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SARS-CoV-2 variants of concern (VOCs) emerged during the COVID-19 pandemic. Here, we used unbiased systems approaches to study the host-selective forces driving VOC evolution. We discovered that VOCs evolved convergent strategies to remodel the host by modulating viral RNA and protein levels, altering viral and host protein phosphorylation, and rewiring virus-host protein-protein interactions. Integrative computational analyses revealed that although Alpha, Beta, Gamma, and Delta ultimately converged to suppress interferon-stimulated genes (ISGs), Omicron BA.1 did not. ISG suppression correlated with the expression of viral innate immune antagonist proteins, including Orf6, N, and Orf9b, which we mapped to specific mutations. Later Omicron subvariants BA.4 and BA.5 more potently suppressed innate immunity than early subvariant BA.1, which correlated with Orf6 levels, although muted in BA.4 by a mutation that disrupts the Orf6-nuclear pore interaction. Our findings suggest that SARS-CoV-2 convergent evolution overcame human adaptive and innate immune barriers, laying the groundwork to tackle future pandemics.
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COVID-19 , SARS-CoV-2 , Humanos , COVID-19/virologia , Imunidade Inata/genética , Pandemias , SARS-CoV-2/genéticaRESUMO
The nonstructural protein 1 (Nsp1) of SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) is a virulence factor that targets multiple cellular pathways to inhibit host gene expression and antiviral response. However, the underlying mechanisms of the various Nsp1-mediated functions and their contributions to SARS-CoV-2 virulence remain unclear. Among the targets of Nsp1 is the mRNA (messenger ribonucleic acid) export receptor NXF1-NXT1, which mediates nuclear export of mRNAs from the nucleus to the cytoplasm. Based on Nsp1 crystal structure, we generated mutants on Nsp1 surfaces and identified an acidic N-terminal patch that is critical for interaction with NXF1-NXT1. Photoactivatable Nsp1 probe reveals the RNA Recognition Motif (RRM) domain of NXF1 as an Nsp1 N-terminal binding site. By mutating the Nsp1 N-terminal acidic patch, we identified a separation-of-function mutant of Nsp1 that retains its translation inhibitory function but substantially loses its interaction with NXF1 and reverts Nsp1-mediated mRNA export inhibition. We then generated a recombinant (r)SARS-CoV-2 mutant on the Nsp1 N-terminal acidic patch and found that this surface is key to promote NXF1 binding and inhibition of host mRNA nuclear export, viral replication, and pathogenicity in vivo. Thus, these findings provide a mechanistic understanding of Nsp1-mediated mRNA export inhibition and establish the importance of this pathway in the virulence of SARS-CoV-2.
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Transporte Ativo do Núcleo Celular , COVID-19 , Proteínas de Transporte Nucleocitoplasmático , RNA Mensageiro , Proteínas de Ligação a RNA , SARS-CoV-2 , Proteínas não Estruturais Virais , Humanos , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidade , SARS-CoV-2/genética , Proteínas não Estruturais Virais/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Animais , COVID-19/virologia , COVID-19/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Replicação Viral , Núcleo Celular/metabolismo , Células Vero , Virulência , Chlorocebus aethiops , Células HEK293RESUMO
Hybrid immunity (vaccination + natural infection) to SARS-CoV-2 provides superior protection to re-infection. We performed immune profiling studies during breakthrough infections in mRNA-vaccinated hamsters to evaluate hybrid immunity induction. The mRNA vaccine, BNT162b2, was dosed to induce binding antibody titers against ancestral spike, but inefficient serum virus neutralization of ancestral SARS-CoV-2 or variants of concern (VoCs). Vaccination reduced morbidity and controlled lung virus titers for ancestral virus and Alpha but allowed breakthrough infections in Beta, Delta and Mu-challenged hamsters. Vaccination primed for T cell responses that were boosted by infection. Infection back-boosted neutralizing antibody responses against ancestral virus and VoCs. Hybrid immunity resulted in more cross-reactive sera, reflected by smaller antigenic cartography distances. Transcriptomics post-infection reflects both vaccination status and disease course and suggests a role for interstitial macrophages in vaccine-mediated protection. Therefore, protection by vaccination, even in the absence of high titers of neutralizing antibodies in the serum, correlates with recall of broadly reactive B- and T-cell responses.
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COVID-19 , SARS-CoV-2 , Animais , Cricetinae , Humanos , Vacina BNT162 , Infecções Irruptivas , COVID-19/prevenção & controle , Mesocricetus , Anticorpos Neutralizantes , Complicações Pós-Operatórias , RNA Mensageiro/genética , Imunidade , Anticorpos Antivirais , VacinaçãoRESUMO
BACKGROUND: Co-morbidity with respiratory viruses including influenza A, cause varying degree of morbidity especially in TB patients compared to general population. This study estimates the risk factors associated with influenza A (H1N1)pdm09 in TB patients with ILI. METHODS: A cohort of tuberculosis (TB) patients who were admitted to and enrolled in a TB Directly Observed Therapy Program (DOTs) in tertiary care hospitals of Lahore (Mayo Hospital and Infectious Disease Hospital) were followed for 12 weeks. At the start of study period, to record influenza-like illness (ILI), a symptom card was provided to all the participants. Every participant was contacted once a week, in person. When the symptoms were reported by the participant, a throat swab was taken for the detection of influenza A (H1N1)pdm09. A nested case control study was conducted and TB patients with ILI diagnosed with influenza A (H1N1)pdm09 by conventional RT-PCR were selected as cases, while those who tested negative by conventional RT-PCR were enrolled as controls. All cases and controls in the study were interviewed face-to-face in the local language. Epidemiological data about potential risk factors were collected on a predesigned questionnaire. Logistic analysis was conducted to identify associated risk factors in TB patients with ILI. RESULTS: From the main cohort of TB patients (n = 152) who were followed during the study period, 59 (39%) developed ILI symptoms; of them, 39 tested positive for influenza A (H1N1)pdm09, while 20 were detected negative for influenza A (H1N1)pdm09. In univariable analysis, four factors were identified as risk factors (p < 0.05). The final multivariable model identified one risk factor (sharing of towels, P = 0.008)) and one protective factor (wearing a face mask, p = < 0.001)) for influenza A (H1N1)pdm09 infection. CONCLUSION: The current study identified the risk factors of influenza A (H1N1)pdm09 infection among TB patients with ILI.
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Vírus da Influenza A Subtipo H1N1 , Influenza Humana , Tuberculose , Humanos , Influenza Humana/epidemiologia , Influenza Humana/virologia , Masculino , Fatores de Risco , Paquistão/epidemiologia , Feminino , Adulto , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Estudos de Casos e Controles , Pessoa de Meia-Idade , Tuberculose/epidemiologia , Tuberculose/complicações , Adulto Jovem , Adolescente , IdosoRESUMO
Semen traits play the vital role in determining the fertility of a broiler breeder flock; however, it can be influenced by several factors. This experiment was carried out to assess some of these factors affecting the semen. A total of 89 male birds and 960 hens of 20-week-old broiler breeder (2215 g ± 7.5%) were divided into two main groups; one was kept in cages (AIC) and another group was kept on deep litter floor (AIF), while both these groups were subjected to AI. The male birds of aforementioned groups (44 males and 480 females) were further divided into 4 sub-groups (11 males and 120 females) to execute different semen collection frequencies i.e., 2, 3, 4, and 5th days' interval. The impact of time duration between semen collection and insemination on sperm kinematics was monitored. The quantitative and qualitative analysis of semen including sperm concentration and sperm kinematics of the collected semen was conducted through a computer-assisted sperm analyzer (CASA) and ONGO machine (working on the CASA principle). Resultantly, the data revealed that the studied parameters of semen were deteriorated with the progression of age of male birds, while the group of males with standard body weight produced the best semen quantitatively and qualitatively followed by overweight particularly during the post peak phase (46-65 = 20 weeks). Although the 3rd day, semen collection frequency was found better for quality, the higher quantity of semen was achieved when males were being collected at the intervals of 4th and 5th day respectively regardless of housing systems. Significant decline in sperm kinematics was recorded with the progression of semen holding duration at the temperature of poultry farm. Furthermore, the highest contamination of E. coli, Salmonella, and Mycoplasma gallisepticum was recorded in the reproductive tract of hens and semen of the AIF group as compared to AIC. Thus, conclusion can be settled that the semen properties are significantly affected by age, body weight, and semen collection intervals in both housing systems, while sperm kinematics is being disrupted with the progression of holding duration. Although housing systems could affect the semen insignificantly, yet lesser contamination was recorded in semen and in the reproductive tract of hens of AIC.
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Galinhas , Sêmen , Animais , Masculino , Feminino , Escherichia coli , Habitação , Espermatozoides , Fertilidade , Peso CorporalRESUMO
Protection from influenza virus infection is canonically associated with antibodies that neutralize the virus by blocking the interaction between the viral hemagglutinin and host cell receptors. However, protection can also be conferred by other mechanisms, including antibody-mediated effector functions. Here, we report the characterization of 22 broadly cross-reactive, nonneutralizing antibodies specific for influenza B virus hemagglutinin. The majority of these antibodies recognized influenza B viruses isolated over the period of 73 years and bind the conserved stalk domain of the hemagglutinin. A proportion of the characterized antibodies protected mice from both morbidity and mortality after challenge with a lethal dose of influenza B virus. Activity in an antibody-dependent cell-mediated cytotoxicity reporter assay correlated strongly with protection, suggesting that Fc-dependent effector function determines protective efficacy. The information regarding mechanism of action and epitope location stemming from our characterization of these antibodies will inform the design of urgently needed vaccines that could induce broad protection against influenza B viruses.IMPORTANCE While broadly protective antibodies against the influenza A virus hemagglutinin have been well studied, very limited information is available for antibodies that broadly recognize influenza B viruses. Similarly, the development of a universal or broadly protective influenza B virus vaccine lags behind the development of such a vaccine for influenza A virus. More information about epitope location and mechanism of action of broadly protective influenza B virus antibodies is required to inform vaccine development. In addition, protective antibodies could be a useful tool to treat or prevent influenza B virus infection in pediatric cohorts or in a therapeutic setting in immunocompromised individuals in conjugation with existing treatment avenues.
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Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Reações Cruzadas/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza B/imunologia , Infecções por Orthomyxoviridae/imunologia , Animais , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Linhagem Celular , Linhagem Celular Tumoral , Cães , Epitopos/imunologia , Feminino , Humanos , Vacinas contra Influenza/imunologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/virologia , Células Sf9RESUMO
BACKGROUND: There is a paucity of literature on a well-defined role of a pharmacist in different aspects of transition of care service (TCS). Although health care institutions have specific details on the discharge process, there is a need for a sustainable TCS with a well-defined role of pharmacists. OBJECTIVE: To describe the impact of a pharmacist-led TCS on acute health care utilization, clinic quality indicators, and identification and resolution of medication-related problems (MRPs). METHODS: A pharmacist-managed TCS service, referred to as the Pharmacist Advancement of Transitions of Care to Home (PATCH) service, was established at an academic medical center, where high-risk patients received a postdischarge phone call from a pharmacist followed by a face-to-face meeting with the pharmacist and the patient's primary care provider (PCP). In a prospective transitions of care group (n = 74), outcomes of patients such as acute health care utilization (an emergency department visit or an inpatient readmission, within 30 days post discharge), clinic quality indicators, and identification and resolution of MRPs were compared to a retrospective control group (n = 87) who received the standard of care. RESULTS: Utilization of acute health care services was significantly lower in the prospective group compared to the retrospective control group (23% vs 41.4%; P = .013). A total of 49 MRPs were discovered in patients who received the TCS. CONCLUSIONS: Pharmacists play an integral role in improving the transitions of care to reduce acute health care utilization. In addition, they may improve care transitions by optimizing clinic quality indicators and by identifying and resolving MRPs.
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Strongyloides hyperinfection syndrome is a rare manifestation caused by the Strongyloides stercoralis parasite and has mortality rates close to 90% if left untreated. Corticosteroids are commonly implicated as a trigger for hyperinfection syndrome in patients with Strongyloides autoinfection, and it has been suggested that even a single dose of corticosteroids can trigger hyperinfection syndrome. Here, we report a case of hyperinfection syndrome eight days after administering a single 8 mg dose of dexamethasone for fetal lung development before a late preterm, emergency cesarean section (C-section) delivery secondary to placental abruption. Prior to the C-section, the patient had been exhibiting signs of autoinfection syndrome, cough, and abdominal pain, for several months. Following corticosteroid administration, she had sequelae of Strongyloides hyperinfection syndrome, including gram-negative bacteremia, undulating fevers, protein wasting enteropathy, and hypersensitivity pneumonitis. Sputum cultures were positive for Strongyloides, and after treatment with ivermectin and albendazole, the patient fully recovered. Strongyloides hyperinfection syndrome is a documented consequence of short courses of corticosteroids. Still, this case is unique because the patient only received a single dose of corticosteroids before developing hyperinfection syndrome. Clinicians must recognize patients at risk for Strongyloides hyperinfection syndrome and understand the risks of administering corticosteroids to patients harboring the parasite.
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Influenza B virus (IBV) strains are one of the components of seasonal influenza vaccines in both trivalent and quadrivalent formulations. The vast majority of these vaccines are produced in embryonated chickens' eggs. While optimized backbones for vaccine production in eggs exist and are in use for influenza A viruses, no such backbones exist for IBVs, resulting in unpredictable production yields. To generate an optimal vaccine seed virus backbone, we have compiled a panel of 71 IBV strains from 1940 to present day, representing the known temporal and genetic variability of IBV circulating in humans. This panel contains strains from the B/Victoria/2/87-like lineage, B/Yamagata/16/88-like lineage and the ancestral lineage that preceded their split to provide a diverse set that would help to identify a suitable backbone which can be used in combination with hemagglutinin (HA) and neuraminidase (NA) glycoproteins from any IBV strain to be incorporated into the seasonal vaccine. We have characterized and ranked the growth profiles of the 71 IBV strains and the best performing strains were used for co-infection of eggs, followed by serial passaging to select for high-growth reassortant viruses. After serial passaging, we selected 10 clonal isolates based on their growth profiles assessed by hemagglutination and plaque-forming units. We then generated reverse genetics systems for the three clones that performed best in growth curves. The selected backbones were then used to generate different reassortant viruses with HA/NA combinations from high and low titer yielding wild type IBV. When the growth profiles of the recombinant reassortant viruses were tested, the low titer yielding HA/NA viruses with the selected backbones yielded higher titers similar to those from high titer yielding HA/NA combinations. The use of these IBV backbones with improved replication in eggs might increase yields for the influenza B virus components of seasonal influenza virus vaccines.
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Nuclear export of influenza A virus (IAV) mRNAs occurs through the nuclear pore complex (NPC). Using the Auxin-Induced Degron (AID) system to rapidly degrade proteins, we show that among the nucleoporins localized at the nucleoplasmic side of the NPC, TPR is the key nucleoporin required for nuclear export of influenza virus mRNAs. TPR recruits the TRanscription and EXport complex (TREX)-2 to the NPC for exporting a subset of cellular mRNAs. By degrading components of the TREX-2 complex (GANP, Germinal-center Associated Nuclear Protein; PCID2, PCI domain containing 2), we show that influenza mRNAs require the TREX-2 complex for nuclear export and replication. Furthermore, we found that cellular mRNAs whose export is dependent on GANP have a small number of exons, a high mean exon length, long 3' UTR, and low GC content. Some of these features are shared by influenza virus mRNAs. Additionally, we identified a 45 nucleotide RNA signal from influenza virus HA mRNA that is sufficient to mediate GANP-dependent mRNA export. Thus, we report a role for the TREX-2 complex in nuclear export of influenza mRNAs and identified RNA determinants associated with the TREX-2-dependent mRNA export.
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Transporte Ativo do Núcleo Celular , Influenza Humana , Orthomyxoviridae , Transporte de RNA , Humanos , Transporte Ativo do Núcleo Celular/genética , Núcleo Celular/metabolismo , Influenza Humana/metabolismo , Poro Nuclear/genética , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Nucleares/metabolismo , Orthomyxoviridae/genética , Transporte de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Hybrid immunity to SARS-CoV-2 provides superior protection to re-infection. We performed immune profiling studies during breakthrough infections in mRNA-vaccinated hamsters to evaluate hybrid immunity induction. mRNA vaccine, BNT162b2, was dosed to induce binding antibody titers against ancestral spike, but inefficient serum virus neutralization of ancestral SARS-CoV-2 or variants of concern (VoCs). Vaccination reduced morbidity and controlled lung virus titers for ancestral virus and Alpha but allowed breakthrough infections in Beta, Delta and Mu-challenged hamsters. Vaccination primed T cell responses that were boosted by infection. Infection back-boosted neutralizing antibody responses against ancestral virus and VoCs. Hybrid immunity resulted in more cross-reactive sera. Transcriptomics post-infection reflects both vaccination status and disease course and suggests a role for interstitial macrophages in vaccine-mediated protection. Therefore, protection by vaccination, even in the absence of high titers of neutralizing antibodies in the serum, correlates with recall of broadly reactive B- and T-cell responses.
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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encodes several proteins that inhibit host interferon responses. Among these, ORF6 antagonizes interferon signaling by disrupting nucleocytoplasmic trafficking through interactions with the nuclear pore complex components Nup98-Rae1. However, the roles and contributions of ORF6 during physiological infection remain unexplored. We assessed the role of ORF6 during infection using recombinant viruses carrying a deletion or loss-of-function (LoF) mutation in ORF6. ORF6 plays key roles in interferon antagonism and viral pathogenesis by interfering with nuclear import and specifically the translocation of IRF and STAT transcription factors. Additionally, ORF6 inhibits cellular mRNA export, resulting in the remodeling of the host cell proteome, and regulates viral protein expression. Interestingly, the ORF6:D61L mutation that emerged in the Omicron BA.2 and BA.4 variants exhibits reduced interactions with Nup98-Rae1 and consequently impairs immune evasion. Our findings highlight the role of ORF6 in antagonizing innate immunity and emphasize the importance of studying the immune evasion strategies of SARS-CoV-2.
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COVID-19 , SARS-CoV-2 , Proteínas Virais , Humanos , COVID-19/virologia , Imunidade Inata , Interferons/genética , Interferons/metabolismo , SARS-CoV-2/genética , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
To enhance understanding of the metabolic indicators of type 2 diabetes mellitus (T2DM) disease pathogenesis and progression, the urinary metabolomes of well characterized rhesus macaques (normal or spontaneously and naturally diabetic) were examined. High-resolution ultra-performance liquid chromatography coupled with the accurate mass determination of time-of-flight mass spectrometry was used to analyze spot urine samples from normal (n = 10) and T2DM (n = 11) male monkeys. The machine-learning algorithm random forests classified urine samples as either from normal or T2DM monkeys. The metabolites important for developing the classifier were further examined for their biological significance. Random forests models had a misclassification error of less than 5%. Metabolites were identified based on accurate masses (<10 ppm) and confirmed by tandem mass spectrometry of authentic compounds. Urinary compounds significantly increased (p < 0.05) in the T2DM when compared with the normal group included glycine betaine (9-fold), citric acid (2.8-fold), kynurenic acid (1.8-fold), glucose (68-fold), and pipecolic acid (6.5-fold). When compared with the conventional definition of T2DM, the metabolites were also useful in defining the T2DM condition, and the urinary elevations in glycine betaine and pipecolic acid (as well as proline) indicated defective re-absorption in the kidney proximal tubules by SLC6A20, a Na(+)-dependent transporter. The mRNA levels of SLC6A20 were significantly reduced in the kidneys of monkeys with T2DM. These observations were validated in the db/db mouse model of T2DM. This study provides convincing evidence of the power of metabolomics for identifying functional changes at many levels in the omics pipeline.
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Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Diabetes Mellitus Experimental/urina , Diabetes Mellitus Tipo 2/urina , Túbulos Renais Proximais/metabolismo , Animais , Betaína/urina , Ácido Cítrico/urina , Glucose/metabolismo , Glicosúria/urina , Humanos , Ácido Cinurênico/urina , Macaca mulatta , Masculino , Metabolômica/métodos , Camundongos , Ácidos Pipecólicos/urina , RNA Mensageiro/metabolismoRESUMO
Common variable immunodeficiency (CVID) is a primary immunodeficiency disorder that causes decreased immunity and increased susceptibility to infections. It affects B lymphocyte differentiation, resulting in predominantly bacterial and less frequently viral, fungal, and protozoal infections. The respiratory and gastrointestinal tracts where antibody defences are essential are usually affected. Individuals with CVID are also predisposed to developing lymphoid and gastrointestinal malignancies. We present two cases with rare infectious and oncological complications of CVID, including a patient with Mycobacterium avium complex-intracellular infection and ovarian cancer, and another patient with group B Streptococcus empyema of the lung with acute myeloid leukaemia. The main objective of this study is to highlight how CVID-induced hypogammaglobulinaemia can lead to rare infections and malignancies. The management of these complications can vary according to severity, but an awareness of their existence is crucial to diagnose them promptly in an already immunocompromised CVID patient.
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Agamaglobulinemia , Imunodeficiência de Variável Comum , Infecção por Mycobacterium avium-intracellulare , Neoplasias Ovarianas , Humanos , Feminino , Imunodeficiência de Variável Comum/complicações , Imunodeficiência de Variável Comum/diagnóstico , Carcinoma Epitelial do Ovário , Complexo Mycobacterium aviumRESUMO
Concerns that infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of coronavirus disease 2019 (COVID-19), may cause new-onset diabetes persist in an evolving research landscape, and precise risk assessment is hampered by, at times, conflicting evidence. Here, leveraging comprehensive single-cell analyses of in vitro SARS-CoV-2-infected human pancreatic islets, we demonstrate that productive infection is strictly dependent on the SARS-CoV-2 entry receptor ACE2 and targets practically all pancreatic cell types. Importantly, the infection remains highly circumscribed and largely non-cytopathic and, despite a high viral burden in infected subsets, promotes only modest cellular perturbations and inflammatory responses. Similar experimental outcomes are also observed after islet infection with endemic coronaviruses. Thus, the limits of pancreatic SARS-CoV-2 infection, even under in vitro conditions of enhanced virus exposure, challenge the proposition that in vivo targeting of ß cells by SARS-CoV-2 precipitates new-onset diabetes. Whether restricted pancreatic damage and immunological alterations accrued by COVID-19 increase cumulative diabetes risk, however, remains to be evaluated.
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COVID-19 , Diabetes Mellitus , Células Secretoras de Insulina , Humanos , Pâncreas , SARS-CoV-2RESUMO
Due to differences in human and murine angiotensin converting enzyme 2 (ACE-2) receptor, initially available SARS-CoV-2 isolates could not infect mice. Here we show that serial passaging of USA-WA1/2020 strain in mouse lungs results in "mouse-adapted" SARS-CoV-2 (MA-SARS-CoV-2) with mutations in S, M, and N genes, and a twelve-nucleotide insertion in the S gene. MA-SARS-CoV-2 infection causes mild disease, with more pronounced morbidity depending on genetic background and in aged and obese mice. Two mutations in the S gene associated with mouse adaptation (N501Y, H655Y) are present in SARS-CoV-2 variants of concern (VoCs). N501Y in the receptor binding domain of viruses of the B.1.1.7, B.1.351, P.1 and B.1.1.529 lineages (Alpha, Beta, Gamma and Omicron variants) is associated with high transmissibility and allows VoCs to infect wild type mice. We further show that S protein mutations of MA-SARS-CoV-2 do not affect neutralization efficiency by human convalescent and post vaccination sera.
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COVID-19 , Evasão da Resposta Imune , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Idoso , Animais , COVID-19/virologia , Humanos , Soros Imunes , Camundongos , Mutação , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
SARS-CoV-2 lineages have diverged into highly prevalent variants termed "variants of concern" (VOCs). Here, we characterized emerging SARS-CoV-2 spike polymorphisms in vitro and in vivo to understand their impact on transmissibility and virus pathogenicity and fitness. We demonstrate that the substitution S:655Y, represented in the gamma and omicron VOCs, enhances viral replication and spike protein cleavage. The S:655Y substitution was transmitted more efficiently than its ancestor S:655H in the hamster infection model and was able to outcompete S:655H in the hamster model and in a human primary airway system. Finally, we analyzed a set of emerging SARS-CoV-2 variants to investigate how different sets of mutations may impact spike processing. All VOCs tested exhibited increased spike cleavage and fusogenic capacity. Taken together, our study demonstrates that the spike mutations present in VOCs that become epidemiologically prevalent in humans are linked to an increase in spike processing and virus transmission.
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COVID-19 , SARS-CoV-2 , Humanos , Mutação , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
We and others have previously shown that the SARS-CoV-2 accessory protein ORF6 is a powerful antagonist of the interferon (IFN) signaling pathway by directly interacting with Nup98-Rae1 at the nuclear pore complex (NPC) and disrupting bidirectional nucleo-cytoplasmic trafficking. In this study, we further assessed the role of ORF6 during infection using recombinant SARS-CoV-2 viruses carrying either a deletion or a well characterized M58R loss-of-function mutation in ORF6. We show that ORF6 plays a key role in the antagonism of IFN signaling and in viral pathogenesis by interfering with karyopherin(importin)-mediated nuclear import during SARS-CoV-2 infection both in vitro , and in the Syrian golden hamster model in vivo . In addition, we found that ORF6-Nup98 interaction also contributes to inhibition of cellular mRNA export during SARS-CoV-2 infection. As a result, ORF6 expression significantly remodels the host cell proteome upon infection. Importantly, we also unravel a previously unrecognized function of ORF6 in the modulation of viral protein expression, which is independent of its function at the nuclear pore. Lastly, we characterized the ORF6 D61L mutation that recently emerged in Omicron BA.2 and BA.4 and demonstrated that it is able to disrupt ORF6 protein functions at the NPC and to impair SARS-CoV-2 innate immune evasion strategies. Importantly, the now more abundant Omicron BA.5 lacks this loss-of-function polymorphism in ORF6. Altogether, our findings not only further highlight the key role of ORF6 in the antagonism of the antiviral innate immune response, but also emphasize the importance of studying the role of non-spike mutations to better understand the mechanisms governing differential pathogenicity and immune evasion strategies of SARS-CoV-2 and its evolving variants. ONE SENTENCE SUMMARY: SARS-CoV-2 ORF6 subverts bidirectional nucleo-cytoplasmic trafficking to inhibit host gene expression and contribute to viral pathogenesis.
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Recurrent toxic shock syndrome (TSS) is uncommon. A certain level of clinical suspicion is indicated with a complex sepsis presentation in the postoperative kidney transplant patient. We present a case of presumed recurrent postoperative TSS in a living kidney transplant recipient. The patient was a 19-year-old Caucasian female with a 4-year prior single episode of toxin-mediated sepsis and chronic kidney disease (CKD) secondary to autosomal recessive Alport's syndrome (confirmed via renal biopsy and genetic testing). She received a human leukocyte antigen (HLA) 2A 2B 1DR MM, CMV -D/-R kidney from her 21-year-old friend. The patient received Campath and IV steroid induction after total cold ischemic time of 170 minutes with 40 minutes of revascularization. On postoperative day (POD) 5, she required re-exploration with reimplantation and stenting of the transplanted ureter. The patient subsequently spiked a fever of 101.6° with a generalized rash prompting collection of blood cultures which demonstrated no growth. Infectious Disease was consulted due to persistent fevers despite IV antibiotics. On POD 12, the patient returned to the operating room (OR) for evacuation of hematoma after decline in Hgb to 5.8 and CT confirmed perinephric hematomas. Kidney biopsy showed no rejection and donor specific antibodies (DSAs) were unremarkable. The patient underwent 1 treatment of empiric plasmapheresis for possible non-HLA antibodies followed by initiation of clindamycin. The patient's condition improved, and she was discharged home with a normal creatinine. Recurrent TSS is rare but should be added to the differential diagnoses of immuno-compromised patients undergoing kidney transplantation with a history of prior toxin-mediated sepsis.