This document discusses diagnostics, treatment, and management of ranavirus. It outlines several diagnostic methods like virus isolation, serology, histopathology, transmission electron microscopy, and PCR. Real-time PCR is commonly used but has limitations. Proper sample selection and testing the right samples is important to accurately detect ranavirus infections, especially subclinical infections. Both mortality events and disease surveillance require considering potential co-infections and the ability to differentiate ranavirus strains. Control of ranavirus involves regulatory practices, vaccination for small populations, and treatment of individuals, which is debated for persistent infections. Reintroduction programs necessitate easy strain differentiation of detected viruses.

1. AN OVERVIEW OF RANAVIRUS
DIAGNOSTICS, TREATMENT AND
MANAGEMENT
Second International Ranavirus
Symposium
July 27-29, 2013
Allan Pessier
Amphibian Disease Laboratory
San Diego Zoo Global
apessier@sandiegozoo.org

2. Introduction
 Diagnostics
 What’s available
 Pitfalls
 Interpretation
 Selecting the right tests
 Treatment and Management
 Wild Populations
 Captive Populations
 Endangered Species and
Reintroduction Programs
BradWilson

3. Sensitivity and Specificity of Diagnostic
Tests
 Gold Standard: The standard accepted diagnostic test for a
condition to which all other diagnostic tests are compared
 Analytical Sensitivity: Lowest concentration of pathogen
DNA that can be detected by the test (PCR).
 Diagnostic Sensitivity: Proportion of true positives
detected.
 Diagnostic Specificity: Proportions of true negatives
detected

4. Pitfalls in Diagnostic Sampling
What affectsyour diagnostic sensitivity and specificity?
 Collection of biologically
relevant samples
 Subclinical infections
 Variation in how samples
are collected
 Sample storage
 PCR Inhibitors
 Contamination

5. Diagnostic Methods

6. Virus Isolation
 Usually tissue samples
 Fish and amphibian cell
lines
 CPE and ID by ELISA, IPX
or PCR
 Greater cost, not widely
available
 Needed for purified
virus, transmission
experiments, best quality
DNA
OIE Gold Standard

7. Serology
 Indirect ELISA
 MarineToads
 GopherTortoises
 May be useful for detection
of prior exposure in
populations (? Adjunct to
PCR surveillance)
 Does not provide
information on current
infection status or RV strain
type

8. Histopathology
 Formalin-fixed tissues
 Evaluate tissues for
lesions consistent with
ranaviral disease
 Rule out other disease
processes
 Immunohistochemistry
 Pathologists are fun

9. Histopathology
 Insensitive for
subclinical infections
 Multicentric necrosis
(liver, spleen, kidney,
skin, hematopoietic
tissue)
 Intracytoplasmic
basophilic inclusion
bodies

13. Transmission Electron Microscopy
 Demonstration of
characteristic
icosohedral virions
 Cell culture or
tissues
 Definitive only for
an iridovirus
 Good for historical
cases

15. Polymerase Chain Reaction (PCR)
 Accessible and fast
 Conventional and
Real-Time
 Major capsid protein
(MCP) is most
common
 Samples include
tissues, blood, oral
and cloacal swabs,
and FFPE

16. PCR Caveats
 Infection vs. Disease
 Viable vs. inactivated
virions
 Encourages tunnel
vision in outbreak
investigation
 Sample selection
(especially naturally
infected animals)
 Contamination

17. Conventional PCR
 Products visualized
on agarose gels
 DNA sequencing to
confirm positives
 Phylogeny of
positive samples*
 Less sensitive than
Real-Time
 Inhibitors
April Johnson

18. “The” Ranavirus and
The Problem with Frog Virus 3
• MCP gene is
highly
conserved
• Different
viruses will have
similar or
identical
sequences
• FV3-like viruses

19. Implications for Research and Animal
Management
 Detection of
unexpected viruses
 Need to be able to
easily differentiate
viruses for
 Prevalence/epidemio
logic studies
 Reintroduction
Programs

20. Real-Time/Taqman PCR
 Great for routine
diagnostics
 High analytical
sensitivity
 No need to seq
positives (Taqman)
 Viral Loads
 MCP-based
 Limits on phylogeny

22. Am I doing the right test(s)?
 Investigation of mortality events
 Did animals die of ranaviral disease
 Co-infections
 Positive PCR doesn’t equal disease
 Population Surveillance and
Management
 High sensitivity
 Adequate samples (type and number)
 Ability to differentiate strains
 Standardization

23. Mortality Events
 Avoid assumptions as
many conditions look
alike
 Subclininical infections
 Co-infections
 Necropsy and
histopathology
 Ancillary testing
April Johnson

24. When there is no pathologist…..
 Save tissues for
multiple types of
diagnostic
investigation
 Fixed tissues or
carcasses for
histopathology (not
frozen)
 Frozen tissues or
carcasses for other
diagnostics

25. Disease Surveillance
 Limitations of PCR for detection of subclinical
infections
 PCRWell-validated for sick animals
 What is the appropriate sample for subclinical infection
 ? Macrophages/leukocytes; kidney
 Adequate sample numbers
 Consideration of diagnostic sensitivity
 Differentiation of strains
 Implications for epidemiology and risk assessments
 Implications for regulatory requirements
 Interpretation of prevalence data

26. Control of Ranavirus Infections
 Regulatory and
Pathogen Pollution
 Adequate tests
 Strain differentiation
 Vaccination
 Small populations
 Reintroduction programs
 Treatment of
individuals

27. Reintroduction/Translocation Programs
 Positive PCR tests on
routine surveillance
 Paralysis even when
animals held in
isolation
 Need for easy strain
differentiation
 ? Role of treatment
protocols for valuable
animals/populations

28. Treatment of Ranavirus Infections
 Guanine analogue
antivirals
(acyclovir, valacyclovir)
 Temperature
elevations
 Questions
 Persistent
infections?