Viruses are obligate intracellular parasites that alter host metabolic machinery to obtain energy and macromolecules that are pivotal for replication. Ranavirus, including the type species of the genus frog virus 3 (FV3), represent an ecologically important group of viruses that infect fish, amphibians, and reptiles. It was established that fatty acid synthesis, glucose, and glutamine metabolism exert roles during iridovirus infections; however, no information exists regarding the role of purine metabolism. In this study, we assessed the impact of exogenously applied purines adenine, adenosine, adenosine 5′-monophosphate (AMP), inosine 5′-monophosphate (IMP), inosine, S-adenosyl-L-homocysteine (SAH), and S-adenosyl-L-methionine (SAM) on FV3 replication. We found that all compounds except for SAH increased FV3 replication in a dose-dependent manner. Of the purines investigated, adenine and adenosine produced the most robust response, increasing FV3 replication by 58% and 51%, respectively. While all compounds except SAH increased FV3 replication, only adenine increased plaque area. This suggests that the stimulatory effect of adenine on FV3 replication is mediated by a mechanism that is at least in part independent from the other compounds investigated. Our results are the first to report a response to exogenously applied purines and may provide insight into the importance of purine metabolism during iridoviral infection.

Cytokinins (CKs) are a group of N⁶-substituted signaling molecules whose biosynthesis and metabolism have been documented in all kingdoms of life, including vertebrates. While their biological relevance in vertebrate systems continues to be elucidated, they have broadly been documented with therapeutic effects in exogenous applications. In this study, we evaluated the virostatic potential of four types of CKs including, N⁶-isopentenyladenine (iP), N⁶-isopentenyladenosine (iPR), N⁶-isopentenyladenosine-5′monophosphate (iPMP), and 2-methylthiol-N⁶-isopentenyladenosine (2MeSiPR) against the ranavirus type species, frog virus 3 (FV3). Following concurrent treatment and infection, iP and iPR reduced viral replication by 33.8% and 59.6%, respectively, in plaque formation assays. A decrease in viral replication was also observed when CK exposure was limited to 12 h prior to infection, where iP and iPR reduced viral replication by 31% and 23.75%, respectively. Treatment with iP and iPR was also marked by 48% and 60% decreases in viral load over 72 h, respectively, as measured in single step growth curves. Plaque morphology was altered in vitro, as iP and iPR treatment increased plaque area by 83% and 112% with lytic zone formation also becoming more prevalent in corresponding treatments. Treatment with iPMP and 2MeSiPR resulted in no effect on viral kinetics in vitro. The results of this study are the first to provide evidence of CK antiviral activity against a DNA virus and highlight the importance of their structure for therapeutic investigations.

Members of the Iridoviridae family, genus Ranavirus, represent a group of globally emerging pathogens of ecological and economic importance. In 2017, an amphibian die-off of wood frogs (Rana sylvatica) and boreal chorus frogs (Pseudacris maculata) was reported in Wood Buffalo National Park, Canada. Isolation and complete genomic sequencing of the tissues of a wood frog revealed the presence of a frog virus 3 (FV3)-like isolate, Rana sylvatica ranavirus (RSR), with a genome size of 105,895 base pairs, 97 predicted open reading frames (ORFs) bearing sequence similarity to FV3 (99.98%) and a FV3-like isolate from a spotted salamander in Maine (SSME; 99.64%). Despite high sequence similarity, RSR had a unique genomic composition containing ORFs specific to either FV3 or SSME. In addition, RSR had a unique 13 amino acid insertion in ORF 49/50L. No differences were found in the in vitro growth kinetics of FV3, SSME, and RSR; however, genomic differences between these isolates were in non-core genes, implicated in nucleic acid metabolism and immune evasion. This study highlights the importance of viral isolation and complete genomic analysis as these not only provide information on ranavirus spatial distribution but may elucidate genomic factors contributing to host tropism and pathogenicity.