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Antiviral and Immunomodulatory Effects of 7-Deaza-2-methyladenosine (7DMA) in a Susceptible Mouse Model of Usutu Virus Infection

Viruses 2025, 17(12), 1639; https://doi.org/10.3390/v17121639

by Rebeca P. F. Rocha^1,2

, Marina A. Fontoura¹

, Fabrício Naciuk¹

, Leonardo C. Oliveira¹, Alice Nagai¹

, Amanda Bellini Silva^1,3

, Alexandre Borin^1,3, Jaqueline S. Felipe^1,3, Marjorie Bruder¹

, Lais D. Coimbra¹ and Rafael Elias Marques^1,*

Reviewer 1:

Armando Arias

Reviewer 2: Anonymous

Reviewer 3: Anonymous

Viruses 2025, 17(12), 1639; https://doi.org/10.3390/v17121639

Submission received: 14 October 2025 / Revised: 10 December 2025 / Accepted: 12 December 2025 / Published: 18 December 2025

(This article belongs to the Special Issue Antiviral Development for Emerging and Re-Emerging Viruses)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This paper by Rocha et al examines the antiviral activity of a promising nucleoside analogue (7DMA) against Usutu virus, an emerging flavivirus that poses a potential threat to humans. To the present date, no drugs have been licensed against USUV, and hence these studies might be relevant to control infection and spread if a spillover ever occurs. These results can be extended to the treatment of other important flaviviruses with a major impact on global health.

The manuscript may be of major interest to those working in the fields of flaviviruses biology and antiviral research. I support its publication in Viruses once the authors have addressed some suggestions listed below

Major point:

The authors observe a significant reduction in the number of splenic lymphocytes in mice treated with 7DMA (Fig 2J, 2K). They argue that this could be due to its potential cytotoxic effect (lines 527 to 534). Specifically, they report that this could be linked to mitochondrial toxicity but show no cytotoxicity assay to prove this. In addition to this, they also show the antiviral activity displayed by this drug in Vero and SH-SY5Y cells, but no viability data in the presence of 7DMA is provided.

There are several kits available (e.g. MTT, Cell Titer, etc) that measure cell viability after exposure to drugs. In particular, MTT specifically quantifies a mitochondrial enzymatic activity that is only maintained in metabolically active cells. I strongly suggest the authors perform a cell viability assay in both Vero and SH-SY5Y treated during 24/48 h in the presence of 7DMA. Ideally, the authors could extrapolate a cytotoxic concentration 50 value in both cell lines that could help better interpret the data.

Minor points and typos:

Fig 1A shows significant differences at both 24 and 48h in Vero cells but no significant differences are found at 24 h in any of the concentrations tested in SH cells. Can you confirm?
To better compare Vero and SH cells in panels C and D (Figure 1), I’d suggest the authors organize the graph in D containing 4 indentations ( e.g. from 10^2 to 10^5
Lines 266 and 269 should read Figure 2D-G. correct the typos
Lines 319-322. These statements are somewhat ambiguous. You mean that 7DMA leads to increased CXLC10 and decreased CSF and LIF, but ONLY in INFECTED animals, right? Please can you rephrase the sentence to be more precise?
You do not explain the suggested mechanism of action for 7DMA. Is a polymerase chain terminator? Dos it cause antiviral mutagenesis such as favipiravir? Is the viral polymerase its main target? Are there any structural studies on its binding site? Please provide more detail here

Author Response

R: We thank Reviewer 1 for his valuable comments on our manuscript.

Major point:

R: As correctly noted by Reviewer 1, it is critical to assess the potential cytotoxic effects of 7DMA in parallel with its antiviral activity. We previously performed MTT viability assays in both Vero and SH-SY5Y cells treated with 7DMA in prior work (Naciuk et al., 2023). In that study, we showed that 7DMA exhibited no significant cytotoxicity in tested cell cultures at concentrations up to 100 μM, the same concentration range used in the present work. We added this reference [16] and a comment to the revised manuscript (lines 219-221). Additionally, the low in vitro cytotoxicity of 7DMA has been independently confirmed by others (see Eyer et al., 2017) in a similar study on West Nile virus.

While we observed that 7DMA shows only minor cytotoxic effects in vitro, we did not assess 7DMA's acute toxicity in vivo. Indeed, clinical development of 7DMA for Hepatitis C treatment but failed on clinical trials due to toxicity-related issues (Carroll et al., 2003; Cho et al., 2020; Naciuk et al., 2023). Our comments in the discussion section (now in lines 549-552) were rewritten to include this limitation, and the hypothesis that reduced splenic lymphocyte counts may be a result of 7DMA toxic effects in vivo.

Minor points and typos:

Fig 1A shows significant differences at both 24 and 48h in Vero cells but no significant differences are found at 24 h in any of the concentrations tested in SH cells. Can you confirm?

R: No, we observed a statistically significant difference in the group treated with 7DMA at a concentration of 100 µM at both 24 and 48h post-infection. We added a *** to the graph in Figure 1B, indicating the statistical significance of 7DMA treatment at 48h, to indicate that treatment at 24h was also significant at the maximum concentration tested.

To better compare Vero and SH cells in panels C and D (Figure 1), I’d suggest the authors organize the graph in D containing 4 indentations ( e.g. from 10^2 to 10^5).

R: The axes in panel D have been adjusted to include four log-scale tick marks (10² to 10⁵), consistent with panel C, to facilitate direct comparison between Vero and SH cells.

Lines 266 and 269 should read Figure 2D-G. correct the typos.

R: The typographical errors have been corrected, and the citation now correctly reads “Figure 2D–G” in lines 266 and 269 of the revised manuscript.

Lines 319-322. These statements are somewhat ambiguous. You mean that 7DMA leads to increased CXLC10 and decreased CSF and LIF, but ONLY in INFECTED animals, right? Please can you rephrase the sentence to be more precise?

R: We rephrased that paragraph (lines 317-322) to remove the ambiguity: “Analysis of cytokines and chemokines expressed in the spleen of IFNAR-/- mice indicated that USUV induced the expression of IL-6 (Figure 3A), CXCL10 (Figure 3B), CXCL1 (Figure 3C), G-CSF (Figure 3D), and LIF (Figure 3H), of which CXCL10 and G-CSF were expressed at the highest levels. Infected mice treated with 7DMA showed increased expression of CXCL10 and reduced expression of G-CSF and LIF. Importantly, 7DMA treatment also led to the expression of GM-CSF (Figure 3E), IL-15 (Figure 3F), and IL-1β (Figure 3G) in USUV-infected mice.”

You do not explain the suggested mechanism of action for 7DMA. Is a polymerase chain terminator? Dos it cause antiviral mutagenesis such as favipiravir? Is the viral polymerase its main target? Are there any structural studies on its binding site? Please provide more detail here

R: 7DMA is a nucleoside analog that is phosphorylated intracellularly by host kinases to its active 5'-triphosphate form. This metabolite acts as a competitive substrate for viral RNA-dependent RNA polymerases and is incorporated into the nascent viral RNA strand. Current evidence suggests that 7DMA functions primarily as a non-obligate chain terminator. Once incorporated, it causes premature termination of RNA synthesis due to structural alterations at the 2'-position (the 2'-C-methyl group), which interferes with further elongation (Barik, 2022; Zmurko et al., 2016). In response, we have expanded the Introduction to include the proposed mechanism of action of 7DMA in more detail (lines 53-56).

Reviewer 2 Report

Comments and Suggestions for Authors

The article is overall clear and reads well. The research hypotheses and objectives should however be better defined.

The conclusions are not completely supported by the data and the interpretation of the results is often disputable (see further). Several biases are not taken into account.

The methodology is clearly explained albeit not always appropriate. Virological and immunological approaches used appear to be well-established in the authors laboratory and the results are solid.

7DMA being a nucleoside analog, its well-known mode of action involves the inhibition of the RNA-dependent RNA polymerase of RNA viruses, including those from the Orthoflavirus genus. It is thus expected to affect the replication of Usutu virus. It is not so clear in this context what is the added value of the “time-of-addition” experiment. 7DMA has logically a stronger effect on viral replication when applied early. Besides, this test is useless to compare the two cell lines. Indeed, the viral stock used has been prepared in Vero cells (it is thus a “Vero-adapted” variant). As a consequence, when used at relatively high MOI (0.1), it quickly reaches the replication plateau in Vero cells (<24h). But, being not adapted to SH-SY5Y cells, it requires more time to reach the plateau in this cell line. So the replication kinetics are not comparable in both cell lines. Logically, there is no effect of 7DMA once the plateau has been reached in Vero cells (because viral replication is limited beyond 24h pi), hence the limited impact at 48H in this cell type. Using lower MOIs and longer kinetics would have helped to highlight that.

One general interpretation of the results could be that the partial inhibition of USUV replication by 7DMA delays the replication kinetics, which is frequently observed with antivirals. Assessing viral replication on a longer period (for instance up to 7 days pi) would have allowed to see if the virus eventually reaches a similar replication plateau. Besides, it would be informative to know whether the process induces the selection of viral escape mutants. In this context, the delay observed in the replication kinetics would correspond to the time needed for the initial selection of these mutants (not considered or even mentioned, and thus not tested, by the authors).

This last hypothesis is in line with the in vivo data. 7DMA does not really protect mice, it only postpones the fatal outcome by 2 to 3 days. It would be tempting to suggest that this delay of 2-3 days is the time needed for the selection of 7DMA-resistant viral mutants. It remains also possible that stopping the 7DMA treatment on day 6 then allowed the virus to replicate freely and induce the mortality of mice on days 7 to 9 (did the author test the possibility of treating beyond day 6?). In any case, comparing untreated and treated mice only on days 3 and 5 drastically limits the interpretation of the data. The conclusions made by the authors about the immunomodulatory effects of 7DMA are not completely supported by the data. First, should the “delay” hypothesis prove true, one should compare the whole kinetics between untreated and treated mice (the same expression pattern as observed in untreated mice on day 5 could only appear on day 7 or 8 in treated mice). Next, does 7DMA really have immunomodulatory effects per se, or is it a mere consequence of the lower viral replication (more likely)? It all comes down to the antiviral activity of 7DMA. It remains true that a lower viral replication in the early stages of infection may change the host-pathogen dynamics and, hence, the host innate immune response. But this is a quite different interpretation from that made by the authors.

Similarly, the following conclusion “We did not observe significant tissue damage or pathology in USUV-infected mouse brains, suggesting that systemic infection and disease are the major components leading to mortality in this model.” is another personal interpretation by the authors not supported by the data. Viral loads are higher in the brain than in other organs, meaning it is a major site of viral replication. Did the authors perform IHC for viral antigens to visualize the extent of viral replication in brain tissues? Did they use more refined approaches (such as apoptosis tests and markers, or cytotoxicity assays, for instance) to evaluate the actual damage to brain cells? It is very likely that cell mortality is extensive at the time of death in these mice.

All in all, this study includes very interesting results obtained using robust methods but which has major flaws in the experimental design (due to poorly defined research questions) and the interpretation of the data. I would suggest to broaden the kinetic study, especially in vivo up to day 8 in 7DMA-treated mice. It might not be needed to repeat all the extensive analyses made on days 3 and 5 post-infection, but at least to follow up some key parameters to better define whether 7DMA only induces a time-shift in the host response and pathology, or if it really changes the host-virus dynamics. I would encourage the authors to be more cautious to avoid any overinterpretation of the data.

Author Response

In this manuscript entitled “Antiviral and Immunomodulatory effects of 7-deaza-2-methyladenosine (7DMA) in a susceptible mouse model of Usutu virus infection”, the authors assess the impact of 7DMA on Usutu virus replication in vitro and in vivo. A detailed analysis of the effects of 7DMA treatment on the host cytokine/chemokine and cellular responses to infection is also presented. The article is clear overall and reads well. The research hypotheses and objectives should, however, be better defined. The conclusions are not completely supported by the data, and the interpretation of the results is often disputable (see further). Several biases are not taken into account. The methodology is clearly explained albeit not always appropriate. Virological and immunological approaches used appear to be well-established in the author’s laboratory and the results are solid.

R: We thank Reviewer 2 for such a detailed evaluation of our manuscript. We made several changes to the manuscript to address his/her concerns.

R: We acknowledge that assessing longer replication kinetics and using lower MOIs could provide additional insight into whether 7DMA delays viral replication or whether viral loads eventually reach a similar plateau. However, our experimental design focused specifically on early and intermediate stages of infection, which are the most relevant windows for evaluating the antiviral effects of nucleoside analogs. In our time-of-addition assays, 7DMA retained antiviral activity up to 18 hours post-infection, suggesting prolonged intracellular action consistent with the expected mechanism of delayed replication under nucleoside analog pressure (Figure 1C, 1D). Although inhibition was not observed at later stages (≥20 hours post-infection), these findings define the compound’s effective temporal window and align with what is expected based on its known mode of action.

7DMA is indeed a nucleoside analogue with a well-known proposed mechanism of action and activity against a range of arboviruses. However, this mechanism of action is inferred from original studies dated from when 7DMA was being developed as a candidate hepatitis C treatment. Importantly, different nucleoside analogues have been tested against various orthoflaviviruses, and showed various levels of potency and cytotoxicity, even when their RdRp active sites are highly conserved (Eyer et al., 2018). Supporting our observations, a comparative study of 14 nucleoside analogs reported that 7-deaza-7-fluoro-2′-C-methyladenosine, a close structural analog of 7DMA, exhibited potent activity against JEV and all DENV serotypes (Zandi et al., 2019). However, other analogs known to inhibit HCV had little to no efficacy in Vero cells. These differences were attributed to both viral polymerase variation and host cell-specific factors such as differential prodrug metabolism and uptake, highlighting the value of using both Vero and SH-SY5Y cells to assess compound activity across distinct cellular environments (Zandi et al., 2019). Regardless of how confident we were in our hypotheses (that 7DMA would exert the expected antiviral activity, and be protective against USUV infection in vivo), we would have to generate the results to back up our claims, resulting in this manuscript. As stated in lines 238-241, the greater efficacy of 7DMA at early timepoints is consistent with its established mechanism of action as an inhibitor of the viral RdRp. This assay not only indicates that 7DMA may act against USUV using the same mechanism of action but also determines the stage of the USUV replication cycle in which the compound is most effective. The biology of USUV is far less characterized than other Orthoflaviviruses, and we contributed towards a better understanding of how nucleoside analogues can be used against USUV.

Regarding the reviewer’s concern about the use of a Vero-adapted viral stock, prior work has shown that a single passage is unlikely to drive significant adaptation, as related flaviviruses such as WNV and SLEV typically require more than 10 serial passages to exhibit measurable replicative advantages (Ciota et al., 2007). We note that our USUV stocks were generated from a single round of propagation in Vero CCL81 cells, with supernatants collected at 96 h p.i. Viral titers were determined by plaque assay. We have clarified this point in the Methods section (lines 82-85).

R: We appreciate the reviewer’s suggestion that the observed delay in replication kinetics may reflect the initial selection of viral escape mutants, which was not evaluated in our study. However, we consider this explanation unlikely given the experimental conditions. Previous work by Eyer et al. demonstrated that resistance to 7DMA in tick-borne encephalitis virus required extended exposure and serial passaging over at least 12 days under increasing drug pressure (Eyer et al., 2017). In contrast, our in vitro assays were limited to shorter timeframes and did not apply continuous selective pressure that would support the emergence of resistant variants.

Moreover, resistance-associated mutations to nucleoside analogs, such as those reported in hepatitis C virus models (Mejer et al., 2020) are often linked to a loss in replicative fitness. When treatment is withdrawn, wild-type viruses typically regain dominance due to their superior fitness. These observations suggest that the emergence of resistant variants during the short window of 7DMA exposure used in our experiments is unlikely.

R: Regarding the reviewer’s suggestion that the observed delay in mortality may result from the selection of resistant viral variants, we consider this explanation unlikely given the timeframe and experimental conditions of our study. As discussed earlier, Mejer et al. demonstrated in hepatitis C virus models that resistance-associated variants selected under nucleoside analogue pressure tend to have reduced replication fitness compared to wild-type strains (Mejer et al., 2020). It has been shown that, once treatment is discontinued, these less fit variants are typically outcompeted by the wild-type virus, which rapidly re-establishes dominance (Berger et al., 2015; Eyer et al., 2017).

In our study, 7DMA treatment was interrupted at day 6 p.i.. The limited treatment duration, combined with the absence of extended selective pressure, makes the emergence of resistant variants unlikely. That said, we recognize that extending treatment beyond day 6 or including later sampling points (e.g., days 7–9) could offer further insight into whether 7DMA provides sustained protection or merely delays disease progression. However, prolonged treatment could also introduce confounding factors, such as potential drug-induced toxicity or additional immunomodulatory effects. Addressing these questions would require a distinct experimental design with dedicated toxicological assessments, which fall beyond the scope of the current study. This limitation has also been noted in the revised manuscript (lines 454–460).

It's important to note that our study shows that in vivo treatment with the nucleoside analogue 7DMA up to day 6 p.i. delayed mortality in IFNAR^-/- mice by 2–3 days, reduced viral loads and altered inflammatory profiles across tissues. As described in lines 553-561: "In summary, our study demonstrates that treatment with the nucleoside analogue 7DMA delays disease progression, reduces viral loads, and is associated with alterations in the inflammatory profile across tissues…”. It's important to note that in the current study, we used IFNAR^-/- mice, which are extremely susceptible to viral infections. Therefore, the fact that 7DMA treatment delayed mortality by 2–3 days, despite a high infection dose (10⁴ PFU), highlights a meaningful therapeutic effect, especially considering the acute and lethal nature of disease progression in this model.

The conclusions made by the authors about the immunomodulatory effects of 7DMA are not completely supported by the data. First, should the “delay” hypothesis prove true, one should compare the whole kinetics between untreated and treated mice (the same expression pattern as observed in untreated mice on day 5 could only appear on day 7 or 8 in treated mice). Next, does 7DMA really have immunomodulatory effects per se, or is it a mere consequence of the lower viral replication (more likely)? It all comes down to the antiviral activity of 7DMA. It remains true that a lower viral replication in the early stages of infection may change the host-pathogen dynamics and, hence, the host innate immune response. But this is a quite different interpretation from that made by the authors.

R: We emphasize that 7DMA demonstrated clear antiviral efficacy against USUV in vivo, even under stringent conditions. In our study, mice infected with a high viral inoculum (10⁴ PFU) exhibited a 2–3 day delay in mortality following 7DMA treatment. This is particularly significant considering the acute and lethal disease progression in IFNAR^-/- mice, which are highly susceptible to arboviral infections. For context, a previous study evaluating favipiravir treatment in USUV-infected mice used a much lower inoculum (10¹ PFU) and similarly observed a 3-day delay in mortality after six days of treatment (Segura Guerrero et al., 2018). Therefore, the severity of disease in our model and the high viral dose used further support the therapeutic relevance of our findings.

As previously mentioned, we acknowledge that extending treatment beyond day 6 p.i. would have provided additional insight into the long-term antiviral efficacy of 7DMA. For example, in a ZIKV model, 7DMA was administered starting 1 hour pre-infection and continued for 10 days, resulting in a longer delay in disease progression. A continued treatment would require a dedicated experimental design including pharmacokinetic and toxicological endpoints, which are beyond the scope of the present study. This limitation is now discussed in the revised manuscript (Discussion, lines 454-460).

The efficacy of 7DMA in reducing viral loads across different tissues was demonstrated and highlighted in Figures 2D–G (lines 263-269). As shown in Figures 3A–H, 4A–F, and 5A–D, the inflammatory profile resulting from USUV infection-only is characterized by increased levels of the cytokines and chemokines IL-6, CXCL10, CXCL1, G-CSF, and LIF in the spleen; IL-1α, CXCL10, CXCL5, G-CSF, and CXCL1 in the liver; and CXCL10, G-CSF, CXCL1, and CCL11 in the brain. However, some cytokines and chemokines were detected exclusively in the USUV-infected 7DMA-treated group, including GM-CSF, IL-15, and IL-1β (Figures 3E, F, and G) in the spleen, and IFN-γ in the liver. This finding suggests that, in addition to its direct antiviral action, 7DMA may modulate specific immune pathways in the context of infection. If we were solely observing an indirect effect of a reduced USUV load in tissues, we would observe only a reduction in cytokine and chemokine levels in 7DMA-treated animals. We do not propose that 7DMA acts as a direct immunomodulator but affects the balance between antiviral immunity and immunopathology.

Finally, we agree with the reviewer that our current dataset does not allow us to definitively distinguish between direct immunoregulatory effects of 7DMA and secondary changes due to reduced viral replication. To clarify this point, we have revised the Introduction (lines 70-76), Results (lines 279-283), and Discussion (lines 558-560) to reflect that the observed changes in cytokine and chemokine profiles may be indirect consequences of antiviral activity rather than evidence of direct immunomodulation. These revisions aim to present a more accurate and balanced interpretation of our findings in alignment with the reviewer’s feedback.

Similarly, the following conclusion “We did not observe significant tissue damage or pathology in USUV-infected mouse brains, suggesting that systemic infection and disease are the major components leading to mortality in this model.” is another personal interpretation by the authors not supported by the data. Viral loads are higher in the brain than in other organs, meaning it is a major site of viral replication. Did the authors perform IHC for viral antigens to visualize the extent of viral replication in brain tissues? Did they use more refined approaches (such as apois tests and markers, or cytotoxicity assays, for instance) to evaluate the actual damage to brain cells? It is very likely that cell mortality is extensive at the time of death in these mice.

R: Please refer to Figure 2D, E F and G, which altogether show that the spleen and brain reach similar infectious viral loads at day 5 p.i., which is also comparable to the viremia. We did not perform immunohistochemistry for viral antigens or apoptosis-specific assays such as cleaved caspase-3 or TUNEL staining. The lack of inflammatory infiltrates and microglial activation, reduced proinflammatory mediator expression in comparison to other tissues and the lack of indicators of brain pathology indicate little tissue damage in USUV-infected IFNAR^-/- mice. In the absence of type I interferon signaling, neuroinflammatory responses are often blunted (Cao, 2024; Rocha et al., 2021), reducing the likelihood of immune-mediated tissue damage. As discussed in lines 469-481 of the revised manuscript, this altered immune context may contribute to the absence of histopathological signs of brain injury observed in our study as it has been discussed by others.

Importantly, we observed no signs of microglial activation, which is a widely accepted marker of neuroinflammation and neuropathology in models of neurodegenerative diseases and viral infections. Similarly, in mice infected with SLEV, accumulation of cleaved caspase-3 and neuronal apoptosis has been observed primarily in the presence of microglial activation, even when viral antigens are present in the brain (Segura Guerrero et al., 2018). Taken together, the absence of both microglial reactivity and histological abnormalities in our study supports the conclusion that significant brain cell damage did not occur under the conditions tested.

R: We thank the reviewer for their constructive feedback. As suggested, extending the in vivo analysis beyond day 6 post-infection would indeed provide valuable insights into the long-term effects of 7DMA treatment. However, due to the high lethality of the model used, in which one hundred percent of IFNAR^-/- mice succumb to infection by day 6, our study focused on early and intermediate timepoints, specifically days 3 and 5 post-infection. As noted in the revised manuscript, addressing this question would require a different experimental design, including extended treatment duration and evaluation of drug-related toxicity, which are beyond the scope of the current study. Also, we have carefully revised our text to present a more cautious and balanced interpretation of the data.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript by Rocha et al. entitled “Antiviral and Immunomodulatory effects of 7-deaza-2-methyladenosine (7DMA) in a susceptible mouse model of Usutu virus infection” presents data on the effects of treatment with 7DMA on USUV infection, using both cell cultures and mice. The cell lines used are Vero CCL-81 derived from African green monkey kidney and SH-SY5Y derived from human neuroblastoma. The mice used in the in vivo studies were IFNAR^-/-, which are more susceptible to USUV infection. In both the in vitro and in vivo models, the authors observed a protective effect of treatment with 7DMA in USUV infection. In the in vivo model, they observed no damage to the tissues analyzed as a result of infection. One of the conclusions of the study is that “The characterization of the protective effects of 7DMA indicated that treatment also altered immunological aspects of disease development, further increasing the expression of mediators such as CXCL10, IL-15, and IFN-γ, and increasing neutrophil recruitment to target organs.”

After reading the manuscript, I have certain doubts, which I list below as major points:

1. The main one relates to the conclusion summarized in the abstract of the manuscript regarding the relationship between treatment and immune response. With the data presented, I think it is not possible to differentiate between the direct effect of 7DMA and the indirect effect of treatment through decreased viral replication. I believe that the authors cannot separate the direct effects from the indirect ones with the experimental strategy proposed, and this should be noted in the manuscript. The text should be clarified by introducing this uncertainty.

In relation to this point, is the difference observed in Figure 2H when comparing USUV+vehicle with USUV+7DMA a direct consequence of the treatment or an indirect consequence of improved survival? The same applies to the differences in Figure 3P and the associated text in lines 334-337. Also text in lines 369-372.

2. The authors use doses of 50 mg/kg body weight of 7DMA in the treatment of mice. Why this dose and not another? The authors should add a sentence and references to establish the basis for this therapeutic dose. On the other hand, analyzing the effect of different doses (there are studies that use up to 75 mg/kg of weight) would help to see if there is a dose-response effect between the treatment and the cytokine profile. Another way to try to separate the direct effects of treatment from the indirect ones would be to use another type of competitive inhibitor in parallel and analyze the cytokine profile.

I understand that this would considerably increase the experimental work, so replying to the comment in point 1 would be sufficient.

3. 7MDA is not commercially available but is synthesized by colleagues in organic chemistry. In this case, I believe it would be necessary to show, even if only as supplementary information, the synthesis parameters indicating the purity level and structure of the compound obtained.

4. Studies of treatment effectiveness in cell lines are conducted with drug concentrations between 3.125 microM and 100 microM over periods of 24 and 48 hours. However, no cell viability data are presented for this range of concentrations and times, and this is essential information that the authors must provide. This will allow us to know that, under the conditions used in the experiment, cell viability is not altered.

5. The recommended representation of data when using a nonparametric test, such as Kruskal-Wallis, is with the median and interquartile range. The statistical data for values p=0.08 and p=0.09 shown in Figure 1C (and others) are not very robust. I understand that you would like to compare the results in Figures 1C and 1D, but in this case, it is only a trend without statistical significance.

6. What is the viral load detection limit (understood as PFU/mL) in different tissues? If you know this, it would be good information to include in the methodology section of the manuscript. Having the genome copy data (obtained by RT-qPCR) in those same tissues would provide very robust confirmation of the viral load data. Is it still possible?

7. Lines 325-327 indicate differences between the mock vehicle and the USUV vehicle. However, no significant differences are indicated between these values in graphs 3J and 3K, as it is indicated in graphs 3I, 3L, and 3M. The text or graphics would need to be modified.

8. Figures 5J and 5K show an increment trend not only for USUV-infected and treated, but also, although to a lesser extent, for untreated USUV. In both cases, the value obtained is higher in USUV than in mock.

Author Response

After reading the manuscript, I have certain doubts, which I list below as major points:

The main one relates to the conclusion summarized in the abstract of the manuscript regarding the relationship between treatment and immune response. With the data presented, I think it is not possible to differentiate between the direct effect of 7DMA and the indirect effect of treatment through decreased viral replication. I believe that the authors cannot separate the direct effects from the indirect ones with the experimental strategy proposed, and this should be noted in the manuscript. The text should be clarified by introducing this uncertainty.

R: We thank the reviewer for this important observation. As suggested, we have revised the Discussion section to clarify that the current experimental design does not allow us to distinguish whether the immunological changes observed in 7DMA-treated animals are due to direct immunomodulatory effects of the compound or are secondary to reduced viral replication (lines 558-560). This clarification has been included explicitly in the revised manuscript (lines 279-283). We believe this addresses the concern and aligns the interpretation of our findings with the limitations of the study.

The authors use doses of 50 mg/kg body weight of 7DMA in the treatment of mice. Why this dose and not another? The authors should add a sentence and references to establish the basis for this therapeutic dose. On the other hand, analyzing the effect of different doses (there are studies that use up to 75 mg/kg of weight) would help to see if there is a dose-response effect between the treatment and the cytokine profile. Another way to try to separate the direct effects of treatment from the indirect ones would be to use another type of competitive inhibitor in parallel and analyze the cytokine profile.

I understand that this would considerably increase the experimental work, so replying to the comment in point 1 would be sufficient.

R: The dosage was determined based on previous literature (Jacobs et al., 2019; Morrey & Siddharthan, 2025; Zmurko et al., 2016). In the study by Morrey and Siddharthan., the authors briefly assessed the antiviral effect of 7DMA in a Usutu virus infection model using doses of 7.5, 23, and 75 mg per kg. They reported that the highest dose, 75 mg per kg, was the most effective in reducing viral load and preventing mortality, although they did not further investigate the underlying mechanisms or host responses. We selected a dose of 50 mg per kg based on prior evidence of efficacy and safety at this level in other viral infection models, including Zika virus. While higher or lower doses are certainly possible, we considered 50 mg per kg to be a reasonable and safe choice for evaluating both antiviral and immunopathogenic outcomes. We have now included a statement in the Methods section to clarify the rationale for this dosage (lines 139-140). Regarding the use of another type of competitive inhibitor, there is no other antiviral compound with reported antiviral activity against USUV in a different chemical class/ mechanism of action, which prevented us from including it in the study.

7MDA is not commercially available but is synthesized by colleagues in organic chemistry. In this case, I believe it would be necessary to show, even if only as supplementary information, the synthesis parameters indicating the purity level and structure of the compound obtained.

R: We appreciate the reviewer’s comment. The synthesis and characterization of 7DMA, including details on purity and structural validation, are described in detail in our previously published work (Naciuk et al., 2023), as referenced in the Methods section (lines 104-106).

Studies of treatment effectiveness in cell lines are conducted with drug concentrations between 3.125 microM and 100 microM over periods of 24 and 48 hours. However, no cell viability data are presented for this range of concentrations and times, and this is essential information that the authors must provide. This will allow us to know that, under the conditions used in the experiment, cell viability is not altered.

R: We thank the reviewer for this observation. We would like to clarify that MTT viability assays in both Vero and SH-SY5Y cells treated with 7DMA were previously performed and reported by our group (Naciuk et al., 2023). In that study, 7DMA showed no significant cytotoxicity at concentrations up to 100 µM, consistent with the exposure range used in the current experiments. To reinforce this point, we have now referenced and summarized these findings in the revised manuscript (lines 219-221).

The recommended representation of data when using a nonparametric test, such as Kruskal-Wallis, is with the median and interquartile range. The statistical data for values p=0.08 and p=0.09 shown in Figure 1C (and others) are not very robust. I understand that you would like to compare the results in Figures 1C and 1D, but in this case, it is only a trend without statistical significance.

R: Figure modified as suggested.

What is the viral load detection limit (understood as PFU/mL) in different tissues? If you know this, it would be good information to include in the methodology section of the manuscript. Having the genome copy data (obtained by RT-qPCR) in those same tissues would provide very robust confirmation of the viral load data. Is it still possible?

R: We modified the methods to include the detection limit of the plaque-forming assay, as suggested. We agree with the reviewer that RT-qPCR data would improve our work; these samples were also processed for the ELISA multiplex analysis, which rendered them unusable for qPCR.

Lines 325-327 indicate differences between the mock vehicle and the USUV vehicle. However, no significant differences are indicated between these values in graphs 3J and 3K, as it is indicated in graphs 3I, 3L, and 3M. The text or graphics would need to be modified.

R: Description of results has been modified as suggested.

Figures 5J and 5K show an increment trend not only for USUV-infected and treated, but also, although to a lesser extent, for untreated USUV. In both cases, the value obtained is higher in USUV than in mock.

R: Indeed, we confirm a trend towards increased leukocyte counts in the brains of USUV-infected, and further in USUV-infected mice treated with 7DMA. Although the average values for each group are visually clear and interesting, the statistical analysis indicated no differences between the groups. We interpreted these findings as more evidence suggesting that USUV-induced brain immunopathology is discrete in this model.

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Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Now I consider the manuscript is acceptable for its publication in Viruses

Author Response

We appreciate the positive response for the reviewer. Thanks for taking the time and effort to review our work.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors overall appropriately addressed my concerns.

Since it was not obvious in the first version of the manuscript, I would recommend to the authors to include key references supporting the fact that the selection of viral escape mutants is unlikely during a single cell passage. It is a key point for the interpretation of the data.

Besides, I do not agree with the authors regarding the lack of lesions in the brain. Neuronal loss can occur without gliosis or microglial activation. It can be difficult to observe without specific methods. Given the relatively high viral loads in the brain, and given the well-known neurotropism of this virus, it would be more accurate to remain cautious. It is true that a multisystemic disease is observed, but it cannot be ruled out based on the data presented that some significant cellular damage occurs in the brain as well. Please bring some nuance in the interpretation and discussion.

I trust the authors to take these further comments into consideration and to implement minor corrections to the manuscript accordingly.

Author Response

The authors overall appropriately addressed my concerns.

R: We made additional changes to the manuscript to address Reviewer 2 concerns. To minimize adaptation to cell culture, we used a single passage of virus, referred in line 107, and added related bibliography to the statement (Ciota et al., 2007). Regarding the concern about viral escape mutants, we included the requirement of extended exposure and reference on lines 61 and 62 (Eyer et al., 2017). Finally, we discuss on lines 522 - 526 that our experimental conditions do not attend to the requirements to the generation of 7DMA-resistant variants.

I trust the authors to take these further comments into consideration and to implement minor corrections to the manuscript accordingly.

R: We agree with Reviewer 2 that we cannot rule out neuronal cell damage in this model, due to the lack of specific methods to assess it. USUV infection induces neuronal damage in wild-type mice (Rocha et al., 2025). However, we lack this kind of data in IFNAR^-/- mice studies, as USUV pathogenicity is strain-dependent and may (Clé et al., 2020) or may not induce neuronal damage (Kuchinsky et al., 2020). We addressed this topic with supporting literature and the technical limitations in lines 477 – 484.

Reviewer 3 Report

Comments and Suggestions for Authors

All comments have been answered satisfactorily.

Author Response

We appreciate the positive response for the reviewer. Thanks for taking the time and effort to review our work.

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Antiviral and Immunomodulatory Effects of 7-Deaza-2-methyladenosine (7DMA) in a Susceptible Mouse Model of Usutu Virus Infection

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