RESUMEN
The unprecedented scale of the COVID-19 pandemic and the rapid evolution of SARS-CoV-2 variants underscore the need for broadly active inhibitors with a high barrier to resistance. The coronavirus main protease (Mpro) is an essential cysteine protease required for viral polyprotein processing and is highly conserved across human coronaviruses. Pomotrelvir is a novel Mpro inhibitor that has recently completed a phase 2 clinical trial. In this report, we demonstrated that pomotrelvir is a potent competitive inhibitor of SARS-CoV-2 Mpro with high selectivity against human proteases. In the enzyme assay, pomotrelvir is also active against Mpro proteins derived from human coronaviruses CoV-229E, CoV-OC43, CoV-HKU1, CoV-NL63, MERS, and SARS-CoV. In cell-based SARS-CoV-2 replicon and SARS-CoV-2 infection assays, pomotrelvir has shown potent inhibitory activity and is broadly active against SARS-CoV-2 clinical isolates including Omicron variants. Many resistance substitutions of the Mpro inhibitor nirmatrelvir confer cross-resistance to pomotrelvir, consistent with the finding from our enzymatic analysis that pomotrelvir and nirmatrelvir compete for the same binding site. In a SARS-CoV-2 infection assay, pomotrelvir is additive when combined with remdesivir or molnupiravir, two nucleoside analogs targeting viral RNA synthesis. In conclusion, our results from the in vitro characterization of pomotrelvir antiviral activity support its further clinical development as an alternative COVID-19 therapeutic option.
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COVID-19 , Coronavirus Humano 229E , Humanos , SARS-CoV-2 , Pandemias , Antivirales/farmacología , Inhibidores de ProteasasRESUMEN
Coronaviruses (CoVs) encode nonstructural proteins 1-16 (nsps 1-16) which form replicase complexes that mediate viral RNA synthesis. Remdesivir (RDV) is an adenosine nucleoside analog antiviral that inhibits CoV RNA synthesis. RDV resistance mutations have been reported only in the nonstructural protein 12 RNA-dependent RNA polymerase (nsp12-RdRp). We here show that a substitution mutation in the nsp13-helicase (nsp13-HEL A335V) of the betacoronavirus murine hepatitis virus (MHV) that was selected during passage with the RDV parent compound confers partial RDV resistance independently and additively when expressed with co-selected RDV resistance mutations in the nsp12-RdRp. The MHV A335V substitution did not enhance replication or competitive fitness compared to WT MHV and remained sensitive to the active form of the cytidine nucleoside analog antiviral molnupiravir (MOV). Biochemical analysis of the SARS-CoV-2 helicase encoding the homologous substitution (A336V) demonstrates that the mutant protein retained the ability to associate with the core replication proteins nsps 7, 8, and 12 but had impaired helicase unwinding and ATPase activity. Together, these data identify a novel determinant of nsp13-HEL enzymatic activity, define a new genetic pathway for RDV resistance, and demonstrate the importance of surveillance for and testing of helicase mutations that arise in SARS-CoV-2 genomes. IMPORTANCE Despite the development of effective vaccines against COVID-19, the continued circulation and emergence of new variants support the need for antivirals such as RDV. Understanding pathways of antiviral resistance is essential for surveillance of emerging variants, development of combination therapies, and for identifying potential new targets for viral inhibition. We here show a novel RDV resistance mutation in the CoV helicase also impairs helicase functions, supporting the importance of studying the individual and cooperative functions of the replicase nonstructural proteins 7-16 during CoV RNA synthesis. The homologous nsp13-HEL mutation (A336V) has been reported in the GISAID database of SARS-CoV-2 genomes, highlighting the importance of surveillance of and genetic testing for nucleoside analog resistance in the helicase.
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COVID-19 , Virus de la Hepatitis Murina , Animales , Ratones , Humanos , Nucleósidos/farmacología , Vacunas contra la COVID-19 , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Replicación Viral/genética , Tratamiento Farmacológico de COVID-19 , Mutación , Virus de la Hepatitis Murina/genética , Antivirales/farmacología , Antivirales/química , ARN Polimerasa Dependiente del ARN/metabolismo , ARN , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismoRESUMEN
Pathological effects of apoptosis associated with viral infections of the central nervous system are an important cause of morbidity and mortality. Reovirus is a neurotropic virus that causes apoptosis in neurons, leading to lethal encephalitis in newborn mice. Reovirus-induced encephalitis is diminished in mice with germ line ablation of NF-κB subunit p50. It is not known whether the proapoptotic function of NF-κB is mediated by neural-cell-intrinsic (neural-intrinsic) processes, NF-κB-regulated cytokine production by inflammatory cells, or a combination of both. To determine the contribution of cell type-specific NF-κB signaling in reovirus-induced neuronal injury, we established mice that lack NF-κB p65 expression in neural cells using the Cre/loxP recombination system. Following intracranial inoculation of reovirus, 50% of wild-type (WT) mice succumbed to infection, whereas more than 90% of mice lacking neural cell NF-κB p65 (Nsp65-/-) survived. While viral loads in brains of WT and Nsp65-/- mice were comparable, histological analysis revealed that reovirus antigen-positive areas in the brains of WT mice displayed increased immunoreactivity for cleaved caspase-3, a marker of apoptosis, relative to Nsp65-/- mice. These data suggest that neural-intrinsic NF-κB-dependent factors are essential mediators of reovirus neurovirulence. RNA sequencing analysis of reovirus-infected brain cortices of WT and Nsp65-/- mice suggests that NF-κB activation in neuronal cells upregulates genes involved in innate immunity, inflammation, and cell death following reovirus infection. A better understanding of the contribution of cell type-specific NF-κB-dependent signaling to viral neuropathogenesis could inform development of new therapeutics that target and protect highly vulnerable cell populations. IMPORTANCE Viral encephalitis contributes to illness and death in children and adults worldwide and has limited treatment options. Identifying common host factors upregulated by neurotropic viruses can enhance an understanding of virus-induced neuropathogenesis and aid in development of therapeutics. Although many neurotropic viruses activate NF-κB during infection, mechanisms by which NF-κB regulates viral neuropathogenesis and contributes to viral encephalitis are not well understood. We established mice in which NF-κB expression is ablated in neural tissue to study the function of NF-κB in reovirus neurovirulence and identify genes activated by NF-κB in response to reovirus infection in the central nervous system. Encephalitis following reovirus infection was dampened in mice lacking neural cell NF-κB. Reovirus induced a chemokine profile in the brain that was dependent on NF-κB signaling and was similar to chemokine profiles elicited by other neurotropic viruses. These data suggest common underlying mechanisms of encephalitis caused by neurotropic viruses and potentially shared therapeutic targets.
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Encefalitis Viral , Neuronas , Infecciones por Reoviridae , Reoviridae , Animales , Ratones , Apoptosis/genética , Apoptosis/inmunología , Quimiocinas/inmunología , Encefalitis Viral/inmunología , Encefalitis Viral/virología , Neuronas/inmunología , FN-kappa B/genética , FN-kappa B/metabolismo , Reoviridae/inmunología , Reoviridae/patogenicidad , Infecciones por Reoviridae/inmunología , Infecciones por Reoviridae/virología , Interacciones Microbiota-Huesped/genética , Interacciones Microbiota-Huesped/inmunologíaRESUMEN
The nucleoside analog remdesivir (RDV) is a Food and Drug Administration-approved antiviral for treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Thus, it is critical to understand factors that promote or prevent RDV resistance. We passaged SARS-CoV-2 in the presence of increasing concentrations of GS-441524, the parent nucleoside of RDV. After 13 passages, we isolated three viral lineages with phenotypic resistance as defined by increases in half-maximal effective concentration from 2.7- to 10.4-fold. Sequence analysis identified nonsynonymous mutations in nonstructural protein 12 RNA-dependent RNA polymerase (nsp12-RdRp): V166A, N198S, S759A, V792I, and C799F/R. Two lineages encoded the S759A substitution at the RdRp Ser759-Asp-Asp active motif. In one lineage, the V792I substitution emerged first and then combined with S759A. Introduction of S759A and V792I substitutions at homologous nsp12 positions in murine hepatitis virus demonstrated transferability across betacoronaviruses; introduction of these substitutions resulted in up to 38-fold RDV resistance and a replication defect. Biochemical analysis of SARS-CoV-2 RdRp encoding S759A demonstrated a roughly 10-fold decreased preference for RDV-triphosphate (RDV-TP) as a substrate, whereas nsp12-V792I diminished the uridine triphosphate concentration needed to overcome template-dependent inhibition associated with RDV. The in vitro-selected substitutions identified in this study were rare or not detected in the greater than 6 million publicly available nsp12-RdRp consensus sequences in the absence of RDV selection. The results define genetic and biochemical pathways to RDV resistance and emphasize the need for additional studies to define the potential for emergence of these or other RDV resistance mutations in clinical settings.
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Antivirales , Tratamiento Farmacológico de COVID-19 , Farmacorresistencia Viral , ARN Polimerasa Dependiente del ARN , SARS-CoV-2 , Adenosina Monofosfato/análogos & derivados , Alanina/análogos & derivados , Animales , Antivirales/farmacología , Farmacorresistencia Viral/genética , Humanos , Ratones , Mutación/genética , ARN Viral/genética , ARN Polimerasa Dependiente del ARN/genética , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/genéticaRESUMEN
Recombination is proposed to be critical for coronavirus (CoV) diversity and emergence of SARS-CoV-2 and other zoonotic CoVs. While RNA recombination is required during normal CoV replication, the mechanisms and determinants of CoV recombination are not known. CoVs encode an RNA proofreading exoribonuclease (nsp14-ExoN) that is distinct from the CoV polymerase and is responsible for high-fidelity RNA synthesis, resistance to nucleoside analogues, immune evasion, and virulence. Here, we demonstrate that CoVs, including SARS-CoV-2, MERS-CoV, and the model CoV murine hepatitis virus (MHV), generate extensive and diverse recombination products during replication in culture. We show that the MHV nsp14-ExoN is required for native recombination, and that inactivation of ExoN results in decreased recombination frequency and altered recombination products. These results add yet another critical function to nsp14-ExoN, highlight the uniqueness of the evolved coronavirus replicase, and further emphasize nsp14-ExoN as a central, completely conserved, and vulnerable target for inhibitors and attenuation of SARS-CoV-2 and future emerging zoonotic CoVs.
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Tratamiento Farmacológico de COVID-19 , Infecciones por Coronavirus/tratamiento farmacológico , Exorribonucleasas/farmacología , SARS-CoV-2/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Antivirales/farmacología , COVID-19/virología , Infecciones por Coronavirus/virología , Exorribonucleasas/genética , Humanos , Recombinación Genética/efectos de los fármacos , SARS-CoV-2/patogenicidad , Proteínas no Estructurales Virales/genética , Replicación Viral/genéticaAsunto(s)
Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Vacunas contra la COVID-19/inmunología , COVID-19/inmunología , SARS-CoV-2/inmunología , Vacuna nCoV-2019 mRNA-1273 , Adolescente , Adulto , Anciano , COVID-19/prevención & control , Vacunas contra la COVID-19/administración & dosificación , Humanos , Inmunización Secundaria , Persona de Mediana Edad , Glicoproteína de la Espiga del Coronavirus/inmunología , Células TH1/fisiología , Adulto JovenRESUMEN
BACKGROUND: Testing of vaccine candidates to prevent infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in an older population is important, since increased incidences of illness and death from coronavirus disease 2019 (Covid-19) have been associated with an older age. METHODS: We conducted a phase 1, dose-escalation, open-label trial of a messenger RNA vaccine, mRNA-1273, which encodes the stabilized prefusion SARS-CoV-2 spike protein (S-2P) in healthy adults. The trial was expanded to include 40 older adults, who were stratified according to age (56 to 70 years or ≥71 years). All the participants were assigned sequentially to receive two doses of either 25 µg or 100 µg of vaccine administered 28 days apart. RESULTS: Solicited adverse events were predominantly mild or moderate in severity and most frequently included fatigue, chills, headache, myalgia, and pain at the injection site. Such adverse events were dose-dependent and were more common after the second immunization. Binding-antibody responses increased rapidly after the first immunization. By day 57, among the participants who received the 25-µg dose, the anti-S-2P geometric mean titer (GMT) was 323,945 among those between the ages of 56 and 70 years and 1,128,391 among those who were 71 years of age or older; among the participants who received the 100-µg dose, the GMT in the two age subgroups was 1,183,066 and 3,638,522, respectively. After the second immunization, serum neutralizing activity was detected in all the participants by multiple methods. Binding- and neutralizing-antibody responses appeared to be similar to those previously reported among vaccine recipients between the ages of 18 and 55 years and were above the median of a panel of controls who had donated convalescent serum. The vaccine elicited a strong CD4 cytokine response involving type 1 helper T cells. CONCLUSIONS: In this small study involving older adults, adverse events associated with the mRNA-1273 vaccine were mainly mild or moderate. The 100-µg dose induced higher binding- and neutralizing-antibody titers than the 25-µg dose, which supports the use of the 100-µg dose in a phase 3 vaccine trial. (Funded by the National Institute of Allergy and Infectious Diseases and others; mRNA-1273 Study ClinicalTrials.gov number, NCT04283461.).
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Vacunas contra la COVID-19/efectos adversos , Vacunas contra la COVID-19/inmunología , COVID-19/prevención & control , SARS-CoV-2/inmunología , Vacuna nCoV-2019 mRNA-1273 , Anciano , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , COVID-19/inmunología , Vacunas contra la COVID-19/administración & dosificación , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pruebas de Neutralización , Glicoproteína de la Espiga del Coronavirus , Linfocitos T/fisiologíaRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the novel viral disease COVID-19. With no approved therapies, this pandemic illustrates the urgent need for broad-spectrum antiviral countermeasures against SARS-CoV-2 and future emerging CoVs. We report that remdesivir (RDV) potently inhibits SARS-CoV-2 replication in human lung cells and primary human airway epithelial cultures (EC50 = 0.01 µM). Weaker activity is observed in Vero E6 cells (EC50 = 1.65 µM) because of their low capacity to metabolize RDV. To rapidly evaluate in vivo efficacy, we engineered a chimeric SARS-CoV encoding the viral target of RDV, the RNA-dependent RNA polymerase of SARS-CoV-2. In mice infected with the chimeric virus, therapeutic RDV administration diminishes lung viral load and improves pulmonary function compared with vehicle-treated animals. These data demonstrate that RDV is potently active against SARS-CoV-2 in vitro and in vivo, supporting its further clinical testing for treatment of COVID-19.
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BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in late 2019 and spread globally, prompting an international effort to accelerate development of a vaccine. The candidate vaccine mRNA-1273 encodes the stabilized prefusion SARS-CoV-2 spike protein. METHODS: We conducted a phase 1, dose-escalation, open-label trial including 45 healthy adults, 18 to 55 years of age, who received two vaccinations, 28 days apart, with mRNA-1273 in a dose of 25 µg, 100 µg, or 250 µg. There were 15 participants in each dose group. RESULTS: After the first vaccination, antibody responses were higher with higher dose (day 29 enzyme-linked immunosorbent assay anti-S-2P antibody geometric mean titer [GMT], 40,227 in the 25-µg group, 109,209 in the 100-µg group, and 213,526 in the 250-µg group). After the second vaccination, the titers increased (day 57 GMT, 299,751, 782,719, and 1,192,154, respectively). After the second vaccination, serum-neutralizing activity was detected by two methods in all participants evaluated, with values generally similar to those in the upper half of the distribution of a panel of control convalescent serum specimens. Solicited adverse events that occurred in more than half the participants included fatigue, chills, headache, myalgia, and pain at the injection site. Systemic adverse events were more common after the second vaccination, particularly with the highest dose, and three participants (21%) in the 250-µg dose group reported one or more severe adverse events. CONCLUSIONS: The mRNA-1273 vaccine induced anti-SARS-CoV-2 immune responses in all participants, and no trial-limiting safety concerns were identified. These findings support further development of this vaccine. (Funded by the National Institute of Allergy and Infectious Diseases and others; mRNA-1273 ClinicalTrials.gov number, NCT04283461).
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Infecciones por Coronavirus/prevención & control , Pandemias/prevención & control , Neumonía Viral/prevención & control , ARN Mensajero/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Vacunas Virales/uso terapéutico , Vacuna nCoV-2019 mRNA-1273 , Adulto , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Formación de Anticuerpos , Betacoronavirus , COVID-19 , Vacunas contra la COVID-19 , Femenino , Humanos , Inmunización Secundaria , Masculino , SARS-CoV-2 , Linfocitos T/inmunología , Vacunas Virales/efectos adversos , Adulto JovenRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in 2019 as the causative agent of the novel pandemic viral disease COVID-19. With no approved therapies, this pandemic illustrates the urgent need for safe, broad-spectrum antiviral countermeasures against SARS-CoV-2 and future emerging CoVs. We report that remdesivir (RDV), a monophosphoramidate prodrug of an adenosine analog, potently inhibits SARS-CoV-2 replication in human lung cells and primary human airway epithelial cultures (EC 50 = 0.01 µM). Weaker activity was observed in Vero E6 cells (EC 50 = 1.65 µM) due to their low capacity to metabolize RDV. To rapidly evaluate in vivo efficacy, we engineered a chimeric SARS-CoV encoding the viral target of RDV, the RNA-dependent RNA polymerase, of SARS-CoV-2. In mice infected with chimeric virus, therapeutic RDV administration diminished lung viral load and improved pulmonary function as compared to vehicle treated animals. These data provide evidence that RDV is potently active against SARS-CoV-2 in vitro and in vivo , supporting its further clinical testing for treatment of COVID-19.
RESUMEN
Coronaviruses (CoVs) traffic frequently between species resulting in novel disease outbreaks, most recently exemplified by the newly emerged SARS-CoV-2, the causative agent of COVID-19. Here, we show that the ribonucleoside analog ß-d-N4-hydroxycytidine (NHC; EIDD-1931) has broad-spectrum antiviral activity against SARS-CoV-2, MERS-CoV, SARS-CoV, and related zoonotic group 2b or 2c bat-CoVs, as well as increased potency against a CoV bearing resistance mutations to the nucleoside analog inhibitor remdesivir. In mice infected with SARS-CoV or MERS-CoV, both prophylactic and therapeutic administration of EIDD-2801, an orally bioavailable NHC prodrug (ß-d-N4-hydroxycytidine-5'-isopropyl ester), improved pulmonary function and reduced virus titer and body weight loss. Decreased MERS-CoV yields in vitro and in vivo were associated with increased transition mutation frequency in viral, but not host cell RNA, supporting a mechanism of lethal mutagenesis in CoV. The potency of NHC/EIDD-2801 against multiple CoVs and oral bioavailability highlights its potential utility as an effective antiviral against SARS-CoV-2 and other future zoonotic CoVs.
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Antivirales/administración & dosificación , Infecciones por Coronavirus/tratamiento farmacológico , Neumonía Viral/tratamiento farmacológico , Ribonucleósidos/administración & dosificación , Replicación Viral/efectos de los fármacos , Adenosina Monofosfato/administración & dosificación , Adenosina Monofosfato/análogos & derivados , Alanina/administración & dosificación , Alanina/análogos & derivados , Animales , Profilaxis Antibiótica , Betacoronavirus/fisiología , COVID-19 , Línea Celular , Infecciones por Coronavirus/patología , Citidina/administración & dosificación , Citidina/análogos & derivados , Modelos Animales de Enfermedad , Farmacorresistencia Viral , Humanos , Hidroxilaminas , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Coronavirus del Síndrome Respiratorio de Oriente Medio/fisiología , Modelos Moleculares , Mutación/efectos de los fármacos , Pandemias , Neumonía Viral/patología , Cultivo Primario de Células , ARN Viral , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/genética , Distribución Aleatoria , Sistema Respiratorio/citología , SARS-CoV-2RESUMEN
Rotavirus (RV) encounters intestinal epithelial cells amidst diverse microbiota, opening possibilities of microbes influencing RV infection. Although RV clearance typically requires adaptive immunity, we unintentionally generated RV-resistant immunodeficient mice, which, we hypothesized, reflected select microbes protecting against RV. Accordingly, such RV resistance was transferred by co-housing and fecal transplant. RV-protecting microbiota were interrogated by heat, filtration, and antimicrobial agents, followed by limiting dilution transplant to germ-free mice and microbiome analysis. This approach revealed that segmented filamentous bacteria (SFB) were sufficient to protect mice against RV infection and associated diarrhea. Such protection was independent of previously defined RV-impeding factors, including interferon, IL-17, and IL-22. Colonization of the ileum by SFB induced changes in host gene expression and accelerated epithelial cell turnover. Incubation of RV with SFB-containing feces reduced infectivity in vitro, suggesting direct neutralization of RV. Thus, independent of immune cells, SFB confer protection against certain enteric viral infections and associated diarrheal disease.
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Inmunidad Adaptativa/genética , Diarrea/microbiología , Mucosa Intestinal/microbiología , Infecciones por Rotavirus/microbiología , Animales , Antiinfecciosos/farmacología , Bacterias/genética , Bacterias/metabolismo , Diarrea/prevención & control , Diarrea/virología , Heces/microbiología , Regulación de la Expresión Génica/genética , Humanos , Íleon/microbiología , Íleon/patología , Íleon/virología , Interferones/genética , Interleucina-17/genética , Interleucinas/genética , Mucosa Intestinal/patología , Mucosa Intestinal/virología , Ratones , Microbiota/genética , Rotavirus/patogenicidad , Infecciones por Rotavirus/prevención & control , Infecciones por Rotavirus/virología , Interleucina-22RESUMEN
Coronaviruses (CoVs) have emerged from animal reservoirs to cause severe and lethal disease in humans, but there are currently no FDA-approved antivirals to treat the infections. One class of antiviral compounds, nucleoside analogues, mimics naturally occurring nucleosides to inhibit viral replication. While these compounds have been successful therapeutics for several viral infections, mutagenic nucleoside analogues, such as ribavirin and 5-fluorouracil, have been ineffective at inhibiting CoVs. This has been attributed to the proofreading activity of the viral 3'-5' exoribonuclease (ExoN). ß-d-N4-Hydroxycytidine (NHC) (EIDD-1931; Emory Institute for Drug Development) has recently been reported to inhibit multiple viruses. Here, we demonstrate that NHC inhibits both murine hepatitis virus (MHV) (50% effective concentration [EC50] = 0.17 µM) and Middle East respiratory syndrome CoV (MERS-CoV) (EC50 = 0.56 µM) with minimal cytotoxicity. NHC inhibited MHV lacking ExoN proofreading activity similarly to wild-type (WT) MHV, suggesting an ability to evade or overcome ExoN activity. NHC inhibited MHV only when added early during infection, decreased viral specific infectivity, and increased the number and proportion of G:A and C:U transition mutations present after a single infection. Low-level NHC resistance was difficult to achieve and was associated with multiple transition mutations across the genome in both MHV and MERS-CoV. These results point to a virus-mutagenic mechanism of NHC inhibition in CoVs and indicate a high genetic barrier to NHC resistance. Together, the data support further development of NHC for treatment of CoVs and suggest a novel mechanism of NHC interaction with the CoV replication complex that may shed light on critical aspects of replication.IMPORTANCE The emergence of coronaviruses (CoVs) into human populations from animal reservoirs has demonstrated their epidemic capability, pandemic potential, and ability to cause severe disease. However, no antivirals have been approved to treat these infections. Here, we demonstrate the potent antiviral activity of a broad-spectrum ribonucleoside analogue, ß-d-N4-hydroxycytidine (NHC), against two divergent CoVs. Viral proofreading activity does not markedly impact sensitivity to NHC inhibition, suggesting a novel interaction between a nucleoside analogue inhibitor and the CoV replicase. Further, passage in the presence of NHC generates only low-level resistance, likely due to the accumulation of multiple potentially deleterious transition mutations. Together, these data support a mutagenic mechanism of inhibition by NHC and further support the development of NHC for treatment of CoV infections.
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Antivirales/farmacología , Citidina/análogos & derivados , Coronavirus del Síndrome Respiratorio de Oriente Medio/efectos de los fármacos , Coronavirus del Síndrome Respiratorio de Oriente Medio/genética , Virus de la Hepatitis Murina/efectos de los fármacos , Virus de la Hepatitis Murina/genética , Animales , Línea Celular , Línea Celular Tumoral , Chlorocebus aethiops , Infecciones por Coronaviridae/tratamiento farmacológico , Infecciones por Coronaviridae/virología , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/virología , Citidina/farmacología , Farmacorresistencia Viral , Exorribonucleasas/metabolismo , Ratones , Coronavirus del Síndrome Respiratorio de Oriente Medio/metabolismo , Virus de la Hepatitis Murina/metabolismo , Mutagénesis , ARN Polimerasa Dependiente del ARN/metabolismo , Células Vero , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Recent outbreaks of SARS-Coronavirus and MERS-Coronavirus (CoV) have heightened awareness about the lack of vaccines or antiviral compounds approved for prevention or treatment of human or potential zoonotic CoVs. Anti-CoV drug development has long been challenged by the activity of a 3' to 5' proofreading exoribonuclease unique to CoVs. Recently, a promising nucleoside analogue with broad-spectrum activity against CoVs has been identified. This review will discuss progress made in the development of antiviral nucleoside and nucleotide analogues targeting viral RNA synthesis as effective therapeutics against CoV infections and propose promising strategies for combination therapy.
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Antivirales/farmacología , Infecciones por Coronavirus/tratamiento farmacológico , Coronavirus/efectos de los fármacos , Nucleósidos/análogos & derivados , Nucleósidos/farmacología , Animales , Infecciones por Coronavirus/virología , Quimioterapia Combinada , Exorribonucleasas/metabolismo , Humanos , Coronavirus del Síndrome Respiratorio de Oriente Medio/efectos de los fármacos , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
Various sensors that detect double-stranded RNA, presumably of viral origin, exist in eukaryotic cells and induce IFN-responses. Ongoing IFN-responses have also been documented in a variety of human autoimmune diseases including relapsing-remitting multiple sclerosis (RRMS) but their origins remain obscure. We find increased IFN-responses in leukocytes in relapsing-remitting multiple sclerosis at distinct stages of disease. Moreover, endogenous RNAs isolated from blood cells of these same patients recapitulate this IFN-response if transfected into naïve cells. These endogenous RNAs are double-stranded RNAs, contain Alu and Line elements and are transcribed from leukocyte transcriptional enhancers. Thus, transcribed endogenous retrotransposon elements can co-opt pattern recognition sensors to induce IFN-responses in RRMS.
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Elementos Alu/inmunología , Interferones/inmunología , Elementos de Nucleótido Esparcido Largo/inmunología , Esclerosis Múltiple/inmunología , ARN Bicatenario/inmunología , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/patologíaAsunto(s)
Reposicionamiento de Medicamentos , Antagonistas de los Receptores de Hormonas Antidiuréticas/uso terapéutico , Trastornos de Ansiedad/tratamiento farmacológico , Trastorno Depresivo Mayor/tratamiento farmacológico , Industria Farmacéutica , Reposicionamiento de Medicamentos/métodos , Humanos , Indoles/uso terapéutico , Pirrolidinas/uso terapéutico , Receptores de Vasopresinas/uso terapéuticoRESUMEN
Several viruses induce intestinal epithelial cell death during enteric infection. However, it is unclear whether proapoptotic capacity promotes or inhibits replication in this tissue. We infected mice with two reovirus strains that infect the intestine but differ in the capacity to alter immunological tolerance to new food antigen. Infection with reovirus strain T1L, which induces an inflammatory immune response to fed antigen, is prolonged in the intestine, whereas T3D-RV, which does not induce this response, is rapidly cleared from the intestine. Compared with T1L, T3D-RV infection triggered apoptosis of intestinal epithelial cells and subsequent sloughing of dead cells into the intestinal lumen. We conclude that the infection advantage of T1L derives from its capacity to subvert host restriction by epithelial cell apoptosis, providing a possible mechanism by which T1L enhances inflammatory signals during antigen feeding. Using a panel of T1L × T3D-RV reassortant viruses, we identified the viral M1 and M2 gene segments as determinants of reovirus-induced apoptosis in the intestine. Expression of the T1L M1 and M2 genes in a T3D-RV background was sufficient to limit epithelial cell apoptosis and enhance viral infection to levels displayed by T1L. These findings define additional reovirus gene segments required for enteric infection of mice and illuminate the antiviral effect of intestinal epithelial cell apoptosis in limiting enteric viral infection. Viral strain-specific differences in the capacity to infect the intestine may be useful in identifying viruses capable of ameliorating tolerance to fed antigen in autoimmune conditions like celiac disease.IMPORTANCE Acute viral infections are thought to be cleared by the host with few lasting consequences. However, there may be much broader and long-lasting effects of viruses on immune homeostasis. Infection with reovirus, a common, nonpathogenic virus, triggers inflammation against innocuous food antigens, implicating this virus in the development of celiac disease, an autoimmune intestinal disorder triggered by exposure to dietary gluten. Using two reovirus strains that differ in the capacity to abrogate oral tolerance, we found that strain-specific differences in the capacity to replicate in the intestine inversely correlate with the capacity to induce apoptotic death of intestinal epithelial cells, providing a host-mediated process to restrict intestinal infection. This work contributes new knowledge about virus-host interactions in the intestine and establishes a foundation for future studies to define mechanisms by which viruses break oral tolerance in celiac disease.
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Apoptosis/inmunología , Células Epiteliales/inmunología , Mucosa Intestinal/inmunología , Orthoreovirus Mamífero 3/inmunología , Orthoreovirus de los Mamíferos/inmunología , Infecciones por Reoviridae/inmunología , Animales , Antígenos Virales/inmunología , Línea Celular , Cricetinae , Células Epiteliales/patología , Células Epiteliales/virología , Mucosa Intestinal/patología , Mucosa Intestinal/virología , Ratones , Infecciones por Reoviridae/patologíaRESUMEN
BackgroundInfants and young children are particularly susceptible to viral encephalitis; however, the mechanisms are unknown. We determined the age-dependent contribution of innate and adaptive immune functions to reovirus-induced encephalitis in mice.MethodsNewborn wild-type mice, 2-20 days of age, were inoculated with reovirus or diluent and monitored for mortality, weight gain, and viral load. Four- and fifteen-day-old IFNAR-/- and RAG2-/- mice were inoculated with reovirus and similarly monitored.ResultsWeight gain was impaired in mice inoculated with reovirus at 8 days of age or less. Clinical signs of encephalitis were detected in mice inoculated at 10 days of age or less. Mortality decreased when mice were inoculated after 6 days of age. Survival was ≤15% in wild type (WT), RAG2-/-, and IFNAR-/- mice inoculated at 4 days of age. All WT mice, 92% of RAG2-/- mice, and only 48% of IFNAR-/- mice survived following inoculation at 15 days of age.ConclusionsSusceptibility of mice to reovirus-induced disease decreases between 6 and 8 days of age. Enhanced reovirus virulence in IFNAR-/- mice relative to WT and RAG2-/- mice inoculated at 15 days of age suggests that maturation of the type-I interferon response contributes to age-related mortality following reovirus infection.
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Factores de Edad , Proteínas de Unión al ADN/genética , Encefalitis Viral/inmunología , Receptor de Interferón alfa y beta/genética , Infecciones por Reoviridae/inmunología , Inmunidad Adaptativa , Animales , Apoptosis , Encéfalo/metabolismo , Línea Celular , Proteínas de Unión al ADN/inmunología , Regulación Viral de la Expresión Génica , Inmunidad Innata , Interferón Tipo I/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Orthoreovirus de los Mamíferos/genética , Orthoreovirus de los Mamíferos/fisiología , Receptor de Interferón alfa y beta/inmunología , Bazo/metabolismo , Carga Viral , Replicación ViralRESUMEN
Many animal models of disease are suboptimal in their representation of human diseases and lack of predictive power in the success of pivotal human trials. In the context of repurposing drugs with known human safety, it is sometimes appropriate to conduct the "last experiment first," that is, progressing directly to human investigations. However, there are not accepted criteria for when to proceed straight to humans to test a new indication. We propose a specific set of criteria to guide the decision-making around when to initiate human proof of principle without preclinical efficacy studies in animal models. This approach could accelerate the transition of novel therapeutic approaches to human applications.
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Ensayos Clínicos como Asunto/métodos , Toma de Decisiones , Evaluación Preclínica de Medicamentos , Reposicionamiento de Medicamentos/métodos , Animales , Humanos , Modelos AnimalesRESUMEN
Viral infections have been proposed to elicit pathological processes leading to the initiation of T helper 1 (TH1) immunity against dietary gluten and celiac disease (CeD). To test this hypothesis and gain insights into mechanisms underlying virus-induced loss of tolerance to dietary antigens, we developed a viral infection model that makes use of two reovirus strains that infect the intestine but differ in their immunopathological outcomes. Reovirus is an avirulent pathogen that elicits protective immunity, but we discovered that it can nonetheless disrupt intestinal immune homeostasis at inductive and effector sites of oral tolerance by suppressing peripheral regulatory T cell (pTreg) conversion and promoting TH1 immunity to dietary antigen. Initiation of TH1 immunity to dietary antigen was dependent on interferon regulatory factor 1 and dissociated from suppression of pTreg conversion, which was mediated by type-1 interferon. Last, our study in humans supports a role for infection with reovirus, a seemingly innocuous virus, in triggering the development of CeD.