Review Reports

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: [1) the authors seem to be suggesting that the ultrasound ultimately may be useful as an in vivo treatment for reducing viral load, although this is not made clear in the introduction. I would suggest setting up the subject by describing the sequence of experiments needed, as in a) measuring viral load reduction in distilled water (this study), b) measuring viral load reduction in relevant biological matrices (serum, blood, cell culture, etc.), c) confirming reduction in viral infectivity corresponding with the reduction in RNA content, d) confirming mechanism through appropriate microscopy studies, e) establishing in vivo efficacy and safety in animal models.]

Response 1: We deeply appreciate this insight. We agree that the manuscript failed to clearly distinguish between in vivo therapy and the specific nature of the current work (proof-of-concept in a liquid matrix). We realized this ambiguity may arise primarily from the absence of a clearly stated Aim of the Study in the Introduction. Guided by the valued insight of the reviewer, we have adjusted the following:

1. First, we changed the title to the suggested one (A Preliminary Study...) instead of referring to it as a true experimental one, focusing on the preliminary nature of our work.
2. The Aim of the Study is now stated clearly in the abstract and the final paragraph of the introduction: “The primary aim of the current study is to test the mechanical vulnerability of the SARS-CoV-2 virus envelope and spike proteins to focused, high-frequency ultrasound waves (25 MHz) in vitro.”
3. We removed the "sweeping generality" the reviewer flagged at the end of the abstract by noting what was actually performed: targeting SARS-CoV-2 virus particles in an aqueous medium. (These results indicate that ultrasound may offer a promising non-pharmacological approach to destroy or inactivate SARS-CoV-2 variants in an aqueous environment).
4. We have adopted the suggested validation framework in the introduction, noting that our study is Step A research, and clarified the need for further research for validation and generalization.  (The aim of the current study is to test the mechanical vulnerability of the SARS-CoV-2 virus envelope and spike proteins to focused, high frequency ultrasound waves in vitro. Since advanced imaging instruments like Atomic Force Microscopy (AFM) or electron microscopy (EM) were not available for biological samples in the study setting, direct visualization of topographical destruction could not be performed. The experimental design was thus adjusted to monitor changes in the Ct values as an indirect measure of viral disruption. Such findings provide preliminary experimental support (Step A) for ultrasound-mediated viral inactivation or structural disruption (indirectly measured via genomic inactivation) and warrant a sequential future validation process, including testing in biological matrices (e.g., blood) and animal models, and by employing high-resolution imaging modalities such as AFM or cryo-EM to cor-relate acoustic exposure with structural damage. Beyond SARS-CoV-2, if reso-nance-based mechanical disruption is confirmed and validated by such future studies, it will offer a widely applicable, non-pharmacological strategy against other enveloped viruses that impact current and future pandemics.)
5. This is also clearly stated at the end of the Discussion section.

Comments 2: [2) The methodology is inadequately described. Firstly, the derivation of samples from COVID 19 patients must be described, so that Table 1 and the results in the first section make more sense. Secondly, the RT-PCR methodology must be described. How were pre-test samples handled? Were they held for 5 minutes prior to assay to control for temperature or the possible action of nucleases? What temperature was the treatment performed at and was temperature monitored during ultrasound treatment? Can you rule out the presence of nucleases in the distilled water?]

Response 2: We appreciate the reviewer’s valuable comment regarding methodology. We have updated the manuscript to clarify that the de-identified samples were retrospectively obtained from a central repository (stored at 4°C for <2 months); table 1 details their demographics. To ensure the results were not affected by the water matrix or handling time, we have now included data from a parallel sham-control pilot study done for internal validity at time of experiment, using Influenza A and B. These samples showed no significant Ct change (P > 0.05) after the same 5-minute immersion confirming the stability of our protocol. We have updated the methodology section to include these control findings.  Regarding handling, all samples were kept on ice throughout the experiment and processed individually to protect genomic stability, with same-day RT-PCR assays. While continuous monitoring was not used, the 0.1% duty cycle used in the experiment made the thermal effect of ultrasound waves unexpected.  Ct values at diagnosis served as the pretest baseline, adjusted for the dilution factor to ensure an accurate comparison with posttest results.

(De-identified nasopharyngeal samples were retrospectively obtained from the central virology laboratory repository (stored at 4°C). Only samples stored for less than 2 months prior to the study were included. The diagnostic Ct values served as the pretest baseline after being adjusted for the dilution factor (0.5 ml VTM : 2.5 ml water) to ensure an accurate baseline comparison for the posttest results using the following formula: [∆ Ct = log2 (DF)]. To ensure internal validity, sham control (n=10, Influenza A and B) was performed using the same distilled water, dilution, experimental duration (5 minutes), and transducer placement, but without ultrasound exposure. No significant Ct changes were observed (P > 0.05), which excludes potential environmental degradation or nuclease activity. The experiment was conducted at 22°C. To maintain genomic stability, all samples were maintained on ice throughout the experiment and processed individually. Following treatment, a same-day posttest RT-PCR assay was performed for all samples while maintaining the cold-chain process. Continuous sample temperature monitoring was not carried out, as the 0.1% duty cycle ensured a nonthermal, mechanical interaction)

Comments 3: [3) I see no attempt made to rule out the contribution of ultrasound induced heating to the reduction in viral load observed. Was this considered?]

Response 3: Our settings utilized a 1 µs burst duration with a 1 ms trigger interval, resulting in a 0.1% duty cycle. This means the ultrasound is (on) for only 1 microsecond and (off) for 999 microseconds during every 1 millisecond of treatment. This extremely low ratio allows for complete thermal relaxation between bursts. This ensures that the sound energy was converted into mechanical energy rather than thermal energy. This is a standard approach in therapeutic ultrasound to achieve non-thermal, cavitational, or mechanical effects. This is added to methodology to clarify that

(The ultrasound intervention was performed at 25 MHz using a 1 µs burst duration and a 1 ms trigger interval ( 0.1% duty cycle) to ensure a purely mechanical, non-thermal interaction at a calculated acoustic pressure of 107.5 kPa.)

Comments 4: [4) I believe that generalizing the ultrasound effect on a variety of enveloped viruses should be deprioritized so that you can focus on mechanism, efficacy in relevant matrices in vitro, including intact infected cells, and extrapolation to in vivo models, if you believe that this is the ultimate aim for this approach.]

Response 4: We completely agree with the reviewer’s suggestion to prioritize the specific mechanical mechanisms and efficacy of the 25 MHz protocol. As noted in our Discussion, we have focused primarily on the resonance-driven envelope instability of SARS-CoV-2, supported by the computational models of Wierzbicki et al. We have specifically detailed the physical requirements, such as direct immersion to eliminate impedance mismatch and the localized high mechanical stress (107.5 kPa) created by the narrowband focused transducer, to explain the observed Ct shifts. While we mention future generalizations as a final thought, the core of our work remains a focus, in vitro proof-of-concept for the mechanical vulnerability of SARS-CoV-2.

4. Response to Comments on the Quality of English Language

Response: We would like to thank the reviewer for the time and effort spent on such a thorough and detailed analysis of our manuscript. It's been a great pleasure to see your detailed critique of the text that provide strong professional perspective on areas of weakness. We have carefully incorporated your linguistic suggestions throughout the document)

5. Additional clarifications

[We would like to clarify 2 points in terms of the terminology:
1) Consistent with current medical style guides (e.g., AMA, APA) that propose closing prefixes to reduce visual clutter and improve readability, the spelling has been standardized to 'pretest' and 'posttest' as single words (without hyphens) across the manuscript, in tables, and in figures.
2) in accordance with the Sex and Gender Equity in Research (SAGER) guidelines, we have chosen to use the term 'sex' rather than 'gender.' These guidelines recommend using 'sex' when reporting biological data and 'gender' when reporting social or cultural identities.]

Response 1: [I think this is the first report on SARS-CoV-2, showing the significance  of ultrasound treatment to inactivate the virus particles in vitro. SARS-CoV-2 is enveloped virus, which may have frequency-dependent mechanical vulnerability, and may be compromised by broadband, low-intensity ultrasound for long duration. Ultrasound at lower frequency and higher intensities is showing more efficient inactivating effect, when they are combined with chemical sesitizers such as methylene blues. Although we do not know the same effect using other enveloped visuses such as HIV or influenze, this manuscript shows the interesting effect for developing antiviral agents against many human diseases.

Further investigations are needed using the methods in more cnotrolled extracorporeal environments, such as blood or plasma circulation systems. However, I think this manuscript shows very interesting evidenses at the first step for the development of antiviral agents.

] We are grateful to the reviewer for their encouraging comments and for recognizing this study as a first report on the frequency-dependent mechanical vulnerability of SARS-CoV-2. We appreciate the reviewer’s insight regarding the comparison between our high-frequency focused approach and lower-frequency methods involving chemical sensitizers. As noted, while sonodynamic therapy with Methylene Blue is highly effective via chemical pathways, our goal was to isolate the pure mechanical resonance of the viral envelope. Furthermore, we fully agree that testing in controlled extracorporeal environments, such as blood or plasma circulation systems, is the next logical step. We have emphasized this in our future directions (Section 4, point iii) to ensure the path toward clinical development is clear. Thank you for recognizing the potential of this work as a foundation for non-pharmacological antiviral strategies.… Based on the valuable reviewer’s note we update this point at the end of discussion section to .[ (ii) investigate whether the mechanical vulnerability observed in this study, focused on the 25 MHz resonance of SARS-CoV-2, can be generalized on other enveloped viruses with different structural dimensions, such as HIV an influenza virus or if it requires frequency optimization for each specific viral architecture]”