Experimental Evidence that Amphibian Ranaviruses Are Multi-Host Pathogens

Infectious diseases, including those caused by ranaviruses (family Iridoviridae), are among the suspected causes of global amphibian declines. Like many pathogens, ranaviruses appear to infect multiple species. We examined several North American amphibian ranavirus isolates to improve our understanding of the effects these viruses have on host communities. Our study had two objectives. The first was to characterize isolates from epizootics affecting wild amphibian populations and compare them to previously described isolates. The second was to test whether amphibian ranaviruses infect ecologically relevant heterologous species, and if so, document the outcome of exposure. The combined results of restriction endonuclease (RE) digestion analyses, sequence analyses, and experimental challenge trials suggest that two amphibian ranaviruses, Ambystoma tigrinum virus (ATV)-like viruses and Frog Virus 3 (FV3)-like viruses, are distinct viral species with different ecologies. Characterizations revealed that several isolates with identical major capsid protein (MCP) gene sequences have distinctive RE profiles. This suggests that high degrees of similarity in MCP sequences may belie important differences among isolates, and we argue it is imperative to characterize ranavirus isolates beyond sequencing the MCP gene. Results from experimental exposure trials indicate that multiple host species may be involved in the ecology of ATV- and FV3-like viruses, and that each virus is capable of infecting several amphibian species that share breeding habitats. Additionally, field collections revealed FV3-like ranaviruses circulating in Wood Frogs (Rana sylvatica) and ATV-like ranaviruses circulating in Tiger Salamanders (Ambystoma tigrinum diaboli) in the same week at a single breeding pond, highlighting the ecological potential for transmission among different host species. Our research also corroborates a growing body of knowledge that suggests individual host populations may differ in their responses to ranavirus infection, a finding with complex conservation implications. Ultimately, experiments elucidating the dynamics of intra- and inter-specific transmission will be particularly important for understanding the roles that ranaviruses play in their host communities and the threat they pose to amphibian populations.

Figure 1

Restriction endonuclease (RE) digests of ranavirus isolates from ranid frogs and Ambystoma tigrinum. HindIII (A) and XbaI (B) digestions of FV3-like ranavirus isolates from ranid frogs. HindIII (C) and XbaI (D) digestions of ATV-like ranavirus isolates from A. tigrinum. Abbreviations of isolate names are as indicated in Table 1. Molecular weight ladder is indicated on the left of each image (New England Biolabs 1 kb ladder, catalog #N3232S) and is abbreviated LDR. Panels C and D show gels that were photographed at different time points after de-staining. Each panel is a compilation of a single gel that was photographed multiple times; the best image for each isolate was then used for the panel.

Figure 2

Outcome of exposure in Ambystoma tigrinum, Rana sylvatica, R. pipiens, and Hyla regilla exposed to three amphibian ranaviruses. Circles represent all animals in each treatment; colors represent the proportion of animals in each treatment that experienced each outcome. Results for A. tigrinum do not include animals harvested on day 14. All animals in control treatments were PCR negative for virus after 28 days, therefore results not shown. Lightest gray: survived to 28 days post-exposure (p.e.) and PCR negative for ranavirus. Medium gray: survived to 28 days p.e. and PCR positive for ranavirus. Darkest gray: died and PCR positive for ranavirus.

Figure 3

Cumulative mortality of Ambystoma tigrinum, Rana sylvatica, R. pipiens, and Hyla regilla when experimentally exposed to three amphibian ranaviruses (ATV-DAL, FV3-EST, FV3-STN). Plots do not include A. tigrinum harvested 14 days post-exposure. Note that the cumulative mortality plots of FV3-EST and FV3-STN overlay one another in all species. Mortality plots were not generated for treatments in which no animals died.

Contributor Notes

Section Editor: E. Schultz.

¹ School of Life Sciences, Arizona State University, Tempe, Arizona 85287-4601, e-mail: (DMS) danna.schock@asu.edu, e-mail: (JKJ) james.jancovich@asu.edu, e-mail: and (JPC) jcollins@asu.edu. Send reprint requests to DMS.

² Canadian Cooperative Wildlife Health Centre, Department of Veterinary Pathology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, S7N 5B4, Canada, e-mail: trent.bollinger@usask.ca

³ Department of Microbiology, University of Mississippi Medical Center, 2500 North State Street, Jackson, Mississippi 39216, e-mail: vchinchar@microbio.umsmed.edu

Received: 04 Jun 2006

Accepted: 22 Jun 2007