MBL deficiency does not affect inflammatory cell recruitment, but alters expression of inflammatory mediators within the RRV infected muscle.

<p>(A–B) Leukocytes were isolated from the quadriceps muscle of RRV-infected WT (solid circles, n = 3–5/time point) or MBL-DKO (open circles, n = 3–7/time point) mice at 7 or 10 dpi. Cells were characterized and quantified by flow cytometry using the markers described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002586#s4" target="_blank">materials and methods</a>. Total numbers of leukocytes (A) and specific cells types (B) are shown. A single experiment for each time point is shown. Compiled data for three independent experiments is shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002586#ppat.1002586.s004" target="_blank">Fig. S4</a> (7 dpi). *p<0.05 by Mann-Whitney analysis. (C) Relative mRNA expression of C3-dependent inflammatory mediators and cytokines S100A9, S100A8, Arginase I, and IL-6 (top panel) and C3-independent cytokines TNFα, IL-1β, and IL-10 (bottom panel) from quadriceps muscle from RRV-infected WT (solid bar, n = 3) or MBL-DKO (open bar, n = 3) mice by quantitative real-time PCR. Raw data values were normalized to 18S rRNA levels, log-transformed, and are graphically depicted as fold expression over mock-infected mice. Data from a single experiment is shown, but is representative of two independent experiments. ***p<0.001; **p<0.005 by t-test.</p

RRV infection induces MBL deposition onto cells, resulting in MBL-dependent C3 deposition onto infected tissues.

<p>(A) To determine if MBL levels are elevated within the quadriceps muscles of RRV-infected wild-type animals, homogenized quadriceps muscles from either mock- or RRV-infected WT mice at 7 dpi were analyzed by immunoblot analysis using anti-mouse MBL-A, anti-mouse MBL-C, or anti-mouse actin antibodies. Each lane represents an individual mouse and is representative of at least three independent experiments. Densitometry measurements of bands in immunoblot from three different experiments are graphically depicted as arbitrary units normalized to actin (mock n = 4; RRV n = 9). (B) To determine if MBL deposition is enhanced onto RRV-infected cell, differentiated C2C12 murine skeletal muscle cells were infected with RRV, and incubated with either serum from a WT or MBL-DKO mouse for 30 minutes prior to harvesting. Cells were washed, harvested, and lysates were analyzed by immunoblot analysis using anti-mouse MBL-C, anti-RRV, or anti-mouse actin antibodies. Densitometry measurements of bands in immunoblot are graphically depicted as arbitrary units normalized to actin (C) C3 deposition was assessed by IHC using an anti-mouse C3 antibody on quadriceps muscle sections from either mock- or RRV-infected WT or MBL-DKO mice at 7 dpi. C3 positive areas are stained in blue. A representative section from each strain is shown and is representative of two independent experiments. No signal was observed in sections incubated with a control goat IgG antibody.</p

MBL is required for development of severe RRV-induced disease and tissue damage.

<p>(A–B) Twenty-four day old WT C57BL/6 (solid square, n = 6), C1q^−/− (open square, n = 6), fB^−/− (solid circle, n = 5), or MBL-DKO (open circle, n = 6) mice were infected with RRV, scored for hind limb function based on a scale described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002586#s4" target="_blank">Materials and Methods</a>, and assessed for weight loss. Each data point represents the arithmetic mean ±SD and is representative of at least three independent experiments. We performed a Mann-Whitney analysis with multiple comparison corrections (p<0.01 was considered significant) on clinical scores at 10 dpi and a one-way ANOVA analysis with Bonferroni's correction on percent of starting weight at 10 dpi (p<0.05 was considered significant) to determine significance between the various knock-out lines compared to WT **p<0.01; *** p<0.001; n.s. not significant. (C) Tissue pathology and inflammation was examined at 10 dpi by H&E staining of paraffin embedded sections of quadriceps muscle. A representative section from each knockout strain is shown. A section from RRV-infected C3^−/− is shown for comparison. Solid arrowheads point to areas of inflammation; open arrowheads indicate tissue damage (D) To assess damage within the muscle, mock or RRV-infected WT or MBL-DKO mice were injected with EBD at 10 dpi, and frozen sections were generated. EBD positive muscle fibers were identified by fluorescence microscopy. Representative sections of mock- and RRV-infected mice are shown and are representative of two independent experiments.</p

MBL deficiency does not affect viral replication or tropism within infected tissues.

<p>(A–C) Quadriceps muscle (A), ankle joints (B), and serum (C) from RRV-infected WT (solid circles, n = 3–9/time point) or MBL-DKO (open circles, n = 3–8/time point) mice were assayed to determine viral titer at various times post infection. Viral titer was determined by plaque assay on BHK-21 cells. Each data point represents the viral titer from a single animal; data is combined from two independent experiments. *p<0.05 as determined by t-test. (D) Tropism within the quadriceps muscle tissue was determined by <i>in situ</i> hybridization using RRV-specific probe. We did not detect any signal using an EBER-specific probe (data not shown). A representative section from each strain is shown (n = 3 for both WT and MBL-DKO mice).</p

Lung pathology in select preCC mice.

<p>(A). OR63f51—normal parenchyma. (B). OR181f61—airway debris and cuffing and edema surrounding the associated vasculature. (C) OR220f57—denuded airway blocked with debris. (D) OR380f64 –perivascular cuffing including eosinophilia. (E) OR941f69 –alveolitis including hyaline membrane formation, arrows point to hyaline membranes. (F) OR5030f128 –normal airway and associated vasculature.</p

Phenotypic relationships.

<p>Heat map of the relationships between lung pathology, titer and weight loss in the preCC. Yellow indicates positive correlation and blue indicates negative correlation.</p

PreCC and founder strain phenotypes.

<p>(A) Weight loss is shown as percent of starting weight at day four post infection, individual preCC mice are shown in open diamonds and mean values for founders are shown in color. All CAST/EiJ mice died or were humanely euthanized before day four post infection. (B) Log transformed lung titer at day four post infection in individual preCC mice and founders, dashed line indicates the limit of detection at 100 PFU per lung. Individual preCC mice are shown in open diamonds, mean values for the founders are color coded by strain. (C) Lung titer vs. weight loss at day four post infection. Individual preCC mice are indicated in open diamonds, mean values for the founder strains are shown in color. The dashed line indicates the limit of detection at 100 PFU per lung.</p

Transcriptional analysis.

<p>(A) A heat map showing a comparison of functionally enriched biological pathways between <i>Trim55</i>^<i>-/-</i> and C57BL/6J mice at days two and four post infection based on RNA expression levels in the lung. Three mice used per condition, per timepoint. Experiment performed once. Relative expression of all DE genes (log2 FC of 1 or greater and FDR < .05, Trim55^-/- vs C57BL/6J) involved in granulocyte adhesion and diapedesis at day two (B) or four (C) post infection.</p

SARS QTL details.

<p>The four SARS-CoV susceptibility QTLs with genome region, LOD score, significance and percent of phenotypic variation.</p

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