RESUMEN
The SARS-CoV-2 Omicron variant is more immune evasive and less virulent than other major viral variants that have so far been recognized1-12. The Omicron spike (S) protein, which has an unusually large number of mutations, is considered to be the main driver of these phenotypes. Here we generated chimeric recombinant SARS-CoV-2 encoding the S gene of Omicron (BA.1 lineage) in the backbone of an ancestral SARS-CoV-2 isolate, and compared this virus with the naturally circulating Omicron variant. The Omicron S-bearing virus robustly escaped vaccine-induced humoral immunity, mainly owing to mutations in the receptor-binding motif; however, unlike naturally occurring Omicron, it efficiently replicated in cell lines and primary-like distal lung cells. Similarly, in K18-hACE2 mice, although virus bearing Omicron S caused less severe disease than the ancestral virus, its virulence was not attenuated to the level of Omicron. Further investigation showed that mutating non-structural protein 6 (nsp6) in addition to the S protein was sufficient to recapitulate the attenuated phenotype of Omicron. This indicates that although the vaccine escape of Omicron is driven by mutations in S, the pathogenicity of Omicron is determined by mutations both in and outside of the S protein.
Asunto(s)
COVID-19 , Proteínas de la Nucleocápside de Coronavirus , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Factores de Virulencia , Virulencia , Animales , Ratones , Línea Celular , Evasión Inmune , SARS-CoV-2/inmunología , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidad , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Proteínas de la Nucleocápside de Coronavirus/genética , Proteínas de la Nucleocápside de Coronavirus/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/metabolismo , Humanos , Vacunas contra la COVID-19/inmunología , Pulmón/citología , Pulmón/virología , Replicación Viral , MutaciónRESUMEN
Human society is dependent on nature1,2, but whether our ecological foundations are at risk remains unknown in the absence of systematic monitoring of species' populations3. Knowledge of species fluctuations is particularly inadequate in the marine realm4. Here we assess the population trends of 1,057 common shallow reef species from multiple phyla at 1,636 sites around Australia over the past decade. Most populations decreased over this period, including many tropical fishes, temperate invertebrates (particularly echinoderms) and southwestern Australian macroalgae, whereas coral populations remained relatively stable. Population declines typically followed heatwave years, when local water temperatures were more than 0.5 °C above temperatures in 2008. Following heatwaves5,6, species abundances generally tended to decline near warm range edges, and increase near cool range edges. More than 30% of shallow invertebrate species in cool latitudes exhibited high extinction risk, with rapidly declining populations trapped by deep ocean barriers, preventing poleward retreat as temperatures rise. Greater conservation effort is needed to safeguard temperate marine ecosystems, which are disproportionately threatened and include species with deep evolutionary roots. Fundamental among such efforts, and broader societal needs to efficiently adapt to interacting anthropogenic and natural pressures, is greatly expanded monitoring of species' population trends7,8.
Asunto(s)
Antozoos , Arrecifes de Coral , Calor Extremo , Peces , Calentamiento Global , Invertebrados , Océanos y Mares , Agua de Mar , Algas Marinas , Animales , Australia , Peces/clasificación , Invertebrados/clasificación , Calentamiento Global/estadística & datos numéricos , Algas Marinas/clasificación , Dinámica Poblacional , Densidad de Población , Agua de Mar/análisis , Extinción Biológica , Conservación de los Recursos Naturales/tendencias , Equinodermos/clasificaciónRESUMEN
Human coronavirus (HCoV)-NL63 causes respiratory tract infections in humans and employs angiotensin-converting enzyme 2 (ACE2) as a receptor. We sought to establish a mouse model of HCoV-NL63 and determine if prior RV-A1B infection affected HCoV-NL63 replication. HCoV-NL63 was propagated in LLC-MK2 cells expressing human ACE2. RV-A1B was grown in HeLa-H1 cells. C57BL6/J or transgenic mice expressing human ACE2 were infected intranasally with sham LLC-MK2 cell supernatant or 1 x 105 TCID50 units HCoV-NL63. Wild-type mice were infected with 1 x 106 PFU RV-A1B. Lungs were assessed for vRNA, bronchoalveolar lavage (BAL) cells, histology, HCoV-NL63 non-structural protein 3 (nsp3), and host gene expression by next generation sequencing and qPCR. To evaluate sequential infections, mice were infected with RV-A1B followed by HCoV-NL63 infection four days later. We report that hACE2 mice infected with HCoV-NL63 showed evidence of replicative infection with increased levels of vRNA, BAL neutrophils and lymphocytes, peribronchial and perivascular infiltrates, and expression of nsp3. Viral replication peaked three days after infection and inflammation persisted six days after infection. HCoV-NL63-infected hACE2 mice showed increased mRNA expression of IFNs, IFN-stimulated proteins and pro-inflammatory cytokines. Infection with RV-A1B four days before HCoV-NL63 significantly decreased both HCoV-NL63 vRNA levels and airway inflammation. Mice infected with RV-A1B prior to HCoV-NL63 showed increased expression of antiviral proteins compared to sham-treated mice. In conclusion, we established a mouse model of HCoV-NL63 replicative infection characterized by relatively persistent viral replication and inflammation. Prior infection with RV-A1B reduced HCoV-NL63 replication and airway inflammation, indicative of viral interference.
RESUMEN
BACKGROUND: The factors contributing to the accelerated convergent evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are not fully understood. Unraveling the contribution of viral replication in immunocompromised patients is important for the early detection of novel mutations and developing approaches to limit COVID-19. METHODS: We deep sequenced SARS-CoV-2 RNA from 192 patients (64% hospitalized, 39% immunosuppressed) and compared the viral genetic diversity within the patient groups of different immunity and hospitalization status. Serial sampling of 14 patients was evaluated for viral evolution in response to antiviral treatments. RESULTS: We identified hospitalized and immunosuppressed patients with significantly higher levels of viral genetic diversity and variability. Further evaluation of serial samples revealed accumulated mutations associated with escape from neutralizing antibodies in a subset of the immunosuppressed patients treated with antiviral therapies. Interestingly, the accumulated viral mutations that arose in this early Omicron wave, which were not common in the patient viral lineages, represent convergent mutations that are prevalent in the later Omicron sublineages, including the XBB, BA.2.86.1 and its descendent JN sublineages. CONCLUSIONS: Our results illustrate the importance of identifying convergent mutations generated during antiviral therapy in immunosuppressed patients, as they may contribute to the future evolutionary landscape of SARS-CoV-2. Our study also provides evidence of a correlation between SARS-CoV-2 convergent mutations and specific antiviral treatments. Evaluating high-confidence genomes from distinct waves in the pandemic with detailed patient metadata allows for discerning of convergent mutations that contribute to the ongoing evolution of SARS-CoV-2.
Asunto(s)
Antivirales , COVID-19 , Evolución Molecular , Huésped Inmunocomprometido , Mutación , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , SARS-CoV-2/inmunología , Antivirales/uso terapéutico , COVID-19/virología , COVID-19/inmunología , Masculino , Femenino , Persona de Mediana Edad , Anticuerpos Neutralizantes/inmunología , Anciano , Adulto , ARN Viral/genética , Tratamiento Farmacológico de COVID-19 , Variación Genética , FilogeniaRESUMEN
BACKGROUND: Kawasaki disease (KD) is a febrile illness of young childhood that can result in coronary artery aneurysms and death. Coronavirus disease 2019 (COVID-19) mitigation strategies resulted in a marked decrease in KD cases worldwide, supporting a transmissible respiratory agent as the cause. We previously reported a peptide epitope recognized by monoclonal antibodies (MAbs) derived from clonally expanded peripheral blood plasmablasts from 3 of 11 KD children, suggesting a common disease trigger in a subset of patients with KD. METHODS: We performed amino acid substitution scans to develop modified peptides with improved recognition by KD MAbs. We prepared additional MAbs from KD peripheral blood plasmablasts and assessed MAb characteristics that were associated with binding to the modified peptides. RESULTS: We report a modified peptide epitope that is recognized by 20 MAbs from 11 of 12 KD patients. These MAbs predominantly use heavy chain VH3-74; two-thirds of VH3-74 plasmablasts from these patients recognize the epitope. The MAbs were nonidentical between patients but share a common complementarity-determining region 3 (CDR3) motif. CONCLUSIONS: These results demonstrate a convergent VH3-74 plasmablast response to a specific protein antigen in children with KD, supporting one predominant causative agent in the etiopathogenesis of the illness.
Asunto(s)
COVID-19 , Síndrome Mucocutáneo Linfonodular , Humanos , Niño , Epítopos , Formación de Anticuerpos , Anticuerpos Monoclonales , PéptidosRESUMEN
The 5' cap structures of higher eukaryote mRNAs have ribose 2'-O-methylation. Likewise, many viruses that replicate in the cytoplasm of eukaryotes have evolved 2'-O-methyltransferases to autonomously modify their mRNAs. However, a defined biological role for 2'-O-methylation of mRNA remains elusive. Here we show that 2'-O-methylation of viral mRNA was critically involved in subverting the induction of type I interferon. We demonstrate that human and mouse coronavirus mutants lacking 2'-O-methyltransferase activity induced higher expression of type I interferon and were highly sensitive to type I interferon. Notably, the induction of type I interferon by viruses deficient in 2'-O-methyltransferase was dependent on the cytoplasmic RNA sensor Mda5. This link between Mda5-mediated sensing of viral RNA and 2'-O-methylation of mRNA suggests that RNA modifications such as 2'-O-methylation provide a molecular signature for the discrimination of self and non-self mRNA.
Asunto(s)
Infecciones por Coronavirus/metabolismo , Coronavirus/fisiología , ARN Helicasas DEAD-box/metabolismo , Metiltransferasas/metabolismo , Proteínas Virales/metabolismo , Animales , Línea Celular , Coronavirus/patogenicidad , Infecciones por Coronavirus/genética , Infecciones por Coronavirus/inmunología , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/inmunología , Humanos , Inmunidad Innata/genética , Interferón Tipo I/genética , Interferón Tipo I/inmunología , Interferón Tipo I/metabolismo , Helicasa Inducida por Interferón IFIH1 , Metilación , Metiltransferasas/genética , Metiltransferasas/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Viral/metabolismo , Receptor de Interferón alfa y beta/genética , Receptores de Reconocimiento de Patrones/genética , Ribosa/metabolismo , Proteínas Virales/genética , Proteínas Virales/inmunología , Virulencia/genética , Replicación Viral/genéticaRESUMEN
Coronaviruses (CoVs) are positive-sense RNA viruses that can emerge from endemic reservoirs and infect zoonotically, causing significant morbidity and mortality. CoVs encode an endoribonuclease designated EndoU that facilitates evasion of host pattern recognition receptor MDA5, but the target of EndoU activity was not known. Here, we report that EndoU cleaves the 5'-polyuridines from negative-sense viral RNA, termed PUN RNA, which is the product of polyA-templated RNA synthesis. Using a virus containing an EndoU catalytic-inactive mutation, we detected a higher abundance of PUN RNA in the cytoplasm compared to wild-type-infected cells. Furthermore, we found that transfecting PUN RNA into cells stimulates a robust, MDA5-dependent interferon response, and that removal of the polyuridine extension on the RNA dampens the response. Overall, the results of this study reveal the PUN RNA to be a CoV MDA5-dependent pathogen-associated molecular pattern (PAMP). We also establish a mechanism for EndoU activity to cleave and limit the accumulation of this PAMP. Since EndoU activity is highly conserved in all CoVs, inhibiting this activity may serve as an approach for therapeutic interventions against existing and emerging CoV infections.
Asunto(s)
Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/virología , Coronavirus/metabolismo , Endorribonucleasas/metabolismo , Poli U/metabolismo , Proteínas no Estructurales Virales/metabolismo , Animales , Antivirales/farmacología , Línea Celular , Chlorocebus aethiops , Coronavirus/enzimología , Coronavirus/inmunología , Endorribonucleasas/genética , Interacciones Microbiota-Huesped/fisiología , Humanos , Interferones/farmacología , Poli U/química , ARN Viral/genética , ARN Viral/metabolismo , Uridina/química , Células Vero , Proteínas no Estructurales Virales/genética , Replicación Viral/fisiologíaRESUMEN
SARS-CoV-2 can infect multiple organs, including lung, intestine, kidney, heart, liver, and brain. The molecular details of how the virus navigates through diverse cellular environments and establishes replication are poorly defined. Here, we generated a panel of phenotypically diverse, SARS-CoV-2-infectible human cell lines representing different body organs and performed longitudinal survey of cellular proteins and pathways broadly affected by the virus. This revealed universal inhibition of interferon signaling across cell types following SARS-CoV-2 infection. We performed systematic analyses of the JAK-STAT pathway in a broad range of cellular systems, including immortalized cells and primary-like cardiomyocytes, and found that SARS-CoV-2 targeted the proximal pathway components, including Janus kinase 1 (JAK1), tyrosine kinase 2 (Tyk2), and the interferon receptor subunit 1 (IFNAR1), resulting in cellular desensitization to type I IFN. Detailed mechanistic investigation of IFNAR1 showed that the protein underwent ubiquitination upon SARS-CoV-2 infection. Furthermore, chemical inhibition of JAK kinases enhanced infection of stem cell-derived cultures, indicating that the virus benefits from inhibiting the JAK-STAT pathway. These findings suggest that the suppression of interferon signaling is a mechanism widely used by the virus to evade antiviral innate immunity, and that targeting the viral mediators of immune evasion may help block virus replication in patients with COVID-19. IMPORTANCE SARS-CoV-2 can infect various organs in the human body, but the molecular interface between the virus and these organs remains unexplored. In this study, we generated a panel of highly infectible human cell lines originating from various body organs and employed these cells to identify cellular processes commonly or distinctly disrupted by SARS-CoV-2 in different cell types. One among the universally impaired processes was interferon signaling. Systematic analysis of this pathway in diverse culture systems showed that SARS-CoV-2 targets the proximal JAK-STAT pathway components, destabilizes the type I interferon receptor though ubiquitination, and consequently renders the infected cells resistant to type I interferon. These findings illuminate how SARS-CoV-2 can continue to propagate in different tissues even in the presence of a disseminated innate immune response.
Asunto(s)
COVID-19/metabolismo , Interacciones Microbiota-Huesped/fisiología , Quinasas Janus/metabolismo , SARS-CoV-2/metabolismo , Línea Celular , Regulación de la Expresión Génica , Humanos , Evasión Inmune , Inmunidad Innata , Interferón Tipo I/metabolismo , Janus Quinasa 1/metabolismo , Miocitos Cardíacos , Receptor de Interferón alfa y beta/metabolismo , Factor de Transcripción STAT1/metabolismo , Transducción de Señal , TYK2 Quinasa/metabolismo , Replicación ViralRESUMEN
Coronaviruses (CoVs) encode multiple interferon (IFN) antagonists that modulate the host response to virus replication. Here, we evaluated the host transcriptional response to infection with murine coronaviruses encoding independent mutations in one of two different viral antagonists, the deubiquitinase (DUB) within nonstructural protein 3 or the endoribonuclease (EndoU) within nonstructural protein 15. We used transcriptomics approaches to compare the scope and kinetics of the host response to the wild-type (WT), DUBmut, and EndoUmut viruses in infected macrophages. We found that the EndoUmut virus activates a focused response that predominantly involves type I interferons and interferon-related genes, whereas the WT and DUBmut viruses more broadly stimulate upregulation of over 2,800 genes, including networks associated with activating the unfolded protein response (UPR) and the proinflammatory response associated with viral pathogenesis. This study highlights the role of viral interferon antagonists in shaping the kinetics and magnitude of the host response during virus infection and demonstrates that inactivating a dominant viral antagonist, the coronavirus endoribonuclease, dramatically alters the host response in macrophages.IMPORTANCE Macrophages are an important cell type during coronavirus infections because they "notice" the infection and respond by inducing type I interferons, which limits virus replication. In turn, coronaviruses encode proteins that mitigate the cell's ability to signal an interferon response. Here, we evaluated the host macrophage response to two independent mutant coronaviruses, one with reduced deubiquitinating activity (DUBmut) and the other containing an inactivated endoribonuclease (EndoUmut). We observed a rapid, robust, and focused response to the EndoUmut virus, which was characterized by enhanced expression of interferon and interferon-related genes. In contrast, wild-type virus and the DUBmut virus elicited a more limited interferon response and ultimately activated over 2,800 genes, including players in the unfolded protein response and proinflammatory pathways associated with progression of significant disease. This study reveals that EndoU activity substantially contributes to the ability of coronaviruses to evade the host innate response and to replicate in macrophages.
Asunto(s)
Infecciones por Coronavirus/metabolismo , Infecciones por Coronavirus/virología , Coronavirus/fisiología , Endorribonucleasas/metabolismo , Interferones/metabolismo , Macrófagos/metabolismo , Macrófagos/virología , Proteínas no Estructurales Virales/metabolismo , Replicación Viral , Animales , Biología Computacional , Infecciones por Coronavirus/genética , Infecciones por Coronavirus/inmunología , Citocinas/metabolismo , Perfilación de la Expresión Génica , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Mediadores de Inflamación/metabolismo , Macrófagos/inmunología , Ratones , Modelos Biológicos , Mutación , ARN Viral , Respuesta de Proteína DesplegadaRESUMEN
Coronaviruses (CoVs) have repeatedly emerged from wildlife hosts and infected humans and livestock animals to cause epidemics with significant morbidity and mortality. CoVs infect various organs, including respiratory and enteric systems, as exemplified by newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The constellation of viral factors that contribute to developing enteric disease remains elusive. Here, we investigated CoV interferon antagonists for their contribution to enteric pathogenesis. Using an infectious clone of an enteric CoV, porcine epidemic diarrhea virus (icPEDV), we generated viruses with inactive versions of interferon antagonist nonstructural protein 1 (nsp1), nsp15, and nsp16 individually or combined into one virus designated icPEDV-mut4. Interferon-responsive PK1 cells were infected with these viruses and produced higher levels of interferon responses than were seen with wild-type icPEDV infection. icPEDV-mut4 elicited robust interferon responses and was severely impaired for replication in PK1 cells. To evaluate viral pathogenesis, piglets were infected with either icPEDV or icPEDV-mut4. While the icPEDV-infected piglets exhibited clinical disease, the icPEDV-mut4-infected piglets showed no clinical symptoms and exhibited normal intestinal pathology at day 2 postinfection. icPEDV-mut4 replicated in the intestinal tract, as revealed by detection of viral RNA in fecal swabs, with sequence analysis documenting genetic stability of the input strain. Importantly, icPEDV-mut4 infection elicited IgG and neutralizing antibody responses to PEDV. These results identify nsp1, nsp15, and nsp16 as virulence factors that contribute to the development of PEDV-induced diarrhea in swine. Inactivation of these CoV interferon antagonists is a rational approach for generating candidate vaccines to prevent disease and spread of enteric CoVs, including SARS-CoV-2.IMPORTANCE Emerging coronaviruses, including SARS-CoV-2 and porcine CoVs, can infect enterocytes, cause diarrhea, and be shed in the feces. New approaches are needed to understand enteric pathogenesis and to develop vaccines and therapeutics to prevent the spread of these viruses. Here, we exploited a reverse genetic system for an enteric CoV, porcine epidemic diarrhea virus (PEDV), and outline an approach of genetically inactivating highly conserved viral factors known to limit the host innate immune response to infection. Our report reveals that generating PEDV with inactive versions of three viral interferon antagonists, nonstructural proteins 1, 15, and 16, results in a highly attenuated virus that does not cause diarrhea in animals and elicits a neutralizing antibody response in virus-infected animals. This strategy may be useful for generating live attenuated vaccine candidates that prevent disease and fecal spread of enteric CoVs, including SARS-CoV-2.
Asunto(s)
Infecciones por Coronavirus/inmunología , Coronavirus/inmunología , Interferones/inmunología , Virus de la Diarrea Epidémica Porcina/inmunología , Vacunas Atenuadas/inmunología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Animales , Betacoronavirus/inmunología , COVID-19 , Chlorocebus aethiops , Infecciones por Coronavirus/prevención & control , Diarrea/patología , Diarrea/virología , Modelos Animales de Enfermedad , Endorribonucleasas/antagonistas & inhibidores , Heces/virología , Íleon/patología , Inmunidad Innata , Yeyuno/patología , Pandemias , Neumonía Viral/inmunología , Virus de la Diarrea Epidémica Porcina/genética , ARN Viral , ARN Polimerasa Dependiente del ARN , SARS-CoV-2 , Porcinos , Enfermedades de los Porcinos/virología , Células Vero , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/inmunologíaRESUMEN
Coronaviruses express a multifunctional papain-like protease, termed papain-like protease 2 (PLP2). PLP2 acts as a protease that cleaves the viral replicase polyprotein and as a deubiquitinating (DUB) enzyme which removes ubiquitin (Ub) moieties from ubiquitin-conjugated proteins. Previous in vitro studies implicated PLP2/DUB activity as a negative regulator of the host interferon (IFN) response, but the role of DUB activity during virus infection was unknown. Here, we used X-ray structure-guided mutagenesis and functional studies to identify amino acid substitutions within the ubiquitin-binding surface of PLP2 that reduced DUB activity without affecting polyprotein processing activity. We engineered a DUB mutation (Asp1772 to Ala) into a murine coronavirus and evaluated the replication and pathogenesis of the DUB mutant virus (DUBmut) in cultured macrophages and in mice. We found that the DUBmut virus replicates similarly to the wild-type (WT) virus in cultured cells, but the DUBmut virus activates an IFN response at earlier times compared to the wild-type virus infection in macrophages, consistent with DUB activity negatively regulating the IFN response. We compared the pathogenesis of the DUBmut virus to that of the wild-type virus and found that the DUBmut-infected mice had a statistically significant reduction (P < 0.05) in viral titer in liver and spleen at day 5 postinfection (d p.i.), although both wild-type and DUBmut virus infections resulted in similar liver pathology. Overall, this study demonstrates that structure-guided mutagenesis aids the identification of critical determinants of the PLP2-ubiquitin complex and that PLP2/DUB activity plays a role as an interferon antagonist in coronavirus pathogenesis.IMPORTANCE Coronaviruses employ a genetic economy by encoding multifunctional proteins that function in viral replication and also modify the host environment to disarm the innate immune response. The coronavirus papain-like protease 2 (PLP2) domain possesses protease activity, which cleaves the viral replicase polyprotein, and also DUB activity (deconjugating ubiquitin/ubiquitin-like molecules from modified substrates) using identical catalytic residues. To separate the DUB activity from the protease activity, we employed a structure-guided mutagenesis approach and identified residues that are important for ubiquitin binding. We found that mutating the ubiquitin-binding residues results in a PLP2 that has reduced DUB activity but retains protease activity. We engineered a recombinant murine coronavirus to express the DUB mutant and showed that the DUB mutant virus activated an earlier type I interferon response in macrophages and exhibited reduced replication in mice. The results of this study demonstrate that PLP2/DUB is an interferon antagonist and a virulence trait of coronaviruses.
Asunto(s)
Infecciones por Coronavirus/virología , Virus de la Hepatitis Murina/fisiología , Proteínas Virales/genética , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Animales , Interacciones Huésped-Patógeno , Interferón Tipo I/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/virología , Ratones , Modelos Moleculares , Virus de la Hepatitis Murina/patogenicidad , Mutagénesis , Conformación Proteica , Relación Estructura-Actividad , Ubiquitinación , Proteínas Virales/química , Virulencia , Replicación ViralRESUMEN
Understanding the effects of logging and fire on forest soil communities is integral to our knowledge of forest ecology and effective resource management. The resulting changes in soil biota have substantial impacts on forest succession and associated ecosystem processes. We quantified bacterial and fungal abundance, diversity and community composition across a logging and burn severity gradient, approximately one month after fire, in temperate wet eucalypt forests in Tasmania, Australia. Using amplicon sequencing and real-time quantitative PCR of the bacterial 16S rRNA gene and fungal ITS1 region, we demonstrate that (i) burn severity is a strong driver of soil microbial community composition, (ii) logging and high severity burning substantially reduce the biomass and diversity of soil bacteria and fungi, and (iii) the impacts of logging and burning on soil microbial communities are largely restricted to the top 10 cm of soil, with weak impacts on the subsoil. The impacts of disturbance on microbial community composition are greater than the effects of site-to-site edaphic differences. Fire also drives more divergence in community composition than logging alone. Key microbial taxa driving differences in severely burnt soils include bacterial genera implicated in plant-growth promotion and producing antifungal compounds as well as saprotrophic fungi that are also capable of forming ectomycorrhizal associations. Our research suggests that low-moderate severity burns are important for maintaining diversity and biomass in soil microbial communities but having a range of burn severities across a site contributes to the overall diversity of habitat conditions providing for both microbial and plant diversity.
Asunto(s)
Microbiota , Suelo , Australia , Ecosistema , Bosques , Hongos/genética , Microbiota/genética , ARN Ribosómico 16S/genética , Microbiología del Suelo , TasmaniaRESUMEN
Patient satisfaction is important to patient outcomes. Previous attempts to conceptualize satisfaction have often taken an atheoretical approach and focused on doctors' communication skills. Patients are becoming more active health consumers involved in their health care and current definitions of patient satisfaction may not accurately reflect patient expectations about their health consultations. Earlier research found that meeting patients' emotional needs - through empathy and patient-centered communication - is important to patient satisfaction. New research is needed to explore how those needs can be met given the changing trend in patient behaviors and the focus on patient-centredness. This study employed two communication theories - the Willingness to Communicate Model and Communication Accommodation Theory - to consider both patients' communicative decisions, and the intergroup features of the health context that can influence communicative behaviors. Two hundred and fifty-three patients from health clinics in Canada and Australia described what satisfaction meant to them, and identified what aspects of their health consultation were satisfying (or not), and we investigated their perceptions of doctor's emotional expression. Results suggest that patient perceptions of their participation in the consultation predicts their perceptions of doctor emotional expression, and their satisfaction with the consultation. Patients want both emotional and medical needs met in an environment that balances interpersonal and intergroup communication. Our findings suggest the need to expand current definitions of patient satisfaction, patient-centredness and emotional expression. We discuss the implications of these findings for health practitioners and consider future research that addresses the need for more individualized health care.
Asunto(s)
Satisfacción Personal , Relaciones Médico-Paciente , Comunicación , Humanos , Participación del Paciente , Satisfacción del Paciente , PercepciónRESUMEN
BACKGROUND: Kawasaki disease (KD) is the leading cause of childhood acquired heart disease in developed nations and can result in coronary artery aneurysms and death. Clinical and epidemiologic features implicate an infectious cause but specific antigenic targets of the disease are unknown. Peripheral blood plasmablasts are normally highly clonally diverse but the antibodies they encode are approximately 70% antigen-specific 1-2 weeks after infection. METHODS: We isolated single peripheral blood plasmablasts from children with KD 1-3 weeks after onset and prepared 60 monoclonal antibodies (mAbs). We used the mAbs to identify their target antigens and assessed serologic response among KD patients and controls to specific antigen. RESULTS: Thirty-two mAbs from 9 of 11 patients recognize antigen within intracytoplasmic inclusion bodies in ciliated bronchial epithelial cells of fatal cases. Five of these mAbs, from 3 patients with coronary aneurysms, recognize a specific peptide, which blocks binding to inclusion bodies. Sera from 5/8 KD patients dayâ ≥â 8 after illness onset, compared with 0/17 infant controls (Pâ <â .01), recognized the KD peptide antigen. CONCLUSIONS: These results identify a protein epitope targeted by the antibody response to KD and provide a means to elucidate the pathogenesis of this important worldwide pediatric problem.
Asunto(s)
Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Formación de Anticuerpos/genética , Células Sanguíneas/inmunología , Epítopos/inmunología , Síndrome Mucocutáneo Linfonodular/genética , Síndrome Mucocutáneo Linfonodular/inmunología , Femenino , Humanos , Lactante , Masculino , Síndrome Mucocutáneo Linfonodular/epidemiología , Estados Unidos/epidemiologíaRESUMEN
Analysis of temperature-sensitive (ts) mutant viruses is a classic method allowing researchers to identify genetic loci involved in viral replication and pathogenesis. Here, we report genetic analysis of a ts strain of mouse hepatitis virus (MHV), tsNC11, focusing on the role of mutations in the macrodomain (MAC) and the papain-like protease 2 (PLP2) domain of nonstructural protein 3 (nsp3), a component of the viral replication complex. Using MHV reverse genetics, we generated a series of mutant viruses to define the contributions of macrodomain- and PLP2-specific mutations to the ts phenotype. Viral replication kinetics and efficiency-of-plating analysis performed at permissive and nonpermissive temperatures revealed that changes in the macrodomain alone were both necessary and sufficient for the ts phenotype. Interestingly, mutations in the PLP2 domain were not responsible for the temperature sensitivity but did reduce the frequency of reversion of macrodomain mutants. Coimmunoprecipitation studies are consistent with an interaction between the macrodomain and PLP2. Expression studies of the macrodomain-PLP2 portion of nsp3 indicate that the ts mutations enhance proteasome-mediated degradation of the protein. Furthermore, we found that during virus infection, the replicase proteins containing the MAC and PLP2 mutations were more rapidly degraded at the nonpermissive temperature than were the wild-type proteins. Importantly, we show that the macrodomain and PLP2 mutant viruses trigger production of type I interferon in vitro and are attenuated in mice, further highlighting the importance of the macrodomain-PLP2 interplay in viral pathogenesis.IMPORTANCE Coronaviruses (CoVs) are emerging human and veterinary pathogens with pandemic potential. Despite the established and predicted threat these viruses pose to human health, there are currently no approved countermeasures to control infections with these viruses in humans. Viral macrodomains, enzymes that remove posttranslational ADP-ribosylation of proteins, and viral multifunctional papain-like proteases, enzymes that cleave polyproteins and remove polyubiquitin chains via deubiquitinating activity, are two important virulence factors. Here, we reveal an unanticipated interplay between the macrodomain and the PLP2 domain that is important for replication and antagonizing the host innate immune response. Targeting the interaction of these enzymes may provide new therapeutic opportunities to treat CoV disease.
Asunto(s)
Virus de la Hepatitis Murina/genética , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/genética , Animales , Línea Celular , Coronavirus/metabolismo , Infecciones por Coronavirus/metabolismo , Proteasas Similares a la Papaína de Coronavirus , Células HEK293 , Humanos , Inmunidad Innata/inmunología , Interferón Tipo I/metabolismo , Ratones , Papaína/genética , Papaína/metabolismo , Péptido Hidrolasas/metabolismo , Dominios Proteicos , Temperatura , Proteínas no Estructurales Virales/genética , Factores de Virulencia/metabolismoRESUMEN
Identifying viral antagonists of innate immunity and determining if they contribute to pathogenesis are critical for developing effective strategies to control emerging viruses. Previously, we reported that an endoribonuclease (EndoU) encoded by murine coronavirus plays a pivotal role in evasion of host innate immune defenses in macrophages. Here, we asked if the EndoU activity of porcine epidemic diarrhea coronavirus (PEDV), which causes acute diarrhea in swine, plays a role in antagonizing the innate response in porcine epithelial cells and macrophages, the sites of viral replication. We constructed an infectious clone of PEDV-Colorado strain (icPEDV-wt) and an EndoU-mutant PEDV (icPEDV-EnUmt) by changing the codon for a catalytic histidine residue of EndoU to alanine (His226Ala). We found that both icPEDV-wt and icPEDV-EnUmt propagated efficiently in interferon (IFN)-deficient Vero cells. In contrast, the propagation of icPEDV-EnUmt was impaired in porcine epithelial cells (LLC-PK1), where we detected an early and robust transcriptional activation of type I and type III IFNs. Infection of piglets with the parental Colorado strain, icPEDV-wt, or icPEDV-EnUmt revealed that all viruses replicated in the gut and induced diarrhea; however, there was reduced viral shedding and mortality in the icPEDV-EnUmt-infected animals. These results demonstrate that EndoU activity is not required for PEDV replication in immortalized, IFN-deficient Vero cells, but is important for suppressing the IFN response in epithelial cells and macrophages, which facilitates replication, shedding, and pathogenesis in vivo We conclude that PEDV EndoU activity is a key virulence factor that suppresses both type I and type III IFN responses.IMPORTANCE Coronaviruses (CoVs) can emerge from an animal reservoir into a naive host species to cause pandemic respiratory or gastrointestinal diseases with significant mortality in humans or domestic animals. Porcine epidemic diarrhea virus (PEDV), an alphacoronavirus (alpha-CoV), infects gut epithelial cells and macrophages, inducing diarrhea and resulting in high mortality in piglets. How PEDV suppresses the innate immune response was unknown. We found that mutating a viral endoribonuclease, EndoU, results in a virus that activates both the type I interferon response and the type III interferon response in macrophages and epithelial cells. This activation of interferon resulted in limited viral replication in epithelial cell cultures and was associated with reduced virus shedding and mortality in piglets. This study reveals a role for EndoU activity as a virulence factor in PEDV infection and provides an approach for generating live-attenuated vaccine candidates for emerging coronaviruses.
Asunto(s)
Infecciones por Coronavirus , Endorribonucleasas , Interferón Tipo I/inmunología , Virus de la Diarrea Epidémica Porcina , Enfermedades de los Porcinos , Proteínas Virales , Animales , Línea Celular , Infecciones por Coronavirus/enzimología , Infecciones por Coronavirus/genética , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/veterinaria , Endorribonucleasas/genética , Endorribonucleasas/inmunología , Interferón Tipo I/genética , Virus de la Diarrea Epidémica Porcina/enzimología , Virus de la Diarrea Epidémica Porcina/genética , Virus de la Diarrea Epidémica Porcina/inmunología , Porcinos , Enfermedades de los Porcinos/enzimología , Enfermedades de los Porcinos/genética , Enfermedades de los Porcinos/inmunología , Enfermedades de los Porcinos/virología , Proteínas Virales/genética , Proteínas Virales/inmunología , Esparcimiento de Virus/inmunologíaRESUMEN
SARS-coronavirus (CoV) is a zoonotic agent derived from rhinolophid bats, in which a plethora of SARS-related, conspecific viral lineages exist. Whereas the variability of virulence among reservoir-borne viruses is unknown, it is generally assumed that the emergence of epidemic viruses from animal reservoirs requires human adaptation. To understand the influence of a viral factor in relation to interspecies spillover, we studied the papain-like protease (PLP) of SARS-CoV. This key enzyme drives the early stages of infection as it cleaves the viral polyprotein, deubiquitinates viral and cellular proteins, and antagonizes the interferon (IFN) response. We identified a bat SARS-CoV PLP, which shared 86% amino acid identity with SARS-CoV PLP, and used reverse genetics to insert it into the SARS-CoV genome. The resulting virus replicated like SARS-CoV in Vero cells but was suppressed in IFN competent MA-104 (3.7-fold), Calu-3 (2.6-fold) and human airway epithelial cells (10.3-fold). Using ectopically-expressed PLP variants as well as full SARS-CoV infectious clones chimerized for PLP, we found that a protease-independent, anti-IFN function exists in SARS-CoV, but not in a SARS-related, bat-borne virus. This PLP-mediated anti-IFN difference was seen in primate, human as well as bat cells, thus independent of the host context. The results of this study revealed that coronavirus PLP confers a variable virulence trait among members of the species SARS-CoV, and that a SARS-CoV lineage with virulent PLPs may have pre-existed in the reservoir before onset of the epidemic.
Asunto(s)
Cisteína Endopeptidasas/fisiología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/enzimología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/patogenicidad , Proteínas Virales/fisiología , Secuencia de Aminoácidos , Animales , Quirópteros/virología , Chlorocebus aethiops , Proteasas 3C de Coronavirus , Cisteína Endopeptidasas/genética , Reservorios de Enfermedades/virología , Células HEK293 , Especificidad del Huésped , Interacciones Huésped-Patógeno , Humanos , Interferones/antagonistas & inhibidores , Filogenia , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Homología de Secuencia de Aminoácido , Síndrome Respiratorio Agudo Grave/epidemiología , Síndrome Respiratorio Agudo Grave/virología , Ubiquitina/metabolismo , Células Vero , Proteínas Virales/genética , Virulencia/genética , Virulencia/fisiología , Replicación Viral/genética , Replicación Viral/fisiología , Zoonosis/epidemiología , Zoonosis/virologíaRESUMEN
In recent decades, a paradigm shift in forest management and associated policies has led to greater emphasis on harvest practices that retain mature, overstory trees in forest stands that would otherwise be clear-cut. While it is often assumed that the maintenance of compositional and structural complexity, such as that achieved through retention forestry approaches, will also mitigate negative impacts to functional diversity, empirical evidence of this relationship is sparse. We examined the effects of an aggregated retention system on taxonomic and functional diversity in a regenerating aspen-dominated forest. Sampling was conducted along transects arranged to capture the transition from harvested (regenerating) forest to mature, unharvested forest (both intact forest stands and 0.1 ha retention aggregates). We then assessed the magnitude and distance of edge effects on multiple indices of taxonomic and functional diversity as well as functional identity. Twelve years after harvest, the distance and magnitude of edge effects on functional and taxonomic diversity did not differ between the two unharvested patch sizes (intact vs. aggregate); however, intact forest exhibited greater resistance to edge effects and greater depth of edge influence into harvested areas for some traits compared to aggregates. Analyses relying on functional traits were generally applicable across sites within a highly variable forest type, and our results demonstrate the promise of using functional traits to assess management impacts on plant diversity across a landscape. Aggregates maintained some functional attributes associated with interior forest and influenced adjacent regeneration. However, trends in some traits (i.e., shade tolerance and seed mass), particularly in the seedling layer, suggest aggregates of this size provide primarily edge habitat.
Asunto(s)
Agricultura Forestal , Mariposas Nocturnas , Animales , Ecosistema , Bosques , ÁrbolesRESUMEN
In line with global targets agreed under the Convention on Biological Diversity, the number of marine protected areas (MPAs) is increasing rapidly, yet socio-economic benefits generated by MPAs remain difficult to predict and under debate. MPAs often fail to reach their full potential as a consequence of factors such as illegal harvesting, regulations that legally allow detrimental harvesting, or emigration of animals outside boundaries because of continuous habitat or inadequate size of reserve. Here we show that the conservation benefits of 87 MPAs investigated worldwide increase exponentially with the accumulation of five key features: no take, well enforced, old (>10 years), large (>100 km(2)), and isolated by deep water or sand. Using effective MPAs with four or five key features as an unfished standard, comparisons of underwater survey data from effective MPAs with predictions based on survey data from fished coasts indicate that total fish biomass has declined about two-thirds from historical baselines as a result of fishing. Effective MPAs also had twice as many large (>250 mm total length) fish species per transect, five times more large fish biomass, and fourteen times more shark biomass than fished areas. Most (59%) of the MPAs studied had only one or two key features and were not ecologically distinguishable from fished sites. Our results show that global conservation targets based on area alone will not optimize protection of marine biodiversity. More emphasis is needed on better MPA design, durable management and compliance to ensure that MPAs achieve their desired conservation value.
Asunto(s)
Conservación de los Recursos Naturales/estadística & datos numéricos , Ecología/estadística & datos numéricos , Ecosistema , Explotaciones Pesqueras/estadística & datos numéricos , Peces/fisiología , Animales , Organismos Acuáticos/fisiología , Biodiversidad , Biomasa , Conservación de los Recursos Naturales/economía , Conservación de los Recursos Naturales/legislación & jurisprudencia , Conservación de los Recursos Naturales/métodos , Arrecifes de Coral , Ecología/economía , Ecología/legislación & jurisprudencia , Ecología/métodos , Explotaciones Pesqueras/legislación & jurisprudencia , Explotaciones Pesqueras/normas , Biología Marina/economía , Biología Marina/legislación & jurisprudencia , Biología Marina/métodos , Biología Marina/estadística & datos numéricos , Agua de Mar , Tiburones , Dióxido de Silicio , Factores de TiempoRESUMEN
Coronaviruses are positive-sense RNA viruses that generate double-stranded RNA (dsRNA) intermediates during replication, yet evade detection by host innate immune sensors. Here we report that coronavirus nonstructural protein 15 (nsp15), an endoribonuclease, is required for evasion of dsRNA sensors. We evaluated two independent nsp15 mutant mouse coronaviruses, designated N15m1 and N15m3, and found that these viruses replicated poorly and induced rapid cell death in mouse bone marrow-derived macrophages. Infection of macrophages with N15m1, which expresses an unstable nsp15, or N15m3, which expresses a catalysis-deficient nsp15, activated MDA5, PKR, and the OAS/RNase L system, resulting in an early, robust induction of type I IFN, PKR-mediated apoptosis, and RNA degradation. Immunofluorescence imaging of nsp15 mutant virus-infected macrophages revealed significant dispersal of dsRNA early during infection, whereas in WT virus-infected cells, the majority of the dsRNA was associated with replication complexes. The loss of nsp15 activity also resulted in greatly attenuated disease in mice and stimulated a protective immune response. Taken together, our findings demonstrate that coronavirus nsp15 is critical for evasion of host dsRNA sensors in macrophages and reveal that modulating nsp15 stability and activity is a strategy for generating live-attenuated vaccines.