Efficacy of microbicidal actives and formulations for inactivation of Lassa virus in suspension

The World Health Organization’s R&D Blueprint list of priority diseases for 2022 includes Lassa fever, signifying the need for research and development in emergency contexts. This disease is caused by the arenavirus Lassa virus (LASV). Being an enveloped virus, LASV should be susceptible to a variety of microbicidal actives, although empirical data to support this expectation are needed. We evaluated the virucidal efficacy of sodium hypochlorite, ethanol, a formulated dual quaternary ammonium compound, an accelerated hydrogen peroxide formulation, and a p-chloro-m-xylenol formulation, per ASTM E1052-20, against LASV engineered to express green fluorescent protein (GFP). A 10-μL volume of virus in tripartite soil (bovine serum albumin, tryptone, and mucin) was combined with 50 μL of disinfectant in suspension for 0.5, 1, 5, or 10 min at 20–25 °C. Neutralized test mixtures were quantified by GFP expression to determine log10 reduction. Remaining material was passaged on Vero cells to confirm absence of residual infectious virus. Input virus titers of 6.6–8.0 log10 per assay were completely inactivated by each disinfectant within 1–5 min contact time. The rapid and substantial inactivation of LASV suggests the utility of these microbicides for mitigating spread of infectious virus during Lassa fever outbreaks.

and p-chloro-m-xylenol (PCMX) against LASV in suspension in the presence of a tripartite soil load 6 .Organic soil loads are used as the challenge matrix to model virus inactivation by microbicides in relevant matrices, such as human sputum or blood.Use of hard water as diluent for specific actives was included in the study design, because it is a known antagonist to microbicidal activity and is commonly available in the field 7 .
In addition to the methodologies in ASTM E-1052-20, our study was made more stringent by decreasing the volume of the microbicides used during testing and by ruling out the presence of residual infectious LASV in the post-disinfection/post-neutralization samples through use of a safety test (performed in addition to the standard quantification of titer reduction).This safety test involved inoculation of 650 µL of undiluted neutralized test sample onto six-well plates containing Chlorocebus aethiops kidney epithelial (Vero) cells and passaging any cultures found to be negative for GFP at least twice.This was done to evaluate the virucidal efficacy test for the possibility of any residual virus being present at levels lower than the limit of detection of the 50% tissue culture infectious dose (TCID 50 ) titration assay performed in Vero cells per the ASTM standard.

Stock virus preparation.
A characterized stock of LASV-GFP was prepared by exposing ten T75 flasks of Vero cells at ≈80% confluency at a multiplicity of infection of 0.01 viral particles per cell.At 4 d post-infection, essentially all cells within the confluent cell monolayers were observed to express GFP.The flasks were then placed in a freezer at − 70 °C.The frozen flasks were thawed on the following day and the conditioned media was removed and clarified by low-speed centrifugation (4500 × g) for 10 min.The resulting supernatants were pooled and layered onto 20% w/v sucrose cushions prepared in Tris-NaCl-EDTA buffer prepared in house.The pooled supernatants were subjected to centrifugation (≈130,000 × g for 2 h), and the viral pellets obtained were resuspended in MEM containing 2% FCS and 10 units per mL pen/strep to create virus culture medium (VCM).The virus pool obtained was aliquoted into small portions that were frozen at − 70 °C.The stock virus titer was found to be > 9.2 log 10 per mL, as determined by TCID 50 assay, using the method described by Reed and Muench 8 .

Microbicides.
A variety of microbicidal actives or formulations containing microbicidal actives were evaluated for virucidal efficacy against LASV-GFP.The microbicides tested are listed in Table 1, along with sources and the supplied and tested concentrations.

Assessment of microbicide neutralization by chemical reagents and Amicon columns.
Neutralizing reagents or mechanical removal using Amicon filter columns were evaluated for ability to neutralize the virucidal effects of the microbicides to enable investigation of specific contact times, and/or to mitigate the cytotoxic effects of the microbicides on the Vero cells used to assay residual virus.The procedures used are described in Supplemental Materials.
Microbicide virucidal efficacy testing.The LASV-GFP suspension inactivation efficacy testing of the microbicides (Fig. 1) was conducted at ambient temperature using ASTM E-1052-20 5 .A modification to the Table 1.Microbicides/microbicidal formulations and concentrations as supplied and as evaluated.The unformulated microbicidal actives and the formulations tested were prepared in hard water 7 (prepared as 1 L deionized water supplemented with 0.4 g calcium carbonate) on the day of assay.The resulting solutions were inverted to mix and used within 2.5 to 4 h of preparation.Mechanical neutralization using Amicon filter columns.For microbicides that could not be adequately neutralized using VCM alone (AHP, PCMX, and dual QAC), a mechanical filtration procedure via Amicon YM100 columns (UFCS510096; EMD Millipore, Darmstadt, Germany) was used during virucidal efficacy testing (Fig. 2).After the planned contact times, virus-microbicide suspensions were diluted with 500 μL of VCM and immediately eluted through the columns.In accordance with the manufacturer's procedures, the columns were centrifuged for 3 min at 14,000 × g and the flow-through was discarded.To the retentate, 400 μL of fresh VCM were then added to the filter cup, centrifuged for an additional 3 min at 14,000 × g, and the flowthrough was again discarded.To elute retained virus from the column, 500 μL of fresh VCM were added to the filter cup, incubated for 2 min, inverted into a fresh tube, and spun for 2 min at 1000 × g.The final eluted volumes were brought to 1000 μL with VCM for evaluation.A single wash step was performed for the AHP and PCMX, whereas four wash steps were needed for the dual QAC.
In either case (neutralization using VCM alone or VCM plus neutralization using Amicon columns), a 350-µL portion of neutralized test solution was assayed for residual infectious virus titer using a ten-fold dilution scheme in VCM, with 50 µL of each resulting dilution being added to 96-well plates of Vero cells (n = 5 replicate wells per dilution).The inoculated cell monolayers were scored 5 d post-infection for GFP, and virus titers (in units of TCID 50 ) were calculated according to the Reed-Muench method 8 .

Plate safety test.
In addition to the 96-well plate TCID 50 assay described above, neutralized material also was evaluated for low levels of infectious virus in a plate safety test.In this test, which is used when dealing with especially lethal challenge viruses, 650 μL of remaining undiluted neutralized test sample (one sample for each technical replicate) were added to Vero cells in wells of a six-well plate containing 4 mL of VCM.In addition,

Analysis of viral inactivation efficacy.
For each disinfectant, three separate independent assays were conducted with each time point having three technical replicates within each assay.TCID 50 titers for positive virus controls and neutralized microbicide test conditions were determined using the method of Reed and Muench 8 .The log 10 reduction values achieved by the microbicides at given contact times were calculated by subtracting the post-disinfection log 10 TCID 50 values (titers) from the log 10 TCID 50 values obtained for the corresponding positive virus control.Statistical comparison of the mean (n = 5 replicates) viral titers obtained in the neutralization effectiveness studies (Supplemental Fig. S1) was performed using a non-parametric unpaired t-test, with statistical significance set at p < 0.05.

Results
Neutralization effectiveness evaluation.The results from the determination of the effectiveness of the neutralization procedures (chemical or mechanical) are provided in the Supplemental Materials.It was found that 0.5% sodium hypochlorite and 67% ethanol could be adequately neutralized using VCM alone.VCM plus mechanical neutralization using Amicon columns was required for dual QAC, AHP, and PCMX.
Virucidal efficacy of 67% ethanol for LASV.Three replicate evaluations of the efficacy of 67% ethanol (EtOH) (Table 1) for inactivating LASV-GFP virus in suspension were conducted.Contact times of 0.5, 1, 5, and 10 min were evaluated at ambient temperature.A mean LASV-GFP titer of 6.62 log 10 TCID 50 per mL (4.4 × 10 6 TCID 50 per mL) was recovered for the positive control (no disinfectant) (Fig. 4).The post-exposure/neutralization titers for the 67% ethanol condition were 0.42 log 10 TCID 50 per mL for the 0.5-min (30-s) contact time and   www.nature.com/scientificreports/0.42 log 10 TCID 50 per mL for the 1-min contact time.These correspond to reductions of 6.2 log 10 .After 5 and 10 min of contact with 67% ethanol, complete inactivation (≥ 6.6 log 10 ) of LASV-GFP was observed (Fig. 4).Further evidence of the complete inactivation of LASV-GFP following the 5-and 10-min contact times was obtained in the plate safety test.One technical replicate from one assay of the 0.5 min contact time and three technical replicates from two assays of the 1-min contact time displayed GFP in this assay.No evidence of residual infectious virus was obtained from the technical replicates for the 5-and 10-min contact times (Table 3).
Virucidal efficacy of a dual QAC formulation for LASV.Three replicate evaluations of the efficacy of a dual QAC formulation (Table 1) for inactivating LASV-GFP virus in suspension were conducted.Contact times of 0.5, 1, 5, and 10 min were evaluated at ambient temperature.A mean LASV-GFP titer of 8 log 10 TCID 50 per mL was recovered for the positive control (no disinfectant) (Fig. 5).The post-exposure/neutralization titers for the dual QAC conditions were ≤ 1.8 log 10 TCID 50 per mL (the defined limit of detection of the titration assay) for all contact times (Fig. 5).The assay limit of detection was determined by residual cytotoxicity to the Vero cells of the neutralization mixture following elution from the Amicon column.These results indicate a reduction in titer of LASV of ≤ 6.2 log 10 for each contact time.
In the case of the dual QAC formulation, the plate safety test was not able to be conducted, due to the residual cytotoxicity of the undiluted post-exposure/neutralization samples to the Vero cells despite repeated mitigation efforts using multiple filtrations via Amicon columns.
Virucidal efficacy of an AHP formulation for LASV.Three replicate evaluations of the efficacy of an AHP formulation (Table 1) for inactivating LASV-GFP virus in suspension were conducted.Contact times of 0.5, 1, 5, and 10 min were evaluated at ambient temperature.A mean LASV-GFP titer of 7.3 log 10 TCID 50 per mL was recovered for the positive control (no disinfectant) (Fig. 6).The post-exposure/neutralization titer for the AHP condition was 0.17 log 10 TCID 50 per mL for the 0.5-min (30-s) contact time, representing a 7.1 log 10 reduction.After 1, 5, and 10 min of contact with AHP, complete inactivation (≥ 7.3 log 10 ) of LASV-GFP was observed (Fig. 6).
Further evidence of the complete inactivation of LASV-GFP following the 1-, 5-, and 10-min contact times for AHP was obtained in the plate safety test.Two technical replicates from one assay of the 0.5-min (30-s) contact Table 3. Plate safety test for inactivation of Lassa virus by 67% ethanol in suspension.GFP, green fluorescent protein.GFP, green fluorescent protein; + , GFP detected; −, GFP not detected.time displayed GFP in this assay.No evidence of residual infectious virus was obtained from the technical replicates for the 1-, 5-, 10-min contact times (Table 4).
Virucidal efficacy of a PCMX formulation for LASV.Three replicate evaluations of the efficacy of three in-test concentrations (0.04, 0.06, and 0.12%) of PCMX in a commercial formulation (Table 1) for inactivating LASV-GFP virus in suspension were conducted.Contact times of 0.5, 1, 5, and 10 min were evaluated at ambient temperature.Mean LASV-GFP titers of 7.8 log 10 TCID 50 per mL, 7.3 log 10 TCID 50 per mL, and 7.3 log 10 TCID 50 per mL were recovered for the positive control (no disinfectant) conditions for the assay of the 0.12, 0.06, and 0.04% in-test concentrations of PCMX, respectively (Fig. 7).PCMX concentration-dependent inactivation of LASV-GFP was observed at the various contact times.For instance, the post-exposure/neutralization titers for the 0.12, 0.06, and 0.04% PCMX conditions were 0.9 log 10 TCID 50 per mL, 2.3 log 10 TCID 50 per mL, and 3.2 log 10 TCID 50 per mL, respectively, after 0.5-min (30-s) contact time, representing log 10 reductions of 6.9, 5.0, and 4.1 log 10 , respectively.Following a 1-min contact time, the 0.12% PCMX condition had no viable virus remaining post-exposure/neutralization, whereas for the 0.06 and 0.04% PCMX conditions 1.0 and 2.4 log 10 TCID 50 per mL, respectively, were recovered representing log 10 reductions of 6.3 and 4.9 log 10 , respectively.Following a 5-min contact time, the post-exposure/neutralization titers for the 0.06 and 0.04% PCMX conditions were 0.97 and 0.94 log 10 TCID 50 per mL, respectively, representing log 10 reductions of 6.3 and 6.4 log 10 , respectively.Complete inactivation (≥ 7.8 log 10 ) of LASV-GFP was afforded by 0.12% PCMX at contact times of 1, 5, and 10 min.For the 0.06 and 0.04% PCMX concentrations, complete inactivation (≥ 7.3 log 10 ) of LASV-GFP was observed at the 10-min contact time (Fig. 7).
Further evidence of the complete inactivation of LASV-GFP following the 1-, 5-, and 10-min contact times for 0.12% PCMX, and following the 10-min contact times for 0.06 and 0.04% PCMX, was obtained in the plate safety tests (Tables 5, 6, 7).One technical replicate from one assay of the 0.5-min contact time displayed GFP in the plate safety test for 0.12% PCMX, whereas no evidence of residual infectious virus was obtained from the technical replicates for the 1-, 5-, and 10-min contact times (Table 5).
As expected on the basis of the titration assay results, multiple replicates for one or more individual assays for the 0.06 and 0.04% PCMX concentrations demonstrated GFP following the 0.5-, 1-, and 5-min contact times,     www.nature.com/scientificreports/confirming the presence of residual infectious virus in these replicates.No evidence of residual infectious virus was obtained from the technical replicates for the 10-min contact times for the 0.06 and 0.04% PCMX exposures (Tables 6 and 7).

Discussion
Per WHO 9 , Lassa fever is endemic in a number of Western African countries, including Benin, Ghana, Guinea, Liberia, Mali, Sierra Leone, and Nigeria.Cases have been reported as recently as March 2023 in Ghana 9 .Natal mastomys rats (murid Mastomys natalensis) are the primary reservoirs of the causal virus, LASV.Transmission to humans occurs primarily through contact with urine or feces of infected mastomys, but human-to-human transmission via direct contact with blood or bodily fluids may also occur, especially in hospital settings 9 .These considerations suggest that inanimate surface hygiene and liquid inactivation methods for LASV might limit dissemination of the virus to humans.Partly because maximum containment is needed for conduct of inactivation studies on LASV, there is little published information on the efficacy of microbicides for inactivation of this arenavirus.A recent review of the available primary data on efficacy of microbicides against LASV 3 revealed that the limited published data pertained to the inactivation of laboratory specimens intended for diagnostic or histology applications [10][11][12][13][14] .The paucity of virucidal data of microbicides against LASV is also emphasized in a recent review of inactivation of emerging viruses in aqueous phase 15 .Our study was intended to resolve this knowledge gap, supplying efficacy information for commonly used microbicidal actives and formulations applicable to inactivation of LASV in liquid suspension.
The United States Environmental Protection Agency (US EPA) recognizes that microbicidal efficacy data may not be available for newly emerging viruses, especially those requiring BSL-4 laboratories for handling the viruses safely.The EPA, therefore, enacted a policy in 2016 enabling efficacy claims against emerging viruses to be made without having provided registration data specifically for those viruses.In its Guidance to Registrants 16 , the EPA has made note of the hierarchy of pathogen susceptibility to microbicides [17][18][19][20][21] in recognizing that efficacy against one enveloped virus implies efficacy against other enveloped viruses.The EPA policy provides a "process that can be used to identify effective disinfectant products for use against emerging viral pathogens and to permit registrants to make limited claims of their product's efficacy against such pathogens." 16 .The guidance outlines "a voluntary two stage process, involving product label amendments and modified terms of registration and applies only to emerging viruses" 16 .
The EPA policy provides inanimate surface hygiene and liquid inactivation alternatives, helpful for use during virus disease outbreaks.Despite this, obtaining empirical data for specific emerging viruses is required for assurance of efficacy against the more lethal viruses.On the basis of information 3 derived from testing enveloped viruses (such as, Ebola virus and SARS-CoV-2), lipid-disrupting agents-including ethanol, quaternary ammonium compounds (such as the dual QAC compound evaluated), and phenolics (such as PCMX)-were expected to be effective against other enveloped viruses, such as LASV.Certain microbicidal actives and formulations were considered mechanistically to be protein-denaturing agents (ethanol, PCMX, AHP, and sodium hypochlorite) or genome-degrading agents (ethanol, AHP, and sodium hypochlorite).In fact, our study found that these agents caused rapid (i.e., within 30 s contact time) and highly effective (≥ 6 log 10 ) inactivation of LASV-GFP when tested in suspension in a tripartite soil load with hard water as diluent to simulate field use of dilutable products, including PCMX and sodium hypochlorite (Table 1).
The standardized ASTM E-1052-20 methodology 5 is based on demonstrating a reduction in infectious virus titer after exposure to a test microbicide.These data are then available for making EPA disinfectant efficacy claims.For instance, the EPA stated the following in its 2012 disinfectant product guidance 22 that "The product should demonstrate complete inactivation of the virus at all dilutions.If cytotoxicity is present, the virus control titer should be increased to demonstrate a ≥ 3 log 10 reduction in viral titer beyond the cytotoxic level." For disinfectants that are non-cytotoxic to the cellular infectivity assays used for demonstrating efficacy, a 4-log 10 reduction in viral titer is typically considered to be effective.However, as we have previously done when dealing with especially lethal viruses, such as Ebola virus 23,24 , we extended the stringency of the assay for detecting residual infectious virus post-exposure to the microbicides by conducting the plate safety test.The latter enabled any infectious virus remaining post-exposure/neutralization to amplify in Vero cells in a six-well plate format, with up to two passages onto fresh cells performed for negative wells.This additional test was used to confirm that conditions scored negative in the TCID 50 titration assay were, in fact, free of infectious virus.
In a recently published preprint 25 , Shaffer et al. have reported on the persistence of LASV Josiah and Sauerwald isolates on hard surfaces and in water.Approximately 1.9 log 10 reduction in titer per day was observed on high-density polyethylene (HDPE) and stainless steel surfaces for the Josiah isolate, and approximately 1.2 log 10 per day on these surfaces for the Sauerwald isolate.These data indicate that surface contamination with infectious LASV could persist for days, depending on the initial titer of the deposited virus.Decay rates for the two isolates in deionized water (0.1 to 0.15 log 10 per day) and wastewater (0.6 to 0.8 log 10 per day) were observed 25 .Inactivation of the two LASV isolates by sodium hypochlorite (1, 5, or 10 mg/L [ppm]) was concentration dependent, with the Sauerwald isolate displaying greater susceptibility to inactivation, with no reasons for this difference being offered in the paper 25 .Greater than 4 log 10 inactivation of LASV occurred within 5 min contact time with 1 to 10 mg/L [ppm] sodium hypochlorite for each isolate 25 .These sodium hypochlorite concentrations are quite low, compared to the concentration used in our study (0.5% [5000 ppm]), and to concentrations proposed previously for use against LASV (0.5-1%) 26 .
In conclusion, we have provided empirical evidence of the virucidal efficacy of commonly employed microbicidal actives (ethanol and sodium hypochlorite) and formulations of microbicidal actives (AHP, PCMX, and dual QAC) for LASV.Each of these, at the appropriate concentration and contact time, was capable of reducing

Figure 1 .
Figure 1.Schematic representation of the suspension inactivation efficacy testing methodology performed using neutralization by VCM (minimal essential medium + 2% fetal calf serum + 10 units per mL penicillin/ streptomycin) alone.The entire procedure was performed three times for each microbicide evaluated, in three technical replicates each, as depicted.

Figure 2 .
Figure 2. Schematic representation of the suspension inactivation efficacy testing methodology using VCM (minimal essential medium + 2% fetal calf serum + 10 units per mL penicillin/streptomycin) and mechanical neutralization using Amicon Spin Columns.The entire procedure was performed three times for each microbicide evaluated, in three technical replicates each, as depicted.

Figure 3 .
Figure 3. Efficacy of 0.5% sodium hypochlorite for inactivating Lassa virus in suspension.Error bars indicate standard deviation of the mean for n = 3 independent studies with 3 technical replicates each.

Figure 4 .
Figure 4. Efficacy of 67% ethanol for inactivating Lassa virus in suspension.Error bars indicate standard deviation of the mean for n = 3 independent studies with 3 technical replicates each.

Figure 5 .
Figure 5. Efficacy of a 2% dual quaternary ammonium compound (QAC) formulation for inactivating Lassa virus in suspension.The limit of detection of the titration assay (1.8 × 10 1 TCID 50 per mL) is indicated by the solid blue horizontal line.Error bars indicate standard deviation of the mean for n = 3 independent studies with 3 technical replicates each.

Figure 6 .Table 4 .
Figure 6.Efficacy of an accelerated hydrogen peroxide formulation (AHP; 1:40 dilution) for inactivating Lassa virus in suspension.Error bars indicate standard deviation of the mean for n = 3 independent studies with 3 technical replicates each.

Figure 7 .
Figure 7. Efficacy of p-chloro-m-xylenol (PCMX) at test concentrations of 0.04, 0.06, and 0.12% for inactivating Lassa virus in suspension.Error bars indicate standard deviation of the mean for n = 3 independent studies with 3 technical replicates each.

Table 2 .
Safety plate test for inactivation of Lassa virus by 0.5% sodium hypochlorite in suspension.GFP, green fluorescent protein; + , GFP detected; −, GFP not detected.