Cryopreservation of Paramecium bursaria Chlorella Virus-1 during an active infection cycle of its host

Abstract

20Best practices in laboratory culture management often include cryopreservation of microbiota, but 21 this can be challenging with some virus particles. By preserving viral isolates researchers can mitigate 22 genetic drift and laboratory-induced selection, thereby maintaining genetically consistent strains between 23 experiments. To this end, we developed a method to cryopreserve the model, green-alga infecting virus, 24 Paramecium bursaria Chlorella virus 1 (PBCV-1). We explored cryotolerance of the infectivity of this virus 25 particle, whereby freezing without cryoprotectants was found to maintain the highest infectivity (~2.5%). 26We then assessed the cryopreservation potential of PBCV-1 during an active infection cycle in its Chlorella 27 variabilis NC64A host, and found that virus survivorship was highest (69.5 ± 16.5 %) when the infected host 28 is cryopreserved during mid-late stages of infection (i.e., coinciding with virion assembly). The most 29 optimal condition for cryopreservation was observed at 240 minutes post-infection. Overall, utilizing the 30 cell as a vehicle for viral cryopreservation resulted in 24.9 -30.1 fold increases in PBCV-1 survival based 31 on 95% confidence intervals of frozen virus particles and virus cryopreserved at 240 minutes post-infection.32 Given that cryoprotectants are often naturally produced by psychrophilic organisms, we suspect that 33 cryopreservation of infected hosts may be a reliable mechanism for virus persistence in non-growth 34 permitting circumstances in the environment, such as ancient permafrosts. 35 Introduction 36Viruses are abundant components of all biological systems and they likely infect every lineage of 37 eukaryotic algae. Their impact is most readily noticed following infection and lysis of abundant bloom 38 forming algae (1-3), though lytic activity of all algal viruses contributes to significant biomass recycling via 39 the 'viral shunt' (4). To date, 65 eukaryotic algal viruses have been isolated and developed as laboratory 40 strains (5, 6). Most of these are maintained through serial propagation on their respective hosts. Though 41 this has been effective for culturing many strains over the last few decades (7, 8), each passage allows for 42 genetic mutations that can accumulate in a population (9), leading to a deviation from a standard 'wild-43 type.' Moreover, it is imperative to control evolution following the development of genetically tractable 44 algal hosts (10) and (ultimately) virus systems. A protocol for successful virus cryobiological preservation 45 would offer an opportunity to maintain genotypically consistent virus stocks. 46Cryopreservation is not a new concept in biological sciences. For most protocols, it involves 47 controlled cooling of biota to sub-freezing temperatures to achieve biological cessation while preserving 48 viability. This most often manifests as slow-cooling at a rate of 1° C / min in the presence of 49 osmoprotectant(s) (e.g., dimethylsulfoxide (DMSO), glycerol) for long-term storage at -130° C or bel...