Transmission dynamics of an insect-specific flavivirus in a naturally infected Culex pipiens laboratory colony and effects of co-infection on vector competence for West Nile virus

Abstract

We established a laboratory colony of Culex pipiens mosquitoes from eggs collected in Colorado and discovered that mosquitoes in the colony are naturally infected with Culex flavivirus (CxFV), an insect-specific flavivirus. In this study we examined transmission dynamics of CxFV and effects of persistent CxFV infection on vector competence for West Nile virus (WNV). We found that vertical transmission is the primary mechanism for persistence of CxFV in Cx. pipiens, with venereal transmission potentially playing a minor role. Vector competence experiments indicated possible early suppression of WNV replication by persistent CxFV infection in Cx. pipiens. This is the first description of insect-specific flavivirus transmission dynamics in a naturally infected mosquito colony and the observation of delayed dissemination of superinfecting WNV suggests that the presence of CxFV may impact the intensity of enzootic transmission of WNV and the risk of human exposure to this important pathogen.

Figure 1

Culex flavivirus titers, shown as log₁₀ RNA copies per individual, for different life stages of Culex pipiens (Colorado colony, naturally infected with CxFV). Titers were determined by qRT-PCR, using CxFV-specific primers. Specimens were tested individually, except for eggs, which were tested as rafts. (BF = blood-fed)

Figure 2

Culex flavivirus growth curves in the absence or presence of West Nile virus superinfection. C6/36 cell cultures were infected with Culex flavivirus at a dose of 0.1 RNA copy/well and 48 h later with WNV at doses of 0.1 or 0.01 pfu/well. CxFV RNA in cell culture medium was titrated by qRT-PCR with CxFV-specific primers. Culex flavivirus growth curves in cells challenged with WNV were compared to those in cells infected with CxFV alone using linear regression and were not significantly different. (48a = 48h titer, pre-WNV challenge and 48b = 48h titer, post-WNV challenge and medium change). Solid line, CxFV alone; dashed line, CxFV + WNV MOI=0.1; dotted line, CxFV + WNV MOI=0.01.

Figure 3

West Nile virus growth curves in C6/36 cells in absence or presence of co-infection with Culex flavivirus. C6/36 cells were infected with CxFV at a dose of 0.1 RNA copy/well and 48h later were challenged with WNV at doses of 0.01 pfu/well (A) and 0.1 pfu/well (B). West Nile virus titers in cell culture medium were determined by plaque assay in Vero cells. West Nile virus growth curves in cells co-infected with CxFV were compared to growth curves in cells infected with WNV alone using linear regression and were significantly different (WNV MOI 0.01, p < 0.001 and WNV MOI 0.1, p = 0.042). Titers were also compared at each time-point using the Wilcoxon rank-sum test. Solid lines, WNV alone, MOI=0.01(A), MOI=0.1(B); dashed lines, WNV + CxFV. *Statistically significant at α ≤ 0.05.

Figure 3

West Nile virus growth curves in C6/36 cells in absence or presence of co-infection with Culex flavivirus. C6/36 cells were infected with CxFV at a dose of 0.1 RNA copy/well and 48h later were challenged with WNV at doses of 0.01 pfu/well (A) and 0.1 pfu/well (B). West Nile virus titers in cell culture medium were determined by plaque assay in Vero cells. West Nile virus growth curves in cells co-infected with CxFV were compared to growth curves in cells infected with WNV alone using linear regression and were significantly different (WNV MOI 0.01, p < 0.001 and WNV MOI 0.1, p = 0.042). Titers were also compared at each time-point using the Wilcoxon rank-sum test. Solid lines, WNV alone, MOI=0.01(A), MOI=0.1(B); dashed lines, WNV + CxFV. *Statistically significant at α ≤ 0.05.