Co-Infection of Mosquitoes with Chikungunya and Dengue Viruses Reveals Modulation of the Replication of Both Viruses in Midguts and Salivary Glands of Aedes aegypti Mosquitoes

Abstract

Arthropod-borne virus (arbovirus) infections cause several emerging and resurgent infectious diseases in humans and animals. Chikungunya-affected areas often overlap with dengue-endemic areas. Concurrent dengue virus (DENV) and chikungunya virus (CHIKV) infections have been detected in travelers returning from regions of endemicity. CHIKV and DENV co-infected Aedes albopictus have also been collected in the vicinity of co-infected human cases, emphasizing the need to study co-infections in mosquitoes. We thus aimed to study the pathogen-pathogen interaction involved in these co-infections in DENV/CHIKV co-infected Aedes aegypti mosquitoes. In mono-infections, we detected CHIKV antigens as early as 4 days post-virus exposure in both the midgut (MG) and salivary gland (SG), whereas we detected DENV serotype 2 (DENV-2) antigens from day 5 post-virus exposure in MG and day 10 post-virus exposure in SG. Identical infection rates were observed for singly and co-infected mosquitoes, and facilitation of the replication of both viruses at various times post-viral exposure. We observed a higher replication for DENV-2 in SG of co-infected mosquitoes. We showed that mixed CHIKV and DENV infection facilitated viral replication in Ae. aegypti. The outcome of these mixed infections must be further studied to increase our understanding of pathogen-pathogen interactions in host cells.

Keywords: Aedes aegypti; arbovirus; chikungunya; co-infection; dengue.

Figure 1

Distribution of viral antigens in midguts infected by chikungunya virus (CHIKV) and dengue virus serotype 2 (DENV-2). Midguts (MG) were collected at days 4, 7, 11, and 15 post-virus exposure for CHIKV and 4, 6, 11, and day 14 post-virus exposure for DENV-2 as indicated in panels (A,B) respectively. Viral antigens were labelled using anti-CHIKV 3E4 (panel (A) in red) or anti-DENV-2 4E11 (panel (B) in green) monoclonal antibodies. Nuclei were stained with DAPI and the actin network with Alexafluor 488 (panel (A) in green) or 633 (panel (B) in magenta) phalloidin. The distribution of CHIKV antigen in a CHIKV-infected midgut at day 3 post-virus exposure was shown in panels (C,D). Muscular tissue stained in green with Alexafluor 488 phalloidin (C,D); CHIKV antigens labelled in red with Cy3-labelled 3E4 anti-E protein (C,D) and nuclei in blue (DAPI) (C). (D) shows a surperimposition of a transmission light picture to an immunofluorescence picture. The distribution of DENV-2 antigens in the posterior part of the midgut at day 3 post-virus exposure was shown in panel E. The muscular tissue is shown in magenta (Alexafluora 633 phalloidin), viral antigens in green (4E11 monoclonal antibody) and nuclei in blue (DAPI).

Figure 2

Distribution overtime of viral antigen in salivary glands infected by CHIKV and DENV-2. SG were collected at days 4, 6, 8, and 13 post-virus exposure for CHIKV and days 3, 7, 10, and 12 post-virus exposure for DENV-2 as indicated in panels A and B respectively. Viral antigens were labelled using anti-CHIKV 3E4 (panel (A) in red) or anti-DENV-2 4E11 (panel (B) in green) monoclonal antibodies. Nuclei were stained with DAPI and the actin network with Alexafluor 488 (panel (A)) or 633 (panel (B)) phalloidin; (C) CHIKV-infected salivary gland at day 11 post-virus exposure. CHIKV is labelled in red (Cy3-labelled 3E4 anti-E protein), the actin network in green (Alexafluor 488 phalloidin), and the nuclei in blue (DAPI). The salivary channel is shown by an arrow; (D) DENV-2 infected salivary gland at high magnification. The virus is labelled in green (4E11 anti-E protein monoclonal antibody) and nuclei in blue (DAPI).

Figure 2

Distribution overtime of viral antigen in salivary glands infected by CHIKV and DENV-2. SG were collected at days 4, 6, 8, and 13 post-virus exposure for CHIKV and days 3, 7, 10, and 12 post-virus exposure for DENV-2 as indicated in panels A and B respectively. Viral antigens were labelled using anti-CHIKV 3E4 (panel (A) in red) or anti-DENV-2 4E11 (panel (B) in green) monoclonal antibodies. Nuclei were stained with DAPI and the actin network with Alexafluor 488 (panel (A)) or 633 (panel (B)) phalloidin; (C) CHIKV-infected salivary gland at day 11 post-virus exposure. CHIKV is labelled in red (Cy3-labelled 3E4 anti-E protein), the actin network in green (Alexafluor 488 phalloidin), and the nuclei in blue (DAPI). The salivary channel is shown by an arrow; (D) DENV-2 infected salivary gland at high magnification. The virus is labelled in green (4E11 anti-E protein monoclonal antibody) and nuclei in blue (DAPI).

Figure 3

Detection of viral antigens in CHIKV/DENV-2 co-infected midguts and salivary glands at various days post-virus exposure. (A,C) Midgut at days 7 and 10 post-viral exposure respectively. CHIKV is labelled in red (Cy3-labelled 3E4 monoclonal antibody), DENV-2 in green (4E11 anti-E monoclonal antibody), nuclei in blue (DAPI), and the actin network in magenta (Alexafluor 633 phalloidin); (B,D) Salivary gland at days 10 and 13 post-viral exposure respectively. DENV-2 antigen is shown in green (4E11 anti-E monoclonal antibody), CHIKV antigen in red (Cy3-labelled 3E4 monoclonal antibody), and nuclei in blue (DAPI).

Figure 3

Detection of viral antigens in CHIKV/DENV-2 co-infected midguts and salivary glands at various days post-virus exposure. (A,C) Midgut at days 7 and 10 post-viral exposure respectively. CHIKV is labelled in red (Cy3-labelled 3E4 monoclonal antibody), DENV-2 in green (4E11 anti-E monoclonal antibody), nuclei in blue (DAPI), and the actin network in magenta (Alexafluor 633 phalloidin); (B,D) Salivary gland at days 10 and 13 post-viral exposure respectively. DENV-2 antigen is shown in green (4E11 anti-E monoclonal antibody), CHIKV antigen in red (Cy3-labelled 3E4 monoclonal antibody), and nuclei in blue (DAPI).

Figure 4

Immunohistochemical (IHC) staining of CHIKV in paraffin-embedded mosquitoes at various times post-virus exposure. CHIKV antigen stains brownish-red. A non-infected mosquito was used as control to determine non-specific staining using 3E4 anti-CHIKV monoclonal antibody.

Figure 5

IHC staining of DENV-2 in paraffin-embedded mosquitoes at various times post-virus exposure. DENV-2 antigen stains brownish-red. A non-infected mosquito was used as control to determine non-specific staining using 4E11 anti-DENV-2 monoclonal antibody.

Figure 6

Distribution of CHIKV and DENV-2 antigens in mono-infected MG, SG and eggs by IHC at 7 dpve. (A,B) MG of CHIKV and DENV-2 infected mosquitoes, respectively, Scale bar = 20 µm; (C,D) SG of CHIKV and DENV-2 infected mosquitoes, respectively, Scale bar = 20 µm; (E,F) Eggs of CHIKV and DENV-2 infected mosquitoes, Scale bar = 100 µm. Antigens are stained brown.

Figure 7

Distribution of CHIKV and DENV-2 antigens in the MG and SG of co-infected mosquitoes at 7 and 10 days post-virus exposure. Serial sections of the same organs were stained against CHIKV and DENV-2 antigens. (A,B) CHIKV and DENV-2 infected MG, respectively at 7 dpve, Scale bar = 20 µm; (C,D) CHIKV and DENV-2 infected SG respectively at 7 dpve, Scale bar = 40 µm; (E,F) CHIKV and DENV-2 infected salivary glands at 10 dpve, Scale bar = 40 µm. Antigens are stained in brownish-red.

Figure 8

Replication kinetics of CHIKV and DENV-2 in mono or dually infected MG and SG. Viral RNA copy number was determined by RT-qPCR at the indicated times post-virus exposure. (A,B) CHIKV RNA was determined in MG (A) and SG (B) of mosquitoes mono-infected by CHIKV (●) or co-infected by CHIKV and DENV-2 (■), respectively); (C,D) DENV-2 RNA was determined in MG (A) and SG (B) of mosquitoes mono-infected by DENV-2 (●) or co-infected by DENV-2 and CHIKV (■). Eight mosquitoes were dissected at each time point and three replicates were performed. * indicates statistical differences between mon-infected and co-infected organs.