Culex flavivirus and West Nile virus mosquito coinfection and positive ecological association in Chicago, United States

Abstract

Culex flavivirus (CxFV) is an insect-specific flavivirus globally distributed in mosquitoes of the genus Culex. CxFV was positively associated with West Nile virus (WNV) infection in a case-control study of 268 mosquito pools from an endemic focus of WNV transmission in Chicago, United States. Specifically, WNV-positive Culex mosquito pools were four times more likely also to be infected with CxFV than were spatiotemporally matched WNV-negative pools. In addition, mosquito pools from residential sites characterized by dense housing and impermeable surfaces were more likely to be infected with CxFV than were pools from nearby urban green spaces. Further, 6/15 (40%) WNV-positive individual mosquitoes were also CxFV positive, demonstrating that both viruses can coinfect mosquitoes in nature. Phylogenetic analysis of CxFV from Chicago demonstrated a pattern similar to WNV, consisting of low global viral diversity and lack of geographic clustering. These results illustrate a positive ecological association between CxFV and WNV, and that coinfection of individual mosquitoes can occur naturally in areas of high flaviviral transmission. These conclusions represent a challenge to the hypothesis of super-infection exclusion in the CxFV/WNV system, whereby an established infection with one virus may interfere with secondary viral infection with a similar virus. This study suggests that infection with insect-specific flaviviruses such as CxFV may not exclude secondary infection with genetically distinct flaviviruses such as WNV, and that both viruses can naturally coinfect mosquitoes that are epidemic bridge vectors of WNV to humans.

FIG. 1.

Phylogenetic tree of partial nonstructural protein 5 sequences from Culex flavivirus (CxFV), West Nile virus (WNV), and other viruses in the genus Flavivirus (CxFV and WNV clades highlighted). An initial neighbor-joining tree was constructed using FastTree version 2.0.1 (Price et al. 2009) and a Jukes-Cantor + CAT model, to serve as a guide tree for more robust Bayesian phylogenetic inference. The substitution model used in the analysis was estimated using jModelTest (Posada 2008) and was of the form GTR + Γ with the following parameters: nucleotide frequencies A = 0.3035; C = 0.2506; G = 0.2734; T = 0.1725; substitution rates AC = 1.7354, AG = 2.4902, AT = 1.6292, CG = 0.9655, CT = 7.2558, and GT = 1; and Γ (gamma distribution of among-site rate variation) = 0.4130. Phylogenetic analyses were performed using this model and the Bayesian method available in MrBayes v3.1.2 (Huelsenbeck and Ronquist 2001) with two parallel runs of four Markov Chain Monte Carlo (MCMC) chains each for 600,000 generations, with subsampling every 100th generation. Stationarity was assessed at effective sample sizes >400 (469 and 559, respectively, for each Markov Chain Monte Carlo (MCMC) chain), using Tracer v1.5 (Drummond and Rambaut 2007) and a burn-in value of 10%. Posterior probability support is shown next to the nodes of the tree. GenBank accession numbers are in parentheses following taxon names; newly generated CxFV sequences from the Chicago study area are in bold, with reference numbers. The scale bar indicates genetic divergence (nucleotide substitutions per site).