Name: Evaluating the Effect of Coated Filters and Pre-Filters on the Infectivity and Concentration of Aerosolized Phi6 Virus (Video) | JoVE Journal
Uploaded: 2026-05-29T13:45:00.000+00:00
Duration: 9 min 32 s
Description: Learn how copper and silver-based coatings on air filters inactivate aerosolized Phi6 virus using a bioaerosol chamber. This method evaluates antiviral efficacy for improved indoor air filtration technologies.

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5 minutes

Sari Korhonen is thanked for excellent technical assistance. Pavlina Gregorova is acknowledged for advice on the qRT-PCR protocol. We thank the late Professor Dennis H. Bamford for his invaluable input in designing the chamber setup and for all his support throughout the experiments. Dr. A.-P. Hyv&#228;rinen is thanked for providing a helping hand in setting up the aerosol test chamber. The facilities and expertise of the Instruct- HiLIFE Biocomplex unit at the University of Helsinki, a member of Instruct-ERIC Centre Finland, FINStruct, and Biocenter Finland are gratefully acknowledged. The study was funded by the Research Council of Finland, COVID special funding 335681, and funding 368144 to N.S.A. The Research Council of Finland is acknowledged for ACCC Flagship funding (grant no. 337552). We disclose the funding support from Clean Touch Medical LTD, M-Filter LTD, and UniqAir LTD to finalize the experiments, with no role played by the companies in the experimental design, execution, reporting, or in writing any part of the manuscript.

Virus, strains, growth media, and infectious virus counts 
The bacteriophage Phi6, originally provided to the research group by Dr. Anne K. Vidaver40, was propagated using Pseudomonas syringae pv. phaseolicola HB10Y as the host bacterium. The host was cultivated in Luria–Bertani (LB) medium at 22 °C. Phi6 agar stocks were prepared from frozen material through serial dilution, following the protocol of Bamford et al. (1995)41, and used for both surface viability assays and virus purification.
Purification and concentration of Phi6 were performed according to the protocol developed by Bamford et al. (1995)41. The resulting suspension was diluted in 20 mM potassium phosphate (K₃PO₄) buffer containing 1 mM magnesium chloride (MgCl₂), adjusted to pH 7.2, to yield a final titer of 5 × 1010–1011 plaque-forming units per milliliter (PFU/mL). This prepared suspension was subsequently used for aerosol generation experiments.
Enumeration of infectious viral particles was performed using standard double-layer agar overlay plaque assay, which was optimized for bacteriophage Phi642. Briefly, first serial dilutions in LB medium were prepared for each virus- or control sample, after which to 100 µL of each dilution, 200 µL of host bacterium suspension and 3 mL of soft agar were added. The soft agar mixtures were overlayed on top of LB agar plates and the plates were incubated at 22 °C overnight. Plaques are then counted and number of PFU/mL were calculated as ( plaques × dilution factor ) / volume plated.
qRT-PCR analysis 
Sample preparation for quantitative reverse transcription real-time polymerase chain reaction (qRT-PCR) followed the protocol of Gregorova et al. (2022)43. Standard curves for P2 and P8 RNA were generated using a 10-fold dilution series ranging from 102 to 109 molecules per 5 µL. Each dilution was processed identically to experimental samples, including chloroform addition, vortexing, centrifugation at 10000 × g for 1 min at 22 °C, and cycling (cycle and primer information in Supplementary Table 1 and Supplementary Table 2). Negative controls were prepared by substituting 5 µL of ultrapure water for the RNA template. qRT-PCR data were analyzed using the specific design and analysis software (see Table of Materials).
Aerosol chamber setup 
A custom-built, autoclavable aerosol chamber was developed for virus aerosolization and filter testing, modeled after a previously described system31. The experimental chamber consisted of a cylindrical glass body with a matching lid, an internal metal scaffold, and insulating gaskets to ensure an airtight seal (see Table of Materials and Supplementary Figure 1).
The overall study setup, including the chamber, is shown in Figure 1. To ensure a stable and reproducible input of viral aerosols, a nebulizer was used in combination with an air pump (see Table of Materials).
Within the chamber, the nebulizer generated an aerosol flow of 8 L/min. Approximately three-quarters of this flow was directed to the exhaust through a HEPA filter, while the remaining one-quarter (2 L/min) entered the chamber. This split was designed to reduce humidity during prolonged measurements. Aerosols were introduced into the chamber via a metal funnel, which directed the flow onto the filter or pre-filter surface.
Aerosol experiments were performed within a biological safety cabinet. After passing through the chamber, airflow was dehumidified in a desiccator to protect the HEPA filter and flow meter from excessive moisture. An external pump further supported the airflow, creating negative pressure within the system and ensuring efficient exhaust through the outlet port.
<img alt="HEPA filtration diagram, virus aerosol setup; nebulizer, pump, flow meter; air purification study." class="xfigimg" src="/files/ftp_upload/70877/70877fig1.jpg" data-altupdatedat="1787025648000" data-alt="Figure 1"> 
Figure 1: Schematic of the experimental aerosol chamber and sample collection. <a href="https://www.jove.com/files/ftp_upload/70877/70877fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Study design 
This study evaluated the anti-viral properties of coatings and filter/ pre-filter materials listed in Table 1, Supplementary Table 3, and Supplementary Table 4 (also see Table of Materials). The investigation was conducted in two stages, surface tests and pre-filter tests in the aerosol chamber.
In surface tests, all coating materials were first assessed on flat test pieces to determine their anti-viral activity. The coated test pieces were provided by Clean Touch Medical LTD (Table 1 and Table of Materials). All coatings are based on copper and/or silver active components embedded in proprietary matrices; differences between formulations primarily relate to metal composition, concentration, and surface compatibility (e.g., flat surfaces vs porous filters) (Supplementary Table 3).
Pre-filter tests in the aerosol chamber: Among the available coatings, only copper- and silver-based coating VS-B could be applied to pre-filters during the study. Pre-filters treated with VS-B were therefore examined in the aerosol chamber to assess their anti-viral performance under aerosol exposure conditions. Photographs of all filters and pre-filters used in the study are provided in Supplementary Figure 2.
Anti-viral efficacy was quantified by two complementary methods: plaque assay to measure the number of infectious viral particles (see section “Virus, strains, growth media, and infectious virus counts”), and quantitative PCR (qPCR) to determine viral genome copy numbers (see section ”qRT-PCR analysis”).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Filters and prefilters</td>
			<td rowspan="2">Filter provided by</td>
			<td>Coatings</td>
		</tr>
		<tr>
			<td> (All provided by Clean Touch Medical)</td>
		</tr>
		<tr>
			<td>Polyester prefilter FMR-5, uncoated (FMR-5)</td>
			<td rowspan="5">M-filter LTD</td>
			<td rowspan="7">Experimental copper-based surface agent A6 (A6) 
			Experimental copper-based surface agent B4 (B4) 
			Copper and silver surface agent Viral Safe (VS) 
			Copper and silver surface agent Viral Safe B (VS B)</td>
		</tr>
		<tr>
			<td>Polyester prefilter FMR-5, coated with VS B (FMR-5 VS B)</td>
		</tr>
		<tr>
			<td>Polyester prefilter S-11, uncoated (S-11)</td>
		</tr>
		<tr>
			<td>Polyester prefilter S-11, coated with VS B (S-11 VS B)</td>
		</tr>
		<tr>
			<td>Double-layer glass fiber filter GF-2, uncoated, (HEPA-type filter) (GF-2)</td>
		</tr>
		<tr>
			<td>Bulpren prefilter BP-6, 6 mm, uncoated (BP-6)</td>
			<td rowspan="2">Clean Touch Medical LTD</td>
		</tr>
		<tr>
			<td>Bulpren prefilter BP-6, 6 mm, coated with VS B (BP-6 VS B)</td>
		</tr>
	</tbody>
</table>
Table 1: Filters and surface coatings used in this study.
Efficiency of anti-viral surface coatings 
A surface viability assay was developed to evaluate the persistence of viral infectivity on different coating materials. Briefly, 20 µL of Phi6 stock (see section “Virus, strains, growth media, and infectious virus counts”) was pipetted onto the coated test pieces and evenly spread over a 2 cm × 2 cm area using a sterile glass triangle. Samples were incubated in a biosafety cabinet at 22 °C for 1 min, 5 min, 1 h, or 3 h.
Viruses were recovered by adding 180 µL of fresh LB broth to the surface and washing by repeated pipetting. A plastic Petri dish served as the control surface. All experiments were conducted at 22 °C. The number of infectious viral particles was quantified using a plaque assay41.
Effect of anti-viral coating VS-B on filter/ pre-filter efficacy 
Virus collection and filter efficiency testing were conducted using either an impaction method with a petri dish containing 20 mM HEPES buffer (pH 7.2) or an all-glass swirling impinger (hereafter referred to as “the impinger”; see Table of Materials)44. For impaction sampling, a petri dish was positioned directly beneath the filter to maximize particle deposition (Figure 1). In experiments with GF‑2 filters that had been in use for 3 months, the impinger was employed instead of the petri dish to enhance sensitivity. The impinger is a glass device equipped with three nozzles and requires an external pump for air sampling into liquid. Airflow through the nozzles generates a swirling motion of the collection liquid along the inner wall, facilitating gentle particle removal. In this configuration, airflow through the filter was maintained at 2 L/min, and upon exiting the chamber, an additional valve increased the total air influx to 10 L/min. Aerosols were captured in 5 mL of 20 mM HEPES buffer (pH 7.2), with evaporated buffer replenished every 30 min. For the impinger experiments, both the petri dish and the bulpren pre-filter (used for airflow equalization; Supplementary Figure 1 and Supplementary Figure 2B) were removed.
The detailed sampling protocol is presented in Table 2. Viral aerosols were collected by recovering 1 mL of suspension from Petri dishes containing 20 mL of 20 mM HEPES buffer (pH 7.2). Of this, 800 µL was used for qRT‑PCR analysis and 200 µL for virus titration. Each filter or pre-filter was tested in triplicate, and control runs without a filter/pre-filter were performed for 15 min before and after each chamber experiment. Following the pre‑experiment control, the filter or pre-filter of interest was inserted into the chamber, which was operated for 3 h, after which the filter/pre-filter was removed, and a post‑experiment control run was conducted. qRT‑PCR and virus titration samples were collected every 30 min, yielding eight qRT‑PCR and eight titration samples per experiment. In the impinger experiments, titration samples were collected only once at the end of the 3 h run. Each qRT‑PCR sample was snap‑frozen in liquid nitrogen and stored at -80 °C until further use, while virus titration samples were processed immediately after the experiment using plaque assay. The virus solution used in the nebulizer was titrated both before and after each experiment to monitor stability.
Three different pre-filters, BP-6, S-11, and FMR-5, and the GF-2 filter (Table 1 and Supplementary Figure 2, Supplementary Table 4) were coated with VS-B. Pre-filters consisted of synthetic fibrous materials with varying structural densities, primarily designed for coarse particle removal and airflow stabilization, whereas the GF-2 filter is a high-efficiency glass fiber filter with HEPA-type performance. Pre-filters without coating and a setup without a filter were tested in the aerosol chamber for comparison.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Time point</td>
			<td>Setup</td>
			<td>Samples</td>
		</tr>
		<tr>
			<td>Control</td>
			<td>0-15 min</td>
			<td>No filter/prefilter, Petri dish with 20 mL of HEPES</td>
			<td rowspan="8">200 μl for titration, 800 μL frozen for qRT-PCR</td>
		</tr>
		<tr>
			<td rowspan="6">Experiment</td>
			<td>0-30 min</td>
			<td>Filter/prefilter of interest, new Petri dish with 20 mL of HEPES</td>
		</tr>
		<tr>
			<td>30-60 min</td>
			<td>Filter/prefilter of interest, same Petri dish with 19 mL of HEPES</td>
		</tr>
		<tr>
			<td>60-90 min</td>
			<td>Filter/prefilter of interest, same Petri dish with 18 mL of HEPES</td>
		</tr>
		<tr>
			<td>90-120 min</td>
			<td>Filter/prefilter of interest, same Petri dish with 17 mL of HEPES</td>
		</tr>
		<tr>
			<td>120-150 min</td>
			<td>Filter/prefilter of interest, same Petri dish with 16 mL of HEPES</td>
		</tr>
		<tr>
			<td>150-180 min</td>
			<td>Filter/prefilter of interest, same Petri dish with 15 mL of HEPES</td>
		</tr>
		<tr>
			<td>Control</td>
			<td>0-15 min</td>
			<td>No filter/prefilter, new Petri dish with 20 mL of HEPES</td>
		</tr>
	</tbody>
</table>
Table 2: Timeline of sample collection.
Effect of anti-viral coating VS on used filters 
The durability of the anti-viral effect on HEPA-type GF-2 filters coated with the copper–silver surface agent Viral Safe (VS) was assessed by measuring the filtering efficiency of filters that had been in use for 3 months, based on both infectious virus counts and viral genome copy numbers. For these experiments, four air purifiers (see Table of Materials), each equipped with a HEPA filter and an activated charcoal layer, were employed. The filters had been operated for a 3‑month period in two restaurants before collection and analysis: one was a fine-dining restaurant, and the other was a staff cafeteria at a factory (more information in Supplementary Figure 3 and Supplementary Figure 4). The performance of these used filters was compared to that of clean, uncoated GF‑2 filters.
Prior to chamber testing, the used filters were removed from the air purifier metal cages, unfolded, and cut into appropriately sized pieces for the aerosol chamber inside a laminar flow hood to ensure biosafety and minimize cross‑contamination. The GF‑2 filters collected from fine-dining restaurant and a staff cafeteria were then tested in the experimental setup described in section “study design”, using a modified 5 mL impinger for aerosol collection.
In addition to anti-viral efficiency, the physical surface properties of the filters were evaluated. Lipophilicity was assessed by applying 50 µL droplets of rapeseed oil, while hydrophobicity was determined by applying 50 µL droplets of deionized water to cut‑out filter strips. Contact angles were documented photographically following the procedure of Xiao and Wiesner (2012)45. Water repellence was quantified using water drop penetration time (WDPT) measurements as described by Lichner et al. (2006), with all values exceeding 7200 s46.
Statistical analysis 
Statistical analysis was performed using standardized statistical software (see Table of Materials) to evaluate the significance of observed variations between tested coating materials and filters. Statistical analysis was performed in two steps for all experiments: the Kruskal-Wallis H test and Games-Howell post-hoc test. The Kruskal-Wallis H test was used to assess whether there were significant differences observed, and the specific variation between sample types was determined using multiple mean comparisons with the Games-Howell post hoc test.
Coating efficiency tests were statistically analyzed by comparing the decrease in infectious virus concentrations at 1 min and 5 min relative to the starting concentrations. Due to the aim of using the coatings on filters, we searched for statistically significant deviation in virus viability compared to the control within the first minutes of the test.
Statistical analysis determining significant differences in pre-filters’ efficiency with and without coating in terms of infectious count and qPCR results was performed for each time point, 30 min, 60 min, 90 min, 120 min, 150 min, and 180 min, separately. The data were then grouped by pre-filter type and the presence of coating on the pre-filter surface. Similarly, the results of the used GF-2 filters were assessed for efficiency in terms of virus inactivation and filtration efficiency by grouping data by location and the presence of coating on the filter surface.

The authors declare no conflict of interest.

In this study, we present a protocol for assessing the efficiency of anti-viral filter and pre-filter surface coatings in a microbiological aerosol chamber. The produced chamber offers a controlled, enclosed system suitable for aseptic microbiological analyses, as it is autoclavable, portable, transparent, and small-scale (3 L) compared to some previously described chambers31,50,51.
All coating materials tested in this study were confirmed to possess anti-viral activity (Figure 2A), with the experimental copper-based surface coating B4 demonstrating the highest efficacy, reducing virus infectivity below the limit of detection within 10 min. Copper-based coating A6 followed closely, achieving complete viral inactivation in 25 min (Figure 2A). These findings are consistent with previous reports on the anti-viral properties of copper and silver17,18,20,21,22,52. No virus inactivation was observed during the first 5 min prior to droplet desiccation, indicating that early reductions in infectivity were largely due to drying rather than the coating itself (control, Figure 2A, Figure 2B)36. Coatings B4 and A6 exhibited faster inactivation kinetics compared to VS and VS-B coatings, whereas VS-B required up to 60 min to reach the detection limit (Figure 2A). Pre-filters with B4 and A6 coatings were unavailable; therefore, VS-B was selected for subsequent filter aerosol experiments.
Results from this study emphasize that HEPA filters and pre-filters effectively reduce the number of viruses passing through them even in the absence of an anti-viral coating. Mechanical stress on aerosolized viruses during filtration led to a 1.0&#8211;2.0 log reduction in viral infectivity (from ~8 &#215; 105 &#8211;&#160;1 &#215; 107 PFU/mL to ~5 &#215; 104 &#8211;&#160;105, Figure 3A). This reduction was consistently observed across all pre-filter tests, supporting previous findings50,53,54. For the GF-2 HEPA-type filter, the loss of viral infectivity was even greater, with no detectable infectious particles within the method&#8217;s sensitivity limits (Figure 3A). The lower infectivity relative to total genome copy numbers suggests that filtration imposes mechanical damage on the virus particles, a phenomenon particularly pronounced for the enveloped Phi6 virus. As enveloped viruses are more susceptible to mechanical disruption, this difference may be smaller for non-enveloped viruses55.
In aerosol tests, coatings had no statistically significant effect on the filtration performance of pre-filters. This likely reflects limited contact between the coating and viruses passing through the filter, producing similar results for coated and uncoated pre-filters (Figure 3B, Figure 4B). The calculated sub-second residence times suggest that anti-viral inactivation kinetics must occur on a very rapid timescale to be effective under realistic airflow conditions. Additionally, our results suggest that the tested coatings do not exhibit the electrostatic affinity characteristic of copper ions toward negatively charged biological molecules, which could otherwise attract viruses to the filter and reduce their passage56. Therefore, the primary benefit of pre-filter coatings may lie in their post-capture self-sanitizing properties, as demonstrated in surface tests, rather than in enhancing the mechanical filtration efficiency. Importantly, the experiments conducted in this study represent single-pass filtration conditions, where aerosolized particles interact with the filter only once. This results in very short residence times and limited virus&#8211;surface contact, which likely reduces the observable impact of anti-viral coatings. In contrast, real-world air purification systems typically operate under recirculating conditions, where repeated air passage increases cumulative contact and may enhance coating effectiveness.
Our results indicate that GF-2 filters demonstrated improved removal of infectious viruses after three months of continuous use in air purifiers at the fine-dining restaurant and staff cafeteria (Supplementary Figure 3 and Supplementary Figure 4) compared to the unused filters (Figure 5). This improvement may be attributed to several factors. First, the accumulation of dust and other particulates has been reported to inactivate viruses57. Second, the progressive buildup of indoor air pollutants can densify the filter structure, potentially enhancing its efficacy temporarily57,58,59. Notably, the presence of coatings did not significantly affect virus infectivity or qRT-PCR-detected genome copy numbers. The passage of non-infectious virus particles was also similar regardless of filter coatings or usage, indicating that the observed filtration efficiency of the GF-2 HEPA-type filters and pre-filters primarily reflects their mechanical properties. Nevertheless, the accumulation of filtered particles on the GF-2 filter surface had a pronounced effect in reducing the number of infectious viruses passing through (Figure 5).
To understand the differences in filter performance observed in Figure 5 and Figure 6, additional tests were conducted. All filters exhibited strong hydrophobicity when exposed to water droplets, whereas oil droplet testing revealed notable differences, with VS-coated filters displaying pronounced lipophilicity. The underlying mechanism for this variation remains unclear, but these results suggest that filter age or usage history may not be the primary determinants of performance. Instead, the type of surface pretreatment may alter the physical properties of the filter, consequently affecting virus retention and passage. Collectively, these findings indicate that anti-viral coatings provide functional benefits beyond mechanical filtration, most notably through their self-sanitizing potential, which may reduce health risks for personnel handling used filters.
While this protocol provides a robust framework for evaluating anti-viral filtration, several limitations should be considered. First, the enveloped bacteriophage Phi6 was used as a surrogate for human respiratory pathogens. Although Phi6 shares structural and chemical similarities with coronaviruses, differences in environmental stability and surface proteins may influence exact inactivation kinetics. Second, while some coatings demonstrated significant efficacy, this study evaluated a limited set of formulations under specific aerosol conditions; future research should broaden the scope to include a wider range of antimicrobial agents. Finally, the experiments were conducted within a controlled aerosol chamber with regulated airflow and humidity. While these conditions ensure reproducibility, they do not fully replicate the complex, turbulent airflow and fluctuating environmental parameters found in real-world indoor environments.
Overall, this study demonstrates that while GF-2 glass fiber filters and pre-filters efficiently remove infectious viruses, particularly after prolonged use due to particle densification, the primary role of copper- and silver-based coatings is the inactivation of viruses upon direct contact. These findings underscore the robustness of mechanical filtration in controlling airborne viruses and suggest that future studies should evaluate the combination of anti-viral coatings with complementary interventions, such as UV irradiation, to further reduce transmission risk. pre-filter60,61. Also, future field studies integrating these coatings into existing building ventilation systems will be essential to validate their performance in situ.

Understanding of airborne viral transmission has advanced substantially during the COVID-19 pandemic. Given that indoor environments present markedly higher risks for transmission than outdoor settings, there is a compelling need to develop more effective protective strategies1,2. In this context, high-performance air conditioning and air purification systems constitute essential components for mitigating virus transmission in enclosed spaces.
Air filtration is one of the most prevalent methods used in air purification systems, relying on the intrinsic physical filtering qualities and multilayer filtering3,4,5. This often includes a pre-filter, a coarse filter that captures large particles; a medium-efficiency filter, for example, a carbon filter; and a high-efficiency particulate air (HEPA) filter that removes at least 99.97% of the particles with the most penetrating particle size of 0.3 &#181;m6,7. The physical properties of the filters contain many antimicrobial benefits. It has been shown earlier that porous materials can inactivate viruses by trapping and drying them8,9,10. Hydrophobicity or hydrophilicity can also be utilized correspondingly. Viruses with hydrophobic coats favor hydrophobic surfaces for sorption, while hydrophilic surfaces attract hydrophilic viruses11,12. In addition, anti-viral surface coating can be used to further enhance purification. The coating of filters aims to inactivate pathogens immediately when they touch the filter13,14. These features could increase operational safety, especially in hospitals, but also in public environments during epidemics and pandemics14.
For practical applications, the inactivating effect of the filter coatings must be rapid enough to be utilized in flow-through air. Thus, the surface area for direct contact between the coating and virus particles should be sufficient, and the coating should be able to inactivate the virus within the given contact time. Heavy metals, such as copper and silver, are known to possess anti-viral effects15,16,17. This anti-viral effect has been observed for viruses such as poliovirus, vaccinia virus, herpes simplex 1, Phi6, papovirus SV-40, adenovirus, vesicular stomatitis virus, SARS-coronavirus, baculovirus, and feline infectious peritonitis virus18,19,20,21,22. Mechanisms include the release of copper and silver ions and the generation of reactive oxygen species (ROS), which react with virus proteins and oxidize nucleic acids23,24,25,26,27,28,29,30.
An aerosol filter test chamber with adjustable aerosol and virus sampling properties was developed for this research, based on a previous chamber setup presented by Sofieva et al. (2022) (see Figure 1 and Supplementary Figure 1)31. The adapted chamber was designed as a closed system in which model viruses for pathogenic respiratory disease-causing viruses are impacted by the filters, assessing the anti-viral efficiency of the coated and uncoated filters. The enveloped bacteriophage Phi6 was selected as a surrogate for respiratory viruses because it is a well-studied and widely used model suitable for viral aerosol studies. As Phi6 is non-pathogenic to humans and classified as Biosafety Level 1 (BSL-1), it allows for the safe and practical assessment of anti-viral coatings within a Biosafety Level 2 (BSL-2) laboratory environment in accordance with institutional safety guidelines. Furthermore, Phi6 shares structural similarities with enveloped viruses that cause respiratory illnesses, such as severe acute respiratory syndrome coronaviruses 1 and 2 (SARS-CoV-1 and SARS-CoV-2). Its lipid envelope also confers a specific sensitivity to desiccation and chemical inactivation, providing a representative model for evaluating the efficacy of antimicrobial surface coatings and filtration systems against enveloped pathogens32,33,34,35,36,37,38,39.
The objective of this study was first to analyze the anti-viral efficacy of the selected filter coating materials containing copper and silver by surface through surface testing with Phi6 bacteriophage prior to their use in air filtration. Subsequently, these copper- and silver-based surface treatment materials were assessed on air pre-filters to evaluate their capacity to enhance both filtration and elimination properties against airborne viruses.

<table><tbody><tr><td>Aerosol chamber glass cylinder and lid</td><td>Laborexin Oy</td><td>N/A</td><td>Custom-made glass cylinder and lid for aerosol containment.</td></tr><tr><td>Aerosol chamber inner metal scaffold</td><td>Clean Touch Medical Ltd.</td><td>N/A</td><td>Custom-made stainless steel support structure for filter mounting.</td></tr><tr><td>Aerosol chamber lid insulating gaskets</td><td>Etra Oy (Etola Group)</td><td>N/A</td><td>Custom-made rubber gaskets for airtight sealing.</td></tr><tr><td>Air pump, WI 53082</td><td>Thomas Industries Inc.</td><td>N/A</td><td>Diaphragm pump used to maintain regulated airflow through the chamber.</td></tr><tr><td>BioSampler</td><td>SKC Inc.</td><td>https://international.skcinc.com/products/air/bioaerosol-samplers/biosampler</td><td>All-glass swirling impinger used for high-efficiency collection of airborne viruses.</td></tr><tr><td>Coated test pieces</td><td>Clean Touch Medical Ltd.</td><td>www.cleantouchmedical.com</td><td>Gloss paper slides coated with antimicrobial formulations for surface efficacy testing.</td></tr><tr><td>CompAir Pro NE-C900 nebulizer</td><td>Omron Healthcare</td><td>NE-C900&amp;nbsp;</td><td>High-output compressor nebulizer used for viral aerosol generation.</td></tr><tr><td>SPSS Statistics software</td><td>IBM Corp.</td><td>https://www.ibm.com/products/spss</td><td>Statistical analysis software, version 28.0.</td></tr><tr><td>QuantStudio Design and Analysis Software</td><td>Thermo Fisher Scientific</td><td>https://www.thermofisher.com/in/en/home/technical-resources/software-downloads.html#pcr</td><td>Software used for qRT-PCR data analysis, version 1.3.1.</td></tr><tr><td>UniqAir PRO air purifier</td><td>UniqAir Oy</td><td>https://www.uniqair.fi/</td><td>Commercial-grade air purification unit used for chamber scavenging.</td></tr></tbody></table>

evaluating the effect of coated filters and pre-filters on the infectivity and concentration of aerosolized phi6 virus

Evaluating the Effect of Coated Filters and Pre-Filters on the Infectivity and Concentration of Aerosolized Phi6 Virus

Development of efficient indoor air filtration technologies has gained prominence following the coronavirus disease 2019 (COVID-19) pandemic. The goal of this study is to evaluate the anti-viral efficacy of various copper and silver-based surface coatings for air filters using a standardized bioaerosol chamber and the enveloped bacteriophage Phi6 as a surrogate for respiratory viruses. Surface testing confirmed that all coatings exhibited anti-viral activity, with the specific copper-based coating formulation (B4) achieving complete inactivation of Phi6 virus within 10 min, A6 in 25 min, and VS-B in 60 min; early reductions in infectivity were primarily due to droplet desiccation. Aerosol experiments showed that high-efficiency particulate air (HEPA) filters and pre-filters substantially reduced viral infectivity even without coatings, with mechanical filtration causing a 1.0&#8211;2.0 log reduction, and high-efficiency grade GF-2 glass fiber filters eliminating detectable infectious virus. Coatings did not significantly enhance filtration efficiency, likely due to limited virus contact with coated surfaces. Used GF-2 filters demonstrated improved virus removal, attributed to particle accumulation and filter densification, while coatings did not alter the copy number of viruses passing through. VS-B coated filters exhibited lipophilic properties, suggesting additional functional benefits. This methodology highlights that while filter efficiency is primarily driven by mechanical entrapment and particle accumulation, the integrated surface coatings provide a self-sanitizing mechanism by inactivating trapped viral particles; the integration of ultraviolet (UV) light is proposed as a potential future research direction to enhance system efficacy further.

Here, we present a study to evaluate the anti-viral efficacy of copper- and silver-based surface coatings on air filters. Using a bioaerosol chamber and Phi6 bacteriophage, this method quantifies viral inactivation and mechanical filtration efficiency for self-sanitizing indoor air technologies.

Development of efficient indoor air filtration technologies has gained prominence following the coronavirus disease 2019 (COVID-19) pandemic. The goal of ...

We investigate the effect of pre-filters and antiviral coatings in reducing airborne virus infectivity and concentrations in indoor settings. Current approaches miss realistic airflow dynamics. Our protocol recreates aerosol conditions and measures both infective viruses and total viral load.To begin, dilute the purified phi6 bacteriophage stock suspension in 20 millimolar potassium phosphate buffer containing one millimolar magnesium chloride adjusted to pH 7.2. Assemble all the filter, pre-filter, and antiviral coating materials within the biosafety cabinet to perform the surface tests. Assess the antiviral activity of all coating materials based on copper, or copper and silver active components embedded in proprietary matrices, namely A6, B4, VS and VS B on flat test pieces.Next, pipette 20 microliters of diluted phi6 bacteriophage stock solution onto coated test pieces to perform the surface viability assay. Spread the sample evenly over a two by two centimeters area using a sterile glass triangle. Incubate the samples at 22 degrees Celsius up to four time points, including one minute, five minutes, one hour, or three hours.Add 180 microliters of fresh LB broth to the surface to recover the viruses after each time point. Wash the surface by repeated pipetting 10 to 20 times to collect the sample. Use a plastic Petri dish as control surface.Quantify antiviral efficacy of the surface coatings using plaque assay to measure the number of infectious viral particles and quantitative reverse transcriptase polymerase chain reaction, or qRT-PCR, to determine viral genome copy numbers. Dilute the purified and concentrated phi6 bacteriophage suspension as demonstrated previously to obtain a viral concentration in the range of 2.5 times 10 to the power of 10, to 4.08 times 10 to the power of 11 plaque-forming units per milliliter. Next, assemble the experimental aerosol chamber using a cylindrical glass body with a matching lid.Insert the internal metal scaffold into the chamber and secure insulating gaskets to ensure an airtight seal. Use a nebulizer in combination with an air pump to generate a stable and reproducible input of viral aerosols. Titrate the virus solution used in the nebulizer before and after each experiment to monitor stability.Set the aerosol flow rate to eight liters per minute within the chamber. To reduce humidity during prolonged measurements, direct approximately three quarters of the aerosol flow to the exhaust through a high-efficiency particulate air, or HEPA, filter. Allow the remaining one quarter of the flow corresponding to two liters per minute to enter the chamber.Use a funnel to direct the aerosol flow toward the filter or pre-filter surface within the chamber. Dehumidify the outgoing airflow using a desiccator to protect the HEPA filter and flow meter from excessive moisture. Using the external pump, create negative pressure within the system, and further support airflow to ensure efficient exhaust through the outlet port.Evaluate the antiviral properties of uncoated or VS B-coated polyester and bulpren pre-filters alongside double-layer glass fiber materials within the aerosol chamber. To evaluate the antiviral effect by impaction sampling, position a Petri dish containing 20 millimolar HEPES buffer at pH 7.2 directly beneath the testing pre-filter to maximize particle deposition. Alternatively, to test the antiviral effect of GF2 filters, set up a triple-nozzle all-glass swirling impinger connected to an external pump for aerosol collection.Maintain airflow through the testing pre-filter or filter at two liters per minute. Increase total air influx to 10 liters per minute using an additional valve after the chamber outlet. Capture the aerosols in five milliliters of 20 millimolar HEPES buffer.And replenish evaporated buffer every 30 minutes. For triplicate sampling, perform control runs without pre-filters or filters for 15 minutes before and after each experiment. After the pre-experiment control run, insert the filter or pre-filter into the chamber and operate the system for three hours.Collect viral aerosols by recovering one milliliter of suspension from Petri dishes containing 20 milliliters of buffer every 30 minutes. Remove the filter and conduct a post-experiment control run. Collect samples every 30 minutes during the experiment to obtain eight qRT-PCR and titration samples each per experiment.In impinger experiments, collect titration samples only once at the end of the three-hour run. Use 800 microliters of the collected sample for qRT-PCR analysis and the remaining 200 microliters for virus titration. Snap freeze qRT-PCR samples in liquid nitrogen and store them at 80 degrees Celsius.Process virus titration samples immediately using plaque assay. The surface viability assays demonstrated that all tested coatings successfully inactivated the virus with significant reductions observed within the first five minutes. The copper-based surface coating, B4, achieved the fastest reduction, decreasing infectious counts by three orders of magnitude within five minutes, and reaching complete inactivation with detection limit below 100 plaque-forming units per milliliter by 15 minutes.A6 coating achieved total virus inactivation within 25 minutes, and VS B within 60 minutes. VS coating exhibited the slowest inactivation rate among the tested materials. The studied pre-filters BP6, S11, and FMR5 were equally effective in lowering the number of infectious viruses, passing through them by 1.5 to 2 orders of magnitude compared to the no-filter control.The GF2 filters demonstrated superior performance, lowering the viral load by at least six orders of magnitude, and no infectious phi6 virus plaques were detected within the detection limit. The VS B coating did not significantly affect infectious virus filtration efficacy compared to uncoated pre-filters. The qRT-PCR results showed that pre-filters lowered the viral genome copy numbers by up to two orders of magnitude.The GF2 filter showed a decrease of up to four orders of magnitude compared to the no-filter control. While the differences between pre-filters and control remained insignificant during the first 30 minutes, the mechanical filtering efficiency increased from the 60-minute time point. Additionally, the application of the VS B coating had no measurable effect on the number of viral genomes passing through the filtration media.This protocol allows researchers to quantify viral inactivation, filtration efficiency, and differences between infectious particles and total viral load. A key challenge is very short virus surface contact time during airflow, which limits the effectiveness of antiviral coatings. Future studies can explore combining coatings with UV or testing performance in real-world ventilation systems and with diverse viruses.

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JoVE Journal

Biology

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