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Middle East respiratory syndrome coronavirus (MERS-CoV) is a beta coronavirus that emerged in 2012, causing severe pneumonia and renal failure. MERS-CoV encodes five accessory proteins. Some of them have been shown to interfere with host antiviral immune response. However, the roles of protein 8b in innate immunity and viral virulence was rarely studied. Here, we introduced individual MERS-CoV accessory protein genes into the genome of an attenuated murine coronavirus (Mouse hepatitis virus, MHV), respectively, and found accessory protein 8b could enhance viral replication in vivo and in vitro and increase the lethality of infected mice. RNA-seq analysis revealed that protein 8b could significantly inhibit type I interferon production (IFN-I) and innate immune response in mice infected with MHV expressing protein 8b. We also found that MERS-CoV protein 8b could initiate from multiple internal methionine sites and at least three protein variants were identified. Residues 1-23 of protein 8b was demonstrated to be responsible for increased virulence in vivo. In addition, the inhibitory effect on IFN-I of protein 8b might not contribute to its virulence enhancement as aa1-23 deletion did not affect IFN-I production in vitro and in vivo. Next, we also found that protein 8b was localized to the endoplasmic reticulum (ER)/Golgi membrane in infected cells, which was disrupted by C-terminal region aa 88-112 deletion. This study will provide new insight into the pathogenesis of MERS-CoV infection. IMPORTANCE Multiple coronaviruses (CoV) cause severe respiratory infections and become global public health threats such as SARS-CoV, MERS-CoV, and SARS-CoV-2. Each coronavirus contains different numbers of accessory proteins which show high variability among different CoVs. Accessory proteins are demonstrated to play essential roles in pathogenesis of CoVs. MERS-CoV contains 5 accessory proteins (protein 3, 4a, 4b, 5, 8b), and deletion of all four accessory proteins (protein 3, 4a, 4b, 5), significantly affects MERS-CoV replication and pathogenesis. However, whether ORF8b also regulates MERS-CoV infection is unknown. Here, we constructed mouse hepatitis virus (MHV) recombinant virus expressing MERS-CoV protein 8b and demonstrated protein 8b could significantly enhance the virulence of MHV, which is mediated by N-terminal domain of protein 8b. This study will shed light on the understanding of pathogenesis of MERS-CoV infection.
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Coronavirus del Síndrome Respiratorio de Oriente Medio/fisiología , Virus de la Hepatitis Murina/fisiología , Dominios y Motivos de Interacción de Proteínas , Proteínas Reguladoras y Accesorias Virales/genética , Animales , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/virología , Interacciones Huésped-Patógeno/inmunología , Inmunidad Innata , Ratones , Mortalidad , Proteínas Reguladoras y Accesorias Virales/química , Tropismo Viral , Virulencia/genética , Factores de Virulencia/genéticaRESUMEN
Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe acute respiratory disease in humans. MERS-CoV strains from early epidemic clade A and contemporary epidemic clade B have not been phenotypically characterized to compare their abilities to infect cells and mice. We isolated the clade B MERS-CoV ChinaGD01 strain from a patient infected during the South Korean MERS outbreak in 2015 and compared the phylogenetics and pathogenicity of MERS-CoV EMC/2012 (clade A) and ChinaGD01 (clade B) in vitro and in vivo Genome alignment analysis showed that most clade-specific mutations occurred in the orf1ab gene, including mutations that were predicted to be potential glycosylation sites. Minor differences in viral growth but no significant differences in plaque size or sensitivity to beta interferon (IFN-ß) were detected between these two viruses in vitro ChinaGD01 virus infection induced more weight loss and inflammatory cytokine production in human DPP4-transduced mice. Viral titers were higher in the lungs of ChinaGD01-infected mice than with EMC/2012 infection. Decreased virus-specific CD4+ and CD8+ T cell numbers were detected in the lungs of ChinaGD01-infected mice. In conclusion, MERS-CoV evolution induced changes to reshape its pathogenicity and virulence in vitro and in vivo and to evade adaptive immune response to hinder viral clearance.IMPORTANCE MERS-CoV is an important emerging pathogen and causes severe respiratory infection in humans. MERS-CoV strains from early epidemic clade A and contemporary epidemic clade B have not been phenotypically characterized to compare their abilities to infect cells and mice. In this study, we showed that a clade B virus ChinaGD01 strain caused more severe disease in mice, with delayed viral clearance, increased inflammatory cytokines, and decreased antiviral T cell responses, than the early clade A virus EMC/2012. Given the differences in pathogenicity of different clades of MERS-CoV, periodic assessment of currently circulating MERS-CoV is needed to monitor potential severity of zoonotic disease.
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Infecciones por Coronavirus/virología , Genotipo , Interacciones Huésped-Patógeno , Coronavirus del Síndrome Respiratorio de Oriente Medio/fisiología , Adulto , Animales , Modelos Animales de Enfermedad , Genoma Viral , Interacciones Huésped-Patógeno/inmunología , Humanos , Interferón Tipo I/farmacología , Masculino , Ratones , Coronavirus del Síndrome Respiratorio de Oriente Medio/clasificación , Coronavirus del Síndrome Respiratorio de Oriente Medio/aislamiento & purificación , Coronavirus del Síndrome Respiratorio de Oriente Medio/patogenicidad , Filogenia , ARN Viral , Linfocitos T/inmunología , Linfocitos T/metabolismo , Virulencia , Replicación Viral/efectos de los fármacos , Replicación Viral/genética , Secuenciación Completa del GenomaRESUMEN
Omicron, as the emerging variant with enhanced vaccine tolerance, has sharply disrupted most therapeutic antibodies. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) belongs to the subgenus Sarbecovirus, members of which share high sequence similarity. Herein, we report one sarbecovirus antibody, 5817, which has broad-spectrum neutralization capacity against SARS-CoV-2 variants of concern (VOCs) and SARS-CoV, as well as related bat and pangolin viruses. 5817 can hardly compete with six classes of receptor-binding-domain-targeted antibodies grouped by structural classifications. No obvious impairment in the potency is detected against SARS-CoV-2 Omicron and subvariants. The cryoelectron microscopy (cryo-EM) structure of neutralizing antibody 5817 in complex with Omicron spike reveals a highly conserved epitope, only existing at the receptor-binding domain (RBD) open state. Prophylactic and therapeutic administration of 5817 potently protects mice from SARS-CoV-2 Beta, Delta, Omicron, and SARS-CoV infection. This study reveals a highly conserved cryptic epitope targeted by a broad sarbecovirus neutralizing antibody, which would be beneficial to meet the potential threat of pre-emergent SARS-CoV-2 VOCs.
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Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo , Animales , Ratones , Anticuerpos ampliamente neutralizantes , Microscopía por Crioelectrón , Anticuerpos Neutralizantes , Epítopos , Anticuerpos AntiviralesRESUMEN
To establish a multidimensional nomogram model for predicting progression-free survival (PFS) and risk stratification in patients with advanced nasopharyngeal carcinoma (NPC). This retrospective cross-sectional study included 156 patients with advanced NPC who underwent dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Radiomic features were extracted from the efflux rate constant (Ktrans ) and extracellular extravascular volume (Ve ) mapping derived from DCE-MRI. Least absolute shrinkage and selection operator (LASSO) Cox regression analysis was applied for feature selection. The Radscore was constructed using the selected features with their respective weights in the LASSO Cox regression analysis. A nomogram model combining the Radscore and clinical factors was built using multivariate Cox regression analysis. The C-index was used to assess the discrimination power of the Radscore and nomogram. The Kaplan-Meier method was used for survival analysis. Of the 360 radiomic features, 28 were selected (7, 6, and 15 features extracted from Ktrans , Ve, and Ktrans +Ve images, respectively). The combined Radscore k trans +Ve (C-index, 0.703, 95% confidence interval [CI]: 0.571-0.836) showed higher efficacy in predicting the prognosis of advanced NPC than Radscore k trans (C-index, 0.693; 95% CI, 0.560-0.826) and Radscore Ve (C-index, 0.614; 95% CI, 0.481-0.746) did. Multivariable Cox regression analysis revealed clinical stage, T stage, and treatment with nimotuzumab as risk factors for PFS. The nomogram established by Radscore k trans +Ve and risk factors (C-index, 0.732; 95% CI: 0.599-0.864) was better than Radscore k trans +Ve in predicting PFS in patients with advanced NPC. A lower Radscore k trans +Ve (HR 3.5584, 95% CI 2.1341-5.933), lower clinical stage (hazard ratio [HR] 1.5982, 95% CI 0.5262-4.854), lower T stage (HR 1.4365, 95% CI 0.6745-3.060), and nimotuzumab (NTZ) treatment (HR 0.7879, 95% CI 0.4899-1.267) were associated with longer PFS. Kaplan-Meier analysis showed a lower PFS in the high-risk group than in the low-risk group (p<0.0001). The nomogram based on combined pretreatment DCE-MRI radiomics features, NTZ, and clinicopathological risk factors may be considered as a noninvasive imaging marker for predicting individual PFS in patients with advanced NPC.
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The current COVID-19 pandemic, caused by SARS-CoV-2, poses a serious public health threat. Effective therapeutic and prophylactic treatments are urgently needed. Angiotensin-converting enzyme 2 (ACE2) is a functional receptor for SARS-CoV-2, which binds to the receptor binding domain (RBD) of SARS-CoV-2 spike protein. Here, we developed recombinant human ACE2-Fc fusion protein (hACE2-Fc) and a hACE2-Fc mutant with reduced catalytic activity. hACE2-Fc and the hACE2-Fc mutant both efficiently blocked entry of SARS-CoV-2, SARS-CoV, and HCoV-NL63 into hACE2-expressing cells and inhibited SARS-CoV-2 S protein-mediated cell-cell fusion. hACE2-Fc also neutralized various SARS-CoV-2 strains with enhanced infectivity including D614G and V367F mutations, as well as the emerging SARS-CoV-2 variants, B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.1 (Kappa), and B.1.617.2 (Delta), demonstrating its potent and broad-spectrum antiviral effects. In addition, hACE2-Fc proteins protected HBE from SARS-CoV-2 infection. Unlike RBD-targeting neutralizing antibodies, hACE2-Fc treatment did not induce the development of escape mutants. Furthermore, both prophylactic and therapeutic hACE2-Fc treatments effectively protected mice from SARS-CoV-2 infection, as determined by reduced viral replication, weight loss, histological changes, and inflammation in the lungs. The protection provided by hACE2 showed obvious dose-dependent efficacy in vivo. Pharmacokinetic data indicated that hACE2-Fc has a relative long half-life in vivo compared to soluble ACE2, which makes it an excellent candidate for prophylaxis and therapy for COVID-19 as well as for SARS-CoV and HCoV-NL63 infections.
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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic of Coronavirus disease 2019 (COVID-19). However, the microbial composition of the respiratory tract and other infected tissues as well as their possible pathogenic contributions to varying degrees of disease severity in COVID-19 patients remain unclear. Between 27 January and 26 February 2020, serial clinical specimens (sputum, nasal and throat swab, anal swab and feces) were collected from a cohort of hospitalized COVID-19 patients, including 8 mildly and 15 severely ill patients in Guangdong province, China. Total RNA was extracted and ultra-deep metatranscriptomic sequencing was performed in combination with laboratory diagnostic assays. We identified distinct signatures of microbial dysbiosis among severely ill COVID-19 patients on broad spectrum antimicrobial therapy. Co-detection of other human respiratory viruses (including human alphaherpesvirus 1, rhinovirus B, and human orthopneumovirus) was demonstrated in 30.8% (4/13) of the severely ill patients, but not in any of the mildly affected patients. Notably, the predominant respiratory microbial taxa of severely ill patients were Burkholderia cepacia complex (BCC), Staphylococcus epidermidis, or Mycoplasma spp. (including M. hominis and M. orale). The presence of the former two bacterial taxa was also confirmed by clinical cultures of respiratory specimens (expectorated sputum or nasal secretions) in 23.1% (3/13) of the severe cases. Finally, a time-dependent, secondary infection of B. cenocepacia with expressions of multiple virulence genes was demonstrated in one severely ill patient, which might accelerate his disease deterioration and death occurring one month after ICU admission. Our findings point to SARS-CoV-2-related microbial dysbiosis and various antibiotic-resistant respiratory microbes/pathogens in hospitalized COVID-19 patients in relation to disease severity. Detection and tracking strategies are needed to prevent the spread of antimicrobial resistance, improve the treatment regimen and clinical outcomes of hospitalized, severely ill COVID-19 patients.
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Disease progression prediction and therapeutic drug target discovery for Coronavirus disease 2019 (COVID-19) are particularly important, as there is still no effective strategy for severe COVID-19 patient treatment. Herein, we performed multi-platform omics analysis of serial plasma and urine samples collected from patients during the course of COVID-19. Integrative analyses of these omics data revealed several potential therapeutic targets, such as ANXA1 and CLEC3B. Molecular changes in plasma indicated dysregulation of macrophage and suppression of T cell functions in severe patients compared to those in non-severe patients. Further, we chose 25 important molecular signatures as potential biomarkers for the prediction of disease severity. The prediction power was validated using corresponding urine samples and plasma samples from new COVID-19 patient cohort, with AUC reached to 0.904 and 0.988, respectively. In conclusion, our omics data proposed not only potential therapeutic targets, but also biomarkers for understanding the pathogenesis of severe COVID-19.
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Tratamiento Farmacológico de COVID-19 , COVID-19/sangre , Descubrimiento de Drogas , Lipidómica , Proteómica , SARS-CoV-2/metabolismo , Biomarcadores/sangre , Femenino , Humanos , MasculinoRESUMEN
BACKGROUND: Since early February 2021, the causative agent of COVID-19, SARS-CoV-2, has infected over 104 million people with more than 2 million deaths according to official reports. The key to understanding the biology and virus-host interactions of SARS-CoV-2 requires the knowledge of mutation and evolution of this virus at both inter- and intra-host levels. However, despite quite a few polymorphic sites identified among SARS-CoV-2 populations, intra-host variant spectra and their evolutionary dynamics remain mostly unknown. METHODS: Using high-throughput sequencing of metatranscriptomic and hybrid captured libraries, we characterized consensus genomes and intra-host single nucleotide variations (iSNVs) of serial samples collected from eight patients with COVID-19. The distribution of iSNVs along the SARS-CoV-2 genome was analyzed and co-occurring iSNVs among COVID-19 patients were identified. We also compared the evolutionary dynamics of SARS-CoV-2 population in the respiratory tract (RT) and gastrointestinal tract (GIT). RESULTS: The 32 consensus genomes revealed the co-existence of different genotypes within the same patient. We further identified 40 intra-host single nucleotide variants (iSNVs). Most (30/40) iSNVs presented in a single patient, while ten iSNVs were found in at least two patients or identical to consensus variants. Comparing allele frequencies of the iSNVs revealed a clear genetic differentiation between intra-host populations from the respiratory tract (RT) and gastrointestinal tract (GIT), mostly driven by bottleneck events during intra-host migrations. Compared to RT populations, the GIT populations showed a better maintenance and rapid development of viral genetic diversity following the suspected intra-host bottlenecks. CONCLUSIONS: Our findings here illustrate the intra-host bottlenecks and evolutionary dynamics of SARS-CoV-2 in different anatomic sites and may provide new insights to understand the virus-host interactions of coronaviruses and other RNA viruses.
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COVID-19/prevención & control , Genoma Viral/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Polimorfismo de Nucleótido Simple , SARS-CoV-2/genética , COVID-19/virología , Frecuencia de los Genes , Genotipo , Haplotipos , Interacciones Huésped-Patógeno , Humanos , Filogenia , SARS-CoV-2/clasificación , SARS-CoV-2/fisiologíaRESUMEN
We report the complete genome sequences of five human coronavirus NL63 (HCoV-NL63) strains obtained using next-generation sequencing. The five HCoV-NL63 strains were obtained from hospitalized children with severe acute respiratory infection detected in Guangdong, China. This study provides several complete genomes of HCoV-NL63 and improves our understanding of HCoV-NL63 evolution in China.
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Purpose: To determine whether the early assessment of temporal lobe microstructural changes using diffusion kurtosis imaging (DKI) can predict late delayed neurocognitive decline after radiotherapy in nasopharyngeal carcinoma (NPC) patients. Methods and Materials: Fifty-four NPC patients undergoing intensity-modulated radiotherapy (IMRT) participated in a prospective DKI magnetic resonance (MR) imaging study. MR imaging was acquired prior to IMRT (-0), 1 month (-1), and 3 (-3) months after IMRT. Kurtosis (Kmean, Kax, Krad) and Diffusivity (Dmean, Dax, Drad) variables in the temporal lobe gray and white matter were computed. Neurocognitive function tests (MoCA) were administered pre-radiotherapy and at 2 years post-IMRT follow-up. All the patients were divided into neurocognitive function decline (NFD group) and neurocognitive function non-decline groups (NFND group) according to whether the MoCA score declined ≥3 2 years after IMRT. All the DKI metrics were compared between the two groups, and the best imaging marker was chosen for predicting a late delayed neurocognitive decline. Results: Kurtosis (Kmean-1, Kmean-3, Kax-1, Kax-3, Krad-1, and Krad-3) and Diffusivity (Dmean-1 and Dmean-3) of white matter were significantly different between the two groups (p<0.05). Axial Kurtosis (Kax-1, Kax-3) of gray matter was significantly different between the two groups (p<0.05). By receiver operating characteristic (ROC) curves, Kmean-1 of white matter performed best in predicting of MoCA scores delayed decline (p<0.05). The radiation dose was also significantly different between NFD and NFND group (p=0.031). Conclusions: Temporal lobe white matter is more vulnerable to microstructural changes and injury following IMRT in NPC. Metrics derived from DKI should be considered as imaging markers for predicting a late delayed neurocognitive decline. Both temporal lobe white and gray matter show microstructural changes detectable by DKI. The Kmean early after radiotherapy has the best prediction performance.
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Human coronavirus NL63 (HCoV-NL63) is primarily associated with common cold in children, elderly and immunocompromised individuals. Outbreaks caused by HCoV-NL63 are rare. Here we report a cluster of HCoV-NL63 cases with severe lower respiratory tract infection that arose in Guangzhou, China, in 2018. Twenty-three hospitalized children were confirmed to be HCoV-NL63 positive, and most of whom were hospitalized with severe pneumonia or acute bronchitis. Whole genomes of HCoV-NL63 were obtained using next-generation sequencing. Phylogenetic and single amino acid polymorphism analyses showed that this outbreak was associated with two subgenotypes (C3 and B) of HCoV-NL63. Half of patients were identified to be related to a new subgenotype C3. One unique amino acid mutation at I507â L in spike protein receptor binding domain (RBD) was detected, which segregated this subgenotype C3 from other known subgenotypes. Pseudotyped virus bearing the I507â L mutation in RBD showed enhanced entry into host cells as compared to the prototype virus. This study proved that HCoV-NL63 was undergoing continuous mutation and has the potential to cause severe lower respiratory disease in humans.
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Infecciones por Coronavirus , Coronavirus Humano NL63/genética , Infecciones del Sistema Respiratorio/virología , Preescolar , China , Coronavirus Humano NL63/aislamiento & purificación , Genotipo , Humanos , Lactante , FilogeniaRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent for coronavirus 2019 (COVID-19) pneumonia. Little is known about the kinetics, tissue distribution, cross-reactivity, and neutralization antibody response in patients with COVID-19. Two groups of patients with RT-PCR-confirmed COVID-19 were enrolled in this study: 12 severely ill patients in intensive care units who needed mechanical ventilation and 11 mildly ill patients in isolation wards. Serial clinical samples were collected for laboratory detection. Results showed that most of the severely ill patients had viral shedding in a variety of tissues for 20-40 days after onset of disease (8/12, 66.7%), while the majority of mildly ill patients had viral shedding restricted to the respiratory tract and had no detectable virus RNA 10 days after onset (9/11, 81.8%). Mildly ill patients showed significantly lower IgM response compared with that of the severe group. IgG responses were detected in most patients in both the severe and mild groups at 9 days after onset, and remained at a high level throughout the study. Antibodies cross-reactive to SARS-CoV and SARS-CoV-2 were detected in patients with COVID-19 but not in patients with MERS. High levels of neutralizing antibodies were induced after about 10 days after onset in both severely and mildly ill patients which were higher in the severe group. SARS-CoV-2 pseudotype neutralization test and focus reduction neutralization test with authentic virus showed consistent results. Sera from patients with COVID-19 inhibited SARS-CoV-2 entry. Sera from convalescent patients with SARS or Middle East respiratory syndrome (MERS) did not. Anti-SARS-CoV-2 S and N IgG levels exhibited a moderate correlation with neutralization titers in patients' plasma. This study improves our understanding of immune response in humans after SARS-CoV-2 infection.