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1.
Cell ; 184(19): 4848-4856, 2021 09 16.
Artículo en Inglés | MEDLINE | ID: mdl-34480864

RESUMEN

Since the first reports of a novel severe acute respiratory syndrome (SARS)-like coronavirus in December 2019 in Wuhan, China, there has been intense interest in understanding how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in the human population. Recent debate has coalesced around two competing ideas: a "laboratory escape" scenario and zoonotic emergence. Here, we critically review the current scientific evidence that may help clarify the origin of SARS-CoV-2.


Asunto(s)
SARS-CoV-2/fisiología , Animales , Evolución Biológica , COVID-19/virología , Humanos , Laboratorios , SARS-CoV-2/genética , Zoonosis/virología
2.
Nat Immunol ; 23(1): 40-49, 2022 01.
Artículo en Inglés | MEDLINE | ID: mdl-34937928

RESUMEN

SARS-CoV-2 infection is generally mild or asymptomatic in children but a biological basis for this outcome is unclear. Here we compare antibody and cellular immunity in children (aged 3-11 years) and adults. Antibody responses against spike protein were high in children and seroconversion boosted responses against seasonal Beta-coronaviruses through cross-recognition of the S2 domain. Neutralization of viral variants was comparable between children and adults. Spike-specific T cell responses were more than twice as high in children and were also detected in many seronegative children, indicating pre-existing cross-reactive responses to seasonal coronaviruses. Importantly, children retained antibody and cellular responses 6 months after infection, whereas relative waning occurred in adults. Spike-specific responses were also broadly stable beyond 12 months. Therefore, children generate robust, cross-reactive and sustained immune responses to SARS-CoV-2 with focused specificity for the spike protein. These findings provide insight into the relative clinical protection that occurs in most children and might help to guide the design of pediatric vaccination regimens.


Asunto(s)
Anticuerpos Antivirales/inmunología , Coronavirus Humano 229E/inmunología , Coronavirus Humano OC43/inmunología , Protección Cruzada/inmunología , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Inmunidad Adaptativa/inmunología , Adulto , Anticuerpos Neutralizantes/inmunología , COVID-19/inmunología , Vacunas contra la COVID-19/inmunología , Niño , Preescolar , Reacciones Cruzadas/inmunología , Humanos
3.
Nature ; 610(7930): 154-160, 2022 10.
Artículo en Inglés | MEDLINE | ID: mdl-35952712

RESUMEN

The SARS-CoV-2 Delta (Pango lineage B.1.617.2) variant of concern spread globally, causing resurgences of COVID-19 worldwide1,2. The emergence of the Delta variant in the UK occurred on the background of a heterogeneous landscape of immunity and relaxation of non-pharmaceutical interventions. Here we analyse 52,992 SARS-CoV-2 genomes from England together with 93,649 genomes from the rest of the world to reconstruct the emergence of Delta and quantify its introduction to and regional dissemination across England in the context of changing travel and social restrictions. Using analysis of human movement, contact tracing and virus genomic data, we find that the geographic focus of the expansion of Delta shifted from India to a more global pattern in early May 2021. In England, Delta lineages were introduced more than 1,000 times and spread nationally as non-pharmaceutical interventions were relaxed. We find that hotel quarantine for travellers reduced onward transmission from importations; however, the transmission chains that later dominated the Delta wave in England were seeded before travel restrictions were introduced. Increasing inter-regional travel within England drove the nationwide dissemination of Delta, with some cities receiving more than 2,000 observable lineage introductions from elsewhere. Subsequently, increased levels of local population mixing-and not the number of importations-were associated with the faster relative spread of Delta. The invasion dynamics of Delta depended on spatial heterogeneity in contact patterns, and our findings will inform optimal spatial interventions to reduce the transmission of current and future variants of concern, such as Omicron (Pango lineage B.1.1.529).


Asunto(s)
COVID-19 , SARS-CoV-2 , COVID-19/epidemiología , COVID-19/prevención & control , COVID-19/transmisión , COVID-19/virología , Ciudades/epidemiología , Trazado de Contacto , Inglaterra/epidemiología , Genoma Viral/genética , Humanos , Cuarentena/legislación & jurisprudencia , SARS-CoV-2/genética , SARS-CoV-2/crecimiento & desarrollo , SARS-CoV-2/aislamiento & purificación , Viaje/legislación & jurisprudencia
4.
Nature ; 594(7861): 88-93, 2021 06.
Artículo en Inglés | MEDLINE | ID: mdl-33827113

RESUMEN

COVID-19 is a disease with unique characteristics that include lung thrombosis1, frequent diarrhoea2, abnormal activation of the inflammatory response3 and rapid deterioration of lung function consistent with alveolar oedema4. The pathological substrate for these findings remains unknown. Here we show that the lungs of patients with COVID-19 contain infected pneumocytes with abnormal morphology and frequent multinucleation. The generation of these syncytia results from activation of the SARS-CoV-2 spike protein at the cell plasma membrane level. On the basis of these observations, we performed two high-content microscopy-based screenings with more than 3,000 approved drugs to search for inhibitors of spike-driven syncytia. We converged on the identification of 83 drugs that inhibited spike-mediated cell fusion, several of which belonged to defined pharmacological classes. We focused our attention on effective drugs that also protected against virus replication and associated cytopathicity. One of the most effective molecules was the antihelminthic drug niclosamide, which markedly blunted calcium oscillations and membrane conductance in spike-expressing cells by suppressing the activity of TMEM16F (also known as anoctamin 6), a calcium-activated ion channel and scramblase that is responsible for exposure of phosphatidylserine on the cell surface. These findings suggest a potential mechanism for COVID-19 disease pathogenesis and support the repurposing of niclosamide for therapy.


Asunto(s)
Anoctaminas/antagonistas & inhibidores , COVID-19/patología , Fusión Celular , Evaluación Preclínica de Medicamentos , Células Gigantes/efectos de los fármacos , SARS-CoV-2/efectos de los fármacos , Glicoproteína de la Espiga del Coronavirus/antagonistas & inhibidores , Anciano , Anciano de 80 o más Años , Células Epiteliales Alveolares/efectos de los fármacos , Células Epiteliales Alveolares/patología , Células Epiteliales Alveolares/virología , Animales , Anoctaminas/metabolismo , COVID-19/metabolismo , COVID-19/virología , Señalización del Calcio/efectos de los fármacos , Línea Celular , Canales de Cloruro/metabolismo , Chlorocebus aethiops , Femenino , Células Gigantes/metabolismo , Células Gigantes/virología , Humanos , Pulmón/efectos de los fármacos , Pulmón/patología , Pulmón/virología , Masculino , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidad , Glicoproteína de la Espiga del Coronavirus/metabolismo , Replicación Viral/efectos de los fármacos
5.
Nature ; 599(7883): 114-119, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-34488225

RESUMEN

The B.1.617.2 (Delta) variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in the state of Maharashtra in late 2020 and spread throughout India, outcompeting pre-existing lineages including B.1.617.1 (Kappa) and B.1.1.7 (Alpha)1. In vitro, B.1.617.2 is sixfold less sensitive to serum neutralizing antibodies from recovered individuals, and eightfold less sensitive to vaccine-elicited antibodies, compared with wild-type Wuhan-1 bearing D614G. Serum neutralizing titres against B.1.617.2 were lower in ChAdOx1 vaccinees than in BNT162b2 vaccinees. B.1.617.2 spike pseudotyped viruses exhibited compromised sensitivity to monoclonal antibodies to the receptor-binding domain and the amino-terminal domain. B.1.617.2 demonstrated higher replication efficiency than B.1.1.7 in both airway organoid and human airway epithelial systems, associated with B.1.617.2 spike being in a predominantly cleaved state compared with B.1.1.7 spike. The B.1.617.2 spike protein was able to mediate highly efficient syncytium formation that was less sensitive to inhibition by neutralizing antibody, compared with that of wild-type spike. We also observed that B.1.617.2 had higher replication and spike-mediated entry than B.1.617.1, potentially explaining the B.1.617.2 dominance. In an analysis of more than 130 SARS-CoV-2-infected health care workers across three centres in India during a period of mixed lineage circulation, we observed reduced ChAdOx1 vaccine effectiveness against B.1.617.2 relative to non-B.1.617.2, with the caveat of possible residual confounding. Compromised vaccine efficacy against the highly fit and immune-evasive B.1.617.2 Delta variant warrants continued infection control measures in the post-vaccination era.


Asunto(s)
Evasión Inmune , SARS-CoV-2/crecimiento & desarrollo , SARS-CoV-2/inmunología , Replicación Viral/inmunología , Anticuerpos Neutralizantes/inmunología , Vacunas contra la COVID-19/inmunología , Fusión Celular , Línea Celular , Femenino , Personal de Salud , Humanos , India , Cinética , Masculino , Glicoproteína de la Espiga del Coronavirus/metabolismo , Vacunación
6.
PLoS Biol ; 21(5): e3002118, 2023 05.
Artículo en Inglés | MEDLINE | ID: mdl-37228015

RESUMEN

The relationship between prevalence of infection and severe outcomes such as hospitalisation and death changed over the course of the COVID-19 pandemic. Reliable estimates of the infection fatality ratio (IFR) and infection hospitalisation ratio (IHR) along with the time-delay between infection and hospitalisation/death can inform forecasts of the numbers/timing of severe outcomes and allow healthcare services to better prepare for periods of increased demand. The REal-time Assessment of Community Transmission-1 (REACT-1) study estimated swab positivity for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection in England approximately monthly from May 2020 to March 2022. Here, we analyse the changing relationship between prevalence of swab positivity and the IFR and IHR over this period in England, using publicly available data for the daily number of deaths and hospitalisations, REACT-1 swab positivity data, time-delay models, and Bayesian P-spline models. We analyse data for all age groups together, as well as in 2 subgroups: those aged 65 and over and those aged 64 and under. Additionally, we analysed the relationship between swab positivity and daily case numbers to estimate the case ascertainment rate of England's mass testing programme. During 2020, we estimated the IFR to be 0.67% and the IHR to be 2.6%. By late 2021/early 2022, the IFR and IHR had both decreased to 0.097% and 0.76%, respectively. The average case ascertainment rate over the entire duration of the study was estimated to be 36.1%, but there was some significant variation in continuous estimates of the case ascertainment rate. Continuous estimates of the IFR and IHR of the virus were observed to increase during the periods of Alpha and Delta's emergence. During periods of vaccination rollout, and the emergence of the Omicron variant, the IFR and IHR decreased. During 2020, we estimated a time-lag of 19 days between hospitalisation and swab positivity, and 26 days between deaths and swab positivity. By late 2021/early 2022, these time-lags had decreased to 7 days for hospitalisations and 18 days for deaths. Even though many populations have high levels of immunity to SARS-CoV-2 from vaccination and natural infection, waning of immunity and variant emergence will continue to be an upwards pressure on the IHR and IFR. As investments in community surveillance of SARS-CoV-2 infection are scaled back, alternative methods are required to accurately track the ever-changing relationship between infection, hospitalisation, and death and hence provide vital information for healthcare provision and utilisation.


Asunto(s)
COVID-19 , Humanos , COVID-19/epidemiología , SARS-CoV-2 , Teorema de Bayes , Pandemias , Inglaterra/epidemiología , Hospitalización
7.
Nature ; 587(7835): 638-643, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-33208942

RESUMEN

Aquatic birds represent a vast reservoir from which new pandemic influenza A viruses can emerge1. Influenza viruses contain a negative-sense segmented RNA genome that is transcribed and replicated by the viral heterotrimeric RNA polymerase (FluPol) in the context of viral ribonucleoprotein complexes2,3. RNA polymerases of avian influenza A viruses (FluPolA) replicate viral RNA inefficiently in human cells because of species-specific differences in acidic nuclear phosphoprotein 32 (ANP32), a family of essential host proteins for FluPol activity4. Host-adaptive mutations, particularly a glutamic-acid-to-lysine mutation at amino acid residue 627 (E627K) in the 627 domain of the PB2 subunit, enable avian FluPolA to overcome this restriction and efficiently replicate viral RNA in the presence of human ANP32 proteins. However, the molecular mechanisms of genome replication and the interplay with ANP32 proteins remain largely unknown. Here we report cryo-electron microscopy structures of influenza C virus polymerase (FluPolC) in complex with human and chicken ANP32A. In both structures, two FluPolC molecules form an asymmetric dimer bridged by the N-terminal leucine-rich repeat domain of ANP32A. The C-terminal low-complexity acidic region of ANP32A inserts between the two juxtaposed PB2 627 domains of the asymmetric FluPolA dimer, suggesting a mechanism for how the adaptive PB2(E627K) mutation enables the replication of viral RNA in mammalian hosts. We propose that this complex represents a replication platform for the viral RNA genome, in which one of the FluPol molecules acts as a replicase while the other initiates the assembly of the nascent replication product into a viral ribonucleoprotein complex.


Asunto(s)
Microscopía por Crioelectrón , Gammainfluenzavirus/enzimología , Interacciones Huésped-Patógeno , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/metabolismo , Animales , Pollos/virología , Genoma Viral/genética , Células HEK293 , Humanos , Gammainfluenzavirus/genética , Modelos Moleculares , Proteínas Nucleares/ultraestructura , Infecciones por Orthomyxoviridae/genética , Infecciones por Orthomyxoviridae/metabolismo , Infecciones por Orthomyxoviridae/virología , Multimerización de Proteína , ARN Viral/biosíntesis , ARN Viral/genética , Proteínas de Unión al ARN/ultraestructura , ARN Polimerasa Dependiente del ARN/ultraestructura , Células Sf9
8.
Proc Natl Acad Sci U S A ; 120(45): e2308655120, 2023 Nov 07.
Artículo en Inglés | MEDLINE | ID: mdl-37903249

RESUMEN

The ongoing SARS-CoV-2 epidemic was marked by the repeated emergence and replacement of "variants" with genetic and phenotypic distance from the ancestral strains, the most recent examples being viruses of the Omicron lineage. Here, we describe a hamster direct contact exposure challenge model to assess protection against reinfection conferred by either vaccination or prior infection. We found that two doses of self-amplifying RNA vaccine based on the ancestral Spike ameliorated weight loss following Delta infection and decreased viral loads but had minimal effect on Omicron BA.1 infection. Prior vaccination followed by Delta or BA.1 breakthrough infections led to a high degree of cross-reactivity to all tested variants, suggesting that repeated exposure to antigenically distinct Spikes, via infection and/or vaccination drives a cross-reactive immune response. Prior infection with ancestral or Alpha variant was partially protective against BA.1 infection, whereas all animals previously infected with Delta and exposed to BA.1 became reinfected, although they shed less virus than BA.1-infected naive hamsters. Hamsters reinfected with BA.1 after prior Delta infection emitted infectious virus into the air, indicating that they could be responsible for onwards airborne transmission. We further tested whether prior infection with BA.1 protected from reinfection with Delta or later Omicron sublineages BA.2, BA.4, or BA.5. BA.1 was protective against BA.2 but not against Delta, BA.4, or BA.5 reinfection. These findings suggest that cohorts whose only immune experience of COVID-19 is Omicron BA.1 infection may be vulnerable to future circulation of reemerged Delta-like derivatives, as well as emerging Omicron sublineages.


Asunto(s)
COVID-19 , Hepatitis D , Animales , Cricetinae , Infección Irruptiva , Reinfección , Reacciones Cruzadas , Anticuerpos Neutralizantes , Anticuerpos Antivirales
9.
PLoS Pathog ; 19(8): e1011545, 2023 08.
Artículo en Inglés | MEDLINE | ID: mdl-37535672

RESUMEN

New variants of SARS-CoV-2 are continually emerging with mutations in spike associated with increased transmissibility and immune escape. Phenotypic maps can inform the prediction of concerning mutations from genomic surveillance, however most of these maps currently derive from studies using monomeric RBD, while spike is trimeric, and contains additional domains. These maps may fail to reflect interdomain interactions in the prediction of phenotypes. To try to improve on this, we developed a platform for deep mutational scanning using whole trimeric spike. We confirmed a previously reported epistatic effect within the RBD affecting ACE2 binding, that highlights the importance of updating the base spike sequence for future mutational scanning studies. Using post vaccine sera, we found that the immune response of vaccinated individuals was highly focused on one or two epitopes in the RBD and that single point mutations at these positions can account for most of the immune escape mediated by the Omicron BA.1 RBD. However, unexpectedly we found that the BA.1 RBD alone does not account for the high level of antigenic escape by BA.1 spike. We show that the BA.1 NTD amplifies the immune evasion of its associated RBD. BA.1 NTD reduces neutralistion by RBD directed monoclonal antibodies, and impacts ACE2 interaction. NTD variation is thus an important mechanism of immune evasion by SARS-CoV-2. Such effects are not seen when pre-stabilized spike proteins are used, suggesting the interdomain effects require protein mobility to express their phenotype.


Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , Enzima Convertidora de Angiotensina 2/genética , COVID-19/genética , Mutagénesis , Mutación , Fenotipo , Glicoproteína de la Espiga del Coronavirus/genética
10.
PLoS Pathog ; 19(11): e1011589, 2023 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-37934791

RESUMEN

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continued to evolve throughout the coronavirus disease-19 (COVID-19) pandemic, giving rise to multiple variants of concern (VOCs) with different biological properties. As the pandemic progresses, it will be essential to test in near real time the potential of any new emerging variant to cause severe disease. BA.1 (Omicron) was shown to be attenuated compared to the previous VOCs like Delta, but it is possible that newly emerging variants may regain a virulent phenotype. Hamsters have been proven to be an exceedingly good model for SARS-CoV-2 pathogenesis. Here, we aimed to develop robust quantitative pipelines to assess the virulence of SARS-CoV-2 variants in hamsters. We used various approaches including RNAseq, RNA in situ hybridization, immunohistochemistry, and digital pathology, including software assisted whole section imaging and downstream automatic analyses enhanced by machine learning, to develop methods to assess and quantify virus-induced pulmonary lesions in an unbiased manner. Initially, we used Delta and Omicron to develop our experimental pipelines. We then assessed the virulence of recent Omicron sub-lineages including BA.5, XBB, BQ.1.18, BA.2, BA.2.75 and EG.5.1. We show that in experimentally infected hamsters, accurate quantification of alveolar epithelial hyperplasia and macrophage infiltrates represent robust markers for assessing the extent of virus-induced pulmonary pathology, and hence virus virulence. In addition, using these pipelines, we could reveal how some Omicron sub-lineages (e.g., BA.2.75 and EG.5.1) have regained virulence compared to the original BA.1. Finally, to maximise the utility of the digital pathology pipelines reported in our study, we developed an online repository containing representative whole organ histopathology sections that can be visualised at variable magnifications (https://covid-atlas.cvr.gla.ac.uk). Overall, this pipeline can provide unbiased and invaluable data for rapidly assessing newly emerging variants and their potential to cause severe disease.


Asunto(s)
COVID-19 , SARS-CoV-2 , Animales , Cricetinae , Humanos , SARS-CoV-2/genética , Virulencia , Aprendizaje Automático
11.
Cell ; 140(3): 397-408, 2010 Feb 05.
Artículo en Inglés | MEDLINE | ID: mdl-20144762

RESUMEN

RIG-I is a key mediator of antiviral immunity, able to couple detection of infection by RNA viruses to the induction of interferons. Natural RIG-I stimulatory RNAs have variously been proposed to correspond to virus genomes, virus replication intermediates, viral transcripts, or self-RNA cleaved by RNase L. However, the relative contribution of each of these RNA species to RIG-I activation and interferon induction in virus-infected cells is not known. Here, we use three approaches to identify physiological RIG-I agonists in cells infected with influenza A virus or Sendai virus. We show that RIG-I agonists are exclusively generated by the process of virus replication and correspond to full-length virus genomes. Therefore, nongenomic viral transcripts, short replication intermediates, and cleaved self-RNA do not contribute substantially to interferon induction in cells infected with these negative strand RNA viruses. Rather, single-stranded RNA viral genomes bearing 5'-triphosphates constitute the natural RIG-I agonists that trigger cell-intrinsic innate immune responses during infection.


Asunto(s)
ARN Helicasas DEAD-box/inmunología , Proteínas de la Membrana/inmunología , Proteínas del Tejido Nervioso/inmunología , Infecciones por Virus ARN/inmunología , ARN Viral/inmunología , Animales , Línea Celular , Proteína 58 DEAD Box , Perros , Humanos , Interferones/inmunología , Ratones , Virus ARN/fisiología , Receptores de Superficie Celular , Receptores Inmunológicos , Replicación Viral
12.
Anal Chem ; 96(28): 11181-11188, 2024 07 16.
Artículo en Inglés | MEDLINE | ID: mdl-38967089

RESUMEN

The COVID-19 pandemic has highlighted the need for rapid and reliable diagnostics that are accessible in resource-limited settings. To address this pressing issue, we have developed a rapid, portable, and electricity-free method for extracting nucleic acids from respiratory swabs (i.e. nasal, nasopharyngeal and buccal swabs), successfully demonstrating its effectiveness for the detection of SARS-CoV-2 in residual clinical specimens. Unlike traditional approaches, our solution eliminates the need for micropipettes or electrical equipment, making it user-friendly and requiring little to no training. Our method builds upon the principles of magnetic bead extraction and revolves around a low-cost plastic magnetic lid, called SmartLid, in combination with a simple disposable kit containing all required reagents conveniently prealiquoted. Here, we clinically validated the SmartLid sample preparation method in comparison to the gold standard QIAamp Viral RNA Mini Kit from QIAGEN, using 406 clinical isolates, including 161 SARS-CoV-2 positives, using the SARS-CoV-2 RT-qPCR assays developed by the US Centers for Disease Control and Prevention (CDC). The SmartLid method showed an overall sensitivity of 95.03% (95% CI: 90.44-97.83%) and a specificity of 99.59% (95% CI: 97.76-99.99%), with a positive agreement of 97.79% (95% CI: 95.84-98.98%) when compared to QIAGEN's column-based extraction method. There are clear benefits to using the SmartLid sample preparation kit: it enables swift extraction of viral nucleic acids, taking less than 5 min, without sacrificing significant accuracy when compared to more expensive and time-consuming alternatives currently available on the market. Moreover, its simplicity makes it particularly well-suited for the point-of-care where rapid results and portability are crucial. By providing an efficient and accessible means of nucleic acid extraction, our approach aims to introduce a step-change in diagnostic capabilities for resource-limited settings.


Asunto(s)
COVID-19 , ARN Viral , SARS-CoV-2 , Humanos , SARS-CoV-2/aislamiento & purificación , SARS-CoV-2/genética , COVID-19/diagnóstico , COVID-19/virología , ARN Viral/aislamiento & purificación , ARN Viral/análisis , Prueba de Ácido Nucleico para COVID-19/métodos , Prueba de Ácido Nucleico para COVID-19/instrumentación , Manejo de Especímenes/métodos , Prueba de COVID-19/métodos , Prueba de COVID-19/instrumentación , Técnicas de Diagnóstico Molecular/métodos , Configuración de Recursos Limitados
13.
J Virol ; 97(5): e0021323, 2023 05 31.
Artículo en Inglés | MEDLINE | ID: mdl-37074204

RESUMEN

ANP32 proteins, which act as influenza polymerase cofactors, vary between birds and mammals. In mammals, ANP32A and ANP32B have been reported to serve essential but redundant roles to support influenza polymerase activity. The well-known mammalian adaptation PB2-E627K enables influenza polymerase to use mammalian ANP32 proteins. However, some mammalian-adapted influenza viruses do not harbor this substitution. Here, we show that alternative PB2 adaptations, Q591R and D701N, also allow influenza polymerase to use mammalian ANP32 proteins, whereas other PB2 mutations, G158E, T271A, and D740N, increase polymerase activity in the presence of avian ANP32 proteins as well. Furthermore, PB2-E627K strongly favors use of mammalian ANP32B proteins, whereas D701N shows no such bias. Accordingly, PB2-E627K adaptation emerges in species with strong pro-viral ANP32B proteins, such as humans and mice, while D701N is more commonly seen in isolates from swine, dogs, and horses, where ANP32A proteins are the preferred cofactor. Using an experimental evolution approach, we show that the passage of viruses containing avian polymerases in human cells drove acquisition of PB2-E627K, but not in the absence of ANP32B. Finally, we show that the strong pro-viral support of ANP32B for PB2-E627K maps to the low-complexity acidic region (LCAR) tail of ANP32B. IMPORTANCE Influenza viruses naturally reside in wild aquatic birds. However, the high mutation rate of influenza viruses allows them to rapidly and frequently adapt to new hosts, including mammals. Viruses that succeed in these zoonotic jumps pose a pandemic threat whereby the virus adapts sufficiently to efficiently transmit human-to-human. The influenza virus polymerase is central to viral replication and restriction of polymerase activity is a major barrier to species jumps. ANP32 proteins are essential for influenza polymerase activity. In this study, we describe how avian influenza viruses can adapt in several different ways to use mammalian ANP32 proteins. We further show that differences between mammalian ANP32 proteins can select different adaptive changes and are responsible for some of the typical mutations that arise in mammalian-adapted influenza polymerases. These different adaptive mutations may determine the relative zoonotic potential of influenza viruses and thus help assess their pandemic risk.


Asunto(s)
Virus de la Influenza A , Gripe Aviar , Gripe Humana , Proteínas Nucleares , Animales , Perros , Humanos , Ratones , Proteínas de Ciclo Celular/metabolismo , Caballos , Virus de la Influenza A/genética , Virus de la Influenza A/metabolismo , Gripe Aviar/genética , Gripe Humana/genética , Mamíferos , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleotidiltransferasas/metabolismo , Proteínas de Unión al ARN/metabolismo , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismo , Porcinos , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación Viral
14.
J Med Virol ; 96(3): e29540, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38529542

RESUMEN

The sex disparity in COVID-19 outcomes with males generally faring worse than females has been associated with the androgen-regulated expression of the protease TMPRSS2 and the cell receptor ACE2 in the lung and fueled interest in antiandrogens as potential antivirals. In this study, we explored enzalutamide, an antiandrogen used commonly to treat prostate cancer, as a potential antiviral against the human coronaviruses which cause seasonal respiratory infections (HCoV-NL63, -229E, and -OC43). Using lentivirus-pseudotyped and authentic HCoV, we report that enzalutamide reduced 229E and NL63 entry and infection in both TMPRSS2- and nonexpressing immortalized cells, suggesting a TMPRSS2-independent mechanism. However, no effect was observed against OC43. To decipher this distinction, we performed RNA-sequencing analysis on 229E- and OC43-infected primary human airway cells. Our results show a significant induction of androgen-responsive genes by 229E compared to OC43 at 24 and 72 h postinfection. The virus-mediated effect on AR-signaling was further confirmed with a consensus androgen response element-driven luciferase assay in androgen-depleted MRC-5 cells. Specifically, 229E induced luciferase-reporter activity in the presence and absence of the synthetic androgen mibolerone, while OC43 inhibited induction. These findings highlight a complex interplay between viral infections and androgen-signaling, offering insights for disparities in viral outcomes and antiviral interventions.


Asunto(s)
Andrógenos , Benzamidas , Coronavirus Humano 229E , Nitrilos , Feniltiohidantoína , Masculino , Femenino , Humanos , Andrógenos/metabolismo , Andrógenos/farmacología , Antagonistas de Andrógenos/farmacología , Antagonistas de Andrógenos/metabolismo , Estaciones del Año , Antivirales/farmacología , Antivirales/metabolismo , Luciferasas
15.
Nucleic Acids Res ; 50(10): 5713-5725, 2022 06 10.
Artículo en Inglés | MEDLINE | ID: mdl-35639917

RESUMEN

The segmented negative-sense RNA genome of influenza A virus is assembled into ribonucleoprotein complexes (RNP) with viral RNA-dependent RNA polymerase and nucleoprotein (NP). It is in the context of these RNPs that the polymerase transcribes and replicates viral RNA (vRNA). Host acidic nuclear phosphoprotein 32 (ANP32) family proteins play an essential role in vRNA replication by mediating the dimerization of the viral polymerase via their N-terminal leucine-rich repeat (LRR) domain. However, whether the C-terminal low-complexity acidic region (LCAR) plays a role in RNA synthesis remains unknown. Here, we report that the LCAR is required for viral genome replication during infection. Specifically, we show that the LCAR directly interacts with NP and this interaction is mutually exclusive with RNA. Furthermore, we show that the replication of a short vRNA-like template that can be replicated in the absence of NP is less sensitive to LCAR truncations compared with the replication of full-length vRNA segments which is NP-dependent. We propose a model in which the LCAR interacts with NP to promote NP recruitment to nascent RNA during influenza virus replication, ensuring the co-replicative assembly of RNA into RNPs.


Asunto(s)
Proteínas Nucleares , ARN Viral , Genoma Viral , Proteínas Nucleares/metabolismo , Proteínas de la Nucleocápside/genética , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Fosfoproteínas/genética , ARN Viral/genética , ARN Viral/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Replicación Viral/genética
16.
Clin Infect Dis ; 76(4): 658-666, 2023 02 18.
Artículo en Inglés | MEDLINE | ID: mdl-35913410

RESUMEN

BACKGROUND: We explore severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody lateral flow immunoassay (LFIA) performance under field conditions compared to laboratory-based electrochemiluminescence immunoassay (ECLIA) and live virus neutralization. METHODS: In July 2021, 3758 participants performed, at home, a self-administered Fortress LFIA on finger-prick blood, reported and submitted a photograph of the result, and provided a self-collected capillary blood sample for assessment of immunoglobulin G (IgG) antibodies using the Roche Elecsys® Anti-SARS-CoV-2 ECLIA. We compared the self-reported LFIA result to the quantitative ECLIA and checked the reading of the LFIA result with an automated image analysis (ALFA). In a subsample of 250 participants, we compared the results to live virus neutralization. RESULTS: Almost all participants (3593/3758, 95.6%) had been vaccinated or reported prior infection. Overall, 2777/3758 (73.9%) were positive on self-reported LFIA, 2811/3457 (81.3%) positive by LFIA when ALFA-reported, and 3622/3758 (96.4%) positive on ECLIA (using the manufacturer reference standard threshold for positivity of 0.8 U mL-1). Live virus neutralization was detected in 169 of 250 randomly selected samples (67.6%); 133/169 were positive with self-reported LFIA (sensitivity 78.7%; 95% confidence interval [CI]: 71.8, 84.6), 142/155 (91.6%; 95% CI: 86.1, 95.5) with ALFA, and 169 (100%; 95% CI: 97.8, 100.0) with ECLIA. There were 81 samples with no detectable virus neutralization; 47/81 were negative with self-reported LFIA (specificity 58.0%; 95% CI: 46.5, 68.9), 34/75 (45.3%; 95% CI: 33.8, 57.3) with ALFA, and 0/81 (0%; 95% CI: 0, 4.5) with ECLIA. CONCLUSIONS: Self-administered LFIA is less sensitive than a quantitative antibody test, but the positivity in LFIA correlates better than the quantitative ECLIA with virus neutralization.


Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , COVID-19/diagnóstico , Autoevaluación , Sensibilidad y Especificidad , Anticuerpos Antivirales , Inmunoensayo/métodos
17.
Eur J Immunol ; 52(11): 1768-1775, 2022 11.
Artículo en Inglés | MEDLINE | ID: mdl-36106692

RESUMEN

SARS-CoV-2 is a newly emerged coronavirus, causing the global pandemic of respiratory coronavirus disease (COVID-19). The type I interferon (IFN) pathway is of particular importance for anti-viral defense and recent studies identified that type I IFNs drive early inflammatory responses to SARS-CoV-2. Here, we use a mouse model of SARS-CoV-2 infection, facilitating viral entry by intranasal recombinant Adeno-Associated Virus (rAAV) transduction of hACE2 in wildtype (WT) and type I IFN receptor-1 deficient (Ifnar1-/- ) mice, to study the role of type I IFN signalling and innate immune responses during SARS-CoV-2 infection. Our data show that type I IFN signalling is essential for inducing anti-viral effector responses to SARS-CoV-2, control of virus replication, and to prevent enhanced disease. Furthermore, hACE2-Ifnar1-/- mice had increased gene expression of the chemokine Cxcl1 and airway infiltration of neutrophils as well as reduced and delayed production of monocyte-recruiting chemokine CCL2. hACE2-Ifnar1-/- mice showed altered recruitment of inflammatory myeloid cells to the lung upon SARS-CoV-2 infection, with a shift from Ly6C+ to Ly6C- expressing cells. Together, our findings suggest that type I IFN signalling deficiency results in a dysregulated innate immune response to SARS-CoV-2 infection.


Asunto(s)
COVID-19 , Inmunidad Innata , Receptor de Interferón alfa y beta , Animales , Ratones , COVID-19/inmunología , Interferón Tipo I , Pandemias , Receptor de Interferón alfa y beta/genética , SARS-CoV-2
18.
J Virol ; 96(21): e0117822, 2022 11 09.
Artículo en Inglés | MEDLINE | ID: mdl-36226985

RESUMEN

Defective viral genomes (DVGs), which are generated by the viral polymerase in error during RNA replication, can trigger innate immunity and are implicated in altering the clinical outcome of infection. Here, we investigated the impact of DVGs on innate immunity and pathogenicity in a BALB/c mouse model of influenza virus infection. We generated stocks of influenza viruses containing the internal genes of an H5N1 virus that contained different levels of DVGs (indicated by different genome-to-PFU ratios). In lung epithelial cells, the high-DVG stock was immunostimulatory at early time points postinfection. DVGs were amplified during virus replication in myeloid immune cells and triggered proinflammatory cytokine production. In the mouse model, infection with the different virus stocks produced divergent outcomes. The high-DVG stock induced an early type I interferon (IFN) response that limited viral replication in the lungs, resulting in minimal weight loss. In contrast, the virus stock with low levels of DVGs replicated to high titers and amplified DVGs over time, resulting in elevated levels of proinflammatory cytokines accompanied by rapid weight loss and increased morbidity and mortality. Our results suggest that the timing and levels of immunostimulatory DVGs generated during infection contribute to H5N1 pathogenesis. IMPORTANCE Mammalian infections with highly pathogenic avian influenza viruses (HPAIVs) cause severe disease associated with excessive proinflammatory cytokine production. Aberrant replication products, such as defective viral genomes (DVGs), can stimulate the antiviral response, and cytokine induction is associated with their emergence in vivo. We show that stocks of a recombinant virus containing HPAIV internal genes that differ in their amounts of DVGs have vastly diverse outcomes in a mouse model. The high-DVG stock resulted in extremely mild disease due to suppression of viral replication. Conversely, the stock that contained low DVGs but rapidly accumulated DVGs over the course of infection led to severe disease. Therefore, the timing of DVG amplification and proinflammatory cytokine production impact disease outcome, and these findings demonstrate that not all DVG generation reduces viral virulence. This study also emphasizes the crucial requirement to examine the quality of virus preparations regarding DVG content to ensure reproducible research.


Asunto(s)
Subtipo H5N1 del Virus de la Influenza A , Virus de la Influenza A , Ratones , Animales , Virus Defectuosos/genética , Virus de la Influenza A/genética , Ratones Endogámicos BALB C , Subtipo H5N1 del Virus de la Influenza A/genética , Genoma Viral , Replicación Viral/genética , Citocinas/genética , Pérdida de Peso/genética , Mamíferos/genética
19.
PLoS Pathog ; 17(6): e1008937, 2021 06.
Artículo en Inglés | MEDLINE | ID: mdl-34061908

RESUMEN

Favipiravir is a nucleoside analogue which has been licensed to treat influenza in the event of a new pandemic. We previously described a favipiravir resistant influenza A virus generated by in vitro passage in presence of drug with two mutations: K229R in PB1, which conferred resistance at a cost to polymerase activity, and P653L in PA, which compensated for the cost of polymerase activity. However, the clinical relevance of these mutations is unclear as the mutations have not been found in natural isolates and it is unknown whether viruses harbouring these mutations would replicate or transmit in vivo. Here, we infected ferrets with a mix of wild type p(H1N1) 2009 and corresponding favipiravir-resistant virus and tested for replication and transmission in the absence of drug. Favipiravir-resistant virus successfully infected ferrets and was transmitted by both contact transmission and respiratory droplet routes. However, sequencing revealed the mutation that conferred resistance, K229R, decreased in frequency over time within ferrets. Modelling revealed that due to a fitness advantage for the PA P653L mutant, reassortment with the wild-type virus to gain wild-type PB1 segment in vivo resulted in the loss of the PB1 resistance mutation K229R. We demonstrated that this fitness advantage of PA P653L in the background of our starting virus A/England/195/2009 was due to a maladapted PA in first wave isolates from the 2009 pandemic. We show there is no fitness advantage of P653L in more recent pH1N1 influenza A viruses. Therefore, whilst favipiravir-resistant virus can transmit in vivo, the likelihood that the resistance mutation is retained in the absence of drug pressure may vary depending on the genetic background of the starting viral strain.


Asunto(s)
Amidas/farmacología , Antivirales/farmacología , Farmacorresistencia Viral/genética , Subtipo H1N1 del Virus de la Influenza A/genética , Infecciones por Orthomyxoviridae/genética , Infecciones por Orthomyxoviridae/transmisión , Pirazinas/farmacología , Animales , Farmacorresistencia Viral/efectos de los fármacos , Hurones , Humanos , Gripe Humana/virología
20.
PLoS Pathog ; 17(5): e1009527, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33956888

RESUMEN

Baloxavir is approved in several countries for the treatment of uncomplicated influenza in otherwise-healthy and high-risk patients. Treatment-emergent viruses with reduced susceptibility to baloxavir have been detected in clinical trials, but the likelihood of widespread occurrence depends on replication capacity and onward transmission. We evaluated the fitness of A/H3N2 and A/H1N1pdm09 viruses with the polymerase acidic (PA) I38T-variant conferring reduced susceptibility to baloxavir relative to wild-type (WT) viruses, using a competitive mixture ferret model, recombinant viruses and patient-derived virus isolates. The A/H3N2 PA/I38T virus showed a reduction in within-host fitness but comparable between-host fitness to the WT virus, while the A/H1N1pdm09 PA/I38T virus had broadly similar within-host fitness but substantially lower between-host fitness. Although PA/I38T viruses replicate and transmit between ferrets, our data suggest that viruses with this amino acid substitution have lower fitness relative to WT and this relative fitness cost was greater in A/H1N1pdm09 viruses than in A/H3N2 viruses.


Asunto(s)
Antivirales/farmacología , Dibenzotiepinas/farmacología , Modelos Animales de Enfermedad , Farmacorresistencia Viral , Virus de la Influenza A/genética , Morfolinas/farmacología , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Piridonas/farmacología , Triazinas/farmacología , Replicación Viral , Sustitución de Aminoácidos , Animales , Femenino , Hurones , Virus de la Influenza A/efectos de los fármacos , Virus de la Influenza A/aislamiento & purificación , Masculino , Infecciones por Orthomyxoviridae/virología
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