Two Cases of Natural Infection of Dengue-2 Virus in Bats in the Colombian Caribbean

Round 1

Reviewer 1 Report

The authors perform an interesting study examining the presence of dengue in bats. There have been numerous studies that have detected genetic material of dengue in bats or evidence of exposure (seroconversion). There has yet to be a successful dengue isolation from bats and the major questions are how are they contributing to transmission cycles as potential alternative reservoirs and what vectors are contributing to transmission. The authors do an excellent job revealing the extent of dengue genomes and presence across a wide variety of tissue types in two individual bats. This paper is suitable for publication, however, there are major revisions required before it can be accepted.

Major revisions

• This paper should be read by a native English speaker. There are several grammar errors and in several instances the language can be more concise. This is not a criticism of the authors, but the draft would flow better with another external reviewer.
• In the discussion, the authors need to spend more time examining the likelihood of bats as a reservoir for dengue. There are several short paragraphs that need to be combined. They discuss the extent of the pathology (which should start the discussion section and is well done and relevant). The end of the discussion section should be focused on how bats are contributing to the maintenance of dengue viruses. In my opinion, these findings represent spillover to dead end hosts by nature of the low infection rate and the catholic feeding behaviour of Aedes albopictus (the most likely sylvatic vector). They shouls also mention what other bat species dengue has been detected in and if it has been found before in the two species in this study. The mention of the bat fly is interesting, but it is important to explain what it means (exposed to circulating dengue virus from haemotophagy, but likely not acting as vectors) and the difficulty incriminating vectors in volant animals (with the exception of blood meal analysis – which has been performed before. Please look at Dr. Rebekah Kent’s work on the involvement of bats in arboviruses in Africa. These would be pertinent to cite/reference.
• The phylogeny needs to be re-run. I do not see any software used to determine the best model for the alignment. ModelFinder is a good tool to use (http://www.iqtree.org/ModelFinder/). Additionally, there are much better approaches to reconstruct phylogenies than UPGMA. This method assumes constant evolution and often poorly performs at determining relationships between sequences. Please use a maximum likelihood approach.
• I am curious why the authors did not mention the collection of blood/sera. Detecting antibodies with an ELISA would demonstrate that the virus is eliciting an immune response from the bats. Better yet would be to test the blood for dengue virus because circulating viremia would indicate that these individual bats were potentially contributing to infecting vector species. Additionally, a qPCR assay would allow for quantification of virus copies per weight of tissue, demonstrating tropism and intensity of infection, however I am unsure if this is available to the authors.

 

Minor revisions

Please note that this is not an all-inclusive list of recommended changes.

Abstract

Line 20: evidence ‘of’ dengue virus

Line 22: A total of 286 bats were captured and euthanized following the ethical protocols of animal experimentation.

Line 23: Duplicate samples of brain, lung spleen, liver, and kidney were collected, with one set stored in Trizol and the other stored in 10% buffered formalin…

Line 31: amplicons were not detected, dengue virus was detected

Line 32: In bats RT-PCR positive for dengue, lesions compatible with viral infections…

 

Introduction

There is no talk about the burden of dengue in Latin America. This would be important to include.

Line 41: I don’t think the 70% of bats are insectivorous is necessary to state

Line 45: Please cite primary references regarding the immune responses of bats to viral infections or if you choose to select a review article, use one that focuses on this topic such as ‘Novel insights in immune systems of bats’ – doi: 10.3389/fimmu.2020.00026

The paragraph from lines 46-59 needs to be re-written. It starts out with emerging infectious diseases, but does not connect that to dengue and then discusses dengue. It feels like there is no theme to that paragraph.

 

Methods

Line 72-73: Bats were initially sedate with an intramuscular injection of atropine (0.11mg/kg) after which they were euthanized with an intracardiac injection of sodium pentobarbitol (0.2ml).

Line 79-80: The RNA was extracted with Trizol and cDNA was synthesized using a reverse…

Line 87-80: were the PCR products sequenced in both forward and reverse directions?

2.3 Phylogenetic analysis:

-what reference sequences were used (are these available in a supplementary table)?

-Please provide a citation for MEGA

-Use model finder on alignment to find the most appropriate model

-Please re-run with a maximum likelihood approach

-were the primers removed from the sequences and were they manually inspected for quality?

-how long was the alignment (base pairs)

-were dengue sequences from bats in other countries used in the alignment. If these are available, I would highly recommend the authors include them in the alignment.

Line 100-101: The microtime was not stained, the tissues were stained. “Four uM thick slices of tissues were cut and stained with hematoxylin-eosin…”

 

Results

The authors need to provide a table of all the bats and species that were collected so we know the prevalence by species.

Line 121: “Dengue was detected in two individual bats, one Carrolia perspicillata and one Phylostomus discolor…” Please note, at the beginning of each section of the manuscript, the full scientific name should be spelled out and not abbreviated.

Figure 2 legend: This needs to be more detailed. The authors should include the method used to make the tree and how many bootstrap replicates were performed and what the minimum bootstrap value shown is. If using an ML phylogenetic reconstruction, only show bootstraps greater than 70

 

Discussion

This is covered in detail in the major revision section. Please state your findings in the first paragraph, then have the pathology findings, and then talk about the implication of dengue in bats (other species, vectors, are they dead end hosts).

Line 195: These findings suggest viral replication of dengue virus in the bat species, however they do not demonstrate viral replication.

Author Response

Minor revisions

Abstract

Line 20: evidence ‘of’ dengue virus. Done.

 

Line 22: A total of 286 bats were captured and euthanized following the ethical protocols of animal experimentation. Done (new line 24).

 

Line 23: Duplicate samples of brain, lung spleen, liver, and kidney were collected, with one set stored in Trizol and the other stored in 10% buffered formalin… Done.

 

Line 31: amplicons were not detected. Done dengue virus was detected.

 

Line 32: In bats RT-PCR positive for dengue, lesions compatible with viral infections Done.

 

“I am curious why the authors did not mention the collection of blood/sera. Detecting antibodies with an ELISA would demonstrate that the virus is eliciting an immune response from the bats. Better yet would be to test the blood for dengue virus because circulating viremia would indicate that these individual bats were potentially contributing to infecting vector species. Additionally, a qPCR assay would allow for quantification of virus copies per weight of tissue, demonstrating tropism and intensity of infection, however I am unsure if this is available to the authors” Appraisers request 2.

 

In the limitations of the present work, we add: Therefore, the sequences detected in the present study are short. Additionally, blood samples for serological assays were not collected.

 

Introduction

There is no talk about the burden of dengue in Latin America. This would be important to include.

 

Line 41: I don’t think the 70% of bats are insectivorous is necessary to state. Delete

Line 45: Please cite primary references regarding the immune responses of bats to viral infections or if you choose to select a review article, use one that focuses on this topic such as ‘Novel insights in immune systems of bats’ – doi: 10.3389/fimmu.2020.00026

 

The paragraph from lines 46-59 needs to be re-written. It starts out with emerging infectious diseases but does not connect that to dengue and then discusses dengue. It feels like there is no theme to that paragraph. Done

 

Methods

Line 72-73: Bats were initially sedate with an intramuscular injection of atropine (0.11mg/kg) after which they were euthanized with an intracardiac injection of sodium pentobarbital (0.2ml).

Done

 

Line 79-80: The RNA was extracted with Trizol and cDNA was synthesized using a reverse…

Done

 

Line 87-80: were the PCR products sequenced in both forward and reverse directions?

Done

 

2.3 Phylogenetic analysis:

-what reference sequences were used (are these available in a supplementary table)?

Check Phylogenetic Analysis (line112-117). Done.

 

Please provide a citation for MEGA. Check Reference 22 was included. Done.

 

Line 100-101: The microtime was not stained, the tissues were stained. “Four uM thick slices of tissues were cut and stained with hematoxylin-eosin…”   Add. Done.

 

Results

The authors need to provide a table of all the bats and species that were collected so we know the prevalence by species.  Done: table was attached

 

Line 121: “Dengue was detected in two individual bats, one Carollia perspicillata and one Phylostomus discolor…” Please note, at the beginning of each section of the manuscript, the full scientific name should be spelled out and not abbreviated.  Done

 

Figure 2 legend: This needs to be more detailed. The authors should include the method used to make the tree and how many bootstrap replicates were performed and what the minimum bootstrap value shown is. If using an ML phylogenetic reconstruction, only show bootstraps greater than 70.  Check Phylogenetic Analysis (line112-117). Done.

 

Discussion

This is covered in detail in the major revision section. Please state your findings in the first paragraph, then have the pathology findings, and then talk about the implication of dengue in bats (other species, vectors, are they dead end hosts). Check Phylogenetic line

 

Line 195: These findings suggest viral replication of dengue virus in the bat species, however they do not demonstrate viral replication.

Done

Reviewer 2 Report

Major comments:

I would like to thank the authors for submitting the manuscript on DENV natural infection in bats in the Columbian Caribbean. While the topic is an important one, the paper has certain limitation. It should be made clear that this is a case report of 2 bats positive for DENV (Title might be misleading). The mention about 286 bats is not relevant for abstract – the authors might mention that in the methods. The abstract also contain details about methodology but the actual figures fail to show all the controls. The authors might want to shorten the methodology in abstract and put relevant control figures in supplementary. In the end of introduction, the authors should clearly mention that this is a study showing 2 PCR positive bats and the pathology witnessed within those bats.

Did the authors do PCR for 6 tissue types from each of the 286 bats – a total of 1716 PCRs, and only samples from 2 bats were positive? If it is so, they should mention the numbers clearly in results section.

For phylogenetic tree, I would suggest a more statistically robust tree i.e. using maximum likelihood tree (with bootstrapping) instead of UPGMA.

It would be crucial to show negative controls/uninfected bat tissue along with the positive immunohistochemistry images (this might be added as supplementary as well).

Discussion (line 176): It is not relevant here to describe why mice inoculation doesn’t show pathological findings as it is not a natural infection. I agree with the authors. However, it would make sense if the authors were to show mice images in the figures and compare with bat tissues.

Bats are known to show minimal pathology towards deadly pathogens (with a few exceptions). It would be interesting for the manuscript to discuss about this in the discussion section.

 

Minor comments:

Line23: mention which pharmacological treatment?

Line 25: change to viral (instead of virus’s)

Line 54: reference?

Line 81: Primers used – the authors might want to consider mentioning the sequences of primers here along with the citation for the same.

Figure 1: No labels for ladder (Lane 10)

Line 181: The reference #37,38 are not clear here?

 

Author Response

 Title.

The words "Two Cases" were added, at the suggestion of the reviewer 2.

 

Minor revisions

Abstract

Minor comments:

 

Line23: mention which pharmacological treatment? Added: with atropine, acepromazine and sodium pentobarbital   

 

Line 25: change to viral (instead of virus’s)

The wording was modified at the suggestion of the reviewer 1. Done

 

Introduction

Line 54: reference? Added: Reference

Gutiérrez, R.L.; Quintero, G.D.C.; Martínez, G.M. Actualización en diagnóstico del dengue: evolución de las técnicas y su aplicación real en la clínica. Medicina & Laboratorio. 2012,18, 9-10, 411-441.

 

Line 81: Primers used – the authors might want to consider mentioning the sequences of primers here along with the citation for the same. Added: 

 

The sequence of the primers referenced in item 16 are shown in the tree. Check Phylogenetic tree.

 

Figure 1: No labels for ladder (Lane 10). Added: Molecular weight marker (lane 10)

 

Line 181: The reference #37,38 are not clear here?. Clear and relevant references. Now are:38, 39. 

 

 

CHANGES IN REFERENCES.

 

1. Ciota. The Role of temperature in transmission of zoonotic arboviruses. Viruses 2019; 11. New reference.

 

2. OPS. Health Information Platform for the Americas. New reference. Because Kunz's reference was deleted.

 

3. SIVIGILA. New reference

 

4. Jones. Global trends in emerging infectious diseases. 2008. Position change now 4, before 7.

 

5. Woolhouse. Host range and emerging and reemerging pathogens. 2005. Position change now 5, before 8.

 

6. Simmonds, P…… ICTV virus taxonomy profile: Flaviviridae.2017. Position change now 6, before 9.

 

7. Feitoza. Dengue infection during pregnancy and adverse maternal, fetal, and infant health outcomes in Rio Branco, Acre State, Brazil, 2007-2012. 2017. Position change now 7, before 10.

 

8. Mattar. Undifferentiated tropical febrile illness in Cordoba, Colombia: Not everything is dengue. 2017. Position change now 7, before 11.

 

9. Gutiérrez. Actualización en diagnóstico del dengue: evolución de las técnicas y su aplicación real en la clínica. 2012. New reference. It was forgotten in the previous shipment.

 

10. 10. Molecular detection and typing of dengue viruses from archived tissues of fatal cases by RT-PCR and Sequencing: Diagnostic and Epidemiologic Implications. 2012. Position change now 10, before 12.

 

11 Ospina. Dengue: Diagnóstico por el laboratorio. 2004. Position change now 11, before 13.

 

12. Jones. The importance of bats as bioindicators. 2009. Position change now 12, before 4.

 

13.. Sánchez-Casas, Palinofagia de los murciélagos del género Glossophaga (Mammalia: Chiroptera) en Mexico. 2000.   Position change now 13, before 16.

 

14. Calderón. Viral Zoonoses that fly with Bats: A 2016. Position change now 14, before 18.

 

15. Banerjee. Novel insights into immune. 2020. New reference. By suggestion of reviewer 1.

 

16. O'Shea. Bat flight and zoonotic viruses. 2014. Position change now 16, before 5.

 

17. Mattar, S.; González, M. The amazing bats: friends, enemies or allies? 2016. Position change now 17, before 6.

 

18. Alvis. Dengue in an area of the Colombian Caribbean, 2003-2010. 2015. Position change now 18, before 14.

 

19. Linares. Mamíferos de Venezuela. Sociedad Conservacionista Audubon de Venezuela, 1998.

Position change now 19, before 15.

 

20. Sánchez. RT-nested-PCR for detection of flaviviruses using degenerated primers and internal

control followed by sequencing for specific identification.  2005. Position change now 20, before

16.

 

21. Ramírez. Pathogenic leptospires in renal tissue of bats from Corrientes, Argentina. 2014.

Position change now 21, before 17.

 

22. Kumar S. MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Mol Biology Evo. 2018. New reference new reference suggested by the reviewer.

 

23. Calderón. Dengue Virus in Bats from Córdoba and Sucre, Colombia. 2019. Position change now 24, before 18.

 

24. Platt. Detection of dengue virus neutralizing antibodies in bats from Costa Rica and Ecuador. 2000. Position change now 24, before 19.

 

25. Aguilar-Setien. Dengue virus in Mexican bats. 2008. Position change now 25, before 20.

 

26. Machain-Williams. Serologic evidence of flavivirus infection in bats in the Yucatan Peninsula of Mexico. 2013. Position change now 26, before 21.

,

27. Sotomayor-Bonilla, J… Dengue virus in bats from southeastern Mexico. 2014. Position change

now 27, before 22.

 

28. Vicente-Santos. Neotropical bats that cohabit with humans function as dead-end hosts for

dengue virus. 2017. Position change now 28, before 23.

    

29. Abundes-Gallegos. Detection of dengue virus in bat flies (Diptera: Streblidae) of common

vampire bats, Desmodus rotundus, in Progreso, Hidalgo, Mexico. 2018. Position change now 29,

before 24.

 

30. Lavergne, A…. Dengue virus infection in neotropical forest mammals: incidental hosts or

potential reservoirs? Incidental host or potencial reservoirs? 2009. Position change now 30, before

25.  

 

31. Amorim. A genetic and pathologic study of a DENV2 clinical isolate capable of inducing

encephalitis and hematological disturbances in immunocompetent mice. 2012. Position change

now 31, before 30.

 

32. Solomon. Dengue and other emerging flaviviruses. 2001. Position change now 32, before 31.

 

33. Saraya. utoimmune causes of encephalitis syndrome in Thailand: Prospective study of 103

patients. 2013. Position change now 33, before 32.

 

 

34. Póvoa. The Pathology of severe dengue in multiple organs of human fatal cases: Histopathology,

ultrastructure and virus replication. 2014. Position change now 34, before 33.

 

35. Rodrigues. Lung in dengue: Computed tomography findings. Position change now 35, before 34.

 

36. Basílio-de-Oliveira. Pathologic study of a fatal case of dengue-3 virus infection in Rio de Janeiro, Br1azil. 2005. Position change now 36, before 35.

 

37. Kularatne. Extensive haemorrhagic necrosis of liver is an unpredictable fatal complication indengue infection: a postmortem study. 2014. Position change now 37, before 36.

 

38. Gonçalves. Imunocompetent mice model for dengue virus infection. 2015. Position change now

38, before 37.

 

39. Kuruvilla. Dengue virus infection and immune response in humanized RAG2(-/-) gamma(c)(-/-)

(RAG-hu) mice. 2017. Position change now 39, before 38.

 

40. An. The pathogenesis of spinal cord involvement in dengue virus infection. 2003. Position

change now 40, before 39

 

41. Desprès. Human isolates of dengue type 1 virus induce apoptosis in mouse neuroblastoma cells.
42. Position change now 41, before 40.

 

42. Samanta. Dengue and its effects on liver. 2015. Position change now 42, before 41.

 

43. Huerre. Liver histopathology and biological correlates in five cases of fatal dengue fever in

Vietnamese children. 2001.  Position change now 43, before 42.

 

44. Lin. Liver injury caused by antibodies against dengue virus nonstructural protein 1 in a murine model. 2008. Position change now 44, before 43.

 

45. Seneviratne. Pathogenesis of liver involvement during dengue viral infections. 2006. Position

change now 45, before 44.

 

46. Lee. Autophagic machinery activated by dengue virus enhances virus replication. 2008. Position

change now 46, before 43.

  

47. Fernando. Patterns and causes of liver involvement in acute dengue infection. 2016. Position

change now 47, before 46.

 

48. Perera-Lecoin. Flavivirus entry receptors: An Update. 2013. Position change now 48, before 47.

  

49. Couvelard. Report of a fatal case of dengue infection with hepatitis: demonstration of dengue

antigens in hepatocytes and liver apoptosis. 1999. Position change now 49, before 48.

 

50. Jessie. Localization of dengue virus in naturally infected human tissues, by immunohistochemistry and in situ hybridization. 2004. Position change now 50, before 49.

 

51. Rathi. Autopsy findings in fatal dengue haemorrhagic fever-06 cases. 2013. Position change

now 51, before 50.

 

52. Boonpucknavig. Glomerular changes in dengue hemorrhagic fever. 1976. Position change now

52, before 51.

 

53. De Brito. Human fatal yellow fever. Immunohistochemical detection of viral antigen in the liver, kidney and heart. 1992. Position change now 53, before 52.

 

54. Halstead. Studies on the pathogenesis of dengue infection in monkeys. II. Clinical laboratory responses to heterologous infection. 1973. Position change now 54, before 53.

 

55. Yauch. Mouse models of dengue virus infection and disease. 2008. Position change now 55,

before 54.  

 

56. De Figueiredo. Mosquitoes infected with dengue viruses in Brazil. 2010. Position change now 56, before 26.

 

57. Jaramillo. Sentinel suveillance for West Nile Virus in culicides and domestic birds in Cordoba.
58. Position change now 57, before 27.

 

58. Parra-Henao. Vectores potenciales de arbovirus en la región de Urabá, noroccidente de

Colombia. 2012. Position change now 58, before 28.

 

59. Hoyos-López. Molecular detection of flaviviruses and alphaviruses in mosquitoes (Diptera: Culicidae) from coastal ecosystems in the Colombian Caribbean. 2016. Position change now

59, before 29.

 

60. Perea-Martínez. Experimental infection of Artibeus intermedius bats with serotype-2 dengue

virus. 2013. New reference.

 

61. Shah. Attempts at experimental infection of the Indian fruit-bat Pteropus giganteus with chikungunya and dengue 2 viruses and antibody survey of bat sera for some viruses. 1966. New reference.

 

62. Reagan. Studies of dengue fever virus in the cave bat (Myotus lucifugus). 1952. New reference.

 

63. Cabrera-Romo. Experimental inoculation of Artibeus jamaicensis bats with dengue virus serotypes 1 or 4 showed no evidence of sustained replication. 2014. New reference.

 

64. Kading. Neutralizing antibodies against flaviviruses, Babanki virus, and Rift Valley fever virus in Ugandan bats. 2018. New reference.

 

65. Fagre. Can Bats Serve as Reservoirs for Arboviruses? 2019. New reference. By suggestion of reviewer 1.

Round 2

Reviewer 1 Report

The authors have adequately addressed the previous comments, but a few minor issues needs edited.

Line 47: The incidence of dengue in Colombia was 255 per 100,000 individuals [3].

Line 82: sedated and not sedate

Line 114: list how many reference sequences were in the alignment

Line 141: ...23 species in six families were caught (Table 1).

Line 144: why is the text bold

Lines 232-240: Please combine the two paragraphs into one about the mosquitoes.

Line 243-244: Megachiroptera and microchiroptera are not used any more as there are taxonomically incorrect (fruit bats and horseshoe bats share a common ancestor). Use Yinpterochiroptera or Yangochiroptera

 

 

 

 

 

Author Response

The authors have adequately addressed the previous comments, but a few minor issues needs edited.

Line 47: The incidence of dengue in Colombia was 255 per 100,000 individuals [3].

Done.

Line 80-82: sedated and not sedate

Done.

Line 114: list how many reference sequences were in the alignment.

Done. Alignment was carried out with thirty-six sequences for different types of DENV.

Line 140: ...23 species in six families were caught (Table 1).

Done. We did the correction.

Line 143: why is the text bold

Done.

Lines 237-243: Please combine the two paragraphs into one about the mosquitoes.

Done. The two paragraphs were combined into one.

Line 247-248: Megachiroptera and microchiroptera are not used any more as there are taxonomically incorrect (fruit bats and horseshoe bats share a common ancestor). Use Yinpterochiroptera or Yangochiroptera.

Done. Corrections were done.

Reviewer 2 Report

I would like to thank the authors for addressing the comments from first revision. However, there are a few more comments:

• The previous comment about including negative control images of Immunohistochemistry has not been addressed.
• Line 212 is not clear to me.
•  

Author Response

I would like to thank the authors for addressing the comments from first revision. However, there are a few more comments:

The previous comment about including negative control images of Immunohistochemistry has not been addressed.

Done. A photography of mice and bats negative controls for immunohistochemistry.

Line 212 is not clear to me.

Done. Line 217-218