Genetic loci regulate Sarbecovirus pathogenesis: A comparison across mice and humans

doi:10.1016/j.virusres.2024.199357

Comparative Study

. 2024 Jun:344:199357.

doi: 10.1016/j.virusres.2024.199357. Epub 2024 Mar 23.

Genetic loci regulate Sarbecovirus pathogenesis: A comparison across mice and humans

Alexandra Schäfer¹, Lisa E Gralinski², Sarah R Leist³, Brea K Hampton⁴, Michael A Mooney⁵, Kara L Jensen³, Rachel L Graham³, Sudhakar Agnihothram³, Sophia Jeng⁶, Steven Chamberlin⁷, Timothy A Bell⁸, D Trevor Scobey³, Colton L Linnertz⁸, Laura A VanBlargan⁹, Larissa B Thackray⁹, Pablo Hock⁸, Darla R Miller¹⁰, Ginger D Shaw¹⁰, Michael S Diamond¹¹, Fernando Pardo Manuel de Villena¹⁰, Shannon K McWeeney¹², Mark T Heise¹³, Vineet D Menachery¹⁴, Martin T Ferris¹⁵, Ralph S Baric¹⁶

Affiliations

¹ Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. Electronic address: aschaefe@email.unc.edu.
² Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. Electronic address: lgralins@email.unc.edu.
³ Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
⁴ Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
⁵ Knight Cancer Institute, Oregon Health & Science University, Portland, OR, USA; Division of Bioinformatics and Computational Biology, Oregon Health & Science University, Portland, OR, USA; Department of Medical Informatics and Clinical Epidemiology, Oregon Health & Science University, Portland, OR, USA.
⁶ Knight Cancer Institute, Oregon Health & Science University, Portland, OR, USA; Oregon Clinical and Translational Research Institute, Oregon Health & Science University, Portland, OR, USA.
⁷ Division of Bioinformatics and Computational Biology, Oregon Health & Science University, Portland, OR, USA; Department of Medical Informatics and Clinical Epidemiology, Oregon Health & Science University, Portland, OR, USA.
⁸ Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
⁹ Department of Medicine, Washington University School of Medicine, St. Louis, MO, USA.
¹⁰ Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
¹¹ Department of Medicine, Washington University School of Medicine, St. Louis, MO, USA; Department of Pathology & Immunology2, Washington University School of Medicine, St. Louis, MO, USA; Department of Molecular Microbiology3, Washington University School of Medicine, St. Louis, MO, USA.
¹² Knight Cancer Institute, Oregon Health & Science University, Portland, OR, USA; Division of Bioinformatics and Computational Biology, Oregon Health & Science University, Portland, OR, USA; Department of Medical Informatics and Clinical Epidemiology, Oregon Health & Science University, Portland, OR, USA; Oregon Clinical and Translational Research Institute, Oregon Health & Science University, Portland, OR, USA.
¹³ Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Rapidly Emerging Antiviral Drug Discovery Initiative, University of North Carolina, Chapel Hill NC, USA.
¹⁴ Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, USA; Institute for Human Infection and Immunity, University of Texas Medical Branch, Galveston TX, USA; Department of Pathology and Center for Biodefense & Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, TX, USA.
¹⁵ Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. Electronic address: mtferris@email.unc.edu.
¹⁶ Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Rapidly Emerging Antiviral Drug Discovery Initiative, University of North Carolina, Chapel Hill NC, USA. Electronic address: rsbaric@email.unc.edu.

PMID: 38508400
PMCID: PMC10981091
DOI: 10.1016/j.virusres.2024.199357

Comparative Study

Genetic loci regulate Sarbecovirus pathogenesis: A comparison across mice and humans

Alexandra Schäfer et al. Virus Res. 2024 Jun.

. 2024 Jun:344:199357.

doi: 10.1016/j.virusres.2024.199357. Epub 2024 Mar 23.

Authors

Affiliations

¹ Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. Electronic address: aschaefe@email.unc.edu.
² Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. Electronic address: lgralins@email.unc.edu.
³ Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
⁴ Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
⁵ Knight Cancer Institute, Oregon Health & Science University, Portland, OR, USA; Division of Bioinformatics and Computational Biology, Oregon Health & Science University, Portland, OR, USA; Department of Medical Informatics and Clinical Epidemiology, Oregon Health & Science University, Portland, OR, USA.
⁶ Knight Cancer Institute, Oregon Health & Science University, Portland, OR, USA; Oregon Clinical and Translational Research Institute, Oregon Health & Science University, Portland, OR, USA.
⁷ Division of Bioinformatics and Computational Biology, Oregon Health & Science University, Portland, OR, USA; Department of Medical Informatics and Clinical Epidemiology, Oregon Health & Science University, Portland, OR, USA.
⁸ Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
⁹ Department of Medicine, Washington University School of Medicine, St. Louis, MO, USA.
¹⁰ Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
¹¹ Department of Medicine, Washington University School of Medicine, St. Louis, MO, USA; Department of Pathology & Immunology2, Washington University School of Medicine, St. Louis, MO, USA; Department of Molecular Microbiology3, Washington University School of Medicine, St. Louis, MO, USA.
¹² Knight Cancer Institute, Oregon Health & Science University, Portland, OR, USA; Division of Bioinformatics and Computational Biology, Oregon Health & Science University, Portland, OR, USA; Department of Medical Informatics and Clinical Epidemiology, Oregon Health & Science University, Portland, OR, USA; Oregon Clinical and Translational Research Institute, Oregon Health & Science University, Portland, OR, USA.
¹³ Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Rapidly Emerging Antiviral Drug Discovery Initiative, University of North Carolina, Chapel Hill NC, USA.
¹⁴ Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, USA; Institute for Human Infection and Immunity, University of Texas Medical Branch, Galveston TX, USA; Department of Pathology and Center for Biodefense & Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, TX, USA.
¹⁵ Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. Electronic address: mtferris@email.unc.edu.
¹⁶ Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Rapidly Emerging Antiviral Drug Discovery Initiative, University of North Carolina, Chapel Hill NC, USA. Electronic address: rsbaric@email.unc.edu.

PMID: 38508400
PMCID: PMC10981091
DOI: 10.1016/j.virusres.2024.199357

Abstract

Coronavirus (CoV) cause considerable morbidity and mortality in humans and other mammals, as evidenced by the emergence of Severe Acute Respiratory CoV (SARS-CoV) in 2003, Middle East Respiratory CoV (MERS-CoV) in 2012, and SARS-CoV-2 in 2019. Although poorly characterized, natural genetic variation in human and other mammals modulate virus pathogenesis, as reflected by the spectrum of clinical outcomes ranging from asymptomatic infections to lethal disease. Using multiple human epidemic and zoonotic Sarbecoviruses, coupled with murine Collaborative Cross genetic reference populations, we identify several dozen quantitative trait loci that regulate SARS-like group-2B CoV pathogenesis and replication. Under a Chr4 QTL, we deleted a candidate interferon stimulated gene, Trim14 which resulted in enhanced SARS-CoV titers and clinical disease, suggesting an antiviral role during infection. Importantly, about 60 % of the murine QTL encode susceptibility genes identified as priority candidates from human genome-wide association studies (GWAS) studies after SARS-CoV-2 infection, suggesting that similar selective forces have targeted analogous genes and pathways to regulate Sarbecovirus disease across diverse mammalian hosts. These studies provide an experimental platform in rodents to investigate the molecular-genetic mechanisms by which potential cross mammalian susceptibility loci and genes regulate type-specific and cross-SARS-like group 2B CoV replication, immunity, and pathogenesis in rodent models. Our study also provides a paradigm for identifying susceptibility loci for other highly heterogeneous and virulent viruses that sporadically emerge from zoonotic reservoirs to plague human and animal populations.

Keywords: Host susceptibility loci; Pathogenesis; SARS-CoV; SARS-CoV-2; Zoonotic CoV.

PubMed Disclaimer

Conflict of interest statement

Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig 1 — **Fig. 1**
Spike phylogeny of representative coronaviruses. The Spike protein sequences of selected coronaviruses were aligned and phylogenetically compared. Coronavirus genera are grouped by classic subgroup designations (1a-b, 2a-d, 3, and 4). PECoV is designated as 1b* because of its distinctive grouping compared with more conserved proteins. Branches in each tree are labeled with consensus support values (in%). The clades for Sarbecoviruses (clades Ia, Ib, and II) are indicated. Sequences were aligned using free end gaps with the Blosum62 cost matrix, and the tree was constructed using the neighbor-joining method based on the multiple sequence alignment in Geneious Prime. Numbers following the underscores in each sequence correspond to the GenBank Accession number. The radial phylogram was exported from Geneious and then rendered for publication using Adobe Illustrator CC 2020.

Fig 2 — **Fig. 2**
**Overview of the individual Collaborative Cross derived mapping crosses used in the studies. a.** The Collaborative Cross is comprised of eight founder strains: A/J, C57BL/6 J, 129S1/SvlmJ, NOD/ItJ, NZO/HILtJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ. An eight-way intercrossing followed by ∼20 sister mating generations resulted in the CC-RI (recombinant inbred) lines. During the sister breeding, so-called pre-CC mice, mice which had not fully reached inbred status yet were used for proof-of-concept experiments. For other mapping studies CC F1 intercrosses (CC-RIX) and CC-F2 crosses were used. The study design for the CC-RIX (b.) and the pre-CC and CC-F2 (c.) mapping studies are shown. Figures were created with Biorender.com.

Fig 3 — **Fig. 3**
**Phenotypic distribution of disease phenotypes after SARS-CoV MA15 and HKU3-MA infection in the CC-RIX panel**. Disease phenotype distribution after infection in the CC-RIX. Groups of 3 mice per CC-RIX strain were infected with either 5 × 10³ PFU SARS-CoV MA15 and followed for 28 days to record disease outcomes (**a-g**). A set of 3 SARS-CoV MA15-infected mice per CC-RIX strain were then used for a heterologous challenged with 1 × 10⁵ PFU HKU3-MA on day 28 post-infection and followed for 4 days for disease (h). a. Percentage survival (0dpi-4dpi, SARS-CoV MA15), b. Lung titer at 2 dpi (SARS-CoV MA15), c. PenH lung function at 2 dpi (respiratory metric for enhanced pause, SARS-CoV MA15), d. Rpef lung function at 2 dpi (respiratory metric for the ratio of time to peak expiratory flow (PEF) relative to total expiratory time, SARS-CoV MA15), total IgG (e) and IgG1 (f) at 32 dpi (SARS-CoV MA15) g. Frequency of lung infiltrating CD8⁺ Dendritic cells at 4 dpi (SARS-CoV MA15), h. Weight loss at 4 dpi (HKU3-MA), Each dot represents the mean value of an individual CC-RIX strain (n = 3 mice per strain).

Fig 4 — **Fig. 4**
**Phenotypic distributions, genomics scans, and allele effects maps for 3 traits across the CC-RIX. a.** (left to right) Log₁₀ SARS-CoV MA15 lung titer at 2 dpi, percentage survival following HKU3-MA infection, and percentage of starting weight at 4 dpi with HKU3-MA. For all 3 panels, the CC-RIX strains are sorted by ascending 2 dpi SARS-CoV MA15 lung titers with each RIX having a row on the x-axis, and the replicate animals from each RIX occupying that row, showing the general lack of correlation between coronavirus disease responses. X-axis legends are over the panels. b. Genome scans showing the LOD traces (see methods), as well as significance thresholds (p = 0.33 (orange) and p = 0.2 (green)) for the same traits listed above. We identified (left to right) *HrS13* (Chr16) for SARS-CoV MA15 titer, *HrS11* (Chr5) and *HrS10* (Chr13) for HKU3-MA mortality, and *HrS12* for HKU3-MA weight loss. c. Allele effect plots showing the estimated phenotypic effect of the founder haplotypes at each of the peaks (shown at the bottom as the zoomed in LOD trace for chromosomes where we mapped QTL) at each of these loci showing causal haplotypes for *HrS13* (where 129S1/SvImJ (pink) and PWK/PhJ (red) alleles cause a reduced titer, *HrS10* where CAST/EiJ (green) and PWK/PhJ (red) alleles cause increased mortality, *HrS11* where a 129S1/SvImJ (pink) allele causes increased mortality, and *HrS12* where a PWK/PhJ (red) allele causes decreased weight loss. d. We identified relationships between *HrS10* and SARS-CoV MA15-related weight loss and clinical disease (shown is weight loss at 4 dpi), where mice which had the PWK or CAST haplotypes were given a score of 1, and all other founder haplotypes given a score of 0), as well as *HrS11* and SARS-CoV MA15 titer (shown here is titer at 2 dpi), 0 = low response haplotype, 1 = 1 copy of the high mortality 129s1 haplotype. Each dot represents data from an individual animal.

Fig 5 — **Fig. 5**
**Identification of major effect locus on chromosome 4 and of *Trim14* as a susceptibility gene during SARS-CoV MA15 and SARS-CoV-2 MA10 infection. a.** Phenotypic distribution and genomic scan for *HrS23* (4dpi weight loss in CC003xCC053-F2), b.HrS23 (4dpi lung congestion score in CC03xCC053-F2), and c. HrS24 (overall mortality in CC011xCC074-F2). d. Lung tissue from *Trim14^Δ47/Δ4*⁷ were found to lack detectable *Trim14* mRNA, likely due to nonsense-mediated decay, as measured by RT-qPCR in comparison to littermates (n = 2 for *Trim14^Δ47/Δ4*⁷ and C57BL/6 J, respectively). e. SNPs in CC003 and CC053 as well as CC011 and CC074 in the *Trim14* gene; Contributing haplotypes: CC003, WSB and CC053, CAST; CC011, PWK and CC074, S129; UTR- untranslated region. Infection of *Trim14^Δ47/Δ47* mice (n = 25; n = 9 for 2 and 4dpi, n = 7 for 7dpi) with SARS-CoV MA15 showed significantly more weight loss (f.) and an increase in viral load (g.) and congestion score in the lung (h.) compared to C57BL/6 J mice (n = 20; n = 10 for 2dpi, n = 6 for 4dpi, n = 4 for 7dpi) over the course of a 7-day infection. i. Cytokine/chemokine distribution in the lung of *Trim14^Δ47/Δ47* and C57BL/6 J at 4dpi of infection (n = 6 for *Trim14^Δ47/Δ4* and n = 6 for C57BL/6 J). j. Composition of lung infiltrating immune cells in the lung of *Trim14^Δ47/Δ4* mice and C57BL/6NJ control mice (n = 3 for each). A similar trend of infection progression was observed in *Trim14^Δ47/Δ47* mice infected with SARS-CoV-2 MA10; n = 10 *Trim14^Δ47/Δ47* and n = 10 C57BL/6 J for weight loss (k.), viral load (l.), and congestion score (m.). Data was analyzed via student t-test, *p < 0.05, **p < 0.01, ***p < 0.005).

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References

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[1] Abiola O., et al. The nature and identification of quantitative trait loci: a community's view. Nat. Rev. Genet. 2003;4(11):911–916. - PMC - PubMed

[2] Abiola O., et al. The nature and identification of quantitative trait loci: a community's view. Nat. Rev. Genet. 2003;4(11):911–916. - PMC - PubMed

[3] Ahmad T., et al. Coronavirus Disease 2019 (COVID-19) pandemic and economic impact. Pak. J. Med. Sci. 2020;36(COVID19–S4):S73–S78. - PMC - PubMed

[4] Ahmad T., et al. Coronavirus Disease 2019 (COVID-19) pandemic and economic impact. Pak. J. Med. Sci. 2020;36(COVID19–S4):S73–S78. - PMC - PubMed

[5] Annan A., et al. Human betacoronavirus 2c EMC/2012-related viruses in bats, Ghana and Europe. Emerg. Infect. Dis. 2013;19(3):456–459. - PMC - PubMed

[6] Annan A., et al. Human betacoronavirus 2c EMC/2012-related viruses in bats, Ghana and Europe. Emerg. Infect. Dis. 2013;19(3):456–459. - PMC - PubMed

[7] Anthony S.J., et al. Further evidence for bats as the evolutionary source of middle east respiratory syndrome Coronavirus. mBio. 2017;8(2) - PMC - PubMed

[8] Anthony S.J., et al. Further evidence for bats as the evolutionary source of middle east respiratory syndrome Coronavirus. mBio. 2017;8(2) - PMC - PubMed

[9] Aquino Y., et al. Dissecting human population variation in single-cell responses to SARS-CoV-2. Nature. 2023;621(7977):120–128. - PMC - PubMed

[10] Aquino Y., et al. Dissecting human population variation in single-cell responses to SARS-CoV-2. Nature. 2023;621(7977):120–128. - PMC - PubMed

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Genetic loci regulate Sarbecovirus pathogenesis: A comparison across mice and humans

Affiliations

Genetic loci regulate Sarbecovirus pathogenesis: A comparison across mice and humans

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Abstract

Conflict of interest statement

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Conflict of interest statement

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