Safety concern of recombination between self-amplifying mRNA vaccines and viruses is mitigated in vivo.

Self-amplifying mRNA (SAM) vaccines can be rapidly deployed in the event of disease outbreaks. A legitimate safety concern is the potential for recombination between alphavirus-based SAM vaccines and circulating viruses. This theoretical risk needs to be assessed in the regulatory process for SAM vaccine approval. Herein, we undertake extensive in vitro and in vivo assessments to explore recombination between SAM vaccine and a wide selection of alphaviruses and a coronavirus. SAM vaccines were found to effectively limit alphavirus co-infection through superinfection exclusion, although some co-replication was still possible. Using sensitive cell-based assays, replication-competent alphavirus chimeras were generated in vitro as a result of rare, but reproducible, RNA recombination events. The chimeras displayed no increased fitness in cell culture. Viable alphavirus chimeras were not detected in vivo in C57BL/6J, Rag1-/- and Ifnar-/- mice, in which high levels of SAM vaccine and alphavirus co-replicated in the same tissue. Furthermore, recombination between a SAM-spike vaccine and a swine coronavirus was not observed. In conclusion we state that although the ability of SAM vaccines to recombine with alphaviruses might be viewed as an environmental safety concern, several key factors substantially mitigate against in vivo emergence of chimeric viruses from SAM vaccine recipients.

Selective lignin arylation for biomass fractionation and benign bisphenols.

Lignocellulose is mainly composed of hydrophobic lignin and hydrophilic polysaccharide polymers, contributing to an indispensable carbon resource for green biorefineries1,2. When chemically treated, lignin is compromised owing to detrimental intra- and intermolecular crosslinking that hampers downstream process3,4. The current valorization paradigms aim to avoid the formation of new C-C bonds, referred to as condensation, by blocking or stabilizing the vulnerable moieties of lignin5-7. Although there have been efforts to enhance biomass utilization through the incorporation of phenolic additives8,9, exploiting lignin's proclivity towards condensation remains unproven for valorizing both lignin and carbohydrates to high-value products. Here we leverage the proclivity by directing the C-C bond formation in a catalytic arylation pathway using lignin-derived phenols with high nucleophilicity. The selectively condensed lignin, isolated in near-quantitative yields while preserving its prominent cleavable ß-ether units, can be unlocked in a tandem catalytic process involving aryl migration and transfer hydrogenation. Lignin in wood is thereby converted to benign bisphenols (34-48 wt%) that represent performance-advantaged replacements for their fossil-based counterparts. Delignified pulp from cellulose and xylose from xylan are co-produced for textile fibres and renewable chemicals. This condensation-driven strategy represents a key advancement complementary to other promising monophenol-oriented approaches targeting valuable platform chemicals and materials, thereby contributing to holistic biomass valorization.

Microplastics dysregulate innate immunity in the SARS-CoV-2 infected lung.

Introduction: Global microplastic (MP) pollution is now well recognized, with humans and animals consuming and inhaling MPs on a daily basis, with a growing body of concern surrounding the potential impacts on human health. Methods: Using a mouse model of mild COVID-19, we describe herein the effects of azide-free 1 µm polystyrene MP beads, co-delivered into lungs with a SARS-CoV-2 omicron BA.5 inoculum. The effect of MPs on the host response to SARS-CoV-2 infection was analysed using histopathology and RNA-Seq at 2 and 6 days post-infection (dpi). Results: Although infection reduced clearance of MPs from the lung, virus titres and viral RNA levels were not significantly affected by MPs, and overt MP-associated clinical or histopathological changes were not observed. However, RNA-Seq of infected lungs revealed that MP exposure suppressed innate immune responses at 2 dpi and increased pro-inflammatory signatures at 6 dpi. The cytokine profile at 6 dpi showed a significant correlation with the 'cytokine release syndrome' signature observed in some COVID-19 patients. Discussion: The findings are consistent with the recent finding that MPs can inhibit phagocytosis of apoptotic cells via binding of Tim4. They also add to a growing body of literature suggesting that MPs can dysregulate inflammatory processes in specific disease settings.

Integrated network toxicology, molecular docking, and in vivo experiments to elucidate molecular mechanism of aflatoxin B1 hepatotoxicity.

Due to the rise in temperature and sea level caused by climate change, the detection rate of aflatoxin B1 (AFB1) in food crops has increased dramatically, and the frequency and severity of aflatoxicosis in humans and animals are also increasing. AFB1 has strong hepatotoxicity, causing severe liver damage and even cancer. However, the mechanism of AFB1 hepatotoxicity remains unclear. By integrating network toxicology, molecular docking and in vivo experiments, this research was designed to explore the potential hepatotoxicity mechanisms of AFB1. Thirty-three intersection targets for AFB1-induced liver damage were identified using online databases. PI3K/AKT1, MAPK, FOXO1 signaling pathways, and apoptosis were significantly enriched. In addition, the proteins of ALB, AKT1, PIK3CG, MAPK8, HSP90AA1, PPARA, MAPK1, EGFR, FOXO1, and IGF1 exhibited good affinity with AFB1. In vivo experiments, significant pathological changes occurred in the liver of mice. AFB1 induction increased the expression levels of EGFR, ERK, and FOXO1, and decreased the expression levsls of PI3K and AKT1. Moreover, AFB1 treatment caused an increase in Caspase3 expression, and a decrease in Bcl2/Bax ratio. By combining network toxicology with in vivo experiments, this study confirms for the first time that AFB1 promotes the FOXO1 signaling pathway by inactivating PI3K/AKT1 and activating EGFR/ERK signaling pathways, hence aggravating hepatocyte apoptosis. This research provides new strategies for studying the toxicity of environmental pollutants and new possible targets for the development of hepatoprotective drugs.

Epitaxial Growth of Two-Dimensional MWW Zeolite.

Two-dimensional (2D) zeolite, with a high aspect ratio, has more open skeletons and accessible active sites than its three-dimensional (3D) counterpart. However, traditional methods of obtaining 2D zeolites often cause structural damage and widespread skeleton defects, hindering efficient selectivity in molecular separation. In this study, we present, for the first time, a direct epitaxial synthesis of 2D zeolite (Epi-MWW) guided by hexagonal boron nitride (h-BN) with a coincidence matching of site lattices to MWW zeolite. The as-grown Epi-MWW zeolite possesses a high crystallinity and intact hexagonal 2D morphology, with an average thickness of 10 nm and an aspect ratio of over 50. Thanks to its excellent molecular accessibility, the diffusion time constants of o-xylene (OX) and p-xylene (PX) are as 12 and 133 times higher than those of conventional MCM-22, respectively; the PX/OX selectivity of Epi-MWW is 7.4 times better than MCM-22 as calculated by the ideal adsorbed solution theory.

SARS-CoV-2 omicron BA.5 and XBB variants have increased neurotropic potential over BA.1 in K18-hACE2 mice and human brain organoids.

The reduced pathogenicity of the omicron BA.1 sub-lineage compared to earlier variants is well described, although whether such attenuation is retained for later variants like BA.5 and XBB remains controversial. We show that BA.5 and XBB isolates were significantly more pathogenic in K18-hACE2 mice than a BA.1 isolate, showing increased neurotropic potential, resulting in fulminant brain infection and mortality, similar to that seen for original ancestral isolates. BA.5 also infected human cortical brain organoids to a greater extent than the BA.1 and original ancestral isolates. In the brains of mice, neurons were the main target of infection, and in human organoids neuronal progenitor cells and immature neurons were infected. The results herein suggest that evolving omicron variants may have increasing neurotropic potential.

Rapid inactivation and sample preparation for SARS-CoV-2 PCR-based diagnostics using TNA-Cifer Reagent E.

RT-qPCR remains a key diagnostic methodology for COVID-19/SARS-CoV-2. Typically, nasal or saliva swabs from patients are placed in virus transport media (VTM), RNA is extracted at the pathology laboratory, and viral RNA is measured using RT-qPCR. In this study, we describe the use of TNA-Cifer Reagent E in a pre-clinical evaluation study to inactivate SARS-CoV-2 as well as prepare samples for RT-qPCR. Adding 1 part TNA-Cifer Reagent E to 5 parts medium containing SARS-CoV-2 for 10 min at room temperature inactivated the virus and permitted RT-qPCR detection. TNA-Cifer Reagent E was compared with established column-based RNA extraction and purification methodology using a panel of human clinical nasal swab samples (n = 61), with TNA-Cifer Reagent E showing high specificity (100%) and sensitivity (97.37%). Mixtures of SARS-CoV-2 virus and TNA-Cifer Reagent E could be stored for 3 days at room temperature or for 2 weeks at 4°C without the loss of RT-qPCR detection sensitivity. The detection sensitivity was preserved when TNA-Cifer Reagent E was used in conjunction with a range of VTM for saliva samples but only PBS (Gibco) and Amies Orange for nasal samples. Thus, TNA-Cifer Reagent E improves safety by rapidly inactivating the virus during sample processing, potentially providing a safe means for molecular SARS-CoV-2 testing outside traditional laboratory settings. The reagent also eliminates the need for column-based and/or automated viral RNA extraction/purification processes, thereby providing cost savings for equipment and reagents, as well as reducing processing and handling times.

A novel metallothionein gene HcMT from halophyte shrub Halostachys caspica respond to cadmium and sodium stress.

Cadmium (Cd) and sodium (Na) are two of the most phytotoxic metallic elements causing environmental and agricultural problems. Metallothioneins (MTs) play an important role in the adaptation to abiotic stress. We previously isolated a novel type 2 MT gene from Halostachys caspica (H. caspica), named HcMT, which responded to metal and salt stress. To understand the regulatory mechanisms controlling HcMT expression, we cloned the HcMT promoter and characterized its tissue-specific and spatiotemporal expression patterns. ß-Glucuronidase (GUS) activity analysis showed that the HcMT promoter was responsive to CdCl2, CuSO4, ZnSO4 and NaCl stress. Therefore, we further investigated the function of HcMT under abiotic stress in yeast and Arabidopsis thaliana (Arabidopsis). In CdCl2, CuSO4 or ZnSO4 stress, HcMT significantly enhanced the metal ions tolerance and accumulation in yeast through function as a metal chelator. Moreover, the HcMT protein also protected yeast cells from NaCl, PEG and hydrogen peroxide (H2O2) toxicity with less effectiveness. However, transgenic Arabidopsis carrying HcMT gene only displayed tolerance to CdCl2 and NaCl, accompanying by higher content of Cd2+ or Na+ and lower H2O2, compared to wild-type (WT) plants. Next, we demonstrated that the recombinant HcMT protein has the ability to bind Cd2+ and the potential of scavenging ROS (reactive oxygen species) in vitro. This result further confirmed that the role of HcMT to influence plants to CdCl2 and NaCl stress may bind metal ions and scavenge ROS. Overall, we described the biological functions of HcMT and developed a metal- and salt-inducible promoter system for using in genetic engineering.

Computer-aided shape features extraction and regression models for predicting the ascending aortic aneurysm growth rate.

OBJECTIVE: ascending aortic aneurysm growth prediction is still challenging in clinics. In this study, we evaluate and compare the ability of local and global shape features to predict the ascending aortic aneurysm growth. MATERIAL AND METHODS: 70 patients with aneurysm, for which two 3D acquisitions were available, are included. Following segmentation, three local shape features are computed: (1) the ratio between maximum diameter and length of the ascending aorta centerline, (2) the ratio between the length of external and internal lines on the ascending aorta and (3) the tortuosity of the ascending tract. By exploiting longitudinal data, the aneurysm growth rate is derived. Using radial basis function mesh morphing, iso-topological surface meshes are created. Statistical shape analysis is performed through unsupervised principal component analysis (PCA) and supervised partial least squares (PLS). Two types of global shape features are identified: three PCA-derived and three PLS-based shape modes. Three regression models are set for growth prediction: two based on gaussian support vector machine using local and PCA-derived global shape features; the third is a PLS linear regression model based on the related global shape features. The prediction results are assessed and the aortic shapes most prone to growth are identified. RESULTS: the prediction root mean square error from leave-one-out cross-validation is: 0.112 mm/month, 0.083 mm/month and 0.066 mm/month for local, PCA-based and PLS-derived shape features, respectively. Aneurysms close to the root with a large initial diameter report faster growth. CONCLUSION: global shape features might provide an important contribution for predicting the aneurysm growth.

Protection of taraxasterol against acetaminophen-induced liver injury elucidated through network pharmacology and in vitro and in vivo experiments.

BACKGROUND: Drug-induced liver injury (DILI) is primarily caused by drugs or their metabolites. Acetaminophen (APAP) is an over-the-counter antipyretic analgesic that exhibits high hepatotoxicity when used for long-term or in overdoses. Taraxasterol is a five-ring triterpenoid compound extracted from traditional Chinese medicinal herb Taraxacum officinale. Our previous studies have demonstrated that taraxasterol exerts protective effects on alcoholic and immune liver injuries. However, the effect of taraxasterol on DILI remains unclear. HYPOTHESIS/PURPOSE: This study aimed to elucidate the effects and mechanisms of action of taraxasterol on APAP-induced liver injury using network pharmacology and in vitro and in vivo experiments. METHODS: Online databases of drug and disease targets were used to screen the targets of taraxasterol and DILI, and a protein-protein interaction network (PPI) was constructed. Core target genes were identified using the tool of Analyze of Cytoscape, gene ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment analyses were performed. Oxidation, inflammation and apoptosis were evaluated to determine the effect of taraxasterol on APAP-stimulated liver damage in AML12 cells and mice. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting were used to explore the potential mechanisms of taraxasterol against DILI. RESULTS: Twenty-four intersection targets for taraxasterol and DILI were identified. Among them, 9 core targets were identified. GO and KEGG analysis showed that core targets are closely related to oxidative stress, apoptosis, and inflammatory response. The in vitro findings showed that taraxasterol alleviated mitochondrial damage in AML12 cells treated with APAP. The in vivo results revealed that taraxasterol alleviated pathological changes in the livers of mice treated with APAP and inhibited the activity of serum transaminases. Taraxasterol increased the activity of antioxidants, inhibited the production of peroxides, and reduced inflammatory response and apoptosis in vitro and in vivo. Taraxasterol promoted Nrf2 and HO-1 expression, suppressed JNK phosphorylation, and decreased the Bax/Bcl-2 ratio and caspase-3 expression in AML12 cells and mice. CONCLUSION: By integrating network pharmacology with in vitro and in vivo experiments, this study indicated that taraxasterol inhibits APAP-stimulated oxidative stress, inflammatory response and apoptosis in AML12 cells and mice by regulating the Nrf2/HO-1 pathway, JNK phosphorylation, and apoptosis-related protein expression. This study provides a new evidence for the use of taraxasterol as a hepatoprotective drug.

Comparative Efficacy of Mayaro Virus-Like Particle Vaccines Produced in Insect or Mammalian Cells.

Mayaro virus (MAYV) is a mosquito-transmitted alphavirus that causes often debilitating rheumatic disease in tropical Central and South America. There are currently no licensed vaccines or antiviral drugs available for MAYV disease. Here, we generated Mayaro virus-like particles (VLPs) using the scalable baculovirus-insect cell expression system. High-level secretion of MAYV VLPs in the culture fluid of Sf9 insect cells was achieved, and particles with a diameter of 64 to 70 nm were obtained after purification. We characterize a C57BL/6J adult wild-type mouse model of MAYV infection and disease and used this model to compare the immunogenicity of VLPs from insect cells with that of VLPs produced in mammalian cells. Mice received two intramuscular immunizations with 1 µg of nonadjuvanted MAYV VLPs. Potent neutralizing antibody responses were generated against the vaccine strain, BeH407, with comparable activity seen against a contemporary 2018 isolate from Brazil (BR-18), whereas neutralizing activity against chikungunya virus was marginal. Sequencing of BR-18 illustrated that this virus segregates with genotype D isolates, whereas MAYV BeH407 belongs to genotype L. The mammalian cell-derived VLPs induced higher mean neutralizing antibody titers than those produced in insect cells. Both VLP vaccines completely protected adult wild-type mice against viremia, myositis, tendonitis, and joint inflammation after MAYV challenge. IMPORTANCE Mayaro virus (MAYV) is associated with acute rheumatic disease that can be debilitating and can evolve into months of chronic arthralgia. MAYV is believed to have the potential to emerge as a tropical public health threat, especially if it develops the ability to be efficiently transmitted by urban mosquito vectors, such as Aedes aegypti and/or Aedes albopictus. Here, we describe a scalable virus-like particle vaccine against MAYV that induced neutralizing antibodies against a historical and a contemporary isolate of MAYV and protected mice against infection and disease, providing a potential new intervention for MAYV epidemic preparedness.

Assessment of shape-based features ability to predict the ascending aortic aneurysm growth.

The current guidelines for the ascending aortic aneurysm (AsAA) treatment recommend surgery mainly according to the maximum diameter assessment. This criterion has already proven to be often inefficient in identifying patients at high risk of aneurysm growth and rupture. In this study, we propose a method to compute a set of local shape features that, in addition to the maximum diameter D, are intended to improve the classification performances for the ascending aortic aneurysm growth risk assessment. Apart from D, these are the ratio DCR between D and the length of the ascending aorta centerline, the ratio EILR between the length of the external and the internal lines and the tortuosity T. 50 patients with two 3D acquisitions at least 6 months apart were segmented and the growth rate (GR) with the shape features related to the first exam computed. The correlation between them has been investigated. After, the dataset was divided into two classes according to the growth rate value. We used six different classifiers with input data exclusively from the first exam to predict the class to which each patient belonged. A first classification was performed using only D and a second with all the shape features together. The performances have been evaluated by computing accuracy, sensitivity, specificity, area under the receiver operating characteristic curve (AUROC) and positive (negative) likelihood ratio LHR+ (LHR-). A positive correlation was observed between growth rate and DCR (r = 0.511, p = 1.3e-4) and between GR and EILR (r = 0.472, p = 2.7e-4). Overall, the classifiers based on the four metrics outperformed the same ones based only on D. Among the diameter-based classifiers, k-nearest neighbours (KNN) reported the best accuracy (86%), sensitivity (55.6%), AUROC (0.74), LHR+ (7.62) and LHR- (0.48). Concerning the classifiers based on the four shape features, we obtained the best accuracy (94%), sensitivity (66.7%), specificity (100%), AUROC (0.94), LHR+ (+∞) and LHR- (0.33) with support vector machine (SVM). This demonstrates how automatic shape features detection combined with risk classification criteria could be crucial in planning the follow-up of patients with ascending aortic aneurysm and in predicting the possible dangerous progression of the disease.

Taraxasterol alleviates aflatoxin B1-induced liver damage in broiler chickens via regulation of oxidative stress, apoptosis and autophagy.

Aflatoxin B1 (AFB1) is the most dangerous and abundant mycotoxin, which is toxic to almost all animals, and poultry is more sensitive to AFB1 toxicity. Ingesting AFB1-contaminated feed can cause significant liver damage and brings serious harm to poultry, which greatly restricts the development of the poultry industry. The present research was implemented to explore the intervention effect and its mechanism of taraxasterol on liver damage induced by AFB1 in broiler chickens. The liver damage model in broiler chickens was established by feeding 0.5 mg/kg AFB1 feed, and taraxasterol (25, 50 and 100 mg/kg BW, respectively) was given in the drinking water for 21 days. The growth performance, liver function, oxidative stress, apoptosis and autophagy were evaluated. The results showed that taraxasterol increased BW and reduced feed-to-gain ratio of broiler chickens induced by AFB1. Taraxasterol improved the levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyltransferase (GGT), total bilirubin (TBIL) and alkaline phosphatase (ALP), and attenuated hepatic histopathological changes induced by AFB1. Meantime, taraxasterol down-regulated cytochrome P450 (CYP450) enzyme system CYP1A1 and CYP2A6 mRNA expression, inhibited the overproduction of reactive oxygen species (ROS) and malondialdehyde (MDA), and enhanced the activities of antioxidant enzymes glutathione (GSH) and catalase (CAT) and the content of antioxidant superoxide dismutase (SOD) of the liver in broiler chickens induced by AFB1. Furthermore, taraxasterol up-regulated the mRNA and protein expression of hepatic nuclear factor E2 related factor 2 (Nrf2), heme oxygenase 1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO1), and down-regulated the expression of hepatic kelch like ECH associated protein 1 (Keap1) induced by AFB1 in Keap1/Nrf2 signaling pathway. The ultrastructural observation and RT-qPCR results found that taraxasterol inhibited apoptosis of hepatocytes, up-regulated the expression of B-cell lymphoma-2 (Bcl-2) mRNA and down-regulated the expression of Bax and caspase3 mRNA. Further, taraxasterol restored the autophagy of hepatocytes and down-regulated the mRNA expression of phosphatidylinositol 3-kinase K (PI3K), protein kinase B (AKT) and mammalian target of rapamycin (mTOR) in AFB1-induced liver of broiler chickens. The above results indicate that taraxasterol alleviates liver damage induced by AFB1 in broiler chickens through regulation of Keap1/Nrf2 signaling pathway to exert its antioxidant effect, mitochondrial apoptosis pathway to improve anti-apoptotic ability and PI3K/AKT/mTOR pathway to restore autophagy.

IFITM3 is a host restriction factor that inhibits porcine transmissible gastroenteritis virus infection.

Interferon-induced transmembrane proteins (IFITMs) play an important role in the innate immune response triggered by viral infection. Transmissible gastroenteritis virus (TGEV) causes severe diarrhea, vomiting and dehydration in piglets, resulting in huge economic losses to the swine industry. In this study, we showed that IFITM3 inhibits the replication of TGEV and interferes with the binding of TGEV to PK15 cells. Moreover, the inhibitory effect of IFITM3 on TGEV circumvents the upregulation of inflammatory cytokines. Subsequently, we found that the M22A mutant loses part of the antiviral effect of IFITM3 on TGEV; in contrast, the K24A mutant enhances the antiviral effect of IFITM3. Notably, our data shows a synergistic effect between IFITM3 and CQ, which further amplifies the antiviral effect against TGEV.

Warmer ambient air temperatures reduce nasal turbinate and brain infection, but increase lung inflammation in the K18-hACE2 mouse model of COVID-19.

Warmer climatic conditions have been associated with fewer COVID-19 cases. Herein we infected K18-hACE2 mice housed at the standard animal house temperature of ∼22 °C, or at ∼31 °C, which is considered to be thermoneutral for mice. On day 2 post infection, RNA-Seq analyses showed no significant differential gene expression lung in lungs of mice housed at the two temperatures, with almost identical viral loads and type I interferon responses. There was also no significant difference in viral loads in lungs on day 5, but RNA-Seq and histology analyses showed clearly elevated inflammatory signatures and infiltrates. Thermoneutrality thus promoted lung inflammation. On day 2 post infection mice housed at 31 °C showed reduced viral loads in nasal turbinates, consistent with increased mucociliary clearance at the warmer ambient temperature. These mice also had reduced virus levels in the brain, and an ensuing amelioration of weight loss and a delay in mortality. Warmer air temperatures may thus reduce infection of the upper respiratory track and the olfactory epithelium, resulting in reduced brain infection. Potential relevance for anosmia and neurological sequelae in COVID-19 patients is discussed.

Zika virus noncoding RNA cooperates with the viral protein NS5 to inhibit STAT1 phosphorylation and facilitate viral pathogenesis.

All flaviviruses, including Zika virus, produce noncoding subgenomic flaviviral RNA (sfRNA), which plays an important role in viral pathogenesis. However, the exact mechanism of how sfRNA enables viral evasion of antiviral response is not well defined. Here, we show that sfRNA is required for transplacental virus dissemination in pregnant mice and subsequent fetal brain infection. We also show that sfRNA promotes apoptosis of neural progenitor cells in human brain organoids, leading to their disintegration. In infected human placental cells, sfRNA inhibits multiple antiviral pathways and promotes apoptosis, with signal transducer and activator of transcription 1 (STAT1) identified as a key shared factor. We further show that the production of sfRNA leads to reduced phosphorylation and nuclear translocation of STAT1 via a mechanism that involves sfRNA binding to and stabilizing viral protein NS5. Our results suggest the cooperation between viral noncoding RNA and a viral protein as a novel strategy for counteracting antiviral responses.

Mouse models of COVID-19 recapitulate inflammatory pathways rather than gene expression.

How well mouse models recapitulate the transcriptional profiles seen in humans remains debatable, with both conservation and diversity identified in various settings. Herein we use RNA-Seq data and bioinformatics approaches to analyze the transcriptional responses in SARS-CoV-2 infected lungs, comparing 4 human studies with the widely used K18-hACE2 mouse model, a model where hACE2 is expressed from the mouse ACE2 promoter, and a model that uses a mouse adapted virus and wild-type mice. Overlap of single copy orthologue differentially expressed genes (scoDEGs) between human and mouse studies was generally poor (≈15-35%). Rather than being associated with batch, sample treatment, viral load, lung damage or mouse model, the poor overlaps were primarily due to scoDEG expression differences between species. Importantly, analyses of immune signatures and inflammatory pathways illustrated highly significant concordances between species. As immunity and immunopathology are the focus of most studies, these mouse models can thus be viewed as representative and relevant models of COVID-19.

Evolution of ACE2-independent SARS-CoV-2 infection and mouse adaption after passage in cells expressing human and mouse ACE2.

Human ACE2 Human angiotensin converting enzyme 2 (hACE2) is the key cell attachment and entry receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with the original SARS-CoV-2 isolates unable to use mouse ACE2 (mACE2). Herein we describe the emergence of a SARS-CoV-2 strain capable of ACE2-independent infection and the evolution of mouse-adapted (MA) SARS-CoV-2 by in vitro serial passaging of virus in co-cultures of cell lines expressing hACE2 and mACE2. MA viruses evolved with up to five amino acid changes in the spike protein, all of which have been seen in human isolates. MA viruses replicated to high titers in C57BL/6J mouse lungs and nasal turbinates and caused characteristic lung histopathology. One MA virus also evolved to replicate efficiently in several ACE2-negative cell lines across several species, including clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) ACE2 knockout cells. An E484D substitution is likely involved in ACE2-independent entry and has appeared in only ≈0.003 per cent of human isolates globally, suggesting that it provided no significant selection advantage in humans. ACE2-independent entry reveals a SARS-CoV-2 infection mechanism that has potential implications for disease pathogenesis, evolution, tropism, and perhaps also intervention development.