Analysis of Coxsackievirus B5 Infections in the Central Nervous System in Brazil: Insights into Molecular Epidemiology and Genetic Diversity

Round 1

Reviewer 1 Report

This is a well written manuscript describing novel genotypes of coxsackievirus B5 that were circulating in Brazil from 2005 to 2018. The authors focus on the VP1 protein of CVB5 and find that two novel genotypes were predominant during the time period assessed. These can be traced back to virus outbreaks in Haiti, France and Turkey. This manuscript will be of broad interest to those tracking CVB5 evolution from both an epidemiologic and basic virus biology standpoint. I have very few criticisms of the manuscript as written. 

The overall fitness of CVB5 virus can most likely be linked to multiple proteins generated by the virus. I think it would be helpful to show how other viral proteins have evolved in these isolates. Understanding how other capsid proteins or non-capsid proteins are altered over time would help to put into context the changes observed in VP1. 

Virus replication in picornaviruses is known to be error prone. Additional discussion on the impact of low fidelity replication would help to improve this study. Not only in the context of the genotypes discovered but also in the techniques used to propagate the virus. The generation of the virus supernatants used for  analysis were amplified in multiple cells lines. It is possible that amplification of these viruses in different cell lines alone could alter the genome sequence and lead to the identification of novel sequences that were not due to circulation in human populations. Providing information on what cell lines were used to propagate each of the viruses or discussing any potential impact would improve this. 

Other comments:

1. In Figure 1 it is not clear what the color scale is denoting. There are no instances of samples being yellow. The scale should be adjusted to reflect potential differences between samples.
2. For Figure 2, include specific text in the legend describing/denoting what the red bars represent. 
3. In Figure 4, it is difficult to discriminate the 3 different green colors that the authors used to specify VP1, VP2 and VP3. Please change these to provide higher contrast. 

Author Response

Dear Editor,

We would like to thank the referees for the valuable and helpful comments, which enriched our paper “Analysis of coxsackievirus B5 infections in the central nervous system in Brazil: insights into molecular epidemiology and genetic diversity”.

We inform that all changes recommended by the reviewers were fully addressed, as detailed below:

 

Response to Reviewer 1 Comments

 Point 1: This is a well written manuscript describing novel genotypes of coxsackievirus B5 that were circulating in Brazil from 2005 to 2018. The authors focus on the VP1 protein of CVB5 and find that two novel genotypes were predominant during the time period assessed. These can be traced back to virus outbreaks in Haiti, France and Turkey. This manuscript will be of broad interest to those tracking CVB5 evolution from both an epidemiologic and basic virus biology standpoint. I have very few criticisms of the manuscript as written.

The overall fitness of CVB5 virus can most likely be linked to multiple proteins generated by the virus. I think it would be helpful to show how other viral proteins have evolved in these isolates. Understanding how other capsid proteins or non-capsid proteins are altered over time would help to put into context the changes observed in VP1. 

Virus replication in picornaviruses is known to be error prone. Additional discussion on the impact of low fidelity replication would help to improve this study. Not only in the context of the genotypes discovered but also in the techniques used to propagate the virus. The generation of the virus supernatants used for analysis were amplified in multiple cells lines. It is possible that amplification of these viruses in different cell lines alone could alter the genome sequence and lead to the identification of novel sequences that were not due to circulation in human populations. Providing information on what cell lines were used to propagate each of the viruses or discussing any potential impact would improve this.

Response 1: Dear reviewer, we are grateful for your suggestions. The dynamic changes occurring in the genome of the enteroviruses, consequence of cumulative mutations and/or recombination events have led to the emergence of strains with distinct fitness/virulence, and with unpredictable pathogenicity and clinically unknown outcomes. Indeed, these changes are not limited to structural proteins like VP1 (the main capsid protein), but also to non-structural proteins that have a significant impact on fitness and virulence (Ang et al., 2021). Analysis involving other proteins from Brazilian isolates, besides VP1 are in progress for a better understanding of the changes observed and their biological impact on viral fitness and genetic diversity.

Regarding the virus replication, as a Regional Reference Centre for Enteroviruses of the Brazilian Ministry of Health, our investigation was conducted in compliance with the scope of the Global Enterovirus Surveillance Program, as part of the WHO and National Public Health responses in support to the Polio Eradication Initiatives. In this regard, the clinical specimens received from patients with clinical illness indicative of acute flaccid paralysis (AFP) or meningitis should be inoculated in both cell lines, RD and L20B cell lines or RD cell line, respectively (WHO., 2015; Ramalho et al., 2019; Sousa et al., 2020). In order to increase the isolation efficiency, we also used the Hep2C cell line. As a result, the viral supernatants used for investigation were not amplified in numerous cell lines, but rather in a single cell line that showed a cytopathic effect, and the supernatants were harvested and kept until typing.

Despite that, we cannot disregard the possibility that the impact of the isolating in cell cultures alter the genome sequence and lead to the identification of novel sequences that were not due to circulation in human populations. In fact, RNA viruses typically encode their own RNA-dependent RNA polymerase enzyme that has low fidelity of replication and absence of proofreading. This combination can result in high mutation/substitution frequencies, allowing viruses to adapt quickly to host cells. Analysis carried out through the Bayesian coalescent molecular clock revealed that the inferred substitution enterovirus rates were in the range of 0.41x10^-2 to 3.07x10^-2 substitutions per site per year (Lukashev & Vakulenko, 2017). In the specific case of CVB5, this rate was 0.77x10^-2. As a result, we believe that the substitution observed in Brazilian CVB5 isolates, can be due to circulation in the population. Anyway, this issue was addressed into discussion section (lines 363-366).

Point 2: In Figure 1 it is not clear what the color scale is denoting. There are no instances of samples being yellow. The scale should be adjusted to reflect potential differences between samples.

Response 2: This point was addressed as suggested and the respective legend was also adjusted as:

Figure 1. Phylogenetic reconstruction of CVB5 VP1 gene (849 bp), based on Brazilian isolates from meningitis and acute flaccid paralysis cases (represented in blue) and their closely related representative genotypes (colored) and sequences. In the temporal maximum clade credibility (MCC) tree, the posterior probabilities are shown in color and size scale – the node circle size is proportional to posterior probability support. The strain name, year of sampling and GenBank accession numbers are also presented. 

 

Point 3: For Figure 2, include specific text in the legend describing/denoting what the red bars represent.

Response 3: This point was addressed as suggested

 

Point 4:  In Figure 4, it is difficult to discriminate the 3 different green colors that the authors used to specify VP1, VP2 and VP3. Please change these to provide higher contrast. 

Response 4: This point was addressed as suggested

References:

Ang, P.Y.; Chong, C.W.H.; Alonso, S. Viral determinants that drive Enterovirus-A71 fitness and virulence. Emerg Microbes Infect. 2021, 10(1), 713-724.

WHO. Enterovirus Surveillance Guidelines – guidelines for enterovirus surveillance in support of the polio eradication. Regional Office for Europe: World Health Organization; 2015. p. 2015.

Ramalho, E.; Sousa I.Jr.; Burlandy, F., et al. Identification and Phylogenetic Characterization of Human Enteroviruses Isolated from Cases of Aseptic Meningitis in Brazil, 2013-2017. Viruses. 2019, 11, 690.

Sousa, I.P.Jr.; Oliveira, M.L.A.; Burlandy, F.M.; Machado, R.S.; Oliveira, S.S.; Tavare,s F.N.; Gomes-Neto. F.; da Costa, E.V.; da Silva, E.E. Molecular characterization and epidemiological aspects of non-polio enteroviruses isolated from acute flaccid paralysis in Brazil: a historical series (2005-2017). Emerg Microbes Infect. 2020, 9, 2536-2546.

Lukashev AN, Vakulenko YA. Molecular evolution of types in non-polio enteroviruses. J Gen Virol. 2017 Dec;98(12):2968-2981. 

Author Response File: Author Response.pdf

Reviewer 2 Report

v    Suggestions to Author/s 1.Dear Dr. Ivanildo P. de Suosa Jr., as a selected reviewer, I made the prompt check of your article: “Analysis of Coxsackievirus B5 infections in the central nervous system in Brazil: Insights into molecular epidemiology and ge netic diversity.” and found it to be excellent, acceptable for publication. 2. I have to congratulate you for the really excellent scientific article! 3. During the prompt check of your article, the following smaller typing mistakes were found. You are kindly asked to correct them as follows: Line No.:     Correction: add / remove  21             Delete: we;   Add: authors;  27             Add: an;  29             Delete: using; Add: by;  44             Delete: now;  45             Delete: have been; Add: were;  58             Delete: have been; Add: were;  291           Delete: have been; Add: were;  292           Delete: have been; Add: were;  293           Delete: we; Add: were;  307           Delete: have been; Add: were;  309           Delete: belonging; Add: belongs;  313           Delete: we;  Add: authors;  314           Add: it was;  321           Delete: our; Add: The authors;  323           Add: authors;  361           Delete: We; Add: The authors’ ;

 

Comments for author File: Comments.pdf

Author Response

Response to Reviewer 2 Comments

 

Point 1: .Dear Dr. Ivanildo P. de Suosa Jr., as a selected reviewer, I made the prompt check of your article: “Analysis of Coxsackievirus B5 infections in the central nervous system in Brazil: Insights into molecular epidemiology and ge netic diversity.” and found it to be excellent, acceptable for publication.

2. I have to congratulate you for the really excellent scientific article!
3. During the prompt check of your article, the following smaller typing mistakes were found.

You are kindly asked to correct them as follows:

 Line No.:     Correction: add / remove

 21             Delete: we;   Add: authors;

 27             Add: an;

 29             Delete: using; Add: by;

 44             Delete: now;

 45             Delete: have been; Add: were;

 58             Delete: have been; Add: were;

 291           Delete: have been; Add: were;

 292           Delete: have been; Add: were;

 293           Delete: we; Add: were;

 307           Delete: have been; Add: were;

 309           Delete: belonging; Add: belongs;

 313           Delete: we;  Add: authors;

 314           Add: it was;

 321           Delete: our; Add: The authors;

 323           Add: authors;

 361           Delete: We; Add: The authors’ ;

  

Response 1: Dear reviewer, we are grateful for your suggestions. We tried to address all points as suggested.

Author Response File: Author Response.pdf