RESUMO
The mosquito midgut functions as a key interface between pathogen and vector. However, studies of midgut physiology and associated virus infection dynamics are scarce, and in Culex tarsalis - an extremely efficient vector of West Nile virus (WNV) - nonexistent. We performed single-cell RNA sequencing on Cx. tarsalis midguts, defined multiple cell types, and determined whether specific cell types are more permissive to WNV infection. We identified 20 cell states comprised of 8 distinct cell types, consistent with existing descriptions of Drosophila and Aedes aegypti midgut physiology. Most midgut cell populations were permissive to WNV infection. However, there were higher levels of WNV RNA (vRNA) in enteroendocrine cells and cells enriched for mitochondrial genes, suggesting enhanced replication in these populations. In contrast, proliferating intestinal stem cell (ISC) populations had the lowest levels of vRNA, a finding consistent with studies suggesting ISC proliferation in the midgut is involved in viral control. Notably, we did not detect significant WNV-infection induced upregulation of canonical mosquito antiviral immune genes (e.g., AGO2, R2D2, etc.) at the whole-midgut level. Rather, we observed a significant positive correlation between immune gene expression levels and vRNA in individual cells, suggesting that within midgut cells, high levels of vRNA may trigger antiviral responses. Our findings establish a Cx. tarsalis midgut cell atlas, and provide insight into midgut infection dynamics of WNV by characterizing cell-type specific enhancement/restriction of, and immune response to, infection at the single-cell level.
RESUMO
Vector competence (VC) refers to the efficiency of pathogen transmission by vectors. Each step in the infection of a mosquito vector constitutes a barrier to transmission that may impose bottlenecks on virus populations. West Nile virus (WNV) is maintained by multiple mosquito species with varying VC. However, the extent to which bottlenecks and VC are linked is poorly understood. Similarly, quantitative analyses of mosquito-imposed bottlenecks on virus populations are limited. We used molecularly barcoded WNV to quantify tissue-associated population bottlenecks in three variably competent WNV vectors. Our results confirm strong population bottlenecks during mosquito infection that are capable of dramatically reshaping virus population structure in a non-selective manner. In addition, we found that mosquitoes with differing VC uniquely shape WNV population structure: highly competent vectors are more likely to contribute to the maintenance of rare viral genotypes. These findings have important implications for arbovirus emergence and evolution.