More Related Content
What's hot
What's hot (20)
Viewers also liked
Viewers also liked (17)
Similar to SGrant - Committee meeting presentation - March 6
Similar to SGrant - Committee meeting presentation - March 6 (20)
SGrant - Committee meeting presentation - March 6
- 1. Tracking the spread and host specificity of FV3 Ranaviruses and Frog Virus 3 Samantha Grant Supervisors: Dr. Christopher Kyle & Dr. Craig Brunetti
- 2. Family Iridoviridae Genus Ranavirus Frog Virus 3 Map of ranavirus distribution in amphibians
- 3. What has been done?
- 4. The next step: My project Can we detect FV3 in Ontario waterbodies with eDNA? Is there variation of FV3 in Ontario waterbodies?
- 5. 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?
- 6. 1. Genetic variation exists within FV3 Hypotheses Prediction: If we sequence waterbodies, then we will find multiple variants of FV3
- 7. 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
- 8. 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
- 9. Sample design ā¢ eDNA samples: ā¢ 20+ sites ā¢ Three visits in summer ā¢ Frog swabs: ā¢ Maximum 10 frogs per site ā¢ Inside of mouths ā¢ Target 3 species
- 10. eDNA processing ā¢ Water samples (3) ā¢ 3 repeats per sample ā¢ 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
- 11. Frog samples: Whatās out there? Act as standards for whatās in the water: ā¢ Extract, quantify with qPCR, sequence ā¢ Is there variability of ranaviruses in different species of frogs? ā¢ Spatial differences? ā¢ Temporal differences?
- 12. Areas to look at on the viral genome: ā¢ Major capsid protein ā« Ranavirus discrimination (10% variability between all) ā¢ Open Reading Frames (ORFs): ā« Variation between closely related FV3 ranaviruses ā« Highly variable sequences ORF 66L of FV3 and 5 FV3-like ranaviruses ORF 50L of FV3 and 5 FV3-like ranaviruses
- 13. Primer Optimization 1. Optimize on cultured FV3 2. Optimize on infected tadpoles 3. Test filtration, extraction, and amplification from water Developing primer and probe sets: ā¢ MCP region for eDNA (70bp) ā¢ MCP region for frogs and eDNA (200-300bp) ā¢ ORFs for eDNA and frogs
- 14. 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 -
- 15. What will this tell us? Can we track the virus base on genetic relatedness? Are there signs of gene flow between ponds?
- 16. ā¢ Further validate eDNA for FV3 detection ā¢ Gain insight on: ā« Pathogen movement and spread ā« Host specificity ā¢ Future work: ā« Genome analysis ā« Preventing the spread Implications Pond 1 Pond 3 Pond 4 Pond 2
- 17. Plan of action Step Time Objective Primer optimization February ā May 2016 Develop, obtain, optimize primers Field samples May ā July 2016 Collect samples, extract, quantitate, amplify, sequence (eDNA and frog) Metabarcoding September 2016 ā January 2017 Create libraries, barcodes, sequence on 454 Conclusions and writing December 2016 ā May 2017 Phylogeography of FV3, begin writing Anticipated completion August 2017 Approximately four chapters
- 18. Questions?
- 19. Develop barcodes to tag different ponds How to differentiate between different ponds for 454: Pond 1 Pond 3 Pond 2 ACTGTGATGC CTGATTAGGA GGCTATGATT Pond 1 Pond 2 Pond 3 Barcodes Target sequences TGACATTATCAā¦ TGACATTATCAā¦ TGACATAATCA ā¦ TGACATTATCAā¦ TGACATTATCAā¦ TGACATTATCAā¦ TGACATTATGA ā¦ TGACATTATCAā¦ TGATATTATCAā¦ ACTGTGATGC ACTGTGATGC ACTGTGATGC CTGATTAGGA CTGATTAGGA CTGATTAGGA GGCTATGATT GGCTATGATT GGCTATGATT
- 21. Where is the virus coming from? ā¢ Track the virus based on genetic relatedness ā¢ Is the virus moving from pond to pond?
- 23. Wood frog (Rana sylvatica) Bull frog (Rana catesbeiana)Leopard frog (Rana pipiens) Green frog (Rana clamitans)
Editor's Notes
- Introduce self Looking at different strains of an amphibian pathogen and how they vary in different Central Ontario ponds
- Currently many wildlife diseases that are growing because of anthropogrenic influences, including pollution, hab. Frogs are in a lot of danger right now. Greater need to survey disease and develop tools to do so so we can midigate negative effects by maximizing the use of our limited conservation resources Amphibians are currently subjected to many diseases including Ranaviruses and chytrid fungus, causing mass die-offs, global decline of amphibian populations, huge threat to biodiversity and the ecosystem that humans thrive on 105 species of amphibians alone infected FV3 of ranavirus of the family iridoviridae Best understood ranavirus Infect amphibians, fish, and reptiles Many viruses that are like FV3, very genetically similar, found across the globe Found everywhere, all continents except 1, large threat Transfer: direct, cannibalism, WATER AND SEDIMENT
- Plenty of work has been searching for the presence or absence (SWABBING) of the virus in different species or ponds, however itās determined to be everywhere basically One way to do this is with environmental DNA, filtering and extracting the virus from water which is non-invasive and gives pos samples without needed to find infected individuals However many issues with eDNA and not quite validated for ranaviruses, still many errors with false positives, false negatives, and contamination, unknown sensitivity thresholds and detection levels Thereās still questions based on how and where the virus is moving, and how it changes on a spatial and temporal scale. Transmission may be related to host species or landscape around ponds, however little is known about that either. More recently, there has been work examining the genomes of different species of ranaviruses to determine locations where there is variability that might have to do with infectiousness or persistence We know about presence absence, that itās found in water, and that there are different strains, but what can we do with that?
- So, how am I planning on doing this? I am planning on seeing if there is genetic variation of FV3 in Ontario ponds, and comparing the variation to different ponds, in order to see if there are similarities or differences within and between ponds, which may give us insight on how the virus is moving.
- We can look at the strains of the virus and where they are in the environment, and how they vary based on location. We know that the virus lasts longer in water than sediment, so weāll be more likely to find the virus in water, which is where we will sample for variation. If there is FV3 host specificity, can it explain the gene flow in the ponds?
- Read hypothesis and prediction We want to be able to sequence the different strains in order to make any comparisons with them TEMPORAL AND GEOGRAPHICAL
- Read hypothesis and prediction The level of variation in the ponds may tell us where the viral strains have moved from based on levels
- Read hypothesis and predictions Host specificity can give us insight about how the virus may be evolving to better infect their target hosts, however we only want to confirm or deny host specificity, we wonāt be looking at the genes or infecting individuals.
- - My work will primarily be focused on eDNA, and I am planning on receiving water samples from around southern-central Ontario, both by collecting myself and having collaborators from the Murray lab gathering others. However some of my work will include infected frogs or tadpoles to gain an idea of what is infecting the amphibians and if there are any geographical differences. Some of these samples Iām hoping to receive from Dr. David Lesbarreres and possibly others from Guelph university, however they have not found any samples yet.
- MICROMETER
- Frog samples, want to see whatās infecting the frogs and if thereās variability between species Extract, quantify with qPCR at MCP and ORFs We can also see if thereās spatial differences in infections, if frogs in Kingston waterbodies are being infected with different variations compared to frogs from Peterborough. And we can also determine if thereās differences in infections based on the time of the season, maybe thereās a strain thatās infecting primarily in the spring and another thatās infecting during the mid summer.
- Once I have the samples, there are 3 regions of the viral genome I will be looking at: The first is the major capsid protein. What cytochrome B is to mammals, the MCP is to ranaviruses. With this 1500bp region, not only can I determine which ranavirus is there, but I can also look at variability within the MCPs that belong to FV3 The other two regions were taken from Morrison et al, which are STR regions that have been seen to vary in repeats based on what isolate they are found in. These are the two regions that I will be focusing on. Real Time quantitative PCR to determine if there is virus or not Amplifies target DNA, whichever there is the most of Presence absence
- First Iām developing primers on the MCP that are specific to 15 ranaviruses and an ideal size for eDNA, between 200-300bp SHAYNA WORKING ON THRESHOLD DETECTION
- - Explain metabarcoding Purpose is looking past presence absence and instead sequence all variations in the water Enrich for the viral DNA (8 cycles), PCR again to ligate adapters, barcodes, and primers, the PCR last to amp everything
- The ultimate question that I want to answer is to gain insight on where the virus is coming from how itās moved throughout the environment Does the virus primarily infect because itās lasting in the water? Does the virus reside in certain frogs as their reservoir host and is that strain the primary strain in that pond? Or are reinfections happening because an infected individual brings the virus to a new pond? We may not be able to outright answer these questions, but I propose that my work will help us gain insight to the answers by looking at the strains and seeing if the variation in the genetics is similar and if they can be traced back to a single strain. We can potentially track the virus and its movements by looking at the relatedness of the strains of FV3, comparing them to a reference strain from databases online such as GenBank, and then compare the differences of the strains and see which ones may be more evolved, which may show locations where itās recently moved. The location of the most original strain, or less mutated, may be where it resides and may persist each season in the water, or in reservoirs such as adult frogs. In order to gain insight on these movements, we will have to examine these strains and see how much variation there is.
- Strengthen the protocols for eDNA and FV3 with careful controls and detection sensibility thresholds Confirm the power of metabarcoding for FV3 diversity detection in waterbodies Gain insight on the movement and spread of FV3 based on genetic relatedness If there is host specificity of different species and strains for future studies Other future work includes complete genome analysis of different strains and continuing work on preventing the spread of the virus
- Optimize primers with synthetic FV3 Obtain field samples and extract Process viral DNA
- CHANGE SEQUENCES ON BOTTOM TO SAMPLE PRIMER BARCODES SOMETHING POP UP TO GIVE A HUGE LIBRARY AS PRODUCT WITHH BARCODES ON
- Lithobates pipiens species decline in past 20 years
- The ultimate question that I want to answer is to gain insight on where the virus is coming from how itās moved throughout the environment Does the virus primarily infect because itās lasting in the water? Does the virus reside in certain frogs as their reservoir host and is that strain the primary strain in that pond? Or are reinfections happening because an infected individual brings the virus to a new pond? We may not be able to outright answer these questions, but I propose that my work will help us gain insight to the answers by looking at the strains and seeing if the variation in the genetics is similar and if they can be traced back to a single strain. We can potentially track the virus and its movements by looking at the relatedness of the strains of FV3, comparing them to a reference strain from databases online such as GenBank, and then compare the differences of the strains and see which ones may be more evolved, which may show locations where itās recently moved. The location of the most original strain, or less mutated, may be where it resides and may persist each season in the water, or in reservoirs such as adult frogs. In order to gain insight on these movements, we will have to examine these strains and see how much variation there is.