 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 yourThanks for your attention! Questions?attention! Questions?

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