Abstract
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Psoralation: A Novel Method of Viral Inactivation for Containment Research
Careful selection, validation, and verification of inactivation processes are critical to work with high-consequence pathogens. The ability to work with inactivated materials at a lower containment level protects the researcher, reduces logistical and ergonomics issues associated with high/maximum-containment work, and expands the ability of researchers to use diagnostic and investigative technologies not readily available at biosafety level 3 (BSL-3) or BSL-4. This article presents a novel method of viral inactivation that permits sterility testing and is an example of productive collaboration among researchers and biosafety professionals. (Full disclosure: In this installment, I am presenting a paper for which I am a contributing author.)
This project evolved from a risk assessment discussion between a biosafety officer and a principal investigator (PI). The research discussed was an extension of a study indicating that a group of compounds called psoralens could be used in conjunction with ultraviolet (UV) light to inactivate Dengue virus. Inactivation in such a manner still allowed the virus to retain immunogenic properties and to elicit T-cell responses in vaccinated mice similar to the wild-type agent. 1 The risk assessment included discussion regarding the containment level of the work, and the PI stressed that the material subjected to psoralen inactivation would be suitable for removal from BSL-3 or BSL-4 to lower containment. As the authors discussed the nature of psoralens with respect to inactivation and sterility testing of risk group 3 (RG3) and RG4 viral agents, they realized that psoralation was inherently different from traditional viral inactivation methods (eg, TRIzol LS) in that it was not damaging to the nucleic acid itself and was not cytotoxic. While initial research was finalized, an additional project was initiated to investigate the biosafety implications of psoralen inactivation across virus families.
Psoralens are a group of small photoreactive compounds able to penetrate phospholipid bilayers and intercalate between pyrimidine residues. Upon exposure to UV-A radiation, they cause cross-linking and inhibition of viral replication, and their nontoxic nature and preservation of antigenic epitopes have led to their use in a number of medical applications. This particular study employed the use of psoralen inactivation across multiple RNA virus families and determined the antigenicity of the treated samples, as well as the suitability of the samples for molecular-based applications, such as polymerase chain reaction and genomic sequencing. 2
The investigators subjected selected RNA viruses to 10 to 20 μg/mL of a psoralen (4′-aminomethyl-trioxsalen) and multiple UV-A radiation energy levels for various time points to characterize virus-specific inactivation parameters. The viruses investigated included members of the following families: Alphaviridae (Venezuelan equine encephalitis virus), Arenaviridae (Lassa virus, Junin virus), Bunyaviridae (Rift Valley fever virus, Crimean-Congo hemorrhagic fever virus), Coronaviridae (Middle East respiratory syndrome virus), Filoviridae (Ebola virus, Marburg virus), Flaviviridae (Dengue virus, West Nile virus, Yellow Fever virus, St. Louis encephalitis virus), and Orthomyxoviridae (H1N1p, H1N1, H3N2, influenza B). While the concentration of psoralen, UV-A energy level, and exposure time required for inactivation were found to vary across the families (dose dependent), all viral stocks tested were successfully inactivated and sterility tested. Furthermore, the inactivated viruses performed well with respect to assays requiring intact epitopes and viral RNA.
While some progress has been made in recent years, the field of biosafety well understands that applied biosafety research is still needed. This paper presents a novel inactivation method for RG3/RG4 viruses and is an example of how biosafety professionals can work with PIs toward improving research quality and safety. I hope that this paper encourages biosafety professionals in approaching researchers to promote peer-reviewed publication of biosafety-related data.