Table 1.
qRT-PCR primers and probes used for detection and quantification of CxFV Izabal RNA.
Figure 1.
Correlation of CxFV Izabal qRT-PCR with C6/36 cell plaque titration.
Samples from each dilution were split such that each dilution was quantified by both qRT-PCR and plaque assay. In this figure, the CxFV Izabal qRT-PCR assay used primers E92F and E151R and probe E112P. Results for other CxFV Izabal primer/probe sets are similar (data not shown).
Figure 2.
Replication of CxFV Izabal and KRV in C6/36 cells.
Figure 3.
Replication of CxFV Izabal in Cx. quinquefasciatus Sebring mosquitoes.
Values at each time point represent the mean ± standard error of three to five mosquitoes.
Figure 4.
Replication of WNV in CxFV Izabal (+) and CxFV Izabal (−) C6/36 cells.
CxFV (+) cells were infected two days prior to re-infection with WNV.
Figure 5.
Replication of WNV in CxFV Izabal (+) and CxFV Izabal (-) Cx. quinquefasciatus Sebring mosquitoes.
CxFV Izabal (+) mosquitoes were inoculated intrathoracically with 3.3 log10 pfu CxFV Izabal. Mock-inoculated mosquitoes were inoculated with an empty glass capillary needle, and CxFV (-) mosquitoes were not inoculated. Seven days post inoculations, all three groups were administered a WNV-infectious blood meal of 6.3 log10 pfu/mL. The average WNV titer in CxFV Izabal (+) mosquitoes was significantly less than in mock-inoculated specimens at 0 DPI (Student's t-test, p = 0.016), denoted by an asterisk. At 4 DPI the average WNV titer in CxFV Izabal (+) mosquitoes was significantly higher than in CxFV (-) specimens (Student's t-test, p = 0.0048), denoted by an asterisk. Values at each time point represent the mean ± standard error of two to five mosquitoes.
Table 2.
WNV Infection, dissemination, and transmission rates in Culex quinquefasciatus either infected sequentially with CxFV Izabal and WNV, or uninfected with CxFV Izabal.
Table 3.
Mean WNV titer in Cx. quinquefasciatus bodies and salivary expectorates of specimens either infected or uninfected intrathoracically with CxFV Izabal.
Figure 6.
WNV transmission by CxFV Izabal (+) and CxFV Izabal (-) mosquito colonies.
Percentage of Cx. quinquefasciatus transmitting WNV nine days following intrathoracic inoculation with either WNV alone or WNV + CxFV Izabal. For the Honduras colony, a significantly higher percentage of mosquitoes inoculated simultaneously with both viruses transmitted WNV than mosquitoes that were inoculated with WNV alone (Fisher's exact test, p = 0.0014).
Figure 7.
Localization of CxFV Izabal and WNV to the midgut in co-infected mosquitoes.
A) WNV-infected muscle tissue of Cx. quinquefasciatus Sebring, harvested 9 DPI. WNV stained with AlexaFluor 488 (green). B) Focus of CxFV Izabal infection on the midgut of Cx. quinquefasciatus. CxFV Izabal stained with AlexaFluor 488. C) Midgut of Cx. quinquefasciatus infected simultaneously with both WNV and CxFV Izabal by intrathoracic inoculation. CxFV Izabal stained with AlexaFluor 594 (red) and WNV with AlexaFluor 488 (green). D) Close-up view of co-infected midgut from panel C showing midgut epithelial cells infected with WNV and CxFV Izabal. CxFV Izabal stained with AlexaFluor 594 (red) and WNV with Alexa 488 (green).
Figure 8.
Localization of CxFV Izabal and WNV to head tissues in co-infected mosquitoes.
A) Uninfected head tissues of Cx. quinquefasciatus stained with AlexaFluor 594 (red). White arrow depicts non-specific staining of debris. B) Head tissues of CxFV Izabal-infected Cx. quinquefasciatus, harvested 7 DPI. CxFV Izabal stained with AlexaFluor 594. C) Head tissues of WNV-infected Cx. quinquefasciatus, harvested 9 DPI. WNV stained with AlexaFluor 488 (green). D) Co-infected head tissues of Cx. quinquefasciatus. Mosquito inoculated simultaneously with CxFV Izabal and WNV, harvested 9 DPI. CxFV Izabal stained with AlexaFluor 594 (red) and WNV with Alexa 488 (green). White arrow denotes non-specific staining of debris.