SECOND INTERNATIONAL SYMPOSIUM ON RANAVIRUSES:: A NORTH AMERICAN HERPETOLOGICAL PERSPECTIVE

Ranaviruses are large double stranded DNA viruses of poikilothermic vertebrates including amphibians, reptiles and fish. In North America, ranaviral disease and ranavirus-related die-off events have been documented in all three classes. Ranaviruses are found worldwide, appear to be emerging in some regions, and are increasingly recognized as a threat to many species

THIRD INTERNATIONAL SYMPOSIUM ON RANAVIRUSES:: ADVANCING THE UNDERSTANDING OF THE THREAT OF RANAVIRUSES TO NORTH AMERICAN HERPETOFAUNA

Members of the genus Ranavirus, one of five genera withinthe family Iridoviridae, encompass a group of large, doublestrandedDNA viruses that infect all three classes of ectothermicvertebrates: fish, amphibians, and reptiles. Ranaviruses areglobally emerging pathogens that cause considerable morbidityand mortality among diverse populations. In North America,ranavirus epizootics are regularly reported in wild and culturedfish, amphibian, and reptile populations

ANALYSIS OF THE DEOXYNUCLEOSIDE KINASE GENE FROM RANAVIRUS GENOMES SUGGESTS A NEW MODEL FOR PHYLOGENETIC STUDY

Ranavirus is a viral genus in the Iridoviridae containing double stranded DNA viruses responsible for rising mortality rates and population declines of amphibian and reptile populations in diverse regions throughout the world. The disease caused by Ranavirus infection can be quite severe, manifesting as edema, soft tissue hemorrhage, organ failure, and in many cases ultimately the death of the host. Due to geographical dispersement and the diversity of host organisms, determining what strain of Ranavirus is present within a host is important for understanding the threat it may pose. Often, the major capsid protein is used to determine what strain or species a ranavirus isolate belongs to, however, this is a relatively conserved gene across Ranavirus species and may not be as informative as we initially had hoped. We built Maximum Likelihood phylogenetic trees in MEGA 7 for full Ranavirus genomes, the major capsid protein gene and the previously unexplored deoxynucleoside kinase gene, from sequences obtained from ranavirus isolates found on GenBank. The trees produced had high bootstrap values (n = 1000) and showed similar branching patterns between isolates. These finding support the utility of the deoxynucleoside kinase gene as an alternative or additional locus for phylogenetic analysis of Ranavirus isolates

SELECT CORE GENES OF RANAVIRUS SUGGEST NEW MODELS FOR FUTURE PHYLOGENETIC STUDIES**

Ranavirus is a genus of double-stranded DNA viruses in the family Iridiovirdae. Ranaviruses can affect a multitude of ectothermic vertebrates including fish, amphibians and reptiles. Infection can cause cutaneous and systemic hemorrhaging, organ failure, and result in mortality events for certain economic and ecologically significant species. Because of its detrimental effect of host species, it is imperative to examine ranaviral evolution in order to develop containment strategies and regulations. Our study aims to find an applicable gene for phylogenetic studies by using candidates from the 26 Ranavirus core genes. From these, ribonuclease reductase small subunit, ribonuclease III, the proliferating nuclear cell antigen, and deoxynucleoside kinase were chosen for analysis due to their high conservation across Ranavirus species and strains. Preliminary studies using other genes and full genomes suggest appropriate phylogenetic models that parallel the viral evolution seen within the full genomes of 20 Ranavirus isolates

ALTERNATIVE GENES FOR EXPLORING RANAVIRUS PHYLOGENETICS: FOUR CORE GENES COMPARED TO THE MAJOR CAPSID PROTEIN.**

The Iridoviridae is a family of virus that infects invertebrates and poikilothermic vertebrates such as amphibians, reptiles, and fish. Ranavirus is one of the five genera within the Iridoviridae. Ranavirus infections have been found on every continent except Antarctica. The ranaviruses have 26 core genes, but our study focuses on the utility of: an immediate early protein ICP-46 [open reading frame (ORF) 91R in Frog virus 3 (FV3)]; a transcription elongation factor TIIS (ORF 81R in FV3); Evrl/Air family protein (ORF 88R in FV3); and a hypothetical/putative protein of unknown function (ORF 94R in FV3) as alternative genes for phylogenetic reconstruction in this group. Gene sequences were obtained from GenBank, aligned using MAFFT, and trees were constructed in MEGA using the Neighbor-Joining method (with 1000 Bootstraps). The phylogenetic trees made from the 4 core genes will be compared to trees built in the same manner using the major capsid protein sequence (MCP, ORF 90R in FV3). The MCP gene is commonly used to reconstruct phylogenetic relationships for ranaviruses because of its conserved nature. This is part of a larger project which seeks to understand which of the 26 core genes are the most phylogenetically informative

USING IRIDOVIRUS CORE GENES TO TEST KNOWN PHYLOGENETIC RELATIONSHIPS BETWEEN AMBYSTOMA TIGRINUM VIRUS STRAINS FROM THE WESTERN USA**

Ranaviruses are emerging infections in ectothermic vertebrates. They are known to affect over 145 species of amphibians globally and are members of the Iridoviridae. Iridoviruses have 26 core genes that are conserved between genera of the viruses. In this study we examined five of the 26 core iridovirus genes in 15 strains of Ambystoma tigrunum virus (ATV) from the western USA, which are known to have local/geographical variation in several other genes which are not in the core genes of iridoviruses. Here we examined the Hypothetical Protein-Clostridium tenani (Open Reading Frame, ORF 11L), the Immediate Early Protein ICP-4 (ORF 13L), Myristilated Membrane Protein A (ORF 51L), Myristilated Membrane Protein B (ORF 1L) and Transcription Elongation Factor TIIS (ORF 24L) as potential alternatives to non-core genes for phylogenetic reconstruction of closely related strains. Sequence data obtained from GenBank and analyzed using a MAFTT server and MEGA software, will be compared visually for homology and utility as alternate genes for discerning local adaptation in these ATV strains. Based on initial data from other core genes, we expect to find that these are too highly homologous to be informative over small geographical scales within the same genus of ranaviruses

IRIDOVIRUS CORE GENES: SUITABLE TARGETS FOR EXAMINING LOCAL ADAPTATION?**

Ranaviruses belong to the viral family Iridoviridae and are globally distributed emerging pathogens in amphibians, fish, and reptiles. Outbreaks of ranaviral disease have led to population declines and even local extirpations in some species. Iridoviruses have 26 core genes that are found within all 6 species. Here we explore the utility of five different core genes from 15 unique strains of Ambystoma tigrinum virus (ATV) that have, in a previous study performed by others, been shown to have local variation. ATV was originally isolated from Sonoran tiger salamanders, and has since been isolated Abystomatid species in the southwestern USA. To perform our study, we pulled the five gene sequences for each of the 15 strains of ATV from GenBank. Each gene was aligned using a MAFTT server, and the aligned sequences were then imported into MEGA where a nucleotide best fit model was run for each set of data. The best fit model was used to build maximum likelihood phylogenetic trees for each of the 5 genes. These trees were then compared with a known tree from a previous study, the full genome trees, and a tree build using the major capsid protein (the gene most often used to recreate phylogenetic relationships in ranaviruses). Based on preliminary data from other core genes, we do not expect to find much sequence variation, and hence we should see poorly resolved trees. Therefore, this subset of the core Iridovirus genes may not be informative for phylogenetic work at small geographical scales

LOOKING FOR LOCAL ADAPTATIONS: ARE A SUBSET OF THE IRIDOVIRUS CORE GENES SUITABLE FOR RECONSTRUCTING PHYLOGENETIC RELATIONSHIPS IN AMBYSTOMA TIGRINUM VIRUS ISOLATES FROM THE SOUTHWESTERN USA?**

Ambystoma tigrinum virus (ATV) is a species of Ranavirus, which is a member of the viral family Iridoviridae. Ranaviruses are globally emerging pathogens of amphibians, fish and reptiles. However, ATV appears to be predominantly located in the Southwestern USA where it affects amphibians. It has previously been established that ATV has local variation within the Southwestern USA using a subset of its genes, however not all of the genes used were core genes. Iridoviruses have 26 core genes that are considered to be conserved, and these genes have not all been explored as possible markers for local variation. Here we will examine the phylogeny created by five different Iridovirus core genes (ORF 10L, 25R, 38R, 58R, and 80R) and compare this to the known phylogeny that includes other loci that are not members of the core for 15 strains of ATV. This will let us determine the utility of these core genes as markers for local variation. We plan to do this by obtaining the sequences of each ORF from GenBank. The sequences will then be aligned using a MAFTT server. The aligned sequences will be imported into MEGA 6, and a nucleotide substitution model best-fit will be run. The best-fit substitution model will then be used with the aligned sequences to build maximum likelihood trees for each locus. Based on preliminary analyses, we expect that there will be little to no variation between the different strains of ATV and that we will not be able to discern local variation using these conserved genes

SYNTENY AND PHYLOGENETIC SIGNAL ANALYSIS OF 19 DIFFERENT STRAINS ENCOMPASSING SIX SPECIES OF RANAVIRUS**

Ranaviruses are globally emerging pathogens of amphibians, reptiles and fish. There are currently 6 different species of ranavirus that have been identified. Here, we examine the synteny profiles of 19 different strains of ranavirus, encompassing representatives from each species, using R (all gene sequences were obtained from GenBank). Using Decipher’s FindSynteny function, we noted the homologous regions of the 19 strains, then the AlignSynteny function was used to align these pairwise comparisons. This also ordered the hits into blocks based on their ordinal position sequence. From this a synteny map was created using R. From the synteny map, we note that Singapore Grouper Iridovirus (SGIV) is the most divergent Ranavirus and may be worthy of species status. In other studies, SGIV is also known to be divergent from most other ranaviruses. Within each species of ranavirus, the genomes are collinear. Additionally, using a known tree based on the full genomes of the 19 different strains of ranavirus, we plan to determine the phylogenetic signal of each of the 26 iridovirus core genes. To do this we will use various R packages that have been developed for morphological traits and adapt them to use for molecular data. We expect that these core genes will have relatively low phylogenetic signal because they are conserved genes

IS THE MYRISTILATED MEMBRANE PROTEIN OF RANAVIRUSES AN ACCEPTABLE ALTERNATIVE FOR PHYLOGENETIC ANALYSIS?**

Ranaviruses are globally distributed emerging infections of ectothermic and poikilothermic vertebrates. They are responsible for countless morbidity and mortality events around the globe, and they are known to affect several endangered species. Therefore, understanding the evolutionary relationships between different strains of Ranavirus becomes important if we are to predict the consequences of their emergence. Past best practice for classifying different Ranavirus isolates has mainly been based on the major capsid protein (MCP), which is a highly conserved gene. However, this approach does not appear to capture the full sequence diversity of isolates. Twenty-six core genes have been identified for ranaviruses, some of which may better demonstrate the true phylogentic relationships between different isolates. Here, we use the myristilated membrane protein (open reading frame 2L in Frog virus 3; MMP) as an alternative to the MCP gene for constructing Ranavirus evolutionary trees. We will compare trees obtained using full length genomes, only the MCP gene sequences, and only the MMP gene sequences. Trees will be built using the Neighbor-Joining method (1000 Bootstraps) and an appropriate nucleotide substitution model in MEGA 6. Trees will then be compared visually for similarities and differences