Avian Flavivirus Enters BHK-21 Cells by a Low pH-Dependent Endosomal Pathway
Round 1
Reviewer 1 Report
The authors provide evidence about the entry mechanisms for a new flavivirus, Duck Tembusu virus. This virus is potentially significant due to its spread in birds in China and potential for infection of humans. The experiments focus on showing that entry is via low pH endosomal pathway, using BHK cells. Extensive editing for grammar/clarity is needed for the manuscript to be fully understandable; in some cases it is clear what is intended and there’s just a missing word or unneeded plural word, but in other instances, the exact meaning of a statement is not easily discernable.
broad comments:
-more detail on the virus (e.g., move statement about discovery in China from discussion into introduction, how does it spread?, more details about significance to humans/zoonotic potential). Essentially, give the reader a broader understanding of why knowledge about this virus is important.
-background or more citation on relevance of BHK cells in this context, to justify use of these cells and only these cells. Would results be expected to be any different in another cell type, or in vivo? Especially given reference to JEV, and work showing JEV enters different cells through different mechanisms-so this topic should at least be mentioned.
specific comments:
-what is the siCtrl?
-line96: is it accurate to say that infectious particles were measured with RT-qPCR?
-while the accession number is provided, a sentence or two on the relevance of the virus strain used would be helpful for readers outside the field (e.g. origin species, is it virulent, how many times cell culture passaged
Author Response
Dear Editor,
Thank you for your efforts on our manuscript. We have revised the manuscript according to the reviewer’s comments and answered each question. Based on the opinions of three reviewers, we updated Fig.1G and H, 2, 3A-C and 6C. For details, please see our positive responses below. Secondly, we have updated linguistics issues from MDPI English editing experts to critically review the manuscript and revise the grammar and language. The red highlights in the latest manuscript and make-up version show those revised details. If you have any question, please feel free to contact me.
Yours sincerely,
Bin Zhou, DVM, PhD, Professor
viruses-626136
Comments from the editors and reviewers:
The authors provide evidence about the entry mechanisms for a new flavivirus, Duck Tembusu virus. This virus is potentially significant due to its spread in birds in China and potential for infection of humans. The experiments focus on showing that entry is via low pH endosomal pathway, using BHK cells. Extensive editing for grammar/clarity is needed for the manuscript to be fully understandable; in some cases it is clear what is intended and there’s just a missing word or unneeded plural word, but in other instances, the exact meaning of a statement is not easily discernable.
broad comments:
-more detail on the virus (e.g., move statement about discovery in China from discussion into introduction, how does it spread?, more details about significance to humans/zoonotic potential). Essentially, give the reader a broader understanding of why knowledge about this virus is important.
Response: Thank you for the suggestion. We supplemented some details introducting the important role in zoonosis. For details, please see “Discussion section” in the latest manuscript.
-background or more citation on relevance of BHK cells in this context, to justify use of these cells and only these cells. Would results be expected to be any different in another cell type, or in vivo? Especially given reference to JEV, and work showing JEV enters different cells through different mechanisms-so this topic should at least be mentioned.
Response: Thank you for the suggestion. Even though Flaviviruses utilize several endocytic pathways to enter host cells, including macropinocytosis/phagocytosis, clathrin-mediated endocytosis, caveola/cholesterol-dependent uptake, and clathrin- and caveola-independent endocytosis, clathrin-mediated endocytosis is believed to be the major route of flavivirus cell entry. Therefore, Duck Tembusu virus can use the different pathways to enter the host cells. However, our present work found that Duck Tembusu virus enters BHK-21 cells through a clathrin-dependent pathway. In the future, we will work whether Duck Tembusu virus has caveola-dependent endocytosis. The information above was supplemented in “Discussion section” of the latest manuscript.
specific comments:
-what is the siCtrl?
Response: Thank you for the suggestion. It is a small interference RNA control previously described in our article published in journal of virology.
Zhang, Y.-N.; Liu, Y.-Y.; Xiao, F.-C.; Liu, C.-C.; Liang, X.-D.; Chen, J.; Zhou, J.; Baloch, A. S.; Kan, L.; Zhou, B.; Qiu, H.-J., Rab5, Rab7, and Rab11 Are Required for Caveola-Dependent Endocytosis of Classical Swine Fever Virus in Porcine Alveolar Macrophages. Journal of Virology 2018, 92, (15), e00797-18.
-line96: is it accurate to say that infectious particles were measured with RT-qPCR?
Response: Thank you for the suggestion. We have changed “the infectious particles” into “the viral load” in the latest manuscript.
-while the accession number is provided, a sentence or two on the relevance of the virus strain used would be helpful for readers outside the field (e.g. origin species, is it virulent, how many times cell culture passaged.
Response: Thank you for the suggestion. DTMUV XZ-2012 is a virulent strain and in vivo studies were reported previously (Sun X, Liu E, Iqbal A, Wang T, Wang X, Haseeb A, Ahmed N, Yang P, Chen Q.The dynamic distribution of duck Tembusu virus in the spleen of infected shelducks. BMC Vet Res. 2019,15(1):112. ). Also, Genbank accession number of this strain is KM188953.
Reviewer 2 Report
Baloch et al investigated the viral entry process of DTMUV mainly focusing on the pH-dependent activation of E enabling the fusion between viral and endosomal membrane.
Major points:
Due to poor English it was not possible to review this manuscript adequately. There are many grammar errors and some paragraphs are written in an incomprehensible way. The entire text has to be reviewed in order to correct any linguistic deficiencies and grammatical inconsistencies. Some examples are given below:
- line 129-130 “Our findings depicted that DTMUV entry into cells were inhibited by chloroquine, NH4Cl, and Baf A1 but unable to prevent cell binding with DTMUV”.
- line 188-189 “Infectivity of the virions inactivated, those enters through a low-pH pathway when pre-treated with acid.”
- 259-260 “DTMUV is RN virus with single strand and +ve sense, produces pathogenicity similar to that of other flaviviruses”
The entire material and methods section does not provide sufficient information to reproduce the experimental setup. Further, many sentences are hard to understand due to linguistic issues.
Examples:
- line 71: The concentrations used for each inhibitor should be mentioned in the corresponding paragraph.
- line 83: “For virus titration, subconfluenz cell monolayers grown in 6 well plates were infected with the serial diluted virus for one hour internalization at 37°C in CO2.” The incubation in CO2 is not clear for me.
- line 95: “…and then added…” It is unclear what has been added
- line 116: Please indicated all antibodies used in the corresponding paragraph
The figure legends are not written in a proper way and lead to confusion.
- Fig. 1: The figure legend described the panels A, C and E as entry and binding and B, D and F as infection. This is confusing as I expected A-C to show the effect on binding and entry and D-F to show the effect on replication/infectivity.
- Fig. 1G: “Confocal miscroscopy showed the inhibition of the drugs.” The drugs/ inhibitors are not inhibited.
- Fig. 3: Fig legend for D) is missing
Minor points:
- Fig. 1B): The y-axis shows relative JEV units instead of DTMUV
- Fig. 2: The decreased expression of DTMUV E shown in the Western Blot is not clear. The protein band intensity seems to be similar for siV-ATPase and siCtrl.
- Fig. 6 C): “A significant decrease of DTMUV E protein was noticed in the siCHC-transfected cells…” As already mentioned for Fig. 2, this effect is not detectable in the presented Western Blot.
Author Response
Dear Editor,
Thank you for your efforts on our manuscript. We have revised the manuscript according to the reviewer’s comments and answered each question. Based on the opinions of three reviewers, we updated Fig.1G and H, 2, 3A-C and 6C. For details, please see our positive responses below. Secondly, we have updated linguistics issues through MDPI English editing experts to critically review the manuscript and revise the grammar and language. The red highlights in the latest manuscript and make-up version show those revised details. If you have any question, please feel free to contact me.
Yours sincerely,
Bin Zhou, DVM, PhD, Professor
viruses-626136
Comments from the editors and reviewers:
Baloch et al investigated the viral entry process of DTMUV mainly focusing on the pH-dependent activation of E enabling the fusion between viral and endosomal membrane.
Major points:
Due to poor English it was not possible to review this manuscript adequately. There are many grammar errors and some paragraphs are written in an incomprehensible way. The entire text has to be reviewed in order to correct any linguistic deficiencies and grammatical inconsistencies. Some examples are given below:
- line 129-130 “Our findings depicted that DTMUV entry into cells were inhibited by chloroquine, NH4Cl, and Baf A1 but unable to prevent cell binding with DTMUV”.
Response: Thank you for the suggestion. We have rewrite the sentences as follow: We found that chloroquine, NH4Cl, and Baf A1 inhibited DTMUV entry into cells but did not inhibit DTMUV binding to cells.
- line 188-189 “Infectivity of the virions inactivated, those enters through a low-pH pathway when pre-treated with acid.”
Response: Thank you for the suggestion. We have changed the sentences as follow: Acidic pretreatment of virions that enter cells via a low-pH pathway inactivates their infectivity.
- 259-260 “DTMUV is RN virus with single strand and +ve sense, produces pathogenicity similar to that of other flaviviruses”
Response: Thank you for the suggestion. We have changed the sentences as follow: DTMUV is an RNA virus with a single strand and positive-sense, which produces pathogenicity similar to that of other Flaviviruses
The entire material and methods section does not provide sufficient information to reproduce the experimental setup. Further, many sentences are hard to understand due to linguistic issues.
Response: Thank you for the suggestion. We have modified “Material and Method section. Linguistic issues in the manuscript have resolved through MDPI English editing services.
Examples:
- line 71: The concentrations used for each inhibitor should be mentioned in the corresponding paragraph.
Response: Thank you for the suggestion. We showed the concentrations of drugs in details in line 135-140 of “3.1. DTMUV Entry is pH Dependent”.
- line 83: “For virus titration, subconfluenz cell monolayers grown in 6 well plates were infected with the serial diluted virus for one hour internalization at 37°C in CO2.” The incubation in CO2 is not clear for me.
Response: Thank you for the suggestion. We supplemented this information as follow: After serial dilutions cells were infected and kept for one hour at 37 oC with 5% carbon dioxide (CO2) incubator.
- line 95: “…and then added…” It is unclear what has been added
Response: Thank you for the suggestion. We have changed the sentences as follow: Briefly, a time of addition assay was also undertaken. Cells were infected with an MOI of 0.5 for 1 h, and lysosomotropic agents were added at 0, 1, 3 and 6 h. After 9 hpi RT-qPCR and Confocal microscopy were performed.
- line 116: Please indicated all antibodies used in the corresponding paragraph
Response: Thank you for the suggestion. In line 116 we have added antibodies information as follows. Rabbit anti-clathrin (P1663) CST and mouse anti-V-ATPase B 1/2 antibody (sc-271832) Santa Cruz Biotechnology Inc. Rabbit anti-clathrin (P1663) CST and mouse anti-V-ATPase B 1/2 antibody (sc-271832) were obtained from Santa Cruz Biotechnology Inc. DTMUV E monoclonal antibody was a kind gift from Prof. Renyong Jia (Sichuan Agricultural University).
The figure legends are not written in a proper way and lead to confusion.
Response: Thank you for the suggestion. We have updated all the figure legends. For details, please see the corresponding section.
- Fig. 1: The figure legend described the panels A, C and E as entry and binding and B, D and F as infection. This is confusing as I expected A-C to show the effect on binding and entry and D-F to show the effect on replication/infectivity.
Response: Thank you for the suggestion. We have modified figure 1 legends A, B and C binding and entry, D, E and F as an infection.
- Fig. 1G: “Confocal miscroscopy showed the inhibition of the drugs.” The drugs/ inhibitors are not inhibited.
Response: Thank you for the suggestion. We have changed the sentences as follow: confocal microscopy and western blotting also showed that the DTMUV E proteins were decreased in a dose-dependent manner as compared with control (Fig. 1G, and H).
- Fig. 3: Fig legend for D) is missing
Response: Thank you for the suggestion. We supplemented the legend of Fig.3D. For details, please see the corresponding section.
Minor points:
- Fig. 1B): The y-axis shows relative JEV units instead of DTMUV
Response: Thank you for the suggestion. We have changed the y-axis of Fig. 1B.
- Fig. 2: The decreased expression of DTMUV E shown in the Western Blot is not clear. The protein band intensity seems to be similar for siV-ATPase and siCtrl.
Response: Thank you for the suggestion. As shown in the updated Fig. 2: The decreased expression of DTMUV E has been updated, and the decreased expression results better than before. For details, please see the corresponding section.
- Fig. 6 C): “A significant decrease of DTMUV E protein was noticed in the siCHC-transfected cells…” As already mentioned for Fig. 2, this effect is not detectable in the presented Western Blot.
Response: Thank you for the suggestion. As shown in the updated Fig. 6 C, the results were updated and the level of protein expression of siCHC and control now are very clear.
Reviewer 3 Report
Summary
The authors of the paper focus their study on the Duck Tembusu virus (DTMUV) and the way to enter and trigger its own replication in mammal cell. DTMUV belongs to the Flaviviridae family, it was recently discovered in China and has a high negative impact in avian production in case of outbreak. But the biology of this virus still be relatively unknown. Author’s model is Baby Hamster Kidney cells line (BHK-21). For the authors, it’s interesting to focus research on this kind of cell line as in vitro model because BHK-21 is frequently used to produce veterinary vaccine against DTMUV. Authors are interesting in the endosomal pathway as a route of entry of the virus and the importance of the pH in the triggering DTMUV replication.
Overall impression/ Broad comments
The research treated in this article takes place in a new field of investigation. At this time; few teams are working on different DTMUV. It’s very interesting that the authors focus their research on this virus because its biology remains globally blurred. The entry step is particularly crucial for Flavivirus to trigger replication and spreading in the host, so it’s important to know well this critical step of the replication cycle of the DTMUV. On this point, this study is setting on a rising field of research on a very recent Flavivirus impacting human and animal health in Asia.
Despite previous strength points, the manuscript presents many weaknesses particularly the quality of presentation and technical applications.
About quality of presentation, the manuscript presents some editing mistakes as lack of space between two words, repeated words (line 64), missing of italic font for genus and species, missing of abbreviations, presentation of provider inhomogeneous, units inhomogeneous (for instance: hour and h), mismatch between figure legend, text and figure. Also, I found unappropriated reference in the text.
The introduction part is very light in information. No reference about the description of flavivirus structure. Authors used a general reference (ref n°2) to describe DTMUV. Then they used specific DTMUV reference to describe general behavior of Flavivirus (ref 10 for instance) or described the entry of Flavivirus with DTMUV reference article and wrote the sentence after that the mechanism of DTMUV is unknown yet.
A very big mistake for scientists who work on arbovirus was written line 48 and 49, where authors classified Chickungunya virus as a Flavivirus.
In material and methods part, some technics are unwell described to allow reproduction of experiments.
Particularly the technic of RT-qPCR used by the authors. About this technic, only 2 sentences to describe it. Authors referred to 2 references (14 and 15) but these ones didn’t describe the RT-qPCR (no primer sequences and PCR conditions in these references), these are general review articles. No list of primers used was provided by authors for the qPCR even in supplementary data. My main concern is the techniques used by the authors, they explained using the 2-DDCt to calculate the amount of viral RNA copies, but the 2-DDCt technic allows to calculate a fold difference or induction/decrease of expression, not an absolute viral RNA copy number. For the later, known RNA copy number reference needs to be used to be able to express the RNA copy number. So, I think the authors do a mistake in each figure legends writing “…to check the viral RNA copy numbers…” for me, they check the increasing or decreasing of the relative quantity of the virus, not the absolute quantity; They must change all figure legends and well explain how they did the qPCR and calculated the fold change of virus.
Plaque assay technic isn’t enough detailed particularly the staining step with crystal violet. But the major problem is no results and figures in this study described titration of the virus by Plaque assay as a result. Authors used in every steps RT-qPCR to follow up the replication of the virus. If plaque assay is used only to have the titer of the virus to calculate the MOI it’s not necessary to write this paragraph in the manuscript.
The part “Time of addition assay” could be added to the “cell infection and drug treatments”.
“siRNA transfection” part. Authors used siRNA technic to knockdown expression of CHC but they missed to explain what is the siRNA control in the material and methods part.
“Western Blotting” part. No information on the antibodies used for detection. Please, add a description and provider references.
In the results part, the authors have the tendency to conclude prior to describe the results in each part.
In each figure legend, they wrote “* ,P<0.05 and **, P<0.01” but in the figure only ** appeared apart for the figure 4. In figure 5, it’s lacked statistical analysis explanation in the text. In Fig 3, no statistic stars on the top of the bars in the graphic but authors wrote “* ,P<0.05 and **, P<0.01” in the figure legend.
In figure legend 1, authors mismatched E and B in the description of results.
In the part 3.4, the MOI used for the experiment doesn’t fit with the figure legend. Pleased, be clear with the MOI used in this study. Same part, authors present results as Fig 5 A and 5B, but in the main text there are not reference to A or B.
In part 3.5, authors referred to previous study using JEV in PK-15 and C6/36 (ref 24, 25) but the references were about Herpes virus in Hela and CHO cells. Then, authors used siRNA technic to knockdown expression of CHC but they missed to explain what is the siRNA control in the material and methods part.
Finally, in the discussion the authors do not develop enough the importance of their results in comparison with the literature, the overall impression of this part is like a summary of the results. Authors have to take step back and to put their results in perspective with the literature of the field and develop better the perspective of research and further development on DTMUV. For instance, they used BHK-21, but what about entry step of this virus using Human cells, avian cells or insect cells?
Authors used BHK21 both to produce virus and to do experiments. May I have some feedback of authors about the possible impact on the results (adaptation of the virus to the cells line for instance)?
Authors used RT-qPCR and indirect detection (E protein) to follow up the replication of the virus. I suggested to add experiments with plaque assay titration of the virus to confirm an effective replication of the virus with production of replicative virus particles.
In paragraph line 264 to 276, authors referred to their own previous work on JEV (line 268) but miss to write the reference, that not sound well for a scientific article. The statements line 275 and 276 is not well write.
In the paragraph line 277 to 284, authors referred to several viruses but they used only one reference on JEV.
In my point of view, the discussion needs to be improved to better highlight the results and to show well the further perspective of research on DTMUV.
Despite the interest of the topic and further innovation/prospective usage of this work, the number of mistakes is harmful for the message of the paper.
My overall impression in this manuscript is it was not well reviewed and edited prior submission.
Major issues and specific comments
Introduction,
Line 34: ref 2 is not appropriated
Line 42: the sentence “Moreover…”is not clear enough
Line 48: ref 10 is not appropriated
Line 49: CHIKV is not a Flavivirus !!! or virus classification changed…
Line 49: ref 11 12 are not appropriated. Ref 11 is about DTMUV not about DENV, WNV or JEV
Line 53: ref 13 is not appropriated. The sentence is about Flavivirus but this ref is about DTMUV. Please, used a general ref about receptor mediated entry of Flavivirus or change the sentence.
Materials and Methods,
Line 65: edit well 37°C as -80°C Line 68
Line 63: for provider use homogenous presentation: (provider, city, country) for instance. But same presentation in all the manuscript)
Line 64: authors only wrote (GIBCO). Referred to the previous comments and so on for others providers in the text.
Line 72: add the abbreviation used later in the text for Bafilomycin here (Baf A1).
Line 75: edit hour. Edit 37°C as -80°C line 68
Line 77: ref 14 and 15 are clearly not appropriated
Line 79: “MOI 0.05” different than in figure legend
Results
In figure 1.B: JEV is written in the ordinate axis legend!! Authors worked in DTMUV!
Line 147: mistake in the text “… (A, C, and E)…” be replaced with (A, B and C)
Line 151: mistake in the text “… (B, D, and F)…” be replaced with (D, E and F)
Line 152: mistake in the MOI value
Line 156: remove “*, P<0.05;”, not in the graph
Line 168: remove “*, P<0.05;”, not in the graph. Please, check it in every figure legend.
Figure 3: add statistical analysis
Line 186: checked statistical analysis.
Line 200: remove “RNA copy number” or be clearer with the RT-qPCR technic employed in this study. Same comment for every figure legend using RT-qPCR.
Line 212: MOI mismatched with the figure legend 5A. Checked the description of results A and B.
Line 215: add A and B fig the figure 5 to better understand results.
Line 230: ref 24 and 25 are clearly not appropriated to JEV. Remove or change
Discussion
Line 259: corrected “… +ve” in a good English
Line 269: add reference to this statement
Line 280: add reference to this statement not only one on JEV
Line 285: add reference to this statement
Minor issues
From ethical general guideline for publication: Authors should state when and where they obtained the cells and virus, giving the date and the name of the researcher, cell line repository, or commercial source (company) who provided the cells or virus, as appropriate.
Author Response
Dear Editor,
Thank you for your efforts on our manuscript. We have revised the manuscript according to the reviewer’s comments and answered each question. Based on the opinions of three reviewers, we updated Fig.1G and H, 2, 3A-C and 6C. For details, please see our positive responses below. Secondly, we have updated linguistics issues through MDPI English editing experts to critically review the manuscript and revise the grammar and language. The red highlights in the latest manuscript and make-up version show those revised details. If you have any question, please feel free to contact me.
Yours sincerely,
Bin Zhou, DVM, PhD, Professor
Viruses-626136
Comments from the editors and reviewers:
Summary
The authors of the paper focus their study on the Duck Tembusu virus (DTMUV) and the way to enter and trigger its own replication in mammal cell. DTMUV belongs to the Flaviviridae family, it was recently discovered in China and has a high negative impact in avian production in case of outbreak. But the biology of this virus still be relatively unknown. Author’s model is Baby Hamster Kidney cells line (BHK-21). For the authors, it’s interesting to focus research on this kind of cell line as in vitro model because BHK-21 is frequently used to produce veterinary vaccine against DTMUV. Authors are interesting in the endosomal pathway as a route of entry of the virus and the importance of the pH in the triggering DTMUV replication.
Overall impression/ Broad comments
The research treated in this article takes place in a new field of investigation. At this time; few teams are working on different DTMUV. It’s very interesting that the authors focus their research on this virus because its biology remains globally blurred. The entry step is particularly crucial for Flavivirus to trigger replication and spreading in the host, so it’s important to know well this critical step of the replication cycle of the DTMUV. On this point, this study is setting on a rising field of research on a very recent Flavivirus impacting human and animal health in Asia.
Despite previous strength points, the manuscript presents many weaknesses particularly the quality of presentation and technical applications.
Response: Thank you for the suggestion. We have modified all the required weaknesses and quality presentation in our manuscript. We have updated linguistics issues through MDPI English editing experts to critically review the manuscript and revise the grammar and language.
About quality of presentation, the manuscript presents some editing mistakes as lack of space between two words, repeated words (line 64), missing of italic font for genus and species, missing of abbreviations, presentation of provider inhomogeneous, units inhomogeneous (for instance: hour and h), mismatch between figure legend, text and figure. Also, I found unappropriated reference in the text.
Response: Thank you for the suggestion. We have changed line 64 wording space and repeated words. All units are homogeneous as per suggestion hour to h. Figure legends and all cited references were checked one by one in updated manuscript. For details, please see the corresponding section.
The introduction part is very light in information. No reference about the description of flavivirus structure. Authors used a general reference (ref n°2) to describe DTMUV. Then they used specific DTMUV reference to describe general behavior of Flavivirus (ref 10 for instance) or described the entry of Flavivirus with DTMUV reference article and wrote the sentence after that the mechanism of DTMUV is unknown yet.
Response: Thank you for the suggestion. We have added reference 2 as flavivirus structure description and reference 10 according to the statement as follows.
Mukhopadhyay, S.; Kuhn, R. J.; Rossmann, M. G., A structural perspective of the flavivirus life cycle. Nature Reviews Microbiology 2005, 3, (1), 13.
9.Wang, J.; Lei, C.-Q.; Ji, Y.; Zhou, H.; Ren, Y.; Peng, Q.; Zeng, Y.; Jia, Y.; Ge, J.; Zhong, B., Duck Tembusu virus nonstructural protein 1 antagonizes IFN-β signaling pathways by targeting VISA. The Journal of Immunology 2016, 197, (12), 4704-4713.
10.Jiang, T.; Liu, J.; Deng, Y.-Q.; Su, J.-L.; Xu, L.-J.; Liu, Z.-H.; Li, X.-F.; Yu, X.-D.; Zhu, S.-Y.; Gao, G. F., Development of RT-LAMP and real-time RT-PCR assays for the rapid detection of the new duck Tembusu-like BYD virus. Archives of virology 2012, 157, (12), 2273-2280.
A very big mistake for scientists who work on arbovirus was written line 48 and 49, where authors classified Chickungunya virus as a Flavivirus.
Response: Thank you for the suggestion. We have deleted some information of chickungunya (line 49).
In material and methods part, some technics are unwell described to allow reproduction of experiments.
Particularly the technic of RT-qPCR used by the authors. About this technic, only 2 sentences to describe it. Authors referred to 2 references (14 and 15) but these ones didn’t describe the RT-qPCR (no primer sequences and PCR conditions in these references), these are general review articles. No list of primers used was provided by authors for the qPCR even in supplementary data. My main concern is the techniques used by the authors, they explained using the 2-DDCt to calculate the amount of viral RNA copies, but the 2-DDCt technic allows to calculate a fold difference or induction/decrease of expression, not an absolute viral RNA copy number. For the later, known RNA copy number reference needs to be used to be able to express the RNA copy number. So, I think the authors do a mistake in each figure legends writing “…to check the viral RNA copy numbers…” for me, they check the increasing or decreasing of the relative quantity of the virus, not the absolute quantity; They must change all figure legends and well explain how they did the qPCR and calculated the fold change of virus.
Plaque assay technic isn’t enough detailed particularly the staining step with crystal violet. But the major problem is no results and figures in this study described titration of the virus by Plaque assay as a result. Authors used in every steps RT-qPCR to follow up the replication of the virus. If plaque assay is used only to have the titer of the virus to calculate the MOI it’s not necessary to write this paragraph in the manuscript.
Response: Thank you for the suggestion. Plaque assay was performed for virus titration. In updated manuscript, we have deleted plaque assay paragraph, as you were suggested us. Primer sequences were added in the section of RT-qPCR protocol, and as for 2-ΔΔCt, please refer to reference 24.
Livak, K. J.; Schmittgen, T. D., Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCTmethod. methods 2001, 25, (4), 402-408.
The part “Time of addition assay” could be added to the “cell infection and drug treatments”.
Response: Thank you for the suggestion. We have merged time of addition assay in the cell infection and drug treatments paragraph.
“siRNA transfection” part. Authors used siRNA technic to knockdown expression of CHC but they missed to explain what is the siRNA control in the material and methods part.
Response: Thank you for the suggestion. In updated manuscript, The negative-control siRNA (sc-37007) from Santa Cruz Biotechnology Inc was described in line 106 and 107.
“Western Blotting” part. No information on the antibodies used for detection. Please, add a description and provider references.
Response: Thank you for the suggestion. Line 117 to 119, we have added antibodies information and provider reference. Rabbit anti-clathrin (P1663) CST and mouse anti-V-ATPase B 1/2 antibody (sc-271832) Santa Cruz Biotechnology Inc. DTMUV E monoclonal antibody is a kind gift of Prof.Renyong Jia from Sichuan Agricultural University.
In the results part, the authors have the tendency to conclude prior to describe the results in each part.
Response: Thank you for the suggestion. We removed the discussion from the results. In addition, we discussed the results in depth. For details, please see the discussion section.
In each figure legend, they wrote “* ,P<0.05 and **, P<0.01” but in the figure only ** appeared apart for the figure 4. In figure 5, it’s lacked statistical analysis explanation in the text. In Fig 3, no statistic stars on the top of the bars in the graphic but authors wrote “* ,P<0.05 and **, P<0.01” in the figure legend.
Response: Thank you for the suggestion. In updated figure legends, we confirmed “* ,P<0.05 and **, P<0.01” in Fig.1, 2,4,5,6. For details, please see the corresponding section.
In figure legend 1, authors mismatched E and B in the description of results.
Response: Thank you for the suggestion. We have modified figure 1 legends in line 150 E and line 154 B according to figure sequence described in legends.
In the part 3.4, the MOI used for the experiment doesn’t fit with the figure legend. Pleased, be clear with the MOI used in this study. Same part, authors present results as Fig 5 A and 5B, but in the main text there are not reference to A or B.
Response: Thank you for the suggestion. For replication we used MOI 0.1 while for binding and entry we used MOI 1. Fig 5A and B reference were added in the main text.
In part 3.5, authors referred to previous study using JEV in PK-15 and C6/36 (ref 24, 25) but the references were about Herpes virus in Hela and CHO cells. Then, authors used siRNA technic to knockdown expression of CHC but they missed to explain what is the siRNA control in the material and methods part.
Response: Thank you for the suggestion. In updated manuscript all reference cited according to the statement.
Yang, S.; He, M.; Liu, X.; Li, X.; Fan, B.; Zhao, S., Japanese encephalitis virus infects porcine kidney epithelial PK15 cells via clathrin-and cholesterol-dependent endocytosis. Virology journal 2013, 10, (1), 258. Chuang, C.-K.; Yang, T.-H.; Chen, T.-H.; Yang, C.-F.; Chen, W.-J., Heat shock cognate protein 70 isoform D is required for clathrin-dependent endocytosis of Japanese encephalitis virus in C6/36 cells. Journal of General Virology 2015, 96, (4), 793-803.
Finally, in the discussion the authors do not develop enough the importance of their results in comparison with the literature, the overall impression of this part is like a summary of the results. Authors have to take step back and to put their results in perspective with the literature of the field and develop better the perspective of research and further development on DTMUV. For instance, they used BHK-21, but what about entry step of this virus using Human cells, avian cells or insect cells?
Response: Thank you for the suggestion.We supplemented some details introducting the important role in zoonosis. In addition, even though Flaviviruses utilize several endocytic pathways to enter host cells, including macropinocytosis/phagocytosis, clathrin-mediated endocytosis, caveola/cholesterol-dependent uptake, and clathrin- and caveola-independent endocytosis, clathrin-mediated endocytosis is believed to be the major route of flavivirus cell entry. Therefore, DTMUV can use the different pathways to enter the host cells. However, our present work found that DTMUV enters BHK-21 cells through a clathrin-dependent pathway. In the future, we will work whether DTMUV has caveola-dependent endocytosis. The information above was supplemented in “Discussion section” of the latest manuscript.
Authors used BHK21 both to produce virus and to do experiments. May I have some feedback of authors about the possible impact on the results (adaptation of the virus to the cells line for instance)?
Response: Thank you for the suggestion. BHK21 cells are preferred host cells of flaviviruses. Except for DTMUV, the infection mechanism of other flaviviruses in BHK21 cells is very clear. Now there is no report of DTMUV infecting BHK21. In our study, BHK21 cells were selected to study the endocytosis of DTMUV.
Authors used RT-qPCR and indirect detection (E protein) to follow up the replication of the virus. I suggested to add experiments with plaque assay titration of the virus to confirm an effective replication of the virus with production of replicative virus particles.
Response: Thank you for the suggestion. In our study, we used RT-qPCR (viral load) and indirect immunofluorescence test (protein level) to assess DTMUV replication after endocytosis. These two assays are enough to perform for effective replication of the virus.
In paragraph line 264 to 276, authors referred to their own previous work on JEV (line 268) but miss to write the reference, that not sound well for a scientific article. The statements line 275 and 276 is not well write.
Response: Thank you for the suggestion. We have added our previous work reference as follows.
Liu, C.-C.; Zhang, Y.-N.; Li, Z.-Y.; Hou, J.-X.; Zhou, J.; Kan, L.; Zhou, B.; Chen, P.-Y., Rab5 and Rab11 are required for clathrin-dependent endocytosis of Japanese encephalitis virus in BHK-21 cells. Journal of virology 2017, 91, (19), e01113-17.
In the paragraph line 277 to 284, authors referred to several viruses but they used only one reference on JEV.
Response: Thank you for the suggestion. In updated manuscript, we have incorporated four references as follow.
Miller, J. L.; Weed, D. J.; Lee, B. H.; Pritchard, S. M.; Nicola, A. V., Low-pH Endocytic Entry of the Porcine Alphaherpesvirus Pseudorabies Virus. Journal of Virology 2019, 93, (2), e01849-18. Wang, S.; Liu, H.; Zu, X.; Liu, Y.; Chen, L.; Zhu, X.; Zhang, L.; Zhou, Z.; Xiao, G.; Wang, W., The ubiquitin-proteasome system is essential for the productive entry of Japanese encephalitis virus. Virology 2016, 498, 116-127. Pastenkos, G.; Lee, B.; Pritchard, S. M.; Nicola, A. V., Bovine herpesvirus 1 entry by a low-pH endosomal pathway. Journal of virology 2018, 92, (20), e00839-18. Cheng, S.; Yan, W.; Gu, W.; He, Q., The ubiquitin-proteasome system is required for the early stages of porcine circovirus type 2 replication. Virology 2014, 456, 198-204.
In my point of view, the discussion needs to be improved to better highlight the results and to show well the further perspective of research on DTMUV.
Response: Thank you for the suggestion. Response: Thank you for the suggestion. We supplemented some details introducting the important role in zoonosis. For details, please see “Discussion section” in the latest manuscript.
Despite the interest of the topic and further innovation/prospective usage of this work, the number of mistakes is harmful for the message of the paper.
My overall impression in this manuscript is it was not well reviewed and edited prior submission.
Response: Thank you for the suggestion. We have updated weakness in our manuscript according to valuable suggestion and manuscript updated by MDPI English editing services.
Major issues and specific comments
Introduction,
Line 34: ref 2 is not appropriated
Response: Thank you for the suggestion. We updated this ref in the list as follow.
Cao, Z.; Zhang, C.; Liu, Y.; Ye, W.; Han, J.; Ma, G.; Zhang, D.; Xu, F.; Gao, X.; Tang, Y., Tembusu virus in ducks, China. Emerging infectious diseases 2011, 17, (10), 1873.
Line 42: the sentence “Moreover…”is not clear enough
Response: Thank you for the suggestion. We deleted this word.
Line 48: ref 10 is not appropriated
Response: Thank you for the suggestion. We updated this ref in the list as follow.
Wang, J.; Lei, C.-Q.; Ji, Y.; Zhou, H.; Ren, Y.; Peng, Q.; Zeng, Y.; Jia, Y.; Ge, J.; Zhong, B., Duck Tembusu virus nonstructural protein 1 antagonizes IFN-β signaling pathways by targeting VISA. The Journal of Immunology 2016, 197, (12), 4704-4713. Jiang, T.; Liu, J.; Deng, Y.-Q.; Su, J.-L.; Xu, L.-J.; Liu, Z.-H.; Li, X.-F.; Yu, X.-D.; Zhu, S.-Y.; Gao, G. F., Development of RT-LAMP and real-time RT-PCR assays for the rapid detection of the new duck Tembusu-like BYD virus. Archives of virology 2012, 157, (12), 2273-2280.
Line 49: CHIKV is not a Flavivirus !!! or virus classification changed…
Response: Thank you for the suggestion. We have deleted chickungunya virus.
Line 49: ref 11 12 are not appropriated. Ref 11 is about DTMUV not about DENV, WNV or JEV
Response: Thank you for the suggestion. We have updated all the reference.
Burke, D.; Monath, T., Fields virology. Fields virology. Philadelphia: Lippincott Williams & Wilkins 2001, 1043-125. Smit, J.; Moesker, B.; Rodenhuis-Zybert, I.; Wilschut, J., Flavivirus cell entry and membrane fusion. Viruses 2011, 3, (2), 160-171.
Line 53: ref 13 is not appropriated. The sentence is about Flavivirus but this ref is about DTMUV. Please, used a general ref about receptor mediated entry of Flavivirus or change the sentence.
Response: Thank you for the suggestion. We have inserted the following references.
Brindley, M. A.; Maury, W., Endocytosis and a low-pH step are required for productive entry of equine infectious anemia virus. Journal of virology 2005, 79, (23), 14482-14488. Jin, S.; Zhang, B.; Weisz, O. A.; Montelaro, R. C., Receptor-mediated entry by equine infectious anemia virus utilizes a pH-dependent endocytic pathway. Journal of virology 2005, 79, (23), 14489-14497. Sieczkarski, S. B.; Whittaker, G. R., Dissecting virus entry via endocytosis. Journal of General Virology 2002, 83, (7), 1535-1545. Nicola, A. V.; Aguilar, H. C.; Mercer, J.; Ryckman, B.; Wiethoff, C. M., Virus entry by endocytosis. Advances in virology 2013.
Materials and Methods,
Line 65: edit well 37°C as -80°C Line 68
Response: Thank you for the suggestion. We have changed as per comments.
Line 63: for provider use homogenous presentation: (provider, city, country) for instance. But same presentation in all the manuscript)
Line 64: authors only wrote (GIBCO). Referred to the previous comments and so on for others providers in the text.
Response: Thank you for the suggestion. We supplemented all the reagents (provider, city, country) in the updated manuscript. For details, please see the corresponding section.
Line 72: add the abbreviation used later in the text for Bafilomycin here (Baf A1).
Response: Thank you for the suggestion. we have added abbreviation.
Line 75: edit hour. Edit 37°C as -80°C line 68
Response: Thank you for the suggestion. It was changed in the updated manuscript.
Line 77: ref 14 and 15 are clearly not appropriated
Response: Thank you for the suggestion. We have inserted the following references.
Qiu, X.; Yu, Y.; Yu, S.; Zhan, Y.; Wei, N.; Song, C.; Sun, Y.; Tan, L.; Ding, C., Development of strand-specific real-time RT-PCR to distinguish viral RNAs during Newcastle disease virus infection. The Scientific World Journal 2014, 2014.
Line 79: “MOI 0.05” different than in figure legend
Response: Thank you for the suggestion. We have changed MOI from 0.5 to 0.1 for replication.
Results
In figure 1.B: JEV is written in the ordinate axis legend!! Authors worked in DTMUV!
Response: Thank you for the suggestion. In updated manuscripts we have changed JEV instead of DTMUV.
Line 147: mistake in the text “… (A, C, and E)…” be replaced with (A, B and C)
Response: Thank you for the suggestion. We have modified figure 1 legends A, B and C binding and entry, D, E and F as an infection.
Line 151: mistake in the text “… (B, D, and F)…” be replaced with (D, E and F)
Response: Thank you for the suggestion. We have replaced according to sequence (D, E and F).
Line 152: mistake in the MOI value
Response: Thank you for the suggestion. For replication we used MOI 0.1 while for binding and entry we used MOI 1.
Line 156: remove “*, P<0.05;”, not in the graph;Line 168: remove “*, P<0.05;”, not in the graph. Please, check it in every figure legend.
Figure 3: add statistical analysis;Line 186: checked statistical analysis.
Response: Thank you for the suggestion. In updated figure legends, we confirmed “* ,P<0.05 and **, P<0.01” in Fig.1,2,3,4,5,6. For details, please see the corresponding section.
Line 200: remove “RNA copy number” or be clearer with the RT-qPCR technic employed in this study. Same comment for every figure legend using RT-qPCR.
Response: Thank you for the suggestion. We have changed “the infectious particles” into “the viral load” in the latest manuscript.
Line 212: MOI mismatched with the figure legend 5A. Checked the description of results A and B.
Response: Thank you for the suggestion. We have checked and added correct MOI.
Line 215: add A and B fig the figure 5 to better understand results.
Response: Thank you for the suggestion. Fig 5A and B added in the main text.
Line 230: ref 24 and 25 are clearly not appropriated to JEV. Remove or change
Response: Thank you for the suggestion. We have updated references.
Yang, S.; He, M.; Liu, X.; Li, X.; Fan, B.; Zhao, S., Japanese encephalitis virus infects porcine kidney epithelial PK15 cells via clathrin-and cholesterol-dependent endocytosis. Virology journal 2013, 10, (1), 258. Chuang, C.-K.; Yang, T.-H.; Chen, T.-H.; Yang, C.-F.; Chen, W.-J., Heat shock cognate protein 70 isoform D is required for clathrin-dependent endocytosis of Japanese encephalitis virus in C6/36 cells. Journal of General Virology 2015, 96, (4), 793-803.
Discussion
Line 259: corrected “… +ve” in a good English
Response: Thank you for the suggestion. We have added positive in English.
Line 269: add reference to this statement
Response: Thank you for the suggestion. We have inserted reference.
38 Liu, C.-C.; Zhang, Y.-N.; Li, Z.-Y.; Hou, J.-X.; Zhou, J.; Kan, L.; Zhou, B.; Chen, P.-Y., Rab5 and Rab11 are required for clathrin-dependent endocytosis of Japanese encephalitis virus in BHK-21 cells. Journal of virology 2017, 91, (19), e01113-17.
Line 280: add reference to this statement not only one on JEV
Response: Thank you for the suggestion. In above comments response we have added four references according to the statements.
Line 285: add reference to this statement
Response: Thank you for the suggestion. We have inserted two reference based on statements.
Liu, C.-C.; Zhang, Y.-N.; Li, Z.-Y.; Hou, J.-X.; Zhou, J.; Kan, L.; Zhou, B.; Chen, P.-Y., Rab5 and Rab11 are required for clathrin-dependent endocytosis of Japanese encephalitis virus in BHK-21 cells. Journal of virology 2017, 91, (19), e01113-17. Zhang, Y.-N.; Liu, Y.-Y.; Xiao, F.-C.; Liu, C.-C.; Liang, X.-D.; Chen, J.; Zhou, J.; Baloch, A. S.; Kan, L.; Zhou, B.; Qiu, H.-J., Rab5, Rab7, and Rab11 Are Required for Caveola-Dependent Endocytosis of Classical Swine Fever Virus in Porcine Alveolar Macrophages. Journal of Virology 2018, 92, (15), e00797-18.
Minor issues
From ethical general guideline for publication: Authors should state when and where they obtained the cells and virus, giving the date and the name of the researcher, cell line repository, or commercial source (company) who provided the cells or virus, as appropriate.
Response: Thank you for the suggestion. Cells were purchased from ATCC and virus was provided by Prof. Rui-bing Cao from Nanjing Agricultural University.
Round 2
Reviewer 1 Report
Thank you for addressing all of the reviewer comments.
Author Response
Thank you for your approval of our response. In addition, we have also modified some languages appropriately.
Reviewer 2 Report
The authors addressed the reviewer´s comments.
Author Response
Thank you for your approval of our reply. In addition, we have also modified some languages appropriately.
Reviewer 3 Report
Summary
The authors of the paper focus their study on the Duck Tembusu virus (DTMUV) and the way to enter and trigger its own replication in mammal cell. DTMUV belongs to the Flaviviridae family, it was recently discovered in China and has a high negative impact in avian production in case of outbreak. But the biology of this virus still be relatively unknown. Author’s model is Baby Hamster Kidney cells line (BHK-21). For the authors, it’s interesting to focus research on this kind of cell line as in vitro model because BHK-21 is frequently used to produce veterinary vaccine against DTMUV. Authors are interesting in the endosomal pathway as a route of entry of the virus and the importance of the pH in the triggering DTMUV replication.
Overall impression/ Broad comments
The research treated in this article takes place in a new field of investigation. At this time; few teams are working on different DTMUV. It’s very interesting that the authors focus their research on this virus because its biology remains globally blurred. The entry step is particularly crucial for Flavivirus to trigger replication and spreading in the host, so it’s important to know well this critical step of the replication cycle of the DTMUV. On this point, this study is setting on a rising field of research on a very recent Flavivirus impacting human and animal health in Asia.
In this reviewed 2nd version of the manuscript, despite corrections, the manuscript still presents some correction to in order to improve the quality of the paper.
About quality of presentation, the manuscript presents some editing mistakes (the “of” in “pathogenesis of and…” line 43 ),
Presentation of provider still be inhomogeneous (line 62 versus line 63 for instance), in all the materiel and method part authors have used different style, with sometime only brand, sometime brand and country.
Units sometime inhomogeneous (for instance: line 101: “15 min” and line 114 “10_min”)
Also, I still found unappropriated reference in the text.
For instance, Line 35: the reference 5 is inappropriate. It’s a paper on rapid detection of Tembusu virus, authors site a paragraph of the introduction of this paper but it still not has any information supported by a reference of economic loss, only statement.
Line 49: reference 18 and 19 is about the same virus in the same paper the same year, choose only one or a paper on another virus.
Line 77: no need ref 22/23 here.
Line 99: replace “for one h” with “for 1 h”
Line 108: “Invitrogen” with “I” in capital.
Line 114: replace “10_min” by “10 min” same syntax than line 68
In the part 3.4, Authors present first Fig5.B then 5.A, it’s not a logic presentation.
Authors used BHK21 both to produce virus and to do experiments. May I have some feedback of authors about the possible impact on the results (adaptation of the virus to the cells line for instance)?
Sorry, but I don’t understand the sentence line 284-285 “DTMUV… to understand”. To understand what?
Authors have answered to almost the main remarks but it still be corrections to do.
Major issues and specific comments
Introduction,
Line 43: remove of after pathogenesis
Line 48: ref 10 is not appropriated
Materials and Methods,
Line 63: for provider use homogenous presentation: (provider, city, country) for instance. But same presentation in all the manuscript)
Line 64: authors only wrote (GIBCO). What is the city, country of provider? Referred to the previous comments and so on for others providers in the text.
Line 72: homogenize the syntax for provider: (SIGMA, location)
Line 77: why add these references?
Line 114: replace “10_min”
Results
Line 240: add treatment or other term “…of CPZ treatment on …
Line 252: remove “-“ before “…-RT-qPCR…”
Discussion
Line 268: write “… transmission medium…” for transmission by mosquito isn’t very good expression. Please change with other term, as “…principal vector….”
Author Response
Dear Editor,
Thank you for your efforts on our manuscript. We have revised the manuscript according to the reviewer comments and answered each question point-to-point. For details, please see our positive responses below. The blue highlights in the latest manuscript and make-up version show those revised details. If you have any question, please feel free to contact me.
Yours sincerely,
Bin Zhou, DVM, PhD, Professor
Viruses-626136
Comments from the editors and reviewers:
Summary
The authors of the paper focus their study on the Duck Tembusu virus (DTMUV) and the way to enter and trigger its own replication in mammal cell. DTMUV belongs to the Flaviviridae family, it was recently discovered in China and has a high negative impact in avian production in case of outbreak. But the biology of this virus still be relatively unknown. Author’s model is Baby Hamster Kidney cells line (BHK-21). For the authors, it’s interesting to focus research on this kind of cell line as in vitro model because BHK-21 is frequently used to produce veterinary vaccine against DTMUV. Authors are interesting in the endosomal pathway as a route of entry of the virus and the importance of the pH in the triggering DTMUV replication.
Overall impression/ Broad comments
The research treated in this article takes place in a new field of investigation. At this time; few teams are working on different DTMUV. It’s very interesting that the authors focus their research on this virus because its biology remains globally blurred. The entry step is particularly crucial for Flavivirus to trigger replication and spreading in the host, so it’s important to know well this critical step of the replication cycle of the DTMUV. On this point, this study is setting on a rising field of research on a very recent Flavivirus impacting human and animal health in Asia.
In this reviewed 2nd version of the manuscript, despite corrections, the manuscript still presents some correction to in order to improve the quality of the paper.
Response: Thank you for the suggestion. We have modified the manuscripts according to the comments point-to-point.
About quality of presentation, the manuscript presents some editing mistakes (the “of” in “pathogenesis of and…” line 43),
Response: Thank you for the suggestion. We have deleted “of” from line 43 in the updated manuscript..
Presentation of provider still be inhomogeneous (line 62 versus line 63 for instance), in all the materiel and method part authors have used different style, with sometime only brand, sometime brand and country.
Response: Thank you for the suggestion. We have added fetal bovine serum information (FBS, GIBCO, Grand island, NY, USA), Antibiotics (GIBCO, Grand island, NY, USA). Line 74 and 75 Sigma Saint Louis, USA and Cayman, Michigan, USA.
Units sometime inhomogeneous (for instance: line 101: “15 min” and line 114 “10_min”)
Response: Thank you for the suggestion. We have deleted 10_min. now “15 min” and “10 min” are homogeneous.
Also, I still found unappropriated reference in the text.
For instance, Line 35: the reference 5 is inappropriate. It’s a paper on rapid detection of Tembusu virus, authors site a paragraph of the introduction of this paper but it still not has any information supported by a reference of economic loss, only statement.
Response: Thank you for the suggestion. We have inserted new reference according to the statement as follows.
Liu, P.; Lu, H.; Li, S.; Wu, Y.; Gao, G. F.; Su, J., Duck egg drop syndrome virus: an emerging Tembusu-related flavivirus in China. Sci China Life Sci 2013, 56, (8), 701-10.
Line 49: reference 18 and 19 is about the same virus in the same paper the same year, choose only one or a paper on another virus.
Response: Thank you for the suggestion. We have removed reference 19 in the updated manuscript..
Line 77: no need ref 22/23 here.
Response: Thank you for the suggestion. We have deleted both reference in the updated manuscript.
Line 99: replace “for one h” with “for 1 h”
Response: Thank you for the suggestion. We have replaced “one h” into “1 h”.
Line 108: “Invitrogen” with “I” in capital.
Response: Thank you for the suggestion. We have changed ‘I’ in capital
Line 114: replace “10_min” by “10 min” same syntax than line 68
Response: Thank you for the suggestion. We have replaced 10 min.
In the part 3.4, Authors present first Fig5.B then 5.A, it’s not a logic presentation.
Response: Thank you for the suggestion. We have replaced the results Fig 5A first then Fig 5B.
Authors used BHK21 both to produce virus and to do experiments. May I have some feedback of authors about the possible impact on the results (adaptation of the virus to the cells line for instance)?
Response: We agree with the suggestion of the author. Since BHK21 cells are often used to study the model cell line of DTMUV, we believe that the cells are very suitable for virus proliferation, which leads us to first study the mechanism of virus entry on this commonly used cell line. Later, we will study the infection mechanism of the virus on duck cells. From this, we can know whether the different cell lines will lead to the different mechanisms of entry.
Sorry, but I don’t understand the sentence line 284-285 “DTMUV… to understand”. To understand what?
Response: Thank you for the suggestion. We have changed the sentence as follows, DTMUV is traverses a particular endocytic pathway, which is similar to most Flaviviruses.
Authors have answered to almost the main remarks but it still be corrections to do.
Major issues and specific comments
Introduction,
Line 43: remove of after pathogenesis
Response: Thank you for the suggestion. We have deleted “of” from line 43.
Line 48: ref 10 is not appropriated
Response: Thank you for the suggestion. We have removed reference 10.
Materials and Methods,
Line 63: for provider use homogenous presentation: (provider, city, country) for instance. But same presentation in all the manuscript)
Response: Thank you for the suggestion. We have added the information line. FBS, GIBCO, Invitrogen, Carlsbad, CA, USA
Line 64: authors only wrote (GIBCO). What is the city, country of provider? Referred to the previous comments and so on for others providers in the text.
Response: Thank you for the suggestion. We have added information GIBCO, Grand island, NY, USA.
Line 72: homogenize the syntax for provider: (SIGMA, location)
Response: Thank you for the suggestion. We have added information Sigma, Saint Loius, USA
Line 77: why add these references?
Response: Thank you for the suggestion. We have deleted both references in the updated manuscript as you were suggested in above comment.
Line 114: replace “10_min”
Response: Thank you for the suggestion. We have replaced and responded in above comments
Results
Line 240: add treatment or other term “…of CPZ treatment on …
Response: Thank you for the suggestion. We have added CPZ “treatment” on DTMUV.
Line 252: remove “-“ before “…-RT-qPCR…”
Response: Thank you for the suggestion. We have deleted “-“.
Discussion
Line 268: write “… transmission medium…” for transmission by mosquito isn’t very good expression. Please change with other term, as “…principal vector….”
Response: Thank you for the suggestion. We have replaced transmission medium into principal vector.
