The influence of the pre-membrane and envelope proteins on structure, pathogenicity and tropism of tick-borne encephalitis virus

  1. Ebba Rosendal
  2. Kyrylo Bisikalo
  3. Stefanie M.A. Willekens
  4. Marie Lindgren
  5. Jiří Holoubek
  6. Pavel Svoboda
  7. Amanda Lappalainen
  8. Ebba Könighofer
  9. Ekaterina Mirgorodskaya
  10. Rickard Nordén
  11. Federico Morini
  12. William Rosenbaum
  13. Daniel Růžek
  14. Ulf Ahlgren
  15. Maria Anastasina
  16. Andres Merits
  17. Sarah J Butcher
  18. Emma Nilsson
  19. Anna K Överby

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Abstract

Tick-borne encephalitis virus (TBEV) is a neurotropic flavivirus that causes thousands of human infections annually. Viral tropism in the brain is determined by the presence of necessary receptors, entry factors and the ability of the virus to overcome host defenses. The viral structural proteins, pre-membrane (prM) and envelope (E), play an important role in receptor binding, membrane fusion, particle maturation, and antibody neutralization. To understand how these proteins influence virus distribution and tropism in the brain, we generated a chimeric virus harboring the prM and ectodomain of E from TBEV in the background of the low pathogenic Langat virus (LGTV). We solved the atomic structures of both the chimeric virus and LGTV to compare them to the known TBEV structure. We show that this chimeric virus remains low-pathogenic, while being structurally and antigenically similar to TBEV. Using 3D optical whole brain imaging combined with immunohistochemistry, we found that both LGTV and the chimeric virus primarily infect cerebral cortex, with no significant differences in their localization or tropism. In contrast, TBEV shows high infection of the cerebellum and strong preference towards Purkinje cells, indicating that the non-structural proteins are important for determining TBEV tropism in the brain. Together, this provides new insights into the roles of the structural and non-structural proteins of tick-borne flaviviruses.

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    Reviewer #1

    Evidence, reproducibility and clarity

    Summary:

    • In this study, authors investigate the impact of pre-membane (prM) and envelope (E) proteins of tick-borne encephalitis virus (TBEV) on viral distribution and tropism, mostly in the brain.*
    • To do so, authors use high resolution imaging of whole mouse brain after infection by either LGTV, a low pathogenic orthoflavivirus also transmitted by ticks, TBEV, or TBEV/LGTV chimeric virus where prM and E of TBEV are inserted in a LGTV background.*
    • Structural and antigenic characterization of the chimeric virus reveal that it remains a low pathogenic virus exhibiting TBEV structural and antigenic features.*
    • Those viruses are then used to infect wt or mavs -/- mice and viral propagation / tropism is explored, revealing that LGTV and LGTVT:prM predominantly infect cerebral cortex while TBEV infects cerebellum.*
    • Authors work at characterizing their viruses is nicely done and convincing, showing that LGTVT:prM replicated just like LGTV, and exhibited increased viral spread in cellulo.*
    • However LGTVT:prM appear to be less pathogenic in vivo and its brain tropism in mavs -/- mice seems to be similar to wt LGTV virus, stressing the fact that the role of structural proteins prM/E is only modest in TBEV specific tropism to cerebellum.*

    * Major comments:*

    • It is stated in the introduction that prior work on LGTV/TBEV chimera have already been done, and that both LGTV and LGTV/TBEV are neuroinvasive and neurovirulent in animal models. In this study, both LGTV and LGTVT:prM fails to establish infection in wt mouse model. Were previous published data on LGTV and derivatives also only performed in mavs, or ifnar deficient mice? The previous studies referred to in the manuscript (ref 21 and 23) are both using wt mice of younger age, 3.5 and 3 weeks respectively. It is known that age influences immune status, and some of the experiments in these previous studies are performed in even younger animals (3 to 8 days suckling mice) likely for this specific reason. The different mice strains in these studies may also influence their susceptibility to infection.

    • *While LGTV and LGTVT:prME fails to result in symptomatic infection in wt mice in our study, a certain level of localized infection is likely taking place and the outcome will depend on the immune status of the animals (age/immune deficiencies). What we tried to highlight in the manuscript was that the relative pathogenicity (TBEV/LGTV The fact that the whole "tropism" part of the study is performed in mavs -/- mice limits the impact of the study as escape from innate immune response is central in shaping viral tropism. Authors should advertise more this fact (absent from the abstract) and discuss more the links between LGTV / TBEV and innate immune response (escape mechanisms and NS proteins, implication of prM in controlling MDA5, MAVS)

    Thank you for pointing out the lack of clarity. All the tropism studies, figure 4 and 5, were done in adult WT mice infected i.c. to allow the virus to surpass the initial barrier of peripheral immune response and establish infection in the brain. We have now stressed this in the result section and in the relevant figure legends.

    Minor comments:

    • Figures need some re-working:*
    • Figure 1 :
    • 1D : only the difference between TBEV and LGTVT:prME is shown. Plotting the difference LGTV / LGTVT:prM would be a nice upgrade.* Thank you for this suggestion. However, as there is no statistical difference between LGTV and ChLGTV in Fig 1D we have maintained the figure as originally made.

    • Figure 2 : Numbering in the panels is wrong (2j in the text is 2K, 2H is 2I, ...) and should be corrected. Thank you, this has been corrected in the figure.

    • Figure 3 : Route of infection could be added to figure labels for more clarity. Thank you, we have added this to the figure.

    • Figure 4A : Labelling the Mock panel with areas of concern in the brain(Cerebrum, Cerebellum, ...) would help a lot readers not familiar with brain anatomy. We agree that adding these labels improves the clarity and accessibility of the figure and have added this to 4A.

    • Figure 4 E : images are too small to be convincing. What is staining Iba-1 is not mentioned in the figure legend. Thank you, we have added the explanation that microglia were stained by Iba1 and increased the size of the images in Figure 4. Additionally, co-staining of viral antigens and the neuronal marker UCHL-1 has been added as the new Figure 4E and Iba-1 staining moved to 4F.

    Significance

    Prior studies already described the generation and characterization of TBEV/LGTV chimeric viruses.

    • The main addition of this paper to the field is the use of impressive high-resolution imaging of whole mouse brains, to explore viral infection and tropism in the brain.*
    • However, presented data remain mostly descriptive, and experiments are performed in a model that may not be optimal to study tropism. As the ability of the virus to escape type I interferon participates to tropism, the fact that infections are only performed in mavs -/- mice limits the relevance of those findings.*

    We agree that studying tropism in MAVS-/- mice might be misleading and that is why the whole tropism study was performed in adult WT mice, we have clarified in the text that these data are from WT mice. In addition to the significance of this study in highlighting the respective contribution of structural proteins and the immune response in shaping tropism, this study also provides a __well-characterized chimeric virus __with a safety profile comparable to LGTV while retaining key structural and antigenic features of TBEV, model that has already helped advance studies on flavivirus receptor interactions and structural dynamics.

    Reviewer #2

    Evidence, reproducibility and clarity

    In the manuscript entitled "The influence of the pre-membrane and envelope proteins on structure,

    • pathogenicity and tropism of tick-borne encephalitis virus" Ebba Rosendal and colleagues present a wealth of data regarding generation and characterisation of a chimeric LGTV virus with TBEV structural proteins, comparing this virus to both LGTV and TBEV across a number of different basic and advanced readouts. They present interesting data regarding the ability of the LGTV-TBEV chimera to spread cell-cell, and the prolonged survival of immunocompromised mice compared with LGTV, which the authors associate with reduced replication in the periphery. As well as an overall increased ability of TBEV to replicate in vitro, and lead to mortality in WT mice in vivo, TBEV was found to be able to infect the cerebellum, whilst this region was rarely infected by LGTV and the chimera. The authors also demonstrate the cross-reactivity of these three viruses via neutralization using serum of TBEV vaccinated individuals.*

    * General comment:*

    • In general, I am impressed by the amount of work and breadth of techniques included in this manuscript, which I think speaks to the benefit of multidisciplinary collaboration. However, in my opinion, some points are lacking. My primary concerns lie with the in vivo experiments. The comparison of LGTV and the chimera at the same timepoints isn't ideal as the shift in mortality means these animals are at a different stage of disease at different time points. Whilst this is interesting in itself, it leaves questions about viral titres and tropism of i.p. inoculated animals at end points, in addition to the exclusion of serum titre analysis, the strength of discussion regarding peripheral replication and its potential impact on neuroinvasion/virulence is weakened. Further, claims of neuronal infection are made in figure 4 in total absence of a neuron marker. If the authors wish to claim cell-specific tropism, the cell-specific markers must be included. For figures dependent upon fluorescent imaging, further clarification as to what the AU axes indicate would aid in better interpretation of the data, especially regarding comparison of cerebellar layers for TBEV infection (described in more detail in my specific comments). Finally, In general, I think some opportunities are missed to describe the big picture of potential applicability/impact/translatability of the results obtained, especially the conclusions can be expanded to better highlight this.*

    Thank you for these very relevant comments and suggestions. In line with these, we have now added a later timepoint (8 days) for LGTV:prME in IPS1-/- mice to better understand the kinetics of the chimeric virus at later time points (Figure 3). Additionally, we have added a neuronal marker in figure 4. The explanation of quantification of the fluorescence data is described in detail in the material and method, where the concept of this arbitrary unit (AU) used for quantification is described.

    Specific points:

    • • Line 67: "It" is a bit of a shaky antecedent - assumedly the authors are referring to tropism, but would be good to state this, as they could also be referring to the underlying mechanisms of pathology. i.e. Tropism is determined by....*

    We agree here and have specified this accordingly.

    • Line 70 - Low pathogenicity in which species? All? Humans? The sentence refers to mice as there has not been any human clinical case with LGTV. We have added that to the text.

    • Line 79 - Strange wording - "and which viral factors influence tropism" is sufficient Corrected accordingly.

    • Line 82 - What does "low pathogenic" mean in this context? Good survivability? No clinical signs? We have clarified in the text that this is referring to similarity to the pathogenicity of LGTV.

    • Line 95: Good to mention in the text the cell type in which the foci are seen We agree, this information has been added to the main text in addition to the figure legend.

    • Line 133 - What is the rationale for the different TBEV strains used? (Kuutsalo-14 here but 93/783 before) We compare the structure of our chimeric virus with the previously published Kuutsalo-14 strain (ref 25). The use of 93/783 in this study is to ensure the same strain of TBEV is used as was used to generate LGTV:prME and to compare the chimeric virus to infectious clones of the parental viruses rescued and passaged in the same way as the chimeric virus itself to ensure differences observed is indeed due to the genetic factors.

    • Line 175/Figure 3 - Why these time points and not later ones for the LGTV chimera? I understand the early time points for replication in the periphery, but would also be good to see brain titres around day 14 when the survival of the chimera inoculated mice decreases quite rapidly. Further, imaging at timepoints at which mortality is somewhat comparable (meaning that virus is likely in the brain) would enable additional readouts to characterise neurovirulence such as cell death markers etc. and allow for a more solid comparative characterisation. Thank you for bringing this to our attention. The figure 3E is displaying data for MAVS-/- mice infected with 10^5 FFU, where the some animals meet end-point criteria already around day 7-9. To address this comment, we have added an additional timepoint at day 8 (seven animals) to explore the trend in viral loads in the brain. However, we refrain from analyzing later time points as this would require a high number of starting animals to ensure adequate numbers surviving to e.g. the suggested day 14, which is not in line with RRR.

    • Interestingly, there is not significant increase in viral loads of LGTV:prME infected animals between day 6 and 8. In line with this, IF imaging analysis of brains from later end-point animals (day 10-14) has shown limited staining of viral antigen in the brain (data not included in manuscript but could be provided to reviewers if requested). This suggests that inflammation is driving the pathology in these animals rather than uncontrolled viral replication. This has also been noted in the text. The tropism and imaging is done in WT mice infected i.c.. and the time/infectious dose has been adjusted to ensure similar clinical manifestation as presented in supplemental Figure 2A. These mice are then euthanized around day 5-6 and processed for brain imaging, line 189.

    • Line 174-182/Figure 3 - Why were serum titres not included in these experiments? These would help to strengthen your argument. (also nice to look at neutralisation in this context, though maybe not essential thanks to your data in figure 2). Viral serum titers have been analyzed previously in MAVS-/- mice in Kurhade et al 2016, and they are high at day 2 and go down to almost detection limit day 4, meaning earlier drop than in peripheral organ and was not included in these experiments. For neutralization, the included time points for the experiments in Figure 3E-H the time points are too short for robust detection of IgG antibody responses.

    • Line 183 - Good to overtly state that this is via i.c. inoculation and the justification for use of this route, and that the mice are assumedly WT. I understand LGTV struggles to get to the brain in mice, but is this representative of how neurotropism looks in animals inoculated via a more "natural" route for TBEV? We appreciate the comment and we have clarified that WT mice are i.c. inoculated. Since we wanted to compare the three viruses, we needed to use an inoculation route that is working for all three viruses. While the tropism after peripheral infection of TBEV is a very interesting question, it remains outside the scope of this study as this cannot be compared with LGTV in WT mice.

    • Figure 4B - What could account for the large variation seen in the TBEV group? This is a very good question that is difficult to answer. Although these are inbred mice, we have previously seen that there are differences in infection rate between different mice using whole brain imaging (Chotiwan et al 2023).

    • Line 200-201 - This image doesn't answer the question of tropism, but contributes to that of microglial activation. A neuronal marker should be included to surmise the cell type infected, rather than using staining for a viral protein to indicate cell morphology/type. Also, the justification for use of the microglial marker over neuronal is lacking, especially as microglia are not mentioned anywhere in the discussion. Also, see suggestion regarding cell death markers above. Thank you for this suggestion we have added a neuronal marker. We have also clarified in the text that we confirm the infection pattern in rhinal cortex with confocal microscopy. Microglia activation has been added to the discussion.

    • Line 203/Figure 4E - Are these images quantifiable? Are any differences observed between the viruses? Quantification of microglial activation is sensitive to imaging quality and area of imaging and requires large sample sets to ensure validity in the conclusions. Here we do not observe any clear differences nor claim that the microglia activation is different between the different viral strains.

    • Line 210 - Bit strange to mention figure 4D again after figure 4E, and I also couldn't spot reference to figure 4F? Thank you for pointing this out the Figure 4D should be Figure 4E, this has been corrected.

    • Are both figures 5A and 5C required for the message you wish to get across? I would suggest either only use 5C or only include the white matter/grey matter comparison for TBEV, in combination with 5A. Thank you we have now removed the mock, LGTV and LGTVT:prME from fig 5C to more clearly communicate the message of difference in infection between GM and WM for TBEV specifically.

    • Figure 5D: does the method of quantification you use/the conclusions you arrive at account for cell size/number? The Purkinje cell bodies are very large and the virus signal in these cells looks saturated - however within the granular layer the nuclei are much smaller but have what seem like large foci of NS5 positivity. Though the overall signal is likely much lower, how does relative distribution look when you account for cell size/number or a binary positive/negative quantification? Relatedly, does the primary anti-NS5 antibody have the same affinity for both LGTV and TBEV NS5? The quantification of OPT in figure 5C is not at the level of single cell resolution but rather virus signal over mock. We agree the cells in the cerebellum has different sizes but we do not claim that the Purkinje layer is more infected compared to the granular cell layer, only that Purkinje cells are infected which is relevant in human TBE.

    NS5 antibody is raised against a peptide in the TBEV NS5 protein which is highly conserved. The aa identity between TBEV and LGTV is 93%, we have not seen a difference in the staining between the different viruses using this antibody.

    • Line 242: Please clarify what you mean by "higher infection" - higher titres? Higher fluorescent signal? We have added "as measured by stronger fluorescent signal" to better explain what we mean with higher infection.

    • Line 242: Can you really say anything about replication here? Infection, yes, but the AU readout and lack of multiple time points doesn't allow for much of an insight into replication, especially when TBEV was left out of the comparison in figure 3F, though even this did not look at live virus. We have changed the wording to infected cells.

    • Line 269-271: Exactly what I was wondering and maybe worth discussing a bit more - is there appropriate literature that you could cite here? We were unsure about the specific concern raised by the reviewer in this comment and, therefore, have not made any changes. If the reviewer could clarify their request, we would be happy to address it accordingly.

    • Line 274-275: Also mosquito borne viruses. See nice paper related to impact of TBEV vaccination on ADE for mosquito borne flaviviruses. Very interesting and would increase the impact of this point. https://doi.org/10.1038/s41467-024-45806-x Thank you for this suggestion we have added this point into the discussion.

    • Line 290-291: Are clinical signs associated with cerebellar injury common for TBEV patients? i.e. does this have translatability to human disease and diagnosis? We have now added some information about cerebellum symptoms in human TBE infection to the discussion.

    • Line 308 conclusions; Your point about the potential use of the chimera for vaccine research/to understand cross-reactivity is worth reiterating here, and potentially something about "highlighting the role of non-structural proteins on tropism determination" Thank you for these suggestions we have now added these aspects in the conclusions.

    • Methods: whilst I realise the statistics are described in the figure legends, it is usually customary to include a short statistics section in the methods to indicate which program was used and why certain statistical tests were chosen, e.g. in figure 1 you use both parametric and non-parametric testing. Thank you for this suggestion. We have added a section describing the statistics in the methods.

    Significance

    Broad ranging characterisation of a novel chimera which has potential applications for vaccine/cross-reactivity research and highlights a key role of non-structural proteins in the determination of viral fitness and tropism. Some limitations regarding cell-specific tropism and kinetics of neuroinvasion and neurovirulence. Likely of interest for basic researchers from range of disciplines within arbovirology.

    • Expertise: arboviruses, imaging, neurovirulence, animal models*
    • Limited expertise: in-depth structural biology, therefore my comments on figure 2 are limited.*

    __Reviewer #3 (Evidence, reproducibility and clarity (Required)): __

    • SUMMARY: The authors generated an LGTV chimeric virus harboring the prM and ectodomain of E from TBEV. Aim of the study is to understand how the virals structural proteins influence the distribution and tropism of the virus in the brain. They solved the atomic structures of LGTV and the chimeric virus demonstrating that the chimeric virus is structurally and antigenically similar to TBEV. In vivo experiments demonstrate that the chimeric virus is less pathogenic than LGTV. Finally using 3D whole brain OPT imaging techniques the authors demonstrate that the three viruses show a similar viral distribution in cerebral cortex with the rhnial cortex being the primary site of cortical infection for all viruses. In general TBEV exhibit higher infection rates and is more widespread in the brain, particularly in cerebellum, compared to LGTV and the chimeric virus. The authors concluded that the distribution and tropism of LGTV and TBEV are not solely dependent on receptor tropism. *

    * MAJOR COMMENTS: *

    • The conclusions are supported by the data.*

    • However, I think the work can be improved if the authors investigate the differences in the antiviral response induced by the chimeric virus compared to LGTV. The authors speculate that the non-structural proteins may play a role in shaping tropism, likely through their immunomodulating role. These data become especially important if you consider that in the experiments of fig 1 the chimeric virus behave similar to the LGTV wt with even an advantage in cell-to-cell spread but in the in vivo experiments with MAVS-/- mice the chimeric virus behave differently, being less pathogenic than LGTV suggesting that the chimeric virus could not escape the antiviral response even in MAVS-/- condition. We thank the reviewer for this suggestion. In line with this we have now added Ifnb1 and Rsad2 RNA levels in different peripheral organs and we see that early on in infection most mice infected with LGTVT:prME show higher upregulation of these genes. These data have been added as a new panel F and G in figure 3.

    • Moreover, in the discussion, line 270 the authors speculate that the observed attenuation could also be due to sub-optial interactions between TBEV prM and C and transmembrane domain of LGTV E. I think it is important to explain and justify why they decided to do not include C protein of TBEV in the chimeric virus, as well as the transmembrane domain of E. The rational for not using the C protein of TBEV is that we did not want to reduce the RNA to C interaction which, could affect the packaging or encapsidation. In line with this, previous research on chimeric flaviviruses has shown that exchanging the prM-E proteins are usually well tolerated while exchanging the C-protein may lead to attenuation or even failure to rescue the virus.

    • Finally, the authors first used A549 cells for studying the kinetics and viral spread of the chimeric virus in vitro. Than they switch to A549-/- cells for studying structure and antigenicity. The different pathogenicity was assessed in Mavs-/- mice but lastly they used mice WT for the 3D whole brain OPT imaging. I found this discrepancy confusing. The authors should justify, including the explanation in the text, why they switch from WT to A549-/- from experiment to experiment. A549 cells were used in the spread and kinetic study because it is an IFN competent cell type which TBEV and LGTV grows well in. The structural studies were performed in A549 MAVS cells because the lack of MAVS results in higher virus titers. The ability of these cells to produce large amount of virus while grown without serum greatly facilitated the purification protocols for cry-EM and mass spectrometry analysis. This has been highlighted in the text of both the material and method and very briefly in the result.

    The pathogenicity with peripheral infection can only be done with MAVS-/- mice as they are more sensitive to LGTV and it is a lethal model. Adult WT mice are resistant to LGTV infection i.p.. As the immune response is important in shaping the tropism, a direct comparison of the viruses is best analyzed in a WT mouse model.

    MINOR COMMENTS:

      • Line 96 - "recombinant parental LGTV" and "recombinant TBEV", the word recombinant is misused in the sentence.* We have removed recombinant.

    • Line 143-144-145 - I believe the authors are referring to Fig 2I and not 2H as written. Moreover, the authors should clarify if all the experiemtns of fig 2 have been performed in A549-/- cells or only the one of fig 2I All experiments in figure 2 are performed in A549 MAVS-/- as highlighted in the material and methods.

    • Line 158 - to be change "Fig 2I" with "fig 2J" Corrected

    • Line 159 - as above: fig 2J to be change with figure 2k Corrected

    *Significance: *

    • The authors designed a chimeric low pathogenic model virus to study the importance of the structural proteins in determing viral tropism and pathogenicity. The strengths of this work is that they combined the use of the chimeric virus with in vivo experiments and 3D whole brain OPT imaging. Integrating together these tools and assays the authors provided an example of complete investigation method for studying neuroinvasive viruses. *

    • My field of expertise: virus-host interaction, at molecular level.*

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    Referee #3

    Evidence, reproducibility and clarity

    Summary: The authors generated an LGTV chimeric virus harboring the prM and ectodomain of E from TBEV. Aim of the study is to understand how the virals structural proteins influence the distribution and tropism of the virus in the brain. They solved the atomic structures of LGTV and the chimeric virus demonstrating that the chimeric virus is structurally and antigenically similar to TBEV. In vivo experiments demonstrate that the chimeric virus is less pathogenic than LGTV. Finally using 3D whole brain OPT imaging techniques the authors demonstrate that the three viruses show a similar viral distribution in cerebral cortex with the rhnial cortex being the primary site of cortical infection for all viruses. In general TBEV exhibit higher infection rates and is more widespread in the brain, particularly in cerebellum, compared to LGTV and the chimeric virus. The authors concluded that the distribution and tropism of LGTV and TBEV are not solely dependent on receptor tropism.

    Major Comments: The conclusions are supported by the data.

    However, I think the work can be improved if the authors investigate the differences in the antiviral response induced by the chimeric virus compared to LGTV. The authors speculate that the non-structural proteins may play a role in shaping tropism, likely through their immunomodulating role. These data become especially important if you consider that in the experiments of fig 1 the chimeric virus behave similar to the LGTV wt with even an advantage in cell-to-cell spread but in the in vivo experiments with MAVS-/- mice the chimeric virus behave differently, being less pathogenic than LGTV suggesting that the chimeric virus could not escape the antiviral response even in MAVS-/- condition.

    Moreover, in the discussion, line 270 the authors speculate that the observed attenuation could also be due to sub-optial interactions between TBEV prM and C and transmembrane domain of LGTV E. I think it is important to explain and justify why they decided to do not include C protein of TBEV in the chimeric virus, as well as the transmembrane domain of E.

    Finally, the authors first used A549 cells for studying the kinetics and viral spread of the chimeric virus in vitro. Than they switch to A549-/- cells for studying structure and antigenicity. The different pathogenicity was assessed in Mavs-/- mice but lastly they used mice WT for the 3D whole brain OPT imaging. I found this discrepancy confusing. The authors should justify, including the explanation in the text, why they switch from WT to A549-/- from experiment to experiment.

    Minor comments:

    Line 96 - "recombinant parental LGTV" and "recombinant TBEV", the word recombinant is misused in the sentence.

    Line 143-144-145 - I believe the authors are referring to Fig 2I and not 2H as written. Moreover, the authors should clarify if all the experiemtns of fig 2 have been performed in A549-/- cells or only the one of fig 2I

    Line 158 - to be change "Fig 2I" with "fig 2J"

    Line 159 - as above: fig 2J to be change with figure 2k

    Significance

    The authors designed a chimeric low pathogenic model virus to study the importance of the structural proteins in determing viral tropism and pathogenicity. The strengths of this work is that they combined the use of the chimeric virus with in vivo experiments and 3D whole brain OPT imaging. Integrating together these tools and assays the authors provided an example of complete investigation method for studying neuroinvasive viruses.

    My field of expertise: virus-host interaction, at molecular level.

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    Referee #2

    Evidence, reproducibility and clarity

    In the manuscript entitled "The influence of the pre-membrane and envelope proteins on structure, pathogenicity and tropism of tick-borne encephalitis virus" Ebba Rosendal and colleagues present a wealth of data regarding generation and characterisation of a chimeric LGTV virus with TBEV structural proteins, comparing this virus to both LGTV and TBEV across a number of different basic and advanced readouts. They present interesting data regarding the ability of the LGTV-TBEV chimera to spread cell-cell, and the prolonged survival of immunocompromised mice compared with LGTV, which the authors associate with reduced replication in the periphery. As well as an overall increased ability of TBEV to replicate in vitro, and lead to mortality in WT mice in vivo, TBEV was found to be able to infect the cerebellum, whilst this region was rarely infected by LGTV and the chimera. The authors also demonstrate the cross-reactivity of these three viruses via neutralisation using serum of TBEV vaccinated individuals.

    General comment:

    In general, I am impressed by the amount of work and breadth of techniques included in this manuscript, which I think speaks to the benefit of multidisciplinary collaboration. However, in my opinion, some points are lacking. My primary concerns lie with the in vivo experiments. The comparison of LGTV and the chimera at the same timepoints isn't ideal as the shift in mortality means these animals are at a different stage of disease at different time points. Whilst this is interesting in itself, it leaves questions about viral titres and tropism of i.p. inoculated animals at end points, in addition to the exclusion of serum titre analysis, the strength of discussion regarding peripheral replication and its potential impact on neuroinvasion/virulence is weakened. Further, claims of neuronal infection are made in figure 4 in total absence of a neuron marker. If the authors wish to claim cell-specific tropism, the cell-specific markers must be included. For figures dependent upon fluorescent imaging, further clarification as to what the AU axes indicate would aid in better interpretation of the data, especially regarding comparison of cerebellar layers for TBEV infection (described in more detail in my specific comments). Finally, In general, I think some opportunities are missed to describe the big picture of potential applicability/impact/translatability of the results obtained, especially the conclusions can be expanded to better highlight this.

    Specific points:

    • Line 67: "It" is a bit of a shaky antecedent - assumedly the authors are referring to tropism, but would be good to state this, as they could also be referring to the underlying mechanisms of pathology. i.e. Tropism is determined by....
    • Line 70 - Low pathogenicity in which species? All? Humans?
    • Line 79 - Strange wording - "and which viral factors influence tropism" is sufficient
    • Line 82 - What does "low pathogenic" mean in this context? Good survivability? No clinical signs?
    • Line 95: Good to mention in the text the cell type in which the foci are seen
    • Line 133 - What is the rationale for the different TBEV strains used? (Kuutsalo-14 here but 93/783 before)
    • Line 175/Figure 3 - Why these time points and not later ones for the LGTV chimera? I understand the early time points for replication in the periphery, but would also be good to see brain titres around day 14 when the survival of the chimera inoculated mice decreases quite rapidly. Further, imaging at timepoints at which mortality is somewhat comparable (meaning that virus is likely in the brain) would enable additional readouts to characterise neurovirulence such as cell death markers etc. and allow for a more solid comparative characterisation.
    • Line 174-182/Figure 3 - Why were serum titres not included in these experiments? These would help to strengthen your argument. (also nice to look at neutralisation in this context, though maybe not essential thanks to your data in figure 2)
    • Line 183 - Good to overtly state that this is via i.c. inoculation and the justification for use of this route, and that the mice are assumedly WT. I understand LGTV struggles to get to the brain in mice, but is this representative of how neurotropism looks in animals inoculated via a more "natural" route for TBEV?
    • Figure 4B - What could account for the large variation seen in the TBEV group?
    • Line 200-201 - This image doesn't answer the question of tropism, but contributes to that of microglial activation. A neuronal marker should be included to surmise the cell type infected, rather than using staining for a viral protein to indicate cell morphology/type. Also, the justification for use of the microglial marker over neuronal is lacking, especially as microglia are not mentioned anywhere in the discussion. Also, see suggestion regarding cell death markers above.
    • Line 203/Figure 4E - Are these images quantifiable? Are any differences observed between the viruses?
    • Line 210 - Bit strange to mention figure 4D again after figure 4E, and I also couldn't spot reference to figure 4F?
    • Are both figures 5A and 5C required for the message you wish to get across? I would suggest either only use 5C or only include the white matter/grey matter comparison for TBEV, in combination with 5A.
    • Figure 5D: does the method of quantification you use/the conclusions you arrive at account for cell size/number? The Purkinje cell bodies are very large and the virus signal in these cells looks saturated - however within the granular layer the nuclei are much smaller but have what seem like large foci of NS5 positivity. Though the overall signal is likely much lower, how does relative distribution look when you account for cell size/number or a binary positive/negative quantification? Relatedly, does the primary anti-NS5 antibody have the same affinity for both LGTV and TBEV NS5?
    • Line 242: Please clarify what you mean by "higher infection" - higher titres? Higher fluorescent signal?
    • Line 242: Can you really say anything about replication here? Infection, yes, but the AU readout and lack of multiple time points doesn't allow for much of an insight into replication, especially when TBEV was left out of the comparison in figure 3F, though even this did not look at live virus.
    • Line 269-271: Exactly what I was wondering and maybe worth discussing a bit more - is there appropriate literature that you could cite here?
    • Line 274-275: Also mosquito borne viruses. See nice paper related to impact of TBEV vaccination on ADE for mosquito borne flaviviruses. Very interesting and would increase the impact of this point. https://doi.org/10.1038/s41467-024-45806-x
    •   Line 290-291: Are clinical signs associated with cerebellar injury common for TBEV patients? i.e. does this have translatability to human disease and diagnosis?
    • Line 308 conclusions; Your point about the potential use of the chimera for vaccine research/to understand cross-reactivity is worth reiterating here, and potentially something about "highlighting the role of non-structural proteins on tropism determination"
    • Methods: whilst I realise the statistics are described in the figure legends, it is usually customary to include a short statistics section in the methods to indicate which program was used and why certain statistical tests were chosen, e.g. in figure 1 you use both parametric and non-parametric testing.

    Significance

    Broad ranging characterisation of a novel chimera which has potential applications for vaccine/cross-reactivity research and highlights a key role of non-structural proteins in the determination of viral fitness and tropism. Some limitations regarding cell-specific tropism and kinetics of neuroinvasion and neurovirulence. Likely of interest for basic researchers from range of disciplines within arbovirology.

    Expertise: arboviruses, imaging, neurovirulence, animal models

    Limited expertise: in-depth structural biology, therefore my comments on figure 2 are limited.

  4. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    In this study, authors investigate the impact of pre-membane (prM) and envelope (E) proteins of tick-borne encephalitis virus (TBEV) on viral distribution and tropism, mostly in the brain.

    To do so, authors use high resolution imaging of whole mouse brain after infection by either LGTV, a low pathogenic orthoflavivirus also transmitted by ticks, TBEV, or TBEV/LGTV chimeric virus where prM and E of TBEV are inserted in a LGTV background. Structural and antigenic characterization of the chimeric virus reveal that it remains a low pathogenic virus exhibiting TBEV structural and antigenic features. Those viruses are then used to infect wt or mavs -/- mice and viral propagation / tropism is explored, revealing that LGTV and LGTVT:prM predominantly infect cerebral cortex while TBEV infects cerebellum.
    Authors work at characterizing their viruses is nicely done and convincing, showing that LGTVT:prM replicated just like LGTV, and exhibited increased viral spread in cellulo. However LGTVT:prM appear to be less pathogenic in vivo and its brain tropism in mavs -/- mice seems to be similar to wt LGTV virus, stressing the fact that the role of structural proteins prM/E is only modest in TBEV specific tropism to cerebellum.

    Major comments:

    • It is stated in the introduction that prior work on LGTV/TBEV chimera have already been done, and that both LGTV and LGTV/TBEV are neuroinvasive and neurovirulent in animal models. In this study, both LGTV and LGTVT:prM fails to establish infection in wt mouse model. Were previous published data on LGTV and derivatives also only performed in mavs, or ifnar deficient mice?

    The fact that the whole "tropism" part of the study is performed in mavs -/- mice limits the impact of the study as escape from innate immune response is central in shaping viral tropism. Authors should advertise more this fact (absent from the abstract) and discuss more the links between LGTV / TBEV and innate immune response (escape mechanisms and NS proteins, implication of prM in controlling MDA5, MAVS)

    Minor comments:

    Figures need some re-working :

    Figure 1 :

    1D : only the difference between TBEV and LGTVT:prM is shown. Plotting the difference LGTV / LGTVT:prM would be a nice upgrade.

    Figure 2 : Numbering in the panels is wrong (2j in the text is 2K, 2H is 2I, ...) and should be corrected.

    Figure 3 : Route of infection could be added to figure labels for more clarity.

    Figure 4A : Labelling the Mock pannel with areas of concern in the brain(Cerebrum, Cerebellum, ...) would help a lot readers not familiar with brain anatomy.

    Figure 4 E : images are too small to be convincing. What is staining Iba-1 is not mentioned in the figure legend.

    Significance

    Prior studies already described the generation and characterization of TBEV/LGTV chimeric viruses. The main addition of this paper to the field is the use of impressive high-resolution imaging of whole mouse brains, to explore viral infection and tropism in the brain.

    However, presented data remain mostly descriptive, and experiments are performed in a model that may not be optimal to study tropism. As the ability of the virus to escape type I interferon participates to tropism, the fact that infections are only performed in mavs -/- mice limits the relevance of those findings.

  5. Version published to 10.1101/2024.10.16.618642 on bioRxiv