Premature aging effects on COVID-19 pathogenesis: new insights from mouse models

Premature aging effects on COVID-19 pathogenesis: new insights from mouse models | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Premature aging effects on COVID-19 pathogenesis: new insights from mouse models Wu Haoyu, Liu Meiqin, Sun Jiaoyang, Hong Guangliang, Lin Haofeng, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4316933/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Aug, 2024 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Aging is identified as a significant risk factor for severe coronavirus disease-2019 (COVID-19), often resulting in profound lung damage and mortality. Yet, the biological relationship between aging, aging-related comorbidities, and COVID-19 remains incompletely understood. This study aimed to elucidate the age-related COVID19 pathogenesis using a Hutchinson-Gilford progeria syndrome (HGPS) mouse model with humanized ACE2 receptors. Pathological features were compared between young, aged, and HGPS hACE2 mice following SARS-CoV-2 challenge. We demonstrated that young mice display robust interferon response and antiviral activity, whereas this response is attenuated in aged mice. Viral infection in aged mice results in severe respiratory tract bleeding, likely contributing a higher mortality rate. In contrast, HGPS hACE2 mice exhibit milder disease manifestations characterized by minor immune cell infiltration and dysregulation of multiple metabolic processes. Comprehensive transcriptome analysis revealed both shared and unique gene expression dynamics among different mouse groups. Collectively, our studies evaluated the impact of SARS-CoV-2 infection on progeroid syndromes using a HGPS hACE2 mouse model, which holds promise as a useful tool for investigating COVID-19 pathogenesis in individuals with premature aging. Biological sciences/Immunology/Infection Health sciences/Diseases/Infectious diseases/Viral infection Biological sciences/Physiology/Ageing HGPS Aging hACE2 mice SARS-CoV-2 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Aging is a natural biological process characterized by the gradual decline of physiological functions across multiple tissues 1 , 2 . Several aging-related phenotypes serve as primary risk factors for various diseases including cancer, diabetes, neurodegeneration, cardiovascular diseases, and immune system diseases 1 , 2 . Progeroid syndromes, regarded as premature aging disease, encompass a group of rare genetic disorders that recapitulate multiple physiological aging-associated phenotypes during early development 3 , 4 . Notably, Hutchinson-Gilford progeria syndrome (HGPS) is a rare autosomal dominant genetic disorder primarily attributed to a point mutation in the LMNA gene (c.1824 C < T), resulting in an alternative splicing event and the production of a truncated Lamin A protein known as progerin 5 . Clinical manifestations of HGPS include growth retardation, loss of hair, sclerotic skin, cardiovascular alteration, bone abnormalities, and inflammation 5 , 6 , resembling accelerated aging phenotypes in childhood. On a cellular level, HGPS share many cellular alterations with normal aging, including accumulated DNA damage, mitochondrial dysfunction, genomic instability, telomere aberrations, and loss of heterochromatin. Thus, HGPS serves as a valuable model for studies of aging and aging-related pathogenesis 7 . Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has emerged as a global public health emergency of international concern 8 . Particularly, severe COVID-19 cases, characterized by pneumonia, respiratory failure, short of breath, septic shock, and multiple organ dysfunction, calls for more healthcare and medical resources 9 , 10 . Patients with certain comorbidities are at a high risk of progressing to severe COVID-19, and one of the main risk factors is aging 11 . Studies have shown that the severity and fatality rates are notably higher in elderly population as compared to the young, with over 73% deaths occurring in individuals over 65 12 . Despite identifying age-related changes such as low immune response activity contributing to increased susceptibility to infectious diseases among the aged, the specific aging-related phenotypes that contribute most to severe or critical COVID-19, and their underlying mechanisms, remain incompletely understood. SARS-CoV-2 enters host cells by recognizing angiotensin-converting enzyme 2 (ACE2), a protein conserved across different species 13 . However, mice, the most used model for human diseases, are not susceptible to SARS-CoV-2 due to the low affinity of mouse ACE2 for the virus 14 , 15 . To address this inherent resistance, our previous studies focused on establishing a humanized ACE2 mouse model using stem cell-based genome editing and tetraploid compensation 16 . Additionally, mice carrying a point mutation (c.1827C > T, representing LMNA c.1824C > T in human) at exon 11 of Lmna gene mimic the clinical manifestations of human HGPS 17 . These HGPS mice display aging-related phenotypes within 3 months, significantly faster than natural aging 17 . To investigate the response of HGPS mice to COVID-19 infection, in this study we generated an HGPS mouse model based on our hACE2 mouse and subjected these HGPS/hACE2 mice to viral challenge. Transcriptome analysis was conducted to monitor changes throughout the infection, and comparisons were made between young, aged, and HGPS mice in their response to SARS-CoV-2. We found that hACE2 mice carrying HGPS mutation show different viral replication dynamics compared to the young mice, which is similar to the aged mice. Transcriptome analysis identified common biological pathways affected across all animal groups, with HGPS mice also exhibiting metabolic dysregulation during viral infection. Furthermore, we demonstrated that the immune response was significantly impaired in aged and HGPS mice compared to young mice upon viral challenge, while aged mice show severe bleeding in the respiratory tract. Overall, our study evaluates the potential pathological phenomena induced by SARS-CoV-2 infection in HGPS, offering insights for understanding progeria syndrome and informing the treatment of COVID-19 patients with premature aging. Results Generation of HGPS/hACE2 mouse model To generate a mouse strain carrying HGPS variants, we utilized CRISPR based base-editing system to introduce a homozygous c.1827 C < T (p.Gly609Gly) mutation in exon 11 of mouse Lmna gene, equivalent to the c.1824 C < T (p.Gly608Gly) mutation found in human LMNA , in mouse embryonic stem cells (mESCs) with humanized ACE2 18 (Fig. 1 A and Supplementary Fig. 1A). Karyotyping and immunofluorescent staining of Lmna mutant mESCs confirmed correct chromosome arrangements and pluripotency (Supplementary Fig. 1C and D). To avoid the breeding issues and ensure an adequate supply of mice for infection studies, we employed tetraploid complementation using our modified mESCs, and generated humanized ACE2 mice carrying homozygous HGPS variants (Fig. 1 A and C). These mice exhibit an alternative splicing event observed in HGPS patients (Supplementary Fig. 1B), resulting in the absence of Lamin A protein and the presence of the spliced Lamin protein known as progerin (Fig. B). Similar to HGPS patients, HGPS hACE2 mice display small body size, reduced body weight, and shortened life span (Fig. 1 C-E), indicating a premature aging phenotype in these mice. Tissues such as bone, heart, spleen, and thymus exhibit varying levels of defects in HGPS patients, while not much is known for the lung 5 . To further investigate the impact of HGPS on the respiratory system in HGPS mice, we carried out transcriptome analysis of lung tissues from 2-month-old wild type and HGPS/hACE2 mice (Fig. 1 F). Consistent with previous studies, we observed up-regulation of genes involved in P53-mediated DNA damage pathway including Gadd45a , Gadd45b , Gadd45g , Atf3 , Btg2 , and Cdkn1a (Supplementary Fig. 1E). Gene Ontology (GO) analysis revealed that immune-related pathways were more enriched in normal hACE2 mice compared to the HGPS ones, indicating a diminished immune response in HGPS mice (Fig. 1 G). Conversely, multiple developmental pathways such as epidermis, vasculature, bone marrow, and adipose development were more enriched in HGPS compared to normal hACE2 mice, suggesting a premature developmental stage of lung in HGPS mice (Fig. 1 G). Typical marker genes of distinct cell types in lungs show only subtle changes in HGPS/hACE2 compared hACE2 mice, indicating a relatively normal lung function in HGPS/hACE2 mice (Supplementary Fig. 1F). Overall, we have successfully generated a hACE2 mouse model harboring the HGPS mutation, which displays phenotypic similarities to human accelerated aging. Replication and host response to primary infection with SARS-CoV-2 To investigate infection outcomes of HGPS mice, we examined the pathogenesis of SARS-CoV-2 in 2-month-old HGPS/hACE2 mice infected with the original strain of the coronavirus from Wuhan (WIV04). In parallel, age-matched young hACE2 mice (2 months old) and aged hACE2 mice (over 14 months old) were included for virus challenging. Mice received intranasal inoculation with 1x10 5 50% tissue culture infective dose (TCID 50 ) of the virus and lung samples were collected at 1, 3, 5, and 7 days post-infection (dpi) for virus replication measurement, clinical response examination, and transcriptome analysis (Fig. 2 A). Although young, aged, and HGPS mice exhibited distinct expression levels of hACE2 in lung, transcriptome analysis of SARS-CoV-2 revealed that aged and HGPS mice displayed significantly more expression of SARS-CoV-2 genes in lung including E, M, N, S, and ORF genes in lung at 1 dpi, which then decreased dramatically at 3 dpi (Fig. 2 B and Supplementary Fig. 2A-B). In contrast, SARS-CoV-2 genes were detected at 1 dpi in the lungs of young mice, reaching the peaks at 3 dpi (Fig. 2 B and Supplementary Fig. 2A). We observed mild decreased body weight in infected young mice compared to the mock-infected ones, while no changes were detected between infected and uninfected aged mice (Fig. 2 C). HGPS mice exhibited a slight loss of body weight in both infected and uninfected groups (Fig. 2 C). In sum, these findings suggest that SARS-CoV-2 replicates differentially in the lungs of young, aged, and HGPS/hACE2 mice, likely resulting in varied responses upon viral infection among different mouse groups. Aged hACE2 mice displayed more severe pathological phenotypes induced by SARS-CoV-2 infection To further evaluate the outcomes of viral infection in different mouse group, we conducted histopathological analysis which illustrated distinct degrees of lung damage among the infected young, aged and HGPS/hACE2 mice. In uninfected control animals, slight inflammation was observed in aged and HGPS mice, characterized by a minor immune cell infiltration, which was not detected in the young mice (Fig. 3 A, 3 E, and 3 I). At 1 dpi, mild changes were observed in the lung tissues of young mice, including the presence of lymphocyte and macrophage infiltration in some alveolar spaces, along with slight thickened alveolar walls (Fig. 3 B). Severe pneumonia developed at 3 dpi in the young mice, characterized by multifocal lesions, bleeding, and massive infiltration with increased number of mixed inflammatory cells at peri-vascular regions (Fig. 3 C). This phenotype became less severe at 5 dpi, with fewer inflammatory cell infiltration and milder thickened alveolar walls compared to those observed at 3 dpi (Fig. 3 D). In consistence with young mice, aged mice displayed hemorrhage and slight thickened alveolar walls at peri-bronchial and peri-vascular regions at 1 dpi, which became more pronounced at 3 dpi (Fig. 3 F-G and Supplementary Fig. 2C). By 5 dpi, we found that two out of three aged mice displayed severe bleeding in the bronchus and pulmonary alveoli, along with inflammatory cell infiltration and fibrin exudation, suggesting a severe COVID-19 phenotype (Fig. 3 G and Supplementary Fig. 2D). Still, all infected mice survived viral infection till euthanized for sample collection at 7 dpi. Notably, these two mice showing severe bleeding phenotype were in poor condition when being euthanized for sample collection. In contrast, HGPS/hACE2 mice did not display a severe phenotype throughout the infection, showing only a slightly increased number of immune cells at peri-bronchial and peri-vascular areas at 1,3 and 5 dpi (Fig. 3 J-L), suggesting a less severe phenotypes in there HGPS mice. Altogether, our histopathological findings demonstrate distinct pathological features induced by SARS-CoV-2 infection between different animal groups, highlighting a severe phenotype in aged mice compared to young hACE2 and HGPS/hACE2 mice. Gene expression dynamics from different groups with viral infection. To comprehensively understand how SARS-CoV-2 induces pathological differences among different mouse groups, we conducted transcriptomic analysis to investigate the dynamics gene expression profiles in the lungs of young, aged, and HGPS hACE2 mice throughout the infection. We classified high variable genes into several clusters based on their expression patterns and calculate the averaged expressing trajectories for each cluster. Subsequently, we performed Gene Ontology (GO) analysis to elucidate the affected biological and molecular pathways for each cluster. Although viral gene expression peaked differentially in the lungs of young compared to aged and HGPS/hACE2 mice, we observed a common response to virus and activation of innate immune upon viral infection transiently at 3 dpi in cluster 1 across all the infected groups (Fig. 4 A, 4 D, and Supplementary Fig. 3A). In young and aged groups, mice showed increased B cell-mediated immunity as well as immunoglobulin production in the lungs at 5 dpi, accompanied by upregulation of genes involved in muscle development in cluster 2 (Fig. 4 B, 4 D, and Supplementary Fig. 3B). This indicates an immediate humoral immune response following viral exposure, which further triggers muscle contraction of lung smooth muscle. Interestingly, this activation occurred earlier in HGPS mouse lungs at 1 dpi (Fig. 4 B), suggesting a distinct immune response to SARS-CoV-2 in HGPS. Viral infection has been reported to cause cilia loss from ciliated cells, resulting in cilia dysfunction in respiratory epithelium 19 – 21 . In agreement with previous findings, we observed a slight downregulation of genes in cluster 3 involved in cilium assembly and movement in young and HGPS mice, which was more pronounced in aged group (Fig. 4 C, 4 D and Supplementary Fig. 3C). This was followed by increased expression of those genes across all groups starting at 3 dpi, suggesting a restoration of function in fluid movement and mucus clearance in the respiratory airway (Fig. 4 C, 4 D and Supplementary Fig. 3C). In addition to the common functional enrichment shared by the three groups, we also identified gene clusters with distinct dynamic patterns unique to each mouse group. For instance, cluster 4 in young mice declined linearly throughout the entire infection and was strongly enriched for genes associated with cell junction assembly, extracellular matrix organization, and cell-matrix adhesion (Fig. 4 E). Moreover, Notch/vascular genes exhibited a rapid decline rapidly in aged mice at 1 dpi, after which a more gradual decline prevailed, indicating functional defects in vascular and endothelial cells in the lungs of aged mice (Fig. 4 E). In infected HGPS mice, genes encoding sulfur, glycoprotein, and liposaccharide metabolic process pathways were featured in cluster 4, exhibiting immediate increase after infection till 3 dpi, followed by a drop towards the end of infection (Fig. 4 F). However, cluster 5 contained genes related to lipid transport and exocytosis regulation pathways who reached their lowest expression level at 3 dpi, followed by a pronounced increase till 7 dpi, showing an opposite trend compared to cluster 4 (Fig. 4 F). These findings suggest SARS-CoV2-induced metabolic dysfunction in infected HGPS lungs. Transcriptome comparison reveal strong immune response in young mice upon viral infection. Next, we focused on infection-induced transcriptomic changes and compared the transcription differences between different mouse groups at each time point post infection. Differential gene expression analysis, together with hierarchical clustering, revealed specifically expressed gene clusters from each group, and GO analysis was applied to these gene sets. Overall, we observed a comparable number of genes between young and aged group at 1, 3, and 5 dpi, while HGPS group displayed subtle changes at 1 and 3 dpi (Fig. 5 A-C). Consistent with our histopathological analysis, expression of genes enriched in virus defense, interferon-beta, and innate immune response pathways was significantly higher in the lungs of young mice throughout the infection, suggesting a more active immune response upon viral infection compared to the aged and HGPS mice (Fig. 5 D). In the aged mice, immune pathways such as immune cell migration, immunoglobulin production, humoral immune response, and cell chemotaxis represented feature genes in the clusters at 1 and 3 dpi (Fig. 5 E). Additionally, we found pathways related to hemorrhage, including wound healing, blood coagulation, and erythrocyte development, were significantly enriched in aged mice, further confirming the bleeding phenotype observed in the lungs of aged mice in the histopathological study (Fig. 5 E and Supplementary Fig. 4). We did not obverse a significant pathway enriched in HGPS mice at 1 and 3 dpi, likely due to the low numbers of unique genes (36 genes for 1 dpi and 53 genes for 3 dpi) in this group. Whereas at 5 dpi, 332 genes were more activated in the lungs of HGPS mice. These genes primarily belong to pathways associated with entrainment of circadian clock, regulation of blood circulation, and hormone transport. This suggests a dysregulation of circadian rhythms in HGPS mice, although the enrichment in this pathway was less pronounced (Fig. 5 F). Altogether, our results demonstrated that young mice, which have a normal immune system to protect against virus, display the highest immune response activation upon SARS-CoV-2 infection compared to aged and HGPS mice. Conversely, viral infection only causes mild pathological phenomena in HGPS mice, while aged mice exhibit lung hemorrhage when challenged by SARS-CoV-2, which could contribute to severe COVID-19. Discussion COVID-19 pandemic has profoundly threatened normal life and health of people worldwide, particularly impacting older adults who are more susceptible to SARS-CoV-2 infection. Therefore, understanding the causal relationship between aging and severe COVID-19 is of great significance for improving preventive measures and therapeutic strategies for the elderly population. In this study, we have investigated the pathological consequences of SARS-CoV-2 infection in a progeria syndrome, HGPS, using a mouse model. We systematically compared the transcriptome landscape of lung tissues from young, aged, and HGPS/hACE2 mice in relation to SARS-CoV-2 infection. Firstly, we demonstrated that innate interferon response and virus defense pathway were more robustly activated in young compared to the aged and HGPS/hACE2 mice, consistent with the findings from other mouse models 22 , 23 . Aging is known to be associated with a functional decline in immune response, likely due to long-term chronic inflammation, as evidenced by our histopathological results 24 . Therefore, this observation further supports the low immune response in the aged and HGPS. Interestingly, while aged mice displayed high rival replication at 1 dpi, they subsequently developed a severe bleeding phenotype in the lungs at 5 dpi, mirroring observations in patients infected with SARS-CoV-2 who display abnormal coagulation profiles 25 , 26 . Severe COVID-19 has been reported from multiple mouse models. Jiang et al used a transgenic mouse model with hACE2 expression driven by human FOXJ1 promoter to study SARS-CoV-2. Body weight loss, heavy lung damage and death were observed in these mice upon viral infection 27 . Dong used an K18-hACE2 mouse model which recapitulates severe COVID-19 with a low viral dose (2 × 10 3 PFU) 28 . Kenneth et al and Jiang er al applied mouse adopted coronavirus on wild type mice and demonstrated severe COVID-19 in the aged mice 29 , 30 . Here, using a different humanized ACE2 strategy, we displayed heavy hemorrhage in our aged hACE2 mice after coronavirus infection, consistent with clinical manifestations seen in COVID-19 patients 31 . Genes involved in cilium assembly and regeneration were activated at 3 dpi from all three groups, indicating a potential restoration of cilial function and clearance mechanisms. Thus, our study suggests that, alongside hypersecretion of mucus gel leading to alveolar and bronchial blockage 32 , bleeding may also contribute to severe functional defect in lung. Aging, together with other aging-related diseases, are identified to be major risk factors for severe COVID19 11 . Since HGPS share several phenotypes with accelerated aging especially atherosclerosis, osteoporosis, and cardiovascular diseases 5 , 33 , 34 , one would assume a severe COVID19-like phenotype from HGPS after viral infection. However, beyond our expectation, HGPS/hACE2 mice only experienced mild pathological outcomes compared to the young and aged mice, indicating that Progeria syndrome cases may not be susceptible to SARS-CoV-2. A few studies investigating the association between genetic variants and COVID-19 susceptibility/severity, identified genes related to cytokines and viral receptors as risk-genes for severe COVID-19 35–37 , whereas progeria variants are not among them. By now, no progeria syndrome patient infected by SARS was reported as severe phenotype, further confirming our hypothesis. Additionally, we also noticed that the other uninfected HGPS hACE2 mice, which are from the same batch of tetraploid complementation as the infected ones, were all died nearly at the end of infection date, suggesting that these infected mice were likely at the end of their lives as well. This could also explain the slight decline of body weight from both uninfected and infect HGPS/hACE2 mice. Thus, viral infection with an early stage of HGPS mice, e.g. at 1 or 1.5 month ago, may worth testing for severe COVID-19. Overall, our study assesses the potential pathological effects triggered by SARS-CoV-2 infection in HGPS, providing valuable insights for understanding progeria syndrome and guiding treatment for SARS-CoV-2 and other variants infected patients with premature aging. Materials and Methods Ethics statement Mouse studies were carried out in an animal biosafety level 3 (ABSL3) facility at Wuhan Institute of Virology, Chinese Academy of Sciences (CAS). All the animal experiments in this study were approved by The Institutional Animal Care and Use in GIBH and the Institutional Review Board of the Wuhan Institute of Virology, CAS. All the procedures involving mice is complied with all relevant ethical regulations (Experimental approvement number, IACUC 2020120). All animal experiments were performed in accordance with ARRIVE guidelines. Cell culture Balb/c mouse embryonic stem cells (ESCs) were derived from dpc 3.5 mouse embryos, and were cultured on feeder cells in Knockout DMEM (Gibco, 10829018) supplemented with 1000 U/mL LIF (Novoprotein, C690), 10% KSR (Gibco, 10828028), NEAA (Gibco, 11140076), Glutamax (Gibco, 35050079), Sodium pyruvate (Gibco, 11360070), beta-mecaptomethanol (sigma, M3148), MEK inhibitor PD0325901 (Holzel Biotech, DC1056, 1 mM) and GSK3 inhibitor CHIR99021 (Holzel Biotech, DC1023, 3 mM). Generation of Lmna mutant hACE2 mESCs Lmna c.1827 C < T variant was introduced into a huminzed ACE2 mES cell line generated from our previous study with slight modification 16 . Briefly, the codon-optimized hACE2 gene was inserted into mouse genome following the same strategy as before (ref16). To target Lmna c.1827, sgRNA (5’-aggagatggatccgcccacc-3’) along with linear targeting donor were transfected into hACE2 mESCs by electroporation. Single colonies were picked 2 days after transfection, and genomic DNA were extracted using DirectPCR Lysis Reagent (VIAGEN, 102-T). Exon 11 of Lmna was amplified using primer set (Forward primer: 5’-agtcagtcccaaactcgctg-3’; Reverse primer: 5’-caagagggactgcaaggagg-3’) and the c.1827 C < T variant was proved by sanger sequencing. Generation of mouse models (tetraploid complementation) Mouse tetraploid embryos used for tetraploid complementation were prepared as reported before 16 . In detail, mESCs cultured at Day 2 were trypsinized until small clumps of cells (15–20 cells per clumps) were seen under the microscope, and then were transferred into microdrops of KSOM medium with 10% FCS under mineral oil. Each clump was placed in a depression in the microdrop. Meanwhile, batches of 30–50 embryos are incubated briefly in acidified Tyrode's solution to dissolve the zona pellucida. Next, two embryos are placed per ES clump for aggregation. All aggregates were incubated overnight at 37C, 5% CO2. After 24 hours of culture, eleven to fifteen embryos are transferred into one uterine horn of a 2.5 dpc pseudopregnant female mouse. Mature CD-1 females are used as pseudopregnant foster mothers with a weight of about 30g. Mice infection The SARS-CoV-2 (IVCAS 6.7512) was prepared as reported before 27 . Male Balb/c mice with different genetic backgrounds (hACE2 and hACE2 plus Lmna c. 1827C < T) and different ages (2 months ago for young and HGPS hACE2 mice, more than 14 months ago for aged hACE2 mice) were treated with tribromoethanol (Avertin, 250mg/kg) and intranasally infected with 1 x 10 5 TCID 50 SARS-CoV-2 in 50 µL DMEM per mouse. The uninfected control mice were inoculated with DMEM only. Mice with different genetic backgrounds were randomly assigned to mock, 1, 3, 5, 7 dpi groups. Mice showing undetectable viral RNA in lung were regarded as a failed infection and were removed from the study. Mice were weighted and observed for clinical signs daily across the infection. 3 mice were euthanized using isoflurane followed by cervical dislocation at 1, 3, 5, 7 dpi per group. Lung tissues were collected for RNA and Hematoxylin and Eosin (H&E) staining. More details can be found in Fig. 2 A and Table 1. Mouse group Mock Infected 1dpi 3dpi 5dpi 7dpi hACE2 mice (2 months) 3 3 3 3 3 (-1) * hACE2 mice (14 months) 3 3 3 3 3 HGPS/hACE2 mice (2 months) 3 3 3 3 3 * One mouse with undetectable viral RNA in lung was removed from the study. H&E staining Lung samples were fixed with 4% paraformaldehyde, followed by paraffin embedment. Fixed lung tissues were cut into 3.5-µm sections for H&E staining following the standard protocol. Transcriptome analysis RNA was isolated from homogenized lung tissue using TRizol with standard protocol. For ployA based mRNA-seq, 1 µg total RNAs were used for library construction. mRNA was enriched and purified using Library Preparation VAHTSTM mRNA Capture Beads (Vazyme, NR401-01). Next, purified mRNAs were fragmented, followed by cDNA synthesis, second-strand synthesis, end repair, adaptor ligation and amplification using VAHTS Universal V8 RNA-seq Library Prep Kit for Illumina (Vazyme, NR605-01) and VAHTS DNA Clean Beads. Libraries were sequenced for on average 20 million pair-end reads. The RNA-seq data processing was performed as described below. To analyze the transcriptome changes, raw reads were first trimmed to remove the adapter contamination, and then aligned to the mouse mm10 genome reference using STAR (2.7.10a) with default settings 38 . FeatureCounts (2.0.1) was used for read assignment with the following parameters, -T 5 -g gene_name -p 39 . DEseq2 was used for data normalization, differential expression analysis, and data visualization 40 . Genes with P-value 1 were considered as differentially expressed genes and used for GO analysis. Time-series analysis was done using Mfuzz package in R (3.18) 41 with detectable genes (more than 10 reads were assigned from all the samples). All data are submitted to GEO under accession GSE264189. Western blotting Mouse lung tissues were snap-frozen in liquid N2 and then homogenized in clod Cell lysis buffer (20 mM Tris-HCl, pH 7.5, containing 150 mM NaCl, 2mM DTT, 50% triglyceride, 100 mM EDTA, 1% SDS, 1% NP40 and 1% Triton X-100) supplemented with Protease inhibitor cocktail (Roche, 4693132001). Total tissue extracts were separated on a 12.5% SDS-PAGE gel (EpiZyme, PG113), and transferred to a PVDF membrane (Millipore, IPVH00010). The following primary antibodies were used: anti-LAMINA/C (CST, 4777S, 1:1000), anti-progerin (abcam, ab66587, 1:1000), Histone 3 (abcam, ab18521, 1:5000). Original scan of the immunoblot is shown in supplementary File1 with molecular mass markers and indicated cropped area. Declarations Acknowledgements We thank Zou Na and Chu Shilong from the animal center of GIBH for the mouse breeding. We thank Prof. Axel Schambach from Hannover Medical School for providing us the codon-optimized hACE2 vector. This work was financially supported by the National Key R&D Program of China (2021YFE0112900, 2023YFF1204701), the Austrian Science Fund (FWF) and ‘Herzfelder’sche Familienstiftung project P35268-B, BMBWF and WTZ-OEAD grant (CN 04/2021), Major Project of Guangzhou National Laboratory (GZNL2023A02005), The National Natural Science Foundation of China (32225012), Science and Technology Projects in Guangzhou (2024A04J4823), Basic Research Project of Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences (GIBHBRP23-02, GIBHBRP23-01), Science and Technology Planning Project of Guangdong Province, China (2023B1212060050, 2023B1212120009, Guangdong Basic and Applied Basic Research Foundation (2021A1515111044) , and Health@InnoHK Program launched by Innovation Technology Commission of the Hong Kong SAR, P. R. China. Author contributions W.H., C.J., and W.G. conceived the study. W.H. designed the experiments, interpreted the data, and prepared the illustrations. 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Kumar, L. & Futschik, M. E. Mfuzz: A software package for soft clustering of microarray data. Bioinformation 2, 5–7 (2007). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 24 Aug, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 03 Jun, 2024 Reviews received at journal 29 May, 2024 Reviews received at journal 24 May, 2024 Reviewers agreed at journal 15 May, 2024 Reviewers agreed at journal 14 May, 2024 Reviewers invited by journal 09 May, 2024 Editor assigned by journal 09 May, 2024 Editor invited by journal 27 Apr, 2024 Submission checks completed at journal 27 Apr, 2024 First submitted to journal 24 Apr, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4316933","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":297279821,"identity":"e8d08fa5-ae2a-4691-bb9d-895be0398533","order_by":0,"name":"Wu Haoyu","email":"","orcid":"","institution":"Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Wu","middleName":"","lastName":"Haoyu","suffix":""},{"id":297279823,"identity":"5971deff-dab3-4204-b4ff-0a54eccd227e","order_by":1,"name":"Liu Meiqin","email":"","orcid":"","institution":"Wuhan Institute of Virology, Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Liu","middleName":"","lastName":"Meiqin","suffix":""},{"id":297279824,"identity":"6f485e86-f9d3-4dc9-b62f-57f03827caff","order_by":2,"name":"Sun Jiaoyang","email":"","orcid":"","institution":"Guangzhou National Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Sun","middleName":"","lastName":"Jiaoyang","suffix":""},{"id":297279825,"identity":"1d58c4ea-ca2f-43ab-95b1-eff77bbb1d26","order_by":3,"name":"Hong Guangliang","email":"","orcid":"","institution":"Guangzhou National Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Hong","middleName":"","lastName":"Guangliang","suffix":""},{"id":297279826,"identity":"a64a2b08-4489-43a7-aead-2ee5a5ded866","order_by":4,"name":"Lin Haofeng","email":"","orcid":"","institution":"Wuhan Institute of Virology, Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Lin","middleName":"","lastName":"Haofeng","suffix":""},{"id":297279827,"identity":"4e2f0870-82f8-4a93-90e7-c8c02cf3b676","order_by":5,"name":"Chen Pan","email":"","orcid":"","institution":"Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Chen","middleName":"","lastName":"Pan","suffix":""},{"id":297279828,"identity":"5082cd24-bf53-4203-a890-0b882d4c1cc3","order_by":6,"name":"Quan Xiongzhi","email":"","orcid":"","institution":"Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Quan","middleName":"","lastName":"Xiongzhi","suffix":""},{"id":297279829,"identity":"78ee1d20-a5ed-4cd9-824b-125210bef2f5","order_by":7,"name":"Wu Kaixin","email":"","orcid":"","institution":"Bioland Laboratory, Guangzhou Regenerative Medicine and Health Guangdong Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Wu","middleName":"","lastName":"Kaixin","suffix":""},{"id":297279830,"identity":"77d86f89-1372-48e8-ac2d-db93ff9d39ec","order_by":8,"name":"Hu Mingli","email":"","orcid":"","institution":"Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Hu","middleName":"","lastName":"Mingli","suffix":""},{"id":297279831,"identity":"276ba922-f937-45ba-810d-34d2ff22a50f","order_by":9,"name":"Yang Xuejie","email":"","orcid":"","institution":"Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Xuejie","suffix":""},{"id":297279832,"identity":"40db9af0-631f-43c6-9516-748033324326","order_by":10,"name":"Ingo Lämmermann","email":"","orcid":"","institution":"University of Natural Resources and Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Ingo","middleName":"","lastName":"Lämmermann","suffix":""},{"id":297279833,"identity":"ea30848d-dd21-445f-8e35-5669dc62120b","order_by":11,"name":"Johannes Grillari","email":"","orcid":"","institution":"The Research Center in Cooperation with AUVA","correspondingAuthor":false,"prefix":"","firstName":"Johannes","middleName":"","lastName":"Grillari","suffix":""},{"id":297279834,"identity":"91ee559a-6bed-473a-8716-6c46439738ce","order_by":12,"name":"Shi Zhengli","email":"","orcid":"","institution":"Wuhan Institute of Virology, Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Shi","middleName":"","lastName":"Zhengli","suffix":""},{"id":297279835,"identity":"a986ab03-f7db-4539-9444-4d03cb0fad92","order_by":13,"name":"Chen Jiekai","email":"","orcid":"","institution":"Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Chen","middleName":"","lastName":"Jiekai","suffix":""},{"id":297279837,"identity":"7389187d-bc58-45d4-a137-12408cc39f87","order_by":14,"name":"Wu Guangming","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAElEQVRIiWNgGAWjYFADZiD+wMAgwyBBihbGGQwMPCRoAeniIUaLwY0E5tc8NXfs1rbzHn5t86uOh392A+OHHwx2eXi0sFnzHHuWvO0wX5p1bh8bj8SdA8ySPQzJxbi0mAG1GPOwHU42O8xjZpzbw8PDcCOBQZqB4UBiA14t/6BaLHskeOSBTv1NQAvzY962w3ZALcaPGX4Y8ICcitcW+zMP2Bjn9h1OANnC2NuQwGN452CbZY9BMk4tku0JzB/efDtsb3b+jPGHH3/q5ORuNx++8aPCDqcWBgb+b6CIAClgk2BsA4kwAtkGONWDAPMHkAMhjD94VY6CUTAKRsEIBQC1yVd7XoXiHQAAAABJRU5ErkJggg==","orcid":"","institution":"Guangzhou National Laboratory","correspondingAuthor":true,"prefix":"","firstName":"Wu","middleName":"","lastName":"Guangming","suffix":""}],"badges":[],"createdAt":"2024-04-24 09:10:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4316933/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4316933/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-70612-2","type":"published","date":"2024-08-24T15:56:58+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":55809722,"identity":"4d01b20d-645a-4f46-ac76-6027177a22e4","added_by":"auto","created_at":"2024-05-03 15:42:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":789413,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of HGPS/hACE2 mice.\u003c/p\u003e\n\u003cp\u003e(A) Schematic diagram of HGPS/hACE2 mouse generation strategy using mES editing combined with tetraploid complementation techniques. (B) Western blot analysis of lung tissues from hACE2 and HGPS/hACE2 mice, showing the presence of Lamin A/C in wild type lung and progerin in HGPS lungs. Histone 3 was used as a loading control. (C) Representative photographs of 2-month-old unedited, heterozygous, and homozygous \u003cem\u003eLmna\u003c/em\u003e c.1827C\u0026lt;T mutant hACE2 mice. (D) Survival curve plot showing the life span of heterozygous and homozygous \u003cem\u003eLmna\u003c/em\u003e mutant mice. (E) Boxplots illustrating the body weight distribution among young, aged, and HGPS Balb/c mice. (F) Volcano plot showing transcriptome changes in the lungs between hACE2 and HGPS/hACE2 mice. Significantly differentially expressed genes are highlighted in green (upregulated in HGPS) and red (upregulated in wild type). (G) Dropblot of GO analysis results derived from differentially expressed gene sets in the lungs of hACE2 and HGPS/hACE2 mice.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-4316933/v1/367d92b7fb263170da003299.png"},{"id":55808244,"identity":"305186c5-e71d-4a93-9155-8febd1b43f3c","added_by":"auto","created_at":"2024-05-03 15:34:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":166695,"visible":true,"origin":"","legend":"\u003cp\u003eSARS-CoV-2 infection and replication dynamics in young, aged, and HGPS hACE2 mice.\u003c/p\u003e\n\u003cp\u003e(A) Experimental scheme of viral infection. Young, aged, and HGPS mice were intranasally infected with 1 x 10\u003csup\u003e5\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e. Lung samples were collected at 1, 3, 5, 7 dpi for RNA and H\u0026amp;E. Mouse body weight were measured for up to 7 days. (B) Expression levels of viral genes (E, S, M, and N genes) in the lungs of infected young, aged, and HGPS mice at 1, 3, 5, and 7 dpi. Data were shown as normalized expression representing normalized read counts from RNA-seq data. Error bars, SEM from at least 2 biological replicates. (C) body weight changes of young, aged, and HGPS mice upon viral infection throughout the whole experiment.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-4316933/v1/d20a78de73ea2137b2575e4d.png"},{"id":55808243,"identity":"cb198ebf-9d0b-4dd8-87c7-66900fd9fe35","added_by":"auto","created_at":"2024-05-03 15:34:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1399507,"visible":true,"origin":"","legend":"\u003cp\u003ePathological changes in young, aged, and HGPS hACE2 mice after SARS-CoV-2 infection.\u003c/p\u003e\n\u003cp\u003e(A-D) H\u0026amp;E staining of the lungs of young hACE2 mice. Mock-infected mice show no lesions. After viral infection, immune cell infiltration (1, 3, and 5 dpi, green arrow), hemorrhage (3 and 5 dpi, yellow arrow), and thickened alveolar walls (3 and 5 dpi, red arrow) were observed at different time points. (E-H) H\u0026amp;E staining of the lungs of aged hACE2 mice. Mock-infected mice show slight immune cell infiltration (green arrow). Upon viral infection, immune cell infiltration (1 dpi, green arrow), heavy bleeding (1, 3, and 5 dpi, yellow arrow), and thickened alveolar walls (1, 3, and 5 dpi, red arrow) were observed at different time points. (I-L) H\u0026amp;E staining of the lungs of HGPS hACE2 mice. Mock-infected mice show slight immune cell infiltration (green arrow). After viral infection, immune cell infiltration (1, 3, and 5 dpi, green arrow) and thickened alveolar walls (1, 3, and 5 dpi, red arrow) were observed at different time points. Black scale bar = 200 μm, and green scale bar = 50 μm.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-4316933/v1/77814094469ec8e618e4896b.png"},{"id":55808242,"identity":"36d4940c-83b2-48c4-b962-557438f84b12","added_by":"auto","created_at":"2024-05-03 15:34:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":734619,"visible":true,"origin":"","legend":"\u003cp\u003etranscriptome changing dynamics of different mouse group upon viral infection.\u003c/p\u003e\n\u003cp\u003e(A-C) Soft clustering of variable genes of young, aged, and HGPS mice after viral infection. (D) Gene Ontology analysis of genes sets corresponding to the gene clusters from (A-C). (E) Soft clustering of variable genes with unique patterns of young (cluster 4) and (cluster 4) aged mice on the left, and corresponding GO analysis results from each cluster on the right. (F) Gene clusters of HGPS group with different trends on the left, together with the GO analysis results on the right.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-4316933/v1/f26f032b96af2ec6a5773f6a.png"},{"id":55808245,"identity":"d137fbf1-ff17-40a4-ab33-004ece48ede7","added_by":"auto","created_at":"2024-05-03 15:34:32","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":413004,"visible":true,"origin":"","legend":"\u003cp\u003eTranscriptome comparison between different mouse group at 1, 3, and 5 dpi.\u003c/p\u003e\n\u003cp\u003e(A-C) Heatmaps illustrating significantly differentially expressed genes from different mouse groups at 1 (A), 3 (B), and 5 (C) dpi. Expression levels are shown as Z-score format. (D-F) GO analysis of genes more active in young group at 1, 3, and 5 dpi (D), more active in HGPS group at 5 dpi (F), and unique to aged group at 1, 3, and 5 dpi (E). Young mice are enriched for genes in virus response and innate immune response pathways. Aged mice show enrichment of immune cell migration as well as blood coagulation pathways. Genes unique to HGPS mice are involved in circadian rhythm pathway.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-4316933/v1/20bd721a425adc017b359f3e.png"},{"id":63300021,"identity":"5728f5aa-9f53-4941-9969-aac76ff4bb70","added_by":"auto","created_at":"2024-08-26 16:09:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3891042,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4316933/v1/700254ea-3286-49f4-a9ad-3cc773f5eecd.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Premature aging effects on COVID-19 pathogenesis: new insights from mouse models","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAging is a natural biological process characterized by the gradual decline of physiological functions across multiple tissues\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Several aging-related phenotypes serve as primary risk factors for various diseases including cancer, diabetes, neurodegeneration, cardiovascular diseases, and immune system diseases\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Progeroid syndromes, regarded as premature aging disease, encompass a group of rare genetic disorders that recapitulate multiple physiological aging-associated phenotypes during early development\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Notably, Hutchinson-Gilford progeria syndrome (HGPS) is a rare autosomal dominant genetic disorder primarily attributed to a point mutation in the \u003cem\u003eLMNA\u003c/em\u003e gene (c.1824 C\u0026thinsp;\u0026lt;\u0026thinsp;T), resulting in an alternative splicing event and the production of a truncated Lamin A protein known as progerin\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Clinical manifestations of HGPS include growth retardation, loss of hair, sclerotic skin, cardiovascular alteration, bone abnormalities, and inflammation\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, resembling accelerated aging phenotypes in childhood. On a cellular level, HGPS share many cellular alterations with normal aging, including accumulated DNA damage, mitochondrial dysfunction, genomic instability, telomere aberrations, and loss of heterochromatin. Thus, HGPS serves as a valuable model for studies of aging and aging-related pathogenesis\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCoronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has emerged as a global public health emergency of international concern\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Particularly, severe COVID-19 cases, characterized by pneumonia, respiratory failure, short of breath, septic shock, and multiple organ dysfunction, calls for more healthcare and medical resources\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Patients with certain comorbidities are at a high risk of progressing to severe COVID-19, and one of the main risk factors is aging\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Studies have shown that the severity and fatality rates are notably higher in elderly population as compared to the young, with over 73% deaths occurring in individuals over 65\u003csup\u003e12\u003c/sup\u003e. Despite identifying age-related changes such as low immune response activity contributing to increased susceptibility to infectious diseases among the aged, the specific aging-related phenotypes that contribute most to severe or critical COVID-19, and their underlying mechanisms, remain incompletely understood.\u003c/p\u003e \u003cp\u003eSARS-CoV-2 enters host cells by recognizing angiotensin-converting enzyme 2 (ACE2), a protein conserved across different species\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. However, mice, the most used model for human diseases, are not susceptible to SARS-CoV-2 due to the low affinity of mouse ACE2 for the virus\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. To address this inherent resistance, our previous studies focused on establishing a humanized ACE2 mouse model using stem cell-based genome editing and tetraploid compensation\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Additionally, mice carrying a point mutation (c.1827C\u0026thinsp;\u0026gt;\u0026thinsp;T, representing \u003cem\u003eLMNA\u003c/em\u003e c.1824C\u0026thinsp;\u0026gt;\u0026thinsp;T in human) at exon 11 of \u003cem\u003eLmna\u003c/em\u003e gene mimic the clinical manifestations of human HGPS\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. These HGPS mice display aging-related phenotypes within 3 months, significantly faster than natural aging\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo investigate the response of HGPS mice to COVID-19 infection, in this study we generated an HGPS mouse model based on our hACE2 mouse and subjected these HGPS/hACE2 mice to viral challenge. Transcriptome analysis was conducted to monitor changes throughout the infection, and comparisons were made between young, aged, and HGPS mice in their response to SARS-CoV-2. We found that hACE2 mice carrying HGPS mutation show different viral replication dynamics compared to the young mice, which is similar to the aged mice. Transcriptome analysis identified common biological pathways affected across all animal groups, with HGPS mice also exhibiting metabolic dysregulation during viral infection. Furthermore, we demonstrated that the immune response was significantly impaired in aged and HGPS mice compared to young mice upon viral challenge, while aged mice show severe bleeding in the respiratory tract. Overall, our study evaluates the potential pathological phenomena induced by SARS-CoV-2 infection in HGPS, offering insights for understanding progeria syndrome and informing the treatment of COVID-19 patients with premature aging.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eGeneration of HGPS/hACE2 mouse model\u003c/h2\u003e \u003cp\u003eTo generate a mouse strain carrying HGPS variants, we utilized CRISPR based base-editing system to introduce a homozygous c.1827 C\u0026thinsp;\u0026lt;\u0026thinsp;T (p.Gly609Gly) mutation in exon 11 of mouse \u003cem\u003eLmna\u003c/em\u003e gene, equivalent to the c.1824 C\u0026thinsp;\u0026lt;\u0026thinsp;T (p.Gly608Gly) mutation found in human \u003cem\u003eLMNA\u003c/em\u003e, in mouse embryonic stem cells (mESCs) with humanized ACE2\u003csup\u003e18\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and Supplementary Fig.\u0026nbsp;1A). Karyotyping and immunofluorescent staining of \u003cem\u003eLmna\u003c/em\u003e mutant mESCs confirmed correct chromosome arrangements and pluripotency (Supplementary Fig.\u0026nbsp;1C and D). To avoid the breeding issues and ensure an adequate supply of mice for infection studies, we employed tetraploid complementation using our modified mESCs, and generated humanized ACE2 mice carrying homozygous HGPS variants (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and C). These mice exhibit an alternative splicing event observed in HGPS patients (Supplementary Fig.\u0026nbsp;1B), resulting in the absence of Lamin A protein and the presence of the spliced Lamin protein known as progerin (Fig. B). Similar to HGPS patients, HGPS hACE2 mice display small body size, reduced body weight, and shortened life span (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-E), indicating a premature aging phenotype in these mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTissues such as bone, heart, spleen, and thymus exhibit varying levels of defects in HGPS patients, while not much is known for the lung\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. To further investigate the impact of HGPS on the respiratory system in HGPS mice, we carried out transcriptome analysis of lung tissues from 2-month-old wild type and HGPS/hACE2 mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Consistent with previous studies, we observed up-regulation of genes involved in P53-mediated DNA damage pathway including \u003cem\u003eGadd45a\u003c/em\u003e, \u003cem\u003eGadd45b\u003c/em\u003e, \u003cem\u003eGadd45g\u003c/em\u003e, \u003cem\u003eAtf3\u003c/em\u003e, \u003cem\u003eBtg2\u003c/em\u003e, and \u003cem\u003eCdkn1a\u003c/em\u003e (Supplementary Fig.\u0026nbsp;1E). Gene Ontology (GO) analysis revealed that immune-related pathways were more enriched in normal hACE2 mice compared to the HGPS ones, indicating a diminished immune response in HGPS mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Conversely, multiple developmental pathways such as epidermis, vasculature, bone marrow, and adipose development were more enriched in HGPS compared to normal hACE2 mice, suggesting a premature developmental stage of lung in HGPS mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Typical marker genes of distinct cell types in lungs show only subtle changes in HGPS/hACE2 compared hACE2 mice, indicating a relatively normal lung function in HGPS/hACE2 mice (Supplementary Fig.\u0026nbsp;1F). Overall, we have successfully generated a hACE2 mouse model harboring the HGPS mutation, which displays phenotypic similarities to human accelerated aging.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eReplication and host response to primary infection with SARS-CoV-2\u003c/h2\u003e \u003cp\u003eTo investigate infection outcomes of HGPS mice, we examined the pathogenesis of SARS-CoV-2 in 2-month-old HGPS/hACE2 mice infected with the original strain of the coronavirus from Wuhan (WIV04). In parallel, age-matched young hACE2 mice (2 months old) and aged hACE2 mice (over 14 months old) were included for virus challenging. Mice received intranasal inoculation with 1x10\u003csup\u003e5\u003c/sup\u003e 50% tissue culture infective dose (TCID\u003csub\u003e50\u003c/sub\u003e) of the virus and lung samples were collected at 1, 3, 5, and 7 days post-infection (dpi) for virus replication measurement, clinical response examination, and transcriptome analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Although young, aged, and HGPS mice exhibited distinct expression levels of \u003cem\u003ehACE2\u003c/em\u003e in lung, transcriptome analysis of SARS-CoV-2 revealed that aged and HGPS mice displayed significantly more expression of SARS-CoV-2 genes in lung including E, M, N, S, and ORF genes in lung at 1 dpi, which then decreased dramatically at 3 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and Supplementary Fig.\u0026nbsp;2A-B). In contrast, SARS-CoV-2 genes were detected at 1 dpi in the lungs of young mice, reaching the peaks at 3 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and Supplementary Fig.\u0026nbsp;2A). We observed mild decreased body weight in infected young mice compared to the mock-infected ones, while no changes were detected between infected and uninfected aged mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). HGPS mice exhibited a slight loss of body weight in both infected and uninfected groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). In sum, these findings suggest that SARS-CoV-2 replicates differentially in the lungs of young, aged, and HGPS/hACE2 mice, likely resulting in varied responses upon viral infection among different mouse groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eAged hACE2 mice displayed more severe pathological phenotypes induced by SARS-CoV-2 infection\u003c/h2\u003e \u003cp\u003eTo further evaluate the outcomes of viral infection in different mouse group, we conducted histopathological analysis which illustrated distinct degrees of lung damage among the infected young, aged and HGPS/hACE2 mice. In uninfected control animals, slight inflammation was observed in aged and HGPS mice, characterized by a minor immune cell infiltration, which was not detected in the young mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). At 1 dpi, mild changes were observed in the lung tissues of young mice, including the presence of lymphocyte and macrophage infiltration in some alveolar spaces, along with slight thickened alveolar walls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Severe pneumonia developed at 3 dpi in the young mice, characterized by multifocal lesions, bleeding, and massive infiltration with increased number of mixed inflammatory cells at peri-vascular regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). This phenotype became less severe at 5 dpi, with fewer inflammatory cell infiltration and milder thickened alveolar walls compared to those observed at 3 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). In consistence with young mice, aged mice displayed hemorrhage and slight thickened alveolar walls at peri-bronchial and peri-vascular regions at 1 dpi, which became more pronounced at 3 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF-G and Supplementary Fig.\u0026nbsp;2C). By 5 dpi, we found that two out of three aged mice displayed severe bleeding in the bronchus and pulmonary alveoli, along with inflammatory cell infiltration and fibrin exudation, suggesting a severe COVID-19 phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG and Supplementary Fig.\u0026nbsp;2D). Still, all infected mice survived viral infection till euthanized for sample collection at 7 dpi. Notably, these two mice showing severe bleeding phenotype were in poor condition when being euthanized for sample collection. In contrast, HGPS/hACE2 mice did not display a severe phenotype throughout the infection, showing only a slightly increased number of immune cells at peri-bronchial and peri-vascular areas at 1,3 and 5 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ-L), suggesting a less severe phenotypes in there HGPS mice. Altogether, our histopathological findings demonstrate distinct pathological features induced by SARS-CoV-2 infection between different animal groups, highlighting a severe phenotype in aged mice compared to young hACE2 and HGPS/hACE2 mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eGene expression dynamics from different groups with viral infection.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo comprehensively understand how SARS-CoV-2 induces pathological differences among different mouse groups, we conducted transcriptomic analysis to investigate the dynamics gene expression profiles in the lungs of young, aged, and HGPS hACE2 mice throughout the infection. We classified high variable genes into several clusters based on their expression patterns and calculate the averaged expressing trajectories for each cluster. Subsequently, we performed Gene Ontology (GO) analysis to elucidate the affected biological and molecular pathways for each cluster. Although viral gene expression peaked differentially in the lungs of young compared to aged and HGPS/hACE2 mice, we observed a common response to virus and activation of innate immune upon viral infection transiently at 3 dpi in cluster 1 across all the infected groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA,\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, and Supplementary Fig.\u0026nbsp;3A). In young and aged groups, mice showed increased B cell-mediated immunity as well as immunoglobulin production in the lungs at 5 dpi, accompanied by upregulation of genes involved in muscle development in cluster 2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB,\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, and Supplementary Fig.\u0026nbsp;3B). This indicates an immediate humoral immune response following viral exposure, which further triggers muscle contraction of lung smooth muscle. Interestingly, this activation occurred earlier in HGPS mouse lungs at 1 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), suggesting a distinct immune response to SARS-CoV-2 in HGPS. Viral infection has been reported to cause cilia loss from ciliated cells, resulting in cilia dysfunction in respiratory epithelium\u003csup\u003e\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. In agreement with previous findings, we observed a slight downregulation of genes in cluster 3 involved in cilium assembly and movement in young and HGPS mice, which was more pronounced in aged group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC,\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD and Supplementary Fig.\u0026nbsp;3C). This was followed by increased expression of those genes across all groups starting at 3 dpi, suggesting a restoration of function in fluid movement and mucus clearance in the respiratory airway (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD and Supplementary Fig.\u0026nbsp;3C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition to the common functional enrichment shared by the three groups, we also identified gene clusters with distinct dynamic patterns unique to each mouse group. For instance, cluster 4 in young mice declined linearly throughout the entire infection and was strongly enriched for genes associated with cell junction assembly, extracellular matrix organization, and cell-matrix adhesion (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Moreover, Notch/vascular genes exhibited a rapid decline rapidly in aged mice at 1 dpi, after which a more gradual decline prevailed, indicating functional defects in vascular and endothelial cells in the lungs of aged mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). In infected HGPS mice, genes encoding sulfur, glycoprotein, and liposaccharide metabolic process pathways were featured in cluster 4, exhibiting immediate increase after infection till 3 dpi, followed by a drop towards the end of infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). However, cluster 5 contained genes related to lipid transport and exocytosis regulation pathways who reached their lowest expression level at 3 dpi, followed by a pronounced increase till 7 dpi, showing an opposite trend compared to cluster 4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). These findings suggest SARS-CoV2-induced metabolic dysfunction in infected HGPS lungs.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTranscriptome comparison reveal strong immune response in young mice upon viral infection.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eNext, we focused on infection-induced transcriptomic changes and compared the transcription differences between different mouse groups at each time point post infection. Differential gene expression analysis, together with hierarchical clustering, revealed specifically expressed gene clusters from each group, and GO analysis was applied to these gene sets. Overall, we observed a comparable number of genes between young and aged group at 1, 3, and 5 dpi, while HGPS group displayed subtle changes at 1 and 3 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-C). Consistent with our histopathological analysis, expression of genes enriched in virus defense, interferon-beta, and innate immune response pathways was significantly higher in the lungs of young mice throughout the infection, suggesting a more active immune response upon viral infection compared to the aged and HGPS mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). In the aged mice, immune pathways such as immune cell migration, immunoglobulin production, humoral immune response, and cell chemotaxis represented feature genes in the clusters at 1 and 3 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Additionally, we found pathways related to hemorrhage, including wound healing, blood coagulation, and erythrocyte development, were significantly enriched in aged mice, further confirming the bleeding phenotype observed in the lungs of aged mice in the histopathological study (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE and Supplementary Fig.\u0026nbsp;4). We did not obverse a significant pathway enriched in HGPS mice at 1 and 3 dpi, likely due to the low numbers of unique genes (36 genes for 1 dpi and 53 genes for 3 dpi) in this group. Whereas at 5 dpi, 332 genes were more activated in the lungs of HGPS mice. These genes primarily belong to pathways associated with entrainment of circadian clock, regulation of blood circulation, and hormone transport. This suggests a dysregulation of circadian rhythms in HGPS mice, although the enrichment in this pathway was less pronounced (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Altogether, our results demonstrated that young mice, which have a normal immune system to protect against virus, display the highest immune response activation upon SARS-CoV-2 infection compared to aged and HGPS mice. Conversely, viral infection only causes mild pathological phenomena in HGPS mice, while aged mice exhibit lung hemorrhage when challenged by SARS-CoV-2, which could contribute to severe COVID-19.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eCOVID-19 pandemic has profoundly threatened normal life and health of people worldwide, particularly impacting older adults who are more susceptible to SARS-CoV-2 infection. Therefore, understanding the causal relationship between aging and severe COVID-19 is of great significance for improving preventive measures and therapeutic strategies for the elderly population. In this study, we have investigated the pathological consequences of SARS-CoV-2 infection in a progeria syndrome, HGPS, using a mouse model. We systematically compared the transcriptome landscape of lung tissues from young, aged, and HGPS/hACE2 mice in relation to SARS-CoV-2 infection. Firstly, we demonstrated that innate interferon response and virus defense pathway were more robustly activated in young compared to the aged and HGPS/hACE2 mice, consistent with the findings from other mouse models\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Aging is known to be associated with a functional decline in immune response, likely due to long-term chronic inflammation, as evidenced by our histopathological results \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Therefore, this observation further supports the low immune response in the aged and HGPS.\u003c/p\u003e \u003cp\u003eInterestingly, while aged mice displayed high rival replication at 1 dpi, they subsequently developed a severe bleeding phenotype in the lungs at 5 dpi, mirroring observations in patients infected with SARS-CoV-2 who display abnormal coagulation profiles \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Severe COVID-19 has been reported from multiple mouse models. Jiang et al used a transgenic mouse model with hACE2 expression driven by human FOXJ1 promoter to study SARS-CoV-2. Body weight loss, heavy lung damage and death were observed in these mice upon viral infection\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Dong used an K18-hACE2 mouse model which recapitulates severe COVID-19 with a low viral dose (2 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e PFU) \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Kenneth et al and Jiang er al applied mouse adopted coronavirus on wild type mice and demonstrated severe COVID-19 in the aged mice\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Here, using a different humanized ACE2 strategy, we displayed heavy hemorrhage in our aged hACE2 mice after coronavirus infection, consistent with clinical manifestations seen in COVID-19 patients \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Genes involved in cilium assembly and regeneration were activated at 3 dpi from all three groups, indicating a potential restoration of cilial function and clearance mechanisms. Thus, our study suggests that, alongside hypersecretion of mucus gel leading to alveolar and bronchial blockage \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, bleeding may also contribute to severe functional defect in lung.\u003c/p\u003e \u003cp\u003eAging, together with other aging-related diseases, are identified to be major risk factors for severe COVID19 \u003csup\u003e11\u003c/sup\u003e. Since HGPS share several phenotypes with accelerated aging especially atherosclerosis, osteoporosis, and cardiovascular diseases\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, one would assume a severe COVID19-like phenotype from HGPS after viral infection. However, beyond our expectation, HGPS/hACE2 mice only experienced mild pathological outcomes compared to the young and aged mice, indicating that Progeria syndrome cases may not be susceptible to SARS-CoV-2. A few studies investigating the association between genetic variants and COVID-19 susceptibility/severity, identified genes related to cytokines and viral receptors as risk-genes for severe COVID-19 \u003csup\u003e35\u0026ndash;37\u003c/sup\u003e, whereas progeria variants are not among them. By now, no progeria syndrome patient infected by SARS was reported as severe phenotype, further confirming our hypothesis. Additionally, we also noticed that the other uninfected HGPS hACE2 mice, which are from the same batch of tetraploid complementation as the infected ones, were all died nearly at the end of infection date, suggesting that these infected mice were likely at the end of their lives as well. This could also explain the slight decline of body weight from both uninfected and infect HGPS/hACE2 mice. Thus, viral infection with an early stage of HGPS mice, e.g. at 1 or 1.5 month ago, may worth testing for severe COVID-19.\u003c/p\u003e \u003cp\u003eOverall, our study assesses the potential pathological effects triggered by SARS-CoV-2 infection in HGPS, providing valuable insights for understanding progeria syndrome and guiding treatment for SARS-CoV-2 and other variants infected patients with premature aging.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEthics statement\u003c/h2\u003e \u003cp\u003eMouse studies were carried out in an animal biosafety level 3 (ABSL3) facility at Wuhan Institute of Virology, Chinese Academy of Sciences (CAS). All the animal experiments in this study were approved by The Institutional Animal Care and Use in GIBH and the Institutional Review Board of the Wuhan Institute of Virology, CAS. All the procedures involving mice is complied with all relevant ethical regulations (Experimental approvement number, IACUC 2020120). All animal experiments were performed in accordance with ARRIVE guidelines.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eBalb/c mouse embryonic stem cells (ESCs) were derived from dpc 3.5 mouse embryos, and were cultured on feeder cells in Knockout DMEM (Gibco, 10829018) supplemented with 1000 U/mL LIF (Novoprotein, C690), 10% KSR (Gibco, 10828028), NEAA (Gibco, 11140076), Glutamax (Gibco, 35050079), Sodium pyruvate (Gibco, 11360070), beta-mecaptomethanol (sigma, M3148), MEK inhibitor PD0325901 (Holzel Biotech, DC1056, 1 mM) and GSK3 inhibitor CHIR99021 (Holzel Biotech, DC1023, 3 mM).\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003eGeneration of Lmna mutant hACE2 mESCs\u003c/h2\u003e \u003cp\u003e \u003cem\u003eLmna\u003c/em\u003e c.1827 C\u0026thinsp;\u0026lt;\u0026thinsp;T variant was introduced into a huminzed ACE2 mES cell line generated from our previous study with slight modification\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Briefly, the codon-optimized hACE2 gene was inserted into mouse genome following the same strategy as before (ref16). To target \u003cem\u003eLmna\u003c/em\u003e c.1827, sgRNA (5\u0026rsquo;-aggagatggatccgcccacc-3\u0026rsquo;) along with linear targeting donor were transfected into hACE2 mESCs by electroporation. Single colonies were picked 2 days after transfection, and genomic DNA were extracted using DirectPCR Lysis Reagent (VIAGEN, 102-T). Exon 11 of \u003cem\u003eLmna\u003c/em\u003e was amplified using primer set (Forward primer: 5\u0026rsquo;-agtcagtcccaaactcgctg-3\u0026rsquo;; Reverse primer: 5\u0026rsquo;-caagagggactgcaaggagg-3\u0026rsquo;) and the c.1827 C\u0026thinsp;\u0026lt;\u0026thinsp;T variant was proved by sanger sequencing.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGeneration of mouse models (tetraploid complementation)\u003c/h2\u003e \u003cp\u003eMouse tetraploid embryos used for tetraploid complementation were prepared as reported before \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In detail, mESCs cultured at Day 2 were trypsinized until small clumps of cells (15\u0026ndash;20 cells per clumps) were seen under the microscope, and then were transferred into microdrops of KSOM medium with 10% FCS under mineral oil. Each clump was placed in a depression in the microdrop. Meanwhile, batches of 30\u0026ndash;50 embryos are incubated briefly in acidified Tyrode's solution to dissolve the zona pellucida. Next, two embryos are placed per ES clump for aggregation. All aggregates were incubated overnight at 37C, 5% CO2. After 24 hours of culture, eleven to fifteen embryos are transferred into one uterine horn of a 2.5 dpc pseudopregnant female mouse. Mature CD-1 females are used as pseudopregnant foster mothers with a weight of about 30g.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMice infection\u003c/h2\u003e \u003cp\u003eThe SARS-CoV-2 (IVCAS 6.7512) was prepared as reported before\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Male Balb/c mice with different genetic backgrounds (hACE2 and hACE2 plus Lmna c. 1827C\u0026thinsp;\u0026lt;\u0026thinsp;T) and different ages (2 months ago for young and HGPS hACE2 mice, more than 14 months ago for aged hACE2 mice) were treated with tribromoethanol (Avertin, 250mg/kg) and intranasally infected with 1 x 10\u003csup\u003e5\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e SARS-CoV-2 in 50 \u0026micro;L DMEM per mouse. The uninfected control mice were inoculated with DMEM only. Mice with different genetic backgrounds were randomly assigned to mock, 1, 3, 5, 7 dpi groups. Mice showing undetectable viral RNA in lung were regarded as a failed infection and were removed from the study. Mice were weighted and observed for clinical signs daily across the infection. 3 mice were euthanized using isoflurane followed by cervical dislocation at 1, 3, 5, 7 dpi per group. Lung tissues were collected for RNA and Hematoxylin and Eosin (H\u0026amp;E) staining. More details can be found in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and Table\u0026nbsp;1.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMouse group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMock\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c6\" namest=\"c3\"\u003e \u003cp\u003eInfected\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1dpi\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3dpi\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5dpi\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7dpi\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ehACE2 mice (2 months)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e3 (-1)\u003c/b\u003e*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ehACE2 mice (14 months)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHGPS/hACE2 mice (2 months)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e* One mouse with undetectable viral RNA in lung was removed from the study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eH\u0026amp;E staining\u003c/h2\u003e \u003cp\u003eLung samples were fixed with 4% paraformaldehyde, followed by paraffin embedment. Fixed lung tissues were cut into 3.5-\u0026micro;m sections for H\u0026amp;E staining following the standard protocol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eTranscriptome analysis\u003c/h2\u003e \u003cp\u003eRNA was isolated from homogenized lung tissue using TRizol with standard protocol. For ployA based mRNA-seq, 1 \u0026micro;g total RNAs were used for library construction. mRNA was enriched and purified using Library Preparation VAHTSTM mRNA Capture Beads (Vazyme, NR401-01). Next, purified mRNAs were fragmented, followed by cDNA synthesis, second-strand synthesis, end repair, adaptor ligation and amplification using VAHTS Universal V8 RNA-seq Library Prep Kit for Illumina (Vazyme, NR605-01) and VAHTS DNA Clean Beads. Libraries were sequenced for on average 20\u0026nbsp;million pair-end reads.\u003c/p\u003e \u003cp\u003eThe RNA-seq data processing was performed as described below. To analyze the transcriptome changes, raw reads were first trimmed to remove the adapter contamination, and then aligned to the mouse mm10 genome reference using STAR (2.7.10a) with default settings \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. FeatureCounts (2.0.1) was used for read assignment with the following parameters, -T 5 -g gene_name -p \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. DEseq2 was used for data normalization, differential expression analysis, and data visualization\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Genes with P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and log2(fold change)\u0026thinsp;\u0026gt;\u0026thinsp;1 were considered as differentially expressed genes and used for GO analysis. Time-series analysis was done using Mfuzz package in R (3.18) \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e with detectable genes (more than 10 reads were assigned from all the samples). All data are submitted to GEO under accession GSE264189.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eMouse lung tissues were snap-frozen in liquid N2 and then homogenized in clod Cell lysis buffer (20 mM Tris-HCl, pH 7.5, containing 150 mM NaCl, 2mM DTT, 50% triglyceride, 100 mM EDTA, 1% SDS, 1% NP40 and 1% Triton X-100) supplemented with Protease inhibitor cocktail (Roche, 4693132001). Total tissue extracts were separated on a 12.5% SDS-PAGE gel (EpiZyme, PG113), and transferred to a PVDF membrane (Millipore, IPVH00010). The following primary antibodies were used: anti-LAMINA/C (CST, 4777S, 1:1000), anti-progerin (abcam, ab66587, 1:1000), Histone 3 (abcam, ab18521, 1:5000). Original scan of the immunoblot is shown in supplementary File1 with molecular mass markers and indicated cropped area.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Zou Na and Chu Shilong from the animal center of GIBH for the mouse breeding. We thank Prof. Axel Schambach from Hannover Medical School for providing us the codon-optimized hACE2 vector. This work was financially supported by the National Key R\u0026amp;D Program of China (2021YFE0112900, 2023YFF1204701), the Austrian Science Fund (FWF) and \u0026lsquo;Herzfelder\u0026rsquo;sche Familienstiftung project P35268-B, BMBWF and WTZ-OEAD grant (CN 04/2021), Major Project of Guangzhou National Laboratory (GZNL2023A02005), The National Natural Science Foundation of China (32225012), Science and Technology Projects in Guangzhou (2024A04J4823), Basic Research Project of Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences (GIBHBRP23-02, GIBHBRP23-01), Science and Technology Planning Project of Guangdong Province, China (2023B1212060050, 2023B1212120009, Guangdong Basic and Applied Basic Research Foundation (2021A1515111044) , and Health@InnoHK Program launched by Innovation Technology Commission of the Hong Kong SAR, P. R. China.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eW.H., C.J., and W.G. conceived the study. W.H. designed the experiments, interpreted the data, and prepared the illustrations. W.K., Q.X., and S.J. prepared the animal models. L.M. and L.H. performed the viral infection and sample collection. \u0026nbsp; H.G., C.P., Y.X., S.Z., I.L., J.G. and H.M. contributed with the data analysis. C.J. and W.G. designed and supervised experiments and provided resources. W.H. wrote the main manuscript text. All authors commented on the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated and analyzed during the current study are available from the corresponding author on reasonable request. All the next-generation-sequencing data are submitted to GEO under accession GSE264189.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eL\u0026oacute;pez-Ot\u0026iacute;n, C., Blasco, M. A., Partridge, L., Serrano, M. \u0026amp; Kroemer, G. The hallmarks of aging. Cell 153, 1194 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eL\u0026oacute;pez-Ot\u0026iacute;n, C., Blasco, M. A., Partridge, L., Serrano, M. \u0026amp; Kroemer, G. Hallmarks of aging: An expanding universe. Cell 186, 243\u0026ndash;278 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSinha, J. K., Ghosh, S. \u0026amp; Raghunath, M. Progeria: A rare genetic premature ageing disorder. 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Bioinformation 2, 5\u0026ndash;7 (2007).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"HGPS, Aging, hACE2 mice, SARS-CoV-2","lastPublishedDoi":"10.21203/rs.3.rs-4316933/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4316933/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAging is identified as a significant risk factor for severe coronavirus disease-2019 (COVID-19), often resulting in profound lung damage and mortality. Yet, the biological relationship between aging, aging-related comorbidities, and COVID-19 remains incompletely understood. This study aimed to elucidate the age-related COVID19 pathogenesis using a Hutchinson-Gilford progeria syndrome (HGPS) mouse model with humanized ACE2 receptors. Pathological features were compared between young, aged, and HGPS hACE2 mice following SARS-CoV-2 challenge. We demonstrated that young mice display robust interferon response and antiviral activity, whereas this response is attenuated in aged mice. Viral infection in aged mice results in severe respiratory tract bleeding, likely contributing a higher mortality rate. In contrast, HGPS hACE2 mice exhibit milder disease manifestations characterized by minor immune cell infiltration and dysregulation of multiple metabolic processes. Comprehensive transcriptome analysis revealed both shared and unique gene expression dynamics among different mouse groups. Collectively, our studies evaluated the impact of SARS-CoV-2 infection on progeroid syndromes using a HGPS hACE2 mouse model, which holds promise as a useful tool for investigating COVID-19 pathogenesis in individuals with premature aging.\u003c/p\u003e","manuscriptTitle":"Premature aging effects on COVID-19 pathogenesis: new insights from mouse models","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-03 15:34:26","doi":"10.21203/rs.3.rs-4316933/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-06-03T08:23:37+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-29T04:33:06+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-24T08:59:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"237132410589147441280337059676646868063","date":"2024-05-15T05:22:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"320584535840263325112777242643091181957","date":"2024-05-15T03:35:30+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-10T00:44:38+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-09T23:04:42+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-04-27T14:32:08+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-27T14:31:04+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-04-24T09:06:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ac691957-90a3-43a9-867e-331d894bc71e","owner":[],"postedDate":"May 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":31361478,"name":"Biological sciences/Immunology/Infection"},{"id":31361479,"name":"Health sciences/Diseases/Infectious diseases/Viral infection"},{"id":31361480,"name":"Biological sciences/Physiology/Ageing"}],"tags":[],"updatedAt":"2024-08-26T15:59:08+00:00","versionOfRecord":{"articleIdentity":"rs-4316933","link":"https://doi.org/10.1038/s41598-024-70612-2","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-08-24 15:56:58","publishedOnDateReadable":"August 24th, 2024"},"versionCreatedAt":"2024-05-03 15:34:26","video":"","vorDoi":"10.1038/s41598-024-70612-2","vorDoiUrl":"https://doi.org/10.1038/s41598-024-70612-2","workflowStages":[]},"version":"v1","identity":"rs-4316933","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4316933","identity":"rs-4316933","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}