Natural Bovine Coronavirus Infection in a Calf Persistently Infected with Bovine Viral Diarrhea Virus: Viral Shedding, Immunological Features and S Gene Variations

Round 1

Reviewer 1 Report

Thank you for your re-submission.

Editing of the English language is required to bring it to a publishable standard.

The discussion now reads well and nothing is being proposed that is too far-reaching. Can I please suggest however, that as authors one should comment on the fact that this is analysis of just one animal. While it is valid to 'suppose' possibilities in your discussion it is also important to acknowledge that the data is generated form one animal and far reaching conclusions can not be drawn. I believe an insertion of such a line after the sentence finishing on line 386 would be appropriate, particularly because, as you point out, your data is in direct conflict with previously published works (reference 28).

Please remove the word 'easily' on line 48 - jumping species should not be described in this way.

Other than these changes, I am happy to recommend the publication of this manuscript.

Author Response

Response to Reviewer 1

Many thanks for the suggestions.

The text has been modified:

-lines 388-389: the suggested sentence was added "... being data generated from the observation of a single animal, ...".

-Line 48: the word "easily" has been removed.

English language has been edited

Author Response File: Author Response.docx

Reviewer 2 Report

Revised version is prefer than the first paper.

Author Response

No action has been required

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Thank you for your submission. I enjoyed reading your manuscript and I commend you and your team for the work carried out.

This is an immunological analysis of a single calf that was co-infected with BVDV and BCoV. BVDV is known to cause immunosuppression in animals and one would suspect that a BVDV infection may lead to persistent or more severe disease as a result of other infections, such as that of BCoV. 

I find the analysis interesting, but I am little concerned about a few details that I will now point to:

1. It is not stated that ethical approval was granted for this study - can you please confirm this and state it in the manuscript.

2. The methodology, as I read it seems fine, although, your method entitled virus isolation is not a method for virus isolation and is rather an assay to detect the presence of infectious virus in a sample - I recommend changing this title.
3. In the results, can you please give more detail in the figure legends. For example, in figure 1 what is TN and TR? What assay was performed here - it says in the legend that BCoV was excreted but this is not a measure of infectious virus so that is incorrect? What is the y-axis? Figure 2, what is the y-axis? Figure 3, what is the y-axis? Figure 6, y-axis? etc.
4. Be careful to define what exactly you are reporting - in your results please differentiate between PCR and assays that measure infectious virus. For example in relation to figure 1, line 243 and 244, you are measuring virus RNA not virus so it is not correct to say high amounts of virus was excreted. In line 278, you refer to BCoV, but really you mean BCoV RNA. Line 279, you are not measuring viral shedding, but virus RNA are you not? Please be accurate in these descriptions. 
5. Please relabel table 2. Remove NS and replace with Ct value.
6. Line 282, please do not use the word 'massive' it is inappropriate, you can just say that the Ct was much lower at this timepoint. Also, again, it is not viral shedding that you are measuring, it is virus RNA.
7. Line 253-255. Can you give more detail here - did you not see any virus induced cpe? Did passaging allow virus to propogate? At what passage was the IF positive?
8. Line 360 – How many samples did you collect at these timepoints? Considering it appears that there is intermittent shedding faecally, is it possible that the sample you collected just happened to not have virus RNA in it, but if you had taken a sample an hour later you might have detected it? In Other words, I don’t agree with the conclusion that viral shedding started later – I don’t think you have conclusive data to say that. All you can say is that it was undetectable in the samples you collected.
9. Line 361 – Can you point me to this data about viral load – I can’t see it.
10. 372 – I don’t see any evidence of a ‘belated transit’ to the GIT and it is incorrect to say this based on the evidence presented.
11. Lines 375 – 382: I’m sorry, I don’t see any evidence presented here to draw these conclusions. For example, there is no evidence presented to rule out the possibility that the animal was co-infected with an enteric version and a respiratory version of the virus around the same time. There is also no evidence presented to say that enteric transmission isn’t important or can happen independent of the respiratory route.
12. Line 381-382: Would it not be more correct to say that there was no strong correlation between Ct value and clinical score over time and that the low Ct coinciding with the highest clinical score is potentially just coincidence, especially considering you are only dealing with one animal and drawing any conclusions based on this one animal is challenging and unlikely to be accurate.
13. Line 388: Massive is used inappropriately here again – you can’t say massive because you have no comparison.
14. Lines 390 – 407: There are some very broad assumptions and conclusions drawn here and I’m sorry to say that I don’t see how you can draw these conclusions based on the evidence presented. Most is pure speculation, founded in minimal data. The data does not highlight the ‘evolutionary plasticity or BCoV’ and you have zero evidence that the changes seen in the protein sequence have any impact on the virus never mind giving it a favourable phenotype in an immunocompromised animal. To be honest, you present zero evidence that the animal even is immunocompromised in the first place.
15. Line 400 – 404: Exposure to the host immune system is a driver of evolution for all viruses, not something unique to BCoV or to the situation described in this manuscript. Monitoring of these sequences and understanding the impact of changes is an obvious and unnecessary conclusion in the context of this manuscript.
16. I struggle with the conclusion section. What is reported here does not underline how little is known about BCoV infection. Also, in the context of BVDV, you have failed to explain how these types of studies would impact on human research – can you please expand on this and maybe identify commonalities already identified?

Because of these issues highlighted here, I can not at this point recommend this manuscript for publication, sorry.

Author Response

1. It is not stated that ethical approval was granted for this study - can you please confirm this and state it in the manuscript.

The study is not an experimental study but describes a clinical case. Therefore, we have received the authorization of the Ministry of Health to carry out experimental study on BCoV infection (The authorization number was added in the text). The authorization of the Ministry of Health is much more important than the Ethical approval from the Department. We can provide the letter of the Ministry of Health authorization.

1. The methodology, as I read it seems fine, although, your method entitled virus isolation is not a method for virus isolation and is rather an assay to detect the presence of infectious virus in a sample - I recommend changing this title.

The method entitled virus isolation was modified in “virus detection” as suggested (Line 191).

1. In the results, can you please give more detail in the figure legends. For example, in figure 1 what is TN and TR? What assay was performed here - it says in the legend that BCoV was excreted but this is not a measure of infectious virus so that is incorrect? What is the y-axis? Figure 2, what is the y-axis? Figure 3, what is the y-axis? Figure 6, y-axis? etc.

The Reviewer is right. The Figures was integrated and modified as suggested.

1. Be careful to define what exactly you are reporting - in your results please differentiate between PCR and assays that measure infectious virus. For example in relation to figure 1, line 243 and 244, you are measuring virus RNA not virus so it is not correct to say high amounts of virus was excreted. In line 278, you refer to BCoV, but really you mean BCoV RNA. Line 279, you are not measuring viral shedding, but virus RNA are you not? Please be accurate in these descriptions. 

The Reviewer is right and the text was modified as suggested. There is no exact correspondence with lines reported, but we have located the signaled points (Lines 268; 270, 273.

1. Please relabel table 2. Remove NS and replace with Ct value.

Table 2 was relabeled as suggested

1. Line 282, please do not use the word 'massive' it is inappropriate, you can just say that the Ct was much lower at this timepoint. Also, again, it is not viral shedding that you are measuring, it is virus RNA.

There is no exact correspondence between signed line and the Reviewer observation. Therefore, the word “massive” in the text was removed and the text was modified (Lines 349, 519).

1. Line 253-255. Can you give more detail here - did you not see any virus induced cpe? Did passaging allow virus to propogate? At what passage was the IF positive?

There is no exact correspondence between signed line and the Reviewer observation. Therefore, the text was modified and integrated as suggested (Lines 283, 284).

1. Line 360 – How many samples did you collect at these timepoints? Considering it appears that there is intermittent shedding faecally, is it possible that the sample you collected just happened to not have virus RNA in it, but if you had taken a sample an hour later you might have detected it? In Other words, I don’t agree with the conclusion that viral shedding started later – I don’t think you have conclusive data to say that. All you can say is that it was undetectable in the samples you collected.

There is no exact correspondence between signed line and the Reviewer observation. Therefore, the text was modified as suggested (Lines 493-494).

1. Line 361 – Can you point me to this data about viral load – I can’t see it.

There is no exact correspondence between signed line and the Reviewer observation. It is possible that the Reviewers highlights line 485. If it was correct, data regarding the viral load (expressed as Ct) are reported in Table 2.

1. 372 – I don’t see any evidence of a ‘belated transit’ to the GIT and it is incorrect to say this based on the evidence presented.

There is no exact correspondence between signed line and the Reviewer observation. Our hypothesis was supported by references datum (22, Saif, 2010). We observed that BCoV was first detected in the nasal discharge and then in the feces. These observations led to the conclusion that the infection route can affect the sequence of infection of the respiratory and intestinal tract. Therefore, if Reviewer disagrees we can delete this paragraph.

1. Lines 375 – 382: I’m sorry, I don’t see any evidence presented here to draw these conclusions. For example, there is no evidence presented to rule out the possibility that the animal was co-infected with an enteric version and a respiratory version of the virus around the same time. There is also no evidence presented to say that enteric transmission isn’t important or can happen independent of the respiratory route.

There is no exact correspondence between signed line and the Reviewer observation. The Authors underline that BCoV was shortly detected in the NSs (T2) and speculate that the role of BCoV as enteric pathogen could be questioned. References datum (9, Vlasova and Saif, 2021) supports the idea that there are enteric and respiratory viruses as members of the same quasispecies able to determine different clinical signs as expression of environmental and host interactions. Therefore, if Reviewer disagrees we can delete this paragraph.

1. Line 381-382: Would it not be more correct to say that there was no strong correlation between Ct value and clinical score over time and that the low Ct coinciding with the highest clinical score is potentially just coincidence, especially considering you are only dealing with one animal and drawing any conclusions based on this one animal is challenging and unlikely to be accurate.

There is no exact correspondence between signed line and the Reviewer observation. The text was modified as suggested and the sentence was deleted.

1. Line 388: Massive is used inappropriately here again – you can’t say massive because you have no comparison.

There is no exact correspondence between signed line and the Reviewer observation. Therefore, we have substitute “massive” with high (Line 519). If necessary we can delete the word.

1. Lines 390 – 407: There are some very broad assumptions and conclusions drawn here and I’m sorry to say that I don’t see how you can draw these conclusions based on the evidence presented. Most is pure speculation, founded in minimal data. The data does not highlight the ‘evolutionary plasticity or BCoV’ and you have zero evidence that the changes seen in the protein sequence have any impact on the virus never mind giving it a favourable phenotype in an immunocompromised animal. To be honest, you present zero evidence that the animal even is immunocompromised in the first place.

There is no exact correspondence between signed line and the Reviewer observation. The Reviewer is right and our conclusions basing on few data. Therefore, Covid-19 infection taught us that immunocompromised patients favor the onset of viral mutations and the emergences of variants (Jensen et al., 2021; Moelling, 2021; Lynch et al., 2021). BVDV persistent infection is characterized by an evident impairment of the immune system (Strong et al., 2015; Walz, 2020; Schweizer et al., 2006; Peterhans and Schweizer, 2013; Liu et al., 2020) e it cannot be ruled out that a similar scenario might happened in immunocompromised cattle.  Based on these observations, and on the well-documented plasticity of Coronaviruses (Laude et al., 1993; Vennema et al., 1998; Guan et al., 2003; Rottier et al., 2005; Song et al., 2005; Vijgen et al., 2005; Decaro and Buonavoglia, 2008; Deacro and Lorusso, 2020; Su et al., 2016), the Authors have speculated that BVDV persistent infection could have played a role on the onset of mutations. Therefore, because our data are based on a single observation, more in deep analysis are required and If Reviewer agrees, we have modulated this observation (Lines 524-525).

1. Line 400 – 404: Exposure to the host immune system is a driver of evolution for all viruses, not something unique to BCoV or to the situation described in this manuscript. Monitoring of these sequences and understanding the impact of changes is an obvious and unnecessary conclusion in the context of this manuscript.

There is no exact correspondence between signed line and the Reviewer observation. The Reviewer is right, but the mutation rates in CoVs, compared to other single-stranded RNA viruses, are moderate to high (average substitution rates being about 10^-4 substitutions per site each year) (Su et al., 2016). The high frequency of RNA recombination is probably the result of the unique mechanism of CoV synthesis, which involves discontinuous transcription and polymerase jumping. It is possible that the viral polymerase associated with the incomplete nascent RNAs, dissociates from its template at a random point, and switches to a homologous site on a different RNA template to complete RNA synthesis by a copy-choice mechanism (Lai, 1992). Our observation is based on these assumptions and, even if based on a single infected animal, are supported by bibliography.

1. I struggle with the conclusion section. What is reported here does not underline how little is known about BCoV infection. Also, in the context of BVDV, you have failed to explain how these types of studies would impact on human research – can you please expand on this and maybe identify commonalities already identified?

We agree with Reviewer observation and our study does not directly impact on human research. We referred to BCoV infection in immunocompromised animals as possible link for the study of SARS-CoV-2 infection in immunocompromised patients. The conclusion section was modified as suggested.

Author Response File: Author Response.docx

Reviewer 2 Report

This paper described a case report of BVDV PI cattle superinfected with BCoV. The indicated results are valuable for clinical, clinicopathological and immunological understanding. Authors showed the difference in S-genes between respiratory and enteric BCoVs from the PI cattle. This is ‘variation’ expressed in the Title, not evolution. Moreover, there is no evidence of evolution of BCoV in this PI cattle. The viruses were detected from nasal and fecal swab at almost the same time. Double infections of respiratory and enteric viruses? There are many unsuitable descriptions in the text as follows

Abbreviation of “persistent infected” is PI, not pi.  

Serial reference number in text should be written as [2-6] not [2,3,4,5,6] (line 52), also lines 56 and67.

Scale units of Y-axis in Figures 1-3 should be indicated such as Fig 5.

184 PMBC > PBMC

250 What are TN and TR in the figure? There is no explanation in the legend.

284 Ct values of NS is not correspond to Fig 1, at T28. Is this correct? or my miss understanding?

291 /ml > /μL  In Fig. 4, this units are used.

Author Response

Reviewer 2

-This paper described a case report of BVDV PI cattle superinfected with BCoV. The indicated results are valuable for clinical, clinicopathological and immunological understanding. Authors showed the difference in S-genes between respiratory and enteric BCoVs from the PI cattle. This is ‘variation’ expressed in the Title, not evolution. Moreover, there is no evidence of evolution of BCoV in this PI cattle. The viruses were detected from nasal and fecal swab at almost the same time. Double infections of respiratory and enteric viruses? There are many unsuitable descriptions in the text as follows

Reviewer is right and the text was modified (variation and not evolution) (Lines 26, 39, 72, 87, 527)

-Abbreviation of “persistent infected” is PI, not pi.  

Reviewer is right and the text was modified (Lines 83, 96, 100, 102, 266, 513).

-Serial reference number in text should be written as [2-6] not [2,3,4,5,6] (line 52), also lines 56 and67.

The Authors Instruction reports: In the text, reference numbers should be placed in square brackets [ ], and placed before the punctuation; for example [1], [1–3] or [1,3].)

-Scale units of Y-axis in Figures 1-3 should be indicated such as Fig 5.

Reviewer is right and the text was modified

-184 PMBC > PBMC

Reviewer is right and the text was modified (Line 208)

-250 What are TN and TR in the figure? There is no explanation in the legend.

Reviewer is right and the text was modified

284 Ct values of NS is not correspond to Fig 1, at T28. Is this correct? or my miss understanding?

Reviewer is right. Nasal shedding was observed at T28. The text was modified

-291 /ml > /μL  In Fig. 4, this units are used.

Reviewer is right. The text was modified

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Hi,

Thank you for the corrections you have made. Unfortunately the major issue with the manuscript remains. It is clear that the conclusions being drawn are far reaching and extensive, but are not based on the data presented. It is good to speculate but the speculation is taken too far in this manuscript and is unjustified.

For example, you say:'Since BCoV was shortly detected in the NSs (T2) and viral load showed higher positivity than feces (Ct + 2.1), our data support the hypothesis that the role of BCoV as enteric pathogen could be questioned and that there are enteric and respiratory viruses as members of the same quasispecies able to determine different clinical signs as expression of environmental and host interactions '. This is completely unjustified based on that small piece of data. One can not draw any such conclusions and to even suggest it is unfair and misleading. Even the Ct value difference - carry out the same PCR on a different machine in a different lab and you will probably get a different result. You need large numbers of animals in a controlled situation to go close to the suppositions you are making and it is unjustified.

You even suggest that the co-infection might cause the evolution of new variants - it might but your data doesn't say so, in fact, by the antibody titres, its difficult to know if the animal is in fact even immunosuppressed. You can't just presume that from an individual animal.

For this reason I am sorry but I can not agree to it's publication - drawing accurate and reasonable conclusions from a body of work is the essence of a good manuscript. This should be presented as a clinical case of interest, no more or used as the basis to attract funding to investigate the situation further.