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Does the introduction explain the objective of the research presented in the preprint?

Yes

Are the methods well-suited for this research?

Somewhat appropriate

Other studies on microwave inactivation of viruses have measured infectivity directly use plaque or TCID50 assays. This study uses a luciferase readout as an indirect measure of infectivity. It would be good to include a control that compares a traditional measure of infectivity against the measurement made using luciferase (or a citation to another study that has made this comparison). Several aspects of the methods are not described in sufficient detail to allow replication. 1. The dimensions of the CPW wave guide and the media volume used to measure the absorption spectra are not provided. 2. The exposure time for treating the virus in the TEM cell is not provided. 3. The experimental conditions (e.g. media volume, media container [cuvette, dish, slide?]) of the virus in the TEM cell are not described.

Are the conclusions supported by the data?

Highly supported

Are the data presentations, including visualizations, well-suited to represent the data?

Somewhat appropriate and clear

I find the data grouping in Fig. 3B unclear. If I understand correctly, multiple individual frequencies were tested (each with five experimental replicates) and have then been grouped into 5 (unequal) frequency bands that are plotted on a bar graph. As frequency is a continuous variable, I think it would be more informative to display the results on a scatter plot without any grouping of test frequencies (testing for significant differences for each frequency does not seem necessary if plotted in this way, fitting inactivation as a function of frequency may be more appropriate). This is important because two frequencies are selected for further testing with an ELISA assay for Fig. 4. The authors note that these resulted in the 'greatest or smallest reductions in infectivity', but as the results of tests at these frequencies are grouped with the results from other frequencies in Fig. 3B, so it is impossible to judge how much greater (or smaller) the results from these frequencies are from the others in the group.

How clearly do the authors discuss, explain, and interpret their findings and potential next steps for the research?

Somewhat clearly

The study found the absorption spectra peaked at 3.1 and 6 GHz, and they obtained the greatest inactivation at the former frequency and the lowest inactivation at the later. I think it would have been good for the discussion to comment on how the results at each absorption peak differed (i.e. one gave the best inactivation, the other the worst). I don't think this has been identified in other literature. 'whether our CPW/VNA system reduces the infectivity of IAV, HIV-1, HCV or other pathogenic enveloped viruses is intriguing but was beyond the scope of this study.' My understanding is that the CPW/VNA system was used to measure the absorption spectra while the TEM cell was used for reducing viral infectivity. If so, this sentence should be corrected.

Is the preprint likely to advance academic knowledge?

Somewhat likely

The preprint is the first to systematically test inactivation at frequencies below 6 GHz (but see doi.org/10.1117/12.2611335), and I agree that is the first to identify a clear mechanism for the reduction in infectivity caused by EM.

Would it benefit from language editing?

No

Would you recommend this preprint to others?

Yes, but it needs to be improved

Better description of the experimental conditions to enable replication (as noted previously).

Is it ready for attention from an editor, publisher or broader audience?

Yes, after minor changes

The authors affiliated with Epirus Inc are the investors of a patent related to the use of EM for inactivating viruses (https://patents.google.com/patent/WO2023200689A1). Most journals would require listing this as a financial conflict of interest.

Competing interests

I provide consulting services for Resonant Health Inc.