Review Reports - GFP-Tagged Erns in Bungowannah Pestivirus: A Tool for Viral Tracking and Functional Studies

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Viruses (ISSN 1999-4915)

Manuscript ID: viruses-4129687

„GFP-Tagged Erns in Bungowannah Pestivirus: A Tool for Viral Tracking and Functional Studies“ by Sara Ezzat and Matthias Schweizer.

Pestiviruses infect a broad range of species and cause significant economic losses in livestock. The Erns protein is a unique feature of pestiviruses because it serves double duty as glycoprotein complex at the surface of the virion but also as a secreted RNase that functions as an interferon (IFN) antagonist. This multifunctionality makes Erns a particularly complex protein and thus it is important to understand its functions during the pestivirus life cycle. By using the high genetic plasticity of Bungowannah pestivirus (BuPV), the authors use the virus to engineer a novel recombinant tool. They generated a recombinant BuPV expressing green fluorescence protein (GFP) fused to the N-terminus of the Erns protein from BVDV NCP7. The authors were able to detect GFP-Erns fusion by fluorescence microscopy and could demonstrate that the fusion remained stable across five serial passages. Furthermore, the recombinant virus infected all tested mammalian cell lines confirming that the generated virus retained the characteristic broad cell tropism of BuPV. However, compared to the parental BuPV stock it replicated more slowly. Finally, the authors confirmed by applying RNase activity assays that the GFP-Erns fusion protein retained its enzymatic function. Taken together, the novel recombinant BuPV-GFP-Erns virus showed stable expression of GFP-Erns during the course of infection, broad infectivity, and preserved its RNAase activity as GFP-Erns fusion.

The data are clear and convincing and the claims are sufficiently supported by data. This work is technically sound and it provides a useful tool for further investigations on the pestivirus life cycle and its pathogenesis. Overall, I feel this work should be published in Viruses. I have outlined a few points for consideration to improve the manuscript. They are specified below.

Figure 2a. The authors showed stability and replication of recombinant BuPV GFP-BVDV ncp7 Erns across serial passages. However, it would be useful to counterstain for viral NS2-3 to see if all NS2-3 positive are also positive for GFP-Erns.

Fig. 2B and 2C. The authors compared viral replication kinetics of the BuPV GFP-ncp7 Erns (p1–p5) in SK-6 cells to the parental BuPV viral stock. Is parental BuPV viral stock referring to BuPV with BuV-Erns or to BuVp with Erns from BVDV NCP7? In other words, it the GFP fusion reducing the replication kinetics or is it the Erns from BVDV NCP7?

Fig. 3A: Is there virus spread in Vero_E6? It doesn’t look like it is. Please comment.

Fig. 3B: I’m a little bit puzzled by the huge difference in TCID₅₀ values in SK6 cells from BuVP stock versus BuVP GFP-ncp7 Erns (1.63E+13 versus 9.24E+06). This is not was we are seeing in Fig. 2B. Please explain.

Author Response

We thank all three reviewers for taking their time to evaluate our manuscript and to provide constructive comments, which helped us to further improve the manuscript. We replied to each comment point-by-point and modified the manuscript accordingly.

Reviewer 1

We thank this reviewer for the concise summary of our report and the very positive evaluation. In the following, we reply to the valuable considerations to improve the article.

Comment 1.1: Figure 2a. The authors showed stability and replication of recombinant BuPV GFP-BVDV ncp7 E^rns across serial passages. However, it would be useful to counterstain for viral NS2-3 to see if all NS2-3 positive are also positive for GFP-E^rns.

Response 1.1: We agree with the reviewer that counterstaining for viral NS2-3 would be nice to have but, in our opinion, would not substantially add new information for our conclusions. In addition, as molecular tools for the ”atypical pestivirus” Bungowannah virus (BuPV) are scarce, we only have a pool of four monoclonal antibodies at our disposal that is able to detect wt BuPV (RAE2020, Apha Scientific). This pool of antibodies stains simultaneously for NS2-3 and E2, both proteins present in large quantities inside infected cells. This would make it impossible to observe a decent double staining with GFP. Finally, the only possibility for the presence of E^rns-GFP in the absence of NS2-3 would be if the soluble, secreted protein would re-enter uninfected cells by endocytosis (Zürcher et al. (2014) J. Virol. 88:13, 7235-7243. doi:10.1128/jvi.00672-14). However, this might not occur to a sufficient extent to observe such strong GFP-signals, as we were never able to stain for E^rns by immunofluorescence microscopy using the supernatant of infected cells but only with affinity-purified protein (Zürcher et al. (2014); Lussi et al. (2021) Viruses-Basel 13:8, 1581. doi:10.3390/v13081581).

Comment 1.2: Fig. 2B and 2C. The authors compared viral replication kinetics of the BuPV GFP-ncp7 E^rns (p1–p5) in SK-6 cells to the parental BuPV viral stock. Is parental BuPV viral stock referring to BuPV with BuPV-E^rns or to BuPV with E^rns from BVDV NCP7? In other words, is the GFP fusion reducing the replication kinetics or is it the E^rns from BVDV NCP7?

Response 1.2: With the term “parental”, we referred to the original virus strain from which we exchanged the E^rns and added the GFP, i.e., the wild-type BuPV. But we fully agree that this might not be obvious and might be confused with a virus that contains bovine E^rns but no GFP. Thus, we specified the term “parental” in chapter 2.1, and we complemented the discussion (end of 3^rd paragraph) to indicate that the bovine E^rns and/or the GFP might be the cause of reduced fitness.

Comment 1.3: Fig. 3A: Is there virus spread in Vero_E6? It doesn’t look like it is. Please comment.

Response 1.3: We agree with the reviewer that without passaging the cells, it is not clear whether the virus spread in Vero-E6 cells. As this was not the purpose of the experiment, we did not passage the various cell types. As the viruses did infect the cells less efficiently, not all cells were infected after48 hours as shown in Fig 3A. But at that time, the cells were already rather confluent, which is known to be unfavorable for viral replication of pestiviruses. However, the fact that we were able to titrate viruses from the supernatant clearly indicates that it was not an abortive infection. We added the latter information in the 2^nd paragraph in chapter 3.3.

Comment 1.4: Fig. 3B: I’m a little bit puzzled by the huge difference in TCID values in SK6 cells from BuPV stock versus BuPV GFP-ncp7 E^rns (1.63E+13 versus 9.24E+06). This is not what we are seeing in Fig. 2B. Please explain.

Response 1.4: Indeed, the differences are rather large, but that’s what we consistently observed. However, in Figure 3B, a 30-times higher MOI with fewer washing steps was applied than in Figure 2B, and for the BuPV GFP-ncp7 E^rns, virus of passage 1 was used, representing a virus that is not adapted to SK-6 cells, in contrast to the parental virus stock. We added this information at the end of chapter 3.3. to explain this astonishing difference.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors!

Your reported the generation of a recombinant Bungowannah pestivirus (BuPV) expressing a GFP-tagged Erns protein derived from BVDV ncp7. You demonstrated stable GFP signal across five passages, broad infectivity in multiple mammalian cell lines, and retained RNase activity of secreted GFP-Erns. The work aims to provide a tractable tool to study Erns trafficking and function. The study addresses a relevant gap and provides a potentially useful tool. The study demonstrates a stable BuPV recombinant expressing GFP-tagged BVDV Erns, useful for tracking Erns localization and assessing function. This is valuable for pestivirus biology and tool development.

I miss any information on Pestivirus L, which should be a closer relative compared to BVDV or CSFV. Maybe this could be included in the discussion.

Well done and written. I did not find any points to be changed in the manuscript!

Author Response

Reviewer 2

We also thank this reviewer for the very positive report and for the compliment “well done and written“.

Comment 2.1: I miss any information on Pestivirus L, which should be a closer relative compared to BVDV or CSFV. Maybe this could be included in the discussion.

Response 2.1: Indeed, Linda pestivirus (LindaV) is the closest relative of BuPV, and both share the peculiarity that till today, they were only isolated in a single country each, i.e., BuPV in Australia and LindaV in Austria. However, as LindaV was first detected in 2015 (reported in 2017), not much research was performed on this virus since then, and the plasticity of the genome compared to BuPV is not known. We added this information in the second to last paragraph in the discussion.

Reviewer 3 Report

Comments and Suggestions for Authors

In general, the work is well structured and presents in-depth research, but there are some aspects that could be improved or expanded to make it even more comprehensive and robust.

The title is clear and describes the main topic of the article: the use of a GFP-labeled Erns protein for functional and tracking studies of the Bungowannah pestivirus.

The abstract provides a concise summary of the study, including the context, objective, main methods, findings and conclusions. It is well organized and informative.

The introduction introduces the scientific context, describing pestiviruses and the role of the Erns protein. The importance of the study and why the pestivirus Bungowannah was chosen as a model are explained. The transition to the study goal is smooth.

Materials and Methods are detailed and clearly describe the steps followed to generate and characterize the recombinant virus. The information is presented in chronological and logical order, facilitating understanding of the experimental process.

The authors must describe all the cell lines used in the study and report their origin.

The authors could insert the genome positions of primes in Table 2.

The results are present systematically, with subsections dealing with specific aspects of the study. The discussion analyses the results obtained, comparisons them with previous studies and proposals implications and future developments. The section is well argued.

The conclusion summarizes the main findings and underlines the importance of the work conducted, highlighting the potential of the recombinant virus as a research tool.

In the "Supplementary Materials" section the link provided to access the supplementary materials is incomplete ("https://www.mdpi.com/article/......"). You must enter the full link.

The References are adequate.

The work focuses exclusively on in vitro experiments. It may be useful in the future to develop in vivo studies to evaluate the behavior of the recombinant virus in a host organ

The manuscript could be accepted for publication after minor revision.

Author Response

Reviewer 3

We also thank the third reviewer for the nice summary and the very positive evaluation. We highly appreciate.

Comment 3.1: The authors must describe all the cell lines used in the study and report their origin.

Response 3.1: We apologize for our omission, but as these were all standard cell lines regularly used at our institute, this information was inadvertently omitted just by negligence. We added the corresponding information with the appropriate references in chapter 2.1.

Comment 3.2: The authors could insert the genome positions of primes in Table 2.

Response 3.2: Thanks for this suggestion. We now added the genome position in Table 1 to indicate the position of the various fragments within the viral genome. This is indeed more informative, and if required, the exact nucleotide position can easily by retrieved by genome alignments. The corresponding GenBank number of the reference strain is now also given in legend to table 1.

Comment 3.3: In the "Supplementary Materials" section the link provided to access the supplementary materials is incomplete ("https://www.mdpi.com/ article/......"). You must enter the full link.

Response 3.3: Upon acceptance (hopefully) of the manuscript for publication, this link will be provided by the journal.

Comment 3.4: The work focuses exclusively on in vitro experiments. It may be useful in the future to develop in vivo studies to evaluate the behavior of the recombinant virus in a host organ.

Response 3.4: We fully agree with the reviewer that this fluorescently labeled virus might be well suited for a number of experimental systems, including vivo studies. However, such in vivo studies were well beyond the scope of the study. Nevertheless, we added the notion that it will hopefully be of use for in vitro as well as in vivo studies at the end of the discussion.