Title: Bacteriophage Φ 6 (Phi6) as a Surrogate for Enveloped Viral pathogens in Standard and Long-lasting Virucidal Ecacy Tests

Development of novel antimicrobials capable of providing long-lasting disinfection on surfaces requires the implementation of new standardized methods to support claims recognized by regulatory authorities. Surrogates for viral pathogens are often desired in order for BSL-2 labs to conduct these new efficacy tests safely and efficiently, however, the knowledge of surrogate suitability for these tests is limited. Here, for the first time, we determine the resistance profile of the bacteriophage Phi6 to quaternary ammonium compounds (QACs) in a variety of test conditions. Additionally, we show that Phi6 can be used to demonstrate the long-lasting virucidal efficacy of a novel antimicrobial, Actizone™ F5 , and that the Phi6 is more resistant to QACs following the UK standard long-lasting disinfection test BSI PAS2424:2014 than Vaccinia virus, which is the marker strain for claims of activity against enveloped viruses in Europe. Surface stability as well as benefits and limitations of Phi6 use relative to other enveloped viruses for antimicrobial testing is also discussed.


Introduction
The ongoing pandemic caused by SARS-CoV-2 1,2 has led to increased interest in enhanced disinfection and longer lasting protection of environmental surfaces against viruses. Enveloped viruses including SARS-CoV-2 are generally considered less stable on inanimate surfaces and more susceptible to disinfectants compared to non-enveloped viruses 3,4,5 . However, recent evidence suggests a surprising ability of enveloped viruses including coronaviruses to persist in the environment 6,7 and remain infectious on various surfaces for up to several days 8,9,10,11 .
Traditional disinfectants approved for use against viruses are not required to demonstrate antimicrobial efficacy beyond the time of application or ability to maintain activity through multiple dry or wet contacts over time. Consequently, field studies have demonstrated that various environmental surfaces treated with traditional products were quickly re-contaminated within a few hours post cleaning and disinfection 12,13,14,15 . These factors combined with everyday challenges of disinfectant application 16,17 leave surfaces and high-touch areas vulnerable to recontamination and onward transmission of surviving pathogens.
To address this technological gap in protection of environmental surfaces, a group of long-lasting disinfectant products have recently emerged for broad consumer and healthcare applications 18,19,20 . The residual efficacy of CAD products are evaluated by the Residual Self-Disinfection (RSD) method in the US and BSI PAS 2424:2014 (2014) in the UK. In contrast to traditional challenge tests, these methods take into account surface abrasion (i.e. repeated touching) and recontamination events that could occur under real-world conditions while staying as close as possible to the practical conditions that are outlined in the standard efficacy tests such as culture preparation, strength and recovery as well as requirements for soil and test surface 4 characteristics. However, both these methods do not provide the option to test viruses, therefore limiting the residual claims possible in these regions mainly to bacterial and yeast species.
On October 14, 2020 the EPA introduced a much anticipated policy innovation which enabled the evaluation and registration of liquid disinfectants with a 24 hr virucidal claim 21 . While there is no universally accepted method to support a residual virucidal claim, the agency has recommended the combined principles of RSD and ASTM E1053 (standard virucidal test) methods as an interim guidance for the study design. The resulting test which we term, herein, as Residual Virucidal Test (RVT) will include the surface abrasion and re-inoculations features of the RSD method while maintaining the standards of virus cultivation, recovery and performance measurements as specified in ASTM E1053.
The objective of this study was twofold. First, to validate an appropriate, practical, and safe surrogate for viral pathogens, and second to use that surrogate to evaluate the residual virucidal efficacy of a novel disinfectant Actizone™ F5 by utilizing the RVT and BSI PAS2424 test methods. Bacteriophage Phi6, being a well-known surrogate for enveloped viruses 22,23 was evaluated as the standard test virus. While the environmental stability and resistance of Phi6 to chemical treatments in relation to some viruses of interest has been studied 3,4,5,24,25,26 comparative resistance of the bacteriophage to QACs has not been reported so far. To this effect, the efficacy of QACs against Phi6 relative to those reported for SARS-like viruses and other pathogenic enveloped viruses is also evaluated. Finally, surface stability as well as benefits and limitations of Phi6 use as a surrogate for antimicrobial testing is also discussed.

Results
Combining our own data regarding Phi6 with data found in the literature, we compared the resistance of Phi6 to the resistance of other enveloped viruses in the presence of a widely-used commercial QACbased disinfectant, Microchem-Plus (MCP™) (Figure 1-2). Our benchmarking of Phi6 demonstrates that the bacteriophage was more resistant to QAC than the bat SARS-like coronavirus, WIV1, when tested in a suspension-based method similar to the EU virucidal test, EN14476 ( Figure 1). While the manufacturer recommended 5% concentration was able to completely deactivate both WIV1 and Phi6 as expected, a 5%/81 dilution of MCP™ was able to reduce WIV1 by between 6.35 and 7. 17 logs, while the same concentration of MCP™ was only able to reduce Phi6 by less than 2.62 logs. A further dilution of MCP™, 5%/243, was ineffective against both viruses, reducing WIV1 by 1.27 logs and Phi6 by 0.04 logs.
We also examined the Phi6 resistance to QAC when tested in a surface-based method similar to ASTM 1053 (Fig. 2). In this case, we only had access to data for a single concentration of MCP™. The manufacturer recommended concentration of 2 oz/gal MCP™ was able to completely deactivate Avian Influenza A(>4.5 LR), Avian Influenza A (H3N2) (>4.5 LR), SARS-Associated Coronavirus (>4.0 LR), and Human Coronavirus (229E) (>3.0 LR). The viral control recovery was higher for Phi6 (7.8 log10) than the other enveloped viruses (4.5-6.5 log10), so while the log10 reduction was high for Phi6 (>5.4 LR), there was not complete deactivation as was seen with the other enveloped viruses.
In addition to examining the resistance of Phi6 to QAC both in suspension and on surfaces, we also evaluated the innate stability of the bacteriophage on surfaces (Fig. 3). Following the protocol described by Chin and colleagues 31 for determining the persistence of SARS-CoV-2 on surfaces, our data shows that in the short term (30 minutes and less), Phi6 is more stable on 6 stainless steel surfaces than SARS-CoV-2. SARS-CoV-2 loses 0.6 logs during the first 30 minutes on a stainless steel surface while Phi6 remains roughly constant (Fig. 3 inset). However, the persistence of Phi6 deteriorates after the first 30 minutes, and it becomes less stable than SARS-CoV-2 after 3 hours.
After determining the suitability of bacteriophage Phi6 as a surrogate for other enveloped viruses, we used Phi6 to test the residual virucidal efficacy of Solvay's long-lasting disinfectant, Actizone™ F5 following the interim guidance provided by the EPA (RSD), and the method described by the BSI (PAS2424) ( Table 1). Solvay's Actizone™ F5 long lasting disinfectant passed the UK standard BSI PAS2424 test by reducing the viral titre of Phi6 on surfaces by at least 3 logs after the surfaces underwent a series of abrasions and inoculations designed to simulate use over a 24-hour period. The viral titre was reduced by an average of 3.06 log10 after a five minute contact time, and by over 5.4 log10 (complete deactivation) after a ten minute contact time. Actizone™ F5 also passed BSI PAS 2424 when tested against vaccinia virus, showing greater than 3.5 log10 reduction and complete deactivation of the virus after a five minute contact time. In addition to testing Actizone™ F5, which is specially designed to provide long lasting disinfection, we also tested a version of Actizone™ F5 without Actizone™P5 (Actizone™P5 is a proprietary polymer which stabilizes the Actizone™ F5 active ingredients on surfaces). The formulation without the polymer did not pass the BSI PAS2424 standard and showed less than 1.43 log10 reduction in viral load. Finally, we also tested Actizone™ F5 against the RVT standard described by the EPA in the recently published interim guidance. Actizone™ F5 passed the RVT protocol, showing greater than 4 log10 reduction in viral titre after a 10 minute contact time. 7

Discussions
In this study, a novel disinfectant, Actizone™ F5 was evaluated for its long lasting, abrasion resistant virucidal activity against bacteriophage Phi6. The evaluations were conducted according to the principles of RSD (EPA US) and BSI PAS2424 (UK) methods which were modified for viruses. Unlike existing virucidal standards, both of these methods subject the disinfectant-treated surface to multiple cycles of alternating wet and dry wiping (abrasions) each interspersed with a challenge consisting of at least 3-4 log10 virus particles. In order to meet the regulatory requirements, the test surface would need to demonstrate a minimum of 3 log10 or 99.9% reduction of the final viral challenge within 10 minutes of exposure. Given the experimental rigor and high performance standards, the products that pass these tests are expected to deliver more durable and longer lasting disinfection of surfaces despite multiple touches or contamination events.
In order to operate with safety as our top priority, and considering our understanding of the suitability of Phi6 as a surrogate for select enveloped viruses, we used Phi6 as our standard test organism in both protocols. In general, stability of a given virus on an inanimate surface is an important prerequisite for its use in carrier based tests. The recovery of viruses need to be sufficient enough to allow investigators to measure the targeted log10 reduction while accounting for the detection limit and errors associated with the assay. Secondly, the surrogate virus ideally should demonstrate similar if not higher levels of tolerance towards the biocidal active in question. However, to our knowledge, the surface stability and efficacy of QACs against Phi6 in relation to other enveloped viruses remain limited. To this effect, we first evaluated the surface stability under relevant environmental and humidity conditions mirroring the conditions tested in SARS-CoV-2 studies 27 . Our data graphed in parallel with this study demonstrate the Phi6 is 8 stable within the first 2 hours, yet viability reduced over a longer period compared to SARS-CoV-2. This finding suggests that Phi6 recovery would be sufficient for most conventional virucidal tests including residual tests which do not require more than 1 hour of drying. On the other hand, challenge tests requiring, for example, more than 2 hr contact time or longer drying times may not be suitable for experimentation with Phi6 as a surrogate for enveloped viruses.
Comparative resistance of Phi6 to quaternary ammonium active was further evaluated according to the standards of ASTM1053 (carrier based) and EN14476 equivalent (suspension) tests.
Micro-Chem Plus™ is a well-known widely used disinfectant with wide spectrum efficacy data available publicly 28  formulation without the proprietary polymer system (Actizone™P5) failed the BSI PAS2424 test with less than 3 log10 reduction. It is proposed that Actizone™P5 polymer with QACs forms a phase stable fluid formulation which upon application forms an abrasion resistant film on surfaces 29 . Upon drying the film acts as an abrasion-resistant barrier that retains actives on the surface that kill bacteria and viruses on-demand. As such, Actizone™P5 technology serves as a safe and inert additive (data on file with Solvay) that will help formulators to achieve a long 9 lasting surface disinfection with standard levels of QACs while minimizing the number of reapplications and risk of chemical exposure during use.
Actizone™ F5 was also tested following the interim guidance provided by the EPA (RSD (RVT)) and passed with a greater than 4 log10 reduction following a 10 minute exposure. In general, the RSD protocol is more challenging than the BSI PAS2424 standard as it requires more abrasions (48 total passes over the surface) and more weight applied to the surface during the abrasions. Although the scientific rationale behind such rigorous abrasion regimen involved in the RSD method is not clear, the abrasion and inoculation procedures of BSI PAS2424 have been justified by a field testing 30 . Regardless, Actizone F5 being efficacious against Phi6 in both RSD and BSI PAS 2424 methods, is likely to be effective against many other enveloped viruses pathogenic to animals. This is to our knowledge, the first demonstration of the Phi6 resistance profile to QACs suggesting that the phage could be an appropriate substitute for many enveloped viruses that are otherwise difficult or highly risky to work with.
Use of safer and easier to handle strains like Phi6 provides additional advantages for investigators. More specifically, Phi6 can be cultivated in a day or two whereas most human and animal viruses require a minimum of 5-7 days of cultivation which increases cost and the experimental turnaround times. Phi6 is also easier to handle and recover: a refrigerated stock of Phi6 will last several months at least without significant reduction of titer, and for contact times of half an hour or less, recovery of viral controls is high (typically <0.5 log10 difference between the applied and recovered titers). In the context of the RVT test, the Gardco® wear tester specified in the protocol (used for abrasion cycles) is a large instrument ( and therefore may not be suitable for operation within the typical biosafety level 3 or level 2 cabinets without

Test strain and culture conditions
Bacteriophage Phi6 (HER102) and its host Pseudomonas syringae HB10Y #3 (HER1102) were purchased from Universite Laval (Quebec City, Quebec, Canada). High titer (~10^9 PFU/mL) phage stock was prepared in broth. Briefly, an aliquot of an overnight P. syringae culture in AOAC nutrient broth (NB) media was diluted into fresh NB and allowed to grow, shaking at room temperature for 4-8 hours. Phage freezer stock was harvested with a sterile toothpick, added to the host culture, and grown overnight at room temperature to lyse completely. The min. The surface was gently scraped with a pipette tip and 100 µL was used for serial dilution and plating as described above. The survival of Phi6 was compared to the survival of SARS-CoV-2 in the same conditions8.

Surface disinfection efficacy tests
Surface disinfection efficacy tests were based on ASTM 1053. A 2 oz/gal concentration of MCP™ was prepared in hard water as described in ASTM 1153. Inoculum was prepared by adding 125 µL BSA, 500 µL mucin, 175 µL yeast extract to 1.7 mL phage stock. 200 µL of the phage suspension was placed on a glass petri plate and dried in a biological safety cabinet. 13 Control films were overlaid with 2 mL phage buffer while test films were overlaid with 2 ml

MCP™.
After a 10 min contact time 2 mL Dey Engley broth was added as a neutralizer and the surface was scraped with a sterile pipette tip to resuspend the phage. The neutralized solutions were serially diluted and plated as described above. The sensitivity of Phi6 was compared to the sensitivity of several enveloped viruses in similar conditions7. In the RSD method, the test surface (stainless steel) tiles were exposed to 100-150 µL of the test substances and placed in a humidity (45-55% RH ) controlled chamber until completely dry.

Residual Virucidal Efficacy Tests
Once the tiles were dry, each tile was inoculated with 10 6 to 10 7 PFU of Phi6 particles and then abraded using a standardized abrasion machine (Gardco wear tester, Gardner, Pompano Beach, sonicated for 1 minute to dislodge surviving phage particles. The resulting mixture was then serially diluted and plated using double overlay agar and incubated as described above.

BSI PAS 2424
In the PAS 2424 test, sterile stainless steel discs were initially inoculated with 10μL of the virus test suspension (~10 8 PFU/mL in dMEM media with 3 g/L bovine serum albumin (BSA) for initial and final inoculations, ~10 6 PFU/mL in dMEM media with 3 g/L bovine serum albumin for reinoculations) and allowed to dry. Following the drying period 100 μL of disinfectant or hard water control was dosed onto the surface of the substrate and left to dry overnight. The dosed substrate then underwent a series of abrasions. A dry abrasion was applied followed by reinoculation of 10 μL of the virus test suspension followed by a drying period. A wet abrasion was then conducted using hard water (pH 7.0 ± 0.2). Once dry a further 10 μL virus reinoculation took place followed by another drying period. This was considered as one abrasion cycle. The process was conducted for a total of three abrasion cycles. The final inoculation was The method conducted to assess for residual virucidal activity was developed by combining aspects of the BS EN 16777:2018 and BS PAS 2424:2014 and is referred to as 'viral PAS 2424' hereon in. In the viral PAS 2424, sterile stainless discs were inoculated with virus test suspension (virus stock mixed 9:1 with 3g/L bovine serum albumin). After drying, within one hour, 100µL of product was dosed onto the substrate surface and left to dry for 24 hours. The dosed substrate then underwent a series of abrasions using a 100% polypropylene wipe and a 210 ± 2 gram weight. A dry abrasion was applied followed by reinoculation of 10µL of the virus test suspension and allowed to dry. A wet abrasion was then conducted using hard water (pH 7.0 ± 0.2). Once dry, a further 10µL of virus test suspension was inoculated and allowed to dry. This was considered as one abrasion cycle. The process was conducted for a total of three abrasion cycles. The final challenge inoculum was 50µL of virus test suspension which was left for a contact time of 5 minutes. Following the contact time, the substrate was immediately transferred to 4950µL of ice-cold DMEM without FBS and vortex mixed (SLS lab basics). The water virus controls were conducted in parallel under the same stated conditions. The virucidal activity of the product was calculated by the decrease in detectable virus titres from the average of the water controls in comparison to the test. A disinfectant or product was deemed as having residual activity if the titre is reduced by at least 3 log10 steps.
Where cytotoxicity of the product was so high that a 3 log10 reduction was not possible the large volume plating (LVP) technique was applied. Following the inactivation assay, a further 1:50 dilution was immediately performed in DMEM (total dilution 1:5000). The resulting mixture was added to eight 96-well plates per assay replicate to assess for residual virus.

Statistical analysis
16 Each data point generated represents the mean ± standard deviation of multiple replicates as indicated. The software used for the data analysis was Microsoft® Excel. The student t-test was used to compute the differences between the samples and the controls with significance assigned to P <0.01. The percent log10 reduction was calculated using the following formula: =(1−10− )*100 where LR is the log10 reduction.

BSI PAS 2424 statistical analysis for vaccinia virus
The tissue culture infective dose 50% per mL (TCID50/mL) of the tests were calculated as a mean of the replicates using the Spearman-Kärber formula. Where LVP was used and no CPE was observed the virus titre was calculated by the Poisson distribution formula. If CPE was observed the Taylor formula was applied.   Table 1 Log10 reduction of either Phi6 or Vaccinia Virus after residual virucidal efficacy testing following the BSI PAS2424 method or the interim guidance based on EPA RSD method. Greater than a 3.0 log10 reduction in viral titer after the specified contact time is required for a regulatory approval. For Phi6 data 4 replicates (n=4) were tested for the RSD method and 5 replicates (n=5) were tested for the BSI PAS2424 method. CD denotes complete deactivation.