RESUMEN
The COVID-19 pandemic urgently needs therapeutic and prophylactic interventions. Here, we report the rapid identification of SARS-CoV-2-neutralizing antibodies by high-throughput single-cell RNA and VDJ sequencing of antigen-enriched B cells from 60 convalescent patients. From 8,558 antigen-binding IgG1+ clonotypes, 14 potent neutralizing antibodies were identified, with the most potent one, BD-368-2, exhibiting an IC50 of 1.2 and 15 ng/mL against pseudotyped and authentic SARS-CoV-2, respectively. BD-368-2 also displayed strong therapeutic and prophylactic efficacy in SARS-CoV-2-infected hACE2-transgenic mice. Additionally, the 3.8 Å cryo-EM structure of a neutralizing antibody in complex with the spike-ectodomain trimer revealed the antibody's epitope overlaps with the ACE2 binding site. Moreover, we demonstrated that SARS-CoV-2-neutralizing antibodies could be directly selected based on similarities of their predicted CDR3H structures to those of SARS-CoV-neutralizing antibodies. Altogether, we showed that human neutralizing antibodies could be efficiently discovered by high-throughput single B cell sequencing in response to pandemic infectious diseases.
Asunto(s)
Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Neutralizantes/aislamiento & purificación , Linfocitos B/inmunología , Infecciones por Coronavirus/inmunología , Neumonía Viral/inmunología , Análisis de la Célula Individual , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Anticuerpos Neutralizantes/administración & dosificación , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/metabolismo , COVID-19 , Convalecencia , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ratones , Pandemias , Análisis de Secuencia de ARN , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , Exones VDJRESUMEN
There is an urgent need for vaccines against coronavirus disease 2019 (COVID-19) because of the ongoing SARS-CoV-2 pandemic. Among all approaches, a messenger RNA (mRNA)-based vaccine has emerged as a rapid and versatile platform to quickly respond to this challenge. Here, we developed a lipid nanoparticle-encapsulated mRNA (mRNA-LNP) encoding the receptor binding domain (RBD) of SARS-CoV-2 as a vaccine candidate (called ARCoV). Intramuscular immunization of ARCoV mRNA-LNP elicited robust neutralizing antibodies against SARS-CoV-2 as well as a Th1-biased cellular response in mice and non-human primates. Two doses of ARCoV immunization in mice conferred complete protection against the challenge of a SARS-CoV-2 mouse-adapted strain. Additionally, ARCoV is manufactured as a liquid formulation and can be stored at room temperature for at least 1 week. ARCoV is currently being evaluated in phase 1 clinical trials.
Asunto(s)
ARN Mensajero/genética , ARN Viral/genética , Vacunas Sintéticas/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Sitios de Unión , Vacunas contra la COVID-19 , Chlorocebus aethiops , Infecciones por Coronavirus/genética , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/prevención & control , Femenino , Células HEK293 , Células HeLa , Humanos , Inmunogenicidad Vacunal , Inyecciones Intramusculares , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos ICR , Nanopartículas/química , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Células TH1/inmunología , Potencia de la Vacuna , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Células Vero , Vacunas Virales/administración & dosificación , Vacunas Virales/genéticaRESUMEN
The World Health Organization has declared SARS-CoV-2 virus outbreak a worldwide pandemic. However, there is very limited understanding on the immune responses, especially adaptive immune responses to SARS-CoV-2 infection. Here, we collected blood from COVID-19 patients who have recently become virus-free, and therefore were discharged, and detected SARS-CoV-2-specific humoral and cellular immunity in eight newly discharged patients. Follow-up analysis on another cohort of six patients 2 weeks post discharge also revealed high titers of immunoglobulin G (IgG) antibodies. In all 14 patients tested, 13 displayed serum-neutralizing activities in a pseudotype entry assay. Notably, there was a strong correlation between neutralization antibody titers and the numbers of virus-specific T cells. Our work provides a basis for further analysis of protective immunity to SARS-CoV-2, and understanding the pathogenesis of COVID-19, especially in the severe cases. It also has implications in developing an effective vaccine to SARS-CoV-2 infection.
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Betacoronavirus/fisiología , Infecciones por Coronavirus/inmunología , Inmunidad Celular , Inmunidad Humoral , Neumonía Viral/inmunología , Adulto , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , COVID-19 , Convalecencia , Infecciones por Coronavirus/sangre , Infecciones por Coronavirus/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pandemias , Neumonía Viral/sangre , Neumonía Viral/patología , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/inmunologíaRESUMEN
The signaling adaptor TRAF3 is a highly versatile regulator of both innate immunity and adaptive immunity, but how its phosphorylation is regulated is still unknown. Here we report that deficiency in or inhibition of the conserved serine-threonine kinase CK1É suppressed the production of type I interferon in response to viral infection. CK1É interacted with and phosphorylated TRAF3 at Ser349, which thereby promoted the Lys63 (K63)-linked ubiquitination of TRAF3 and subsequent recruitment of the kinase TBK1 to TRAF3. Consequently, CK1É-deficient mice were more susceptible to viral infection. Our findings establish CK1É as a regulator of antiviral innate immune responses and indicate a novel mechanism of immunoregulation that involves CK1É-mediated phosphorylation of TRAF3.
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Caseína Cinasa 1 épsilon/inmunología , Inmunidad Innata/inmunología , Interferón beta/inmunología , Factor 3 Asociado a Receptor de TNF/inmunología , Animales , Caseína Cinasa 1 épsilon/antagonistas & inhibidores , Caseína Cinasa 1 épsilon/genética , Ensayo de Inmunoadsorción Enzimática , Células HEK293 , Células HeLa , Herpes Simple/inmunología , Herpesvirus Humano 1/inmunología , Humanos , Interferón Tipo I/biosíntesis , Interferón Tipo I/inmunología , Interferón beta/biosíntesis , Espectrometría de Masas , Ratones , Ratones Noqueados , Fosforilación , Proteínas Serina-Treonina Quinasas , Reacción en Cadena en Tiempo Real de la Polimerasa , Infecciones por Rhabdoviridae/inmunología , Factor 3 Asociado a Receptor de TNF/genética , Ubiquitinación , Vesiculovirus/inmunología , Fiebre del Nilo Occidental/inmunología , Virus del Nilo Occidental/inmunologíaRESUMEN
Omicron (B.1.1.529), the most heavily mutated SARS-CoV-2 variant so far, is highly resistant to neutralizing antibodies, raising concerns about the effectiveness of antibody therapies and vaccines1,2. Here we examined whether sera from individuals who received two or three doses of inactivated SARS-CoV-2 vaccine could neutralize authentic Omicron. The seroconversion rates of neutralizing antibodies were 3.3% (2 out of 60) and 95% (57 out of 60) for individuals who had received 2 and 3 doses of vaccine, respectively. For recipients of three vaccine doses, the geometric mean neutralization antibody titre for Omicron was 16.5-fold lower than for the ancestral virus (254). We isolated 323 human monoclonal antibodies derived from memory B cells in triple vaccinees, half of which recognized the receptor-binding domain, and showed that a subset (24 out of 163) potently neutralized all SARS-CoV-2 variants of concern, including Omicron. Therapeutic treatments with representative broadly neutralizing monoclonal antibodies were highly protective against infection of mice with SARS-CoV-2 Beta (B.1.351) and Omicron. Atomic structures of the Omicron spike protein in complex with three classes of antibodies that were active against all five variants of concern defined the binding and neutralizing determinants and revealed a key antibody escape site, G446S, that confers greater resistance to a class of antibodies that bind on the right shoulder of the receptor-binding domain by altering local conformation at the binding interface. Our results rationalize the use of three-dose immunization regimens and suggest that the fundamental epitopes revealed by these broadly ultrapotent antibodies are rational targets for a universal sarbecovirus vaccine.
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Vacunas contra la COVID-19 , COVID-19 , Células B de Memoria , SARS-CoV-2 , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/aislamiento & purificación , Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/aislamiento & purificación , Anticuerpos Antivirales/uso terapéutico , COVID-19/inmunología , COVID-19/prevención & control , COVID-19/virología , Vacunas contra la COVID-19/administración & dosificación , Vacunas contra la COVID-19/inmunología , Modelos Animales de Enfermedad , Humanos , Células B de Memoria/inmunología , Ratones , Pruebas de Neutralización , SARS-CoV-2/clasificación , SARS-CoV-2/genética , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunologíaRESUMEN
RNA interference (RNAi) functions as a potent antiviral immunity in plants and invertebrates; however, whether RNAi plays antiviral roles in mammals remains unclear. Here, using human enterovirus 71 (HEV71) as a model, we showed HEV71 3A protein as an authentic viral suppressor of RNAi during viral infection. When the 3A-mediated RNAi suppression was impaired, the mutant HEV71 readily triggered the production of abundant HEV71-derived small RNAs with canonical siRNA properties in cells and mice. These virus-derived siRNAs were produced from viral dsRNA replicative intermediates in a Dicer-dependent manner and loaded into AGO, and they were fully active in degrading cognate viral RNAs. Recombinant HEV71 deficient in 3A-mediated RNAi suppression was significantly restricted in human somatic cells and mice, whereas Dicer deficiency rescued HEV71 infection independently of type I interferon response. Thus, RNAi can function as an antiviral immunity, which is induced and suppressed by a human virus, in mammals.
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Enterovirus Humano A/inmunología , Infecciones por Enterovirus/inmunología , Inmunidad , Interferencia de ARN , ARN Viral/inmunología , Animales , Proteínas Argonautas/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Enterovirus Humano A/genética , Células HEK293 , Humanos , Mamíferos , Ratones , Ratones de la Cepa 129 , Ratones Noqueados , Mutación/genética , Ribonucleasa III/metabolismo , Proteínas Virales/inmunologíaRESUMEN
Zika virus (ZIKV) has become a public health threat due to its global transmission and link to severe congenital disorders. The host immune responses to ZIKV infection have not been fully elucidated, and effective therapeutics are not currently available. Herein, we demonstrated that cholesterol-25-hydroxylase (CH25H) was induced in response to ZIKV infection and that its enzymatic product, 25-hydroxycholesterol (25HC), was a critical mediator of host protection against ZIKV. Synthetic 25HC addition inhibited ZIKV infection in vitro by blocking viral entry, and treatment with 25HC reduced viremia and conferred protection against ZIKV in mice and rhesus macaques. 25HC suppressed ZIKV infection and reduced tissue damage in human cortical organoids and the embryonic brain of the ZIKV-induced mouse microcephaly model. Our findings highlight the protective role of CH25H during ZIKV infection and the potential use of 25HC as a natural antiviral agent to combat ZIKV infection and prevent ZIKV-associated outcomes, such as microcephaly.
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Antivirales/farmacología , Hidroxicolesteroles/farmacología , Microcefalia/virología , Infección por el Virus Zika/complicaciones , Animales , Encéfalo/efectos de los fármacos , Modelos Animales de Enfermedad , Técnica del Anticuerpo Fluorescente , Humanos , Macaca mulatta , Ratones , Microscopía Confocal , Internalización del Virus/efectos de los fármacos , Virus Zika/efectos de los fármacos , Virus Zika/fisiologíaRESUMEN
A new coronavirus, known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the aetiological agent responsible for the 2019-2020 viral pneumonia outbreak of coronavirus disease 2019 (COVID-19)1-4. Currently, there are no targeted therapeutic agents for the treatment of this disease, and effective treatment options remain very limited. Here we describe the results of a programme that aimed to rapidly discover lead compounds for clinical use, by combining structure-assisted drug design, virtual drug screening and high-throughput screening. This programme focused on identifying drug leads that target main protease (Mpro) of SARS-CoV-2: Mpro is a key enzyme of coronaviruses and has a pivotal role in mediating viral replication and transcription, making it an attractive drug target for SARS-CoV-25,6. We identified a mechanism-based inhibitor (N3) by computer-aided drug design, and then determined the crystal structure of Mpro of SARS-CoV-2 in complex with this compound. Through a combination of structure-based virtual and high-throughput screening, we assayed more than 10,000 compounds-including approved drugs, drug candidates in clinical trials and other pharmacologically active compounds-as inhibitors of Mpro. Six of these compounds inhibited Mpro, showing half-maximal inhibitory concentration values that ranged from 0.67 to 21.4 µM. One of these compounds (ebselen) also exhibited promising antiviral activity in cell-based assays. Our results demonstrate the efficacy of our screening strategy, which can lead to the rapid discovery of drug leads with clinical potential in response to new infectious diseases for which no specific drugs or vaccines are available.
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Betacoronavirus/química , Cisteína Endopeptidasas/química , Descubrimiento de Drogas/métodos , Modelos Moleculares , Inhibidores de Proteasas/química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/química , Antivirales/química , Antivirales/farmacología , Betacoronavirus/efectos de los fármacos , COVID-19 , Células Cultivadas/virología , Proteasas 3C de Coronavirus , Infecciones por Coronavirus/enzimología , Infecciones por Coronavirus/virología , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Humanos , Pandemias , Neumonía Viral/enzimología , Neumonía Viral/virología , Inhibidores de Proteasas/farmacología , Estructura Terciaria de Proteína , SARS-CoV-2RESUMEN
Because of the low host specificity, Ichthyophthirius multifiliis (Ich) can widely cause white spot disease in aquatic animals, which is extremely difficult to treat. Prior research has demonstrated a considerable impact of concentrated mannan-oligosaccharide (cMOS) on the prevention of white spot disease in goldfish, but the specific mechanism is still unknown. In this study, transcriptome sequencing, histological analysis, immunofluorescence analysis, phagocytosis activity assay and qRT-PCR assay were used to systematically reveal the potential mechanism of cMOS in supporting the resistance of goldfish (Carrasius auratus) to Ich invasion. According to the transcriptome analysis, the gill tissue of goldfish receiving the cMOS diet showed greater expression of mannose-receptor (MRC) related genes, higher phagocytosis activity, up-regulated expression of phagocytosis-related genes and inflammatory-related genes compared with the control, indicating that cMOS can have an effect on phagocytosis and non-specific immunity of goldfish. After the Ich challenge, transcriptome analysis revealed that cMOS fed goldfish displayed a higher level of phagocytic response, whereas non-cMOS fed goldfish displayed a greater inflammatory reaction. Besides, after Ich infection, cMOS-fed goldfish displayed greater phagocytosis activity, a stronger MRC positive signal, higher expression of genes associated with phagocytosis (ABCB2, C3, MRC), and lower expression of genes associated with inflammation (IL-1ß, IL-17, IL-8, TNF-α, NFKB). In conclusion, our experimental results suggest that cMOS may support phagocytosis by binding to MRC on the macrophage cell membrane and change the non-specific immunity of goldfish by stimulating cytokine expression. The results of this study provide new insights for the mechanism of cMOS on parasitic infection, and also suggest phagocytosis-related pathways may be potential targets for prevention of Ich infection.
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Enfermedades de los Peces , Carpa Dorada , Animales , Mananos/farmacología , Citocinas/genética , Macrófagos/metabolismo , FagocitosisRESUMEN
The compound Honokiol, derived from the bark of Magnolia officinalis, possesses the ability to induce apoptosis and inhibit cellular damage caused by reactive oxygen species. The objective of this study was to investigate the toxicological and histopathological effects of Honokiol on zebrafish (Danio rerio) through conducting a semistatic acute toxicity test involving immersion in an Honokiol-containing solution. The results showed that the toxic effects of Honokiol on zebrafish were primarily manifested in the liver and gills. When exposed to 0.6 mg/L of Honokiol, it could lead to liver hemorrhage as well as swelling and necrosis of gill tissues, and high concentrations of Honokiol could trigger inflammatory responses. Additionally, research found that Honokiol could induce apoptosis in liver and gill tissues through the P53 pathway and possessed the ability to enhance antioxidation. The present findings significantly contribute to a more profound understanding of the toxic impact of Honokiol and its underlying mechanism, thereby providing a valuable reference for the future safe utilization of Honokiol and related pharmaceutical advancements.
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Apoptosis , Compuestos de Bifenilo , Lignanos , Hígado , Pez Cebra , Lignanos/farmacología , Lignanos/toxicidad , Animales , Compuestos de Bifenilo/toxicidad , Hígado/efectos de los fármacos , Hígado/patología , Apoptosis/efectos de los fármacos , Branquias/efectos de los fármacos , Branquias/patología , Proteína p53 Supresora de Tumor/metabolismo , Magnolia/química , Compuestos Alílicos , FenolesRESUMEN
Sepsis, a critical condition resulting from the systemic inflammatory response to a severe microbial infection, represents a global public health challenge. However, effective treatment or intervention to prevent and combat sepsis is still lacking. Here, we report that hyodeoxycholic acid (HDCA) has excellent anti-inflammatory properties in sepsis. We discovered that the plasma concentration of HDCA was remarkably lower in patients with sepsis and negatively correlated with the severity of the disease. Similar changes in HDCA levels in plasma and cecal content samples were observed in a mouse model of sepsis, and these changes were associated with a reduced abundance of HDCA-producing strains. Interestingly, HDCA administration significantly decreased systemic inflammatory responses, prevented organ injury, and prolonged the survival of septic mice. We demonstrated that HDCA suppressed excessive activation of inflammatory macrophages by competitively blocking lipopolysaccharide binding to the Toll-like receptor 4 (TLR4) and myeloid differentiation factor 2 receptor complex, a unique mechanism that characterizes HDCA as an endogenous inhibitor of inflammatory signaling. Additionally, we verified these findings in TLR4 knockout mice. Our study highlights the potential value of HDCA as a therapeutic molecule for sepsis.
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Microbioma Gastrointestinal , Sepsis , Animales , Ratones , Inflamación , Lipopolisacáridos , Ratones Endogámicos C57BL , Sepsis/tratamiento farmacológico , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismoRESUMEN
OBJECTIVE: The pathogenesis of sepsis is complex, and the sepsis-induced systemic proinflammatory phase is one of the key drivers of organ failure and consequent mortality. Akkermansia muciniphila (AKK) is recognised as a functional probiotic strain that exerts beneficial effects on the progression of many diseases; however, whether AKK participates in sepsis pathogenesis is still unclear. Here, we evaluated the potential contribution of AKK to lethal sepsis development. DESIGN: Relative abundance of gut microbial AKK in septic patients was evaluated. Cecal ligation and puncture (CLP) surgery and lipopolysaccharide (LPS) injection were employed to establish sepsis in mice. Non-targeted and targeted metabolomics analysis were used for metabolites analysis. RESULTS: We first found that the relative abundance of gut microbial AKK in septic patients was significantly reduced compared with that in non-septic controls. Live AKK supplementation, as well as supplementation with its culture supernatant, remarkably reduced sepsis-induced mortality in sepsis models. Metabolomics analysis and germ-free mouse validation experiments revealed that live AKK was able to generate a novel tripeptide Arg-Lys-His (RKH). RKH exerted protective effects against sepsis-induced death and organ damage. Furthermore, RKH markedly reduced sepsis-induced inflammatory cell activation and proinflammatory factor overproduction. A mechanistic study revealed that RKH could directly bind to Toll-like receptor 4 (TLR4) and block TLR4 signal transduction in immune cells. Finally, we validated the preventive effects of RKH against sepsis-induced systemic inflammation and organ damage in a piglet model. CONCLUSION: We revealed that a novel tripeptide, RKH, derived from live AKK, may act as a novel endogenous antagonist for TLR4. RKH may serve as a novel potential therapeutic approach to combat lethal sepsis after successfully translating its efficacy into clinical practice.
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Sepsis , Receptor Toll-Like 4 , Porcinos , Humanos , Ratones , Animales , Receptor Toll-Like 4/metabolismo , Sepsis/prevención & control , Transducción de Señal , VerrucomicrobiaRESUMEN
The glycan loop of Zika virus (ZIKV) envelope protein (E) contains the glycosylation site and has been well documented to be important for viral pathogenesis and transmission. In the present study, we report that deletions in the E glycan loop, which were recorded in African ZIKV strains previously, have re-emerged in their contemporary Asian lineages. Here, we generated recombinant ZIKV containing specific deletions in the E glycan loop by reverse genetics. Extensive in vitro and in vivo characterization of these deletion mutants demonstrated an attenuated phenotype in an adult A129 mouse model and reduced oral infections in mosquitoes. Surprisingly, these glycan loop deletion mutants exhibited an enhanced neurovirulence phenotype, and resulted in a more severe microcephalic brain in neonatal mouse models. Crystal structures of the ZIKV E protein and a deletion mutant at 2.5 and 2.6 Å, respectively, revealed that deletion of the glycan loop induces encephalitic flavivirus-like conformational alterations, including the appearance of perforations on the surface and a clear change in the topology of the loops. Overall, our results demonstrate that the E glycan loop deletions represent neonatal mouse neurovirulence markers of ZIKV. IMPORTANCE Zika virus (ZIKV) has been identified as a cause of microcephaly and acquired evolutionary mutations since its discovery. Previously deletions in the E glycan loop were recorded in African ZIKV strains, which have re-emerged in the contemporary Asian lineages recently. The glycan loop deletion mutants are not glycosylated, which are attenuated in adult A129 mouse model and reduced oral infections in mosquitoes. More importantly, the glycan loop deletion mutants induce an encephalitic flavivirus-like conformational alteration in the E homodimer, resulting in a significant enhancement of neonatal mouse neurovirulence. This study underscores the critical role of glycan loop deletion mutants in ZIKV pathogenesis, highlighting a need for global virological surveillance for such ZIKV variants.
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Proteínas del Envoltorio Viral , Infección por el Virus Zika , Virus Zika , Animales , Ratones , Modelos Animales de Enfermedad , Polisacáridos/química , Proteínas del Envoltorio Viral/genética , Virulencia , Replicación Viral/genética , Virus Zika/genética , Virus Zika/patogenicidad , Infección por el Virus Zika/virologíaRESUMEN
The geographic range of Zika virus (ZIKV) has expanded from Asia to the Americas, leading to the 2015-2016 pandemic with enhanced neurovirulence. At present, ZIKV is continuously circulating in many Southeast Asian countries. Unfortunately, the persistent evolution of ZIKV in Southeast Asia and its influence on the biological characteristics of the virus remain incompletely understood. In this study, the in vitro and in vivo properties of a new ZIKV isolate obtained from Cambodia in 2019 (CAM/2019) were characterized and compared with those of the Cambodian strain (CAM/2010). Compared with CAM/2010, the CAM/2019 virus showed similar plaque morphology and growth curves in cell cultures and induced comparable viremia and organ viral loads profiles in both BALB/c and A129 (IFNAR1-/- ) mice upon intraperitoneal (i.p.) inoculation. Remarkably, the CAM/2019 virus exhibited enhanced neurovirulence in neonatal mice compared with CAM/2010, with a 74-fold reduction in the 50% lethal dose (LD50 ). Consistently, CAM/2019 produced higher viral loads in the brains of BALB/c neonatal mice than CAM/2010 did. Sequence alignment showed that the CAM/2019 virus has acquired 12 amino acid substitutions, several of which were found to be associated with neurovirulence. In particular, the CAM/2019 virus shared an A1204T substitution in NS2A with the Thai isolate SI-BKK02 that was isolated from a microcephaly case. Taken together, our results indicate that a ZIKV strain isolated with specific mutations has emerged in Cambodia, highlighting the need for extensive molecular and disease surveillance in Cambodia and other Asian countries.
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Infección por el Virus Zika , Virus Zika , Animales , Ratones , Filogenia , Infección por el Virus Zika/epidemiología , Cambodia/epidemiología , Asia/epidemiologíaRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants continue to emerge and cocirculate in humans and wild animals. The factors driving the emergence and replacement of novel variants and recombinants remain incompletely understood. Herein, we comprehensively characterized the competitive fitness of SARS-CoV-2 wild type (WT) and three variants of concern (VOCs), Alpha, Beta and Delta, by coinfection and serial passaging assays in different susceptible cells. Deep sequencing analyses revealed cell-specific competitive fitness: the Beta variant showed enhanced replication fitness during serial passage in Caco-2 cells, whereas the WT and Alpha variant showed elevated fitness in Vero E6 cells. Interestingly, a high level of neutralizing antibody sped up competition and completely reshaped the fitness advantages of different variants. More importantly, single clone purification identified a significant proportion of homologous recombinants that emerged during the passage history, and immune pressure reduced the frequency of recombination. Interestingly, a recombination hot region located between nucleotide sites 22,995 and 28,866 of the viral genomes could be identified in most of the detected recombinants. Our study not only profiled the variable competitive fitness of SARS-CoV-2 under different conditions, but also provided direct experimental evidence of homologous recombination between SARS-CoV-2 viruses, as well as a model for investigating SARS-CoV-2 recombination.
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COVID-19 , SARS-CoV-2 , Animales , Humanos , SARS-CoV-2/genética , Células CACO-2 , Recombinación Homóloga , Glicoproteína de la Espiga del CoronavirusRESUMEN
Elizabethkingia miricola is an emerging opportunistic pathogen that is highly pathogenic in both immunocompromised humans and animals. Once the disease occurs, treatment can be very difficult. Therefore, a deep understanding of the pathological mechanism of Elizabethkingia miricola is the key to the prevention and control of the disease. In this study, we isolated the pathogenic bacteria from bullfrogs with dark skin color, weak limbs, wryneck, and cataracts. Via subsequent morphological observations and a 16S rRNA gene sequence analysis, the pathogen was identified as Elizabethkingia miricola. The histopathological and transmission electron microscopy analysis revealed that the brain was the main target organ. Therefore, brain samples from diseased and healthy bullfrogs were used for the RNA-Seq analysis. The comparative transcriptome analysis revealed that the diseased bullfrog brain was characterized by the immune activation and inflammatory response, which were mediated by the "NOD-like receptor signaling pathway" and the "Toll-like receptor signaling pathway". We also performed qRT-PCR to examine the expression profile of inflammation-related genes, which further verified the reliability of our transcriptome data. Based on the above results, it was concluded that the NOD/Toll-like receptor-related networks that dominate the immune activation and inflammatory response were activated in the brain of Elizabethkingia miricola-infected bullfrogs. This study contributes to the search for therapeutic targets for bullfrog meningitis and provides basic information for establishing effective measures to prevent and control bullfrog meningitis.
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Infecciones por Flavobacteriaceae , Flavobacteriaceae , Meningitis , Animales , Humanos , Rana catesbeiana , ARN Ribosómico 16S/genética , Reproducibilidad de los Resultados , Infecciones por Flavobacteriaceae/microbiología , Infecciones por Flavobacteriaceae/patología , Ranidae , Transducción de SeñalRESUMEN
BACKGROUND: Adenosine is a purine nucleoside involved in regulating bone homeostasis through binding to A1, A2A, A2B, and A3 adenosine receptors (A1R, A2AR, A2BR, and A3R, respectively). However, the underlying mechanisms by which adenosine and receptor subtypes regulate osteoclast differentiation remain uncertain. This study aims to assess the role of exogenous adenosine and receptor subtypes in receptor activator of NF-κB ligand (RANKL)-induced osteoclast formation and explore the underlying molecular mechanisms. METHODS AND RESULTS: The nanofibrous mats incorporated with adenosine exhibited robust ability to facilitate rat critical-size calvarial defect healing with decreased number of osteoclasts. Moreover, exogenous adenosine substantially enhanced the expression of A2AR and suppressed tartrate-resistant acid phosphatase-positive osteoclast formation and expression of osteoclast-related genes Ctsk, NFATc1, MMP9, and ACP5. This enhancement and suppression could be reversed by adding an A2AR antagonist, ZM241385, in RAW264.7 cells. Finally, RNA sequencing showed that the expression of Fos-related antigen 2 (Fra2) was distinctly downregulated through stimulation of adenosine in RAW264.7 cells treated with RANKL. This downregulation was reversed by ZM241385 according to real-time PCR, Western blot, and immunofluorescence analyses. CONCLUSIONS: These findings demonstrated that exogenous adenosine binding to A2AR attenuated osteoclast differentiation via the inhibition of activating protein-1 (AP-1, including Fra2 subunit) pathway both in vitro and in vivo.
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Osteogénesis , Factor de Transcripción AP-1 , Adenosina/metabolismo , Adenosina/farmacología , Animales , Diferenciación Celular , FN-kappa B/metabolismo , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Osteoclastos , Osteogénesis/genética , Ratas , Receptores Purinérgicos P1/metabolismo , Factor de Transcripción AP-1/metabolismoRESUMEN
COVID-19 has spread around the world and caused serious public health and social problems. Although several vaccines have been authorized for emergency use, new effective antiviral drugs are still needed. Some repurposed drugs including Chloroquine, Hydroxychloroquine and Remdesivir were immediately used to treat COVID-19 after the pandemic. However, the therapeutic effects of these drugs have not been fully demonstrated in clinical studies. In this paper, we found an antimalarial drug, Naphthoquine, showed good broad-spectrum anti-coronavirus activity. Naphthoquineinhibited HCoV-229E, HCoV-OC43 and SARS-CoV-2 replication in vitro, with IC50 = 2.05 ± 1.44 µM, 5.83 ± 0.74 µM, and 2.01 ± 0.38 µM, respectively. Time-of-addition assay was also performed to explore at which stage Naphthoquine functions during SARS-CoV-2 replication. The results suggested that Naphthoquine may influence virus entry and post-entry replication. Considering the safety of Naphthoquine was even better than that of Chloroquine, we think Naphthoquine has the potential to be used as a broad-spectrum drug for coronavirus infection.
Asunto(s)
1-Naftilamina/análogos & derivados , Aminoquinolinas/farmacología , Antivirales/farmacología , Coronavirus/efectos de los fármacos , SARS-CoV-2/efectos de los fármacos , 1-Naftilamina/farmacología , Animales , Línea Celular , Chlorocebus aethiops , Coronavirus Humano 229E/efectos de los fármacos , Coronavirus Humano NL63/efectos de los fármacos , Coronavirus Humano OC43/efectos de los fármacos , Humanos , Técnicas In Vitro , Células Vero , Replicación Viral/efectos de los fármacosRESUMEN
In recent years, inhibitors of the BET bromodomain proteins, such as BRD4 inhibitors, have demonstrated robust antitumor activity. JQ-1, a representative small molecular BRD4 inhibitor, is also effective to block PD-1/PD-L1 signaling by significantly decreasing the PD-L1 expression on tumor cells. However, toxicity of BRD4 inhibitors on lymphoid and hematopoietic tissues limits their clinical usage. In this research, we designed and studied an immunogenic BRD4 inhibitor, SZU-119, by coupling JQ-1 with a TLR7 agonist, SZU-101. In vitro, SZU-119 stimulated the production of cytokines in mouse BMDCs and spleen lymphocytes, and inhibited the expression of PD-L1 in mouse B16 tumor cells. In vivo, SZU-119 suppressed the B16 tumor growth at both injected and uninjected sites, and prolonged the survival time of mice. SZU-119 elevated the number of total CD8+ and IFN-γ+ CD8+ T cells in spleens, with greater CTL cytotoxicity to B16 tumor cells. It was also observed that the infiltration of CD8+ T cells was increased in tumors at both local and distant sites, and the PD-L1 expression was decreased in tumor cells at the primary site. In conclusion, we have demonstrated that SZU-119 activated the innate immune cells, kept efficacy of PD-L1 blockade and abrogated immune toxicity, showing more potent antitumor effects than the simple mixture of SZU-101 and JQ-1 in a mouse melanoma model. Our work provides new insights for the development of anti-melanoma drugs that concurrently target innate and adaptive immunity.