Concentration- and Time-Dependent Virucidal Responses of Major Transboundary Animal Disease Viruses to Disinfectants

Abstract

Transboundary animal diseases (TADs) pose persistent threats to global livestock production, and chemical disinfection remains a critical component of biosecurity. However, virucidal efficacy is commonly assessed using single-condition endpoints, limiting comparative interpretation across biologically heterogeneous viruses. In this study, an experimental framework explicitly structured across virus species, disinfectant concentration, and contact time was applied to systematically compare virucidal response patterns across four major TAD viruses—avian influenza virus, African swine fever virus, foot-and-mouth disease virus, and lumpy skin disease virus. Four representative disinfectant active ingredients from distinct chemical classes were evaluated across multiple concentrations and defined contact times using quantitative suspension assays. Virucidal efficacy was quantified using log₁₀ reduction values, and critical concentrations required to achieve ≥4 log₁₀ reduction were derived for comparative analysis. Distinct concentration–response profiles were observed among disinfectant classes, with some ingredients showing relatively consistent activity across viruses, while others exhibited pronounced virus-specific thresholds. Notably, enveloped viruses did not uniformly display higher susceptibility, and extension of contact time enhanced efficacy predominantly in an ingredient-dependent manner. To integrate these multifactorial outcomes at the virus level, a quartile-based analysis was applied, providing a conservative indicator of relative viral resistance across disinfectants and exposure conditions. Overall, these findings demonstrate that virucidal susceptibility is shaped by interactions between disinfectant chemistry and exposure parameters, and support concentration–time-resolved, pattern-based evaluation frameworks—supplemented by quartile-based resistance ranking—beyond single-condition endpoints for assessing disinfectant efficacy against animal viruses.

1. Introduction

Transboundary animal diseases (TADs), including avian influenza (AI), African swine fever (ASF), foot-and-mouth disease (FMD), and lumpy skin disease (LSD), continue to pose major threats to global livestock production, international trade, and food security. Owing to their high transmissibility and substantial socio-economic impact, these diseases are subject to strict international notification and control measures [1,2,3,4]. Regardless of the availability of vaccines or therapeutic interventions, chemical disinfection remains a fundamental component of biosecurity strategies for preventing virus introduction and limiting environmental spread in livestock production systems [5,6,7,8].

Despite being grouped under the common category of TADs, these viruses differ markedly in biological and structural characteristics that are directly relevant to chemical inactivation. Avian influenza virus (AIV) [9,10,11] and foot-and-mouth disease virus (FMDV) [12,13,14] are RNA viruses, whereas African swine fever virus (ASFV) [15,16,17] and lumpy skin disease virus (LSDV) [18,19] possess large double-stranded DNA genomes. In addition, differences in envelope composition, capsid architecture, and environmental stability are expected to influence how rapidly and at what disinfectant concentrations viral inactivation occurs. Consequently, virucidal efficacy cannot be assumed to be uniform across TAD viruses, even when the same disinfectant is applied under nominally identical conditions.

Nevertheless, virucidal efficacy studies for animal viruses have frequently relied on simplified experimental designs, often evaluating a single virus–disinfectant combination at a fixed concentration or a single contact time [6,15,20,21,22]. While such studies are suitable for determining whether predefined efficacy thresholds are achieved, they provide limited information on how virucidal responses change as disinfectant concentration increases or as contact time is extended. As a result, it remains difficult to compare susceptibility among different TAD viruses or to interpret why a disinfectant may appear highly effective against one virus but less effective against another under specific concentration–time conditions.

To overcome these limitations, virucidal efficacy needs to be examined as a function of explicitly defined experimental variables, including virus species, disinfectant concentration, and contact time. In the present study, four major TAD viruses—AIV, ASFV, FMDV, and LSDV—were selected to represent a broad range of genome types, structural features, and environmental resilience. Rather than assessing efficacy at a single endpoint, the study design systematically varied disinfectant concentration and contact time, enabling direct comparison of inactivation kinetics and response patterns among biologically distinct viruses under equivalent experimental conditions.

In parallel, disinfectant active ingredients were selected to represent distinct chemical classes and mechanisms of action, including quaternary ammonium compounds (benzalkonium chloride, BZK) [23,24], aldehyde-based disinfectants (glutaraldehyde, GLT) [25,26], oxidizing agents (potassium peroxymonosulfate, PPMS) [27,28], and organic acids (citric acid, CA) [29,30]. Because these compounds differ in how they interact with viral envelopes, capsids, and nucleic acids, their virucidal effects are expected to depend not only on virus type but also on disinfectant concentration and duration of exposure. Evaluating these active ingredients across multiple concentrations and contact times within a unified experimental matrix allows systematic assessment of how disinfectant chemistry shapes virus-specific inactivation behavior.

The objective of the present study was to generate a virucidal efficacy dataset explicitly structured across virus species, disinfectant concentration, and contact time for four representative TAD viruses and four classes of disinfectant active ingredients. By analyzing how viral inactivation responses change across graded concentrations and defined exposure times, this study compares virus-specific susceptibility patterns under identical experimental conditions. This concentration–time–virus-resolved approach provides a framework for interpreting virucidal efficacy beyond single-condition pass–fail assessments and enables more nuanced comparison of disinfectant performance across heterogeneous animal viruses.

2. Materials and Methods

2.1. Viruses and Cell Lines

Four representative viruses were used in this study: avian influenza virus (AIV; H9N2 strain A/chicken/Republic of Korea/MS96/1996; KVCC-VR1100013), African swine fever virus (ASFV; ASFV-PJ-VaCIn1; KVCC-VR2400015), foot-and-mouth disease virus (FMDV; serotype O, vaccine strain O/SKR/Boeun/2017; KVCC-VR1700004), and lumpy skin disease virus (LSDV; Neethling strain; KVCC-VR2100023). All viruses were obtained from the Korea Veterinary Culture Collection (KVCC, Gimcheon, Republic of Korea) and handled in biosafety level (BSL)-appropriate containment facilities at the Animal and Plant Quarantine Agency (APQA, Gimcheon, Republic of Korea).

AIV was propagated in Madin–Darby canine kidney (MDCK) cells (ATCC^® CCL-34™, Manassas, VA, USA) using a cell culture-based system rather than the conventional embryonated egg method, to maintain consistency across virus propagation platforms for comparative analysis. ASFV, FMDV, and LSDV were propagated in Vero (ATCC^® CCL-81™, Manassas, VA, USA), LFBK (porcine fetal kidney-derived; RRID: CVCL_RX26; KVCC, Republic of Korea), and Madin–Darby bovine kidney (MDBK) (ATCC^® CCL-22™, Manassas, VA, USA) cells, respectively. Infectious titers of all viruses were determined by the tissue culture infectious dose (TCID₅₀) assay, and log reduction values (LRVs) were calculated following disinfectant exposure.

All cell lines were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) or Minimum Essential Medium (MEM) (Corning Inc., Corning, NY, USA) supplemented with 5–10% fetal bovine serum (FBS), Antibiotic–Antimycotic solution (100 U/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL amphotericin B; Gibco, Thermo Fisher Scientific, Waltham, MA, USA), and 200 mM L-glutamine (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). Cultures were incubated at 37 °C in a humidified atmosphere containing 5% CO₂. All virus propagation, titration, and inactivation experiments were conducted in compliance with APQA biosafety regulations and with approval from the institutional biosafety committee.

2.2. Disinfectant Active Ingredients

Four representative active ingredients commonly used in veterinary disinfectants were selected for this study. All reagents were purchased from Sigma–Aldrich (St. Louis, MO, USA) and included benzalkonium chloride (BZK) solution (Cat. No. 63249; ≥50% via Cl, 50% in H₂O), glutaraldehyde (GLT) solution (Cat. No. G5882; Grade I, 25% in H₂O, specially purified for electron microscopy), peroxymonosulfate (PPMS) compound (Oxone^®; Cat. No. 228036; ≥99%), and citric acid (CA) (Cat. No. 251275; ACS reagent grade, ≥99.5%, crystals).

The concentration ranges for each disinfectant and the corresponding ordered levels (L1–L6) were determined through range-finding virucidal screening designed to encompass sub-effective, intermediate, and fully effective conditions relative to the ≥4 log₁₀ reduction criterion. Based on these assessments, CA was tested at 0.375–12.0 g/L, PPMS at 0.25–8.0 g/L, GLT at 0.025–0.8 g/L, and BZK at 0.125–4.0 g/L. Six ordered concentration levels within each range were selected to capture concentration–response patterns with sufficient resolution while maintaining experimental feasibility across multiple viruses and contact times, as summarized in Table 1.

Table 1.

Concentration ranges of disinfectant active ingredients evaluated in this study.

Active Ingredient	Experimental Concentrations (g/L)
Benzalkonium chloride (BZK)	0.125	0.25	0.5	1.0	2.0	4.0
Glutaraldehyde (GLT)	0.025	0.05	0.1	0.2	0.4	0.8
Peroxymonosulfate (PPMS)	0.25	0.5	1.0	2.0	4.0	8.0
Citric acid (CA)	0.375	0.75	1.5	3.0	6.0	12.0
Level *	L1	L2	L3	L4	L5	L6

* Concentration levels (L1–L6) represent ordered, ingredient-specific ranges defined for comparative analysis and do not indicate equivalence of absolute concentrations across compounds.

Each active ingredient was freshly diluted with standard hard water (300 ppm as CaCO₃) to prepare working solutions. All working solutions were prepared immediately prior to testing and used within 24 h. Oxidizing agents such as PPMS were stored in amber glass bottles to minimize light-induced degradation.

Neutralization efficacy and the absence of residual disinfectant activity were confirmed using control recovery assays, ensuring that disinfectant carry-over did not influence viral titration results. Because absolute concentration ranges differed among active ingredients, subsequent analyses and visualizations were performed using ordered concentration levels (L1–L6) to enable comparison of concentration–response patterns without implying direct equivalence of absolute concentrations across compounds.

2.3. Virucidal Efficacy Test Procedure

Virucidal efficacy testing was conducted using a quantitative suspension assay in accordance with the APQA guideline for disinfectant efficacy evaluation, designed to assess the concentration- and time-dependent virucidal activity of disinfectant active ingredients.

Working solutions of each active ingredient were freshly prepared prior to testing. Six concentration levels and three contact times (2, 10, and 30 min) were applied. All experiments were independently conducted in triplicate for each active ingredient and virus.

For each test, the disinfectant working solution and virus suspension were mixed at a 1:1 ratio (500 µL each) to obtain a total reaction volume of 1 mL. The mixture was incubated at 37 °C for the designated contact time (2, 10, or 30 min). Immediately after the contact period, chemical activity was terminated by neutralizing 500 µL of the reaction mixture with an equal volume (500 µL) of cell culture medium containing 10% fetal bovine serum (FBS), used as a neutralizing medium.

The validity and efficiency of the neutralization step, as well as the absence of residual disinfectant activity, were confirmed using neutralization controls and control recovery assays prior to viral titration. The neutralized mixture was then serially tenfold diluted (10⁰–10⁻⁶) and inoculated (100 µL per well) onto host cells appropriate for each virus (MDCK, Vero, LFBK, or MDBK).

Inoculated cells were incubated at 37 °C in a humidified 5% CO₂ atmosphere for 5–7 days, and the presence or absence of cytopathic effect (CPE) was monitored daily. Virus titers were calculated using the Spearman–Kärber method, and virucidal efficacy was determined by comparing the mean viral titers of treated samples with those of untreated virus controls.

According to international standards for virucidal efficacy testing (e.g., EN 14675 [31] and ASTM E1052 [32]), a reduction of ≥4 log₁₀ (corresponding to ≥99.99% inactivation) was considered evidence of effective virucidal activity. To distinguish disinfectant-induced cytotoxicity from virus-induced CPE, parallel cytotoxicity controls were included in which the same concentrations of disinfectant were applied to cells in the absence of virus. Maintenance of cell viability following neutralization was additionally verified under neutralization control conditions.

All procedures were conducted in biosafety level (BSL)-2 or BSL-3 laboratories, depending on the risk classification of each virus, using appropriately certified containment equipment and protocols.

2.4. Data Analysis and Statistical Evaluation

Virucidal efficacy was expressed as the viral reduction value (log₁₀ reduction value, LRV), calculated as the difference between the mean viral titer of the virus control and that of the disinfectant-treated sample. The critical concentration (C*) was defined as an operational screening metric representing the lowest tested concentration achieving ≥4 log₁₀ reduction within the discrete concentration grid, rather than as a continuous estimate of virucidal potency. Failure to achieve the ≥4 log₁₀ reduction threshold within the tested concentration range was treated as a distinct outcome indicative of high viral resistance, rather than as missing data, and was conservatively handled according to the upper limit of the tested range.

For comparative visualization, potency scores were derived from C* values to summarize ingredient- and virus-specific virucidal profiles across contact times. Data are presented as mean ± standard deviation (SD) from three independent experiments.

Differences in virucidal efficacy across active ingredients, concentrations, contact times, and virus species were examined using multifactorial comparative analyses to support pattern-based interpretation rather than formal dose–response modeling. Concentration-dependent trends were evaluated descriptively to facilitate cross-virus and cross-ingredient comparisons of virucidal response patterns. Graphical visualization was performed using line plots, heatmaps, and radar charts.

All statistical analyses and visualizations were conducted using GraphPad Prism 10.2 (GraphPad Software, San Diego, CA, USA) and Microsoft Excel 2021 (Microsoft Corporation, Redmond, WA, USA), with statistical significance set at p < 0.05 where applicable. An overview of the experimental design, including the virus panel, active ingredients, concentration levels, contact times, and replicate structure, is shown in Figure 1.

Figure 1.

Experimental design and overall criterion-based assay framework.

A total of four representative viruses—Avian influenza virus (AIV), African swine fever virus (ASFV), Foot and mouth disease virus (FMDV) and Lumpyskin disease virus (LSDV)—were tested against four active ingredients. Each compound was evaluated at six concentration levels and three contact times with three replicates per condition. Data analysis was based on log reduction value (LRV), with ≥4 log₁₀ reduction (≥99.99%) defined as virucidal activity.

3. Results

3.1. Concentration–Response Patterns Across Viruses and Active Ingredients

Figure 2 illustrates the concentration–response patterns of four disinfectant active ingredients across four viruses using ordered concentration levels (L1–L6) defined independently for each ingredient. This approach emphasizes qualitative differences in response profiles rather than direct comparison of absolute concentrations. The profiles correspond to a 10-min contact time, selected to maximize discrimination of virus- and ingredient-specific response behaviors without widespread saturation.

Figure 2.

Virus-specific concentration–response patterns of disinfectant active ingredients at a fixed contact time of 10 min. Four disinfectant active ingredients—BZK, GLT, PPMS, and CA—were evaluated against AIV, ASFV, FMDV, and LSDV. The x-axis represents ordered concentration levels (L1–L6) defined independently for each ingredient, and the y-axis indicates log₁₀ reduction values (LRVs). Data points represent mean ± SD. The horizontal dashed line denotes the virucidal benchmark of ≥4 log₁₀ reduction.

Across the tested viruses, distinct concentration–response behaviors were observed, ranging from pronounced ingredient-driven divergence to relatively uniform response profiles. This heterogeneity was most evident for FMDV (Figure 2C), where certain ingredients exhibited minimal virucidal activity across the entire concentration range, whereas others achieved consistently high LRVs even at lower concentration levels. A similar, though less extreme, ingredient-dependent divergence was also observed for AIV (Figure 2A), particularly at intermediate concentration levels where abrupt transitions in efficacy occurred.

In contrast, ASFV (Figure 2B) exhibited a comparatively uniform and predictable concentration–response profile across all tested ingredients. LRVs increased in a largely monotonic manner with increasing concentration levels, and all ingredients reached or exceeded the ≥4 log₁₀ reduction benchmark at higher levels under the tested conditions. This pattern indicates broad susceptibility with limited qualitative differentiation among disinfectant classes.

LSDV (Figure 2D) displayed an intermediate response pattern between these two extremes. Virucidal activity increased progressively with concentration for all ingredients, but notable variability was observed in response slopes and transition points, particularly at intermediate levels. Compared with ASFV, LSDV exhibited greater variability in susceptibility to disinfectant ingredients, with a wider spread of virucidal efficacy across the tested chemical classes. In contrast to FMDV, LSDV did not show a pronounced ingredient-specific dichotomy, but rather displayed a continuous distribution of susceptibility across disinfectant classes.

Collectively, these concentration–response profiles demonstrate that virucidal efficacy is shaped by virus- and ingredient-specific response characteristics rather than by concentration level alone. The observed differences in response slopes, transition behavior, and plateau formation provide the basis for subsequent criterion-based quantitative comparison across virus–ingredient combinations.

3.2. Time-Dependent Critical Concentrations (C*) Required for Virucidal Efficacy Across Viruses and Active Ingredients

To complement the concentration–response patterns described in Section 3.1, virucidal efficacy was further evaluated using a criterion-based metric defining the critical concentration (C*) required to achieve a ≥4 log₁₀ reduction in viral titer. As defined in Section 2.4, C* represents a discrete screening metric based on threshold attainment rather than a continuous estimate of virucidal potency. C* values were determined as the lowest tested concentrations meeting the predefined efficacy criterion at contact times of 2, 10, and 30 min and are summarized in Figure 3.

Figure 3.

Time-dependent critical concentrations (C*) required for virucidal efficacy. Heatmaps display the critical concentration (C*) required to achieve a ≥4 log₁₀ reduction in viral titer for each virus–active ingredient combination at contact times of 2, 10, and 30 min. C* was defined as the lowest tested concentration meeting the efficacy criterion. Color intensity represents log₁₀-transformed C* values, with lower values indicating greater virucidal potency. Cells labeled “>max” or “<min” indicate failure to reach or attainment of the ≥4 log₁₀ reduction criterion beyond the tested concentration range, respectively, and an asterisk (*) denotes partial threshold attainment. Grey and white cells represent these boundary conditions and are not mapped to the continuous color scale.

At the 2-min contact time, C* values varied markedly across virus–ingredient combinations, indicating substantial heterogeneity in early virucidal responses (Figure 3A). Several combinations failed to reach the efficacy threshold within the tested concentration range, whereas others achieved threshold attainment at relatively low concentrations. Such non-achievement outcomes were treated as distinct indicators of high viral resistance rather than as missing data. For example, FMDV met the efficacy criterion for CA at or below the lowest tested concentration, whereas BZK did not reach the threshold against FMDV within the tested range. In contrast, GLT showed partial threshold attainment against FMDV, with threshold achievement observed in only a subset of replicates.

Increasing the contact time to 10 min reduced C* values for selected virus–ingredient combinations, reflecting time-dependent shifts in the concentration required to achieve the efficacy criterion (Figure 3B). However, this effect was not uniform across all combinations. Several virus–ingredient pairs exhibited minimal or no change in C* despite incremental increases in LRV across concentration levels. Notably, ASFV did not reach the efficacy threshold for CA at 10 min within the tested concentration range.

Further extension of the contact time to 30 min resulted in limited additional reductions in C* for most combinations (Figure 3C). In many cases, C* values plateaued between 10 and 30 min, indicating that prolonged contact duration did not substantially alter established response patterns. Virus–ingredient combinations that failed to reach the threshold at earlier time points frequently remained ineffective despite extended exposure.

Overall, the C*-based analysis highlights that time-dependent changes in virucidal efficacy are highly virus- and ingredient-specific. By focusing on threshold attainment, this approach documents response features such as delayed threshold crossing, partial efficacy, and persistent failure that are not directly evident from concentration–response curves alone.

3.3. Spectrum-Oriented Comparison of Ingredient-Specific Virucidal Profiles

To compare ingredient-specific virucidal profiles across viruses and contact times, potency scores derived from C* values (minimum concentrations achieving LRV ≥ 4) were visualized using radar plots (Figure 4). This spectrum-oriented representation emphasizes relative response patterns across viruses rather than direct comparison of absolute potency values, enabling qualitative interpretation of ingredient performance under heterogeneous viral conditions.

Figure 4.

Spectrum-oriented comparison of ingredient-specific virucidal profiles across viruses and contact times. Radar plots show potency scores derived from critical concentrations (C*) required to achieve ≥4 log₁₀ reduction for four disinfectant active ingredients (BZK, GLT, PPMS, and CA) against four viruses (AIV, ASFV, FMDV, and LSDV) at contact times of 2, 10, and 30 min. Potency scores were calculated as the inverse of C*, with larger radial distances indicating greater virucidal potency. For combinations not reaching the efficacy criterion, the maximum tested concentration was used for score calculation. Radially expanded profiles are associated with broader and stronger virucidal potency across viruses, whereas compressed or asymmetric profiles reflect virus-selective or limited activity.

Across contact times (2, 10, and 30 min), each active ingredient exhibited a distinct and reproducible polygonal profile, indicating characteristic virus-specific response spectra (Figure 4). These profiles were largely conserved across contact times, with temporal effects manifested primarily as moderate radial shifts along individual viral axes rather than qualitative changes in overall polygon geometry.

GLT displayed consistently expanded and relatively balanced profiles across viruses and contact times, whereas CA produced asymmetric profiles with preferential extension toward FMDV. BZK exhibited profiles oriented predominantly toward AIV, with secondary extension toward ASFV and LSDV and limited extension along the FMDV axis, while PPMS generated comparatively compact and uniform polygons across viruses. These ingredient-specific spectrum patterns were preserved across contact times. From a virus-centered perspective, AIV showed relatively compressed profiles across ingredients, whereas greater separation among ingredient-specific profiles was observed for ASFV and LSDV, with the most pronounced differentiation evident for FMDV. Overall, Figure 4 provides a spectrum-based visualization of ingredient-specific virucidal response patterns that complements the time-resolved C*-based analysis presented in Figure 3.

3.4. Virus Resistance Ranking Based on Quartile Analysis of C* Distributions

To integrate the multifactorial C* dataset into a virus-level comparison, virus resistance ranking was performed based on the distribution of log₁₀-transformed C* values obtained across all tested active ingredients and contact times (Table 2). For each virus, 12 C* values (four active ingredients × three contact times) were included, encompassing a broad range of concentration–time conditions.

Table 2.

Virus resistance ranking based on quartile analysis of log10 (C*) distributions.

Virus	Q1	Median	Q3	Failures (C* > Max)	Resistant Rank *
FMDV	0.602	0.602	1.079	3/12	1 (most resistant)
ASFV	0.602	0.753	0.903	2/12	2
LSDV	0.301	0.602	0.778	0/12	3
AIV	−0.301	0.477	0.602	0/12	4 (least resistant)

* Resistance rank was determined primarily by the upper quartile (Q3), with higher Q3 values indicating greater virus resistance (i.e., higher concentration requirements to achieve ≥4 log₁₀ reduction).

Quartile analysis emphasized upper-quartile (Q3) values as a conservative indicator of virus resistance, reflecting higher concentration requirements under less favorable conditions. FMDV exhibited the highest Q3 value (1.079) among the four viruses and included multiple instances in which the ≥4 log₁₀ reduction criterion was not achieved at the maximum tested concentration. ASFV displayed intermediate resistance (Q3 = 0.903) and also showed failure cases. LSDV showed a lower Q3 value (0.778) with no observed failures, whereas AIV exhibited the lowest resistance, characterized by the lowest Q3 value (0.602) and the absence of failure cases across all conditions.

Based on these quartile-based comparisons, the overall virus resistance ranking was FMDV > ASFV > LSDV > AIV.

4. Discussion

Virucidal susceptibility of transboundary animal disease (TAD) viruses has traditionally been discussed using broad categorical descriptors, such as envelope presence or genome type [7,33,34]. While such classifications provide useful initial guidance, the present findings demonstrate that they offer limited explanatory power when virucidal responses are examined across multiple disinfectant chemistries and exposure conditions. This view is consistent with previous reports showing that the commonly accepted paradigm of greater susceptibility of enveloped viruses is conditional upon disinfectant chemistry, with substantial variability observed among non-enveloped viruses [35]. Consistent with the limitations of endpoint-based evaluations, virucidal resistance emerges not as an intrinsic, fixed viral property but as a context-dependent outcome shaped by interactions among viral structure, disinfectant chemistry, and exposure parameters. Within this framework, the virus strains used in the present study were selected as commonly applied reference models to enable standardized, multi-condition comparison of virucidal response patterns, rather than to exhaustively represent all subtypes or virulent variants.

Across the experimental matrix, foot-and-mouth disease virus (FMDV) exhibited the highest overall resistance, frequently requiring higher disinfectant concentrations to achieve the ≥4 log₁₀ reduction criterion and ranking highest when C* distributions were integrated. This observation is consistent with the widely recognized environmental stability of FMDV, which has led to its classification as one of the most resistant animal viruses to chemical disinfection [36,37,38]. Importantly, this resistance was not uniform across disinfectant classes. Citric acid achieved effective inactivation of FMDV at comparatively low concentrations and short contact times, whereas other active ingredients failed to reach the efficacy threshold even at the highest tested levels. These findings caution against treating FMDV as a universally worst-case target across all disinfectant types and underscore the central role of disinfectant chemistry in shaping apparent resistance profiles.

Beyond FMDV, similar condition-dependent response patterns were evident for other viruses when the experimentally observed concentration–response behaviors were examined. AIV, which is often regarded as broadly susceptible based on its enveloped structure [39,40,41], did not consistently exhibit high sensitivity across the disinfectant-specific concentration–time profiles observed in this study. Although AIV generally showed lower resistance than the DNA viruses, its responses varied substantially among disinfectant chemistries, indicating that envelope presence alone cannot account for the concentration-dependent inactivation patterns revealed by the data. ASFV and LSDV displayed intermediate response profiles across multiple conditions, further illustrating that genome type and envelope status provide only partial explanatory power when interpreting virucidal efficacy across chemically diverse disinfectants.

Disinfectant-centered analysis further clarified these patterns. Glutaraldehyde consistently demonstrated broad and balanced virucidal activity across all tested viruses, consistent with its protein cross-linking mechanism [42,43]. In contrast, organic acid-based disinfection showed pronounced virus selectivity, achieving high efficacy against certain viruses while remaining limited against others. These patterns were preserved across contact times, indicating that virucidal spectra reflect stable interactions between disinfectant chemistry and virus structural properties rather than disinfectant concentration and contact time alone. For example, the high susceptibility of FMDV to citric acid is consistent with its known acid sensitivity and comparatively lower capsid stability under acidic conditions [30,44], whereas the multilayered virion architecture of ASFV may confer greater tolerance to acidic environments and limit the efficacy of organic acid-based disinfection [45].

Contact time refined, but did not override, chemical compatibility. Although extended exposure reduced the critical concentration required for efficacy in selected virus–disinfectant combinations, this effect was neither linear nor universal. In many cases, increasing contact time from 10 to 30 min resulted in little additional reduction in the required concentration, consistent with prior reports indicating that extended exposure alone cannot overcome limitations arising from disinfectant mechanism-of-action characteristics and virus structural properties [46,47,48].

To enable a conservative comparison of virus resistance across the full experimental matrix, resistance ranking was derived from upper-quartile (Q3) C* values rather than mean or median estimates. This distribution-based approach emphasizes worst-case resistance behavior, which is particularly relevant in regulatory and biosecurity contexts where failure under suboptimal conditions carries significant practical implications. Conditions in which the ≥4 log₁₀ reduction criterion was not achieved within the tested concentration range were therefore retained as explicit indicators of high resistance. Based on this integration, FMDV was identified as the most resistant virus overall, followed by ASFV, LSDV, and AIV. From a regulatory perspective, such a distribution-based ranking provides a rational basis for the selection of representative worst-case target or surrogate viruses for conservative disinfectant efficacy evaluation.

The present study was conducted under controlled laboratory conditions using a suspension test system to enable standardized comparison of virucidal response patterns across viruses, disinfectant chemistries, and exposure parameters. Accordingly, the findings should be interpreted as reflecting intrinsic concentration–time relationships rather than direct simulation of field disinfection scenarios. In real-world settings, additional factors such as organic loading, temperature variation, pH fluctuations, and complex environmental matrices may influence disinfectant performance. Future studies incorporating field-relevant conditions or on-site validation are therefore needed to extend the applicability of these laboratory-based findings.

5. Conclusions

This study demonstrates that virucidal susceptibility of major transboundary animal disease viruses is a condition-dependent outcome shaped by viral structure, disinfectant chemistry, and exposure parameters, rather than an intrinsic, fixed viral property. Virus- and ingredient-specific response patterns observed across standardized concentration–time matrices challenge the adequacy of single-condition endpoint evaluations and virus-centric classifications. By emphasizing pattern-based interpretation across multiple concentrations and contact times, this work provides a more realistic and informative framework for disinfectant evaluation and supports informed decision-making in regulatory assessment, surrogate virus selection, and field-level animal health biosecurity.

Abbreviations

The following abbreviations are used in this manuscript:

AIV	Avian influenza virus
ASFV	African swine fever virus
BZK	Benzalkonium chloride
C*	Critical concentration
CA	Citric acid
CPE	Cytopathic effect
FMDV	Foot-and-mouth disease virus
GLT	Glutaraldehyde
LRV	Log reduction value
LSDV	Lumpy skin disease virus
PPMS	Potassium peroxymonosulfate
TAD	Transboundary animal disease

Author Contributions

Conceptualization, S.S. and W.J.; methodology, S.S.; software, S.-H.P.; validation, S.-H.P. and K.-S.S.; formal analysis, K.-S.S.; investigation, S.-H.P.; resources, S.S.; data curation, S.S.; writing—original draft preparation, S.S.; writing—review and editing, S.S.; visualization, S.S.; supervision, S.S.; project administration, W.J.; funding acquisition, W.J. and H.-O.K. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available in the article.

Conflicts of Interest

The authors declare no conflicts of interest. And the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Funding Statement

This research was funded by the Animal and Plant Quarantine Agency, grant number B-1543073-2023-26-01, Republic of Korea.