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# A new cell line to decipher Wolbachia-arbovirus interactions

Vincent Raquin, Claire Valiente Moro, Patrick Mavingui

<figure><img src="image_2.png" /></figure>

## To cite this version:

Vincent Raquin, Claire Valiente Moro, Patrick Mavingui. A new cell line to decipher Wolbachia-arbovirus interactions. 7th international Wolbachia conference, Dec 2012, Oléron, France. hal-04569508

HAL Id: hal-04569508

https://hal.science/hal-04569508

Submitted on 6 May 2024

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Distributed under a Creative Commons Attribution 4.0 International License
# A new cell line to decipher Wolbachia-arbovirus interactions

Vincent Raquin, Claire Valiente Moro, Patrick Mavingui

► To cite this version:

Vincent Raquin, Claire Valiente Moro, Patrick Mavingui. A new cell line to decipher Wolbachia-arbovirus interactions. 7th international Wolbachia conference, Dec 2012, Oléron, France. hal-04569508

HAL Id: hal-04569508

https://hal.science/hal-04569508v1

Submitted on 6 May 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_4.png" /></figure>

# A new mosquito cell line to decipher
## Wolbachia-arbovirus interactions

Vincent RAQUIN¹, Claire VALIENTE MORO¹, Patrick MAVINGUI¹

¹Université de Lyon, Lyon France; Université Lyon 1, Villeurbanne, CNRS, UMR 5557, Ecologie Microbienne, Lyon, France

<figure><img src="image_5.png" /></figure>

web

## INTRODUCTION

Endosymbiosis is a particular form of association between organisms in which one partner, generally a prokaryot, lives inside the body of its eukaryotic host. It has been estimated that 15 to 20% of insect taxa harbour endosymbionts [1], and some of them cannot be cultured on common laboratory media. *Wolbachia* is an obligate intracellular alphaproteobacterium of the *Anaplasmataceae* family, very prevalent among insects including vectors of human and animal diseases. The mosquito *Aedes albopictus*, a vector of dengue and chikungunya viruses, is naturally infected by two *Wolbachia* strains designated *wAlbA* and *wAlbB*. Recent work showed that *Wolbachia* can reduce dengue virus replication/dissemination in its natural host *Aedes albopictus*, but the mechanisms involved remain unclear [2]. Mosquito cell lines can be useful models to explore such mechanisms. Here we report the establishment of *Ae. albopictus* C6/36 cell line with a persistent and high density infection by *Wolbachia* *wAlbB* strain.

## RESULTS

<figure><img src="image_6.png" /><figcaption>Figure 1. Infection dynamic of wAlbB in C6/36 cells over 9 months of culture</figcaption></figure>

Ratio of *wsp/actin* gene copy numbers by quantitative PCR using DNA template of infected cells. (N=3)

*Wolbachia* density varies over passages. High infection rate per host cell (i.e. 70 *wsp* copy per cell) was reached at passage 17 then a continue decline was observed until stabilization at passage 36 (12 *wsp* copy per cell). PCR amplifications on DNA extracted from tetracycline treated cells were negative.

<figure><img src="image_7.png" /><figcaption>(a) Low-magnification micrograph showing a C6/36 cell infected by a large number of *Wolbachia* (arrowheads), dispersed throughout the cytoplasm. N, nucleus. (b) High-magnification micrograph of two bacteria presumably undergoing cell division, close to the membrane of the host cell. W, *Wolbachia*. (c) A *Wolbachia* cell in a large vacuole, surrounded by smaller ones, which could undergo a process of lysis. (d) Low-magnification micrograph of C6/36_wAlbB cell treated with tetracycline, used as control.</figcaption></figure>

## Conclusion

The infection of C6/36 cell line by *wAlbB* was achieved, with a high bacterial density over months. *Wolbachia* was detected in almost 100% of cells, however its density variate among cells and passages. No *Wolbachia* signal was detected in any controls, suggesting that signals come from living bacteria. As C6/36 was not initially infected by *Wolbachia*, it can be used to infect and compare different *Wolbachia* strains under a common genetic background. In addition, the cell line permissivity to a broad range of arboviruses allow multiple infection studies.

## References

[1]: Buchner, P., 1965. Endosymbiosis of Animals with Plant Microorganisms. Interscience Publishers, New York.

[2]: Blagrove, MS., Arias-Goeta, C., Failloux, A.-B., Sinkins SP, 2012. *Wolbachia* strain *wMel* induces cytoplasmic incompatibility and blocks dengue transmission in *Aedes albopictus*. PNAS Jan 3;109(1)

**Acknowledgments:** VR was funded by a PhD fellowship from French Ministère de l'Enseignement Supérieur et de la Recherche. We would like to thank Nelly Burlet (UMR 5558, Lyon, France) for helpful advices in epifluorescence microscopy. This work was funded by ANR-2010-BLAN-170101 ImmunSymbArt and was carried out under the framework of GDRI « Biodiversity and Infectious Diseases in VietNam » of COST Action FA0701.

## METHODS | Development of C6/36_wAlbB cell line

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /><figcaption>Figure 2. Fluorescent *in situ* hybridization on C6/36_wAlbB cells</figcaption></figure>

*Wolbachia* is shown in **red** using rhodamine-labelled 16S rRNA probes, and **cell nuclei** are labelled in **blue** with DAPI. Cells were visualized under an epifluorescence microscope using 63X objective. Bar = 20 µm

From P15 to P28, almost 100% of cells are infected, some of them at a high density. At P36, the number of infected cells and the density per cell decrease. Antibiotic treated cells were used as control (CTRL).

Pictures = Centre Technologique des Microstructures (Lyon 1)

<figure><img src="image_10.png" /></figure>
<figure><img src="image_2.png" /></figure>

# A new mosquito cell line to decipher
## Wolbachia-arbovirus interactions

Vincent RAQUIN¹, Claire VALIENTE MORO¹, Patrick MAVINGUI¹

¹Université de Lyon, Lyon France; Université Lyon 1, Villeurbanne, CNRS, UMR 5557, Ecologie Microbienne, Lyon, France

<figure><img src="image_3.png" /></figure>

web

## INTRODUCTION

Endosymbiosis is a particular form of association between organisms in which one partner, generally a prokaryot, lives inside the body of its eukaryotic host. It has been estimated that 15 to 20% of insect taxa harbour endosymbionts [1], and some of them cannot be cultured on common laboratory media. *Wolbachia* is an obligate intracellular alphaproteobacterium of the *Anaplasmataceae* family, very prevalent among insects including vectors of human and animal diseases. The mosquito *Aedes albopictus*, a vector of dengue and chikungunya viruses, is naturally infected by two *Wolbachia* strains designated *wAlbA* and *wAlbB*. Recent work showed that *Wolbachia* can reduce dengue virus replication/dissemination in its natural host *Aedes albopictus*, but the mechanisms involved remain unclear [2]. Mosquito cell lines can be useful models to explore such mechanisms. Here we report the establishment of *Ae. albopictus* C6/36 cell line with a persistent and high density infection by *Wolbachia* *wAlbB* strain.

## RESULTS

<figure><img src="image_4.png" /><figcaption>**Figure 1.** Infection dynamic of *wAlbB* in C6/36 cells over 9 months of culture</figcaption></figure>

Ratio of *wsp/actin* gene copy numbers by quantitative PCR using DNA template of infected cells. (N=3)

*Wolbachia* density varies over passages. High infection rate per host cell (i.e. 70 *wsp* copy per cell) was reached at passage 17 then a continue decline was observed until stabilization at passage 36 (12 *wsp* copy per cell). PCR amplifications on DNA extracted from tetracycline treated cells were negative.

<figure><img src="image_5.png" /><figcaption>(a) Low-magnification micrograph showing a C6/36 cell infected by a large number of *Wolbachia* (arrowheads), dispersed throughout the cytoplasm. N, nucleus. (b) High-magnification micrograph of two bacteria presumably undergoing cell division, close to the membrane of the host cell. W, *Wolbachia*. (c) A *Wolbachia* cell in a large vacuole, surrounded by smaller ones, which could undergo a process of lysis. (d) Low-magnification micrograph of C6/36 *wAlbB* cell treated with tetracycline, used as control.</figcaption></figure>

## Conclusion

The infection of C6/36 cell line by *wAlbB* was achieved, with a high bacterial density over months. *Wolbachia* was detected in almost 100% of cells, however its density variate among cells and passages. No *Wolbachia* signal was detected in any controls, suggesting that signals come from living bacteria. As C6/36 was not initially infected by *Wolbachia*, it can be used to infect and compare different *Wolbachia* strains under a common genetic background. In addition, the cell line permissivity to a broad range of arboviruses allow multiple infection studies.

## References

[1]: Buchner, P., 1965. Endosymbiosis of Animals with Plant Microorganisms. Interscience Publishers, New York.

[2]: Blagrove, MS., Arias-Goeta, C., Failloux, A.-B., Sinkins SP, 2012. *Wolbachia* strain *wMel* induces cytoplasmic incompatibility and blocks dengue transmission in *Aedes albopictus*. PNAS Jan 3;109(1)

**Acknowledgments:** VR was funded by a PhD fellowship from French Ministère de l'Enseignement Supérieur et de la Recherche. We would like to thank Nelly Burlet (UMR 5558, Lyon, France) for helpful advices in epifluorescence microscopy. This work was funded by ANR-2010-BLAN-170101 ImmunSymbArt and was carried out under the framework of GDRI « Biodiversity and Infectious Diseases in VietNam » of COST Action FA0701.

## METHODS | Development of C6/36_wAlbB cell line

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /><figcaption>**Figure 2.** Fluorescent *in situ* hybridization on C6/36_wAlbB cells</figcaption></figure>

*Wolbachia* is shown in **red** using rhodamine-labelled 16S rRNA probes, and **cell nuclei** are labelled in **blue** with DAPI. Cells were visualized under an epifluorescence microscope using 63X objective. Bar = 20 µm

From P15 to P28, almost 100% of cells are infected, some of them at a high density. At P36, the number of infected cells and the density per cell decrease. Antibiotic treated cells were used as control (CTRL).

Pictures = Centre Technologique des Microstructures (Lyon 1)

<figure><img src="image_8.png" /></figure>