Allelic Variation in the Toll-Like Receptor Adaptor Protein Ticam2 Contributes to SARS-Coronavirus Pathogenesis in Mice

Abstract

Host genetic variation is known to contribute to differential pathogenesis following infection. Mouse models allow direct assessment of host genetic factors responsible for susceptibility to Severe Acute Respiratory Syndrome coronavirus (SARS-CoV). Based on an assessment of early stage lines from the Collaborative Cross mouse multi-parent population, we identified two lines showing highly divergent susceptibilities to SARS-CoV: the resistant CC003/Unc and the susceptible CC053/Unc. We generated 264 F2 mice between these strains, and infected them with SARS-CoV. Weight loss, pulmonary hemorrhage, and viral load were all highly correlated disease phenotypes. We identified a quantitative trait locus of major effect on chromosome 18 (27.1-58.6 Mb) which affected weight loss, viral titer and hemorrhage. Additionally, each of these three phenotypes had distinct quantitative trait loci [Chr 9 (weight loss), Chrs 7 and 12 (virus titer), and Chr 15 (hemorrhage)]. We identified Ticam2, an adaptor protein in the TLR signaling pathways, as a candidate driving differential disease at the Chr 18 locus. Ticam2^-/- mice were highly susceptible to SARS-CoV infection, exhibiting increased weight loss and more pulmonary hemorrhage than control mice. These results indicate a critical role for Ticam2 in SARS-CoV disease, and highlight the importance of host genetic variation in disease responses.

Keywords: Collaborative Cross; F2; MPP; Multi-parent Advanced Generation Inter-Cross (MAGIC); SARS-CoV; Ticam2; host susceptibility genes; multiparental populations.

Figure 1

preCC parent and F1 phenotypes. preCC mice from lines 3067 (n = 1) and 773 (n = 2) were infected with 10⁵ PFU of MA15 and followed for overall pathogenesis as measured by weight loss relative to day zero (A) and virus titer in the lung at day four (B). Two animals from line 773 were received; however, one succumbed to infection at day 3 postinfection. Weight loss (C) and titer (D) were tested in reciprocal F1 mice [CC003×CC053 (n = 7 for WL and n = 4 for titer) and CC053×CC003 (n = 14 for WL and n = 12 for titer)].

Figure 2

F2 phenotypes. F2 mice were infected with 10⁵ PFU of MA15 and monitored for 4 d. Percent starting weight as measured at day 4 is shown in (A) and virus titer is shown in (B). A significant correlation was observed between weight loss and titer as shown in (C). Pulmonary hemorrhage was scored at the time of harvest and is shown in (D).

Figure 3

SARS QTL. QTL analysis using the F2 phenotypes and genotypes revealed multiple QTL. The dashed line indicates a significance value of 0.05 as determined by permutation test.

Figure 4

Interactions between loci driving viral titer responses. (A) Interactions between HrS7 and HrS8. (B) Interactions between HrS7 and HrS5. In both figures, the y-axis is viral titers (log10), while the x-axis shows the HrS7 genotype (A/A= CC003 homozygous A/B is heterozygous, B/B is CC053 homozygous). Within each x-axis class the genotypes of HrS8 (A) or HrS5 (B) are binned left to right (CC003/CC003; CC003/CC053; CC053/CC053).

Figure 5

Allele effects. Phenotypes were broken out based on a homozygous CC053/Unc genotype, a homozygous CC003/Unc genotype, or a heterozygous genotype for day 3 and day 4 weight loss, log₁₀ viral titer, and hemorrhage at the chromosome 18 QTL.

Figure 6

Ticam2 knockouts. Ticam2^−/− (n = 14) and C57BL/6J (n = 12) mice were infected with 10⁵ PFU of MA15 for 4 d. Weight loss data (A) confirms our previously published results (Totura et al. 2015). Vascular cuffing in the lung was scored in a blinded manner (B) and pulmonary hemorrhage was scored at the time of tissue harvest (C). Asterisks indicate a P value < 0.05.