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

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.