Genetic Diversity in the Collaborative Cross Model Recapitulates Human West Nile Virus Disease Outcomes
West Nile virus (WNV) is an emerging neuroinvasive flavivirus that now causes significant morbidity and mortality worldwide. The innate and adaptive immune responses to WNV infection have been well studied in C57BL/6J inbred mice, but this model lacks the variations in susceptibility, immunity, and outcome to WNV infection that are observed in humans, thus limiting its usefulness to understand the mechanisms of WNV infection and immunity dynamics. To build a model of WNV infection that captures human infection outcomes, we have used the Collaborative Cross (CC) mouse model. We show that this model, which recapitulates the genetic diversity of the human population, demonstrates diversity in susceptibility and outcomes of WNV infection observed in humans. Using multiple F1 crosses of CC mice, we identified a wide range of susceptibilities to infection, as demonstrated through differences in survival, clinical disease score, viral titer, and innate and adaptive immune responses in both peripheral tissues and the central nervous system. Additionally, we examined the Oas1b alleles in the CC mice and confirmed the previous finding that Oas1b plays a role in susceptibility to WNV; however, even within a given Oas1b allele status, we identified a wide range of strain-specific WNV-associated phenotypes. These results confirmed that the CC model is effective for identifying a repertoire of host genes involved in WNV resistance and susceptibility. The CC effectively models a wide range of WNV clinical, virologic, and immune phenotypes, thus overcoming the limitations of the traditional C57BL/6J model, allowing genetic and mechanistic studies of WNV infection and immunity in differently susceptible populations. Mouse models of West Nile virus infection have revealed important details regarding the innate and adaptive immune responses to this emerging viral infection. However, traditional mouse models lack the genetic diversity present in human populations and therefore limit our ability to study various disease outcomes and immunologic mechanisms subsequent to West Nile virus infection. In this study, we used the Collaborative Cross mouse model to more effectively model the wide range of clinical, virologic, and immune phenotypes present upon West Nile virus infection in humans.
[…] Following preparation of single-cell suspensions, cells were plated at 1 × 106 cells/well and stained for surface markers for 15 min on ice. For tetramer staining, cells were stained with the WNV NS4b-H2Db tetramer (generated by the Immune Monitoring Lab, Fred Hutchinson Cancer Research Center). Cells were subsequently fixed, permeabilized (Foxp3 fixation/permeabilization concentrate and diluent; eBioscience), and stained intracellularly with antibodies for 30 min on ice. Flow cytometry was performed on a BD LSRII machine and with BD FACSDiva software. Analysis was performed using FlowJo software.The following directly conjugated antibodies were used: CD3-ECD (143-2C11), CD4-BV605 (RM4-5), CD8-BV650 (53-6.7), Foxp3-Alexa700 (FJK-16S), and NS4b class I tetramer-allophycocyanin. The Foxp3 intracellular staining kit (eBioscience) was used for fixation/permeabilization and all intracellular staining. AmCyan Live/Dead stain (Invitrogen) was used in all panels for identification of live cells. […]