Reference
The Collaborative Cross (CC) is a large (~1,000 line) panel of recombinant inbred (RI) mouse strains being developed through a community effort (Churchill et al. 2004). The CC combines the genomes of eight genetically diverse founder strains A/J, C57BL/6J, 129S1/SvImJ, NOD/LtJ, NZO/HlLtJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ to capture nearly 90% of the known variation present in laboratory mice. CC strains are derived using a unique funnel breeding scheme. Once inbred, the RI CC lines can be used to generate thousands of potential 'outbred' but completely reproducible genomes through the generation of recombinant inbred crosses (RIX). The designation 'PreCC' is used to describe a mapping population of CC mice that is still at incipient stages of inbreeding.
CTC (2004), Churchill, G. A., et al.. The Collaborative Cross, a community resource for the genetic analysis of complex traits. Nat Genet. 36, 1133-7.
Experiment
Severe Acute Respiratory Coronavirus (SARS-CoV) emerged in humans in Southeast Asia in 2002 and 2003 after evolving from related coronaviruses circulating in bats. SARS-CoV caused an atypical pneumonia that was fatal in 10% of all patients and 50% of elderly patients. Patients infected with SARS-CoV experienced fever, difficulty breathing and low blood oxygen saturation levels. Severe cases developed diffuse alveolar damage (DAD) and acute respiratory distress syndrome (ARDS) and disease severity was positively associated with increased age. Host genetic background is also thought to influence disease severity but this understanding is complicated by inconsistent sample collection, varying treatment regimens and the limited scope of the SARS epidemic in humans. Existing animal models of SARS-CoV infection have revealed that this lethal pulmonary infection causes a denuding bronchiolitis and severe pneumonia which oftentimes progresses to acute respiratory failure.
Using genetically diverse animals from incipient lines of the Collaborative Cross (CC) mouse panel, the authors demonstrate a greatly expanded range of phenotypes relative to classical mouse models of SARS-CoV infection including lung pathology, weight loss and viral titer. Genetic mapping revealed several loci contributing to differential disease responses.
Mice from the eight founder strains as well as 147 eight- to twenty-week old female PreCC#/Unc mice were infected with 10^5 plaque forming units (PFU) of mouse adapted SARS-CoV, designated MA15. One PreCC#/Unc mouse per line was infected, and for the founder strains at day four n = 2 (A/J), n = 3 (C57BL/6J, 128S1/SvImJ, NOD/ShiLtJ, CAST/EiJ, PWK/PhJ and WSB/EiJ) and n = 5 (NZO/HILtJ). Weight loss was observed over the course of a four day infection. At day four post infection mice were euthanized and tissue collected for assessment of viral load in the lung as well as virus-induced inflammation and pathology.
A wide range of susceptibilities to SARS-CoV infection was found among the founder strains of the CC and the overall heritability of weight changes following SARS-CoV infection determined to have a coefficient of genetic determination of 0.72. NOD/ShiLtJ mice were resistant to infection and gained weight over the course of the experiment. A/J, C57BL/6J, 129S1/SvImJ, and NZO/HILtJ mice experienced moderate and transient weight loss as previously described, while CAST/EiJ, PWK/PhJ and WSB/EiJ mice demonstrated extreme susceptibility to SARS-CoV infection including substantial weight loss and death.
Subsequent dose response studies using the three highly susceptible wild-derived strains indicated an LD50 of between 100 and 500 PFU for CAST/EiJ mice, between 500 PFU and 1000 PFU for PWK/PhJ and between 10^3 and 10^5 PFU for WSB/EiJ mice. PreCC#/Unc mice infected with SARS-CoV ranged from over 30% weight loss by day four post infection to over 10% weight gain, exceeding the range of susceptibilities observed in the founder strains. Additionally, 26 PreCC#/Unc mice (18% of the PreCC#/Unc cohort) succumbed to infection prior to the day four harvest point indicating extreme susceptibility to SARS-CoV infection.
Viral load in the lung at day four post infection was determined for each surviving PreCC#/Unc mouse as well as for each of the founder strains. Viral lung titers showed a heritability of 0.60 as measured by the coefficient of genetic determination amongst the 7 surviving founder strains. Amongst the founder strains, PWK/PhJ mice had the lowest viral loads in the lungs, with 1.75 x 10^3 PFU per lung at day four post infection. PWK/PhJ mice also showed significant weight loss and a low LD50 indicating that viral load was unlikely to be responsible for pathogenesis in these mice. In contrast, C57BL/6J mice had the highest amount of virus at 6.35 x 10^6 PFU per lung. Lung titers in the PreCC#/Unc mice ranged from below the limit of detection (100 PFU/lung) to over 10^8 PFU per lung, greatly exceeding the range of viral loads in the founder strains. Some PreCC#/Unc mice had viral loads in the lung below the 100 PFU limit of detection, despite having substantial weight loss. CAST/EiJ mice are extremely susceptible to SARS-CoV infection and do not survive until the day four post infection timepoint.
The authors genotyped 140 PreCC#/Unc animals at high density, including several that succumbed to infection prior to the scheduled day four harvest. Each PreCC#/Unc animal was genotyped using the Mouse Diversity Array (Affymetrix) at 372,249 well-performing SNPs which were polymorphic across the founder strains. Once genotypes were determined, founder strain haplotype probabilities were computed for all genotyped loci using the HAPPY algorithm. Genetic map positions were based on the integrated mouse genetic map using mouse genome build NCBI Build 37.
QTL mapping was conducted to identify host genome regions containing polymorphisms significantly associated with SARS-induced disease responses. Mapping was performed using the BAGPIPE package to regress each phenotype on the computed haplotypes in the interval between adjacent genotype markers, producing a LOD score in each interval to evaluate significance. For the likely regions of identified QTL peaks, SNP data for the eight founder strains from the Sanger Institute Mouse Genomes Project were downloaded and analyzed as described in Ferris et al. (2013).
Four novel QTL for SARS-related phenotypes were identified:
Hrsq1 (host response to SARS QTL 1, vascular cuffing) maps to Chr 3: 18286790 - 26668414 with genome-wide significance and a peak LOD score of 7.79904. HrS1 accounts for 26% of trait variation. C57BL/6J and WSB/EiJ haplotypes increase vascular cuffing relative to the haplotypes of the other six founder strains at Hrsq1.
Hrsq2 (host response to SARS QTL 2, titer) maps to Chr 16: 31583769 - 36719997 with highly suggestive significance and a peak LOD score of 7.26072. Hrsq2 accounts for 22% of trait variation. The PWK/PhJ haplotype reduces viral titer at Hrsq2.
Hrsq3 (host response to SARS QTL 3, eosinophilia) maps to Chr 15: 72103120 - 75803414 with highly suggestive significance and a peak LOD score of 7.43370. Hrsq3 accounts for 26% of trait variability. The A/J haplotype increases eosinophillic infiltration at Hrsq3.
Hrsq4 (host response to SARS QTL 4, vascular cuffing) maps to Chr 13: 52822984 - 54946286 with genome-wide significance a peak LOD score of 9.06019. Hrsq4 accounts for 21% of trait variability. The CAST/EiJ haplotype reduces vascular cuffing at Hrsq4. Hrsq4 has a moderate peak even without considering Hrsq1 status, suggesting that the Hrsq4 and Hrsq1 interactions are additive.
Analysis of other phenotypes did not lead to discovery of QTL passing the p < 0.01 significance threshold.
Integrating phenotypic and genetic data narrowed Hrsq1 to a single gene, Trim55, an E3 ubiquitin ligase with a role in muscle fiber maintenance. Lung pathology and transcriptomic data from mice genetically deficient in Trim55 were used to validate its role in SARS-CoV-induced vascular cuffing and inflammation.
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