Genetic loci regulate Sarbecovirus pathogenesis: A comparison across mice and humans - PubMed This site needs JavaScript to work properly. Please enable it to take advantage of the complete set of features! Clipboard, Search History, and several other advanced features are temporarily unavailable. Skip to main page content An official website of the United States government Here's how you know The .gov means it’s official. Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site. The site is secure. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely. Log in Show account info Close Account Logged in as: username Dashboard Publications Account settings Log out Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation Search: Search Advanced Clipboard User Guide Save Email Send to Clipboard My Bibliography Collections Citation manager Display options Display options Format Abstract PubMed PMID Save citation to file Format: Summary (text) PubMed PMID Abstract (text) CSV Create file Cancel Email citation Email address has not been verified. Go to My NCBI account settings to confirm your email and then refresh this page. To: Subject: Body: Format: Summary Summary (text) Abstract Abstract (text) MeSH and other data Send email Cancel Add to Collections Create a new collection Add to an existing collection Name your collection: Name must be less than 100 characters Choose a collection: Unable to load your collection due to an error Please try again Add Cancel Add to My Bibliography My Bibliography Unable to load your delegates due to an error Please try again Add Cancel Your saved search Name of saved search: Search terms: Test search terms Would you like email updates of new search results? Saved Search Alert Radio Buttons Yes No Email: (change) Frequency: Monthly Weekly Daily Which day? The first Sunday The first Monday The first Tuesday The first Wednesday The first Thursday The first Friday The first Saturday The first day The first weekday Which day? Sunday Monday Tuesday Wednesday Thursday Friday Saturday Report format: Summary Summary (text) Abstract Abstract (text) PubMed Send at most: 1 item 5 items 10 items 20 items 50 items 100 items 200 items Send even when there aren't any new results Optional text in email: Save Cancel Create a file for external citation management software Create file Cancel Your RSS Feed Name of RSS Feed: Number of items displayed: 5 10 15 20 50 100 Create RSS Cancel RSS Link Copy Full text links Elsevier Science Free PMC article Full text links Actions Cite Collections Add to Collections Create a new collection Add to an existing collection Name your collection: Name must be less than 100 characters Choose a collection: Unable to load your collection due to an error Please try again Add Cancel Permalink Permalink Copy Display options Display options Format AbstractPubMedPMID Page navigation Title & authors Abstract Conflict of interest statement Figures References Publication types MeSH terms Substances Grants and funding LinkOut - more resources Title & authors Abstract Conflict of interest statement Figures References Publication types MeSH terms Substances Grants and funding LinkOut - more resources Comparative Study Virus Res Actions Search in PubMed Search in NLM Catalog Add to Search . 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 Schäfer  1 , Lisa E Gralinski  2 , Sarah R Leist  3 , Brea K Hampton  4 , Michael A Mooney  5 , Kara L Jensen  3 , Rachel L Graham  3 , Sudhakar Agnihothram  3 , Sophia Jeng  6 , Steven Chamberlin  7 , Timothy A Bell  8 , D Trevor Scobey  3 , Colton L Linnertz  8 , Laura A VanBlargan  9 , Larissa B Thackray  9 , Pablo Hock  8 , Darla R Miller  10 , Ginger D Shaw  10 , Michael S Diamond  11 , Fernando Pardo Manuel de Villena  10 , Shannon K McWeeney  12 , Mark T Heise  13 , Vineet D Menachery  14 , Martin T Ferris  15 , Ralph S Baric  16 Affiliations Expand 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 Item in Clipboard Comparative Study Genetic loci regulate Sarbecovirus pathogenesis: A comparison across mice and humans Alexandra Schäfer et al. Virus Res. 2024 Jun. Show details Display options Display options Format Abstract PubMed PMID Virus Res Actions Search in PubMed Search in NLM Catalog Add to Search . 2024 Jun:344:199357. doi: 10.1016/j.virusres.2024.199357. Epub 2024 Mar 23. Authors Alexandra Schäfer  1 , Lisa E Gralinski  2 , Sarah R Leist  3 , Brea K Hampton  4 , Michael A Mooney  5 , Kara L Jensen  3 , Rachel L Graham  3 , Sudhakar Agnihothram  3 , Sophia Jeng  6 , Steven Chamberlin  7 , Timothy A Bell  8 , D Trevor Scobey  3 , Colton L Linnertz  8 , Laura A VanBlargan  9 , Larissa B Thackray  9 , Pablo Hock  8 , Darla R Miller  10 , Ginger D Shaw  10 , Michael S Diamond  11 , Fernando Pardo Manuel de Villena  10 , Shannon K McWeeney  12 , Mark T Heise  13 , Vineet D Menachery  14 , Martin T Ferris  15 , Ralph S Baric  16 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 Item in Clipboard Full text links Cite Display options Display options Format AbstractPubMedPMID 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. Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved. 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 Spike phylogeny of representative coronaviruses.… Fig. 1 Spike phylogeny of representative coronaviruses. The Spike protein sequences of selected coronaviruses were… 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 Overview of the individual Collaborative… Fig. 2 Overview of the individual Collaborative Cross derived mapping crosses used in the studies.… 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 Phenotypic distribution of disease phenotypes… Fig. 3 Phenotypic distribution of disease phenotypes after SARS-CoV MA15 and HKU3-MA infection in the… 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 Phenotypic distributions, genomics scans, and… Fig. 4 Phenotypic distributions, genomics scans, and allele effects maps for 3 traits across the… 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 Identification of major effect locus… Fig. 5 Identification of major effect locus on chromosome 4 and of Trim14 as a… 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 Trim14Δ47/Δ47 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 Trim14Δ47/Δ47 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 Trim14Δ47/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 Trim14Δ47/Δ47 and C57BL/6 J at 4dpi of infection (n = 6 for Trim14Δ47/Δ4 and n = 6 for C57BL/6 J). j. Composition of lung infiltrating immune cells in the lung of Trim14Δ47/Δ4 mice and C57BL/6NJ control mice (n = 3 for each). A similar trend of infection progression was observed in Trim14Δ47/Δ47 mice infected with SARS-CoV-2 MA10; n = 10 Trim14Δ47/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). See this image and copyright information in PMC References 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 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 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 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 Aquino Y., et al. Dissecting human population variation in single-cell responses to SARS-CoV-2. Nature. 2023;621(7977):120–128. - PMC - PubMed Show all 54 references Publication types Research Support, N.I.H., Extramural Actions Search in PubMed Search in MeSH Add to Search Research Support, Non-U.S. Gov't Actions Search in PubMed Search in MeSH Add to Search Comparative Study Actions Search in PubMed Search in MeSH Add to Search MeSH terms Animals Actions Search in PubMed Search in MeSH Add to Search COVID-19 / virology Actions Search in PubMed Search in MeSH Add to Search Coronavirus Infections / genetics Actions Search in PubMed Search in MeSH Add to Search Coronavirus Infections / virology Actions Search in PubMed Search in MeSH Add to Search Disease Models, Animal Actions Search in PubMed Search in MeSH Add to Search Genome-Wide Association Study Actions Search in PubMed Search in MeSH Add to Search Humans Actions Search in PubMed Search in MeSH Add to Search Mice Actions Search in PubMed Search in MeSH Add to Search Quantitative Trait Loci* Actions Search in PubMed Search in MeSH Add to Search SARS-CoV-2 / genetics Actions Search in PubMed Search in MeSH Add to Search Tripartite Motif Proteins / genetics Actions Search in PubMed Search in MeSH Add to Search Virus Replication Actions Search in PubMed Search in MeSH Add to Search Substances Tripartite Motif Proteins Actions Search in PubMed Search in MeSH Add to Search Grants and funding R01 AI157155/AI/NIAID NIH HHS/United States R21 AI145372/AI/NIAID NIH HHS/United States P01 AI167966/AI/NIAID NIH HHS/United States K00 AG073570/AG/NIA NIH HHS/United States U01 AI149644/AI/NIAID NIH HHS/United States U19 AI100625/AI/NIAID NIH HHS/United States R01 AI157253/AI/NIAID NIH HHS/United States U19 AI109761/AI/NIAID NIH HHS/United States UL1 TR002369/TR/NCATS NIH HHS/United States R00 AG049092/AG/NIA NIH HHS/United States Show all 10 grants LinkOut - more resources Full Text Sources Elsevier Science Europe PMC PubMed Central Molecular Biology Databases Mouse Genome Informatics (MGI) Miscellaneous NCI CPTAC Assay Portal Full text links [x] Elsevier Science Free PMC article [x] Cite Copy Download .nbib .nbib Format: AMA APA MLA NLM Send To Clipboard Email Save My Bibliography Collections Citation Manager [x] NCBI Literature Resources MeSH PMC Bookshelf Disclaimer The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). Unauthorized use of these marks is strictly prohibited. Follow NCBI Twitter Facebook LinkedIn GitHub Connect with NLM Twitter SM-Facebook SM-Youtube National Library of Medicine 8600 Rockville Pike Bethesda, MD 20894 Web Policies FOIA HHS Vulnerability Disclosure Help Accessibility Careers NLM NIH HHS USA.gov