Coronaviruses are a family of enveloped positive-sense single-stranded RNA viruses that can cause respiratory diseases in a variety of animal hosts, including humans (Farhud et al., 2022). Over the past two decades, three highly pathogenic human coronaviruses - Severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East Respiratory Syndrome coronavirus (MERS-CoV), and most recently severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) - have triggered global epidemics (Peeri et al., 2020). The emergence of these high-consequence respiratory viruses has spurred global efforts to understand the environmental stability and transmission dynamics of coronaviruses. Bovine coronavirus (BCoV), a member of the Betacoronavirus genus alongside SARS-CoV, MERS-CoV, and SARS-CoV-2, has been widely used as a surrogate for human coronaviruses in environmental studies due to their structural and genomic similarities, and lower pathogenic risk (Kimbrell et al., 2024; Islam et al., 2021; Watanabe et al., 2022; Cantu et al., 2023). Although differences in spike protein sequence and lipid envelope composition exist across coronaviruses, recent studies have directly evaluated BCoV alongside SARS-CoV-2 on common surfaces and in engineered wastewater treatment or concentration systems, demonstrating similar trends in viral decay and genome persistence under comparable conditions (Watanabe et al., 2022; Guérin-Rechdaoui et al., 2022; LaTurner et al., 2021; Graham et al., 2021). Similar to other enveloped RNA viruses, the survival and persistence of BCoV is heavily influenced by environmental factors such as temperature and relative humidity (Wei et al., 2022). A study found a significant decline of SARS-CoV-2 viability at elevated temperatures, with a major loss of infectivity at 40 °C compared to room temperature, and complete inactivation at 55 °C (Harapan et al., 2022). Several other studies also supported this trend, which observed reduced persistence and higher decay rate of infectious viruses at increased temperatures (Dabisch et al., 2021; Sharma et al., 2021; Riddell et al., 2020; Zhang et al., 2022). Relative humidity also plays an important role, however with a more complex effect. Several groups have reported rapid loss of virus viability at higher relative humidity (Chan et al., 2011; Biryukov et al., 2020; van Doremalen et al., 2013), whereas Casanova et al. demonstrated the relationship between virus inactivation and relative humidity being non-monotonic as the virus survival rate was higher at low (20%) and high (80%) relative humidity, compared to that at moderate (50%) relative humidity (Casanova et al., 2010). Besides the effects of temperature and relative humidity, extensive work has detailed coronavirus survival on different types of common surfaces, ranging from non-porous surfaces such as plastic and metals to porous surfaces such as fabrics and paper, and in liquid media (Watanabe et al., 2022; Ren et al., 2020; Aboubakr et al., 2021; Baig et al., 2022). As many coronaviruses are known to transmit through the airborne route, a number of researchers have investigated the infectivity of aerosolized coronaviruses under controlled environmental conditions such as temperature, relative humidity, sunlight, droplet size, and suspension media conditions, despite the challenges involved in capturing viable virus and performing infectivity assays (Dabisch et al., 2021; Doremalen et al., 2020; Smither et al., 2020; Schuit et al., 2020; Ijaz et al., 1985; Pyankov et al., 2018). These efforts highlight the ongoing need for systematic evaluation of the effects of aerosolization and the associated environmental stressors on coronavirus infectivity, an important yet underexplored parameter for understanding transmission risk and persistence of viral aerosols in real-world indoor settings.

The bioaerosol generation and aerosolization process itself imposes mechanical stresses such as shear, impaction, and turbulence on bioaerosols that can compromise their microbial integrity (Smith et al., 2025; Smith and King, 2023). Such mechanical forces are not unique to laboratory settings. In fact, the process of aerosolization is similar to air mixing and circulation generated by fans or the Heating, Ventilation, and Air Conditioning (HVAC) systems commonly present in homes, hospitals, schools, and industrial facilities (Thornton et al., 2022). The use of a fan or high-velocity airflow to mix air in enclosed environments can subject airborne particles to shear stress and impact, which can cause physical damages to the microbes (Redmann et al., 2022). The application to mix large volumes of air is frequently found in critical infrastructures such as meat processing plants, which emerged as hotspots for COVID-19 outbreaks, whose environments are characterized by high indoor ventilation, low temperature, enclosed spaces, and vigorous air circulation (Herstein et al., 2021; Pokora et al., 2021; Zhang and King, 2023). These real-world hotspots highlight the importance of understanding how airborne viruses respond not only to environmental conditions, but also to the stresses imposed during air mixing. Our group has previously shown that aerosolization-induced mechanical stress can alter bacterial physiology by promoting lipid aggregation, upregulating antibiotic resistance genes, and inducing dormancy response (Smith et al., 2025; Smith and King, 2023; Smith and King, 2022). Analogous effects on viral particles remain unexplored. While viruses cannot actively respond to stress in the same way as bacteria, the structural integrity of their envelope and capsid may be compromised during aerosolization or turbulent airborne transport, potentially affecting infectivity.

To address this research gap, we aim to investigate how aerosolization and air mixing affect coronavirus infectivity using BCoV as a surrogate for human coronaviruses. Rather than implicating direct equivalence to SARS-CoV-2, this study uses BCoV to investigate mechanisms governing coronavirus stability during aerosolization and airborne residence, with relevance to indoor environments where mechanical air movement, ventilation, and mixing are common. It is part of a broader research effort to characterize how environmental stressors including aerosolization and airflow entrainment affect airborne bacteria and viruses. By comparing infectious titers and gene copy numbers of bioaerosol samples collected at different time intervals of aerosolization and air mixing, our goal is to better understand the influence of these mechanical stresses on airborne coronavirus survival, especially under conditions that mimic fan-driven or HVAC-mediated aerosol dispersion in real-world indoor environments.

BCoV was nebulized by a Collison nebulizer into a sealed, propeller-mixed chamber for 5, 10, 15, 30, and 45 min, after which the virus aerosols were collected using a WWC sampler. Residual liquid remaining in the nebulizer reservoir after each run was also collected, as this sample represents the virus that underwent mechanical nebulization but remained in liquid suspension rather than entering the aerosol phase. This allows comparison between the effects of nebulization on the liquid suspension and the effects of aerosolization plus airborne residence time on virus infectivity. Residual nebulizer liquid (nebulized virus suspension) was sampled alongside the original stock. Infectious titers expressed as log₁₀ TCID₅₀/mL for stock, nebulized suspensions, and aerosol samples after 5, 10, 15, 30, and 45 min aerosolization are presented in Figure 1. Infectious titers of the stock remained consistent across all runs, ranging from 4.45 to 5.20 log₁₀ TCID₅₀/mL. Nebulized suspensions exhibited similarly stable infectivity, having a lowest infectious titer of 4.45 log₁₀ TCID₅₀/mL and highest infectious titer of 4.86 log₁₀ TCID₅₀/mL. When comparing the stock and nebulized virus suspensions at each aerosolization time point, the nebulized samples had slightly higher infectious titers at 5, 30, and 45 min, and lower infectious titer at 10 and 15 min, although the difference was not statistically significant (p = 0.068). In contrast, WWC-collected aerosols showed markedly reduced titers (1.19 to 2.40 log₁₀ TCID₅₀/mL), falling well below stock and nebulized suspension levels and declining significantly with longer aerosolization (p < 0.0001). This suggests that while the viral infectivity was preserved in the liquid resuspension after nebulization, aerosolization under continuous air mixing imposes stresses that progressively inactivate the viruses.

To assess whether the process of nebulization alone affects BCoV infectivity in the liquid form, infectious titer of the suspension remaining in the nebulizer after 0 (stock), 5, 10, 15, 20, 30, 45, and 60 min of continuous nebulization is shown in Figure 2A. Infectivity fluctuated modestly between 4.32 and 4.70 log₁₀ TCID₅₀/mL without a consistent time-dependent trend. One-way ANOVA with post hoc comparisons confirmed no significant differences across all tested durations (p ≥ 0.22 for all comparisons). The infectivity of the virus aerosol samples collected after 5, 10, 15, 30, and 45 min chamber dispersion was evaluated for the effect of aerosolization and constant air mixing (Figure 2B). The infectivity declined from 2.40 log₁₀ TCID₅₀/mL at the shortest aerosolization duration tested of 5 min to 1.19 log₁₀ TCID₅₀/mL at the longest duration of 45 min. After consistently decreased to 1.42 log₁₀ TCID₅₀/mL at 15 min, the infectivity plateaued to 1.53 log₁₀ TCID₅₀/mL at 30 min, followed by a slower decrease to 1.19 log₁₀ TCID₅₀/mL at 45 min. One-way ANOVA indicated a significant overall effect of aerosolization time on virus infectivity (p < 0.0001), and Dunn’s multiple comparisons revealed a significant difference between 5 min and 45 min (p = 0.0098). The lowest measured titer approached the assay LoD of 1.15 log₁₀ TCID₅₀/mL, underscoring that extended aerosol suspension under mixing conditions substantially diminishes viable virus recovery.

BCoV RNA levels were quantified in the stock, nebulized, and aerosol samples across different aerosolization durations using qRT-PCR (Figure 3). The virus in the stock suspensions used for aerosolization were relatively consistent, ranging from 3.85 × 10⁴ GCN/mL to 8.22 × 10⁴ GCN/mL. Similarly, the corresponding nebulized samples contained lower levels of BCoV between 1.46 × 10⁴ GCN/mL and 6.12 × 10⁴ GCN/mL. The viral RNA content in the nebulized samples was consistently lower than in the corresponding stock samples for each aerosolization duration tested. A paired t-test confirmed this reduction to be statistically significant (p = 0.01), suggesting that the nebulization process itself contributes to a measurable loss or degradation of viral RNA. This decline in RNA concentration may reflect shear stress during nebulization, partial inactivation by impaction, or adsorption to internal surfaces of the nebulizer. The viral RNA levels in aerosol samples collected by the WWC were relatively stable across all time points (p > 0.17), ranging from 2.41 × 10³ to 4.19 × 10³ GCN/m³ of air. This limited variability suggests that while viral RNA is recoverable after aerosolization, its airborne concentration may not decline as sharply as infectivity. These findings indicate that BCoV RNA is more resilient than infectivity during airborne suspension, likely due to the RNA’s inherent stability even after partial virion damage or inactivation.

To further explore the impact of nebulization time on BCoV RNA levels in liquid suspensions, BCoV GCN values were measured after 0 (stock), 5, 10, 15, 20, 30, 45, and 60 min of continuous nebulization (Figure 4A). A pronounced decrease in viral GCN was observed immediately after 5 min of nebulization, with levels dropping from 4.09 × 10⁴ GCN/mL to 1.73 × 10⁴ GCN/mL. However, beyond this initial drop, the viral GCNs remained relatively stable, fluctuating only slightly between 1.51 × 10⁴ GCN/mL and 2.23 × 10⁴ GCN/mL for the remainder of the 60-min nebulization. One-way ANOVA analysis did not find this variation to be statistically significant (p = 0.15), and post hoc comparisons between time points (p ≥ 0.26 for all comparisons) confirmed the absence of meaningful differences. This pattern supports the notion that most of the RNA loss occurs early during nebulization, possibly due to initial mechanical stress, and reaches a steady state thereafter. For WWC-collected BCoV aerosol samples with normalization to the stock, the highest (4.19 × 10³ GCN/m³ of air) and lowest (1.24 × 10³ GCN/m³ of air) viral RNA levels were observed at 5 min and 10 min of aerosolization, respectively (Figure 4B). Although a clear monotonic decrease in GCN with increasing aerosolization time was not observed, a moderate increase in RNA concentration was noted from 15 to 45 min. These differences were not statistically significant based on multiple comparison testing (p ≥ 0.18 for all comparisons), suggesting that RNA levels in the aerosols remained relatively stable and were not strongly affected by extended suspension times under the tested conditions.

Environmental conditions within the testing chamber were monitored throughout the experiments, as temperature and relative humidity are known to influence both virus viability and aerosol sampling efficiency. Across the five aerosolization durations, temperature ranged from 22.6 and 24.1 °C, and the average relative humidity ranged from 55.4 to 69.1% (Figure 5). As expected, an inverse relationship between temperature and relative humidity was observed, consistent with the physics of air’s moisture-holding capacity. This occurs as warmer air can hold more water vapor before becoming saturated. Unless additional moisture is introduced, relative humidity decreases as temperature increases. Despite the continuous generation of aerosols contributing water vapor to the chamber, fluctuations in the temperature still drive the observed opposite trends in relative humidity. Kruskal–Wallis analysis showed a statistically significant overall difference in temperature across aerosolization durations (p = 0.01), but Dunn’s post-hoc tests found no significant pairwise differences (p ≥ 0.20 for all comparisons). This suggests that while temperature varied slightly over time, the differences between individual conditions were modest and unlikely to have biological relevance. On the other hand, relative humidity showed more variation and ANOVA revealed a significant overall difference (p = 0.0003). Dunn’s test identified a significant increase in relative humidity between the 5 min and 45 min time points (p = 0.02), while other comparisons were not significant (p ≥ 0.17), indicating that major changes were limited to select time point.

This study examined the infectivity and RNA stability of aerosolized BCoV across different durations of aerosolization in a mechanically mixed chamber environment. Our results demonstrated that while the nebulization process caused minimal loss of viral infectivity in the liquid suspension, prolonged aerosol residence under mechanical mixing markedly reduced infectious titers of the viruses. In contrast, viral RNA remained comparatively stable throughout the aerosolization period, even as infectivity declined. The observation that pre- and post-nebulized BCoV suspensions exhibited similar infectious titers suggests that the shear and impaction forces inside the Collison nebulizer do not substantially impair virion infectivity. While mechanical shear during the nebulization could induce virion alterations that are not immediately reflected as infectivity loss, prior studies have reported high preservation (over 80%) of viability following Collison nebulization for other enveloped viruses, including influenza A viruses, SARS-CoV-2, and PRD1 bacteriophage (Baig et al., 2022; Niazi et al., 2021; Paton et al., 2021). Nevertheless, sublethal effects may exist that could influence host cell entry or replication, and future studies should include analysis of the structural integrity of the virions. On the other hand, our results revealed a consistent reduction in viral RNA immediately following nebulization in the nebulizer reservoir across all aerosolization durations. This early RNA loss that was most obvious within the first five minutes of aerosol generation likely reflects an initial effect of mechanical stress, including shear or impaction, as well as possible adsorption to internal surfaces (Niazi et al., 2021). Notably, after this rapid initial decrease, RNA levels in the nebulizer reservoir stabilize, fluctuating only modestly between 1.51 × 10⁴ GCN/mL and 2.23 × 10⁴ GCN/mL over 60 min. One potential explanation is that once a subset of genomes is degraded or adsorbed, the remaining RNA achieves a dynamic equilibrium, perhaps protected within intact virions or by equilibrium between genome release and further loss. Similar patterns have been observed in a study using a Collison nebulizer to generate influenza aerosols, where no loss of viable virus or viral RNA was detected in the nebulizer reservoir post-nebulization while airborne titers declined more substantially as airborne viral RNA remained stable (Brown et al., 2015). Another study nebulized transmissible gastroenteritis virus (TGEV) using both Collison and medical nebulizers at varying pressures, and found no significant reduction (less than 10%) in virus titer in the nebulizer suspension before and after nebulization, which is consistent with our findings that the mechanical process of nebulization does not inherently reduce viral infectivity in suspension (Kim et al., 2007). Similar minimal loss of virus viability was found by Hermann et al. after 55 min nebulization of enveloped porcine reproductive and respiratory syndrome virus (PRRSV) (Hermann et al., 2006). These studies along with ours using different types of enveloped viruses suggest that while nebulization generates small respirable droplets suitable for experimental aerosolization, the shear and impaction forces involved in the 6-jet Collison nebulizer do not necessarily compromise the virus envelope or capsid structure. Once airborne, however, BCoV infectivity significantly declined over time from 5 to 45 min of aerosolization with mechanical air mixing. This decline aligns with prior studies on aerosols of various types of coronaviruses including SARS-CoV-2, SARS-CoV, and mouse hepatitis virus (MHV) that have demonstrated similar time-dependent reductions in virus titers within controlled environments (Doremalen et al., 2020; Oswin et al., 2021). Other than the mechanical forces imposed from air mixing, the loss of infectivity in aerosols can also be attributable to a combination of physical and environmental stressors such as droplet evaporation, increased chance of impaction damage, desiccation, and degradation (Brown et al., 2015; Rezaei and Netz, 2021). Despite the markable loss of infectivity at longer aerosolization time, viral RNA levels measured by qRT-PCR remained relatively constant, fluctuating within a narrow range. This disparity suggests that the viral genome is more resilient to airborne degradation than the infectious virion itself, a pattern observed in influenza viruses as well (Brown et al., 2015). A number of studies has shown that RNA from SARS-CoV-2 can persist on surfaces long after viable virus is undetectable (Paton et al., 2021; Puhach et al., 2023; Kasloff et al., 2021; Yamagishi et al., 2020). Our results show that this higher resilience of viral RNA is also relevant in BCoV aerosols, highlighting the potential for molecular methods to overestimate infectious risk if not interpreted in conjunction with culture-based assays. These findings reinforce the importance of pairing qRT-PCR detection with infectivity assays to fully characterize airborne virus viability, particularly in environmental or clinical surveillance contexts.

Environmental conditions within the chamber remained broadly stable yet displayed measurable trends over the course of aerosolization. Temperatures recorded during the five sampling intervals ranged narrowly from 22.6 °C to 24.1 °C, whereas relative humidity increased from 55.4% at 5 min to 69.1% at 45 min. The inverse relationship between temperature and relative humidity reflects the physics of air’s moisture-holding capacity, yet in our closed system, continuous aerosol generation offset some of this effect by introducing additional moisture. The small temperature changes observed over the aerosolization period were statistically detectable but unlikely to have meaningful biological impact. In contrast, relative humidity exhibited a significant overall increase, especially between the 5 min and 45 min time point. Previous studies reported that relative humidity (20% vs. 70%) moderately affected the infectivity of SARS-CoV-2 aerosols, though to a lesser extent than temperature or UV (Dabisch et al., 2021). A model using modified Wells-Riley equations revealed virus-specific responses to relative humidity - the infection risk of airborne influenza increases with relative humidity over 20 to 80%, while SARS-CoV-2 aerosols showed a non-monotonic pattern, having the highest infection risk at 53% relative humidity and declining at both lower and higher levels (Aganovic et al., 2022). Another controlled experiment has also shown that the impact of relative humidity on airborne SARS-CoV-2 infectivity is complex and dependent on the suspension matrix used (Smither et al., 2020). Consistent trends have also been reported for airborne influenza A viruses, where Motos et al. observed that viruses aerosolized in saline remained infectious for the longest time at low relative humidity (<30%) but exhibited rapid infectivity loss at elevated relative humidity (40 to 70%) (Motos et al., 2024). In our study, the gradual increase in relative humidity may have contributed to BCoV inactivation in aerosols but was likely acted jointly with mechanical mixing, evaporative stress, and airborne residence time rather than as an isolated driver of inactivation. Moreover, the relative humidity levels in our tests (55 to 70%) fall within the mid-range evaluated in previous studies and are less likely to exert a pronounced effect on viral stability.

While the findings in this present study suggest that coronaviruses infectivity, using BCoV as a surrogate, declines during prolonged airborne suspension, several limitations should be acknowledged. First, the study was conducted under a specific set of environmental conditions, including a narrow temperature range (22.6 °C to 24.1 °C) and moderate relative humidity (55 to 70%), which may not capture the full spectrum of environmental variability encountered in real-world settings. Second, the use of a sealed, continuously mixed chamber may not fully represent natural airflow dynamics or deposition processes in occupied indoor environments. The propeller mixed chamber does not replicate the linear airflow profiles of HVAC systems. Rather, it provides a controlled environment to examine mechanical mixing and airborne residence under recirculating conditions commonly encountered in occupied indoor spaces with fans or localized ventilation. Prior APS measurements and CFD modeling under identical conditions demonstrated homogeneous aerosol dispersion and stable particle residence times, supporting the interpretation that infectivity loss reflects cumulative airborne stress rather than uneven deposition or selective loss of larger droplets (Baig et al., 2022; Smith et al., 2025). Third, the use of a single virus surrogate BCoV limits the generalizability of findings to other coronaviruses with different structural or physicochemical properties. Lastly, the temporal resolution of aerosol sampling was restricted to discrete time points, which may overlook more transient changes in virus viability. While the nebulized medium did not include complex respiratory fluids such as saliva or mucus, the use of 10x diluted PBS containing 0.08% NaCl approximates the NaCl content of saliva (0.017–0.123%). These limitations highlight the need for future studies that incorporate respiratory matrices, multiple virus strains, and additional environmental variables such as light exposure and air pollutants to enhance our understanding.

In this study, we investigated how aerosolization and airborne suspension affect coronavirus infectivity and RNA persistence under controlled mixed-air conditions, using BCoV as a surrogate. While the nebulization showed minimal effect on the viral infectivity of the virus suspension in the reservoir, continuous air mixing and prolonged residence time led to a progressive loss of virus infectivity in the aerosols. In contrast, viral RNA levels in both reservoir and WWC-collected aerosols remained comparatively stable, underscoring the greater resilience of genomic material and the importance of combining molecular and culture-based assays for accurate risk assessment. Moderate increases in relative humidity and minimal temperature fluctuations in the present study likely played a secondary role in virus inactivation relative to mechanical shear and evaporative stress. These findings advance our understanding of airborne coronavirus persistence and highlight the need for future studies that incorporate broader environmental conditions and human-relevant coronavirus surrogates to inform mitigation strategies for indoor airborne transmission.