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Native Wolbachia from Aedes albopictus Blocks Chikungunya Virus Infection In Cellulo.

Raquin V, Valiente Moro C, Saucereau Y, Tran FH, Potier P, Mavingui P - PLoS ONE (2015)

Bottom Line: To better understand the Wolbachia/CHIKV/Ae. albopictus interaction, we generated a cellular model using Ae. albopictus derived C6/36 cells that we infected with the wAlbB strain.Our results indicate that CHIKV infection is negatively impacted at both RNA replication and virus assembly/secretion steps in presence of wAlbB.More broadly, this put into question the ecological role of Wolbachia symbiont in Ae. albopictus, but also the ability of the CHIKV to counteract Wolbachia's antiviral potential in vivo.

View Article: PubMed Central - PubMed

Affiliation: Université de Lyon, UMR5557 Ecologie Microbienne, CNRS, USC1190 INRA, VetAgro Sup, Université Lyon 1, Villeurbanne, France.

ABSTRACT
Wolbachia, a widespread endosymbiont of terrestrial arthropods, can protect its host against viral and parasitic infections, a phenotype called "pathogen blocking". However, in some cases Wolbachia may have no effect or even enhance pathogen infection, depending on the host-Wolbachia-pathogen combination. The tiger mosquito Aedes albopictus is naturally infected by two strains of Wolbachia, wAlbA and wAlbB, and is a competent vector for different arboviruses such as dengue virus (DENV) and chikungunya virus (CHIKV). Interestingly, it was shown in some cases that Ae. albopictus native Wolbachia strains are able to inhibit DENV transmission by limiting viral replication in salivary glands, but no such impact was measured on CHIKV replication in vivo. To better understand the Wolbachia/CHIKV/Ae. albopictus interaction, we generated a cellular model using Ae. albopictus derived C6/36 cells that we infected with the wAlbB strain. Our results indicate that CHIKV infection is negatively impacted at both RNA replication and virus assembly/secretion steps in presence of wAlbB. Using FISH, we observed CHIKV and wAlbB in the same mosquito cells, indicating that the virus is still able to enter the cell in the presence of the bacterium. Further work is needed to decipher molecular pathways involved in Wolbachia-CHIKV interaction at the cellular level, but this cellular model can be a useful tool to study the mechanism behind virus blocking phenotype induced by Wolbachia. More broadly, this put into question the ecological role of Wolbachia symbiont in Ae. albopictus, but also the ability of the CHIKV to counteract Wolbachia's antiviral potential in vivo.

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Proportion of Wolbachia-infected cells detected by Fluorescence in situ Hybridization.Rhodamine-labelled oligonucleotide probe designed on Wolbachia 16S rRNA gene (red) detected the bacteria in the cytoplasm of the host cell at passages P.15 and P.37 (A). Nuclei of the host cells are shown in blue after DAPI labelling (bars = 20μm). Percentage of cells with a Wolbachia-positive signal in FISH at P.15 and P.37 (B) (Error bars represent the standard deviation of the mean of 50 independent microscope fields from three independent samples).
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pone.0125066.g003: Proportion of Wolbachia-infected cells detected by Fluorescence in situ Hybridization.Rhodamine-labelled oligonucleotide probe designed on Wolbachia 16S rRNA gene (red) detected the bacteria in the cytoplasm of the host cell at passages P.15 and P.37 (A). Nuclei of the host cells are shown in blue after DAPI labelling (bars = 20μm). Percentage of cells with a Wolbachia-positive signal in FISH at P.15 and P.37 (B) (Error bars represent the standard deviation of the mean of 50 independent microscope fields from three independent samples).

Mentions: Previous studies mentioned that the wAlbB strain could be maintained in C6/36 [41,42]. Despite this, wAlbB dynamics of infection in C6/36 remains unknown. The wAlbB cells were purified from Aa23 cells, as they were already adapted to cell line culture. The C6/36 cells tend to grow in adhesive cell clusters, forming patchy monolayers independently of Wolbachia infection (S1 Fig). Two attempts were necessary to obtain Wolbachia infected cells, designated C6/36_wAlbB, with a wsp signal in PCR persisting in cells after several passages (not shown). Electron microscopy of C6/36_wAlbB cells (P.30) revealed the presence of Wolbachia as round-shaped particles of varying size inside the cytoplasm, surrounded by a host cell membrane where the bacteria seem to divide (Fig 1). As expected, no Wolbachia was seen outside a cell, while some bacteria could be released after the lysis of their host cell. In C6/36_TET cells, i.e. cells cured from Wolbachia by tetracycline treatment, no difference in cell aspect was noted compared to Wolbachia-infected cells, despite the absence of Wolbachia infection. The C6/36_wAlbB cells were maintained in continuous culture for 40 passages, corresponding to approximately 5 months. Quantitative PCR analysis showed that the density of Wolbachia was highly dynamic according to the passages (Fig 2), with the lowest density of 0.9 wsp/actin ratio at P.7 to 67.6 wsp/actin ratio at P.17 for the highest. After P.17, Wolbachia's density decreased to remain around 10 wsp/actin ratio from P.36 to P.40. The C6/36_TET cells were negative for Wolbachia infection in qPCR. The FISH also confirmed the absence of Wolbachia in C6/36_TET cells whereas the bacteria were detected in C6/36_wAlbB cytoplasm (Fig 3A), even if the infection did not reach 100% of the cultured cells (Fig 3B). Along with the density of bacteria measured in qPCR, the Wolbachia fluorescent signal decreased from P.15 to P.37 and that goes together with a significantly lower proportion of Wolbachia-infected cells from 92.4% to 45.3% at P.15 and P.37, respectively (P <2.2e-16) (Fig 3B).


Native Wolbachia from Aedes albopictus Blocks Chikungunya Virus Infection In Cellulo.

Raquin V, Valiente Moro C, Saucereau Y, Tran FH, Potier P, Mavingui P - PLoS ONE (2015)

Proportion of Wolbachia-infected cells detected by Fluorescence in situ Hybridization.Rhodamine-labelled oligonucleotide probe designed on Wolbachia 16S rRNA gene (red) detected the bacteria in the cytoplasm of the host cell at passages P.15 and P.37 (A). Nuclei of the host cells are shown in blue after DAPI labelling (bars = 20μm). Percentage of cells with a Wolbachia-positive signal in FISH at P.15 and P.37 (B) (Error bars represent the standard deviation of the mean of 50 independent microscope fields from three independent samples).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4414612&req=5

pone.0125066.g003: Proportion of Wolbachia-infected cells detected by Fluorescence in situ Hybridization.Rhodamine-labelled oligonucleotide probe designed on Wolbachia 16S rRNA gene (red) detected the bacteria in the cytoplasm of the host cell at passages P.15 and P.37 (A). Nuclei of the host cells are shown in blue after DAPI labelling (bars = 20μm). Percentage of cells with a Wolbachia-positive signal in FISH at P.15 and P.37 (B) (Error bars represent the standard deviation of the mean of 50 independent microscope fields from three independent samples).
Mentions: Previous studies mentioned that the wAlbB strain could be maintained in C6/36 [41,42]. Despite this, wAlbB dynamics of infection in C6/36 remains unknown. The wAlbB cells were purified from Aa23 cells, as they were already adapted to cell line culture. The C6/36 cells tend to grow in adhesive cell clusters, forming patchy monolayers independently of Wolbachia infection (S1 Fig). Two attempts were necessary to obtain Wolbachia infected cells, designated C6/36_wAlbB, with a wsp signal in PCR persisting in cells after several passages (not shown). Electron microscopy of C6/36_wAlbB cells (P.30) revealed the presence of Wolbachia as round-shaped particles of varying size inside the cytoplasm, surrounded by a host cell membrane where the bacteria seem to divide (Fig 1). As expected, no Wolbachia was seen outside a cell, while some bacteria could be released after the lysis of their host cell. In C6/36_TET cells, i.e. cells cured from Wolbachia by tetracycline treatment, no difference in cell aspect was noted compared to Wolbachia-infected cells, despite the absence of Wolbachia infection. The C6/36_wAlbB cells were maintained in continuous culture for 40 passages, corresponding to approximately 5 months. Quantitative PCR analysis showed that the density of Wolbachia was highly dynamic according to the passages (Fig 2), with the lowest density of 0.9 wsp/actin ratio at P.7 to 67.6 wsp/actin ratio at P.17 for the highest. After P.17, Wolbachia's density decreased to remain around 10 wsp/actin ratio from P.36 to P.40. The C6/36_TET cells were negative for Wolbachia infection in qPCR. The FISH also confirmed the absence of Wolbachia in C6/36_TET cells whereas the bacteria were detected in C6/36_wAlbB cytoplasm (Fig 3A), even if the infection did not reach 100% of the cultured cells (Fig 3B). Along with the density of bacteria measured in qPCR, the Wolbachia fluorescent signal decreased from P.15 to P.37 and that goes together with a significantly lower proportion of Wolbachia-infected cells from 92.4% to 45.3% at P.15 and P.37, respectively (P <2.2e-16) (Fig 3B).

Bottom Line: To better understand the Wolbachia/CHIKV/Ae. albopictus interaction, we generated a cellular model using Ae. albopictus derived C6/36 cells that we infected with the wAlbB strain.Our results indicate that CHIKV infection is negatively impacted at both RNA replication and virus assembly/secretion steps in presence of wAlbB.More broadly, this put into question the ecological role of Wolbachia symbiont in Ae. albopictus, but also the ability of the CHIKV to counteract Wolbachia's antiviral potential in vivo.

View Article: PubMed Central - PubMed

Affiliation: Université de Lyon, UMR5557 Ecologie Microbienne, CNRS, USC1190 INRA, VetAgro Sup, Université Lyon 1, Villeurbanne, France.

ABSTRACT
Wolbachia, a widespread endosymbiont of terrestrial arthropods, can protect its host against viral and parasitic infections, a phenotype called "pathogen blocking". However, in some cases Wolbachia may have no effect or even enhance pathogen infection, depending on the host-Wolbachia-pathogen combination. The tiger mosquito Aedes albopictus is naturally infected by two strains of Wolbachia, wAlbA and wAlbB, and is a competent vector for different arboviruses such as dengue virus (DENV) and chikungunya virus (CHIKV). Interestingly, it was shown in some cases that Ae. albopictus native Wolbachia strains are able to inhibit DENV transmission by limiting viral replication in salivary glands, but no such impact was measured on CHIKV replication in vivo. To better understand the Wolbachia/CHIKV/Ae. albopictus interaction, we generated a cellular model using Ae. albopictus derived C6/36 cells that we infected with the wAlbB strain. Our results indicate that CHIKV infection is negatively impacted at both RNA replication and virus assembly/secretion steps in presence of wAlbB. Using FISH, we observed CHIKV and wAlbB in the same mosquito cells, indicating that the virus is still able to enter the cell in the presence of the bacterium. Further work is needed to decipher molecular pathways involved in Wolbachia-CHIKV interaction at the cellular level, but this cellular model can be a useful tool to study the mechanism behind virus blocking phenotype induced by Wolbachia. More broadly, this put into question the ecological role of Wolbachia symbiont in Ae. albopictus, but also the ability of the CHIKV to counteract Wolbachia's antiviral potential in vivo.

Show MeSH
Related in: MedlinePlus