Date Published: March 14, 2019
Publisher: Public Library of Science
Author(s): Samantha R. Coy, Alyssa N. Alsante, James L. Van Etten, Steven W. Wilhelm, Susanna A Wood.
Best practices in laboratory culture management often include cryopreservation of microbiota, but this can be challenging with some virus particles. By preserving viral isolates researchers can mitigate genetic drift and laboratory-induced selection, thereby maintaining genetically consistent strains between experiments. To this end, we developed a method to cryopreserve the model, green-alga infecting virus, Paramecium bursaria Chlorella virus 1 (PBCV-1). We explored cryotolerance of the infectivity of this virus particle, whereby freezing without cryoprotectants was found to maintain the highest infectivity (~2.5%). We then assessed the cryopreservation potential of PBCV-1 during an active infection cycle in its Chlorella variabilis NC64A host, and found that virus survivorship was highest (69.5 ± 16.5%) when the infected host is cryopreserved during mid-late stages of infection (i.e., coinciding with virion assembly). The most optimal condition for cryopreservation was observed at 240 minutes post-infection. Overall, utilizing the cell as a vehicle for viral cryopreservation resulted in 24.9–30.1 fold increases in PBCV-1 survival based on 95% confidence intervals of frozen virus particles and virus cryopreserved at 240 minutes post-infection. Given that cryoprotectants are often naturally produced by psychrophilic organisms, we suspect that cryopreservation of infected hosts may be a reliable mechanism for virus persistence in non-growth permitting circumstances in the environment, such as ancient permafrosts.
Viruses are abundant components of all biological systems and they likely infect every lineage of eukaryotic algae. Their impact is most readily noticed following infection and lysis of abundant bloom forming algae [1–3], though lytic activity of all algal viruses contributes to significant biomass recycling via the ‘viral shunt’ . To date, 65 eukaryotic algal viruses have been isolated and developed as laboratory strains [5, 6]. Most of these are maintained through serial propagation on their respective hosts. Though this has been effective for culturing many strains over the last few decades [7, 8], each passage allows for genetic mutations that can accumulate in a population , leading to a deviation from a standard ‘wild-type.’ Moreover, it is imperative to control evolution following the development of genetically tractable algal hosts  and (ultimately) virus systems. Although seed-stock systems can be developed without cryopreservation, many systems are not amenable to this either because the virus particles are degraded during purification efforts or lose their infectivity during storage. Moreover, it can take time to achieve axenic status with new virus isolates, thus making contaminating bacterial activity a significant source of degradation. Thus, a protocol for successful virus cryobiological preservation that is applicable to a wide variety of algae-virus systems would offer an opportunity to universally improve virus management and distribution in the laboratory.
Following the cryopreservation procedures of other algal virus researchers [13–16], we investigated the cryo-potential of the PBCV-1 particle. Cryoprotectant alone treatments elicited a lethal effect: ~87% of the infectious virus particles were inactivated in the presence of these chemicals following 24 hr exposure at 4°C. Given this effect, we decided to freeze PBCV-1 particles at -150°C without any cryoprotectants. This resulted in ~2.5% recovery of the infectious virus population, which was stable for storage periods of up to one year (Fig 1). Seeing room for improvement, we tested the cryo-potential of PBCV-1 in an infected, cell-associated state.
The current maintenance strategy for chloroviruses involves serial propagation on the alga host followed by lysate particle storage at 4°C. Chloroviruses are relatively stable under these conditions, though even PBCV-1 is known to degrade after several years of storage. In any case, many algae-virus systems are less amenable to long-term storage at 4°C. For example, new algae-virus systems are not always quickly made axenic, and are thus susceptible to degradation from contaminating bacteria. On the other hand, viruses propagated on axenic hosts can still degrade. For reasons unknown, chloroviruses are more stable in lysates (bacterial-free) than in particle stocks purified by sucrose density gradients , but they always eventually lose their infectivity. Serial propagation of viruses is therefore often required. Even if this is done infrequently, it can still promote genetic drift and result in deviation from wild-type status. This is concerning for all virus types, though RNA viruses, which have the fastest mutation rates, would be most susceptible [9, 37]. Beyond considering spontaneous, replication-associated errors, chloroviruses encode putative enzymes involved in genomic rearrangements. For example, GIY-YIG mobile endonucleases and an IS607 transposon may be involved in insertions/deletions and/or gene loss/duplications observed in genomic comparisons of chloroviruses [38, 39]. Thus, maintenance of wild-type strains is important for consistency between experiments. Virology labs could follow the microbial culture collection strategy, which typically uses a cryo-banking/seed-stock system for the dissemination of microbial specimens. The purpose of the seed-stock system is to minimize serial propagation of microbiota. The American Type Culture Collection (ATCC) suggests that consumers transfer their cultures no more than five-times after propagation from the thawed culture collection stock. Though a seemingly strict standard, it is not difficult to imagine the consequences of violating this. For example, the United States Pharmacopeia and National Formulary requires test organisms to be maintained this way for routine antibiotic efficacy screens, and non-compliance can undermine therapeutic treatment . Although there is no direct clinical link to maintaining algal viruses this way, the logic is consistent with any research requirements. The cryopreservation protocol described here can help researchers better set up these cryo-banking/seed stock systems.