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RNA interference modulates replication of dengue virus in Drosophila melanogaster cells.

Mukherjee S, Hanley KA - BMC Microbiol. (2010)

Bottom Line: The four serotypes did not differ in mean titer.While serotypes did not differ in their average response to Dcr-2 knockdown, strains within serotypes showed significant differences in their sensitivity to Dcr-2 knockdown.Moreover, knockdown of three additional components of the RNAi pathway, Argonaute 2 (Ago-2), Dcr-1 and Ago-1, also resulted in a significant increase in replication of the two DENV strains tested, and the magnitude of this increase was similar to that resulting from Dcr-2 knockdown.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Biology Program, New Mexico State University, Las Cruces, NM 88003, USA.

ABSTRACT

Background: Mosquito-borne dengue virus (DENV, genus Flavivirus) has emerged as a major threat to global human health in recent decades, and novel strategies to contain the escalating dengue fever pandemic are urgently needed. RNA interference (RNAi) induced by exogenous small interfering RNAs (siRNAs) has shown promise for treatment of flavivirus infections in hosts and prevention of transmission by vectors. However, the impact of RNAi triggered by authentic virus infection on replication of DENV, or any flavivirus, has received little study. The objectives of the current study were threefold: first, to assess the utility of Drosophila melanogaster S2 cells for the study of DENV, second to investigate the impact of multiple enzymes in the RNAi pathway on DENV replication; and third to test for variation in the response of the four serotypes of DENV to modulation of RNAi.

Results: Three strains from each of the four DENV serotypes showed replication in S2 cells following infection at multiplicity of infection (MOI) 0.1 and MOI 10; each strain achieved titers > 4.0 log10pfu/ml five days after infection at MOI 10. The four serotypes did not differ in mean titer. S2 cells infected with DENV-1, 2, 3 or 4 produced siRNAs, indicating that infection triggered an RNAi response. Knockdown of one of the major enzymes in the RNAi pathway, Dicer-2 (Dcr-2), resulted in a 10 to 100-fold enhancement of replication of all twelve strains of DENV in S2 cells. While serotypes did not differ in their average response to Dcr-2 knockdown, strains within serotypes showed significant differences in their sensitivity to Dcr-2 knockdown. Moreover, knockdown of three additional components of the RNAi pathway, Argonaute 2 (Ago-2), Dcr-1 and Ago-1, also resulted in a significant increase in replication of the two DENV strains tested, and the magnitude of this increase was similar to that resulting from Dcr-2 knockdown.

Conclusions: These findings indicate that DENV can replicate in Drosophila S2 cells and that the RNAi pathway plays a role in modulating DENV replication in these cells. S2 cells offer a useful cell culture model for evaluation of the interaction between DENV and the RNAi response.

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Replication of DENV in Drosophila melanogaster S2 cells. A: Titer of 12 strains of DENV 5 days post infection following passage 1 (S2 p1 MOI 10, solid bars) and passage 2 (S2 p2 MOI 10, open bars) in Drosophila melanogaster S2 cells. In passage 1, cells were infected with each virus strain at MOI 10. In passage 2, cells were infected with 500 μl of cell supernatant from passage 1; B: Titer of 12 strains of DENV 5 days pi following infection of S2 cells at MOI 0.1 (S2 p1 MOI 0.1); C: Replication kinetics of DENV-4 Taiwan at MOI 0.1 in Drosophila melanogaster S2 cells.
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Figure 2: Replication of DENV in Drosophila melanogaster S2 cells. A: Titer of 12 strains of DENV 5 days post infection following passage 1 (S2 p1 MOI 10, solid bars) and passage 2 (S2 p2 MOI 10, open bars) in Drosophila melanogaster S2 cells. In passage 1, cells were infected with each virus strain at MOI 10. In passage 2, cells were infected with 500 μl of cell supernatant from passage 1; B: Titer of 12 strains of DENV 5 days pi following infection of S2 cells at MOI 0.1 (S2 p1 MOI 0.1); C: Replication kinetics of DENV-4 Taiwan at MOI 0.1 in Drosophila melanogaster S2 cells.

Mentions: S2 cells were grown to 80% confluency (6.0 log10 cells/well ± 3.1 log10 cells/well) in six-well tissue culture treated plates (BD Falcon). Triplicate wells were infected with each of the 12 C6/36 p1 MOI 0.1 stocks at a specified MOI, based on titer in C6/36 cells (Table 1) divided by the number of S2 cells/well, in a total volume of one ml. Virus was incubated for two hrs at 28°C with occasional, gentle rocking and washed once with one ml of conditioned S2 media. Thereafter three ml of conditioned S2 media was added to each well. S2 cells were infected at MOI 10 and incubated for five days at 28°C after which cell supernatants, designated S2 p1 MOI 10, were collected and frozen as described above. 500 μl from each S2 p1 MOI 10 replicate were then passaged in fresh S2 cells as described above. Given the titers on day five for S2 p1 MOI 10 (Figure 2A), 500 μl of supernatants contained a total of 3.2 - 4.4 log10plaque forming units (log10pfu). Cells were incubated for five days and harvested to yield S2 p2 MOI 10. S2 cells were infected similarly at MOI 0.1 to yield cell supernatants S2 p1 MOI 0.1, but these supernatants were not passaged further. Virus titer in all cell supernatants was determined by serial titration in C6/36 cells as described above.


RNA interference modulates replication of dengue virus in Drosophila melanogaster cells.

Mukherjee S, Hanley KA - BMC Microbiol. (2010)

Replication of DENV in Drosophila melanogaster S2 cells. A: Titer of 12 strains of DENV 5 days post infection following passage 1 (S2 p1 MOI 10, solid bars) and passage 2 (S2 p2 MOI 10, open bars) in Drosophila melanogaster S2 cells. In passage 1, cells were infected with each virus strain at MOI 10. In passage 2, cells were infected with 500 μl of cell supernatant from passage 1; B: Titer of 12 strains of DENV 5 days pi following infection of S2 cells at MOI 0.1 (S2 p1 MOI 0.1); C: Replication kinetics of DENV-4 Taiwan at MOI 0.1 in Drosophila melanogaster S2 cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2874549&req=5

Figure 2: Replication of DENV in Drosophila melanogaster S2 cells. A: Titer of 12 strains of DENV 5 days post infection following passage 1 (S2 p1 MOI 10, solid bars) and passage 2 (S2 p2 MOI 10, open bars) in Drosophila melanogaster S2 cells. In passage 1, cells were infected with each virus strain at MOI 10. In passage 2, cells were infected with 500 μl of cell supernatant from passage 1; B: Titer of 12 strains of DENV 5 days pi following infection of S2 cells at MOI 0.1 (S2 p1 MOI 0.1); C: Replication kinetics of DENV-4 Taiwan at MOI 0.1 in Drosophila melanogaster S2 cells.
Mentions: S2 cells were grown to 80% confluency (6.0 log10 cells/well ± 3.1 log10 cells/well) in six-well tissue culture treated plates (BD Falcon). Triplicate wells were infected with each of the 12 C6/36 p1 MOI 0.1 stocks at a specified MOI, based on titer in C6/36 cells (Table 1) divided by the number of S2 cells/well, in a total volume of one ml. Virus was incubated for two hrs at 28°C with occasional, gentle rocking and washed once with one ml of conditioned S2 media. Thereafter three ml of conditioned S2 media was added to each well. S2 cells were infected at MOI 10 and incubated for five days at 28°C after which cell supernatants, designated S2 p1 MOI 10, were collected and frozen as described above. 500 μl from each S2 p1 MOI 10 replicate were then passaged in fresh S2 cells as described above. Given the titers on day five for S2 p1 MOI 10 (Figure 2A), 500 μl of supernatants contained a total of 3.2 - 4.4 log10plaque forming units (log10pfu). Cells were incubated for five days and harvested to yield S2 p2 MOI 10. S2 cells were infected similarly at MOI 0.1 to yield cell supernatants S2 p1 MOI 0.1, but these supernatants were not passaged further. Virus titer in all cell supernatants was determined by serial titration in C6/36 cells as described above.

Bottom Line: The four serotypes did not differ in mean titer.While serotypes did not differ in their average response to Dcr-2 knockdown, strains within serotypes showed significant differences in their sensitivity to Dcr-2 knockdown.Moreover, knockdown of three additional components of the RNAi pathway, Argonaute 2 (Ago-2), Dcr-1 and Ago-1, also resulted in a significant increase in replication of the two DENV strains tested, and the magnitude of this increase was similar to that resulting from Dcr-2 knockdown.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Biology Program, New Mexico State University, Las Cruces, NM 88003, USA.

ABSTRACT

Background: Mosquito-borne dengue virus (DENV, genus Flavivirus) has emerged as a major threat to global human health in recent decades, and novel strategies to contain the escalating dengue fever pandemic are urgently needed. RNA interference (RNAi) induced by exogenous small interfering RNAs (siRNAs) has shown promise for treatment of flavivirus infections in hosts and prevention of transmission by vectors. However, the impact of RNAi triggered by authentic virus infection on replication of DENV, or any flavivirus, has received little study. The objectives of the current study were threefold: first, to assess the utility of Drosophila melanogaster S2 cells for the study of DENV, second to investigate the impact of multiple enzymes in the RNAi pathway on DENV replication; and third to test for variation in the response of the four serotypes of DENV to modulation of RNAi.

Results: Three strains from each of the four DENV serotypes showed replication in S2 cells following infection at multiplicity of infection (MOI) 0.1 and MOI 10; each strain achieved titers > 4.0 log10pfu/ml five days after infection at MOI 10. The four serotypes did not differ in mean titer. S2 cells infected with DENV-1, 2, 3 or 4 produced siRNAs, indicating that infection triggered an RNAi response. Knockdown of one of the major enzymes in the RNAi pathway, Dicer-2 (Dcr-2), resulted in a 10 to 100-fold enhancement of replication of all twelve strains of DENV in S2 cells. While serotypes did not differ in their average response to Dcr-2 knockdown, strains within serotypes showed significant differences in their sensitivity to Dcr-2 knockdown. Moreover, knockdown of three additional components of the RNAi pathway, Argonaute 2 (Ago-2), Dcr-1 and Ago-1, also resulted in a significant increase in replication of the two DENV strains tested, and the magnitude of this increase was similar to that resulting from Dcr-2 knockdown.

Conclusions: These findings indicate that DENV can replicate in Drosophila S2 cells and that the RNAi pathway plays a role in modulating DENV replication in these cells. S2 cells offer a useful cell culture model for evaluation of the interaction between DENV and the RNAi response.

Show MeSH
Related in: MedlinePlus