A Multitrait Locus Regulates Sarbecovirus Pathogenesis

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Infectious diseases have shaped the human population genetic structure, and genetic variation influences the susceptibility to many viral diseases. However, a variety of challenges have made the implementation of traditional human Genome-wide Association Studies (GWAS) approaches to study these infectious outcomes challenging. In contrast, mouse models of infectious diseases provide an experimental control and precision, which facilitates analyses and mechanistic studies of the role of genetic variation on infection. Here we use a genetic mapping cross between two distinct Collaborative Cross mouse strains with respect to SARS-CoV disease outcomes. We find several loci control differential disease outcome for a variety of traits in the context of SARS-CoV infection. Importantly, we identify a locus on mouse Chromosome 9 that shows conserved synteny with a human GWAS locus for SARS-CoV-2 severe disease. We follow-up and confirm a role for this locus, and identify two candidate genes, CCR9 and CXCR6 that both play a key role in regulating the severity of SARS-CoV, SARS-CoV-2 and a distantly related bat sarbecovirus disease outcomes. As such we provide a template for using experimental mouse crosses to identify and characterize multitrait loci that regulate pathogenic infectious outcomes across species.

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  1. SciScore for 10.1101/2022.06.01.494461: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    EthicsField Sample Permit: All virological studies were conducted under BSL3 conditions, and personnel wore appropriate personal protective gear.
    IACUC: Mouse studies and in vivo infections: Mouse studies were performed at the University of North Carolina (Animal Welfare Assurance #A3410-01) using protocols approved by the UNC Institutional Animal Care and Use Committee (IACUC).
    Sex as a biological variableFirst, F1 mice between CC011 and CC074 were generated by cross males and females in both directions, and then the F2 mice were bred in all 4 possible F1 x F1 combinations, to ensure appropriately balanced sex Chromosome and parent-of-origin effects.
    RandomizationF2 mice (226 males, 177 females) were weaned such that littermates were randomized to different experimental cages to reduce litter- or batch-effects on the study, and mice were transferred at 5-6 weeks of age to the laboratory for infection between 9-12 weeks of age.
    BlindingFor Matute-Bello scoring samples were blinded and three random fields of lung tissue were chosen and scored for the following: (A) neutrophils in alveolar space (none = 0, 1–5 cells = 1, > 5 cells = 2), (B) neutrophils in interstitial space (none = 0, 1–5 cells = 1, > 5 cells = 2), (C) hyaline membranes (none = 0, one membrane = 1, > 1 membrane = 2), (D) Proteinaceous debris in air spaces (none = 0, one instance = 1, > 1 instance = 2), (E) alveolar septal thickening (< 2Å∼ mock thickness = 0, 2–4Å∼ mock thickness = 1, > 4Å∼ mock thickness = 2).
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Cells were incubated with antibodies against the following markers: efluor506 Viability Dye (Thermo Fisher, 65-0866-14), BUV395 anti-CD45 (Clone 30-F11, BD Biosciences), BV711
    suggested: (BD Biosciences Cat# 740725, RRID:AB_2740403)
    Experimental Models: Cell Lines
    For virus titration, the caudal lobe of the right lung was homogenized in PBS, resulting homogenate was serial-diluted and inoculated onto confluent monolayers of Vero E6 cells (ATCC CCL-81), followed by agarose overlay.
    Vero E6
    suggested: None
    Experimental Models: Organisms/Strains
    For studies in genetically defined knockout mice, 15-week old CCR9-/- mice (strain 027041), 15-week old CXCR6-/- mice (strain 005693) 15-week old female C57BL/6NJ mice (strain 005304), and 15-week old C57BL/6J (strain 000664) were purchased from Jackson Laboratory, and the genotype of these mutant mice were confirmed via genotyping on the MiniMUGA array (Neogen, Inc.
    suggested: None
    suggested: None
    suggested: RRID:IMSR_JAX:005304)
    suggested: RRID:IMSR_JAX:000664)
    CXCR6-/-, CCR9-/-, and appropriate C57BL/6 control mice were inoculated intranasally with 1×105 PFU of either SARS-CoV MA15, SARS-CoV-2 MA10, or HKU3-SRBD MA in 50 μl of PBS. Body weight, mortality, and pulmonary function by whole body plethysmography (56) were monitored daily as indicated.
    suggested: None
    Software and Algorithms
    Cells were incubated with antibodies against the following markers: efluor506 Viability Dye (Thermo Fisher, 65-0866-14), BUV395 anti-CD45 (Clone 30-F11, BD Biosciences), BV711
    BD Biosciences
    suggested: (BD Biosciences, RRID:SCR_013311)
    Data were acquired on a flow cytometer (BD-X20; BD Biosciences) and analyzed using FlowJo software (Tree Star) (Figure S5).
    suggested: (FlowJo, RRID:SCR_008520)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.