Limits...
Wolbachia restricts insect-specific flavivirus infection in Aedes aegypti cells

View Article: PubMed Central - PubMed

ABSTRACT

Mosquito-borne viruses are known to cause disease in humans and livestock and are often difficult to control due to the lack of specific antivirals and vaccines. The Wolbachia endosymbiont has been widely studied for its ability to restrict positive-strand RNA virus infection in mosquitoes, although little is known about the precise antiviral mechanism. In recent years, a variety of insect-specific viruses have been discovered in mosquitoes and an interaction with mosquito-borne viruses has been reported for some of them; however, nothing is known about the effect of Wolbachia on insect-specific virus infection in mosquitoes. Here, we show that transinfection of the Drosophila-derived wMelPop Wolbachia strain into Aedes aegypti-derived cells resulted in inhibition and even clearance of the persistent cell-fusing agent flavivirus infection in these cells. This broadens the antiviral activity of Wolbachia from acute infections to persistent infections and from arboviruses to mosquito-specific viruses. In contrast, no effect on the Phasi Charoen-like bunyavirus persistent infection in these cells was observed, suggesting a difference in Wolbachia inhibition between positive- and negative-strand RNA viruses.

No MeSH data available.


Related in: MedlinePlus

Effect of wMelPop on CFAV and PCLV infection in Aag2 cells. (a) Detection of CFAV, Wolbachia or PCLV in Aag2, Aag2wMelPop and two different cultures of Aag2wMelPop cells treated with tetracycline (Aag2wMelPop-tet sets 1 and 2) cells by RT-PCR. Actin was used as loading control. (b) Detection of CFAV or PCLV in C6/36 cells incubated with supernatant of Aag2, Aag2wMelPop or Aag2wMelPop treated with tetracycline (two different cultures, Aag2wMelPop-tet sets 1 and 2) by RT-PCR. Actin was used as a loading control. (c) Quantification of CFAV RNA in Aag2wMelPop (Wol) or Aag2wMelPop treated with tetracycline (Tet) cells after incubation with Aag2 supernatant containing CFAV by SYBR Green. S7 was used as internal control. Relative RNA expression is represented as (CFAV/S7). Error bars show sem from three independent experiments. (d) Quantification of PCLV RNA in Aag2wMelPop (Wol) or Aag2wMelPop treated with tetracycline (Tet) cells, either after incubation with Aag2 supernatant harbouring PCLV or untreated by SYBR Green. S7 was used as an internal control. Relative RNA expression is represented as (PCLV/S7) and mock-infected tetracycline cells were set to 1. Error bars show sem from three independent experiments. *P≤0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5120408&req=5

Figure 2: Effect of wMelPop on CFAV and PCLV infection in Aag2 cells. (a) Detection of CFAV, Wolbachia or PCLV in Aag2, Aag2wMelPop and two different cultures of Aag2wMelPop cells treated with tetracycline (Aag2wMelPop-tet sets 1 and 2) cells by RT-PCR. Actin was used as loading control. (b) Detection of CFAV or PCLV in C6/36 cells incubated with supernatant of Aag2, Aag2wMelPop or Aag2wMelPop treated with tetracycline (two different cultures, Aag2wMelPop-tet sets 1 and 2) by RT-PCR. Actin was used as a loading control. (c) Quantification of CFAV RNA in Aag2wMelPop (Wol) or Aag2wMelPop treated with tetracycline (Tet) cells after incubation with Aag2 supernatant containing CFAV by SYBR Green. S7 was used as internal control. Relative RNA expression is represented as (CFAV/S7). Error bars show sem from three independent experiments. (d) Quantification of PCLV RNA in Aag2wMelPop (Wol) or Aag2wMelPop treated with tetracycline (Tet) cells, either after incubation with Aag2 supernatant harbouring PCLV or untreated by SYBR Green. S7 was used as an internal control. Relative RNA expression is represented as (PCLV/S7) and mock-infected tetracycline cells were set to 1. Error bars show sem from three independent experiments. *P≤0.05.

Mentions: The presence of active PCLV production/infection in Aag2 and Aag2wMelPop cells was further confirmed by RT-PCR and was also detected following the transfer of supernatant from these cells to C6/36 cells, resulting in PCLV-positive C6/36 cells (Fig. 2a, b). CFAV was easily detected by RT-PCR in Aag2 cells, as well as in C6/36 cells incubated with Aag2 supernatant, in contrast to Aag2wMelPop or C6/36 cells incubated with Aag2wMelPop supernatant (Fig. 2a, b). To determine if the presence of wMelPop in Aag2 cells cured the cells from CFAV infection or just strongly inhibited CFAV replication/infection, Aag2wMelPop cells were treated with tetracycline over several passages, resulting in the loss of Wolbachia. The absence of Wolbachia in Aag2wMelPop-tetracyline-treated cells (called Aag2wMelPop-tet) was confirmed by RT-PCR (Fig. 2a). Similar to what is seen in the parental Aag2wMelPop cells, no CFAV could be detected in Aag2wMelPop-tet cells (Fig. 2a), even when a different region of the CFAV genome was used for detection (Fig. S3a) or in C6/36 cells incubated with Aag2wMelPop-tet supernatant (Fig. 2b). In contrast, PCLV was detected in each of these samples (Fig. 2a, b). This suggested that wMelPop transinfection cures Aag2 cells of persistent CFAV infection, but has no effect on PCLV. To exclude the possibility that tetracycline treatment per se inhibits CFAV, Aag2 cells were treated with tetracycline and CFAV levels were monitored over time. No effect on CFAV could be detected in tetracycline-treated Aag2 cells compared to untreated cells (Fig. S3b).


Wolbachia restricts insect-specific flavivirus infection in Aedes aegypti cells
Effect of wMelPop on CFAV and PCLV infection in Aag2 cells. (a) Detection of CFAV, Wolbachia or PCLV in Aag2, Aag2wMelPop and two different cultures of Aag2wMelPop cells treated with tetracycline (Aag2wMelPop-tet sets 1 and 2) cells by RT-PCR. Actin was used as loading control. (b) Detection of CFAV or PCLV in C6/36 cells incubated with supernatant of Aag2, Aag2wMelPop or Aag2wMelPop treated with tetracycline (two different cultures, Aag2wMelPop-tet sets 1 and 2) by RT-PCR. Actin was used as a loading control. (c) Quantification of CFAV RNA in Aag2wMelPop (Wol) or Aag2wMelPop treated with tetracycline (Tet) cells after incubation with Aag2 supernatant containing CFAV by SYBR Green. S7 was used as internal control. Relative RNA expression is represented as (CFAV/S7). Error bars show sem from three independent experiments. (d) Quantification of PCLV RNA in Aag2wMelPop (Wol) or Aag2wMelPop treated with tetracycline (Tet) cells, either after incubation with Aag2 supernatant harbouring PCLV or untreated by SYBR Green. S7 was used as an internal control. Relative RNA expression is represented as (PCLV/S7) and mock-infected tetracycline cells were set to 1. Error bars show sem from three independent experiments. *P≤0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5120408&req=5

Figure 2: Effect of wMelPop on CFAV and PCLV infection in Aag2 cells. (a) Detection of CFAV, Wolbachia or PCLV in Aag2, Aag2wMelPop and two different cultures of Aag2wMelPop cells treated with tetracycline (Aag2wMelPop-tet sets 1 and 2) cells by RT-PCR. Actin was used as loading control. (b) Detection of CFAV or PCLV in C6/36 cells incubated with supernatant of Aag2, Aag2wMelPop or Aag2wMelPop treated with tetracycline (two different cultures, Aag2wMelPop-tet sets 1 and 2) by RT-PCR. Actin was used as a loading control. (c) Quantification of CFAV RNA in Aag2wMelPop (Wol) or Aag2wMelPop treated with tetracycline (Tet) cells after incubation with Aag2 supernatant containing CFAV by SYBR Green. S7 was used as internal control. Relative RNA expression is represented as (CFAV/S7). Error bars show sem from three independent experiments. (d) Quantification of PCLV RNA in Aag2wMelPop (Wol) or Aag2wMelPop treated with tetracycline (Tet) cells, either after incubation with Aag2 supernatant harbouring PCLV or untreated by SYBR Green. S7 was used as an internal control. Relative RNA expression is represented as (PCLV/S7) and mock-infected tetracycline cells were set to 1. Error bars show sem from three independent experiments. *P≤0.05.
Mentions: The presence of active PCLV production/infection in Aag2 and Aag2wMelPop cells was further confirmed by RT-PCR and was also detected following the transfer of supernatant from these cells to C6/36 cells, resulting in PCLV-positive C6/36 cells (Fig. 2a, b). CFAV was easily detected by RT-PCR in Aag2 cells, as well as in C6/36 cells incubated with Aag2 supernatant, in contrast to Aag2wMelPop or C6/36 cells incubated with Aag2wMelPop supernatant (Fig. 2a, b). To determine if the presence of wMelPop in Aag2 cells cured the cells from CFAV infection or just strongly inhibited CFAV replication/infection, Aag2wMelPop cells were treated with tetracycline over several passages, resulting in the loss of Wolbachia. The absence of Wolbachia in Aag2wMelPop-tetracyline-treated cells (called Aag2wMelPop-tet) was confirmed by RT-PCR (Fig. 2a). Similar to what is seen in the parental Aag2wMelPop cells, no CFAV could be detected in Aag2wMelPop-tet cells (Fig. 2a), even when a different region of the CFAV genome was used for detection (Fig. S3a) or in C6/36 cells incubated with Aag2wMelPop-tet supernatant (Fig. 2b). In contrast, PCLV was detected in each of these samples (Fig. 2a, b). This suggested that wMelPop transinfection cures Aag2 cells of persistent CFAV infection, but has no effect on PCLV. To exclude the possibility that tetracycline treatment per se inhibits CFAV, Aag2 cells were treated with tetracycline and CFAV levels were monitored over time. No effect on CFAV could be detected in tetracycline-treated Aag2 cells compared to untreated cells (Fig. S3b).

View Article: PubMed Central - PubMed

ABSTRACT

Mosquito-borne viruses are known to cause disease in humans and livestock and are often difficult to control due to the lack of specific antivirals and vaccines. The Wolbachia endosymbiont has been widely studied for its ability to restrict positive-strand RNA virus infection in mosquitoes, although little is known about the precise antiviral mechanism. In recent years, a variety of insect-specific viruses have been discovered in mosquitoes and an interaction with mosquito-borne viruses has been reported for some of them; however, nothing is known about the effect of Wolbachia on insect-specific virus infection in mosquitoes. Here, we show that transinfection of the Drosophila-derived wMelPop Wolbachia strain into Aedes aegypti-derived cells resulted in inhibition and even clearance of the persistent cell-fusing agent flavivirus infection in these cells. This broadens the antiviral activity of Wolbachia from acute infections to persistent infections and from arboviruses to mosquito-specific viruses. In contrast, no effect on the Phasi Charoen-like bunyavirus persistent infection in these cells was observed, suggesting a difference in Wolbachia inhibition between positive- and negative-strand RNA viruses.

No MeSH data available.


Related in: MedlinePlus