**2.4 Efficacy of DHP for inactivating influenza A H1N1**

H1N1 is a strain of influenza A (family *Orthomyxoviridae*) that was responsible for a 2009 pandemic declared by the World Health Organization (WHO). Like SARS-CoV-2, H1N1 is an enveloped virus, and it has been known to remain infectious on non-porous surfaces, such as glass and stainless steel, for 24–48 hours

[28, 29]. A DHP device was tested against titers of H1N1, with a starting TCID50 of 6.05 log10, in a laboratory biosafety hood to determine if DHP effectively inactivated the virus in comparison to the control condition after 120 minutes exposure (**Tables 1** and **2**).

Aliquots of diluted stock H1N1 were used to inoculate 1″ × 1″ squares on the center of 1″ × 3″ glass slides that had previously been sterilized and autoclaved. The slides were then placed into plastic Petri dishes. Ten slides, in total, were prepared in this way, with duplicates for each timepoint: Time Zero, T = 60 minutes Virus Control, T = 120 minutes Virus Control, T = 60 minutes Virus Test Carrier, T = 120 minutes Virus Test Carrier. Once inoculated with virus, the slides were allowed to dry for 25 minutes at 24°C and 36% relative humidity. The dried carriers were placed in their respective laboratory hoods, one of which was currently being treated with a DHP device that had been operating for 12 hours to precondition the space. The Time Zero samples were immediately collected and eluted with 2 mL of Influenza Infection Medium (EMEM supplemented with 0.125% w/v bovine serum albumin +1 μg/mL TPCK-trypsin + antibiotics). Serial dilutions were then performed to the 10−5 dilution and plated in quadruplicate onto MDCK (dog kidney) monolayers. At the designated timepoints, the T = 60 and the T = 120 samples were harvested and enumerated in an identical fashion to the Time Zero samples. The assay trays were then incubated at 35°C on an orbital rotator (60 rotations/minute) for 60 minutes. Once the virus-host cell adsorption had completed, the trays were removed from incubation, and 1.0 mL of the Influenza Infection Medium was pipetted into each well of the assay plate for each of the samples. The MDBK wells were then incubated for 7 days. All titers were determined using the Spearman-Kärber method [30].

After the incubation was complete, the wells were scored for viral cytopathic effect (CPE), and the Tissue Culture Infectivity Dose at the 50% Endpoint Dilution (TCID50) was calculated for each pair of samples (**Table 2**). In comparison to the control, the DHP-treated samples yielded a ≥ 2.62 log10 reduction in virus titer at 60 minutes and a ≥ 1.87 log10 reduction at 120 minutes. The log10 reduction in titer observed at 60 minutes corresponds to a percent reduction of ≥99.8%, compared to the control condition (**Table 2**) [31].


*a Abbreviations used: ATCC, American Type Culture Collection; CRFK, Crandel-Reese Feline Kidney; EMEM, Eagle's Minimum Essential Media; MDCK, Madin-Darby Canine Kidney; MEM, Minimum Essential Media. b Testing performed at Antimicrobial Test Laboratories, Round Rock, Texas, USA.*

*c Testing performed at ATS Labs, Eagan, MN, USA.*

*d Testing performed at Microchem Laboratory, Round Rock, TX, USA.*

#### **Table 1.**

*Summary of viruses and detector cells used in these efficacy studiesa .*


**Table 2.**

*Inactivation of influenza virus H1N1 over time by exposure to Dry Hydrogen Peroxide (DHP).*

## **2.5 Efficacy of DHP for inactivating feline calicivirus**

Feline calicivirus (FeCV) is a non-enveloped, single-stranded RNA virus (family *Caliciviridae*) that is often used as a surrogate in laboratory testing to simulate human norovirus, a major cause of gastrointestinal hospital-acquired infections (HAIs) [32, 33]. On non-porous surfaces, FeCV has been found to remain viable for 12–72 hours [34]. The efficacy of a prototype DHP device was tested against titers of FeCV, with a starting titer of 6.6 log10 TCID50/mL, over the course of 24 hours (**Tables 1** and **3**).

Aliquots of FeCV (ATCC VR-782) were inoculated onto glass slides with an accompanying organic soil load of ≤1% fetal bovine serum (FBS) to simulate contamination in a physiological matrix. The original titer of the input virus control was approximately 8.0 log10/mL, but after being allowed to dry on the carriers, the FeCV titer had decreased to an average of 6.6 log10/ml. For both the control and treatment groups, duplicate samples were collected at each timepoint (Time zero, T = 2 hours, T = 6 hours, T = 24 hours). After drying of the virus onto the slides was complete, the carriers were placed in their respective biosafety laboratory hoods, and the DHP device was activated in the hood containing the treatment group of samples. Temperature and humidity levels remained between 21 and 24°C and 36–39%, respectively, throughout the duration of the experiment. The test carriers were retrieved and scraped to resuspend the contents at the designated timepoints. Each sample's contents were transferred to a sterile tube and then serially diluted in the test medium (MEM supplemented with inactivated FBS, 100 units/mL penicillin, gentamicin, and 2.5 μg/mL amphotericin B). Once diluted, a cell-based infectivity assay involving Crandel Reese feline kidney (CRFK) cells was used to determine infectious titer.

The average titer (TCID50/mL) for each pair of samples was then calculated (**Table 3**). DHP-treatment resulted in FeCV inactivation (1.5 log10 after 2 hours,


#### **Table 3.**

*Inactivation of feline calicivirus over time by exposure to Dry Hydrogen Peroxide (DHP).*

and 2.8 log10 reduction after 6 hours of exposure time). The 2-hour and 6-hour log10 reductions in infectious titer correspond to 96.8% and 99.8% inactivation, respectively, in comparison to the control condition (**Table 3**) [35].
