RESUMO
The activation of the host adaptive immune system is crucial for eliminating viruses. However, influenza infection often suppresses the innate immune response that precedes adaptive immunity, and the adaptive immune responses are typically delayed. Dendritic cells, serving as professional antigen-presenting cells, have a vital role in initiating the adaptive immune response. In this study, an immuno-stimulating antiviral system (ISAS) is introduced, which is composed of the immuno-stimulating adjuvant lipopeptide Pam3CSK4 that acts as a scaffold onto which it is covalently bound 3 to 4 influenza-inhibiting peptides. The multivalent display of peptides on the scaffold leads to a potent inhibition against H1N1 (EC50 = 20 nM). Importantly, the resulting lipopeptide, Pam3FDA, shows an irreversible inhibition mechanism. The chemical modification of peptides on the scaffold maintains Pam3CSK4's ability to stimulate dendritic cell maturation, thereby rendering Pam3FDA a unique antiviral. This is attributed to its immune activation capability, which also acts in synergy to expedite viral elimination.
Assuntos
Células Dendríticas , Lipopeptídeos , Lipopeptídeos/química , Lipopeptídeos/farmacologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Células Dendríticas/imunologia , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/imunologia , Antivirais/farmacologia , Antivirais/química , Humanos , AnimaisRESUMO
Enteroviruses (EVs) are among the most prevalent viruses worldwide. They are characterized by a high genetic and phenotypic diversity, being able to cause a plethora of symptoms. EV-D68, a respiratory EV, and EV-D94, an enteric EV, represent an interesting paradigm of EV tropism heterogeneity. They belong to the same species, but display distinct phenotypic characteristics and in vivo tropism. Here, we used these two viruses as well as relevant 3D respiratory, intestinal and neural tissue culture models, to highlight key distinctive features of enteric and respiratory EVs. We emphasize the critical role of temperature in restricting EV-D68 tissue tropism. Using transcriptomic analysis, we underscore fundamental differences between intestinal and respiratory tissues, both in the steady-state and in response to infection. Intestinal tissues present higher cell proliferation rate and are more immunotolerant than respiratory tissues. Importantly, we highlight the different strategies applied by EV-D94 and EV-D68 towards the host antiviral response of intestinal and respiratory tissues. EV-D68 strongly activates antiviral pathways while EV-D94, on the contrary, barely induces any host defense mechanisms. In summary, our study provides an insightful characterization of the differential pathogenesis of EV-D68 and EV-D94 and the interplay with their main target tissues.
Assuntos
Enterovirus Humano D , Infecções por Enterovirus , Enterovirus , Infecções Respiratórias , Antígenos Virais , Antivirais , Enterovirus Humano D/fisiologia , Humanos , TropismoRESUMO
Peripheral allogeneic hematopoietic stem cell transplant recipients are the most vulnerable patients to community-acquired respiratory viruses such as respiratory syncytial virus, influenza virus, or others. These patients are likely to develop severe acute viral infections; community-acquired respiratory viruses have also been identified as triggers of bronchiolitis obliterans (BO). BO is a manifestation of pulmonary graft-versus-host disease, most often leading to irreversible ventilatory impairment. To date, there are no data on whether Severe acute respiratory syndrome âcoronavirus 2 (SARS-CoV-2) could be a trigger for BO. Here, we report the first report of a case of bronchiolitis obliterans syndrome following SARS-CoV-2 infection occurring 10 months after allogeneic hematopoietic stem cell transplant with a flare of underlying extra thoracic graft-versus-host disease. This observation provides a new perspective and should be of particular interest to clinicians, suggesting the need for close monitoring of pulmonary function test (PFTs) after SARS-CoV-2 infection. The mechanisms leading to bronchiolitis obliterans syndrome after SARS-CoV-2 infection require further investigation.
Assuntos
Síndrome de Bronquiolite Obliterante , Bronquiolite Obliterante , COVID-19 , Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Humanos , SARS-CoV-2 , Bronquiolite Obliterante/etiologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversosRESUMO
Influenza virus is one of the main causes of respiratory infections worldwide. Despite the availability of seasonal vaccines and antivirals, influenza virus infections cause an important health and economic burden. Therefore, the need to identify alternative antiviral strategies persists. In this study, we identified non-steroidal estrogens as potent inhibitors of influenza virus due to their interaction with the hemagglutinin protein, preventing viral entry. This activity is maintained in vitro, ex vivo, and in vivo. Therefore, we found a new domain to target on the hemagglutinin and a class of compounds that could be further optimized for influenza treatment.
Assuntos
Estrogênios não Esteroides , Influenza Humana , Infecções por Orthomyxoviridae , Orthomyxoviridae , Humanos , Hemaglutininas , Infecções por Orthomyxoviridae/tratamento farmacológico , Infecções por Orthomyxoviridae/prevenção & controle , Orthomyxoviridae/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Antivirais/farmacologiaRESUMO
Heparin has been known to be a broad-spectrum inhibitor of viral infection for almost 70 years, and it has been used as a medication for almost 90 years due to its anticoagulant effect. This nontoxic biocompatible polymer efficiently binds to many types of viruses and prevents their attachment to cell membranes. However, the anticoagulant properties are limiting their use as an antiviral drug. Many heparin-like compounds have been developed throughout the years; however, the reversible nature of the virus inhibition mechanism has prevented their translation to the clinics. In vivo, such a mechanism requires the unrealistic maintenance of the concentration above the binding constant. Recently, we have shown that the addition of long hydrophobic linkers to heparin-like compounds renders the interaction irreversible while maintaining the low-toxicity and broad-spectrum activity. To date, such hydrophobic linkers have been used to create heparin-like gold nanoparticles and ß-cyclodextrins. The former achieves a nanomolar inhibition concentration on a non-biodegradable scaffold. The latter, on a fully biodegradable scaffold, shows only a micromolar inhibition concentration. Here, we report that the addition of hydrophobic linkers to a new type of multifunctional scaffold (dendritic polyglycerol, dPG) creates biocompatible compounds endowed with nanomolar activity. Furthermore, we present an in-depth analysis of the molecular design rules needed to achieve irreversible virus inhibition. The most active compound (dPG-5) showed nanomolar activity against herpes simplex virus 2 (HSV-2) and respiratory syncytial virus (RSV), giving a proof-of-principle for broad-spectrum while keeping low-toxicity. In addition, we demonstrate that the virucidal activity leads to the release of viral DNA upon the interaction between the virus and our polyanionic dendritic polymers. We believe that this paper will be a stepping stone toward the design of a new class of irreversible nontoxic broad-spectrum antivirals.
Assuntos
Nanopartículas Metálicas , Vírus , Anticoagulantes/farmacologia , Antivirais/química , Antivirais/farmacologia , Glicerol , Ouro , Heparina/farmacologia , Polímeros/farmacologiaRESUMO
Viral infections are among the main causes of death worldwide, and we lack antivirals for the majority of viruses. Heparin-like sulfated or sulfonated compounds have been known for decades for their ability to prevent infection by heparan sulfate proteoglycan (HSPG)-dependent viruses but only in a reversible way. We have previously shown that gold nanoparticles and ß-cyclodextrins coated with mercapto-undecane sulfonic acid (MUS) inhibit HSPG-dependent viruses irreversibly while retaining the low-toxicity profile of most heparin-like compounds. In this work, we show that, in stark contrast to heparin, these compounds also inhibit different strains of influenza virus and vesicular stomatitis virus (VSV), which do not bind HSPG. The antiviral action is virucidal and irreversible for influenza A virus (H1N1), while for VSV, there is a reversible inhibition of viral attachment to the cell. These results further broaden the spectrum of activity of MUS-coated gold nanoparticles and ß-cyclodextrins.
Assuntos
Vírus da Influenza A Subtipo H1N1 , Nanopartículas Metálicas , Vírus , Antivirais/farmacologia , Ouro , Heparitina Sulfato/farmacologiaRESUMO
Enterovirus 71 (EV71) causes hand, foot and mouth disease, a mild and self-limited illness that is sometimes associated with severe neurological complications. EV71 neurotropic determinants remain ill-defined to date. We previously identified a mutation in the VP1 capsid protein (L97R) that was acquired over the course of a disseminated infection in an immunocompromised host. The mutation was absent in the respiratory tract but was present in the gut (as a mixed population) and in blood and cerebrospinal fluid (as a dominant species). In this study, we demonstrated that this mutation does not alter the dependence of EV71 on the human scavenger receptor class B2 (SCARB2), while it enables the virus to bind to the heparan sulfate (HS) attachment receptor and modifies viral tropism in cell lines and in respiratory, intestinal and neural tissues. Variants with VP197L or VP197R were able to replicate to high levels in intestinal and neural tissues and, to a lesser extent, in respiratory tissues, but their preferred entry site (from the luminal or basal tissue side) differed in respiratory and intestinal tissues and correlated with HS expression levels. These data account for the viral populations sequenced from the patient's respiratory and intestinal samples and suggest that improved dissemination, resulting from an acquired ability to bind HS, rather than specific neurotropism determinants, enabled the virus to reach and infect the central nervous system. Finally, we showed that iota-carrageenan, a highly sulfated polysaccharide, efficiently blocks the replication of HS-dependent variants in cells and 2D neural cultures. Overall, the results of this study emphasize the importance of HS binding in EV71 pathogenesis and open new avenues for the development of antiviral molecules that may prevent this virus's dissemination.
Assuntos
Proteínas do Capsídeo/genética , Enterovirus Humano A/fisiologia , Doença de Mão, Pé e Boca/virologia , Heparitina Sulfato/metabolismo , Tropismo Viral/genética , Animais , Enterovirus Humano A/patogenicidade , Doença de Mão, Pé e Boca/genética , Doença de Mão, Pé e Boca/metabolismo , Humanos , Proteínas de Membrana Lisossomal/metabolismo , Camundongos , Mutação , Receptores Depuradores/metabolismo , Replicação Viral/genéticaRESUMO
Despite their genetic similarities, enteric and respiratory enteroviruses (EVs) have highly heterogeneous biophysical properties and cause a vast diversity of human pathologies. In vitro differences include acid sensitivity, optimal growth temperature and tissue tropism, which reflect a preferential in vivo replication in the respiratory or gastrointestinal tract and are thus key determinants of EV virulence. To investigate the underlying cause of these differences, we generated chimeras at the capsid-level between EV-D68 (a respiratory EV) and EV-D94 (an enteric EV). Although some chimeras were nonfunctional, EV-D94 with both the capsid and 2A protease or the capsid only of EV-D68 were both viable. Using this latter construct, we performed several functional assays, which indicated that capsid proteins determine acid sensitivity and tropism in cell lines and in respiratory, intestinal and neural tissues. Additionally, capsid genes were shown to also participate in determining the optimal growth temperature, since EV-D94 temperature adaptation relied on single mutations in VP1, while constructs with EV-D68 capsid could not adapt to higher temperatures. Finally, we demonstrate that EV-D68 maintains residual binding-capacity after acid-treatment despite a loss of infectivity. In contrast, non-structural rather than capsid proteins modulate the innate immune response in tissues. These unique biophysical insights expose another layer in the phenotypic diversity of one of world's most prevalent pathogens and could aid target selection for vaccine or antiviral development.
Assuntos
Ácidos/química , Proteínas do Capsídeo/metabolismo , Infecções por Enterovirus/virologia , Enterovirus/fisiologia , Intestinos/virologia , Neurônios/virologia , Sistema Respiratório/virologia , Proteínas do Capsídeo/genética , Enterovirus/classificação , Infecções por Enterovirus/genética , Infecções por Enterovirus/metabolismo , Humanos , Temperatura , Tropismo ViralRESUMO
Viral respiratory infections are usually mild and self-limiting; still they exceptionally result in life-threatening infections in previously healthy children. To investigate a potential genetic cause, we recruited 120 previously healthy children requiring support in intensive care because of a severe illness caused by a respiratory virus. Using exome and transcriptome sequencing, we identified and characterized three rare loss-of-function variants in IFIH1, which encodes an RIG-I-like receptor involved in the sensing of viral RNA. Functional testing of the variants IFIH1 alleles demonstrated that the resulting proteins are unable to induce IFN-ß, are intrinsically less stable than wild-type IFIH1, and lack ATPase activity. In vitro assays showed that IFIH1 effectively restricts replication of human respiratory syncytial virus and rhinoviruses. We conclude that IFIH1 deficiency causes a primary immunodeficiency manifested in extreme susceptibility to common respiratory RNA viruses.
Assuntos
Predisposição Genética para Doença/genética , Síndromes de Imunodeficiência/genética , Helicase IFIH1 Induzida por Interferon/genética , Interferon beta/biossíntese , Vírus Sinciciais Respiratórios/imunologia , Infecções Respiratórias/virologia , Rhinovirus/imunologia , Adenosina Trifosfatases/genética , Pré-Escolar , Cuidados Críticos , Feminino , Variação Genética/genética , Humanos , Síndromes de Imunodeficiência/imunologia , Lactente , Recém-Nascido , Interferon beta/imunologia , Masculino , Estudos Prospectivos , Isoformas de Proteínas/genética , Infecções Respiratórias/imunologia , Replicação Viral/imunologiaRESUMO
Viral infections kill millions yearly. Available antiviral drugs are virus-specific and active against a limited panel of human pathogens. There are broad-spectrum substances that prevent the first step of virus-cell interaction by mimicking heparan sulfate proteoglycans (HSPG), the highly conserved target of viral attachment ligands (VALs). The reversible binding mechanism prevents their use as a drug, because, upon dilution, the inhibition is lost. Known VALs are made of closely packed repeating units, but the aforementioned substances are able to bind only a few of them. We designed antiviral nanoparticles with long and flexible linkers mimicking HSPG, allowing for effective viral association with a binding that we simulate to be strong and multivalent to the VAL repeating units, generating forces (â¼190 pN) that eventually lead to irreversible viral deformation. Virucidal assays, electron microscopy images, and molecular dynamics simulations support the proposed mechanism. These particles show no cytotoxicity, and in vitro nanomolar irreversible activity against herpes simplex virus (HSV), human papilloma virus, respiratory syncytial virus (RSV), dengue and lenti virus. They are active ex vivo in human cervicovaginal histocultures infected by HSV-2 and in vivo in mice infected with RSV.
Assuntos
Antivirais , Materiais Biomiméticos , Herpes Simples/tratamento farmacológico , Herpesvirus Humano 2/metabolismo , Nanopartículas , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Vírus Sinciciais Respiratórios/metabolismo , Animais , Antivirais/química , Antivirais/farmacologia , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Proteoglicanas de Heparan Sulfato/química , Proteoglicanas de Heparan Sulfato/farmacologia , Herpes Simples/metabolismo , Herpes Simples/patologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/química , Nanopartículas/uso terapêutico , Infecções por Vírus Respiratório Sincicial/metabolismo , Infecções por Vírus Respiratório Sincicial/patologiaRESUMO
BACKGROUND: The leading cause of acute illnesses, respiratory viruses, typically cause self-limited diseases, although severe complications can occur in fragile patients. Rhinoviruses (RVs), respiratory enteroviruses (EVs), influenza virus, respiratory syncytial viruses (RSVs), and coronaviruses are highly prevalent respiratory pathogens, but because of the lack of reliable animal models, their differential pathogenesis remains poorly characterized. OBJECTIVE: We sought to compare infections by respiratory viruses isolated from clinical specimens using reconstituted human airway epithelia. METHODS: Tissues were infected with RV-A55, RV-A49, RV-B48, RV-C8, and RV-C15; respiratory EV-D68; influenza virus H3N2; RSV-B; and human coronavirus (HCoV)-OC43. Replication kinetics, cell tropism, effect on tissue integrity, and cytokine secretion were compared. Viral adaptation and tissue response were assessed through RNA sequencing. RESULTS: RVs, RSV-B, and HCoV-OC43 infected ciliated cells and caused no major cell death, whereas H3N2 and EV-D68 induced ciliated cell loss and tissue integrity disruption. H3N2 was also detected in rare goblet and basal cells. All viruses, except RV-B48 and HCoV-OC43, altered cilia beating and mucociliary clearance. H3N2 was the strongest cytokine inducer, and HCoV-OC43 was the weakest. Persistent infection was observed in all cases. RNA sequencing highlighted perturbation of tissue metabolism and induction of a transient but important immune response at 4 days after infection. No majority mutations emerged in the viral population. CONCLUSION: Our results highlight the differential in vitro pathogenesis of respiratory viruses during the acute infection phase and their ability to persist under immune tolerance. These data help to appreciate the range of disease severity observed in vivo and the occurrence of chronic respiratory tract infections in immunocompromised hosts.
Assuntos
Infecções por Vírus de RNA/fisiopatologia , Infecções por Vírus de RNA/virologia , Mucosa Respiratória/virologia , Humanos , Vírus de RNARESUMO
Causing an international outbreak of respiratory disease, Enterovirus D68 quickly entered the closed circle of emerging viral pathogens of public health significance. As rapid and accurate detection of EV-D68 is essential for an efficient clinical management, we designed and validated a new highly efficient one-step quantitative rRT-PCR specific to EV-D68 VP4-VP2 region. With 100% specificity and 95.6% sensitivity to all EV-D68 strains, this new assay can be reliably used to detect and quantify EV-D68 in respiratory samples and represents an interesting additional tool for diagnosis as it targets an original region of the genome.
Assuntos
Enterovirus Humano D/isolamento & purificação , Infecções por Enterovirus/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/métodos , Infecções Respiratórias/virologia , Proteínas Estruturais Virais/genética , Surtos de Doenças , Enterovirus Humano D/genética , Infecções por Enterovirus/epidemiologia , Infecções por Enterovirus/virologia , Humanos , Filogenia , Infecções Respiratórias/diagnóstico , Estações do Ano , Sensibilidade e EspecificidadeRESUMO
Vitamin D has immunomodulatory properties in the defence against pathogens. Its insufficiency is a widespread feature of cystic fibrosis (CF) patients, which are repeatedly suffering from rhinovirus (RV)-induced pulmonary exacerbations.To investigate whether vitamin D has antiviral activity, primary bronchial epithelial cells from CF children were pre-treated with vitamin D and infected with RV16. Antiviral and anti-inflammatory activity of vitamin D was assessed. RV and LL-37 levels were measured in bronchoalveolar lavage (BAL) of CF children infected with RV.Vitamin D reduced RV16 load in a dose-dependent manner in CF cells (10(-7â )M, p<0.01). The antiviral response mediated by interferons remained unchanged by vitamin D in CF cells. Vitamin D did not exert anti-inflammatory properties in RV-infected CF cells. Vitamin D increased the expression of the antimicrobial peptide LL-37 up to 17.4-fold (p<0.05). Addition of exogenous LL-37 decreased viral replication by 4.4-fold in CF cells (p<0.05). An inverse correlation between viral load and LL-37 levels in CF BAL (r=-0.48, p<0.05) was observed.RV replication in primary CF bronchial cells was reduced by vitamin D through the induction of LL-37. Clinical studies are needed to determine the importance of an adequate control of vitamin D for prevention of virus-induced pulmonary CF exacerbations.
Assuntos
Catelicidinas/efeitos dos fármacos , Colecalciferol/farmacologia , Fibrose Cística/metabolismo , Células Epiteliais/efeitos dos fármacos , Rhinovirus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Vitaminas/farmacologia , Adolescente , Peptídeos Catiônicos Antimicrobianos , Brônquios/citologia , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/virologia , Estudos de Casos e Controles , Catelicidinas/metabolismo , Criança , Pré-Escolar , Fibrose Cística/virologia , Células Epiteliais/virologia , Feminino , Humanos , Lactente , Masculino , Reação em Cadeia da Polimerase em Tempo Real , Carga Viral , Vitamina D/farmacologiaRESUMO
UNLABELLED: Recombination is a widespread phenomenon that ensures both the stability and variation of RNA viruses. This phenomenon occurs with different frequencies within species of the Enterovirus genus. Intraspecies recombination is described frequently among non-rhinovirus enteroviruses but appears to be sporadic in rhinoviruses. Interspecies recombination is even rarer for rhinoviruses and mostly is related to ancient events which contributed to the speciation of these viruses. We reported that artificially engineered 5' untranslated region (UTR) interspecies rhinovirus/rhinovirus or rhinovirus/non-rhinovirus enterovirus recombinants are fully viable. Using a similar approach, we demonstrated in this study that exchanges of the P1-2A polyprotein region between members of the same rhinovirus species, but not between members of different species, give rise to competent chimeras. To further assess the rhinovirus intra- and interspecies recombination potential, we used artificially induced recombination by cotransfection of 5'-end-deleted and 3'-end-deleted and replication-deficient genomes. In this system, intraspecies recombination also resulted in viable viruses with high frequency, whereas no interspecies rhinovirus recombinants could be recovered. Mapping intraspecies recombination sites within the polyprotein highlighted recombinant hotspots in nonstructural genes and at gene boundaries. Notably, all recombinants occurring at gene junctions presented in-frame sequence duplications, whereas most intragenic recombinants were homologous. Taken together, our results suggest that only intraspecies recombination gives rise to viable rhinovirus chimeras in the polyprotein coding region and that recombination hotspots map to nonstructural genes with in-frame duplications at gene boundaries. These data provide new insights regarding the mechanism and limitations of rhinovirus recombination. IMPORTANCE: Recombination represents a means to ensure both the stability and the variation of RNA viruses. While intraspecies recombination is described frequently among non-rhinovirus enteroviruses, it seems to occur more rarely in rhinoviruses. Interspecies recombination is even rarer in this virus group and is mostly related to ancient events, which contributed to its speciation. We used engineered chimeric genomes and artificially induced RNA recombination to study experimentally the recombination potential of rhinoviruses and analyze recombination sites. Our results suggest that only intraspecies recombination gives rise to viable chimeras in the polyprotein coding region. Furthermore, characterization of intraspecies chimeras provides new insight into putative recombination hotspots within the polyprotein. In summary, we applied two powerful and complementary experimental approaches to improve current knowledge on rhinovirus recombination.
Assuntos
Quimera/genética , Produtos do Gene env/genética , Transferência Genética Horizontal/genética , Engenharia Genética/métodos , Rhinovirus/genética , Regiões 5' não Traduzidas/genética , Sequência de Bases , Mapeamento Cromossômico , Imunofluorescência , Técnicas de Transferência de Genes , Células HeLa , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
We report a fatal case of acute lower respiratory tract disease with human rhinovirus C (HRV-C) as the unique cause in a 19-month-old girl with a history of repeated episodes of bronchiolitis. HRV-C type 8 nucleic acids were observed in respiratory, stool, and cerebrospinal fluid samples, and infectious virions were isolated from patient serum after inoculation onto reconstituted airway epithelia.
Assuntos
Sangue/virologia , Bronquiolite/etiologia , Infecções por Picornaviridae/diagnóstico , Infecções por Picornaviridae/virologia , Rhinovirus/isolamento & purificação , Viremia/diagnóstico , Viremia/virologia , Bronquiolite/complicações , Líquido Cefalorraquidiano/virologia , Evolução Fatal , Fezes/virologia , Feminino , Humanos , Lactente , Infecções por Picornaviridae/patologia , Sistema Respiratório/virologia , Rhinovirus/classificação , Rhinovirus/genética , Viremia/patologia , Cultura de VírusRESUMO
Enterovirus 71 (EV71) is one of the most virulent enteroviruses, but the specific molecular features that enhance its ability to disseminate in humans remain unknown. We analyzed the genomic features of EV71 in an immunocompromised host with disseminated disease according to the different sites of infection. Comparison of five full-length genomes sequenced directly from respiratory, gastrointestinal, nervous system, and blood specimens revealed three nucleotide changes that occurred within a five-day period: a non-conservative amino acid change in VP1 located within the BC loop (L97R), a region considered as an immunogenic site and possibly important in poliovirus host adaptation; a conservative amino acid substitution in protein 2B (A38V); and a silent mutation in protein 3D (L175). Infectious clones were constructed using both BrCr (lineage A) and the clinical strain (lineage C) backgrounds containing either one or both non-synonymous mutations. In vitro cell tropism and competition assays revealed that the VP197 Leu to Arg substitution within the BC loop conferred a replicative advantage in SH-SY5Y cells of neuroblastoma origin. Interestingly, this mutation was frequently associated in vitro with a second non-conservative mutation (E167G or E167A) in the VP1 EF loop in neuroblastoma cells. Comparative models of these EV71 VP1 variants were built to determine how the substitutions might affect VP1 structure and/or interactions with host cells and suggest that, while no significant structural changes were observed, the substitutions may alter interactions with host cell receptors. Taken together, our results show that the VP1 BC loop region of EV71 plays a critical role in cell tropism independent of EV71 lineage and, thus, may have contributed to dissemination and neurotropism in the immunocompromised patient.
Assuntos
Enterovirus Humano A/fisiologia , Enterovirus Humano A/patogenicidade , Infecções por Enterovirus/virologia , Neurônios/virologia , Proteínas Estruturais Virais/genética , Ligação Viral , Adulto , Substituição de Aminoácidos , Animais , Líquido da Lavagem Broncoalveolar/virologia , Células CACO-2 , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , DNA Viral/genética , Enterovirus Humano A/genética , Enterovirus Humano A/imunologia , Fezes/virologia , Humanos , Hospedeiro Imunocomprometido , Masculino , Mutação , Neuroblastoma , Células Vero , Proteínas Estruturais Virais/sangue , Proteínas Estruturais Virais/líquido cefalorraquidiano , Proteínas Estruturais Virais/imunologiaRESUMO
This study introduces the nanobromhexine lipid particle (NBL) platform designed for effective pulmonary drug delivery. Inspired by respiratory virus transport mechanisms, NBL address challenges associated with mucus permeation and inflammation in pulmonary diseases. Composed of low molecular weight polyethylene glycol-coated lipid nanoparticles with bromhexine hydrochloride, NBL exhibit a size of 118 ± 24 nm, a neutral zeta potential, osmolarity of 358 ± 28 mOsmol/kg, and a pH of 6.5. Nebulizing without leakage and showing no toxicity to epithelial cells, NBL display mucoadhesive properties with a 60% mucin-binding efficiency. They effectively traverse the dense mucus layer of Calu-3 cultures in an air-liquid interface, as supported by a 55% decrease in MUC5AC density and a 29% increase in nanoparticles internalization compared to non-exposed cells. In assessing immunomodulatory effects, NBL treatment in SARS-CoV-2-infected lung cells leads to a 40-fold increase in anti-inflammatory MUC1 gene expression, a proportional reduction in pro-inflammatory IL-6 expression, and elevated anti-inflammatory IL-10 expression. These findings suggest a potential mechanism to regulate the excessive IL-6 expression triggered by virus infection. Therefore, the NBL platform demonstrates promising potential for efficient pulmonary drug delivery and immunomodulation, offering a novel approach to addressing mucus permeation and inflammation in pulmonary diseases.
Assuntos
Pulmão , Nanopartículas , SARS-CoV-2 , Nanopartículas/administração & dosagem , Humanos , Pulmão/metabolismo , SARS-CoV-2/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Imunomodulação , Linhagem Celular , Mucina-1/metabolismo , COVID-19 , Lipídeos/química , Lipídeos/administração & dosagem , Muco/metabolismo , Polietilenoglicóis/química , Células Epiteliais/metabolismo , Células Epiteliais/efeitos dos fármacos , Tratamento Farmacológico da COVID-19 , Mucina-5AC/metabolismo , LipossomosRESUMO
Enterovirus A71 (EV-A71) causes hand, foot, and mouth disease outbreaks with neurological complications and deaths. We previously isolated an EV-A71 variant in the stool, cerebrospinal fluid, and blood of an immunocompromised patient who had a leucine-to-arginine substitution on the VP1 capsid protein, resulting in increased heparin sulfate binding. We show here that this mutation increases the virus's pathogenicity in orally infected mice with depleted B cells, which mimics the patient's immune status, and increases susceptibility to neutralizing antibodies. However, a double mutant with even greater heparin sulfate affinity is not pathogenic, suggesting that increased heparin sulfate affinity may trap virions in peripheral tissues and reduce neurovirulence. This research sheds light on the increased pathogenicity of variant with heparin sulfate (HS)-binding ability in individuals with decreased B cell immunity.
Assuntos
Enterovirus Humano A , Infecções por Enterovirus , Enterovirus , Humanos , Animais , Camundongos , Enterovirus/genética , Enterovirus Humano A/genética , Antígenos Virais/metabolismo , Heparitina Sulfato/metabolismo , Heparina/metabolismoRESUMO
Introduction: Rhinovirus (RV) infections constitute one of the main triggers of asthma exacerbations and an important burden in pediatric yard. However, the mechanisms underlying this association remain poorly understood. Methods: In the present study, we compared infections of in vitro reconstituted airway epithelia originating from asthmatic versus healthy donors with representative strains of RV-A major group and minor groups, RV-C, RV-B, and the respiratory enterovirus EV-D68. Results: We found that viral replication was higher in tissues derived from asthmatic donors for all tested viruses. Viral receptor expression was comparable in non-infected tissues from both groups. After infection, ICAM1 and LDLR were upregulated, while CDHR3 was downregulated. Overall, these variations were related to viral replication levels. The presence of the CDHR3 asthma susceptibility allele (rs6967330) was not associated with increased RV-C replication. Regarding the tissue response, a significantly higher interferon (IFN) induction was demonstrated in infected tissues derived from asthmatic donors, which excludes a defect in IFN-response. Unbiased transcriptomic comparison of asthmatic versus control tissues revealed significant modifications, such as alterations of cilia structure and motility, in both infected and non-infected tissues. These observations were supported by a reduced mucociliary clearance and increased mucus secretion in non-infected tissues from asthmatic donors. Discussion: Altogether, we demonstrated an increased permissiveness and susceptibility to RV and respiratory EV infections in HAE derived from asthmatic patients, which was associated with a global alteration in epithelial cell functions. These results unveil the mechanisms underlying the pathogenesis of asthma exacerbation and suggest interesting therapeutic targets.
RESUMO
Chronic hepatitis C (CHC) is associated with the development of metabolic disorders, including both hepatic and extra-hepatic insulin resistance (IR). Here, we aimed at identifying liver-derived factor(s) potentially inducing peripheral IR and uncovering the mechanisms whereby HCV can regulate the action of these factors. We found ANGPTL4 (Angiopoietin Like 4) mRNA expression levels to positively correlate with HCV RNA (r = 0.46, p < 0.03) and HOMA-IR score (r = 0.51, p = 0.01) in liver biopsies of lean CHC patients. Moreover, we observed an upregulation of ANGPTL4 expression in two models recapitulating HCV-induced peripheral IR, i.e. mice expressing core protein of HCV genotype 3a (HCV-3a core) in hepatocytes and hepatoma cells transduced with HCV-3a core. Treatment of differentiated myocytes with recombinant ANGPTL4 reduced insulin-induced Akt-Ser473 phosphorylation. In contrast, conditioned medium from ANGPTL4-KO hepatoma cells prevented muscle cells from HCV-3a core induced IR. Treatment of HCV-3a core expressing HepG2 cells with PPARγ antagonist resulted in a decrease of HCV-core induced ANGPTL4 upregulation. Together, our data identified ANGPTL4 as a potential driver of HCV-induced IR and may provide working hypotheses aimed at understanding the pathogenesis of IR in the setting of other chronic liver disorders.