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

Abstract

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 – 155 kbp in length and they are predicted to encode around 90–160 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.

The Ambystoma tigrinum virus (ATV) RNase III gene can modulate host PKR activation and interferon production

“…A mutant ATV deleted of the 25R gene (ATVΔ25R) was generated previously (Aron et al, 2016). However, since were able to grow, titer and maintain the small plaque phenotype of ATVΔ25R in the presence of G418, we asked if this mutant virus has reduced pathogenicity in ATV’s natural host, tiger salamanders ( Ambystoma tigrinum ).…”

“…Wild-type Ambystoma tigrinum virus (wtATV), was originally isolated from tiger salamanders in Southern Arizona (Jancovich et al, 1997). Generation of the ATV deletion mutants lacking the RNase III gene (ORF 25R), ATVΔ25R and the homologue of the eukaryotic translation initiation factor 2α subunit (vIF2αH) ATVΔ57R have been described previously (Aron et al, 2016; Jancovich and Jacobs, 2011). Wild-type and mutant ATV were amplified and quantified in FHM cells as described previously (Aron et al, 2016).…”

“…Generation of the ATV deletion mutants lacking the RNase III gene (ORF 25R), ATVΔ25R and the homologue of the eukaryotic translation initiation factor 2α subunit (vIF2αH) ATVΔ57R have been described previously (Aron et al, 2016; Jancovich and Jacobs, 2011). Wild-type and mutant ATV were amplified and quantified in FHM cells as described previously (Aron et al, 2016). Briefly, viral amplification was performed in 100 mm dishes of FHM cells that were infected with a multiplicity of infection of 0.01, rocked for 1 hr and then overlayed with HMEM and 5% FBS.…”

“…However, we are continuing to learn more about the molecular events that allow this interesting group of viruses to infect such a diversity of hosts. Ambystoma tigrinum virus (ATV) was originally isolated from diseased tiger salamanders in Southern Arizona in 1997 (Jancovich et al, 1997) and this ranavirus has been used to further understand ranavirus infection dynamics (Aron et al, 2016; Jancovich and Jacobs, 2011). Genomic sequencing of ATV revealed a number of genes that may enhance viral pathogenesis based on homology to other known proteins (Jancovich et al, 2003).…”

“…Therefore, we hypothesized that the ATV RNase III protein (25R) may also play a role in modulating IFN production and activation of the PKR pathway by its inherent dsRNA endonuclease activity. We have previously generated a mutant ATV deleted of the 25R (ATVΔ25R) gene using a GFP-G418 resistance gene screenable and selectable marker (Aron et al, 2016). ATVΔ25R has a small plaque phenotype in FHM cells but is unstable, reverting back to wtATV once the G418 selection reagent is removed during amplification of the virus.…”