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

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.

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.