Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 121
Filtrar
1.
Anal Chem ; 93(38): 13063-13071, 2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34541844

RESUMO

Short of a vaccine, frequent and rapid testing, preferably at home, is the most effective strategy to contain the COVID-19 pandemic. Herein, we report on single-stage and two-stage molecular diagnostic tests that can be carried out with simple or no instrumentation. Our single-stage amplification is reverse transcription-loop mediated isothermal amplification (RT-LAMP) with custom-designed primers targeting the ORF1ab and the N gene regions of the virus genome. Our new two-stage amplification, dubbed Penn-RAMP, comprises recombinase isothermal amplification (RT-RPA) as its first stage and LAMP as its second stage. We compared various sample preparation strategies aimed at deactivating the virus while preserving its RNA and tested contrived and patient samples, consisting of nasopharyngeal swabs, oropharyngeal swabs, and saliva. Amplicons were detected either in real time with fluorescent intercalating dye or after amplification with the intercalating colorimetric dye LCV, which is insensitive to sample's PH. Our single RT-LAMP tests can be carried out instrumentation-free. To enable concurrent testing of multiple samples, we developed an inexpensive heat block that supports both the single-stage and two-stage amplification. Our RT-LAMP and Penn-RAMP assays have, respectively, analytical sensitivities of 50 and 5 virions/reaction. Both our single- and two-stage assays have successfully detected SARS-CoV-2 in patients with viral loads corresponding to the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) threshold cycle smaller than 32 while operating with minimally processed samples, without nucleic acid isolation. Penn-RAMP provides a 10-fold better sensitivity than RT-LAMP and does not need thermal cycling like PCR assays. All reagents are amenable to dry, refrigeration-free storage. The SARS-CoV-2 test described herein is suitable for screening at home, at the point of need, and in resource-poor settings.


Assuntos
COVID-19 , SARS-CoV-2 , Teste para COVID-19 , Humanos , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , Pandemias , Sistemas Automatizados de Assistência Junto ao Leito , RNA Viral/genética , Sensibilidade e Especificidade
2.
Cell ; 184(19): 4848-4856, 2021 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-34480864

RESUMO

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.


Assuntos
SARS-CoV-2/fisiologia , Animais , Evolução Biológica , COVID-19/virologia , Humanos , Laboratórios , SARS-CoV-2/genética , Zoonoses/virologia
3.
mBio ; 12(4): e0194821, 2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34334001

RESUMO

The origins of the calamitous SARS-CoV-2 pandemic are now the subject of vigorous discussion and debate between two competing hypotheses for how it entered the human population: (i) direct infection from a feral source, likely a bat and possibly with an intermediate mammalian host, and (ii) a lab accident whereby bat isolates infected a researcher, who then passed it to others. Here, we ask whether the tools of science can help resolve the origins question and conclude that while such studies can provide important information, these are unlikely to provide a definitive answer. Currently available data combined with historical precedent from other outbreaks and viewed through the prism of Occam's razor favor the feral source hypothesis, but science can provide only probabilities, not certainty.


Assuntos
COVID-19/transmissão , Vigilância em Saúde Pública/métodos , Zoonoses/transmissão , Zoonoses/virologia , Acidentes de Trabalho , Animais , Quirópteros/virologia , Humanos , SARS-CoV-2
4.
mBio ; 12(4): e0178121, 2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34372695

RESUMO

The 2',5'-oligoadenylate (2-5A)-dependent endoribonuclease, RNase L, is a principal mediator of the interferon (IFN) antiviral response. Therefore, the regulation of cellular levels of 2-5A is a key point of control in antiviral innate immunity. Cellular 2-5A levels are determined by IFN-inducible 2',5'-oligoadenylate synthetases (OASs) and by enzymes that degrade 2-5A. Importantly, many coronaviruses (CoVs) and rotaviruses encode 2-5A-degrading enzymes, thereby antagonizing RNase L and its antiviral effects. A-kinase-anchoring protein 7 (AKAP7), a mammalian counterpart, could possibly limit tissue damage from excessive or prolonged RNase L activation during viral infections or from self-double-stranded RNAs that activate OAS. We show that these enzymes, members of the two-histidine phosphoesterase (2H-PE) superfamily, constitute a subfamily referred here as 2',5'-PEs. 2',5'-PEs from the mouse CoV mouse hepatitis virus (MHV) (NS2), Middle East respiratory syndrome coronavirus (MERS-CoV) (NS4b), group A rotavirus (VP3), and mouse (AKAP7) were investigated for their evolutionary relationships and activities. While there was no activity against 3',5'-oligoribonucleotides, they all cleaved 2',5'-oligoadenylates efficiently but with variable activity against other 2',5'-oligonucleotides. The 2',5'-PEs are shown to be metal ion-independent enzymes that cleave trimer 2-5A (2',5'-p3A3) producing mono- or diadenylates with 2',3'-cyclic phosphate termini. Our results suggest that the elimination of 2-5A might be the sole function of viral 2',5'-PEs, thereby promoting viral escape from innate immunity by preventing or limiting the activation of RNase L. IMPORTANCE Viruses often encode accessory proteins that antagonize the host antiviral immune response. Here, we probed the evolutionary relationships and biochemical activities of two-histidine phosphoesterases (2H-PEs) that allow some coronaviruses and rotaviruses to counteract antiviral innate immunity. In addition, we investigated the mammalian enzyme AKAP7, which has homology and shared activities with the viral enzymes and might reduce self-injury. These viral and host enzymes, which we refer to as 2',5'-PEs, specifically degrade 2',5'-oligoadenylate activators of the antiviral enzyme RNase L. We show that the host and viral enzymes are metal ion independent and exclusively cleave 2',5'- and not 3',5'-phosphodiester bonds, producing cleavage products with cyclic 2',3'-phosphate termini. Our study defines 2',5'-PEs as enzymes that share characteristic conserved features with the 2H-PE superfamily but have specific and distinct biochemical cleavage activities. These findings may eventually lead to pharmacological strategies for developing antiviral drugs against coronaviruses, rotaviruses, and other viruses.


Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Nucleotídeos de Adenina/metabolismo , Endorribonucleases/metabolismo , Coronavírus da Síndrome Respiratória do Oriente Médio/enzimologia , Vírus da Hepatite Murina/enzimologia , Oligorribonucleotídeos/metabolismo , Rotavirus/enzimologia , Animais , Humanos , Imunidade Inata/imunologia , Interferons/imunologia , Camundongos
5.
Clin Chem ; 2021 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-34383886

RESUMO

BACKGROUND: High sensitivity SARS-CoV-2 antigen assays are desirable to mitigate false negative results. Limited data are available to quantify and track SARS-CoV-2 antigen burden in respiratory samples from different populations. METHODS: We developed the Microbubbling SARS-CoV-2 Antigen Assay (MSAA) with smartphone readout, with a limit of detection (LOD) of 0.5 pg/mL (10.6 fmol/L) nucleocapsid (N) antigen or 4000 copies/mL inactivated SARS-CoV-2 virus in nasopharyngeal (NP) swabs. We developed a computer vision and machine learning-based automatic microbubble image classifier to accurately identify positives and negatives, and quantified and tracked antigen dynamics in ICU COVID inpatients and immunocompromised COVID patients. RESULTS: Compared to qualitative RT-PCR methods, the MSAA demonstrated a positive percent agreement (PPA) of 97% (95% confidence interval (CI), 92-99%) and a negative percent agreement (NPA) of 97% (95% CI, 94-100%) in a clinical validation study with 372 residual clinical NP swabs. In immunocompetent individuals, the antigen positivity rate in swabs decreased as days-after-symptom-onset increased, despite persistent nucleic acid positivity. Antigen was detected for longer and variable periods of time in immunocompromised patients with hematologic malignancies. Total microbubble volume, a quantitative marker of antigen burden, correlated inversely with Ct values and days-after-symptom-onset. Viral sequence variations were detected in patients with long duration of high antigen burden. CONCLUSIONS: The MSAA enables sensitive and specific detection of acute infections, quantification and tracking of antigen burden, and may serve as a screening method in longitudinal studies to identify patients who are likely experiencing active rounds of ongoing replication and warrant close viral sequence monitoring.

6.
Genome Biol ; 22(1): 169, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34082799

RESUMO

BACKGROUND: Rapid spread of SARS-CoV-2 has led to a global pandemic, resulting in the need for rapid assays to allow diagnosis and prevention of transmission. Reverse transcription-polymerase chain reaction (RT-PCR) provides a gold standard assay for SARS-CoV-2 RNA, but instrument costs are high and supply chains are potentially fragile, motivating interest in additional assay methods. Reverse transcription and loop-mediated isothermal amplification (RT-LAMP) provides an alternative that uses orthogonal and often less expensive reagents without the need for thermocyclers. The presence of SARS-CoV-2 RNA is typically detected using dyes to report bulk amplification of DNA; however, a common artifact is nonspecific DNA amplification, which complicates detection. RESULTS: Here we describe the design and testing of molecular beacons, which allow sequence-specific detection of SARS-CoV-2 genomes with improved discrimination in simple reaction mixtures. To optimize beacons for RT-LAMP, multiple locked nucleic acid monomers were incorporated to elevate melting temperatures. We also show how beacons with different fluorescent labels can allow convenient multiplex detection of several amplicons in "single pot" reactions, including incorporation of a human RNA LAMP-BEAC assay to confirm sample integrity. Comparison of LAMP-BEAC and RT-qPCR on clinical saliva samples showed good concordance between assays. To facilitate implementation, we developed custom polymerases for LAMP-BEAC and inexpensive purification procedures, which also facilitates increasing sensitivity by increasing reaction volumes. CONCLUSIONS: LAMP-BEAC thus provides an affordable and simple SARS-CoV-2 RNA assay suitable for population screening; implementation of the assay has allowed robust screening of thousands of saliva samples per week.


Assuntos
COVID-19/diagnóstico , RNA Viral/genética , SARS-CoV-2/isolamento & purificação , Teste para COVID-19 , Humanos , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , Sondas de Ácido Nucleico/genética , SARS-CoV-2/genética , Saliva/virologia , Sensibilidade e Especificidade
7.
Proc Natl Acad Sci U S A ; 118(22)2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-34031250

RESUMO

Infection with the flavivirus Zika virus (ZIKV) can result in tissue tropism, disease outcome, and route of transmission distinct from those of other flaviviruses; therefore, we aimed to identify host machinery that exclusively promotes the ZIKV replication cycle, which can inform on differences at the organismal level. We previously reported that deletion of the host antiviral ribonuclease L (RNase L) protein decreases ZIKV production. Canonical RNase L catalytic activity typically restricts viral infection, including that of the flavivirus dengue virus (DENV), suggesting an unconventional, proviral RNase L function during ZIKV infection. In this study, we reveal that an inactive form of RNase L supports assembly of ZIKV replication factories (RFs) to enhance infectious virus production. Compared with the densely concentrated ZIKV RFs generated with RNase L present, deletion of RNase L induced broader subcellular distribution of ZIKV replication intermediate double-stranded RNA (dsRNA) and NS3 protease, two constituents of ZIKV RFs. An inactive form of RNase L was sufficient to contain ZIKV genome and dsRNA within a smaller RF area, which subsequently increased infectious ZIKV release from the cell. Inactive RNase L can interact with cytoskeleton, and flaviviruses remodel cytoskeleton to construct RFs. Thus, we used the microtubule-stabilization drug paclitaxel to demonstrate that ZIKV repurposes RNase L to facilitate the cytoskeleton rearrangements required for proper generation of RFs. During infection with flaviviruses DENV or West Nile Kunjin virus, inactive RNase L did not improve virus production, suggesting that a proviral RNase L role is not a general feature of all flavivirus infections.

8.
J Virol ; 95(12)2021 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-33789998

RESUMO

The COVID-19 pandemic poses a serious global health threat. The rapid global spread of SARS-CoV-2 highlights an urgent need to develop effective therapeutics for blocking SARS-CoV-2 infection and spread. Stimulator of Interferon Genes (STING) is a chief element in host antiviral defense pathways. In this study, we examined the impact of the STING signaling pathway on coronavirus infection using the human coronavirus OC43 (HCoV-OC43) model. We found that HCoV-OC43 infection did not stimulate the STING signaling pathway, but the activation of STING signaling effectively inhibits HCoV-OC43 infection to a much greater extent than that of type I interferons (IFNs). We also discovered that IRF3, the key STING downstream innate immune effector, is essential for this anticoronavirus activity. In addition, we found that the amidobenzimidazole (ABZI)-based human STING agonist diABZI robustly blocks the infection of not only HCoV-OC43 but also SARS-CoV-2. Therefore, our study identifies the STING signaling pathway as a potential therapeutic target that could be exploited for developing broad-spectrum antiviral therapeutics against multiple coronavirus strains in order to face the challenge of future coronavirus outbreaks.IMPORTANCE The highly infectious and lethal SARS-CoV-2 is posing an unprecedented threat to public health. Other coronaviruses are likely to jump from a nonhuman animal to humans in the future. Novel broad-spectrum antiviral therapeutics are therefore needed to control known pathogenic coronaviruses such as SARS-CoV-2 and its newly mutated variants, as well as future coronavirus outbreaks. STING signaling is a well-established host defense pathway, but its role in coronavirus infection remains unclear. In the present study, we found that activation of the STING signaling pathway robustly inhibits infection of HCoV-OC43 and SARS-CoV-2. These results identified the STING pathway as a novel target for controlling the spread of known pathogenic coronaviruses, as well as emerging coronavirus outbreaks.


Assuntos
COVID-19/metabolismo , Coronavirus Humano OC43/metabolismo , Proteínas de Membrana/metabolismo , SARS-CoV-2/metabolismo , Transdução de Sinais , Células A549 , Animais , COVID-19/genética , Chlorocebus aethiops , Coronavirus Humano OC43/genética , Células HEK293 , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , SARS-CoV-2/genética , Células Vero
9.
Proc Natl Acad Sci U S A ; 118(16)2021 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-33811184

RESUMO

Coronaviruses are adept at evading host antiviral pathways induced by viral double-stranded RNA, including interferon (IFN) signaling, oligoadenylate synthetase-ribonuclease L (OAS-RNase L), and protein kinase R (PKR). While dysregulated or inadequate IFN responses have been associated with severe coronavirus infection, the extent to which the recently emerged SARS-CoV-2 activates or antagonizes these pathways is relatively unknown. We found that SARS-CoV-2 infects patient-derived nasal epithelial cells, present at the initial site of infection; induced pluripotent stem cell-derived alveolar type 2 cells (iAT2), the major cell type infected in the lung; and cardiomyocytes (iCM), consistent with cardiovascular consequences of COVID-19 disease. Robust activation of IFN or OAS-RNase L is not observed in these cell types, whereas PKR activation is evident in iAT2 and iCM. In SARS-CoV-2-infected Calu-3 and A549ACE2 lung-derived cell lines, IFN induction remains relatively weak; however, activation of OAS-RNase L and PKR is observed. This is in contrast to Middle East respiratory syndrome (MERS)-CoV, which effectively inhibits IFN signaling and OAS-RNase L and PKR pathways, but is similar to mutant MERS-CoV lacking innate immune antagonists. Remarkably, OAS-RNase L and PKR are activated in MAVS knockout A549ACE2 cells, demonstrating that SARS-CoV-2 can induce these host antiviral pathways despite minimal IFN production. Moreover, increased replication and cytopathic effect in RNASEL knockout A549ACE2 cells implicates OAS-RNase L in restricting SARS-CoV-2. Finally, while SARS-CoV-2 fails to antagonize these host defense pathways, which contrasts with other coronaviruses, the IFN signaling response is generally weak. These host-virus interactions may contribute to the unique pathogenesis of SARS-CoV-2.


Assuntos
Células Epiteliais/imunologia , Células Epiteliais/virologia , Imunidade Inata , Pulmão/patologia , Miócitos Cardíacos/imunologia , Miócitos Cardíacos/virologia , RNA de Cadeia Dupla/metabolismo , SARS-CoV-2/imunologia , Células A549 , Endorribonucleases/metabolismo , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/fisiologia , Nariz/virologia , Replicação Viral , eIF-2 Quinase
10.
PLoS One ; 16(4): e0250158, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33852639

RESUMO

While the world awaits a widely available COVID-19 vaccine, availability of testing is limited in many regions and can be further compounded by shortages of reagents, prolonged processing time and delayed results. One approach to rapid testing is to leverage the volatile organic compound (VOC) signature of SARS-CoV-2 infection. Detection dogs, a biological sensor of VOCs, were utilized to investigate whether SARS-CoV-2 positive urine and saliva patient samples had a unique odor signature. The virus was inactivated in all training samples with either detergent or heat treatment. Using detergent-inactivated urine samples, dogs were initially trained to find samples collected from hospitalized patients confirmed with SARS-CoV-2 infection, while ignoring samples collected from controls. Dogs were then tested on their ability to spontaneously recognize heat-treated urine samples as well as heat-treated saliva from hospitalized SARS-CoV-2 positive patients. Dogs successfully discriminated between infected and uninfected urine samples, regardless of the inactivation protocol, as well as heat-treated saliva samples. Generalization to novel samples was limited, particularly after intensive training with a restricted sample set. A unique odor associated with SARS-CoV-2 infection present in human urine as well as saliva, provides impetus for the development of odor-based screening, either by electronic, chemical, or biological sensing methods. The use of dogs for screening in an operational setting will require training with a large number of novel SARS-CoV-2 positive and confirmed negative samples.


Assuntos
Teste para COVID-19/métodos , COVID-19/diagnóstico , Cães Trabalhadores/psicologia , Animais , COVID-19/urina , Cães , Feminino , Humanos , Masculino , Programas de Rastreamento , Estudo de Prova de Conceito , SARS-CoV-2/isolamento & purificação , Saliva/química , Manejo de Espécimes/métodos , Compostos Orgânicos Voláteis/química
11.
J Clin Microbiol ; 59(5)2021 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-33579730

RESUMO

Community-based health care clinics and hospital outreach services have the potential to expand coronavirus disease 2019 (COVID-19) diagnostics to rural areas. However, reduced specimen stability during extended transport, the absence of a cold chain to centralized laboratories, and biosafety concerns surrounding specimen handling have limited this expansion. In the following study, we evaluated eNAT (Copan Italia, Brescia, Italy) as an alternative transport system to address the biosafety and stability challenges associated with expanding COVID-19 diagnostics to rural and remote regions. In this study, we demonstrated that high-titer severe acute respiratory virus syndrome coronavirus 2 (SARS-CoV-2) lysate placed into eNAT medium cannot be propagated in cell culture, supporting viral inactivation. To account for off-site testing in these settings, we assessed the stability of contrived nasopharyngeal (NP) specimens stored for up to 14 days in various transport media (eNAT, eSwab, viral transport medium [VTM], saline, and phosphate-buffered saline [PBS]) at 4°C, 22 to 25°C, and 35°C. The molecular detection of SARS-CoV-2 was unaffected by sample storage temperature over the 2 weeks when stored in eNAT or PBS (change in cycle threshold, ≤1). In contrast, variable stability was observed across test conditions for other transport media. As eNAT can inactivate SARS-CoV-2, it may support COVID-19 diagnostics at the point of care. Evaluation of compatibility of eNAT with Cepheid Xpert Xpress SARS-CoV-2 assay demonstrated diagnostic accuracy and sensitivity equivalent to those of VTM. Taken together, these findings suggest that the implementation of eNAT as a collection device can expand COVID-19 testing to areas with limited health care access.


Assuntos
Teste para COVID-19 , COVID-19/diagnóstico , Meios de Cultura , Manejo de Espécimes/normas , Humanos , Sensibilidade e Especificidade , Temperatura
12.
mBio ; 12(1)2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33468702

RESUMO

The severe acute respiratory coronavirus 2 (SARS-CoV-2) is the cause of the global outbreak of COVID-19. The epidemic accelerated in Philadelphia, PA, in the spring of 2020, with the city experiencing a first peak of infections on 15 April, followed by a decline through midsummer. Here, we investigate spread of the epidemic in the first wave in Philadelphia using full-genome sequencing of 52 SARS-CoV-2 samples obtained from 27 hospitalized patients collected between 30 March and 17 July 2020. Sequences most commonly resembled lineages circulating at earlier times in New York, suggesting transmission primarily from this location, though a minority of Philadelphia genomes matched sequences from other sites, suggesting additional introductions. Multiple genomes showed even closer matches to other Philadelphia isolates, suggestive of ongoing transmission within Philadelphia. We found that all of our isolates contained the D614G substitution in the viral spike and belong to lineages variously designated B.1, Nextstrain clade 20A or 20C, and GISAID clade G or GH. There were no viral sequence polymorphisms detectably associated with disease outcome. For some patients, genome sequences were determined longitudinally or concurrently from multiple body sites. In both cases, some comparisons showed reproducible polymorphisms, suggesting initial seeding with multiple variants and/or accumulation of polymorphisms after infection. These results thus provide data on the sources of SARS-CoV-2 infection in Philadelphia and begin to explore the dynamics within hospitalized patients.IMPORTANCE Understanding how SARS-CoV-2 spreads globally and within infected individuals is critical to the development of mitigation strategies. We found that most lineages in Philadelphia had resembled sequences from New York, suggesting infection primarily but not exclusively from this location. Many genomes had even nearer neighbors within Philadelphia, indicating local spread. Multiple genome sequences were available for some subjects and in a subset of cases could be shown to differ between time points and body sites within an individual, indicating heterogeneous viral populations within individuals and raising questions on the mechanisms responsible. There was no evidence that different lineages were associated with different outcomes in patients, emphasizing the importance of individual-specific vulnerability.


Assuntos
COVID-19/virologia , SARS-CoV-2/genética , Células A549 , Adulto , Idoso , Idoso de 80 Anos ou mais , Enzima de Conversão de Angiotensina 2/genética , COVID-19/epidemiologia , Feminino , Genoma Viral , Humanos , Masculino , Pessoa de Meia-Idade , New York/epidemiologia , Philadelphia/epidemiologia , Filogenia , Polimorfismo Genético , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/genética
13.
Nat Commun ; 12(1): 469, 2021 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-33473140

RESUMO

Antibody cocktails represent a promising approach to prevent SARS-CoV-2 escape. The determinants for selecting antibody combinations and the mechanism that antibody cocktails prevent viral escape remain unclear. We compared the critical residues in the receptor-binding domain (RBD) used by multiple neutralizing antibodies and cocktails and identified a combination of two antibodies CoV2-06 and CoV2-14 for preventing viral escape. The two antibodies simultaneously bind to non-overlapping epitopes and independently compete for receptor binding. SARS-CoV-2 rapidly escapes from individual antibodies by generating resistant mutations in vitro, but it doesn't escape from the cocktail due to stronger mutational constraints on RBD-ACE2 interaction and RBD protein folding requirements. We also identified a conserved neutralizing epitope shared between SARS-CoV-2 and SARS-CoV for antibody CoV2-12. Treatments with CoV2-06 and CoV2-14 individually and in combination confer protection in mice. These findings provide insights for rational selection and mechanistic understanding of antibody cocktails as candidates for treating COVID-19.


Assuntos
Anticorpos Monoclonais/farmacologia , COVID-19/tratamento farmacológico , SARS-CoV-2/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , COVID-19/virologia , Chlorocebus aethiops , Modelos Animais de Doenças , Feminino , Humanos , Fragmentos de Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/imunologia , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Mutação , Ligação Proteica , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Células Vero
14.
Advers Resil Sci ; : 1-5, 2020 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-33106790

RESUMO

The HEALthy Brain and Child Development (HBCD) study will establish a large cohort of pregnant women from regions of the country significantly affected by the opioid crisis and follow them and their children for at least 10 years. Findings from this cohort will help researchers understand normative childhood brain development as well as the long-term impact of prenatal and postnatal opioid and other drug and environmental exposures. The study will collect data on pregnancy and fetal development; infant and early childhood structural and functional brain imaging; anthropometrics; medical history; family history; biospecimens; and social, emotional, and cognitive development. Knowledge gained from this research will be critical to help predict and prevent some of the known effects of prenatal and postnatal exposure to certain drugs or environmental exposures, including risk for future substance use, mental disorders, and other behavioral and developmental problems. In this special issue, a subset of investigators that received funding for planning grants for the HBCD study provide careful guidelines and frameworks for study design, recruitment and retention of vulnerable populations, culturally sensitive practices, and biospecimen and neurodevelopmental assessment recommendations gathered in feasibility studies that will help inform the full HBCD study planned to begin recruitment in 2022.

15.
Proc Natl Acad Sci U S A ; 117(40): 24802-24812, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-32958664

RESUMO

The oligoadenylate synthetase (OAS)-RNase L system is an IFN-inducible antiviral pathway activated by viral infection. Viral double-stranded (ds) RNA activates OAS isoforms that synthesize the second messenger 2-5A, which binds and activates the pseudokinase-endoribonuclease RNase L. In cells, OAS activation is tamped down by ADAR1, an adenosine deaminase that destabilizes dsRNA. Mutation of ADAR1 is one cause of Aicardi-Goutières syndrome (AGS), an interferonopathy in children. ADAR1 deficiency in human cells can lead to RNase L activation and subsequent cell death. To evaluate RNase L as a possible therapeutic target for AGS, we sought to identify small-molecule inhibitors of RNase L. A 500-compound library of protein kinase inhibitors was screened for modulators of RNase L activity in vitro. We identified ellagic acid (EA) as a hit with 10-fold higher selectivity against RNase L compared with its nearest paralog, IRE1. SAR analysis identified valoneic acid dilactone (VAL) as a superior inhibitor of RNase L, with 100-fold selectivity over IRE1. Mechanism-of-action analysis indicated that EA and VAL do not bind to the pseudokinase domain of RNase L despite acting as ATP competitive inhibitors of the protein kinase CK2. VAL is nontoxic and functional in cells, although with a 1,000-fold decrease in potency, as measured by RNA cleavage activity in response to treatment with dsRNA activator or by rescue of cell lethality resulting from self dsRNA induced by ADAR1 deficiency. These studies lay the foundation for understanding novel modes of regulating RNase L function using small-molecule inhibitors and avenues of therapeutic potential.


Assuntos
Adenosina Desaminase/deficiência , Doenças Autoimunes do Sistema Nervoso/enzimologia , Endorribonucleases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Malformações do Sistema Nervoso/enzimologia , Fenol/farmacologia , 2',5'-Oligoadenilato Sintetase/genética , 2',5'-Oligoadenilato Sintetase/metabolismo , Nucleotídeos de Adenina/metabolismo , Adenosina Desaminase/genética , Doenças Autoimunes do Sistema Nervoso/genética , Doenças Autoimunes do Sistema Nervoso/fisiopatologia , Morte Celular/efeitos dos fármacos , Endorribonucleases/genética , Endorribonucleases/metabolismo , Inibidores Enzimáticos/química , Humanos , Malformações do Sistema Nervoso/genética , Malformações do Sistema Nervoso/fisiopatologia , Oligorribonucleotídeos/metabolismo , Fenol/química , Proteínas de Ligação a RNA/genética
16.
RNA ; 26(12): 1976-1999, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32989044

RESUMO

Coronavirus EndoU inhibits dsRNA-activated antiviral responses; however, the physiologic RNA substrates of EndoU are unknown. In this study, we used mouse hepatitis virus (MHV)-infected bone marrow-derived macrophage (BMM) and cyclic phosphate cDNA sequencing to identify the RNA targets of EndoU. EndoU targeted viral RNA, cleaving the 3' side of pyrimidines with a strong preference for U ↓ A and C ↓ A sequences (endoY ↓ A). EndoU-dependent cleavage was detected in every region of MHV RNA, from the 5' NTR to the 3' NTR, including transcriptional regulatory sequences (TRS). Cleavage at two CA dinucleotides immediately adjacent to the MHV poly(A) tail suggests a mechanism to suppress negative-strand RNA synthesis and the accumulation of viral dsRNA. MHV with EndoU (EndoUmut) or 2'-5' phosphodiesterase (PDEmut) mutations provoked the activation of RNase L in BMM, with corresponding cleavage of RNAs by RNase L. The physiologic targets of EndoU are viral RNA templates required for negative-strand RNA synthesis and dsRNA accumulation. Coronavirus EndoU cleaves U ↓ A and C ↓ A sequences (endoY ↓ A) within viral (+) strand RNA to evade dsRNA-activated host responses.


Assuntos
Vírus da Hepatite Murina/enzimologia , RNA/química , Endorribonucleases Específicas de Uridilato/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Células Cultivadas , Macrófagos/virologia , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Motivos de Nucleotídeos , Ligação Proteica , RNA/metabolismo , Endorribonucleases Específicas de Uridilato/genética , Proteínas não Estruturais Virais/genética
17.
bioRxiv ; 2020 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-32995797

RESUMO

Coronaviruses are adept at evading and/or antagonizing double-stranded RNA-induced host antiviral pathways, including interferon signaling, OAS-RNase L and PKR while robust cytokine responses characterize severe coronavirus disease. Knowledge of how newly emerged SARS-CoV-2 interacts with these pathways is minimal. SARS-CoV-2 readily infects patient-derived nasal epithelial cells and induced pluripotent stem cell-derived alveolar type 2 cells(iAT2) and cardiomyocytes(iCM). Robust activation of interferons or RNase L is not observed, while PKR activation is evident in iAT2 and iCM. In SARS-CoV-2 infected Calu-3 and A549 ACE2 lung derived cell lines, activation of all pathways is observed, similar to a mutant MERS-CoV lacking innate immune antagonists. Moreover, increased replication in RNASEL knockout A549 ACE2 cells, implicates RNase L in restricting SARS-CoV-2. Finally, while SARS-CoV-2 is less adept at antagonizing these host defense pathways compared to other coronaviruses, the innate immune response is still generally weak. These host-virus interactions may contribute to the unique pathogenesis of SARS-CoV-2.

18.
J Virol ; 94(21)2020 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-32817221

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in China at the end of 2019 and has rapidly caused a pandemic, with over 20 million recorded COVID-19 cases in August 2020 (https://covid19.who.int/). There are no FDA-approved antivirals or vaccines for any coronavirus, including SARS-CoV-2. Current treatments for COVID-19 are limited to supportive therapies and off-label use of FDA-approved drugs. Rapid development and human testing of potential antivirals is urgently needed. Numerous drugs are already approved for human use, and subsequently, there is a good understanding of their safety profiles and potential side effects, making them easier to fast-track to clinical studies in COVID-19 patients. Here, we present data on the antiviral activity of 20 FDA-approved drugs against SARS-CoV-2 that also inhibit SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). We found that 17 of these inhibit SARS-CoV-2 at non-cytotoxic concentrations. We directly followed up seven of these to demonstrate that all are capable of inhibiting infectious SARS-CoV-2 production. Moreover, we evaluated two of these, chloroquine and chlorpromazine, in vivo using a mouse-adapted SARS-CoV model and found that both drugs protect mice from clinical disease.IMPORTANCE There are no FDA-approved antivirals for any coronavirus, including SARS-CoV-2. Numerous drugs are already approved for human use that may have antiviral activity and therefore could potentially be rapidly repurposed as antivirals. Here, we present data assessing the antiviral activity of 20 FDA-approved drugs against SARS-CoV-2 that also inhibit SARS-CoV and MERS-CoV in vitro We found that 17 of these inhibit SARS-CoV-2, suggesting that they may have pan-anti-coronaviral activity. We directly followed up seven of these and found that they all inhibit infectious-SARS-CoV-2 production. Moreover, we evaluated chloroquine and chlorpromazine in vivo using mouse-adapted SARS-CoV. We found that neither drug inhibited viral replication in the lungs, but both protected against clinical disease.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Células A549 , Animais , COVID-19 , Cloroquina/farmacologia , Clorpromazina/farmacologia , Aprovação de Drogas , Avaliação Pré-Clínica de Medicamentos , Humanos , Pandemias , SARS-CoV-2 , Resultado do Tratamento , Estados Unidos , United States Food and Drug Administration , Replicação Viral/efeitos dos fármacos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...