Identifying the genetic mechanisms facilitating host range and virulence of a viral pathogen that threatens European amphibian biodiversity

Lead Research Organisation: Zoological Soc London Inst of Zoology
Department Name: Institute of Zoology

Abstract

Amphibians are experiencing catastrophic declines, caused by the emergence of infectious diseases. In Europe, the causative agent is predominantly viral (ranavirus), and our work has shown that emergence of ranavirus in amphibian communities can result in at least three different patterns: the mortality and population decline can be host-specific, it can affect the whole host community or infections can be asymptomatic, co-occurring in areas where lethal infections are common. From analysis of viral isolates we have determined that this variation correlates with viral phylogeney: in Spain, a genetic lineage responsible for amphibian community mortality and decline co-circulates with a genetically distinct asymptomatic lineage; in the UK, a third lineage is associated with death and decline of a single host species (common frogs - infection of other host species is rare and does not lead to significant mortality or decline in spill-over hosts).

Here we propose to take advantage of this novel system to ascertain what genetic factors are responsible for these differences in host range and virulence. Our approach is three-pronged: first, we will sequence 130 ranavirus genomes to catalogue genetic variation amongst the European lineages and identify candidate loci. We will also take a more bottom-up phylogenetic approach, identifying any genetic variants that map with differences in host species in a global panel of ranavirus genomes. Second, we carry out controlled infection experiments to verify the different host range and virulence of different viral isolates in three animal models (two anurans and one caudate chosen to encompass the host range we have observed in Spain). Third, we will generate viral knockouts, targeting candidate regions we have identified in the first two objectives, and test hypotheses regarding gene function using recombinants and wild type virus in our animal models.

Our results will be a crucial contribution to our understanding of why ranaviruses are such lethal pathogens in some circumstances. Ranavirus emergence is on the increase across Europe, and is emerging as a novel threat to amphibian and reptile biodiversity in Latin America and Madagascar, two amphibian and reptile biodiversity hotspots. Our research will more widely inform the epidemiological community as to what genetic factors may be important for viruses to exploit novel hosts. Given that most emerging infections are viral and emergence arises through host jumps, we expect our findings to be of broad interest to this community.

Planned Impact

Our results will provide the pertinent genomic information, host range and virulence data that will enable us to develop diagnostic marker systems for the identification of highly virulent, less virulent and asymptomatic ranavirus lineages. These resources will be of global utility, as ranavirus is an OIE notifiable pathogen yet current tools for detection fail to differentiate between lineages. Ranaviruses are lethal pathogens of fish, amphibians and reptiles - including many economically important (farmed and traded species) as well as taxa significant to biodiversity. Thus, stakeholders interested in the tools we are developing include national and regional veterinary authorities responsible for monitoring notifiable diseases in the trade, wildlife managers assessing risk that ranaviruses pose to wildlife under their care, and the international research community that make up the Global Ranavirus Consortium. This last includes labs identified as equipped for diagnostic identification of ranaviruses. Our experimental investigation of tissue-level infection dynamics as related to pathogen genotype will allow all those who employ molecular diagnostic tools to do so more effectively while incurring less cost per animal.

At the UK level, Garner sits on the UK Wildlife Health Committee with representatives from NGOs (Natural England) and the Government (DEFRA). The outputs from our research are reported to this committee via meetings on a bi-annual basis and provide a scientific basis for decisions on amphibian conservation in the UK. Our Project Partner Bosch informs a similar committee in Spain, where similar efforts are underway to manage amphibian populations with respect to infectious diseases. Both countries are signatories to the OIE and as such decisions regarding amphibian trade and amphibian wildlife management must take into account the relative risk of different genotypes. We can deliver this information directly to the national bodies responsible for making decisions regarding pathogens in trade and movement of potentially infected animals: for example, in the UK the ZSL is tasked with providing quarterly wildlife disease updates to the GB Wildlife Disease Surveillance scheme, which is the UK's reporting scheme that informs the OIE. The ZSL also sits on the Ad Hoc Group on Amphibian Diseases, providing information to the OIE which underpins the classification of diseases as notifiable and guides recommendations as to the risk posed by pathogens according to genotype Our results will provide both the tools and the virulence data necessary for the OIE to start issuing regarding the relative risk of ALRV lineages and how this should feed into appropriate management strategies for traded animals.

Our spatial and genomic data will guide the development of efforts to map the distribution of ranaviruses based on genotypes. We are currently involved in the modification of the GRRS to accommodate virus genotype, alongside projects to genotype ATV ALRVs (Jancovich) and North American FV3s (Waltzek lab, University of Florida).

We also expect our outputs to find a wide academic audience. Our combination of pathogen genomics, multi-host experimental studies of host/tissue specificity and virulence, and our explicit tests of function using knockouts is novel and will be crucial for any current and future academic effort aiming at understanding ranavirus epidemiology, host/parasite interactions, virology, parasite evolution and host/parasite coevolution.

Publications

10 25 50

publication icon
Robert J (2018) Water Contaminants Associated With Unconventional Oil and Gas Extraction Cause Immunotoxicity to Amphibian Tadpoles. in Toxicological sciences : an official journal of the Society of Toxicology

 
Description Early results are indicating that different lineages of ranavirus may have different environmental envelopes for replication. This is currently being investigated. We are near to completed in developing a reliable quantitative PCR diagnostic for ranavirus.

Added 2017: The qPCR is ready for release in two ways: publication should be submitted next month and project technician Leung has requested to present the technique at the Global Ranavirus Consortium meeting this summer (2017).

Added 2018: qPCR publication is out. Further work on temperature using long-term monitoring data and combining in vitro and in vivo assays has revealed a key role for environmental temperature in determining prevalence of infection and severity of disease outcomes. Last, through a collaboration within the IoZ, we have begun to describe an important role for amphibian skin microbial communities in determining prevalence of infection and severity of disease outcomes. Together, these two findings inform our future research plans that we hope will help us develop a disease mitigation strategy for emerging ranaviruses.

Added 2019: We cannot replicate the patterns of mortality caused by CMTV-like ranaviruses in nature in controlled experiments, and our work with Dr Saucedo during his placement has lead us to establish methods for different routes of transmission, and have been granted permission by the HO to infect and freeze-kill tadpoles so we can 'feed' ranaviruses to foraging tadpoles. Should be getting results for this approach next month. Our work on examining virus growth rates in cell culture across temperatures and comparing this to virulence in common frogs is now being used in an NC3Rs-funded studentship and one of our London DTP students to, respectively, examine if cell culture can replace animals for studies of virulence and if ranaviruses evolve in response to climate change. The NC3Rs student is also building on Dr Saucedo's histology work to determine if intervention before the onset of lethal disease in experiments can still yield relevant information regarding virulence through the use of histopathology and qPCR, using the diagnostic developed in 2017.
Exploitation Route Uptake of the qPCR diagnostic in the research community has begun. Our work on temperature and microbiomes will undoubtedly inspire others to investigate the role these factors play in ranavirosis in their study systems.
Sectors Environment

 
Description Through presentations and talks, we have raised awareness of the threat posed to UK amphibians, reptiles and fish by the CMTV-like ranaviruses emerging on the continent.
Sector Environment
Impact Types Cultural

 
Description Reporting of ranavirus infection
Geographic Reach Multiple continents/international 
Policy Influence Type Influenced training of practitioners or researchers
 
Description NERC London DTP studentship
Amount £86,000 (GBP)
Organisation Natural Environment Research Council 
Sector Public
Country United Kingdom
Start 10/2017 
End 09/2021
 
Description State Programme for R&D: Challenges facing Society
Amount € 122,000 (EUR)
Funding ID CGL2015-70070-R 
Organisation Spanish Ministry of Economy and Competitiveness 
Sector Public
Country Spain
Start 09/2016 
End 09/2019
 
Description nerc London DTP
Amount £86,000 (GBP)
Organisation Natural Environment Research Council 
Sector Public
Country United Kingdom
Start 10/2018 
End 04/2022
 
Title Viral load estimation by quantitative PCR 
Description Design of sensitive and specific qPCR methods targeting virus and host loci to be used in tandem to generate viral load estimates 
Type Of Material Technology assay or reagent 
Year Produced 2016 
Provided To Others? Yes  
Impact screening of various sample types for presence and intensity of ranavirus infection in six labs on three continents (as of February 2017) yielding more accurate information on host and geographic distribution of a World Organisation for Animal Health listed pathogen. 
 
Title Citizen science reports of amphibian mortality in the UK between 1991-2010 
Description Records relating to a citizen science report of amphibian mortality in the UK between 1991-2010. The fields cover georeference data and data on the mortality event (numbers of affected animals and signs of disease observed). 
Type Of Material Database/Collection of data 
Year Produced 2016 
Provided To Others? Yes  
Impact Enabled modelling of spread of infectious disease in British frog populations and identification of likely drivers of emergence, which lead to publication of findings: Price SJ, Garner TWJ, Cunningham AA, Langton TES & RA Nichols (2016) Reconstructing the emergence of a lethal infectious disease of wildlife supports a key role for spread through translocations by humans. Proc R Soc B 283: 20160952. http://dx.doi.org/10.1098/rspb.2016.0952 
URL http://rspb.royalsocietypublishing.org/content/283/1839/20160952
 
Title Metadata for Genetic determinants of ranavirus host range and virulence 
Description Raw reads from ranavirus genomes sequenced during this project published with NCBI. Currently archived while we complete and submit the relevant manuscript 
Type Of Material Database/Collection of data 
Year Produced 2019 
Provided To Others? No  
Impact None as of yet, information is archived but still embargoed until we publish 
URL http://www.ncbi.nlm.nih.gov/bioproject/511378
 
Description Gene knockout methodologies 
Organisation University of Rochester
Department Department of Neuroscience
Country United States 
Sector Academic/University 
PI Contribution Dr. Stephen Price visited Jacques Robert's lab at the University of Rochester for there months to train and access support in virus gene knockout methodologies. Whilst there he collaborated in infection experiments and helped analyse data generated by the Robert lab.
Collaborator Contribution Provided training and support in methods essential for meeting one of the project objectives. Provided animals, training and a virus isolate for infection studies.
Impact Creation of four ranavirus (knock-out) recombinants Completed two infection trials Collaboration on a study on effects of fracking on virus infection
Start Year 2015
 
Description Amphibian Week for Edinburgh MVetSci 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact One week online workshop including video lectures, discussion groups, group activity.
Year(s) Of Engagement Activity 2015,2016,2017,2018
URL http://www.ed.ac.uk/schools-departments/vet/studying/postgraduate/taught-programmes/conservation-med...
 
Description Anthropogenic changes in distribution and severity of disease caused by ranaviruses affecting UK common frogs 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited presentation in Symposium 'Amphibian Disease Susceptibility in a Changing World' held at the 9th World Congress of Herpetology in Dunedin, New Zealand. Extensive question period afterwards.
Year(s) Of Engagement Activity 2020
 
Description Departmental seminar at University of Liverpool 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact Invited departmental seminar at University of Liverpool, topic was 'amphibian-associated ranaviruses: the first (and worst?) infectious disease conservation issue for amphibians'
Year(s) Of Engagement Activity 2017
URL https://www.liverpool.ac.uk/integrative-biology/events/
 
Description Did Bd-CAPE drive the Kihansi spray toad (Nectophrynoides asperginis) to extinction in the wild? 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact PDRA presentation on chytrid genome associated with first and only reported decline of amphibians on the African continent. Numerous African researchers engaged afterwards to discuss options for surveys and post-decline investigations of amphibians.
Year(s) Of Engagement Activity 2020
URL http://www.wchnz.com/wch2020
 
Description Graduate student conservation conference (Edinburgh 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Postgraduate students
Results and Impact Presentation on the relative importance of ecology vs evolution for mitigating amphibian infectious diseases
Year(s) Of Engagement Activity 2015
 
Description International Ranavirus Symposium, Budapest - conference talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Regular talk at international conference for researchers and third sector organisations
Year(s) Of Engagement Activity 2017
 
Description International Ranavirus Symposium, Budapest; Keynote talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Keynote talk at international conference for researchers and third sector organisations
Year(s) Of Engagement Activity 2017
 
Description Invited departmental seminar (Uppsala) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Postgraduate students
Results and Impact Invited departmental seminar for Ecology and Evolution department at Uppsala University
Year(s) Of Engagement Activity 2015
 
Description Invited public talk (Richmond) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact I was invited to give a talk about wildlife disease and amphibian conservation to the local Richmond Park nature association
Year(s) Of Engagement Activity 2015
 
Description Invited talk (Silwood Park Seminar Series) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Postgraduate students
Results and Impact Seminar to broad academic audience which sparked new contacts and ongoing discussion afterwards
Year(s) Of Engagement Activity 2017
 
Description Keynote address to the Global Ranavirus Consortium 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Co-presented keynote address on the host and geographic distribution of ranaviruses in Europe and their potential and actual threats to biodiversity
Year(s) Of Engagement Activity 2017
URL https://www.ranavirus.org/2017-4th-international-symposium-on-ranavirus/
 
Description Keynote address to the Herpetological Association of Africa outlining potential threat of ranaviruses to African herpetofauna 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Delivered a keynote address to the annual meeting of the Herpetological Association of Africa outlining the potential threat ranaviruses pose to African herpetofauna
Year(s) Of Engagement Activity 2017
URL http://www.africanherpetology.org/
 
Description Lecture on GRC Ranavirus course 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Live video lecture to raise awareness about ranaviruses among students and professionals.
Year(s) Of Engagement Activity 2016
URL http://www.ranavirus.org/online-course-on-ranavirus-biology/
 
Description Mitigating single pathogen and co-infections that threaten amphibian biodiversity 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact First major scientific event examining how to mitigate single and multiple pathogens in amphibian populations threatened by infectious diseases. Conveners include PIs from 3 NERC standard grants. 2 day symposium involving researchers from across Europe, the Americas and Australasia, and 2 single day workshops, 1 on microbiomes and 1 doing a mock disease intervention. One publication from 2nd workshop. Attendees report overwhelmingly as to quality and reach of event, new collaborations formed and suggestion this become a once every few years event.
Year(s) Of Engagement Activity 2019
 
Description NC3Rs Workshop on Amphibian Welfare 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Third sector organisations
Results and Impact Two day workshop to engage people from different sectors with shared interest in driving change
Year(s) Of Engagement Activity 2017
 
Description Presentation at ZSL scientific event 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Presentation regarding the potential impact of invasive species on emergence of threatening amphibian infectious diseases, delivered as part of the ZSL Scientific Event launching the 2016 Living Planet Report
Year(s) Of Engagement Activity 2016
URL https://www.zsl.org/science/whats-on/the-living-planet-report-2016-threats-pressures-and-addressing-...
 
Description Presentation to stakeholders 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact Presentation to a combined meeting of British Amphibian and Reptile groups (ARGs UK) to inform about the potential risk posed to UK herpetofauna by variants of ranaviruses emerging on the European continent
Year(s) Of Engagement Activity 2016
 
Description Resolving Batrachochytrium dendrobatidis lineage distribution and ecology in South Africa 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact PhD student presentation on developing and applying lineage specific qPCRs to understand lineage interactions in nature. Extensive interest in developing similar tools for other lineages and applying existing tools to European settings.
Year(s) Of Engagement Activity 2020
URL http://www.wchnz.com/wch2020
 
Description School visit 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Presentations and conversations with three and four year olds and pre-school staff about wonderful amphibians and reptiles
Year(s) Of Engagement Activity 2017
 
Description Training on IoZ contribution to training on the London DTP and the Oxford DTP 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact I contributed to our annual event hosting PhD candidates on two NERC-funded DTPs. The core content of my presentation was constructed around current research on my NERC-funded projects, and providing PhD candidates the opportunity to become aware of the opportunities and support that could be provided to them through existing NERC projects. Some of the participants contacted me for further information about methods, contacts and the possibility of involvement in the research programmes
Year(s) Of Engagement Activity 2017