Microwave irradiation as a novel strategy for mitigating airborne transmission of highly pathogenic avian influenza A(H5N1) virus: an optimization study

doi:10.21203/rs.3.rs-4929197/v1

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Microwave irradiation as a novel strategy for mitigating airborne transmission of highly pathogenic avian influenza A(H5N1) virus: an optimization study

https://doi.org/10.21203/rs.3.rs-4929197/v1

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The highly pathogenic avian influenza A(H5N1) virus threatens animal and human health globally. Innovative strategies are needed to reduce airborne transmission and prevent outbreaks. This study investigated the efficacy of microwave inactivation against aerosolized A(H5N1) by (1) identifying the optimal frequency band in 10 min of exposure and (2) evaluating the effect of exposure time.

A(H5N1) was aerosolized and exposed to various microwave frequencies (between 8 and 16 GHz with different ranges) for 10 minutes. Viral titers were quantified using TCID50, and inactivation was assessed by comparing irradiated samples to controls. The 11−13 GHz band resulted in the highest inactivation, with an average of 89% mean reduction in A(H5N1) titer in particular in the range of 11-12 GHZ (pick of efficacy). Considering the overall tests and results, the optimal band (8-12 GHZ) was further tested with 1, 3, and 5-minute exposures. Inactivation was time-dependent, with 5-minute exposure yielding a 94% mean reduction, compared to 58% and 48% for 3 and 1-minute exposures, respectively. Optimized microwave emitters in high-risk environments like poultry farms and veterinary clinics could offer a novel, non-chemical approach to mitigating avian influenza spread and outbreaks.

Biological sciences/Biological techniques

Biological sciences/Biophysics

Biological sciences/Biotechnology

Avian influenza

A(H5N1) virus

Radiated microwaves

Frequency bands

Exposure time

The highly pathogenic avian influenza A(H5N1) virus, a member of the Influenzavirus A genus within the Orthomyxoviridae family, poses a significant global threat to both animal and human health^{1, 2}. Since the identification of a particular viral lineage in domestic geese in Guangdong, China, in 1996, this pathogen has led to severe epizootics in poultry populations throughout Asia, Europe, Africa, and North America³. Of particular concern is the virus’s ability to cross the species barrier and infect humans, with sporadic cases documented in multiple countries following direct contact with infected animals^4–6. Notably, the A(H5N1) virus exhibits a case fatality rate of approximately 50% in affected individuals⁷. Although limited human-to-human transmission has been observed in a small number of family clusters⁸, sustained community spread has not yet been reported. Additionally, this virus has been detected in cattle and their milk, posing a significant risk of transmission to humans^{9, 10}. The potential for A(H5N1) to evolve more efficient human-to-human transmission and trigger a global pandemic remains a serious concern for public health authorities worldwide^{5, 6}. Furthermore, the possibility of genetic reassortment among avian, swine, and human influenza viruses increases the risk of novel strains emerging with enhanced transmissibility¹¹. Exposure to infected poultry or contaminated environments is the primary risk factor for human infections, placing poultry workers, veterinarians, and healthcare workers at increased risk^{12, 13}. In human infections, A(H5N1) targets the alveoli of the lower respiratory tract¹⁴, causing severe viral pneumonia accompanied by a profound dysregulation of the host cytokine response¹⁵. The clinical course is typically rapidly progressive, often leading to acute respiratory distress syndrome and multi-organ failure¹⁶. Consequently, an A(H5N1) outbreak could severely strain healthcare systems^{1, 5, 6}, highlighting the urgent need for measures to control infections in poultry and cattle to reduce the risk of human exposure.

Electromagnetic radiation within the microwave spectrum, spanning frequencies from 300 MHz to 300 GHz, exhibits non-ionizing properties while possessing sufficient energy to induce molecular vibrations in matter¹⁷. Among the diverse applications of this vibrational excitation, the phenomenon of resonant energy transfer from microwave radiation to specific acoustic vibrational modes of viral particles has emerged as a promising avenue for non-chemical decontamination strategies^18–23. This approach shows significant promise for real-time air decontamination, primarily aimed at mitigating the spread of airborne respiratory pathogens^{21, 22}. The mechanism exploits the confined acoustic dipolar mode of resonance in viral particles²⁴. When aerosolized viruses are exposed to microwave radiation at specific frequencies, energy is transferred to the virions’ confined acoustic vibrational modes ^{23, 24}. This energy transfer induces resonance, leading to structural disruption and subsequent inactivation of the viral particles^{23, 24}. Recent studies have demonstrated the effectiveness of this method against various strains of SARS-CoV-2, including the Wuhan, delta, and omicron variants^20–22. Experiments conducted in a controlled bioaerosol system revealed that exposure to microwave radiation resulted in an average reduction of 91.31% in viral titer across these variants²¹. Comparable levels of inactivation were observed for the H1N1 influenza virus, achieving a 90% reduction in viral titer²². However, notable differences in the optimal parameters for inactivation were identified between SARS-CoV-2 and H1N1 influenza virus²². While SARS-CoV-2 showed sensitivity to frequencies up to 12 GHz, the H1N1 influenza virus exhibited susceptibility to higher frequencies, up to 16 GHz²⁰. The duration of exposure also emerged as a critical factor, with SARS-CoV-2 demonstrating a ten-fold reduction in infectivity within one minute^{21, 22}, while the H1N1 influenza virus required five minutes to achieve comparable levels of inactivation²².

Given the ongoing global concern regarding the A(H5N1) virus and its potential impact on animal and human health^{5, 6}, this study was designed to evaluate the efficacy of microwave radiation for inactivating aerosolized A(H5N1) virus particles. The investigation had two primary objectives. First, using a frequency step size approach, we sought to identify the most suitable frequency band to maximize virucidal activity against A(H5N1). Second, we examined how the microwave application time affected the inactivation elicited by microwave illumination.

Experimental setup

The experimental protocol for investigating the effects of microwave radiation on aerosolized A(H5N1) virus comprised five principal stages. Initially, the virus was propagated in Madin-Darby canine kidney (MDCK) cells to generate a high-titer suspension suitable for subsequent aerosolization. This stage was crucial in ensuring a sufficient viral load for the experiments and maintaining consistency across trials. Following the preparatory phase, the viral suspension underwent a controlled aerosolization process to create a fine mist of airborne particles. This step simulated real-world conditions of A(H5N1) viral transmission and allowed for the assessment of microwave radiation effects on suspended viral particles. The aerosolized virus was then subjected to microwave exposure under rigorously controlled conditions. To elucidate the optimal parameters for viral inactivation, a systematic evaluation of various frequency bands was conducted. This approach facilitated the identification of specific frequency ranges that demonstrated maximal efficacy in viral inactivation. To further enhance our understanding of the microwave inactivation process, a temporal optimization study was performed. This experiment aimed to elucidate the relationship between exposure duration and inactivation efficacy, providing insights into the kinetics of A(H5N1) inactivation under microwave radiation.

Propagation of the A(H5N1) virus

The propagation of the A(H5N1) virus was conducted in a Biosafety Level 3 (BSL-3) laboratory at ViroStatics srl, located within the Scientific and Technological Park Porto Conte Ricerche srl (Alghero, Italy). MDCK cells, obtained from the American Type Culture Collection (Manassas, VA, USA), were maintained at 37°C in a 5% CO₂ atmosphere. The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Biowest, Nuaillé, France), 1% penicillin/streptomycin antibiotic solution (Biowest), and 1% L-glutamine (Biowest). Following propagation of the live highly pathogenic avian influenza virus (H5N1) A/ck/Israel/65/10, infectious titers were quantified using the standard 50% tissue culture infectious dose (TCID₅₀) assay in MDCK cells. Upon achieving a high titer (> 1×10⁵ TCID₅₀/mL) of the A(H5N1) strain, a stock suspension was prepared for subsequent aerosolization experiments.

Aerosolization of the A(H5N1) viral suspension

The A(H5N1) viral suspension was aerosolized using a commercially available aerosol generator (Omron, Kyoto, Japan) to produce particles up to 1 µm in size within a 32 L plastic, air-proof container. This aerosolization process was designed to mimic the natural airborne transmission of the virus, simulating the droplets and aerosols that would be produced during respiratory events such as coughing, sneezing, or talking. The use of a sealed container ensured containment of the aerosolized virus for controlled exposure to the subsequent microwave treatment.

Microwave exposure

The aerosolized A(H5N1) virus was subjected to microwave radiation generated by a radio frequency (RF) generator, a custom-designed apparatus previously described in detail^{21, 22}. In brief, this RF system was specifically engineered for controlled microwave radiation delivery in virus inactivation studies. The system comprised the following components: an ultra-wideband frequency-tunable synthesizer capable of operating across the C band to the Ku band, allowing for the testing of a broad range of frequencies; medium power and high-power microwave amplifiers to enhance signal strength; a digital variable attenuator to regulate output power; and embedded software written in C + + on the ESP32 platform using Visual Studio Code, which controlled all possible configurations of the RF components. The final power amplifier of the demonstrator employed cutting-edge 0.15 µm GaN on SiC solid-state high-electron-mobility transistor technology, capable of delivering up to 10 W across an ultra-wideband range. The RF output of the transmitter was connected via an RF cable to a horn antenna, which generated an electromagnetic field with a strength of 200 V/m near the antenna and 40 V/m at the vertices of the chamber containing the aerosolized virus. This setup enabled precise control over the frequency band and exposure time. Prior to each experiment, the system was calibrated using a broadband field meter to ensure accurate assessment of the electromagnetic field’s inactivation potency at specific frequencies²¹.

Optimization of the frequency band

In a series of experiments designed to optimize the frequency band for viral inactivation, aerosolized viral samples were systematically exposed to microwave radiation. Each sample was subjected to a standardized 10-minute microwave exposure period. To comprehensively assess the inactivation efficacy of microwave radiation on aerosolized A(H5N1) virus, seven distinct frequency bands were evaluated: 8 − 10 GHz, 9 − 11 GHz, 10 − 12 GHz, 11 − 13 GHz, 12 − 14 GHz, 13 − 15 GHz, and 14 − 16 GHz. Tests for each frequency band were performed in duplicate. Control samples consisted of aerosolized viral specimens that were not subjected to microwave illumination, with the RF source remaining inactive for the entire 10-minute exposure duration. Following the microwave treatment, the irradiated aerosol was recovered through a process of active impingement¹⁹. This recovery method involved the direct collection of the treated aerosol into a glass collector containing complete medium supplemented with 2% FBS. The glass collector was equipped with an inlet and tangential nozzles, facilitating the efficient suction of air from the plastic chamber when a vacuum was applied at a rate of 12 L/min. This collection process ensured the preservation of the treated viral particles for subsequent analysis and quantification of inactivation efficacy across the various frequency bands tested. Results were expressed using two complementary methods to ensure comprehensive representation of the experimental outcomes. First, mean virus titers were calculated based on the TCID₅₀ assay, providing a quantitative measure of viral infectivity. Second, viral inactivation ratios were computed by comparing the titers of illuminated samples to those of unilluminated controls. These ratios were presented as means derived from a minimum of two independent experiments.

Optimization of the exposure time

After identifying the optimal frequency band for inactivating aerosolized A(H5N1) virus, we investigated the influence of exposure duration on inactivation efficacy. Our objective was to reduce the total exposure time from the initial 10-minute duration employed in preceding experiments, with the primary aim of identifying the minimum microwave illumination time required for effective viral inactivation. To achieve this goal, we evaluated three distinct exposure durations: 5 minutes, 3 minutes, and 1 minute within the previously determined optimal frequency band. Each duration was subjected to triplicate testing. Aerosolized virus samples were subjected to microwave radiation using these parameters, and the residual viral infectivity was subsequently assessed. To quantify the results, we calculated and reported mean virus titers from the TCID₅₀ assay. Additionally, we computed viral inactivation ratios by comparing microwave-exposed samples to unilluminated controls. The ratios were presented as means with corresponding minimum and maximum values, based on at least triplicate experiments.

Efficacy of microwave radiation for inactivating aerosolized A(H5N1) virus across different frequency bands

The inactivation efficacy of microwave radiation on aerosolized A(H5N1) virus as a function of frequency band is summarized in Table 1. A non-linear frequency-dependent effect on viral inactivation was observed, with distinct patterns of efficacy across the tested frequency bands (Figure 1). The most pronounced inactivation was achieved in the 11−13 GHz frequency band (in particular, in the 11-12 GHZ frequency band), which resulted in a mean reduction of 89% in viral titer (range: 88−90%). This was closely followed by the 8−10 GHz band, which demonstrated a mean reduction of 83% (range: 78−88%). The 10−12 GHz band also exhibited significant inactivation, with a mean reduction of 79% (range: 76−83%). Notably, the 9−11 GHz band displayed a somewhat lower, yet still substantial, inactivation efficacy with a mean reduction of 66% (range: 61−70%). In contrast, the higher frequency bands demonstrated a lack of inactivation efficacy. Specifically, the 12−14 GHz, 13−15 GHz, and 14−16 GHz ranges showed minimal to no reduction in viral titer compared to the unirradiated control. The mean viral titers for these frequency bands were comparable to or even slightly higher than the control, although this difference is likely within the margin of experimental error. Based on these findings and the previous success in inactivating SARS-CoV-2^{21, 22}(about 90% of inactivation rate), a frequency band of 8−12 GHz was selected for further evaluation in the optimization of exposure time experiments. This selection was driven by the observed efficacy in this frequency range and its potential to effectively inactivate both A(H5N1) and SARS-CoV-2 viruses, suggesting a broader applicability of this approach across different viral pathogens.

Time-dependent efficacy of microwave radiation for inactivating aerosolized A(H5N1) virus

Following the optimization of the frequency band, we investigated the impact of exposure duration on the efficacy of microwave radiation for inactivating aerosolized A(H5N1) virus. The results, summarized in Table 2, demonstrate a clear time-dependent effect on viral inactivation within the 8−12 GHz frequency band. A positive correlation between exposure time and viral inactivation efficacy was observed (Figure 2). Moving from the starting 10-minute microwave exposure period, the 5-minute exposure demonstrated optimal efficacy, yielding a mean viral titer reduction of 94% (range: 92−95%). The narrow range of outcomes indicates high consistency, suggesting that this duration is sufficient to achieve robust and reproducible viral inactivation.

In contrast, a 3-minute exposure resulted in moderate inactivation, with a mean reduction of 58% (range: 29−74%). The broader range observed in this condition suggests greater variability in outcomes, potentially due to the exposure time approaching a critical threshold for effective inactivation.

Notably, a brief 1-minute exposure yielded a mean reduction of 48% (range: 0−76%). The substantial variability in results, ranging from no effect to significant inactivation, indicates that this duration is insufficient to ensure consistent virucidal activity.

Recent epidemics and pandemics of respiratory viruses – including the 2003 severe acute respiratory syndrome outbreak, the 2009 H1N1 influenza pandemic, the 2012 Middle East respiratory syndrome coronavirus outbreak, and the COVID-19 pandemic caused by SARS-CoV-2²³ – have necessitated a critical reassessment of existing strategies for viral containment and prevention. Notably, a recent Cochrane review suggested that relying exclusively on antiviral medications and vaccines may be insufficient to effectively interrupt or mitigate the spread of acute respiratory viruses²⁴. In response to these challenges, microwave inactivation of airborne microorganisms has emerged as a promising non-chemical technology for viral inactivation^16–22.

To the best of our knowledge this study represents, the first investigation into the inactivation efficacy of microwave illumination against aerosolized avian influenza A(H5N1) virus. Our research, aimed at optimizing the approach to maximize virucidal activity against an airborne pathogen currently under close surveillance^{5, 6}, yielded two principal findings. First, our analysis revealed that the optimal frequency range for inactivating A(H5N1) in an aerosolized state lies between 11 and 13 GHz, resulting in a substantial mean reduction of 89% in viral titer. Second, we unequivocally demonstrated the time-dependent nature of A(H5N1) viral inactivation, revealing a positive correlation between exposure duration and inactivation efficacy.

With respect to viral inactivation in response to the frequency range generated by the RF-wave emission system, A(H5N1) exhibited susceptibility up to 13 GHz. This threshold is lower than our previously observed value for the H1N1 human influenza virus (up to 16 GHz)²⁰ but aligns with the sensitivity of SARS-CoV-2 (up to 12 GHz)¹⁹. Consequently, for subsequent experiments aimed at verifying the effect of exposure time on viral inactivation, we selected an 8 − 12 GHz frequency band to encompass both A(H5N1) and SARS-CoV-2 susceptibility ranges. Importantly, the similar optimal frequency ranges observed for diverse viruses hint at a common biophysical basis for microwave susceptibility among enveloped viruses. This could potentially allow for the development of generalized microwave-based disinfection protocols effective against a wide range of viral threats^{20, 21}. In this regard, the effectiveness of the 8 − 12 GHz frequency band may be attributed to its resonance with the confined acoustic vibrational modes of viral particles, as proposed by the structure resonance energy transfer (SRET) model²². This resonance effect likely induces structural disruptions in viral components, ultimately leading to loss of infectivity. The SRET mechanism, as described by Yang et al.²², suggests that microwaves of the same frequency can resonantly excite the dipolar mode of the confined acoustic vibrations inside virions. This process, known as microwave resonant absorption, is influenced by various factors including the virus’s hydration level, surface charge, size, and surrounding media²¹. Our current findings on the inactivation of aerosolized A(H5N1) virus align with previous studies demonstrating the virucidal effects of non-thermal microwaves against various virions in different media – including SARS-CoV-2 and H1N1 influenza virus in aerosol form^{19, 20,} SARS-CoV-2 in deionized water²⁵, H3N2 influenza virus²² and bovine coronavirus (BCoV)²⁶ in aqueous solutions, human coronavirus HCoV-229E in culture medium²⁷, and the BCoV on dry surfaces²⁸. The time-dependent nature of A(H5N1) inactivation observed in our study corroborates previous observations showing time-dependent inactivation of both SARS-CoV-2 and H1N1 in aerosols using microwave illumination^{19, 20}. Accordingly, our current results demonstrate that longer exposure times lead to more consistent and effective viral inactivation, with the 5-minute exposure striking an optimal balance between high efficacy and practical application time.

Several limitations of this study warrant consideration. The experiments were conducted under controlled laboratory conditions, which may not fully represent real-world scenarios where factors such as humidity, temperature fluctuations, and the presence of organic matter could potentially influence inactivation efficacy. Furthermore, while our research demonstrated the inactivation of aerosolized viruses, additional studies are necessary to evaluate the effectiveness of this approach for viruses on surfaces or in liquid media.

As we continue to address the challenges posed by emerging and re-emerging avian influenza threats^{3, 5, 6}, the investigation of non-thermal microwaves in real-world environments represents a crucial next step. For instance, the implementation of microwave emitters optimized against A(H5N1) could potentially provide continuous disinfection of circulating air in high-risk environments such as poultry farms, processing facilities, and veterinary clinics. This approach could significantly mitigate the risk of airborne transmission in these settings.

This study demonstrates the efficacy of microwave radiation for inactivating aerosolized A(H5N1) virus. The results, demonstrate a clear time-dependent effect on viral inactivation within the 8 − 12 GHz frequency band: the 5-minute exposure demonstrated optimal efficacy, yielding the most consistent and effective viral inactivation (a mean viral titer reduction of 94% - range: 92 − 95%). These findings corroborate previous research on other enveloped viruses, indicating a shared biophysical foundation for microwave susceptibility. This commonality could pave the way for the development of broadly applicable disinfection protocols. While further research is needed to address limitations and explore real-world applications, this non-thermal microwave approach shows promise as a novel strategy for mitigating the spread of airborne viral pathogens, including the highly pathogenic A(H5N1) influenza virus.

Competing Interests

A.M., P.B. and M.L. are employees of Elettronica S.p.A. The other authors have no conflicts of interest to declare.

Author Contribution

A.M. P.B., S.B., G.P.P. contributed to Conceptualization; A.M. P.B., S.B., G.P.P., ASV contributed to Methodology; A.M. P.B., M.L. contributed to Experiment; A.M., P.B., M.L. contributed to Analysis; A.M., P.B., M.L. contributed to Device Fabrication; A.M. contributed to Funding acquisition; S.B., G.P.P., ASV contributed to Supervision; A.M., P.B., M.L. contributed to Writing-drafting; S.B., G.P.P., ASV contributed to Writing-review.

Acknowledgement

This project was sponsored by an unrestricted grant from Elettronica S.p.A. (Rome, Italy)

Data Availability

Access to the datasets generated and analyzed during this study can be provided by the corresponding author upon reasonable request

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Table 1. Effect of microwave frequency bands on A(H5N1) viral titer reduction

Frequency band, GHz	Mean viral titer, TCID₅₀/mL	Mean percentage reduction compared to control
Frequency band, GHz	Mean viral titer, TCID₅₀/mL
Unirradiated control	26300	−
8−10	4390	83
9−11	8970	66
10−12	5480	79
11−13	2870	89
12−14	26800	0
13−15	25700	2
14−16	29400	0

Percentage reduction was calculated relative to the unirradiated control, showing mean values observed across replicates. Abbreviation: TCID₅₀, 50% tissue culture infective dose.

Table 2. Effect of microwave irradiation time on A(H5N1) viral titer reduction

Exposure time, min	Mean viral titer, TCID₅₀/mL	Mean percentage reduction compared to control
Exposure time, min	Mean viral titer, TCID₅₀/mL
Unirradiated control	1670	−
5	100	94
3	698	58
1	861	48

Percentage reduction is calculated relative to the unirradiated control, showing mean values observed across replicates. Abbreviation: TCID₅₀, 50% tissue culture infective dose.

Competing interest reported. A.M., P.B. and M.L. are employees of Elettronica S.p.A. The other authors have no conflicts of interest to declare.

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Microwave irradiation as a novel strategy for mitigating airborne transmission of highly pathogenic avian influenza A(H5N1) virus: an optimization study

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Abstract

Figures

Introduction

Methods

Experimental setup

Propagation of the A(H5N1) virus

Aerosolization of the A(H5N1) viral suspension

Microwave exposure

Optimization of the frequency band

Optimization of the exposure time

Results

Discussion

Conclusions

Declarations

Competing Interests

Author Contribution

Acknowledgement

Data Availability

References

Tables

Additional Declarations

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