Identification of essential and non-essential genes in Ambystoma tigrinum virus

•Develop a standardized protocol and procedure to generate recombination cassettes used to knock-out ranavirus genes.•Optimized the recombination protocol to generate a knock-out ranavirus.•Identified 2 essential, one semi-essential and 2 non-essential genes in Ambystoma tigrinum virus.

Members of the genus Ranavirus (family Iridoviridae) are large double-stranded (ds) DNA viruses that are found world-wide infecting fish, amphibian and reptile ectothermic hosts. Ranavirus genomes range from 105 to 155 kbp in length and they are predicted to encode around 90-125 genes. Currently, our knowledge of the function of ∼50% of these genes is known or inferred based on homology to orthologous genes characterized in other systems; however, the function of the remaining open reading frames (ORFS) is unknown. Therefore, in order to begin to uncover the function of unknown ORFs in ranaviruses we developed a standardized approach to generate a recombination cassette for any ORF in Ambystoma tigrinum virus (ATV). Our standardized approach quickly and efficiently assembles recombination cassettes and recombinant ATV. We have used this approach to identify two essential, one semi-essential and two non-essential genes in ATV.