Frog Virus 3 Genomes Reveal Prevalent Recombination between Ranavirus Lineages and Their Origins in Canada

Abstract

Ranaviruses are pathogens associated with the decline of amphibian populations across much of their distribution. In North America, frog virus 3 (FV3) is a widely distributed pathogen with wild populations of amphibians harboring different lineages and putative recombinants between FV3 and common midwife toad virus (CMTV). These recombinants have higher pathogenicity, and CMTV-derived genes associated with virulence are reported in wild strains in Canada. However, while FV3 is linked to amphibian die-offs in North America, CMTVs have been reported only in commercial frog farms in North America. We sequenced complete genomes of 18 FV3 isolates from three amphibian species to characterize genetic diversity of the lineages in Canada and infer possible recombinant regions. The 18 FV3 isolates displayed different signals of recombination, varying from none to interspersed recombination with previously isolated CMTV-like viruses. In general, most recombination breakpoints were located within open reading frames (ORFs), generating new ORFs and proteins that were a mixture between FV3 and CMTV. A combined spatial and temporal phylogeny suggests the presence of the FV3 lineage in Canada is relatively contemporary (<100 years), corroborating the hypothesis that both CMTV- and FV3-like viruses spread to North America when the international commercial amphibian trade started. Our results highlight the importance of pathogen surveillance and viral dynamics using full genomes to more clearly understand the mechanisms of disease origin and spread. IMPORTANCE Amphibian populations are declining worldwide, and these declines have been linked to a number of anthropogenic factors, including disease. Among the pathogens associated with amphibian mortality, ranaviruses have caused massive die-offs across continents. In North America, frog virus 3 (FV3) is a widespread ranavirus that can infect wild and captive amphibians. In this study, we sequenced full FV3 genomes isolated from frogs in Canada. We report widespread recombination between FV3 and common midwife toad virus (CMTV). Phylogenies indicate a recent origin for FV3 in Canada, possibly as a result of international amphibian trade.

Sublethal effects of wild-type and a vIF-2α-knockout Frog virus 3 on postmetamorphic wood frogs (Rana sylvatica): potential for a stage-specific reservoir

“…least two different FV3-like viruses prevailing in this area (Grant et al 2019;Vilaça et al 2019). These observations, in combination with our experimental results, could indicate that the source population evolved partial immunity against FV3-like ranaviruses due to repeated exposures to the pathogen.…”

“…Field studies as well as experimental exposures to different ranavirus isolates have shown that individuals may sustain sublethal infections (Pearman et al 2004;Gray et al 2007;Greer et al 2009;Miller et al 2009;Hoverman et al 2011) and can shed sufficient amounts of ranavirus virions to infect other individuals (10 3 -10 4 PFU/mL: Brunner et al 2004;Robert et al 2005;Rojas et al 2005). Furthermore, overwintering tadpoles and paedomorphic salamanders can act as reservoir in aquatic habitats (Brunner et al 2004;Gray et al 2009a), and several field studies have identified sublethal infected postmetamorphic amphibians, with only a minority exhibiting pathological signs (Australia and Europe: Cullen et al 1995;Cunningham et al 2007b;Ariel et al 2009b;North America: Gray et al 2009b;Hoverman et al 2011;Vilaça et al 2019). Our findings, as well as reports of re-occurring mass mortality events in other amphibian populations inhabiting semipermanent and ephemeral wetlands (Green et al 2002;Petranka et al 2007;Todd-Thompson 2010), further support a specific life-history stage reservoir theory.…”

“…Four clutches of wood frog eggs were collected from a natural, semipermanent wetland in Wood Buffalo National Park near Fort Smith, Northwest Territories, Canada, on 14 May 2018. The wood frog population at this site had low ranavirus prevalence in 2017 (1/23 in terrestrials (5%) and 0/35 in tadpoles (0%); Bienentreu 2019) and the ranavirus was identified as FV3-like strain (Vilaça et al 2019). Each egg clutch was individually packed in a new 3.7 L Ziploc bag filled with pond water and then placed into a cooler filled halfway with water and ice packs.…”

Sublethal Effects of Wild-Type and a vIF-2&alpha;-Knockout <em>Frog virus 3</em> on Post-Metamorphic Wood Frogs (<em>Rana sylvatica</em>)

“…However, Ranavirus has been commonly observed in amphibians in the area [32,34], with two confirmed die-off events at wetlands 7 and 29 km away in 2017 [28], and mortality occurring among wood frog tadpoles within 3.5 km in 2009 [49]. Additionally, sequencing led to the identification of two different FV3-like viruses in amphibian populations in the research area [33,50].…”

Amphibian Disease Ecology: Are We Just Scratching the Surface?

“…In general, the outcomes of epizootics in amphibian communities strongly depend on the respective pathogen strain (Brunner et al 2015;Berger et al 2016). Different pathogens are highly variable in their virulence (commonly measured as the time to the host's death; Berger et al 2005;Brunner and Collins 2009;Farrer et al 2011;Morrison et al 2014), and recent studies have identified the circulation of highly recombinant pathogen lineages in wild populations (Farrer et al 2011;Claytor et al 2017;O'Hanlon et al 2018;Vilaça et al 2019). The origins of the different Bd lineages, as well as Bsal, are in northeast Asia, where they infect native amphibians without causing severe epizootics, and most individuals sustain sublethal infections (Martel et al 2014;O'Hanlon et al 2018).…”