Modeling Host Genetic Regulation of Influenza Pathogenesis in the Collaborative Cross

<div><p>Genetic variation contributes to host responses and outcomes following infection by influenza A virus or other viral infections. Yet narrow windows of disease symptoms and confounding environmental factors have made it difficult to identify polymorphic genes that contribute to differential disease outcomes in human populations. Therefore, to control for these confounding environmental variables in a system that models the levels of genetic diversity found in outbred populations such as humans, we used incipient lines of the highly genetically diverse Collaborative Cross (CC) recombinant inbred (RI) panel (the pre-CC population) to study how genetic variation impacts influenza associated disease across a genetically diverse population. A wide range of variation in influenza disease related phenotypes including virus replication, virus-induced inflammation, and weight loss was observed. Many of the disease associated phenotypes were correlated, with viral replication and virus-induced inflammation being predictors of virus-induced weight loss. Despite these correlations, pre-CC mice with unique and novel disease phenotype combinations were observed. We also identified sets of transcripts (modules) that were correlated with aspects of disease. In order to identify how host genetic polymorphisms contribute to the observed variation in disease, we conducted quantitative trait loci (QTL) mapping. We identified several QTL contributing to specific aspects of the host response including virus-induced weight loss, titer, pulmonary edema, neutrophil recruitment to the airways, and transcriptional expression. Existing whole-genome sequence data was applied to identify high priority candidate genes within QTL regions. A key host response QTL was located at the site of the known anti-influenza <i>Mx1</i> gene. We sequenced the coding regions of <i>Mx1</i> in the eight CC founder strains, and identified a novel <i>Mx1</i> allele that showed reduced ability to inhibit viral replication, while maintaining protection from weight loss.</p> </div

Candidate genes within QTL regions.

<p>For <i>HrI2</i>, (A) refers to genes with a private A/J SNP, (C) refers to genes with a private 129S1/SvImJ SNP. Unmarked genes had both A/J and 129S1/SvImJ SNPs.</p

QTL identified in the pre-CC population.

<p>QTL identified in the pre-CC population.</p

A novel <i>Mx1</i> allele differentially impacts host response to influenza.

<p>The founder strain alleles at <i>Mx1</i> were grouped based on their phenotypic effects into three functionally distinct classes corresponding to <i>domesticus</i> (<i>dom</i>: A/J, C57BL6/J, 129s1/SvImJ, NOD/HiLtJ and WSB/EiJ), <i>castaneus</i> (<i>cast</i>: CAST/EiJ) and <i>musculus</i> (<i>mus</i>: PWK/PhJ and NZO/ShILtJ). Points shown are individual pre-CC animals with these haplotypes, mean bars are shown for each class. These functionally distinct classes were separable based upon differences in (A) D4 weight and (B) Log titer, with the heterozygous classes showing intermediate phenotypes. Across the pre-CC population, homozygous <i>dom</i> animals had severe weight loss and high titers. Homozygous <i>mus</i> animals showed little weight loss and low titers. Homozygous <i>cast</i> animals showed little weight loss, but had intermediate viral titers. Brackets between groups represent significant differences (* = p<0.05, ** = p<0.003) based on Tukey's HSD. We found no difference by qPCR (C) in expression of <i>Mx1</i> at 2 days post-infection following influenza infection in a strain from each of these three functional classes. By sequencing <i>Mx1</i> (D), we were able to identify five haplotypes across the eight founder strains (Haplotype 1 = A/J, C57BL/6J, 129S1/SvImJ, NOD/HiLtJ; Haplotype 2 = WSB/EiJ; Haplotype 3 = PWK/PhJ; Haplotype 4 = NZO/HiLtJ; Haplotype 5 = CAST/EiJ). Arrows indicate locations of polymorphisms, with small arrows indicating non-coding changes, and large arrows indicating coding changes. Colors correspond to the founder strains having those polymorphisms (brown = multiple strains possess mutation). Grey exons indicate those not transcribed due to either deletion and frameshift, or insertion and early stop codon.</p

Phenotypes measured in the pre-CC and founder strains.

<p>Phenotypes measured in the pre-CC and founder strains.</p

Transcriptional Modules across the pre-CC population.

<p>(A) highly variable transcripts from across the pre-CC population were grouped into modules (sets of transcripts that are connected and similarly expressed (shown here by heat map intensity) within individuals). Modules are given colored bars below them for visual clarity. (B) Modules were correlated with different disease phenotypes.</p

Genetic variation at <i>HrI3</i> contributes to variation in pulmonary edema in an independent set of Collaborative Cross lines.

<p>Following the identification of <i>HrI3</i>, we infected animals from fully inbred Collaborative Cross lines, where each line was homozygous for a single founder allele at <i>HrI3</i>. (A) We found a significant effect of genotype at <i>HrI3</i> on the extent and severity of pulmonary edema at four days post infection. Mild (B) and Severe (C) pulmonary edema can be seen at 200× magnification in animals from this experiment. Pulmonary edema was scored on the basis of evidence of transudates accumulating in the alveolar spaces (denoted by star marks in panel C).</p

Diverse disease-associated phenotypes across the pre-CC population.

<p>Pre-CC mice showed a wide range of variation in phenotypes including D4 weight (Y-axis histogram, A and B), Log titer (X-axis histogram, A) and Airway Inflammation (X-axis histogram, B). In addition, strong correlations existed between D4 weight and both (A) Log titer and (B) Airway Inflammation across the pre-CC population (black diamonds). Despite these correlations, individual pre-CC mice showed unique combinations of disease phenotypes (e.g. low Log titer and severe D4 weight loss) not present in the founder strains of the CC (colored circles: A/J (n = 11) = yellow, C57BL/6J (n = 6) = grey, 129S1/SvImJ (n = 5) = pink, NOD/ShiLtJ (n = 5) = dk. Blue, NZO/HILtJ (n = 12) = lt. blue, CAST/EiJ (n = 5) = green, PWK/PhJ (n = 5) = red, WSB/EiJ (n = 5) = purple).</p

Diverse disease pathologies present across the pre-CC population.

<p>Histopathological examination of lung sections following IAV infection showed a diverse range of phenotypes. Each image is a single 100× magnification image of the lung section of a single pre-CC mouse (strain ID, D4 weight, and log titer (BDL = below detectable limit) are listed over each image). Disease phenotypes were scored for aspects of the damage to, and inflammatory cell infiltration around the airways (A), inflammatory cell infiltration around the vasculature (B), and damage and inflammatory cell infiltration in the alveolar spaces (C). Note that the image of OR219 shows a relatively healthy looking lung, and is useful as a baseline image.</p