Tracking the spread and host specificity of FV3

Ranaviruses and Frog Virus 3

Samantha GrantSupervisors: Dr. Christopher Kyle & Dr. Craig Brunetti

Family IridoviridaeGenus Ranavirus

Frog Virus 3

Map of ranavirus distribution in amphibians

What has been done?

The next step: My project

Can we detect FV3 in Ontario waterbodies

with eDNA?

Is there variation of FV3 in Ontario waterbodies?

Are there different variations of FV3 infecting different species of frog?

The next step: My project

Are patterns of viral gene flow among waterbodies

influenced by host dispersal?

1. Genetic variation exists within FV3

Hypotheses

Prediction: If we sequence waterbodies, then we will find

multiple variants of FV3

2. Genetic variation of FV3 exists among different waterbodies

Hypotheses

Prediction: If we sequence FV3 variants in multiple ponds,

then the genetic variation of the virus will vary between

each location

3. Viral gene flow patterns are influenced by host specificity

Hypotheses

Prediction: If we sequence FV3 from different frog species, there will be patterns that

indicate gene flow

Sample design•eDNA samples:

•20+ sites•Three visits in summer

•Frog swabs:•Maximum 10 frogs per site• Inside of mouths•Target 3 species

eDNA processing•Water samples (3)

•3 repeats per sample•Varying locations

Collect 250mL water samples

Filter through 0.1um filter paper

to catch virus

Quantify with qPCR to

determine if virus present

Extract virus from filter

Frog samples: What’s out there?Act as standards for what’s in the water:•Extract, quantify with qPCR, sequence• Is there variability of ranaviruses in

different species of frogs?•Spatial differences?•Temporal differences?

May June July Aug

Areas to look at on the viral genome:

•Major capsid protein▫Ranavirus discrimination

(10% variability between all)

•Open Reading Frames (ORFs):▫Variation between closely

related FV3 ranaviruses▫Highly variable sequences

ORF 66L of FV3 and 5 FV3-like ranaviruses

ORF 50L of FV3 and 5 FV3-like ranaviruses

Primer Optimization

1. Optimize on cultured FV32. Optimize on infected tadpoles3. Test filtration, extraction, and amplification from water

Developing primer and probe sets:•MCP region for eDNA (70bp)•MCP region for frogs and eDNA (200-300bp)•ORFs for eDNA and frogs

Metabarcoding and 454 pyrosequencing

Consensus (Sanger) vs total variation (metabarcoding)Variant 1 -Variant 2 -Variant 3 -Variant 4 -Variant 5 -Variant 6 -Variant 7 -Variant 8 -

What will this tell us?

Can we track the virus base on genetic relatedness?

Are there signs of gene flow between ponds?

•Further validate eDNA for FV3 detection•Gain insight on:▫Pathogen movement and spread▫Host specificity

•Future work:▫Genome analysis ▫Preventing the spread

Implications

Pond 1

Pond 3 Pond 4

Pond 2

Plan of actionStep Time Objective

Primer optimization February – May 2016 Develop, obtain, optimize primers

Field samples May – July 2016 Collect samples, extract, quantitate, amplify, sequence (eDNA and frog)

Metabarcoding September 2016 – January 2017

Create libraries, barcodes, sequence on 454

Conclusions and writing December 2016 – May 2017

Phylogeography of FV3, begin writing

Anticipated completion August 2017 Approximately four chapters

Questions?

Develop barcodes to tag different pondsHow to differentiate between different ponds for 454:

Pond 1

Pond 3

Pond 2

ACTGTGATGC

CTGATTAGGA

GGCTATGATT

Pond 1

Pond 2

Pond 3

BarcodesTarget

sequences

TGACATTATCA…

TGACATTATCA…

TGACATAATCA…TGACATTATCA…

TGACATTATCA…

TGACATTATCA…

TGACATTATGA…TGACATTATCA…

TGATATTATCA…

ACTGTGATGC

ACTGTGATGC

ACTGTGATGC

CTGATTAGGA

CTGATTAGGA

CTGATTAGGA

GGCTATGATT

GGCTATGATT

GGCTATGATT

Where is the virus coming from?

• Track the virus based on genetic relatedness

• Is the virus moving from pond to pond?

Tadpole species ID

Wood frog (Rana sylvatica)

Bull frog (Rana catesbeiana)Leopard frog (Rana pipiens)

Green frog (Rana clamitans)