Evidence for multiple recent host species shifts among the Ranaviruses (family Iridoviridae)

Abstract

Members of the genus Ranavirus (family Iridoviridae) have been recognized as major viral pathogens of cold-blooded vertebrates. Ranaviruses have been associated with amphibians, fish, and reptiles. At this time, the relationships between ranavirus species are still unclear. Previous studies suggested that ranaviruses from salamanders are more closely related to ranaviruses from fish than they are to ranaviruses from other amphibians, such as frogs. Therefore, to gain a better understanding of the relationships among ranavirus isolates, the genome of epizootic hematopoietic necrosis virus (EHNV), an Australian fish pathogen, was sequenced. Our findings suggest that the ancestral ranavirus was a fish virus and that several recent host shifts have taken place, with subsequent speciation of viruses in their new hosts. The data suggesting several recent host shifts among ranavirus species increase concern that these pathogens of cold-blooded vertebrates may have the capacity to cross numerous poikilothermic species barriers and the potential to cause devastating disease in their new hosts.

FIG. 1.

Annotation of the EHNV genome showing the order and orientation of ORFs. The EHNV genome was annotated as described in Materials and Methods. Each arrow represents an ORF in the right or left orientation. Conserved iridovirus ORFs (C) and putative virulence ORFs (V) are indicated. The brackets enclose the region of consecutively oriented ORFs and the arrows indicate the locations of genomic inversions compared to FV3.

FIG. 2.

Phylogenetic analysis of EHNV ORF 87L. Homologous sequences to the EHNV DHFR gene (ORF 87L) were obtained by BLASTP analysis. The neighbor-joining tree was determined using MEGA4, and it is shown with statistical support indicating the robustness of the inferred branching pattern, as assessed using the bootstrap test. The accession number for each gene in the phylogeny is given in Materials and Methods.

FIG. 3.

Concatenated phylogeny of 26 conserved iridovirus sequences. Phylogenetic relationships of 26 conserved open reading frames from the 13 completely sequenced iridovirus genomes are shown. The neighbor-joining tree obtained using MEGA4 is shown, with statistical support indicating the robustness of the inferred branching pattern, as assessed using the bootstrap test. The sequences used for this analysis are described in Tables S1 and S3 in the supplemental material. The most recent common ancestors (MRCAs) are indicated at particular branch points on the phylogeny.

FIG. 4.

Dot plot analysis of EHNV versus ATV. The genomic sequence of EHNV was compared to that of ATV by dot plot analysis (JDotter [www.biovirus.org/]). The dot plot comparison of EHNV with ATV shows unique sequences in the EHNV genome.

FIG. 5.

Dot plot analysis of EHNV versus other ranaviruses. The genomic sequence of EHNV was compared to those of FV3 and SGIV by dot plot analysis (JDotter [www.biovirus.org/]). (A) Comparison of the EHNV genome to the FV3 genome. (B) Dot plot comparison of EHNV and SGIV genomic sequences.

FIG. 6.

Dot plot analysis of SGIV compared to EHNV and FV3. Dot plots were generated comparing SGIV to EHNV (A) and FV3 (B). Colinear segments were sequentially numbered along the SGIV genome. Consecutively ordered segments along the EHNV and FV3 genomes are circled, while inverted segments are boxed.

FIG. 7.

Model of ranavirus genomic rearrangements. Using the dot plot analysis shown in Fig. 5, consecutively ordered colinear segments were arranged diagrammatically. Comparing the order and orientation of the colinear segments, the rearrangements observed in Fig. 4 occurred in the FV3-like virus lineage and not in the ATV-like virus lineage.

FIG. 8.

Ranavirus multiple-species jump hypotheses. Throughout the majority of evolutionary history, the iridoviruses have been restricted to fish species. Based on recent genomic sequence information, we hypothesize that the most recent common ancestor of the ranaviruses was a fish virus (MRCA A). In addition, we hypothesize that there have been evolutionarily recent host shifts. We propose two hypotheses to explain these multiple recent host shifts among the amphibian-like ranaviruses. (A) One hypothesis suggests that the most recent common ancestor of the ALRVs was a fish virus (MRCA B) and that a jump occurred from fish to frogs, with a subsequent jump from frogs to turtles. In addition, if the most recent common ancestor of the ATV-like viruses was a fish virus (MRCA C), then another jump from fish to salamanders occurred. (B) An alternative hypothesis suggests that the most recent common ancestor of the ranaviruses was a fish virus (MRCA A) and that a jump occurred from fish into tetrapod amphibians (MRCA B). At this time, it is unclear if the shift in host species was from fish to frogs, fish to salamanders, or both. A subsequent host shift occurred from frogs to turtles, as well as a jump from salamanders back into fish.