RESUMEN
Since May 2023, a novel combination of neuraminidase mutations, I223V + S247N, has been detected in influenza A(H1N1)pdm09 viruses collected in countries spanning 5 continents, mostly in Europe (67/101). The viruses belong to 2 phylogenetically distinct groups and display ≈13-fold reduced inhibition by oseltamivir while retaining normal susceptibility to other antiviral drugs.
Asunto(s)
Antivirales , Farmacorresistencia Viral , Subtipo H1N1 del Virus de la Influenza A , Gripe Humana , Neuraminidasa , Oseltamivir , Filogenia , Oseltamivir/farmacología , Oseltamivir/uso terapéutico , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H1N1 del Virus de la Influenza A/genética , Humanos , Antivirales/farmacología , Antivirales/uso terapéutico , Gripe Humana/virología , Gripe Humana/tratamiento farmacológico , Gripe Humana/epidemiología , Neuraminidasa/antagonistas & inhibidores , Neuraminidasa/genética , Farmacorresistencia Viral/genética , MutaciónRESUMEN
Epigenetic memory for signal-dependent transcription has remained elusive. So far, the concept of epigenetic memory has been largely limited to cell-autonomous, preprogrammed processes such as development and metabolism. Here we show that IFNß stimulation creates transcriptional memory in fibroblasts, conferring faster and greater transcription upon restimulation. The memory was inherited through multiple cell divisions and led to improved antiviral protection. Of â¼2,000 IFNß-stimulated genes (ISGs), about half exhibited memory, which we define as memory ISGs. The rest, designated nonmemory ISGs, did not show memory. Surprisingly, mechanistic analysis showed that IFN memory was not due to enhanced IFN signaling or retention of transcription factors on the ISGs. We demonstrated that this memory was attributed to accelerated recruitment of RNA polymerase II and transcription/chromatin factors, which coincided with acquisition of the histone H3.3 and H3K36me3 chromatin marks on memory ISGs. Similar memory was observed in bone marrow macrophages after IFNγ stimulation, suggesting that IFN stimulation modifies the shape of the innate immune response. Together, external signals can establish epigenetic memory in mammalian cells that imparts lasting adaptive performance upon various somatic cells.
Asunto(s)
Células de la Médula Ósea/inmunología , División Celular/inmunología , Epigénesis Genética/inmunología , Inmunidad Innata , Interferón beta/inmunología , Macrófagos/inmunología , Transducción de Señal/inmunología , Transcripción Genética/inmunología , Animales , Células de la Médula Ósea/citología , División Celular/genética , Cromatina/genética , Cromatina/inmunología , Histonas/genética , Histonas/inmunología , Interferón beta/genética , Macrófagos/citología , Ratones , Ratones Mutantes , ARN Polimerasa II/genética , ARN Polimerasa II/inmunología , Transducción de Señal/genética , Factores de Transcripción/genética , Factores de Transcripción/inmunologíaRESUMEN
Susceptibility of influenza A viruses to baloxavir can be affected by changes at amino acid residue 38 in the polymerase acidic (PA) protein. Information on replicative fitness of PA-I38-substituted viruses remains sparse. We demonstrated that substitutions I38L/M/S/T not only had a differential effect on baloxavir susceptibility (9- to 116-fold) but also on in vitro replicative fitness. Although I38L conferred undiminished growth, other substitutions led to mild attenuation. In a ferret model, control viruses outcompeted those carrying I38M or I38T substitutions, although their advantage was limited. These findings offer insights into the attributes of baloxavir-resistant viruses needed for informed risk assessment.
Asunto(s)
Antivirales/uso terapéutico , Farmacorresistencia Viral/genética , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/genética , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Oxazinas/uso terapéutico , Piridinas/uso terapéutico , Tiepinas/uso terapéutico , Triazinas/uso terapéutico , Replicación Viral/genética , Sustitución de Aminoácidos , Animales , Dibenzotiepinas , Modelos Animales de Enfermedad , Perros , Hurones , Secuenciación de Nucleótidos de Alto Rendimiento , Células de Riñón Canino Madin Darby , Masculino , Pruebas de Sensibilidad Microbiana , Morfolinas , Infecciones por Orthomyxoviridae/virología , Piridonas , ARN Polimerasa Dependiente del ARN/genética , Estaciones del Año , Resultado del Tratamiento , Proteínas Virales/genéticaRESUMEN
Baloxavir showed broad-spectrum in vitro replication inhibition of 4 types of influenza viruses (90% effective concentration range 1.2-98.3 nmol/L); susceptibility pattern was influenza A Ë B Ë C Ë D. This drug also inhibited influenza A viruses of avian and swine origin, including viruses that have pandemic potential and those resistant to neuraminidase inhibitors.
Asunto(s)
Antivirales/farmacología , Gammainfluenzavirus/efectos de los fármacos , Virus de la Influenza A/efectos de los fármacos , Virus de la Influenza B/efectos de los fármacos , Oxazinas/farmacología , Piridinas/farmacología , Tiepinas/farmacología , Thogotovirus/efectos de los fármacos , Triazinas/farmacología , Animales , Pollos/virología , Dibenzotiepinas , Perros , Humanos , Gripe Aviar/tratamiento farmacológico , Gripe Aviar/virología , Gripe Humana/tratamiento farmacológico , Gripe Humana/virología , Células de Riñón Canino Madin Darby/virología , Pruebas de Sensibilidad Microbiana , Morfolinas , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Infecciones por Orthomyxoviridae/veterinaria , Infecciones por Orthomyxoviridae/virología , Piridonas , Porcinos/virología , Enfermedades de los Porcinos/tratamiento farmacológico , Enfermedades de los Porcinos/virologíaRESUMEN
The anti-influenza therapeutic baloxavir targets cap-dependent endonuclease activity of polymerase acidic (PA) protein. We monitored baloxavir susceptibility in the United States with next generation sequencing analysis supplemented by phenotypic one-cycle infection assay. Analysis of PA sequences of 6,891 influenza A and B viruses collected during 2016/17 and 2017/18 seasons showed amino acid substitutions: I38L (two A(H1N1)pdm09 viruses), E23G (two A(H1N1)pdm09 viruses) and I38M (one A(H3N2) virus); conferring 4-10-fold reduced susceptibility to baloxavir.
Asunto(s)
Sustitución de Aminoácidos/efectos de los fármacos , Antivirales/farmacología , Farmacorresistencia Viral/genética , Inhibidores Enzimáticos/farmacología , Subtipo H3N2 del Virus de la Influenza A/efectos de los fármacos , Virus de la Influenza B/efectos de los fármacos , Gripe Humana/tratamiento farmacológico , Oxazinas/farmacología , Piridinas/farmacología , Tiepinas/farmacología , Triazinas/farmacología , Sustitución de Aminoácidos/genética , Antivirales/uso terapéutico , Dibenzotiepinas , Humanos , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/aislamiento & purificación , Virus de la Influenza B/genética , Virus de la Influenza B/aislamiento & purificación , Gripe Humana/epidemiología , Gripe Humana/virología , Pruebas de Sensibilidad Microbiana , Morfolinas , Piridonas , Estaciones del Año , Vigilancia de Guardia , Estados Unidos , Proteínas Virales/genéticaRESUMEN
Actively transcribed genes are enriched with the histone variant H3.3. Although H3.3 deposition has been linked to transcription, mechanisms controlling this process remain elusive. We investigated the role of the histone methyltransferase Wolf-Hirschhorn syndrome candidate 1 (WHSC1) (NSD2/MMSET) in H3.3 deposition into interferon (IFN) response genes. IFN treatment triggered robust H3.3 incorporation into activated genes, which continued even after cessation of transcription. Likewise, UV radiation caused H3.3 deposition in UV-activated genes. However, in Whsc1(-/-) cells IFN- or UV-triggered H3.3 deposition was absent, along with a marked reduction in IFN- or UV-induced transcription. We found that WHSC1 interacted with the bromodomain protein 4 (BRD4) and the positive transcription elongation factor b (P-TEFb) and facilitated transcriptional elongation. WHSC1 also associated with HIRA, the H3.3-specific histone chaperone, independent of BRD4 and P-TEFb. WHSC1 and HIRA co-occupied IFN-stimulated genes and supported prolonged H3.3 incorporation, leaving a lasting transcriptional mark. Our results reveal a previously unrecognized role of WHSC1, which links transcriptional elongation and H3.3 deposition into activated genes through two molecularly distinct pathways.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Chaperonas de Histonas/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Proteínas de Ciclo Celular/genética , Células Cultivadas , Cromatina/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/efectos de la radiación , Chaperonas de Histonas/genética , N-Metiltransferasa de Histona-Lisina/genética , Histonas/genética , Interferón beta/farmacología , Ratones , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Elongación de la Transcripción Genética , Factores de Transcripción/genética , Rayos UltravioletaRESUMEN
BACKGROUND: Understanding respiratory syncytial virus (RSV) global epidemiology is important to inform future prevention strategies. METHODS: Hospitalized infants <1-year-old with acute illness were enrolled prospectively in Albania, Jordan, Nicaragua, and Philippines during respiratory seasons in 2015-2017. Medical chart review, parental interview, and post-discharge follow up were conducted. Respiratory specimens were tested using real-time RT-PCR for RSV. Infant characteristics associated with very severe illness (intensive care unit [ICU] admission or receipt of supplemental oxygen) were assessed using logistic regression to adjust for potential confounders (age, sex, study site, and preterm birth). RESULTS: Of 3634 enrolled hospitalized infants, 1129 (31%) tested positive for RSV. The median age of RSV-positive infants was 2.7 (IQR: 1.4-6.1) months and 665 (59%) were male. Very severe illness in 583 (52%) RSV-positive infants was associated with younger age (aOR 4.1, 95% CI: 2.6-6.5 for 0-2 compared to 9-11-months; P < .01), low weight-for-age z-score (aOR 1.9, 95% CI: 1.2-2.8; P < .01), ICU care after birth (aOR 1.6, 95% CI: 1.0-2.5; P = .048), and cesarean delivery (aOR 1.4, 95% CI: 1.0-1.8; P = .03). RSV subgroups A and B co-circulated at all sites with alternating predominance by year; subgroup was not associated with severity (aOR 1.0, 95% CI: 0.8-1.4). Nine (0.8%) RSV-positive infants died during admission or within ≤30 days of discharge, of which 7 (78%) were <6-months-old. CONCLUSIONS: RSV was associated with nearly a third of infant acute illness hospitalizations in four middle-income countries during the respiratory season, where, in addition to young age, factors including low weight-for-age might be important predictors of severity. RSV prevention strategies targeting young infants could substantially reduce RSV-associated hospitalizations in middle-income countries.
Asunto(s)
Nacimiento Prematuro , Infecciones por Virus Sincitial Respiratorio , Virus Sincitial Respiratorio Humano , Femenino , Lactante , Recién Nacido , Humanos , Masculino , Enfermedad Aguda , Cuidados Posteriores , Países en Desarrollo , Alta del Paciente , HospitalizaciónRESUMEN
Year-round virological characterization of circulating epidemic influenza viruses is conducted worldwide to detect the emergence of viruses that may escape pre-existing immunity or acquire resistance to antivirals. High throughput phenotypic assays are needed to complement the sequence-based analysis of circulating viruses and improve pandemic preparedness. The recent entry of a polymerase inhibitor, baloxavir, into the global market further highlighted this need. Here, we optimized a cell-based assay that considerably streamlines antiviral and antigenic testing by replacing lengthy immunostaining and imaging procedures used in current assay with measuring the enzymatic activity of nascent neuraminidase (NA) molecules expressed on the surface of virus-infected cells. For convenience, this new assay was named IRINA (Influenza Replication Inhibition Neuraminidase-based Assay). IRINA was successfully validated to assess inhibitory activity of baloxavir on virus replication by testing a large set (>150) of influenza A and B viruses, including drug resistant strains and viruses collected during 2017-2022. To test its versatility, IRINA was utilized to evaluate neutralization activity of a broadly reactive human anti-HA monoclonal antibody, FI6, and post-infection ferret antisera, as well as the inhibition of NA enzyme activity by NA inhibitors. Performance of IRINA was tested in parallel using respective conventional assays. IRINA offers an attractive alternative to current phenotypic assays, while maintaining reproducibility and high throughput capacity. Additionally, the improved turnaround time may prove to be advantageous when conducting time sensitive studies, such as investigating a new virus outbreak. This assay can meet the needs of surveillance laboratories by providing a streamlined and cost-effective approach for virus characterization.
Asunto(s)
Gripe Humana , Neuraminidasa , Animales , Humanos , Reproducibilidad de los Resultados , Farmacorresistencia Viral , Hurones , Replicación Viral , Antivirales/farmacología , Antivirales/uso terapéutico , Inhibidores Enzimáticos/farmacología , Oseltamivir/farmacologíaRESUMEN
Global analysis of the susceptibility of influenza viruses to neuraminidase (NA) inhibitors (NAIs) and the polymerase acidic (PA) inhibitor (PAI) baloxavir was conducted by five World Health Organization Collaborating Centres for Reference and Research on Influenza during two periods (May 2018-May 2019 and May 2019-May 2020). Combined phenotypic and NA sequence-based analysis revealed that the global frequency of viruses displaying reduced or highly reduced inhibition (RI or HRI) or potential to show RI/HRI by NAIs remained low, 0.5% (165/35045) and 0.6% (159/26010) for the 2018-2019 and 2019-2020 periods, respectively. The most common amino acid substitution was NA-H275Y (N1 numbering) conferring HRI by oseltamivir and peramivir in A(H1N1)pdm09 viruses. Combined phenotypic and PA sequence-based analysis showed that the global frequency of viruses showing reduced susceptibility to baloxavir or carrying substitutions associated with reduced susceptibility was low, 0.5% (72/15906) and 0.1% (18/15692) for the 2018-2019 and 2019-2020 periods, respectively. Most (n = 61) of these viruses had I38âT/F/M/S/L/V PA amino acid substitutions. In Japan, where baloxavir use was highest, the rate was 4.5% (41/919) in the 2018-2019 period and most of the viruses (n = 32) had PA-I38T. Zoonotic viruses isolated from humans (n = 32) in different countries did not contain substitutions in NA associated with NAI RI/HRI phenotypes. One A(H5N6) virus had a dual substitution PA-I38V + PA-E199G, which may reduce susceptibility to baloxavir. Therefore, NAIs and baloxavir remain appropriate choices for the treatment of influenza virus infections, but close monitoring of antiviral susceptibility is warranted.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A , Gripe Humana , Sustitución de Aminoácidos , Antivirales/farmacología , Antivirales/uso terapéutico , Dibenzotiepinas , Farmacorresistencia Viral/genética , Endonucleasas/genética , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Humanos , Subtipo H1N1 del Virus de la Influenza A/genética , Virus de la Influenza B , Morfolinas , Neuraminidasa/genética , Neuraminidasa/uso terapéutico , Oseltamivir/farmacología , Oseltamivir/uso terapéutico , Piridonas , TriazinasRESUMEN
Pimodivir exerts an antiviral effect on the early stages of influenza A virus replication by inhibiting the cap-binding function of polymerase basic protein 2 (PB2). In this study, we used a combination of sequence analysis and phenotypic methods to evaluate pimodivir susceptibility of influenza A viruses collected from humans and other hosts. Screening PB2 sequences for substitutions previously associated with reduced pimodivir susceptibility revealed a very low frequency among seasonal viruses circulating in the U.S. during 2015-2020 (<0.03%; 3/11,934) and among non-seasonal viruses collected in various countries during the same period (0.2%; 18/8971). Pimodivir potently inhibited virus replication in two assays, a single-cycle HINT and a multi-cycle FRA, with IC50 values in a nanomolar range. Median IC50 values determined by HINT were similar for both subtypes of seasonal viruses, A(H1N1)pdm09 and A(H3N2), across three seasons. Human seasonal viruses with PB2 substitutions S324C, S324R, or N510K displayed a 27-317-fold reduced pimodivir susceptibility by HINT. In addition, pimodivir was effective at inhibiting replication of a diverse group of animal-origin viruses that have pandemic potential, including avian viruses of A(H5N6) and A(H7N9) subtypes. A rare PB2 substitution H357N was identified in an A(H4N2) subtype poultry virus that displayed >100-fold reduced pimodivir susceptibility. Our findings demonstrate a broad inhibitory activity of pimodivir and expand the existing knowledge of amino acid substitutions that can reduce susceptibility to this investigational antiviral.
Asunto(s)
Antivirales/farmacología , Virus de la Influenza A/efectos de los fármacos , Piridinas/farmacología , Pirimidinas/farmacología , Pirroles/farmacología , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Proteínas Virales/antagonistas & inhibidores , Animales , Farmacorresistencia Viral , Inhibidores Enzimáticos/farmacología , Humanos , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/efectos de los fármacos , Subtipo H3N2 del Virus de la Influenza A/genética , Subtipo H7N9 del Virus de la Influenza A/efectos de los fármacos , Subtipo H7N9 del Virus de la Influenza A/genética , Virus de la Influenza A/genética , Gripe Humana/virología , Pruebas de Sensibilidad Microbiana , Infecciones por Orthomyxoviridae/virología , ARN Polimerasa Dependiente del ARN/genética , Proteínas Virales/genética , Replicación Viral/efectos de los fármacosRESUMEN
BACKGROUND: The cotton rat (genus Sigmodon) is an essential small animal model for the study of human infectious disease and viral therapeutic development. However, the impact of the host microbiome on infection outcomes has not been explored in this model, partly due to the lack of a comprehensive characterization of microbial communities across different cotton rat species. Understanding the dynamics of their microbiome could significantly help to better understand its role when modeling viral infections in this animal model. RESULTS: We examined the bacterial communities of the gut and three external sites (skin, ear, and nose) of two inbred species of cotton rats commonly used in research (S. hispidus and S. fulviventer) by using 16S rRNA gene sequencing, constituting the first comprehensive characterization of the cotton rat microbiome. We showed that S. fulviventer maintained higher alpha diversity and richness than S. hispidus at external sites (skin, ear, nose), but there were no differentially abundant genera. However, S. fulviventer and S. hispidus had distinct fecal microbiomes composed of several significantly differentially abundant genera. Whole metagenomic shotgun sequencing of fecal samples identified species-level differences between S. hispidus and S. fulviventer, as well as different metabolic pathway functions as a result of differential host microbiome contributions. Furthermore, the microbiome composition of the external sites showed significant sex-based differences while fecal communities were not largely different. CONCLUSIONS: Our study shows that host genetic background potentially exerts homeostatic pressures, resulting in distinct microbiomes for two different inbred cotton rat species. Because of the numerous studies that have uncovered strong relationships between host microbiome, viral infection outcomes, and immune responses, our findings represent a strong contribution for understanding the impact of different microbial communities on viral pathogenesis. Furthermore, we provide novel cotton rat microbiome data as a springboard to uncover the full therapeutic potential of the microbiome against viral infections.
RESUMEN
Baloxavir, a new antiviral drug targeting cap-dependent endonuclease activity of polymerase acidic (PA) protein of influenza viruses, is now approved in multiple countries. Several substitutions at isoleucine 38 in PA protein (e.g., PA-I38T) have been associated with decreased baloxavir susceptibility in vitro and in vivo. In recent years, next generation sequencing (NGS) analysis and pyrosequencing have been used by CDC and U.S. Public Health Laboratories to monitor drug susceptibility of influenza viruses. Here we described an improved pyrosequencing assay for detecting influenza A viruses carrying substitutions at PA-38. Cyclic and customized orders of nucleotide dispensation were evaluated, and pyrosequencing results were compared to those generated using NGS. Our data showed that the customized nucleotide dispensation has improved the pyrosequencing assay performance in identification of double mixtures (e.g., PA-38I/T); however, identification of PA-38 variants in triple mixtures remains a challenge. While NGS analysis indicated the presence of PA-I38K in one clinical specimen and isolate, our attempts to detect this mutation by pyrosequencing or recover the virus carrying PA-I38K in cell culture were unsuccessful, raising a possibility of a rarely occurring sequencing error. Overall, pyrosequencing provides a convenient means to detect baloxavir resistant influenza viruses when NGS is unavailable or a faster turnaround time is required.
Asunto(s)
Antivirales/farmacología , Dibenzotiepinas/farmacología , Farmacorresistencia Viral/genética , Virus de la Influenza A/efectos de los fármacos , Virus de la Influenza A/genética , Morfolinas/farmacología , Piridonas/farmacología , Triazinas/farmacología , Sustitución de Aminoácidos , Animales , Perros , Genoma Viral , Secuenciación de Nucleótidos de Alto Rendimiento , Virus de la Influenza A/clasificación , Células de Riñón Canino Madin Darby , Replicación Viral/efectos de los fármacosRESUMEN
BACKGROUND & OBJECTIVE: Recombinant DNA technology allows expression of the human papillomavirus (HPV) major capsid protein (L1) in heterologous expression systems and the recombinant protein self assembles to virus-like particles (VLP). We took up this study to produce recombinant HPV-16 L1 in yeast, establish the process of recombinant L1 derived VLP preparation and develop an ELISA using VLP as the antigen for serological evaluation of anti HPV-16 L1 antibody status. METHODS: Complete HPV-16 L1 was amplified from genomic DNA of an esophageal cancer biopsy, cloned and the protein was expressed in a galactose-inducible Saccharomyces cerevisiae expression system. Self assembled VLP was purified by a two-step density gradient centrifugation process and the VLP preparation used to test its suitability in developing an ELISA. RESULTS: The recombinant protein was predominantly a ~55 KD species with distinct immunoreactivity and formed VLP as confirmed by electron microscopy. An ELISA using the VLP showed its efficacy in appropriate immunoreactivity to serum/plasma IgG. INTERPRETATION & CONCLUSION: Recombinant HPV-16 capsid protein derived VLP was produced and the VLP antigen based ELISA can be used to probe serological association of HPV with different clinical conditions. The VLP technology can be improved further and harnessed for future vaccine development efforts in the country.
Asunto(s)
Proteínas de la Cápside/inmunología , Papillomavirus Humano 16/inmunología , Proteínas Oncogénicas Virales/inmunología , Anticuerpos Antivirales/análisis , Antígenos Virales/biosíntesis , Antígenos Virales/genética , Proteínas de la Cápside/biosíntesis , Proteínas de la Cápside/genética , Ensayo de Inmunoadsorción Enzimática , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/ultraestructura , Humanos , Proteínas Oncogénicas Virales/biosíntesis , Proteínas Oncogénicas Virales/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Saccharomyces cerevisiae/genéticaRESUMEN
Gastrin-releasing peptide (GRP) is an evolutionarily well-conserved neuropeptide that was originally recognized for its ability to mediate gastric acid secretion in the gut. More recently, however, GRP has been implicated in pulmonary lung inflammatory diseases including bronchopulmonary dysplasia, chronic obstructive pulmonary disease, emphysema, and others. Antagonizing GRP or its receptor mitigated lethality associated with the onset of viral pneumonia in a well-characterized mouse model of influenza. In mice treated therapeutically with the small-molecule GRP inhibitor, NSC77427, increased survival was accompanied by decreased numbers of GRP-producing pulmonary neuroendocrine cells, improved lung histopathology, and suppressed cytokine gene expression. In addition, in vitro studies in macrophages indicate that GRP synergizes with the prototype TLR4 agonist, lipopolysaccharide, to induce cytokine gene expression. Thus, these findings reveal that GRP is a previously unidentified mediator of influenza-induced inflammatory disease that is a potentially novel target for therapeutic intervention.
Asunto(s)
Péptido Liberador de Gastrina/metabolismo , Subtipo H1N1 del Virus de la Influenza A/fisiología , Subtipo H3N2 del Virus de la Influenza A/fisiología , Gripe Humana/inmunología , Pulmón/patología , Macrófagos/inmunología , Células Neuroendocrinas/metabolismo , Infecciones por Orthomyxoviridae/inmunología , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Péptido Liberador de Gastrina/antagonistas & inhibidores , Humanos , Inmunidad , Masculino , Ratones , Ratones Endogámicos C57BL , Pirimidinas/farmacología , Sigmodontinae , Transducción de Señal , Receptor Toll-Like 4/metabolismoRESUMEN
Host-derived "danger-associated molecular patterns" (DAMPs) contribute to innate immune responses and serve as markers of disease progression and severity for inflammatory and infectious diseases. There is accumulating evidence that generation of DAMPs such as oxidized phospholipids and high-mobility-group box 1 (HMGB1) during influenza virus infection leads to acute lung injury (ALI). Treatment of influenza virus-infected mice and cotton rats with the Toll-like receptor 4 (TLR4) antagonist Eritoran blocked DAMP accumulation and ameliorated influenza virus-induced ALI. However, changes in systemic HMGB1 kinetics during the course of influenza virus infection in animal models and humans have yet to establish an association of HMGB1 release with influenza virus infection. To this end, we used the cotton rat model that is permissive to nonadapted strains of influenza A and B viruses, respiratory syncytial virus (RSV), and human rhinoviruses (HRVs). Serum HMGB1 levels were measured by an enzyme-linked immunosorbent assay (ELISA) prior to infection until day 14 or 18 post-infection. Infection with either influenza A or B virus resulted in a robust increase in serum HMGB1 levels that decreased by days 14 to 18. Inoculation with the live attenuated vaccine FluMist resulted in HMGB1 levels that were significantly lower than those with infection with live influenza viruses. RSV and HRVs showed profiles of serum HMGB1 induction that were consistent with their replication and degree of lung pathology in cotton rats. We further showed that therapeutic treatment with Eritoran of cotton rats infected with influenza B virus significantly blunted serum HMGB1 levels and improved lung pathology, without inhibiting virus replication. These findings support the use of drugs that block HMGB1 to combat influenza virus-induced ALI.IMPORTANCE Influenza virus is a common infectious agent causing serious seasonal epidemics, and there is urgent need to develop an alternative treatment modality for influenza virus infection. Recently, host-derived DAMPs, such as oxidized phospholipids and HMGB1, were shown to be generated during influenza virus infection and cause ALI. To establish a clear link between influenza virus infection and HMGB1 as a biomarker, we have systematically analyzed temporal patterns of serum HMGB1 release in cotton rats infected with nonadapted strains of influenza A and B viruses and compared these patterns with a live attenuated influenza vaccine and infection by other respiratory viruses. Towards development of a new therapeutic modality, we show herein that blocking serum HMGB1 levels by Eritoran improves lung pathology in influenza B virus-infected cotton rats. Our study is the first report of systemic HMGB1 as a potential biomarker of severity in respiratory virus infections and confirms that drugs that block virus-induced HMGB1 ameliorate ALI.
Asunto(s)
Biomarcadores/sangre , Proteína HMGB1/sangre , Infecciones por Orthomyxoviridae/patología , Infecciones por Picornaviridae/patología , Infecciones por Virus Sincitial Respiratorio/patología , Suero/química , Animales , Disacáridos/administración & dosificación , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Factores Inmunológicos/administración & dosificación , Infecciones por Orthomyxoviridae/diagnóstico , Infecciones por Picornaviridae/diagnóstico , Infecciones por Virus Sincitial Respiratorio/diagnóstico , Sigmodontinae , Fosfatos de Azúcar/administración & dosificación , Resultado del TratamientoRESUMEN
The role of the histone chaperone SPT6 in mammalian cells is not fully understood. Here, we investigated the involvement of SPT6 in type I interferon (IFN)-induced transcription in murine fibroblasts. In RNA-seq analysis, Spt6 siRNA attenuates about half of ~ 200 IFN-stimulated genes (ISGs), while not affecting housekeeping genes. ISGs with high mRNA induction are more susceptible to Spt6 siRNA than those with lower levels of induction. ChIP analysis shows that SPT6 is recruited to highly inducible, Spt6 siRNA-sensitive ISGs, but not to other siRNA-insensitive ISGs. Furthermore, SPT6 recruitment is abrogated in cells lacking the histone methyltransferase NSD2. In co-IP experiments, SPT6 interacts with NSD2. In summary, SPT6 facilitates IFN-induced transcription, highlighting its critical role in gene activation.
Asunto(s)
Regulación de la Expresión Génica/fisiología , N-Metiltransferasa de Histona-Lisina/fisiología , Interferón Tipo I/fisiología , Factores de Transcripción/fisiología , Transcripción Genética/fisiología , Animales , Células Cultivadas , Inmunoprecipitación de Cromatina , N-Metiltransferasa de Histona-Lisina/metabolismo , Interferón Tipo I/metabolismo , Ratones , Unión Proteica , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Análisis de Secuencia de ARN , Factores de Transcripción/metabolismoRESUMEN
Respiratory syncytial virus (RSV) is the most common cause of bronchiolitis in infants. Maternal immunization is an option to increase maternal antibody levels and protect infants from infection. Here we assess the efficacy of virus-like particle (VLP) vaccine candidates containing stabilized pre-fusion (pre-F) or post-fusion (post-F) conformations of the RSV F protein and the attachment RSV G protein in a maternal immunization model using cotton rats. VLP vaccines containing RSV F and G proteins strongly boost pre-existing RSV immunity in dams preventing their perinatal drop in immunity. Boosting is stronger for the pre-F VLP than for the post-F VLP or purified subunit F protein vaccines, giving an advantage on mothers' protection. VLP immunization of dams provides significant protection to pups from RSV challenge and reduced pulmonary inflammation. Collectively, our results show that a VLP vaccine with RSV F and G proteins is safe and effective for maternal and adult vaccination.
Asunto(s)
Inmunidad Materno-Adquirida , Infecciones por Virus Sincitial Respiratorio/prevención & control , Vacunas contra Virus Sincitial Respiratorio/inmunología , Virus Sincitial Respiratorio Humano/inmunología , Animales , Anticuerpos Antivirales/inmunología , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunización , Pulmón/inmunología , Pulmón/virología , Masculino , Ratas , Infecciones por Virus Sincitial Respiratorio/inmunología , Infecciones por Virus Sincitial Respiratorio/virología , Vacunas contra Virus Sincitial Respiratorio/administración & dosificación , Vacunas contra Virus Sincitial Respiratorio/genética , Virus Sincitial Respiratorio Humano/genética , Sigmodontinae , Proteínas del Envoltorio Viral/administración & dosificación , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Proteínas Virales de Fusión/administración & dosificación , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/inmunologíaRESUMEN
Acute respiratory infection (ARI) with respiratory syncytial virus (RSV) is the most common cause of both hospitalizations and mortality in young infants worldwide. Repeat infections with RSV are common throughout life in both pediatric and elderly populations. Thus far, cotton rats (Sigmodon hispidus) are found to be the best animal model to study RSV infection. However, the lack of a cotton rat reference genome limits genome-wide host gene expression studies. We constructed the first lung tissue de novo transcriptome for the cotton rat. Cotton rat lung tissue transcripts were assigned to 12,211 unique UniProt genes, which were then utilized to profile the host immune response after RSV infection. Differential expression analysis showed up-regulation of host genes involved in cellular functions including defense responses to viral infection and immune system processes. A number of transcripts were downregulated during the later stage of infection. A set of transcripts unique to RSV-infected cotton rats was identified. To validate RNA-Seq data of three such transcripts (TR453762, TR529629, and TR5333), their expression was confirmed by quantitative real-time polymerase chain reaction.
Asunto(s)
Inmunidad Innata/genética , Infecciones por Virus Sincitial Respiratorio/genética , Sigmodontinae/genética , Transcriptoma/genética , Animales , Regulación de la Expresión Génica/inmunología , Genoma , Interacciones Huésped-Parásitos/genética , Interacciones Huésped-Parásitos/inmunología , Pulmón/metabolismo , Pulmón/virología , Ratas , Infecciones por Virus Sincitial Respiratorio/inmunología , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitiales Respiratorios/inmunología , Virus Sincitiales Respiratorios/patogenicidad , Sigmodontinae/inmunología , Sigmodontinae/virologíaRESUMEN
Maternal immunization directed to control RSV infection in newborns and infants is an appealing vaccination strategy currently under development. In this work we have modeled maternal vaccination against RSV in cotton rats (CR) to answer two fundamental questions on maternal vaccine safety. We tested (i), whether a known, unsafe RSV vaccine (i.e., FI-RSV Lot 100 vaccine) induces vaccine enhanced disease in the presence of passively transferred, RSV maternal immunity, and (ii) whether the same FI-RSV vaccine could induce vaccine enhanced disease in CR litters when used to immunize their RSV-primed mothers. Our data show that FI-RSV immunization of pups with subsequent RSV infection results in vaccine-enhanced disease independent of whether the pups were born to RSV-seropositive or RSV-seronegative mothers, and that FI-RSV immunization of RSV-seropositive mothers does not present a health risk to either the mother or the infant. Our study also raises a novel concern regarding infant immunization, namely that "safe" RSV vaccines (e.g., live RSV administered intramuscularly) may induce vaccine-enhanced disease in RSV-infected pups born to seropositive mothers. Finally, we describe for the first time a sharp decrease in RSV neutralizing antibody titers in immunized seropositive CR at the time of delivery. This decline may reflect maternal immune suppression, potentially pinpointing a window of increased vulnerability to RSV infection that could be alleviated by effective immunization of expectant mothers.
Asunto(s)
Evaluación Preclínica de Medicamentos , Infecciones por Virus Sincitial Respiratorio/prevención & control , Vacunas contra Virus Sincitial Respiratorio/efectos adversos , Vacunas contra Virus Sincitial Respiratorio/inmunología , Virus Sincitiales Respiratorios/inmunología , Animales , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/epidemiología , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/patología , Femenino , Inyecciones Intramusculares , Embarazo , Vacunas contra Virus Sincitial Respiratorio/administración & dosificación , SigmodontinaeRESUMEN
Dysregulated Toll-like receptor (TLR)-4 activation is involved in acute systemic sepsis, chronic inflammatory diseases, such as atherosclerosis and diabetes, and in viral infections, such as influenza infection. Thus, therapeutic control of the TLR4 signalling pathway is of major interest. Here we tested the activity of the small-molecule synthetic TLR4 antagonist, FP7, in vitro on human monocytes and monocyte-derived dendritic cells (DCs) and in vivo during influenza virus infection of mice. Our results indicate that FP7 antagonized the secretion of proinflammatory cytokines (IL-6, IL-8, and MIP-1ß) by monocytes and DCs (IC50 < 1 µM) and prevented DC maturation upon TLR4 activation by ultrapure lipopolysaccharide (LPS). FP7 selectively blocked TLR4 stimulation, but not TLR1/2, TLR2/6, or TLR3 activation. TLR4 stimulation of human DCs resulted in increased glycolytic activity that was also antagonized by FP7. FP7 protected mice from influenza virus-induced lethality and reduced both proinflammatory cytokine gene expression in the lungs and acute lung injury (ALI). Therefore, FP7 can antagonize TLR4 activation in vitro and protect mice from severe influenza infection, most likely by reducing TLR4-dependent cytokine storm mediated by damage-associated molecular patterns (DAMPs) like HMGB1.