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Journal of Hospital Infection
Volume 166, December 2025, Pages 5-11
Stability of Andes virus and its inactivation by WHO-recommended hand-rub formulations and surface disinfectants
Author links open overlay panelB.E. Nilsson-Payant a b c, R.F. Dafi d e f, S. Krüger g, M. Rosenthal g h i, D. Todt j k l, M.M. Addo d e f, E. Steinmann l m, T.L. Meister d e f
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https://doi.org/10.1016/j.jhin.2025.08.010Get rights and content
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Summary
Background
Hantaviruses are responsible for thousands of infections globally. In the absence of vaccines or targeted treatments for severe diseases such as haemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome, medical care focuses solely on managing symptoms. As a result, preventive hygiene measures, such as disinfection, are essential to reduce transmission and minimize the virus's impact on human health.
Aim
The aim of this study was to assess the effectiveness of commonly used disinfectants in inactivating Andes virus (ANDV) and evaluate the virus's stabilty to support evidence-based hygiene strategies.
Methods
The virucidal activity of hand antiseptics against ANDV was assessed using a quantitative suspension test in accordance with the European guideline EN14476. Surface disinfection efficacy was evaluated according to EN16777 and virus stability was assessed on stainless steel.
Conclusion
These findings indicate that ANDV has lower environmental stability compared to other enveloped viruses and can be effectively inactivated by all disinfectants tested. This data can inform risk assessments, especially in areas where hantavirus outbreaks are common, and may guide cleaning protocols for both healthcare environments and high-risk public spaces.
Keywords
Hantavirus
ANDV
Disinfection
Hand-rub formulations
Surface disinfection
ANDV stability
Stainless steel
Introduction
Hantaviruses can cause serious illnesses such as haemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS). These viruses are geographically widespread and can generally be classified as ‘Old World’ hantaviruses, which are predominantly found in Europe and Asia, and ‘New World’ hantaviruses, which are primarily found in the Americas [1].
The primary mode of transmission is through contact with infected rodents, which shed the virus in their faeces, urine, and saliva. Humans primarily become infected by inhaling aerosolized particles from contaminated dust or food [1]. Globally, it is estimated that up to 200,000 hantavirus infections occur each year, with almost 90% of cases being reported in China, highlighting the virus's status as a significant emerging infectious disease [2]. In Europe, 4048 hantavirus cases were reported in 2019, with Germany accounting for 38% of these cases [3]. However, due to the asymptomatic nature or generic flu-like symptoms, it is thought that actual human case numbers are significantly higher. While hantaviruses were initially regarded as strictly zoonotic, reports suggests that the New World Andes virus (ANDV) can also be transmitted between humans [1,4,5]. The risk of ANDV transmission between humans increases with prolonged close contact with symptomatic patients, raising the likelihood of nosocomial infections in healthcare settings [4]. Indeed, sporadic nosocomial transmission events occurred in several hospitals [1].
Efforts to develop medical countermeasures are ongoing, but, with no effective vaccines currently available, hygiene and infection control measures are especially important [1]. Currently there are no standardized guidelines for ANDV decontamination.
Therefore, the aim of this study was to compare the virucidal efficacy of World Health Organization (WHO)-recommended hand-rub formulations, based on ethanol and 2-propanol, against ANDV. This study also aimed to investigate the stability of ANDV on stainless steel and to review its inactivation by surface disinfectants.
Methods
Cell and virus culture
VeroE6 cells (ATCC CRL-1586) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin at 37 °C and 5% CO2.
ANDV isolate Chile-9717869 (donated by Piet Maes, KU Leuven, Belgium) was propagated in VeroE6 cells in DMEM supplemented with only 2% FBS and virus-containing medium was collected at 7 and 10 days post infection (dpi). Cell debris were removed by centrifugation (4000 g, 10 min, 4 °C) and virus stocks were concentrated using Amicon Ultra-15 centrifugal filter units (100 kDa molecular weight cut-off).
Immuno-focus assays
Infectious viral titres were determined by immuno-focus assays in VeroE6 cells. In brief, virus stocks and recovered virus solutions were serially diluted 10-fold (100 to 10−5) and used to infect VeroE6 cells for 1 h at 37 °C. Cells were subsequently overlayed with either a semi-liquid 1.2% Avicel overlay in Minimum Essential Medium (MEM) supplemented with 4% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin, 10 mM HEPES and 1×MEM non-essential amino acid solution (NEAA) or 2.8% methylcellulose diluted 1:3 with DMEM supplemented with 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin, 1×NEAA and 200 mmol/mL l-glutamine and incubated at 37 °C. At 7 or 12 dpi, overlays were removed and cells were fixed with 4% formaldehyde in phosphate-buffered saline (PBS) for 30 min at room temperature prior to permeabilization in 0.5% Triton X-100 in water for 30 min at room temperature. Cells were stained using the broadly cross-reactive Tula virus (TULV) 1 monoclonal antibody detecting hantavirus nucleoprotein or a cross-reactive Puumala virus (PUUV) monoclonal antibody and an IRDye 800CW-conjugated secondary antibody or an anti-mouse horseradish peroxidase-conjugated secondary antibody, respectively [6]. Near-infrared fluorescence signal of stained plaques was imaged on an Odyssey CLx imaging system (LI-COR) and analysed using the Image Studio software (LI-COR) or foci were counted using brightfield microscopy. Focus-forming units (ffu)/mL were determined.
Quantitative suspension assay
Virucidal activity of WHO-recommended hand-rub formulations I and II (Table I) were assessed based on European guideline EN14476, as described previously [7,8]. Specifically, eight parts of disinfectant or cell culture medium for the untreated control (UTC) were mixed with one-part interfering substance (bovine serum albumin (BSA), final concentration 0.3 g/L, clean condition) and one-part ANDV and incubated for 30 s at room temperature (21 °C). Virus titration by immuno-plaque assay was performed on Vero E6 cells to determine remaining infectious viral titres. Cell viability was assessed following a similar protocol using one-part of 1×PBS instead of virus. WHO formulations I and II were tested at final concentrations of 20%, 30%, 40%, 60% and 80%. The experiment was performed in three independent replicates.
Table I. Inactivation capacity of WHO-recommended hand rub formulations I and II
Concentrations refer to those used in the quantitative suspension test; ‘incubation time’ indicates the duration of virus exposure to each respective concentration.
ANDV stability testing
Stainless steel discs (2 cm diameter discs, article no. 4174–3000; GK Formblech GmbH, Berlin, Germany) were decontaminated in 70% (v/v) ethanol for 15 min and subsequently contaminated with 50 μL virus solution containing nine parts ANDV (9×105 ffu) and one-part interfering substance (BSA, final concentration 0.3 g/L). After visible drying of the inoculum (1 h), the steel discs were stored in the dark at ∼21 °C. Virus was recovered at indicated timepoints post contamination (0 h, 8 h, 1 d, 2 d, 3 d, and 5 d post contamination) by transferring three steel discs of each condition into a 25 mL container harbouring 1.5 mL cell culture medium (without FCS) and subsequent vortexing for 60 s. Immuno-focus assays were performed on VeroE6 cells to determine the remaining infectious viral titres.
Inactivation of ANDV by surface disinfectants
Five different commercially available surface disinfectants were tested regarding their potential to inactivate ANDV. Depending on their active compounds (Table II), the disinfectants can be subdivided into alcohol-based products: Bacillol AF (450 mg/g 1-propanol, 250 mg/g 2-propanol, 47 mg/g ethanol), Antifect N liquid (250 mg/g ethanol, 350 mg/g 2-propanol), aldehyde-based products: Kohrsolin FF (50 mg/g glutaraldehyde, 30 mg/g benzylalkyldimethylammonium chloride, 30 mg/g didecyldimethylammonium chloride), Incidin Rapid (98 mg/g glutaraldehyde, 50 mg/g alkyldimethylbenzylammonium chloride, 50 mg/g didecyldimethylammonium chloride) and hydrogen peroxide-based products: Incidin OxyFoam (15 mg/g hydrogen peroxide).
Table II. Inactivation capacity of surface disinfectants
Concentrations refer to those used during the inactivation testing; ‘incubation time’ indicates the duration of virus exposure to each respective surface disinfectant.
Stainless steel discs were decontaminated as mentioned above. Subsequently, the stainless steel discs were contaminated with 50 μL virus solution containing nine parts ANDV and one part interfering substance (BSA, final concentration 0.3 g/L, clean condition). After visible drying of the inoculum (1 h), 100 μL of surface disinfectant was applied on to the carrier and incubated at indicated concentrations for indicated timeperiods at room temperature (21 °C, Table II). Hereafter, the specimens were transferred into a 25 mL container harbouring 2 mL cell culture medium (without FCS) and vortexed for 60 s to resuspend the virus. Immuno-plaque assays were performed on VeroE6 cells to determine the remaining infectious viral titres. Cell viability was assessed following a similar protocol using 100 μL of 1×PBS instead of disinfectant.
Statistical analysis
Inactivation kinetics of ANDV by WHO formulations I and II were compared to other enveloped viruses such as Zika virus, Ebola virus and Chikungunya virus as well as the reference virus Modified Vaccinia Ankara using robust Hill non-linear dose–response fit. This approach allows us to express inactivation efficacy on a standardized scale ranging from 0 to 100% inactivation, making cross-virus comparisons feasible. Results
Efficient inactivation of ANDV by WHO-recommended hand-rub formulations I and II
Hand antisepsis plays a central role in infection prevention and control, particularly for viruses capable of indirect transmission. Since viral susceptibility to alcohol varies depending on structural and biochemical properties, it is essential to evaluate the inactivation kinetics of individual pathogens.
Here, we evaluated the inactivation capacity of WHO-recommended hand-rub formulations I and II based on ethanol and 2-propanol, respectively (Figure 1). In a quantitative suspension test according to EN14476, ANDV was exposed to WHO-recommended hand-rub formulations I and II for 30 s at room temperature. Subsequently remaining infectious viral loads were determined by immuno-plaque assay.
Figure 1. Inactivation of Andes virus (ANDV) by WHO-recommended hand rub formulations I (A) and II (B). ffu, focus-forming units; UTC, untreated control. Light brown bars: viral titres recovered after exposure to the disinfectants. Each data point indicates one biological replicate. All experiments were performed three times. (C, D) Comparison of inactivation efficacy to other enveloped viruses: EBOV (Ebola virus), CHIKV (Chikungunya virus), HCV (hepatitis C virus), YFV (yellow fever virus), and ZIKV (Zika virus) as well as the reference virus MVA (Modified Vaccinia Ankara) using robust non-linear fitting.
Forty percent of WHO-recommended hand-rub formulation I effectively reduced infectious viral loads to the lower limit of detection (18.18 ffu/mL), whereas 30% did not affect ANDV infectivity (Figure 1A). Formulation II was even more effective, fully reducing viral titres at a concentration as low as 30% (Figure 1B).
Using a robust Hill non-linear regression model, the viral titres of previously tested viruses were normalized to compare their inactivation by varying concentrations of the WHO-recommended hand-rub formulations I and II. The comparison included several enveloped viruses: modified vaccina Ankara (MVA), the reference virus for enveloped viruses, Ebola virus (EBOV), Chikungunya virus (CHIKV), hepatitis C virus (HCV), Yellow Fever virus (YFV), and Zika virus (ZIKV) [[9], [10], [11]]. For both formulations, MVA and CHIKV showed the lowest susceptibility, requiring concentrations above 40% (formulation I) and 30% (formulation II) for complete inactivation (below the limit of quantification). EBOV and HCV were moderately more susceptible, whereas YFV, ZIKV, and ANDV were readily inactivated by 30% of formulation I and just over 20% of formulation II (Figure 1C and D). These data suggest that although all the viruses are classified as enveloped viruses, their susceptibility to alcohols varies significantly, indicating that hygiene measures cannot be universally applied but must be tailored to each specific virus.
Stability of ANDV on stainless steel discs
To better understand the potential for indirect transmission of ANDV, its stability on inanimate surfaces was investigated under controlled environmental conditions. Surface stability is a key parameter when assessing the risk of fomite-mediated transmission, particularly in healthcare and other shared environments.
Therefore, we assessed the environmental stability of ANDV on stainless steel by applying a virus suspension (9×105 ffu/50 μL) to stainless steel discs. After visible drying of the inoculum (1 h), the steel discs were stored in the dark at ∼21 °C and infectious virus was quantified at multiple timepoints (0 h, 8 h, 1 d, 2 d, 3 d, and 5 d post contamination). Viral titres remained relatively stable during the first 8 h but declined significantly – by nearly 3 log10 ffu/mL – after 24 h. After 5 days, detectable levels of infectious virus were still present, corresponding to a half-life of 23.61 h of dried virus on stainless steel at room temperature (Figure 2A).
Figure 2. (A) Stability of Andes virus (ANDV) on stainless steel at room temperature (∼21 °C). Light brown area displays the 95% confidence interval. ffu, focus-forming units. (B–D) Inactivation of ANDV by surface disinfectants. UTC, untreated control. The light brown bars show viral titres recovered after exposure to the surface disinfectants. The dotted black line indicates the lower limit of detection simultaneously representing the cell toxicity of each product. Each data point indicates one biological replicate. The experiment was performed in three independent replicates.
Surface disinfectants inactivate ANDV
To comprehensively assess potential infection control measures, the efficacy of commonly used surface disinfectants against ANDV was evaluated. This was prompted by our finding that infectious ANDV can remain viable on stainless steel surfaces for up to 5 days, highlighting the importance of effective surface decontamination strategies.
The experiment was performed according to the European guideline EN16777, which outlines standardized procedures for testing the virucidal efficacy of disinfectants on surfaces. In brief, ANDV solution was dried on stainless steel discs and subsequently two alcohol-based, two aldehyde-based, and one hydrogen peroxide-based disinfectant were applied to assess viral inactivation.
Within the exposure time and concentration specified by each manufacturer, all disinfectants tested were highly effective against ANDV. All of which achieved complete inactivation of ANDV, reducing viral loads to the lower limit of detection (Figure 2B–D). This suggests that these disinfectants are reliable tools for inactivating ANDV on inanimate surfaces, helping reduce transmission risk in both healthcare and other high-contact environments.
Discussion
In recent years, viruses from both known and newly identified species caused localized outbreaks and even global pandemics. Often, science struggles to keep pace in the development of new treatments or vaccines. In the interim, hygiene measures frequently serve as the primary line of defence. These practices vary depending on the specific virus; however, hand and surface disinfection consistently play an important role, especially in healthcare settings, to help prevent transmission. Although nosocomial transmission of ANDV is not frequent, it has been documented [1]. The detection of viral RNA in various body fluids of infected individuals indicates a potential for such transmission and underscores the necessity of considering it as a viable route of infection. Consequently, information about preventive hygiene measures is essential, as these measures may also apply to other members of the Bunvaviricetes class.
Specific data on disinfection for certain hantaviruses, such as ANDV, Hantaan virus (HTNV) or PUUV, remain limited. Thus, we tested inactivation of ANDV by WHO-recommended hand-rub formulations I and II based on ethanol and 2-propanol, respectively, to inform risk assessments aimed at minimizing human-to-human transmission. Both formulations effectively inactivated ANDV within 30 s of exposure. Even reducing concentrations to 40% and 30% for formulations I and II, respectively, was sufficient to completely abolish the detection of infectious virus, placing ANDV among a group of viruses less resistant to alcohol such as ZIKV, YFV, and RSV. These viruses share the common feature of being enveloped, making them highly susceptible to alcohol, which disrupts the viral envelope and renders the virus non-infectious. Interestingly, a previous study found that HTNV partially survives 30% but not 40% ethanol treatment for 2 min at room temperature [12].
Additional factors that influence the likelihood of direct transmission include environmental stability of the virus. Here, we found that ANDV exhibits stability on stainless steel similar to SARS-CoV-2, while the half-life of mpox virus (MPXV) ranges between 6–7 days [13,14]. A previous study found that the Old World hantaviruses PUUV and TULV exhibited significantly increased stability in solution compared to being dried on glass surfaces [15]. Similarly, another study detected infectious HTNV in solutions up to nine days when stored at room temperature, compared to a 90% loss of infectious loads after 90 min and no detectable virus after 24 h after drying on steel discs [12]. By contrast, PUUV was shown to retain its infectivity for 12–15 days at room temperature in secreted faeces of experimentally infected bank voles, the natural host reservoir for the virus, suggesting that virus stability differs notably depending on the environmental context to which the virus is exposed [15]. In general, lowering the temperature can significantly enhance the stability of viruses [14]. At 37 °C, HTNV, SFSV, and CCHFV in solution remained infectious for at least 8 days, 18 days, and 7 h, respectively. At 20 °C, the periods of infectivity increased to 9, 40, and 11 days. At 4 °C, survival was further prolonged to 96, 528, and 15 days, respectively [12]. Similarly, PUUV and TULV remained infectious for 5–11 days at room temperature and up to 18 days at 4 °C, but were completely inactivated within 24 h at 37 °C [15]. Other environmental factors such as humidity, light exposure, pH level but also protein contamination affect virus stability [14,[16], [17], [18], [19], [20]]. Furthermore, the type of surface material and its porosity also influence virus stability and recovery rates. Thus, viruses on paper, cotton, and wood may be less stable than viruses on stainless steel, plastic, or glass [13]. Future studies are required to assess the stability of ANDV on other surfaces and to evaluate the risk of infection, which would also require investigating the minimum infectious dose.
Given that the majority of viruses tested can survive on inanimate surfaces for a certain period of time depending on different viral and environmental factors, contaminated surfaces pose a significant risk of transmission. Therefore, we tested commercially available surface disinfectants based on alcohol, aldehyde, and hydrogen peroxide to help reduce this transmission risk. By contrast with viruses such as MPXV, ANDV was effectively inactivated by every surface disinfectant tested [14]. While aldehyde-based disinfectants were among the effective options, they are generally not recommended for routine surface disinfection due to toxicological concerns. Commercial disinfectants such as Clidox®, Halamid-d®, and Virkon® S have also been shown to be effective for decontaminating surfaces potentially contaminated with PUUV [21].
In conclusion, our data suggest that ANDV is efficiently inactivated by all disinfectants tested, validating their potential utility in the context of the healthcare system and outbreak scenarios. ANDV may also serve as a use case for other members of the Bunyaviricetes class. However, though these laboratory-based assessments provide important preliminary data, real-world application requires further validation. Future studies should include standardized in-vivo efficacy testing, such as the four-field test (EN 16615) for surface disinfectants, and the fingerpad method developed by Sattar for evaluating the performance of hand antiseptics under practical conditions [22].
Volume 166, December 2025, Pages 5-11
Stability of Andes virus and its inactivation by WHO-recommended hand-rub formulations and surface disinfectants
Author links open overlay panelB.E. Nilsson-Payant a b c, R.F. Dafi d e f, S. Krüger g, M. Rosenthal g h i, D. Todt j k l, M.M. Addo d e f, E. Steinmann l m, T.L. Meister d e f
Show more
Add to Mendeley
Share
Cite
https://doi.org/10.1016/j.jhin.2025.08.010Get rights and content
Under a Creative Commons license
Open access
Summary
Background
Hantaviruses are responsible for thousands of infections globally. In the absence of vaccines or targeted treatments for severe diseases such as haemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome, medical care focuses solely on managing symptoms. As a result, preventive hygiene measures, such as disinfection, are essential to reduce transmission and minimize the virus's impact on human health.
Aim
The aim of this study was to assess the effectiveness of commonly used disinfectants in inactivating Andes virus (ANDV) and evaluate the virus's stabilty to support evidence-based hygiene strategies.
Methods
The virucidal activity of hand antiseptics against ANDV was assessed using a quantitative suspension test in accordance with the European guideline EN14476. Surface disinfection efficacy was evaluated according to EN16777 and virus stability was assessed on stainless steel.
Conclusion
These findings indicate that ANDV has lower environmental stability compared to other enveloped viruses and can be effectively inactivated by all disinfectants tested. This data can inform risk assessments, especially in areas where hantavirus outbreaks are common, and may guide cleaning protocols for both healthcare environments and high-risk public spaces.
Keywords
Hantavirus
ANDV
Disinfection
Hand-rub formulations
Surface disinfection
ANDV stability
Stainless steel
Introduction
Hantaviruses can cause serious illnesses such as haemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS). These viruses are geographically widespread and can generally be classified as ‘Old World’ hantaviruses, which are predominantly found in Europe and Asia, and ‘New World’ hantaviruses, which are primarily found in the Americas [1].
The primary mode of transmission is through contact with infected rodents, which shed the virus in their faeces, urine, and saliva. Humans primarily become infected by inhaling aerosolized particles from contaminated dust or food [1]. Globally, it is estimated that up to 200,000 hantavirus infections occur each year, with almost 90% of cases being reported in China, highlighting the virus's status as a significant emerging infectious disease [2]. In Europe, 4048 hantavirus cases were reported in 2019, with Germany accounting for 38% of these cases [3]. However, due to the asymptomatic nature or generic flu-like symptoms, it is thought that actual human case numbers are significantly higher. While hantaviruses were initially regarded as strictly zoonotic, reports suggests that the New World Andes virus (ANDV) can also be transmitted between humans [1,4,5]. The risk of ANDV transmission between humans increases with prolonged close contact with symptomatic patients, raising the likelihood of nosocomial infections in healthcare settings [4]. Indeed, sporadic nosocomial transmission events occurred in several hospitals [1].
Efforts to develop medical countermeasures are ongoing, but, with no effective vaccines currently available, hygiene and infection control measures are especially important [1]. Currently there are no standardized guidelines for ANDV decontamination.
Therefore, the aim of this study was to compare the virucidal efficacy of World Health Organization (WHO)-recommended hand-rub formulations, based on ethanol and 2-propanol, against ANDV. This study also aimed to investigate the stability of ANDV on stainless steel and to review its inactivation by surface disinfectants.
Methods
Cell and virus culture
VeroE6 cells (ATCC CRL-1586) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin at 37 °C and 5% CO2.
ANDV isolate Chile-9717869 (donated by Piet Maes, KU Leuven, Belgium) was propagated in VeroE6 cells in DMEM supplemented with only 2% FBS and virus-containing medium was collected at 7 and 10 days post infection (dpi). Cell debris were removed by centrifugation (4000 g, 10 min, 4 °C) and virus stocks were concentrated using Amicon Ultra-15 centrifugal filter units (100 kDa molecular weight cut-off).
Immuno-focus assays
Infectious viral titres were determined by immuno-focus assays in VeroE6 cells. In brief, virus stocks and recovered virus solutions were serially diluted 10-fold (100 to 10−5) and used to infect VeroE6 cells for 1 h at 37 °C. Cells were subsequently overlayed with either a semi-liquid 1.2% Avicel overlay in Minimum Essential Medium (MEM) supplemented with 4% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin, 10 mM HEPES and 1×MEM non-essential amino acid solution (NEAA) or 2.8% methylcellulose diluted 1:3 with DMEM supplemented with 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin, 1×NEAA and 200 mmol/mL l-glutamine and incubated at 37 °C. At 7 or 12 dpi, overlays were removed and cells were fixed with 4% formaldehyde in phosphate-buffered saline (PBS) for 30 min at room temperature prior to permeabilization in 0.5% Triton X-100 in water for 30 min at room temperature. Cells were stained using the broadly cross-reactive Tula virus (TULV) 1 monoclonal antibody detecting hantavirus nucleoprotein or a cross-reactive Puumala virus (PUUV) monoclonal antibody and an IRDye 800CW-conjugated secondary antibody or an anti-mouse horseradish peroxidase-conjugated secondary antibody, respectively [6]. Near-infrared fluorescence signal of stained plaques was imaged on an Odyssey CLx imaging system (LI-COR) and analysed using the Image Studio software (LI-COR) or foci were counted using brightfield microscopy. Focus-forming units (ffu)/mL were determined.
Quantitative suspension assay
Virucidal activity of WHO-recommended hand-rub formulations I and II (Table I) were assessed based on European guideline EN14476, as described previously [7,8]. Specifically, eight parts of disinfectant or cell culture medium for the untreated control (UTC) were mixed with one-part interfering substance (bovine serum albumin (BSA), final concentration 0.3 g/L, clean condition) and one-part ANDV and incubated for 30 s at room temperature (21 °C). Virus titration by immuno-plaque assay was performed on Vero E6 cells to determine remaining infectious viral titres. Cell viability was assessed following a similar protocol using one-part of 1×PBS instead of virus. WHO formulations I and II were tested at final concentrations of 20%, 30%, 40%, 60% and 80%. The experiment was performed in three independent replicates.
Table I. Inactivation capacity of WHO-recommended hand rub formulations I and II
| I | 80% ethanol (v/v) 1.45% glycerol (v/v) 0.125% hydrogen peroxide (v/v) |
20, 30, 40, 60, 80 | 30 |
| II | 75% 2-propanol (v/v) 1.45% glycerol (v/v) 0.125% hydrogen peroxide (v/v) |
20, 30, 40, 60, 80 | 30 |
ANDV stability testing
Stainless steel discs (2 cm diameter discs, article no. 4174–3000; GK Formblech GmbH, Berlin, Germany) were decontaminated in 70% (v/v) ethanol for 15 min and subsequently contaminated with 50 μL virus solution containing nine parts ANDV (9×105 ffu) and one-part interfering substance (BSA, final concentration 0.3 g/L). After visible drying of the inoculum (1 h), the steel discs were stored in the dark at ∼21 °C. Virus was recovered at indicated timepoints post contamination (0 h, 8 h, 1 d, 2 d, 3 d, and 5 d post contamination) by transferring three steel discs of each condition into a 25 mL container harbouring 1.5 mL cell culture medium (without FCS) and subsequent vortexing for 60 s. Immuno-focus assays were performed on VeroE6 cells to determine the remaining infectious viral titres.
Inactivation of ANDV by surface disinfectants
Five different commercially available surface disinfectants were tested regarding their potential to inactivate ANDV. Depending on their active compounds (Table II), the disinfectants can be subdivided into alcohol-based products: Bacillol AF (450 mg/g 1-propanol, 250 mg/g 2-propanol, 47 mg/g ethanol), Antifect N liquid (250 mg/g ethanol, 350 mg/g 2-propanol), aldehyde-based products: Kohrsolin FF (50 mg/g glutaraldehyde, 30 mg/g benzylalkyldimethylammonium chloride, 30 mg/g didecyldimethylammonium chloride), Incidin Rapid (98 mg/g glutaraldehyde, 50 mg/g alkyldimethylbenzylammonium chloride, 50 mg/g didecyldimethylammonium chloride) and hydrogen peroxide-based products: Incidin OxyFoam (15 mg/g hydrogen peroxide).
Table II. Inactivation capacity of surface disinfectants
| Antifect N liquid | 250 mg/g ethanol, 350 mg/g 2-propanol |
100% | 30 s |
| Bacillol AF | 450 mg/g 1-propanol, 250 mg/g 2-propanol, 47 mg/g ethanol |
100% | 30 s |
| Kohrsolin FF | 50 mg/g glutaraldehyde, 30 mg/g benzylalkyldimethyl-ammonium chloride, 30 mg/g didecyldimethyl-ammonium chloride |
0.5% | 5 min |
| Incidin Rapid | 98 mg/g glutaraldehyde, 50 mg/g alkyldimethylbenzyl-ammonium chloride, 50 mg/g didecyldimethyl-ammonium chloride |
0.5% | 5 min |
| Incidin OxyFoam | 15 mg/g hydrogen peroxide | 100% | 30 s |
Stainless steel discs were decontaminated as mentioned above. Subsequently, the stainless steel discs were contaminated with 50 μL virus solution containing nine parts ANDV and one part interfering substance (BSA, final concentration 0.3 g/L, clean condition). After visible drying of the inoculum (1 h), 100 μL of surface disinfectant was applied on to the carrier and incubated at indicated concentrations for indicated timeperiods at room temperature (21 °C, Table II). Hereafter, the specimens were transferred into a 25 mL container harbouring 2 mL cell culture medium (without FCS) and vortexed for 60 s to resuspend the virus. Immuno-plaque assays were performed on VeroE6 cells to determine the remaining infectious viral titres. Cell viability was assessed following a similar protocol using 100 μL of 1×PBS instead of disinfectant.
Statistical analysis
Inactivation kinetics of ANDV by WHO formulations I and II were compared to other enveloped viruses such as Zika virus, Ebola virus and Chikungunya virus as well as the reference virus Modified Vaccinia Ankara using robust Hill non-linear dose–response fit. This approach allows us to express inactivation efficacy on a standardized scale ranging from 0 to 100% inactivation, making cross-virus comparisons feasible. Results
Efficient inactivation of ANDV by WHO-recommended hand-rub formulations I and II
Hand antisepsis plays a central role in infection prevention and control, particularly for viruses capable of indirect transmission. Since viral susceptibility to alcohol varies depending on structural and biochemical properties, it is essential to evaluate the inactivation kinetics of individual pathogens.
Here, we evaluated the inactivation capacity of WHO-recommended hand-rub formulations I and II based on ethanol and 2-propanol, respectively (Figure 1). In a quantitative suspension test according to EN14476, ANDV was exposed to WHO-recommended hand-rub formulations I and II for 30 s at room temperature. Subsequently remaining infectious viral loads were determined by immuno-plaque assay.
Figure 1. Inactivation of Andes virus (ANDV) by WHO-recommended hand rub formulations I (A) and II (B). ffu, focus-forming units; UTC, untreated control. Light brown bars: viral titres recovered after exposure to the disinfectants. Each data point indicates one biological replicate. All experiments were performed three times. (C, D) Comparison of inactivation efficacy to other enveloped viruses: EBOV (Ebola virus), CHIKV (Chikungunya virus), HCV (hepatitis C virus), YFV (yellow fever virus), and ZIKV (Zika virus) as well as the reference virus MVA (Modified Vaccinia Ankara) using robust non-linear fitting.
Forty percent of WHO-recommended hand-rub formulation I effectively reduced infectious viral loads to the lower limit of detection (18.18 ffu/mL), whereas 30% did not affect ANDV infectivity (Figure 1A). Formulation II was even more effective, fully reducing viral titres at a concentration as low as 30% (Figure 1B).
Using a robust Hill non-linear regression model, the viral titres of previously tested viruses were normalized to compare their inactivation by varying concentrations of the WHO-recommended hand-rub formulations I and II. The comparison included several enveloped viruses: modified vaccina Ankara (MVA), the reference virus for enveloped viruses, Ebola virus (EBOV), Chikungunya virus (CHIKV), hepatitis C virus (HCV), Yellow Fever virus (YFV), and Zika virus (ZIKV) [[9], [10], [11]]. For both formulations, MVA and CHIKV showed the lowest susceptibility, requiring concentrations above 40% (formulation I) and 30% (formulation II) for complete inactivation (below the limit of quantification). EBOV and HCV were moderately more susceptible, whereas YFV, ZIKV, and ANDV were readily inactivated by 30% of formulation I and just over 20% of formulation II (Figure 1C and D). These data suggest that although all the viruses are classified as enveloped viruses, their susceptibility to alcohols varies significantly, indicating that hygiene measures cannot be universally applied but must be tailored to each specific virus.
Stability of ANDV on stainless steel discs
To better understand the potential for indirect transmission of ANDV, its stability on inanimate surfaces was investigated under controlled environmental conditions. Surface stability is a key parameter when assessing the risk of fomite-mediated transmission, particularly in healthcare and other shared environments.
Therefore, we assessed the environmental stability of ANDV on stainless steel by applying a virus suspension (9×105 ffu/50 μL) to stainless steel discs. After visible drying of the inoculum (1 h), the steel discs were stored in the dark at ∼21 °C and infectious virus was quantified at multiple timepoints (0 h, 8 h, 1 d, 2 d, 3 d, and 5 d post contamination). Viral titres remained relatively stable during the first 8 h but declined significantly – by nearly 3 log10 ffu/mL – after 24 h. After 5 days, detectable levels of infectious virus were still present, corresponding to a half-life of 23.61 h of dried virus on stainless steel at room temperature (Figure 2A).
Figure 2. (A) Stability of Andes virus (ANDV) on stainless steel at room temperature (∼21 °C). Light brown area displays the 95% confidence interval. ffu, focus-forming units. (B–D) Inactivation of ANDV by surface disinfectants. UTC, untreated control. The light brown bars show viral titres recovered after exposure to the surface disinfectants. The dotted black line indicates the lower limit of detection simultaneously representing the cell toxicity of each product. Each data point indicates one biological replicate. The experiment was performed in three independent replicates.
Surface disinfectants inactivate ANDV
To comprehensively assess potential infection control measures, the efficacy of commonly used surface disinfectants against ANDV was evaluated. This was prompted by our finding that infectious ANDV can remain viable on stainless steel surfaces for up to 5 days, highlighting the importance of effective surface decontamination strategies.
The experiment was performed according to the European guideline EN16777, which outlines standardized procedures for testing the virucidal efficacy of disinfectants on surfaces. In brief, ANDV solution was dried on stainless steel discs and subsequently two alcohol-based, two aldehyde-based, and one hydrogen peroxide-based disinfectant were applied to assess viral inactivation.
Within the exposure time and concentration specified by each manufacturer, all disinfectants tested were highly effective against ANDV. All of which achieved complete inactivation of ANDV, reducing viral loads to the lower limit of detection (Figure 2B–D). This suggests that these disinfectants are reliable tools for inactivating ANDV on inanimate surfaces, helping reduce transmission risk in both healthcare and other high-contact environments.
Discussion
In recent years, viruses from both known and newly identified species caused localized outbreaks and even global pandemics. Often, science struggles to keep pace in the development of new treatments or vaccines. In the interim, hygiene measures frequently serve as the primary line of defence. These practices vary depending on the specific virus; however, hand and surface disinfection consistently play an important role, especially in healthcare settings, to help prevent transmission. Although nosocomial transmission of ANDV is not frequent, it has been documented [1]. The detection of viral RNA in various body fluids of infected individuals indicates a potential for such transmission and underscores the necessity of considering it as a viable route of infection. Consequently, information about preventive hygiene measures is essential, as these measures may also apply to other members of the Bunvaviricetes class.
Specific data on disinfection for certain hantaviruses, such as ANDV, Hantaan virus (HTNV) or PUUV, remain limited. Thus, we tested inactivation of ANDV by WHO-recommended hand-rub formulations I and II based on ethanol and 2-propanol, respectively, to inform risk assessments aimed at minimizing human-to-human transmission. Both formulations effectively inactivated ANDV within 30 s of exposure. Even reducing concentrations to 40% and 30% for formulations I and II, respectively, was sufficient to completely abolish the detection of infectious virus, placing ANDV among a group of viruses less resistant to alcohol such as ZIKV, YFV, and RSV. These viruses share the common feature of being enveloped, making them highly susceptible to alcohol, which disrupts the viral envelope and renders the virus non-infectious. Interestingly, a previous study found that HTNV partially survives 30% but not 40% ethanol treatment for 2 min at room temperature [12].
Additional factors that influence the likelihood of direct transmission include environmental stability of the virus. Here, we found that ANDV exhibits stability on stainless steel similar to SARS-CoV-2, while the half-life of mpox virus (MPXV) ranges between 6–7 days [13,14]. A previous study found that the Old World hantaviruses PUUV and TULV exhibited significantly increased stability in solution compared to being dried on glass surfaces [15]. Similarly, another study detected infectious HTNV in solutions up to nine days when stored at room temperature, compared to a 90% loss of infectious loads after 90 min and no detectable virus after 24 h after drying on steel discs [12]. By contrast, PUUV was shown to retain its infectivity for 12–15 days at room temperature in secreted faeces of experimentally infected bank voles, the natural host reservoir for the virus, suggesting that virus stability differs notably depending on the environmental context to which the virus is exposed [15]. In general, lowering the temperature can significantly enhance the stability of viruses [14]. At 37 °C, HTNV, SFSV, and CCHFV in solution remained infectious for at least 8 days, 18 days, and 7 h, respectively. At 20 °C, the periods of infectivity increased to 9, 40, and 11 days. At 4 °C, survival was further prolonged to 96, 528, and 15 days, respectively [12]. Similarly, PUUV and TULV remained infectious for 5–11 days at room temperature and up to 18 days at 4 °C, but were completely inactivated within 24 h at 37 °C [15]. Other environmental factors such as humidity, light exposure, pH level but also protein contamination affect virus stability [14,[16], [17], [18], [19], [20]]. Furthermore, the type of surface material and its porosity also influence virus stability and recovery rates. Thus, viruses on paper, cotton, and wood may be less stable than viruses on stainless steel, plastic, or glass [13]. Future studies are required to assess the stability of ANDV on other surfaces and to evaluate the risk of infection, which would also require investigating the minimum infectious dose.
Given that the majority of viruses tested can survive on inanimate surfaces for a certain period of time depending on different viral and environmental factors, contaminated surfaces pose a significant risk of transmission. Therefore, we tested commercially available surface disinfectants based on alcohol, aldehyde, and hydrogen peroxide to help reduce this transmission risk. By contrast with viruses such as MPXV, ANDV was effectively inactivated by every surface disinfectant tested [14]. While aldehyde-based disinfectants were among the effective options, they are generally not recommended for routine surface disinfection due to toxicological concerns. Commercial disinfectants such as Clidox®, Halamid-d®, and Virkon® S have also been shown to be effective for decontaminating surfaces potentially contaminated with PUUV [21].
In conclusion, our data suggest that ANDV is efficiently inactivated by all disinfectants tested, validating their potential utility in the context of the healthcare system and outbreak scenarios. ANDV may also serve as a use case for other members of the Bunyaviricetes class. However, though these laboratory-based assessments provide important preliminary data, real-world application requires further validation. Future studies should include standardized in-vivo efficacy testing, such as the four-field test (EN 16615) for surface disinfectants, and the fingerpad method developed by Sattar for evaluating the performance of hand antiseptics under practical conditions [22].
