**2.6 Efficacy of DHP for inactivating MS2 bacteriophage**

MS2 is a single-stranded non-enveloped RNA bacteriophage that often infects *Escherichia coli* (*E. coli*), and has been used as a surrogate for human norovirus and other non-enveloped viruses. MS2 bacteriophage has been shown to survive on non-porous surfaces for 4–10 days, which is aligned with the length of time norovirus can survive under similar conditions [36, 37]. The efficacy of a DHP device against airborne MS2 bacteriophage was investigated over the course of 4 hours (**Tables 1** and **4**).

This trial was conducted in an aerobiology chamber with a volume of ~30 m3 to simulate the conditions of the DHP device's intended use more accurately. The test inoculum containing a titer (~5.0 log10/mL) of MS2 bacteriophage strain 15597-B1 was split equally and added to two separate nebulizers within the test chamber. These nebulizers were then activated inside the chamber for 60 minutes before the Time Zero sample collection occurred, using an SKC bio-sampler (500 L) equipped with phosphate buffered saline. The sample was then serially diluted and plated in 50% Tryptic Soy Agar (TSA) containing *E. coli* to facilitate the accurate enumeration of the remaining infectious MS2 bacteriophage. Subsequent samples were then collected each hour for the following four hours, with no DHP present, to serve as the no-treatment control. Once the chamber had been adequately decontaminated, the solutions containing the MS2 bacteriophage were again added to the nebulizers. The DHP device was activated after the collection of the Time Zero sample, and subsequent sample collections were performed identically to the control samples. All plated samples were then incubated for 24 hours, and the plaque-forming units (PFU) of MS2 were enumerated. A reduction in log10 PFU relative to the untreated control condition is indicative of extent of inactivation.

The Time Zero samples yielded counts of 5.84 × 104 and 5.83 × 104 PFU for the control and DHP-treated groups, respectively. After an hour of exposure to DHP, the count of plaques formed by destroyed *E. coli* decreased by 3.54 log10 to 1.70 × 101 PFU, whereas the corresponding untreated control sample decreased by 0.83 log10 to 8.61 × 103 PFU. Compared to the untreated control, DHP achieved a 2.71 log10 reduction in infectious airborne MS2 bacteriophage titer after 1 hour of exposure, which corresponds to a 99.8% reduction (**Table 4**) [38].


#### **Table 4.**

*Plaque-forming units (PFU)/mL for E. coli infected with MS2 bacteriophage over time after exposure to Dry Hydrogen Peroxide (DHP).*
