Evaluation of Ranavirus Major Capsid Protein Gene Sequence Mutations in United States East Coast Frog and Tadpole Populations

Infectious disease is a leading cause of death in ectothermic taxa including amphibians, reptiles, and bony fish. Frog Virus 3 (FV3) Ranavirus is a prominent pathogen that infects frog and tadpole populations with lethal consequences, especially for pre-metamorphosed individuals. Wood frogs (Lithobates sylvaticus) are particularly susceptible to FV3 infection and mortality events. Conservation of this animal is a critical effort for the maintenance of these ecologically significant animals. Amphibians contribute to the regulation of insect pest levels, energy transfer between aquatic and terrestrial habitats, algae community productivity and function, and vertebrate biomass. Understanding genetic variations of FV3 is crucial to understanding the evolution of this virus and host susceptibility. Diagnostic testing is key for preventing the spread of the virus and reducing the mass mortality events observed in tadpole populations. Populations studied originate from 5 US states, Delaware, Maryland, New Jersey, Pennsylvania, and Virginia. This study explored the validity of current Real Time-PCR diagnostic testing methods and sought out genetic variations that may account for inconsistent diagnostic results brought forth by previous studies. Despite symptom observations in the field and positive fluorescence during diagnostic testing, 416 individuals have been deemed negative as they did not meet the melting temperature (Tm) criteria currently in place. This study presents findings that support the hypothesis that many, if not all, of these individuals are in fact positive for the virus. Due to the prior conflicting diagnostic results the Tm range currently in place may not account for new mutations within a ~530 base pair targeted region of the viral major capsid protein (MCP) gene sequence. Molecular techniques were used to evaluate the amplified DNA of the targeted region of the MCP gene. Traditional PCR and gel electrophoresis results provided evidence of infection in 41 individuals from nine populations. This was followed by sequence analysis of the PCR product for 22 of those individuals. Sequence results confirmed Ranavirus infections in 15 individuals from seven populations. A single base pair mutation was located in six individuals from a New Jersey population