RESUMEN
Viral RNA-dependent RNA polymerases (RdRps) are a target for broad-spectrum antiviral therapeutic agents. Recently, we demonstrated that incorporation of the T-1106 triphosphate, a pyrazine-carboxamide ribonucleotide, into nascent RNA increases pausing and backtracking by the poliovirus RdRp. Here, by monitoring enterovirus A-71 RdRp dynamics during RNA synthesis using magnetic tweezers, we identify the "backtracked" state as an intermediate used by the RdRp for copy-back RNA synthesis and homologous recombination. Cell-based assays and RNA sequencing (RNA-seq) experiments further demonstrate that the pyrazine-carboxamide ribonucleotide stimulates these processes during infection. These results suggest that pyrazine-carboxamide ribonucleotides do not induce lethal mutagenesis or chain termination but function by promoting template switching and formation of defective viral genomes. We conclude that RdRp-catalyzed intra- and intermolecular template switching can be induced by pyrazine-carboxamide ribonucleotides, defining an additional mechanistic class of antiviral ribonucleotides with potential for broad-spectrum activity.
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Pirazinas/química , Virus ARN/genética , ARN Viral/genética , ARN Polimerasa Dependiente del ARN/genética , Recombinación Genética , Ribonucleótidos/química , Animales , Antivirales , Catálisis , Células Cultivadas , Técnicas Genéticas , Genoma , Genoma Viral , Recombinación Homóloga , Humanos , Cinética , Ratones , Ratones Transgénicos , Simulación de Dinámica Molecular , Mutagénesis , Nucleótidos/genética , Conformación Proteica , ARN/química , ARN Polimerasa Dependiente del ARN/metabolismo , RNA-Seq , Transgenes , VirulenciaRESUMEN
The influenza A virus (IAV) consists of 8 single-stranded, negative-sense viral RNA (vRNA) segments. After infection, vRNA is transcribed, replicated, and wrapped by viral nucleoprotein (NP) to form viral ribonucleoprotein (vRNP). The transcription, replication, and nuclear export of the viral genome are regulated by the IAV protein, NS2, which is translated from spliced mRNA transcribed from viral NS vRNA. This splicing is inefficient, explaining why NS2 is present in low abundance after IAV infection. The levels of NS2 and its subsequent accumulation are thought to influence viral RNA replication and vRNP nuclear export. Here we show that NS2 is ubiquitinated at the K64 and K88 residues by K48-linked and K63-linked polyubiquitin (polyUb) chains, leading to the degradation of NS2 by the proteasome. Additionally, we show that a host deubiquitinase, OTUB1, can remove polyUb chains conjugated to NS2, thereby stabilizing NS2. Accordingly, knock down of OTUB1 by siRNA reduces the nuclear export of vRNP, and reduces the overall production of IAV. These results collectively demonstrate that the levels of NS2 in IAV-infected cells are regulated by a ubiquitination-deubiquitination system involving OTUB1 that is necessary for optimal IAV replication.
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Cisteína Endopeptidasas , Virus de la Influenza A , Proteínas no Estructurales Virales , Replicación Viral , Animales , Perros , Humanos , Cisteína Endopeptidasas/metabolismo , Cisteína Endopeptidasas/genética , Enzimas Desubicuitinizantes/metabolismo , Células HEK293 , Virus de la Influenza A/metabolismo , Gripe Humana/metabolismo , Gripe Humana/virología , ARN Viral/metabolismo , ARN Viral/genética , Ubiquitinación , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/genética , Replicación Viral/fisiología , Línea Celular , Células Vero , Chlorocebus aethiopsRESUMEN
The emergence of SARS-CoV-2, which is responsible for the COVID-19 pandemic, has highlighted the need for rapid characterization of viral mechanisms associated with cellular pathogenesis. Viral UTRs represent conserved genomic elements that contribute to such mechanisms. Structural details of most CoV UTRs are not available, however. Experimental approaches are needed to allow for the facile generation of high-quality viral RNA tertiary structural models, which can facilitate comparative mechanistic efforts. By integrating experimental and computational techniques, we herein report the efficient characterization of conserved RNA structures within the 5'UTR of the HCoV-OC43 genome, a lab-tractable model coronavirus. We provide evidence that the 5'UTR folds into a structure with well-defined stem-loops (SLs) as determined by chemical probing and direct detection of hydrogen bonds by NMR. We combine experimental base-pair restraints with global structural information from SAXS to generate a 3D model that reveals that SL1-4 adopts a topologically constrained structure wherein SLs 3 and 4 coaxially stack. Coaxial stacking is mediated by short linker nucleotides and allows SLs 1 to 2 to sample different cojoint orientations by pivoting about the SL3,4 helical axis. To evaluate the functional relevance of the SL3,4 coaxial helix, we engineered luciferase reporter constructs harboring the HCoV-OC43 5'UTR with mutations designed to abrogate coaxial stacking. Our results reveal that the SL3,4 helix intrinsically represses translation efficiency since the destabilizing mutations correlate with increased luciferase expression relative to wildtype without affecting reporter mRNA levels, thus highlighting how the 5'UTR structure contributes to the viral mechanism.
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Regiones no Traducidas 5' , Coronavirus Humano OC43 , ARN Viral , Coronavirus Humano OC43/genética , Luciferasas/genética , Dispersión del Ángulo Pequeño , Difracción de Rayos X , ARN Viral/genéticaRESUMEN
Various vaccine platforms were developed and deployed against the COVID-19 disease. The Fc-mediated functions of IgG antibodies are essential in the adaptive immune response elicited by vaccines. However, the long-term changes of protein subunit vaccines and their combinations with messenger RNA (mRNA) vaccines are unknown. A total of 272 serum and plasma samples were collected from individuals who received first to third doses of the protein subunit Medigen, the mRNA (BNT, Moderna), or the adenovector AstraZeneca vaccines. The IgG subclass level was measured using enzyme-linked immunosorbent assay, and Fc-N glycosylation was measured using liquid chromatography coupled to tandem mass spectrometry. Antibody-dependent-cellular-phagocytosis (ADCP) and complement deposition (ADCD) of anti-spike (S) IgG antibodies were measured by flow cytometry. IgG1 and 3 reached the highest anti-S IgG subclass level. IgG1, 2, and 4 subclass levels significantly increased in mRNA- and Medigen-vaccinated individuals. Fc-glycosylation was stable, except in female BNT vaccinees, who showed increased bisection and decreased galactosylation. Female BNT vaccinees had a higher anti-S IgG titer than that of males. ADCP declined in all groups. ADCD was significantly lower in AstraZeneca-vaccinated individuals. Each vaccine produced specific long-term changes in Fc structure and function. This finding is critical when selecting a vaccine platform or combination to achieve the desired immune response.
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Anticuerpos Antivirales , Vacunas contra la COVID-19 , COVID-19 , Inmunoglobulina G , SARS-CoV-2 , Vacunas de Subunidad , Vacunas de ARNm , Humanos , Inmunoglobulina G/sangre , Femenino , Anticuerpos Antivirales/sangre , Masculino , COVID-19/prevención & control , COVID-19/inmunología , SARS-CoV-2/inmunología , SARS-CoV-2/genética , Adulto , Persona de Mediana Edad , Vacunas contra la COVID-19/inmunología , Vacunas de Subunidad/inmunología , Vacunas de Subunidad/genética , Vacunas de Subunidad/administración & dosificación , Glicosilación , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , Anciano , ARN Mensajero/genética , Adulto Joven , Vacunas de Subunidades ProteicasRESUMEN
The coronavirus disease 2019 (COVID-19) pandemic has sparked widespread concern globally, particularly with the Omicron variant and its sub-lineages emerging as the predominant cause of infection for nearly two years. Taiwan's successful containment of COVID-19, underscored by broad vaccine coverage, the utilization of anti-viral therapeutics, and timely response strategies, has resulted in reduced excess mortality. Moreover, there is a crucial need for a phased exit strategy, balancing efforts to curtail disease transmission with the mitigation of socioeconomic impacts from rigorous measures. In this review, we examined the evolution and the epidemiological landscape of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron sub-variants in Taiwan as well as other countries of the world. We also critically evaluated the effectiveness of COVID-19 vaccines against various SARS-CoV-2 variants. Additionally, we addressed the advantages of heterologous immunization strategies, fluctuations in neutralizing antibody titers, and complexities in establishing protective correlates among swiftly mutating viral variants.
RESUMEN
Since April 2022, waves of SARS-CoV-2 Omicron variant cases have surfaced in Taiwan and spread throughout the island. Using high-throughput sequencing of the SARS-CoV-2 genome, we analyzed 2,405 PCR-positive swab samples from 2,339 persons and identified the Omicron BA.2.3.7 variant as a major lineage within recent community outbreaks in Taiwan.
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COVID-19 , Humanos , Taiwán/epidemiología , COVID-19/epidemiología , SARS-CoV-2/genética , Brotes de EnfermedadesRESUMEN
BACKGROUND: Children are susceptible to severe or fatal enterovirus 71 (EV71) infections. We aimed to evaluate the efficacy, safety, and immunogenicity of EV71vac, an aluminium phosphate-adjuvanted inactivated EV71 vaccine in children aged 2-71 months. METHODS: We did a randomised, double-blinded, placebo-controlled, phase 3 trial at five hospitals in Taiwan and two in Vietnam. Children aged 2-71 months were stratified by country and age, and randomly assigned (1:1) to receive two doses of EV71vac or placebo via intramuscular injection 56 days apart. Children aged 2-23 months received a third booster dose on day 366. The primary endpoint was the clinical efficacy of the total vaccinated cohort against EV71-associated diseases during the follow-up period, from 14 days after the second dose to when 15 cases of EV71 infections were confirmed in the per-protocol population. Our safety analysis included all participants who received at least one dose of EV71vac. This trial is registered with ClinicalTrials.gov, NCT03865238, and is complete. FINDINGS: Between April 23 and Dec 25, 2019, of 3663 children assessed, 3061 were randomly assigned, of whom 3049 were vaccinated: 1521 children in the EV71vac group and 1528 in the placebo group. By May 20, 2021, our primary efficacy analysis included 2959 children, with 1476 children in the EV71vac group and 1483 children in the placebo group. The vaccine efficacy of EV71vac was 96·8% (95% CI 85·5-100) against EV71 associated diseases (p<0·0001). The percentage of participants who reported solicited adverse events were similar in both groups: 865 (56·9%) in the EV71vac group and 852 (55·8%) in the placebo group. Almost all reported solicited adverse events were mild and self-limited. INTERPRETATION: EV71vac is safe, well-tolerated, and highly effective in preventing EV71 associated diseases in children aged 2-71 months. FUNDING: Medigen Vaccine Biologics and A+ Industrial Innovative R&D Program of the Ministry of Economic Affairs, Taiwan.
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Enterovirus Humano A , Infecciones por Enterovirus , Enterovirus , Adyuvantes Inmunológicos , Anticuerpos Antivirales , Niño , Método Doble Ciego , Infecciones por Enterovirus/prevención & control , Humanos , Lactante , Vacunas de Productos Inactivados/efectos adversosRESUMEN
[This corrects the article DOI: 10.1371/journal.ppat.1009352.].
RESUMEN
Serological and plasmablast responses and plasmablast-derived IgG monoclonal antibodies (MAbs) have been analysed in three COVID-19 patients with different clinical severities. Potent humoral responses were detected within 3 weeks of onset of illness in all patients and the serological titre was elicited soon after or concomitantly with peripheral plasmablast response. An average of 13.7% and 3.5% of plasmablast-derived MAbs were reactive with virus spike glycoprotein or nucleocapsid, respectively. A subset of anti-spike (10 of 32) antibodies cross-reacted with other betacoronaviruses tested and harboured extensive somatic mutations, indicative of an expansion of memory B cells upon SARS-CoV-2 infection. Fourteen of 32 anti-spike MAbs, including five anti-receptor-binding domain (RBD), three anti-non-RBD S1 and six anti-S2, neutralised wild-type SARS-CoV-2 in independent assays. Anti-RBD MAbs were further grouped into four cross-inhibiting clusters, of which six antibodies from three separate clusters blocked the binding of RBD to ACE2 and five were neutralising. All ACE2-blocking anti-RBD antibodies were isolated from two recovered patients with prolonged fever, which is compatible with substantial ACE2-blocking response in their sera. Finally, the identification of non-competing pairs of neutralising antibodies would offer potential templates for the development of prophylactic and therapeutic agents against SARS-CoV-2.
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Anticuerpos Antivirales/inmunología , COVID-19/inmunología , SARS-CoV-2/inmunología , Adulto , Enzima Convertidora de Angiotensina 2/antagonistas & inhibidores , Enzima Convertidora de Angiotensina 2/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/genética , Células Productoras de Anticuerpos/inmunología , Sitios de Unión , Epítopos , Humanos , Inmunoglobulina G/inmunología , Nucleocápside/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunologíaRESUMEN
BACKGROUND: The association between M segment splicing and pathogenicity remains ambiguous in human influenza A viruses. In this study, we aimed to investigate M splicing in various human influenza A viruses and characterize its physiological roles by applying the splicing inhibitor, herboxidiene. METHODS: We examined the M splicing of human H1N1 and H3N2 viruses by comparing three H1N1 and H3N2 strains, respectively, through reverse transcriptase-polymerase chain reaction (RT-PCR) analyses. We randomly selected M sequences of human H1N1, H2N2, and H3N2 viruses isolated from 1933 to 2020 and examined their phylogenetic relationships. Next, we determined the effects of single nucleotide variations on M splicing by generating mutant viruses harboring the 55C/T variant through reverse genetics. To confirm the importance of M2 splicing in the replication of H1N1 and H3N2, we treated infected cells with splicing inhibitor herboxidiene and analyzed the viral growth using plaque assay. To explore the physiological role of the various levels of M2 protein in pathogenicity, we challenged C57BL/6 mice with the H1N1 WSN wild-type strain, mutant H1N1 (55T), and chimeric viruses including H1N1 + H3wt and H1N1 + H3mut. One-tailed paired t-test was used for virus titer calculation and multiple comparisons between groups were performed using two-way analysis of variance. RESULTS: M sequence splice site analysis revealed an evolutionarily conserved single nucleotide variant C55T in H3N2, which impaired M2 expression and was accompanied by collinear M1 and mRNA3 production. Aberrant M2 splicing resulted from splice-site selection rather than a general defect in the splicing process. The C55T substitution significantly reduced both M2 mRNA and protein levels regardless of the virus subtype. Consequently, herboxidiene treatment dramatically decreased both the H1N1 and H3N2 virus titers. However, a lower M2 expression only attenuated H1N1 virus replication and in vivo pathogenicity. This attenuated phenotype was restored by M replacement of H3N2 M in a chimeric H1N1 virus, despite low M2 levels. CONCLUSIONS: The discrepancy in M2-dependence emphasizes the importance of M2 in human influenza A virus pathogenicity, which leads to subtype-specific evolution. Our findings provide insights into virus adaptation processes in humans and highlights splicing regulation as a potential antiviral target.
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Subtipo H1N1 del Virus de la Influenza A , Virus de la Influenza A , Gripe Humana , Animales , Ratones , Humanos , Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/genética , Filogenia , Ratones Endogámicos C57BL , Nucleótidos , Gripe Humana/tratamiento farmacológico , Gripe Humana/genéticaRESUMEN
Enterovirus A71 (EV-A71) and many members of the Picornaviridae family are neurotropic pathogens of global concern. These viruses are primarily transmitted through the fecal-oral route, and thus suitable animal models of oral infection are needed to investigate viral pathogenesis. An animal model of oral infection was developed using transgenic mice expressing human SCARB2 (hSCARB2 Tg), murine-adapted EV-A71/MP4 virus, and EV-A71/MP4 virus with an engineered nanoluciferase gene that allows imaging of viral replication and spread in infected mice. Next-generation sequencing of EV-A71 genomes in the tissues and organs of infected mice was also performed. Oral inoculation of EV-A71/MP4 or nanoluciferase-carrying MP4 virus stably induced neurological symptoms and death in infected 21-day-old weaned mice. In vivo bioluminescence imaging of infected mice and tissue immunostaining of viral antigens indicated that orally inoculated virus can spread to the central nervous system (CNS) and other tissues. Next-generating sequencing further identified diverse mutations in viral genomes that can potentially contribute to viral pathogenesis. This study presents an EV-A71 oral infection murine model that efficiently infects weaned mice and allows tracking of viral spread, features that can facilitate research into viral pathogenesis and neuroinvasion via the natural route of infection. IMPORTANCE Enterovirus A71 (EV-A71), a positive-strand RNA virus of the Picornaviridae, poses a persistent global public health problem. EV-A71 is primarily transmitted through the fecal-oral route, and thus suitable animal models of oral infection are needed to investigate viral pathogenesis. We present an animal model of EV-A71 infection that enables the natural route of oral infection in weaned and nonimmunocompromised 21-day-old hSCARB2 transgenic mice. Our results demonstrate that severe disease and death could be stably induced, and viral invasion of the CNS could be replicated in this model, similar to severe real-world EV-A71 infections. We also developed a nanoluciferase-containing EV-A71 virus that can be used with this animal model to track viral spread after oral infection in real time. Such a model offers several advantages over existing animal models and can facilitate future research into viral spread, tissue tropism, and viral pathogenesis, all pressing issues that remain unaddressed for EV-A71 infections.
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Sistema Nervioso Central/virología , Enterovirus Humano A/patogenicidad , Infecciones por Enterovirus/complicaciones , Proteínas de Membrana de los Lisosomas/genética , Boca/virología , Enfermedades del Sistema Nervioso/virología , Receptores Depuradores/genética , Animales , Modelos Animales de Enfermedad , Enterovirus Humano A/genética , Infecciones por Enterovirus/patología , Infecciones por Enterovirus/virología , Genoma Viral , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Tropismo Viral , Replicación Viral , DesteteRESUMEN
An outbreak of the hand-foot-mouth disease with severe neurological cases, mainly caused by the genotype C1 enterovirus A71 (EV-A71), occurred in Taiwan between 2018 and early 2019. In the recent decade, the most dominant EV-A71 genotypes in Taiwan were B5 and C4 but changed to C1 in 2018. Antibody-mediated immunity plays a key role in limiting the EV-A71 illness in humans. However, the level of neutralizing activities against genotype C1 virus by human polyclonal and monoclonal antibodies (MAbs) remains largely unclear. In the study, we demonstrated that that 39% (9 in 23) of post-infection sera from the genotype B5- or C4-infected patients in 2014-2017 exhibit reduced titers with the 2018-2019 genotype C1 viruses than with the earlier B5 and C4 viruses tested. This finding with polyclonal sera is confirmed with human MAbs derived from genotype B5 virus-infected individuals. The 2018-2019 genotype C1 virus is resistant to the majority of canyon-targeting human MAbs, which may be associated with the residue change near or at the bottom of the canyon region on the viral capsid. The remaining three antibodies (16-2-11B, 16-3-4D, and 17-1-12A), which target VP1 S241 on the 5-fold vertex, VP3 E81 on the 3-fold plateau and VP2 D84 on the 2-fold plateau of genotype C1 viral capsid, respectively, retained neutralizing activities with variable potencies. These neutralizing antibodies were also found to be protective against a lethal challenge of the 2018-2019 genotype C1 virus in an hSCARB2-transgenic mice model. These results indicate that the EV-A71-specific antibody response may consist of a fraction of poorly neutralizing antibodies against 2018-2019 genotype C1 viruses among a subset of previously infected individuals. Epitope mapping of protective antibodies that recognize the emerging genotype C1 virus has implications for anti-EV-A71 MAbs and the vaccine field.
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Antígenos Virales/genética , Enterovirus Humano A/genética , Variación Genética , Genoma Viral , Genotipo , Enfermedad de Boca, Mano y Pie/genética , Animales , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/genética , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Niño , Preescolar , Enterovirus Humano A/inmunología , Enterovirus Humano A/aislamiento & purificación , Femenino , Enfermedad de Boca, Mano y Pie/epidemiología , Enfermedad de Boca, Mano y Pie/inmunología , Humanos , Masculino , Ratones , Ratones Transgénicos , TaiwánRESUMEN
ZFP36L1, a CCCH-type zinc finger protein, is an RNA-binding protein that participates in controlling cellular mRNA abundance and turnover by posttranscriptional regulation. Here, we demonstrated that ZFP36L1 has an important role in host defense against influenza A virus (IAV) infection. Overexpression of ZFP36L1 reduced IAV replication via translational repression of HA, M and NS RNA segment transcripts. IAV infection upregulated cellular ZFP36L1 expression, and endogenous ZFP36L1 knockdown significantly enhanced IAV replication. ZFP36L1 directly binds to IAV NS1 mRNA in the cytoplasm and blocks the expression and function of NS1 protein. Mutation of CCCH-type zinc finger domains of ZFP36L1 lost its antiviral potential and NS1 mRNA binding. Thus, ZFP36L1 can act as a host innate defense by targeting HA, M and NS mRNA transcripts to suppress viral protein translation.
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Factor 1 de Respuesta al Butirato/metabolismo , Proteínas de la Matriz Viral/genética , Proteínas no Estructurales Virales/genética , Células A549 , Animales , Sitios de Unión , Factor 1 de Respuesta al Butirato/química , Factor 1 de Respuesta al Butirato/genética , Perros , Células HEK293 , Humanos , Virus de la Influenza A/metabolismo , Virus de la Influenza A/fisiología , Células de Riñón Canino Madin Darby , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de la Matriz Viral/metabolismo , Proteínas no Estructurales Virales/metabolismo , Replicación ViralRESUMEN
Enterovirus 71 (EV71) is the major etiological agent contributing to the development of hand-foot-mouth disease (HFMD). There are not any global available vaccines or antibody drugs against EV71 released yet. In this study, we perform the virus immunization in a cost-effective and convenient approach by preparing virus particles from size exclusion and immunization of chicken. Polyclonal yolk-immunoglobulin (IgY) was simply purified from egg yolk and monoclonal single-chain variable fragments (scFv) were selected via phage display technology with two scFv libraries containing 6.0 × 106 and 1.3 × 107 transformants. Specific clones were enriched after 5 rounds of bio-panning and four identical genes were classified after the sequence analysis. Moreover, the higher mutation rates were revealed in the CDR regions, especially in the CDR3. IgY showed specific binding activities to both EV71-infected and Coxsackievirus 16-infected cell lysates and high infectivity inhibitory activity of EV71. However, while IgY detected a 37 kDa protein, the selected scFv seemingly detected higher size proteins which could be cell protein instead of EV71 proteins. Despite the highly effective chicken antibody generation, the purity of virus particles prepared by size exclusion is the limitation of this study, and further characterization should be carried out rigorously.
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Enterovirus Humano A , Enterovirus , Enfermedad de Boca, Mano y Pie , Anticuerpos de Cadena Única , Animales , Virión/genética , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/metabolismo , Yema de Huevo , PollosRESUMEN
The pathogenic human enterovirus EV-A71 has raised serious public health concerns. A hallmark of EV-A71 infection is the distortion of host transcriptomes in favour of viral replication. While high-throughput approaches have been exploited to dissect these gene dysregulations, they do not fully capture molecular perturbations at the single-cell level and in a physiologically relevant context. In this study, we applied a single-cell RNA sequencing approach on infected differentiated enterocyte cells (C2BBe1), which model the gastrointestinal epithelium targeted initially by EV-A71. Our single-cell analysis of EV-A71-infected culture provided several lines of illuminating observations: 1) This systems approach demonstrated extensive cell-to-cell variation in a single culture upon viral infection and delineated transcriptomic differences between the EV-A71-infected and bystander cells. 2) By analysing expression profiles of known EV-A71 receptors and entry facilitation factors, we found that ANXA2 was closely correlated in expression with the viral RNA in the infected population, supporting its role in EV-A71 entry in the enteric cells. 3) We further catalogued dysregulated lncRNAs elicited by EV-A71 infection and demonstrated the functional implication of lncRNA CYTOR in promoting EV-A71 replication. Viewed together, our single-cell transcriptomic analysis illustrated at the single-cell resolution the heterogeneity of host susceptibility to EV-A71 and revealed the involvement of lncRNAs in host antiviral response.
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Enterovirus Humano A/patogenicidad , Interacciones Huésped-Patógeno/genética , Transcriptoma , Células Cultivadas , Enterocitos/metabolismo , Enterocitos/patología , Enterocitos/virología , Enterovirus Humano A/genética , Enterovirus Humano A/inmunología , Infecciones por Enterovirus/genética , Infecciones por Enterovirus/inmunología , Infecciones por Enterovirus/patología , Infecciones por Enterovirus/virología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Interacciones Huésped-Patógeno/inmunología , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Mucosa Intestinal/virología , ARN Largo no Codificante/genética , Análisis de la Célula Individual , Replicación Viral/genéticaRESUMEN
There is conclusive evidential support for the existence of virus-derived small RNA (vsRNA) in mammals. Two types of vsRNA have been reported from picornaviruses. The first is virus-derived short-interfering RNA (vsiRNA) that is processed from viral double-stranded RNA intermediates during RNA replication. The other is small RNA derived from the highly base-paired single-stranded genomic region, e.g. the internal ribosome entry site (IRES) of picornaviruses. vsiRNA interacts with the Argonaute protein to control viral RNA replication through the process of RNA interference. However, the function of structure-based vsRNA is largely unknown. We previously identified vsRNA1 generated from the enterovirus-A71 (EV-A71) IRES region by the endogenous enzyme Dicer. Exogenous vsRNA1 can inhibit IRES activity both in vivo and in vitro, hence viral replication is inhibited. Here we describe key methods used to characterize vsRNA, including annotation by next-generation sequencing, abundance measurement by Northern blotting, determination of Dicer-dependence by gel-shift assay and in vitro cleavage assay, and the inhibitory effect on IRES activity via in vitro translation assay.
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Northern Blotting/métodos , Enterovirus Humano A/genética , Genoma Viral , ARN Viral/análisis , Animales , Línea Celular Tumoral , ARN Helicasas DEAD-box , Electroforesis en Gel de Poliacrilamida/métodos , Ensayo de Cambio de Movilidad Electroforética/métodos , Regulación Viral de la Expresión Génica , Humanos , Sitios Internos de Entrada al Ribosoma/genética , Ratones , Interferencia de ARN , ARN Interferente Pequeño , ARN Viral/metabolismo , Ribonucleasa III , Análisis de Secuencia de ARN/métodos , Replicación Viral/genéticaRESUMEN
Coxsackievirus A16 (CA16) is one of the major causative agents of hand, foot, and mouth disease (HFMD). Children aged <5 years are the most affected by CA16 HFMD globally. Although clinical symptoms of CA16 infections are usually mild, severe complications, such as aseptic meningitis or even death, have been recorded. Currently, no vaccine or antiviral therapy for CA16 infection exists. Single-chain variable fragment (scFv) antibodies significantly inhibit viral infection and could be a potential treatment for controlling the infection. In this study, scFv phage display libraries were constructed from splenocytes of a laying hen immunized with CA16-infected lysate. The pComb3X vector containing the scFv genes was introduced into ER2738 Escherichia coli and rescued by helper phages to express scFv molecules. After screening with five cycles of bio-panning, an effective scFv antibody showing favorable binding activity to proteins in CA16-infected lysate on ELISA plates was selected. Importantly, the selected scFv clone showed a neutralizing capability against the CA16 virus and cross-reacted with viral proteins in EV71-infected lysate. Intriguingly, polyclonal IgY antibody not only showed binding specificity against proteins in CA16-infected lysate but also showed significant neutralization activities. Nevertheless, IgY-binding protein did not cross-react with proteins in EV71-infected lysate. These results suggest that the IgY- and scFv-binding protein antibodies provide protection against CA16 viral infection in in vitro assays and may be potential candidates for treating CA16 infection in vulnerable young children.
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Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Pollos/inmunología , Enterovirus/inmunología , Animales , Especificidad de Anticuerpos , Línea Celular Tumoral , Humanos , Anticuerpos de Cadena Única/inmunología , Vacunas Virales/inmunologíaRESUMEN
BACKGROUND: Enterovirus A71 (EV-A71) has been noted for its tendency to lead to neurological manifestations in young children and infants. Although the alimentary tract has been identified as the primary replication site of this virus, how EV-A71 replicates in the gut and is transmitted to other organs remains unclear. METHODS: By using differentiated C2BBe1 cells as a model, we observed that intestinal epithelial cells (IECs) were permissive to EV-A71 infection, and viral particles were released in a nonlytic manner. RESULTS: The coexistence of active caspase 3 and EV-A71 protein was observed in the infected undifferentiated C2BBe1 and RD cells but not in the infected differentiated C2BBe1 cells. Furthermore, EV-A71 infection caused differentiated C2BBe1 and intestinal organoids to secrete exosomes containing viral components and have the ability to establish active infection. Inhibition of the exosome pathway decreased EV-A71 replication and release in IECs and increased the survival rates of infected animals. CONCLUSIONS: Our findings showed that EV-A71 is able to be actively replicated in enterocytes, and that the exosome pathway is involved in the nonlytic release of viral particles, which may be useful for developing antiviral strategies.
Asunto(s)
Enterovirus Humano A/fisiología , Células Epiteliales/metabolismo , Células Epiteliales/virología , Exosomas/metabolismo , Animales , Diferenciación Celular , Enterovirus , Enterovirus Humano A/genética , Enterovirus Humano A/crecimiento & desarrollo , Infecciones por Enterovirus/virología , Humanos , Ratones Transgénicos , ARN Viral/genética , ARN Viral/metabolismo , Replicación ViralRESUMEN
We discovered that neuropilin 1 (NRP1) is a new receptor candidate to mediate enterovirus A71 (EVA71) into cells. In the engineered form as a decoy receptor, NRP1 was able to recognize and neutralize EVA71 but not enterovirus D68 or coxsackievirus B3 (CVB3). NRP1 recognizes EVA71 through a novel domain on the VP3 capsid protein. The principle in the design, engineering, and refinement of the NRP1-based decoy receptor described in this study represents a general and well-suited antiviral strategy.
Asunto(s)
Enterovirus Humano A , Infecciones por Enterovirus , Enterovirus , Enterovirus Humano A/genética , Humanos , Neuropilina-1/genética , Receptores Virales/genéticaRESUMEN
In the past few decades, enterovirus A71 (EVA71) has caused devastating outbreaks in the Asia-Pacific region, resulting in serious sequelae in infected young children. No preventive or therapeutic interventions are currently available for curing EVA71 infection, highlighting a great unmet medical need for this disease. Here, we showed that one novel single-domain antibody (sdAb), F1, isolated from an immunized llama, could alleviate EVA71 infection both in vitro and in vivo We also confirmed that the sdAb clone F1 recognizes EVA71 through a novel conformational epitope comprising the highly conserved region of VP3 capsid protein by using competitive-binding and overlapping-peptide enzyme-linked immunosorbent assays (ELISAs). Because of the virion's icosahedral structure, we reasoned that adjacent epitopes must be clustered within molecular ranges that may be simultaneously bound by an engineered antibody with multiple valency. Therefore, two single-domain binding modules (F1) were fused to generate an sdAb-in-tandem design so that the capture of viral antigens could be further increased by valency effects. We showed that the tetravalent construct F1×F1-hFc, containing two sdAb-in-tandem on a fragment crystallizable (Fc) scaffold, exhibits more potent neutralization activity against EVA71 than does the bivalent sdAb F1-hFc by at least 5.8-fold. We also demonstrated that, using a human scavenger receptor class B member 2 (hSCARB2) transgenic mouse model, a half dose of the F1×F1-hFc provided better protection against EVA71 infection than did the F1-hFc. Thus, our study furnishes important insights into multivalent sdAb engineering against viral infection and provides a novel strategic deployment approach for preparedness of emerging infectious diseases such as EVA71.