RESUMEN
Viral respiratory infections are a common cause of severe disease, especially in infants, people who are immunocompromised, and in the elderly. Neutrophils, an important innate immune cell, infiltrate the lungs rapidly after an inflammatory insult. The most well-characterized effector mechanisms by which neutrophils contribute to host defense are largely extracellular and the involvement of neutrophils in protection from numerous bacterial and fungal infections is well established. However, the role of neutrophils in responses to viruses, which replicate intracellularly, has been less studied. It remains unclear whether and, by which underlying immunological mechanisms, neutrophils contribute to viral control or confer protection against an intracellular pathogen. Furthermore, neutrophils need to be tightly regulated to avoid bystander damage to host tissues. This is especially relevant in the lung where damage to delicate alveolar structures can compromise gas exchange with life-threatening consequences. It is inherently less clear how neutrophils can contribute to host immunity to viruses without causing immunopathology and/or exacerbating disease severity. In this review, we summarize and discuss the current understanding of how neutrophils in the lung direct immune responses to viruses, control viral replication and spread, and cause pathology during respiratory viral infections.
Asunto(s)
Interacciones Huésped-Patógeno , Neutrófilos/inmunología , Neutrófilos/metabolismo , Infecciones por Respirovirus/etiología , Infecciones por Respirovirus/metabolismo , Respirovirus/fisiología , Inmunidad Adaptativa , Animales , Biomarcadores , Comunicación Celular , Coinfección , Citocinas/metabolismo , Resistencia a la Enfermedad/genética , Resistencia a la Enfermedad/inmunología , Susceptibilidad a Enfermedades , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunidad Innata , Infiltración Neutrófila/genética , Infiltración Neutrófila/inmunología , Infecciones por Respirovirus/patología , Índice de Severidad de la Enfermedad , Replicación ViralRESUMEN
Respiratory viruses remain a significant cause of morbidity and mortality in the human population, underscoring the importance of ongoing basic research into virus-host interactions. However, many critical aspects of infection are difficult, if not impossible, to probe using standard cell lines, 2D culture formats, or even animal models. In vitro systems such as airway epithelial cultures at air-liquid interface, organoids, or 'on-chip' technologies allow interrogation in human cells and recapitulate emergent properties of the airway epithelium-the primary target for respiratory virus infection. While some of these models have been used for over thirty years, ongoing advancements in both culture techniques and analytical tools continue to provide new opportunities to investigate airway epithelial biology and viral infection phenotypes in both normal and diseased host backgrounds. Here we review these models and their application to studying respiratory viruses. Furthermore, given the ability of these systems to recapitulate the extracellular microenvironment, we evaluate their potential to serve as a platform for studies specifically addressing viral interactions at the mucosal surface and detail techniques that can be employed to expand our understanding.
Asunto(s)
Interacciones Huésped-Patógeno , Mucosa Respiratoria/virología , Infecciones por Respirovirus/metabolismo , Infecciones por Respirovirus/virología , Respirovirus/fisiología , Comunicación Celular , Técnicas de Cultivo de Célula , Células Cultivadas , Espacio Extracelular/metabolismo , Modelos Biológicos , Organoides , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patología , Infecciones por Respirovirus/patología , Ingeniería de Tejidos , ViriónRESUMEN
BACKGROUND: Caprine parainfluenza virus type 3 (CPIV3) is major pathogen of goat herds causing serious respiratory tract disease and economic losses to the goat industry in China. We analyzed the differential proteomics of CPIV3-infected Madin-Darby bovine kidney (MDBK) cells using quantitative iTRAQ coupled LC-MS/MS. In addition, four DEPs were validated by qRT-PCR and western blot analysis. RESULTS: Quantitative proteomics analysis revealed 163 differentially expressed proteins (DEPs) between CPIV3-infected and mock-infected groups (p-value < 0.05 and fold change > 1.2), among which 91 were down-regulated and 72 were up-regulated. Gene ontology (GO) analysis showed that these DEPs were involved in molecular functions, cellular components and biological processes. Biological functions in which the DEPs were involved in included diseases, genetic information processing, metabolism, environmental information processing, cellular processes, and organismal systems. STRING analysis revealed that four heat shock proteins (HSPs) included HSPA5, HSPA1B, HSP90B1 and HSPA6 may be associated with proliferation of CPIV3 in MDBK cells. qRT-PCR and western blot analysis showed that the selected HSPs were identical to the quantitative proteomics data. CONCLUSION: To our knowledge, this is the first report of the proteomic changes in MDBK cells after CPIV3 infection.
Asunto(s)
Proteínas de Choque Térmico/metabolismo , Proteómica , Infecciones por Respirovirus/veterinaria , Respirovirus/fisiología , Animales , Western Blotting , Bovinos , Línea Celular , Cromatografía Liquida , Perfilación de la Expresión Génica , Proteínas de Choque Térmico/genética , Riñón/virología , Reacción en Cadena en Tiempo Real de la Polimerasa , Respirovirus/genética , Infecciones por Respirovirus/genética , Infecciones por Respirovirus/metabolismo , Espectrometría de Masas en Tándem , Replicación Viral/fisiologíaRESUMEN
Caprine parainfluenza virus type 3 (CPIV3) is a newly emerging pathogenic respiratory agent infecting both young and adult goats, and it was identified in eastern China in 2013. Cellular microRNAs (miRNAs) have been reported to be important modulators of the intricate virus-host interactions. In order to elucidate the role of miRNAs in madin-darby bovine kidney (MDBK) cells during CPIV3 infection. In this study, we performed high-throughput sequencing technology to analyze small RNA libraries in CPIV3-infected and mock-infected MDBK cells. The results showed that a total of 249 known and 152 novel candidate miRNAs were differentially expressed in MDBK cells after CPIV3 infection, and 22,981 and 22,572 target genes were predicted, respectively. In addition, RT-qPCR assay was used to further confirm the expression patterns of 13 of these differentially expressed miRNAs and their mRNA targets. Functional annotation analysis showed these up- and downregulated target genes were mainly involved in MAPK signaling pathway, Jak-STAT signaling pathway, Toll-like receptor signaling pathway, p53 signaling pathway, focal adhesion, NF-kappa B signaling pathway, and apoptosis, et al. To our knowledge, this is the first report of the comparative expression of miRNAs in MDBK cells after CPIV3 infection. Our finding provides information concerning miRNAs expression profile in response to CPIV3 infection, and offers clues for identifying potential candidates for antiviral therapies against CPIV3.
Asunto(s)
Enfermedades de los Bovinos/genética , Riñón/metabolismo , MicroARNs/genética , Infecciones por Respirovirus/genética , Infecciones por Respirovirus/veterinaria , Respirovirus/fisiología , Animales , Bovinos , Enfermedades de los Bovinos/metabolismo , Enfermedades de los Bovinos/virología , Línea Celular , Perfilación de la Expresión Génica , Riñón/virología , MicroARNs/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Respirovirus/genética , Infecciones por Respirovirus/metabolismo , Infecciones por Respirovirus/virologíaRESUMEN
Despite increased understanding of how viral infection is involved in asthma exacerbations, it is less clear which viruses are involved and to what extent they contribute to asthma exacerbations. Here, we sought to determine the prevalence of different respiratory viruses during asthma exacerbations. Systematic computerized searches of the literature up to June 2017 without language limitation were performed. The primary focus was on the prevalence of respiratory viruses, including AdV (adenovirus), BoV (bocavirus), CoV (coronavirus), CMV (cytomegalovirus), EnV (enterovirus), HSV (herpes simplex virus), IfV (influenza virus), MpV (metapneumovirus), PiV (parainfluenzavirus), RV (rhinovirus) and RSV (respiratory syncytial virus) during asthma exacerbations. We also examined the prevalence of viral infection stratified by age, geographic region, type of respiratory secretion, and detection method. Sixty articles were included in the final analysis. During asthma exacerbations, the mean prevalence of AdV, BoV, CoV, CMV, EnV, HSV, IfV, MpV, PiV, RV and RSV was 3.8%, 6.9%, 8.4%, 7.2%, 10.1%, 12.3%, 10.0%, 5.3%, 5.6%, 42.1% and 13.6%, respectively. EnV, MPV, RV and RSV were more prevalent in children, whereas AdV, BoV, CoV, IfV and PiV were more frequently present in adults. RV was the major virus detected globally, except in Africa. RV could be detected in both the upper and lower airway. Polymerase chain reaction was the most sensitive method for detecting viral infection. Our findings indicate the need to develop prophylactic polyvalent or polyvirus (including RV, EnV, IfV and RSV) vaccines that produce herd immunity and reduce the healthcare burden associated with virus-induced asthma exacerbations.
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Asma/epidemiología , Sistema Respiratorio/virología , Infecciones del Sistema Respiratorio/epidemiología , Virosis/epidemiología , Adenoviridae/patogenicidad , Adenoviridae/fisiología , África/epidemiología , Factores de Edad , Américas/epidemiología , Asia/epidemiología , Asma/complicaciones , Asma/virología , Coronavirus/patogenicidad , Coronavirus/fisiología , Citomegalovirus/patogenicidad , Citomegalovirus/fisiología , Enterovirus/patogenicidad , Enterovirus/fisiología , Europa (Continente)/epidemiología , Bocavirus Humano/patogenicidad , Bocavirus Humano/fisiología , Humanos , Metapneumovirus/patogenicidad , Metapneumovirus/fisiología , Prevalencia , Virus Sincitial Respiratorio Humano/patogenicidad , Virus Sincitial Respiratorio Humano/fisiología , Infecciones del Sistema Respiratorio/complicaciones , Infecciones del Sistema Respiratorio/virología , Respirovirus/patogenicidad , Respirovirus/fisiología , Rhinovirus/patogenicidad , Rhinovirus/fisiología , Simplexvirus/patogenicidad , Simplexvirus/fisiología , Virosis/complicaciones , Virosis/virologíaRESUMEN
Respiratory syncytial virus (RSV) is the most important viral agent of severe pediatric respiratory tract disease worldwide, but it lacks a licensed vaccine or suitable antiviral drug. A live attenuated chimeric bovine/human parainfluenza virus type 3 (rB/HPIV3) was developed previously as a vector expressing RSV fusion (F) protein to confer bivalent protection against RSV and HPIV3. In a previous clinical trial in virus-naive children, rB/HPIV3 was well tolerated but the immunogenicity of wild-type RSV F was unsatisfactory. We previously modified RSV F with a designed disulfide bond (DS) to increase stability in the prefusion (pre-F) conformation and to be efficiently packaged in the vector virion. Here, we further stabilized pre-F by adding both disulfide and cavity-filling mutations (DS-Cav1), and we also modified RSV F codon usage to have a lower CpG content and a higher level of expression. This RSV F open reading frame was evaluated in rB/HPIV3 in three forms: (i) pre-F without vector-packaging signal, (ii) pre-F with vector-packaging signal, and (iii) secreted pre-F ectodomain trimer. Despite being efficiently expressed, the secreted pre-F was poorly immunogenic. DS-Cav1 stabilized pre-F, with or without packaging, induced higher titers of pre-F specific antibodies in hamsters, and improved the quality of RSV-neutralizing serum antibodies. Codon-optimized RSV F containing fewer CpG dinucleotides had higher F expression, replicated more efficiently in vivo, and was more immunogenic. The combination of DS-Cav1 pre-F stabilization, optimized codon usage, reduced CpG content, and vector packaging significantly improved vector immunogenicity and protective efficacy against RSV. This provides an improved vectored RSV vaccine candidate suitable for pediatric clinical evaluation.IMPORTANCE RSV and HPIV3 are the first and second leading viral causes of severe pediatric respiratory disease worldwide. Licensed vaccines or suitable antiviral drugs are not available. We are developing a chimeric rB/HPIV3 vector expressing RSV F as a bivalent RSV/HPIV3 vaccine and have been evaluating means to increase RSV F immunogenicity. In this study, we evaluated the effects of improved stabilization of F in the pre-F conformation and of codon optimization resulting in reduced CpG content and greater pre-F expression. Reduced CpG content dampened the interferon response to infection, promoting higher replication and increased F expression. We demonstrate that improved pre-F stabilization and strategic manipulation of codon usage, together with efficient pre-F packaging into vector virions, significantly increased F immunogenicity in the bivalent RSV/HPIV3 vaccine. The improved immunogenicity included induction of increased titers of high-quality complement-independent antibodies with greater pre-F site Ø binding and greater protection against RSV challenge.
Asunto(s)
Portadores de Fármacos , Vacunas contra Virus Sincitial Respiratorio/inmunología , Respirovirus/fisiología , Proteínas Virales de Fusión/inmunología , Virión/metabolismo , Ensamble de Virus , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Codón , Cricetinae , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/inmunología , Estabilidad Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Vacunas contra Virus Sincitial Respiratorio/administración & dosificación , Vacunas contra Virus Sincitial Respiratorio/genética , Respirovirus/genética , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Proteínas Virales de Fusión/química , Proteínas Virales de Fusión/genética , Virión/genéticaRESUMEN
UNLABELLED: Respiratory paramyxoviruses, including the highly prevalent human parainfluenza viruses, cause the majority of childhood croup, bronchiolitis, and pneumonia, yet there are currently no vaccines or effective treatments. Paramyxovirus research has relied on the study of laboratory-adapted strains of virus in immortalized cultured cell lines. We show that findings made in such systems about the receptor interaction and viral fusion requirements for entry and fitness-mediated by the receptor binding protein and the fusion protein-can be drastically different from the requirements for infection in vivo. Here we carried out whole-genome sequencing and genomic analysis of circulating human parainfluenza virus field strains to define functional and structural properties of proteins of circulating strains and to identify the genetic basis for properties that confer fitness in the field. The analysis of clinical strains suggests that the receptor binding-fusion molecule pairs of circulating viruses maintain a balance of properties that result in an inverse correlation between fusion in cultured cells and growth in vivo. Future analysis of entry mechanisms and inhibitory strategies for paramyxoviruses will benefit from considering the properties of viruses that are fit to infect humans, since a focus on viruses that have adapted to laboratory work provides a distinctly different picture of the requirements for the entry step of infection. IMPORTANCE: Mechanistic information about viral infection-information that impacts antiviral and vaccine development-is generally derived from viral strains grown under laboratory conditions in immortalized cells. This study uses whole-genome sequencing of clinical strains of human parainfluenza virus 3-a globally important respiratory paramyxovirus-in cell systems that mimic the natural human host and in animal models. By examining the differences between clinical isolates and laboratory-adapted strains, the sequence differences are correlated to mechanistic differences in viral entry. For this ubiquitous and pathogenic respiratory virus to infect the human lung, modulation of the processes of receptor engagement and fusion activation occur in a manner quite different from that carried out by the entry glycoprotein-expressing pair of laboratory strains. These marked contrasts in the viral properties necessary for infection in cultured immortalized cells and in natural host tissues and animals will influence future basic and clinical studies.
Asunto(s)
Sistema Respiratorio/virología , Respirovirus/fisiología , Internalización del Virus , Animales , Genoma Viral , Humanos , Respirovirus/aislamiento & purificación , Respirovirus/patogenicidad , Respirovirus/ultraestructura , Infecciones por Respirovirus/virología , Análisis de Secuencia de ADN , Sigmodontinae , VirulenciaRESUMEN
Parainfluenza virus 3 (PIV-3) is a common viral infection not only in humans, but also in many other species. Serological evidence suggests that nearly 100 % of children in the United States have been infected with PIV-3 by 5 years of age. Similarly, in cattle, PIV-3 is commonly associated with bovine respiratory disease complex. A novel dolphin PIV-3 (TtPIV-1) was described by Nollens et al. in 2008 from a dolphin that was diagnosed with an unknown respiratory illness. At that time, TtPIV-1 was found to be most similar to, but distinct from, bovine PIV-3 (BPIV-3). In the present study, similar viral growth kinetics and pro-inflammatory cytokine (IL-1ß, IL-6, and CXCL8) production were seen between BPIV-3 and TtPIV-1 in BEAS-2B, MDBK, and Vero cell lines. Initial nomenclature of TtPIV-1 was based on partial sequence of the fusion and RNA polymerase genes. Based on the similarities we saw with the in vitro work, it was important to examine the TtPIV-1 genome in more detail. Full genome sequencing and subsequent phylogenetic analysis revealed that all six viral genes of TtPIV-1 clustered within the recently described BPIV-3 genotype B strains, and it is proposed that TtPIV-1 be re-classified with BPIV-3 genotype B strains.
Asunto(s)
Respirovirus/clasificación , Respirovirus/aislamiento & purificación , Animales , Delfín Mular/virología , Línea Celular , Análisis por Conglomerados , Citocinas/análisis , Genoma Viral , Datos de Secuencia Molecular , Filogenia , ARN Viral/genética , Respirovirus/genética , Respirovirus/fisiología , Infecciones por Respirovirus/veterinaria , Análisis de Secuencia de ADN , Homología de Secuencia , Cultivo de Virus , Replicación ViralRESUMEN
Macrophages are an important cell type for regulation of immunity, and can play key roles in virus pathogenesis. Here we address the effect of infection of primary human macrophages with the related paramyxoviruses Parainfluenza virus 5 (PIV5) and Mumps virus (MuV). Monocyte-derived macrophages infected with PIV5 or MuV showed very little cytopathic effect, but were found to be defective in migration toward a gradient of chemokines such as macrophage colony stimulating factor (MCSF) and vascular endothelial growth factor (VEGF). For MuV infection, the inhibition of migration required live virus infection, but was not caused by a loss of chemokine receptors on the surface of infected cells. MuV-mediated inhibition of macrophage chemotaxis was through a soluble factor released from infected cells. MuV infection enhanced secretion of TNF-α, but not macrophage inhibitory factor (MIF). Antibody inhibition and add-back experiments demonstrated that TNF-α was both necessary and sufficient for MuV-mediate chemotaxis inhibition.
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Inhibición de Migración Celular/efectos de los fármacos , Macrófagos/virología , Virus de la Parotiditis/fisiología , Respirovirus/fisiología , Factor de Necrosis Tumoral alfa/farmacología , Quimiotaxis/efectos de los fármacos , Interacciones Huésped-Patógeno , Humanos , Evasión Inmune , Oxidorreductasas Intramoleculares/metabolismo , Factor Estimulante de Colonias de Macrófagos/inmunología , Factor Estimulante de Colonias de Macrófagos/farmacología , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Macrófagos/inmunología , Cultivo Primario de Células , Receptores de Quimiocina/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Factor A de Crecimiento Endotelial Vascular/inmunología , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
The paper gives the results of monitoring the circulation of influenza viruses in the 2010-2011 season, that covers the second year of circulation of pandemic A(H1N1)v virus strains, and their interaction with seasonal A (H3N2) and B strains. Unlike the previous season, the beginning of an increase in morbidity was recorded in January 2011; its peak in the most of contiguous areas was noted at 5-7 weeks of 2011, with its further decline to threshold levels at week 11 of 2011. Preschool and school children were most involved in the epidemic process. Three influenza virus strains (A(H1N1)v, A(H3N2), and B) were found to circulate. Differences were found in the level of participation of the isolated strains in individual areas of the Russian Federation. Detailed typing of the isolated strains determined the compliance of the vast majority of them with vaccine viruses. The pandemic influenza A(H1N1)v virus strains retained their susceptibility to oseltamivir and were resistant to rimantadine. The participation of non-influenza acute respiratory viral infection pathogens was estimated as follows: 11.9% for parainfluenza viruses, 5.9% for adenoviruses, and 3.5% for PC viruses, and 0.7% for pneumonia Mycoplasma, which was comparable with the previous epidemic seasons.
Asunto(s)
Infecciones por Adenoviridae/epidemiología , Gripe Humana/epidemiología , Pandemias , Infecciones por Respirovirus/epidemiología , Academias e Institutos , Adenoviridae/efectos de los fármacos , Adenoviridae/fisiología , Infecciones por Adenoviridae/tratamiento farmacológico , Infecciones por Adenoviridae/virología , Adolescente , Adulto , Antivirales/administración & dosificación , Antivirales/uso terapéutico , Niño , Preescolar , Coinfección , Farmacorresistencia Viral , Humanos , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H1N1 del Virus de la Influenza A/fisiología , Subtipo H3N2 del Virus de la Influenza A/efectos de los fármacos , Subtipo H3N2 del Virus de la Influenza A/fisiología , Virus de la Influenza B , Gripe Humana/tratamiento farmacológico , Gripe Humana/virología , Oseltamivir/administración & dosificación , Oseltamivir/uso terapéutico , Respirovirus/efectos de los fármacos , Respirovirus/fisiología , Infecciones por Respirovirus/tratamiento farmacológico , Infecciones por Respirovirus/virología , Rimantadina/administración & dosificación , Rimantadina/uso terapéutico , Federación de Rusia/epidemiología , Estaciones del AñoRESUMEN
Human parainfluenza viruses (HPIVs) are uncommon, yet high-risk pathogens after hematopoietic stem cell transplant (HCT). We evaluated 5178 pediatric and adult patients undergoing HCT between 1974 and 2010 to determine the incidence, risk factors, response to treatment, and outcome of HPIV infection as well as any change in frequency or character of HPIV infection over time. HPIV was identified in 173 patients (3.3%); type 3 was most common (66%). HPIV involved upper respiratory tract infection (URTI; 57%), lower respiratory tract infection (LRTI; 9%), and both areas of the respiratory tract (34%), at a median of 62 days after transplantation. In more recent years, HPIV has occurred later after HCT, whereas the proportion with nosocomial infection and mortality decreased. Over the last decade, HPIV was more common in older patients and in those receiving reduced intensity conditioning (RIC). RIC was a significant risk factor for later (beyond day +30). HPIV infections, and this association was strongest in patients with URTI. HCT using a matched unrelated donor (MURD), mismatched related donor (MMRD), age 10 to 19 years, and graft-versus-host disease (GVHD) were all risk factors for HPIV infections. LRTI, early (<30 days), age 10 to 19 years, MMRD, steroid use, and coinfection with other pathogens were risk factors for mortality. The survival of patients with LRTI, especially very early infections, was poor regardless of ribavirin treatment. HPIV incidence remains low, but may have delayed onset associated with RIC regimens and improving survival. Effective prophylaxis and treatment for HPIV are needed.
Asunto(s)
Enfermedad Injerto contra Huésped/epidemiología , Trasplante de Células Madre Hematopoyéticas , Infecciones por Paramyxoviridae/epidemiología , Infecciones del Sistema Respiratorio/epidemiología , Acondicionamiento Pretrasplante , Adolescente , Adulto , Factores de Edad , Antivirales/uso terapéutico , Niño , Preescolar , Enfermedad Injerto contra Huésped/tratamiento farmacológico , Enfermedad Injerto contra Huésped/mortalidad , Enfermedad Injerto contra Huésped/virología , Histocompatibilidad , Humanos , Inmunosupresores/uso terapéutico , Incidencia , Infecciones por Paramyxoviridae/tratamiento farmacológico , Infecciones por Paramyxoviridae/mortalidad , Infecciones del Sistema Respiratorio/tratamiento farmacológico , Infecciones del Sistema Respiratorio/mortalidad , Infecciones del Sistema Respiratorio/virología , Respirovirus/efectos de los fármacos , Respirovirus/fisiología , Estudios Retrospectivos , Ribavirina/uso terapéutico , Factores de Riesgo , Análisis de Supervivencia , Estados Unidos/epidemiología , Donante no EmparentadoRESUMEN
BACKGROUND: Reliable estimates of disease burden associated with respiratory viruses are keys to deployment of preventive strategies such as vaccination and resource allocation. Such estimates are particularly needed in tropical and subtropical regions where some methods commonly used in temperate regions are not applicable. While a number of alternative approaches to assess the influenza associated disease burden have been recently reported, none of these models have been validated with virologically confirmed data. Even fewer methods have been developed for other common respiratory viruses such as respiratory syncytial virus (RSV), parainfluenza and adenovirus. METHODS AND FINDINGS: We had recently conducted a prospective population-based study of virologically confirmed hospitalization for acute respiratory illnesses in persons <18 years residing in Hong Kong Island. Here we used this dataset to validate two commonly used models for estimation of influenza disease burden, namely the rate difference model and Poisson regression model, and also explored the applicability of these models to estimate the disease burden of other respiratory viruses. The Poisson regression models with different link functions all yielded estimates well correlated with the virologically confirmed influenza associated hospitalization, especially in children older than two years. The disease burden estimates for RSV, parainfluenza and adenovirus were less reliable with wide confidence intervals. The rate difference model was not applicable to RSV, parainfluenza and adenovirus and grossly underestimated the true burden of influenza associated hospitalization. CONCLUSION: The Poisson regression model generally produced satisfactory estimates in calculating the disease burden of respiratory viruses in a subtropical region such as Hong Kong.
Asunto(s)
Hospitalización/estadística & datos numéricos , Modelos Estadísticos , Orthomyxoviridae/fisiología , Infecciones del Sistema Respiratorio/epidemiología , Infecciones del Sistema Respiratorio/virología , Adenoviridae/fisiología , Hong Kong/epidemiología , Humanos , Distribución de Poisson , Análisis de Regresión , Reproducibilidad de los Resultados , Virus Sincitiales Respiratorios/fisiología , Respirovirus/fisiologíaRESUMEN
Paramyxovirus matrix (M) proteins organize virus assembly, functioning as adapters that link together viral ribonucleoprotein complexes and viral glycoproteins at infected cell plasma membranes. M proteins may also function to recruit and manipulate host factors to assist virus budding, similar to retroviral Gag proteins. By yeast two-hybrid screening, angiomotin-like 1 (AmotL1) was identified as a host factor that interacts with the M protein of parainfluenza virus 5 (PIV5). AmotL1-M protein interaction was observed in yeast, in transfected mammalian cells, and in virus-infected cells. Binding was mapped to a 83-amino acid region derived from the C-terminal portion of AmotL1. Overexpression of M-binding AmotL1-derived polypeptides potently inhibited production of PIV5 VLPs and impaired virus budding. Expression of these polypeptides moderately inhibited production of mumps VLPs, but had no effect on production of Nipah VLPs. siRNA-mediated depletion of AmotL1 protein reduced PIV5 budding, suggesting that this interaction is beneficial to paramyxovirus infection.
Asunto(s)
Proteínas de la Membrana/metabolismo , Mapeo de Interacción de Proteínas , Respirovirus/fisiología , Proteínas de la Matriz Viral/metabolismo , Ensamble de Virus , Angiomotinas , Interacciones Huésped-Patógeno , Humanos , Unión Proteica , Técnicas del Sistema de Dos HíbridosRESUMEN
The entry of enveloped viruses into host cells is accomplished by fusion of the viral envelope with the target cell membrane. For the paramyxovirus parainfluenza virus type 5 (PIV-5), this fusion involves an attachment protein (HN) and a class I viral fusion protein (F). We investigated the effect of 20 different combinations of 12 amino-acid substitutions within functional domains of the PIV-5 F glycoprotein, by performing cell surface expression measurements, quantitative fusion and syncytia assays. We found that combinations of mutations conferring an autonomous phenotype with mutations leading to an increased fusion activity were compatible and generated functional PIV-5 F proteins. The addition of mutations in the heptad-repeat domains led to both autonomous and hyperfusogenic phenotypes, despite the low cell surface expression of the corresponding mutants. Such engineering approach may prove useful not only for deciphering the fundamental mechanism behind viral-mediated membrane fusion but also in the development of potential therapeutic applications.
Asunto(s)
Evolución Molecular Dirigida , Respirovirus/fisiología , Proteínas Virales de Fusión/fisiología , Internalización del Virus , Sustitución de Aminoácidos/genética , Animales , Línea Celular , Células Gigantes/virología , Haplorrinos , Humanos , Mutagénesis , Respirovirus/genética , Proteínas Virales de Fusión/genéticaRESUMEN
Respiratory syncytial (RSV) and parainfluenza (PIV) viruses are primary causes of acute bronchiolitis and wheezing illnesses in infants and young children. To further understand inflammation in the airways following infection, we tested for the presence of matrix metalloproteinases (MMP) and natural tissue inhibitors of MMP (TIMP) in primary and established human cell lines, and in the nasopharyngeal secretions (NPS) of human infants infected with RSV or PIV. Using ELISA and multiplex-based assays, MMP-9 and TIMP-1 proteins were, respectively, detected in 66/67 and 67/67 NPS. During PIV or RSV infection TIMP-1 concentrations were associated with hypoxic bronchiolitis. TIMP-1 amounts were also negatively correlated with O2 saturation, and positively correlated with IL-6, MIP-1alpha, and G-CSF amounts following RSV infection. IL-6, MIP-1alpha, and G-CSF were negatively correlated with O2 saturation during RSV infection. Acute respiratory tract disease was not associated with MMP-9 protein/protease activity. Additional studies using real-time quantitative PCR suggested that MMP-9 mRNA copy numbers were elevated in normal human bronchial epithelial (NHBE) cells infected with RSV, while TIMP-1 and TIMP-2 were not increased. However, ELISA did not reveal MMP-9 protein in the NHBE cell culture supernatants. Hence, the data implied that airway epithelial cells were not the primary source of MMP or TIMP following paramyxovirus infection. Taken together, the data suggested that paramyxovirus infection perturbs MMP-9/TIMP-1 homeostasis that in turn may contribute to the severity of respiratory tract disease.
Asunto(s)
Metaloproteinasa 9 de la Matriz/metabolismo , Infecciones por Virus Sincitial Respiratorio/metabolismo , Virus Sincitiales Respiratorios/fisiología , Infecciones del Sistema Respiratorio/metabolismo , Infecciones por Respirovirus/metabolismo , Respirovirus/fisiología , Inhibidores Tisulares de Metaloproteinasas/metabolismo , Línea Celular Tumoral , Quimiocina CCL3 , Quimiocina CCL4 , Femenino , Factor Estimulante de Colonias de Granulocitos/biosíntesis , Humanos , Lactante , Interleucina-6/biosíntesis , Proteínas Inflamatorias de Macrófagos/biosíntesis , Masculino , Nasofaringe/metabolismo , Oxígeno/metabolismo , Mucosa Respiratoria/metabolismo , Infecciones del Sistema Respiratorio/virologíaRESUMEN
OBJECTIVES: To explore the effects of ectodomain sequences between HR1 and HR2 of F1 protein on the specific interaction with its homologous hemagglutinin-neuraminidase (HN) in paramyxoviruses. METHODS: Site-directed mutagenesis was used to obtain mutants containing new enzyme sites on the F genes of Newcastle disease virus (NDV) and human parainfluenza virus (hPIV), and four DNA segments located between the HR1 and HR2 (NDV F-1, hPIV F-1, NDV F-2 and hPIV F-2) were obtained by cutting mutant F genes with specific endonucleases. Gene recombination was used to get chimeric F proteins NDV-C1 and hPIV-C1 by exchanging NDV F-1 and hPIV F-1 each other, and NDV-C2 and hPIV-C2 were also obtained by the same way. All the mutants and chimeric F proteins were co-expressed with their homologous or heterologous HN proteins in eukaryocytes. The fusion functions were assayed with Giemsa staining and reporter gene method for qualitative and quantitative analyses, respectively. The cell surface expression of F proteins was assayed with fluorescence-activated cell sorter (FACS) for quantitative analysis. RESULTS: All the mutants of F proteins had the same functions as their relevant wild types. Chimeric F proteins NDV-C1 and hPIV-C1 had 76.34 and 65.82% of fusion activities, and NDV-C2 and hPIV-C2 had 96.25 and 93.78% of fusion activities, respectively, as compared with their relevant wild types. The analysis of FACS indicated that all the mutants and chimeric F proteins had almost the same expression efficiencies as their relevant wild types. CONCLUSIONS: The segments of NDV F-1 and hPIV F-1 were important for their specific membrane fusion, but NDV F-2 and hPIV F-2 were not.
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Virus de la Enfermedad de Newcastle/fisiología , Paramyxoviridae/crecimiento & desarrollo , Respirovirus/fisiología , Proteínas Virales de Fusión/fisiología , Internalización del Virus , Citometría de Flujo , Genes Reporteros , Proteína HN/fisiología , Histocitoquímica , Inmunohistoquímica , Microscopía , Mutagénesis Sitio-Dirigida , Virus de la Enfermedad de Newcastle/genética , Paramyxoviridae/genética , Recombinación Genética , Respirovirus/genética , Proteínas Virales de Fusión/genética , Proteínas Virales/fisiología , beta-Galactosidasa/análisis , beta-Galactosidasa/genéticaRESUMEN
Toll-like receptors (TLR) are an important component in the innate immune response to a wide variety of pathogens. Recently, a series of studies has addressed the hypothesis that TLR4 also participates in the host innate response against respiratory syncytial virus (RSV), the leading cause of lower respiratory tract infections in infants and young children. In most of the studies available, RSV, which is not a natural pathogen of mice, has been systematically used in mouse models of human bronchiolitis, with conflicting results. Pneumonia virus of mice (PVM), a member of the pneumovirus genus, shares many similarities with RSV. The serological and structural relationships that exist between them suggest that the immune response to these viruses may be similar in their respective natural hosts. To determine the role of TLR4 in host defense against PVM, TLR4-competent and TLR4-deficient mice were intranasally infected with PVM. Variation of body weight, pulmonary function values, histopathology, and pulmonary viral loads were analyzed. None of the investigated clinical, functional, histological and virological parameters was different between strains, which demonstrates that the sensitivity of the mouse to its natural pneumovirus infection is independent of the presence or absence of TLR4 sensing.
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Pulmón/metabolismo , Pulmón/virología , Infecciones por Pneumovirus/metabolismo , Respirovirus/fisiología , Receptor Toll-Like 4/metabolismo , Animales , Peso Corporal , Femenino , Regulación de la Expresión Génica , Pulmón/patología , Ratones , Respiración , Receptor Toll-Like 4/genéticaRESUMEN
Detection of viral antigens and isolation methods has long been used for the diagnosis of respiratory virus infections. The objective was to determine the ability of HuH7 cells to support the replication of prototype and wild strains of respiratory viruses. The cell culture-adapted strains of influenza viruses A and B, parainfluenza viruses 1-4, respiratory syncytial viruses A and B, both strains of the human metapneumoviruses, numerous rhinoviruses, most of the adenoviruses, coronaviruses 229E and OC43, and a number of enteroviruses (poliovirus type 3, coxsackie virus B1, echovirus type 30) replicate in HuH7. The kinetic study of the replication of influenza A and B viruses showed that there were infected cells in HuH7 and MDCK lines as early as 24 hr post-infection. However, the replication of influenza A and B viruses was more rapid and intense on MDCK cells than on HuH7 cells. During the three winters of 1999-2000, 2000-2001, and 2001-2002, of the 1,226 (23.3%) direct fluorescent assay-positive nasal aspirates from children admitted to hospital, 788 were positive for respiratory syncytial virus, 228 for influenza virus, 133 for parainfluenza virus, and 77 for adenovirus. Of the 4,032 direct fluorescent assay-negative nasal aspirates, 571 virus isolates were identified by using HuH7 cell culture; 272 rhinoviruses, 100 influenza viruses A and B, 85 enteroviruses, 40 adenoviruses, 35 coronaviruses, 31 parainfluenza viruses, and 10 respiratory syncytial viruses. Interestingly, 100/328 (30.5%) influenza viruses A and B, 40/189 (21.1%) adenoviruses, and 31/164 (19%) parainfluenza viruses type 1-3, not detected by direct fluorescent assay, were identified by isolation in HuH7 cell culture.
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Línea Celular Tumoral/virología , Infecciones del Sistema Respiratorio/virología , Replicación Viral/fisiología , Coronavirus/fisiología , Humanos , Virus de la Influenza A/fisiología , Virus de la Influenza B/fisiología , Metapneumovirus/fisiología , Virus Sincitiales Respiratorios/fisiología , Respirovirus/fisiología , Rhinovirus/fisiologíaRESUMEN
The Respiratory Syncytial Virus 2003 symposium took place from 8th-11th November 2003 in Stone Mountain, Georgia, and brought together more than 200 international investigators engaged in RSV research. RSV biology, pathogenesis, and clinical data, as well as RSV vaccines and antivirals, were addressed in the meeting, and this review will aim to briefly summarize and discuss the implications of new findings. The meeting also served as the inauguration of the Robert M. Chanock Award for lifetime achievement in RSV research, an award named in honor of the person who started the field of RSV research by recovering the first human RS virus from infants with severe bronchiolitis in 1956.
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Virus Sincitiales Respiratorios/inmunología , Respirovirus/inmunología , Vacunas Virales/inmunología , Animales , Humanos , Virus Sincitiales Respiratorios/patogenicidad , Respirovirus/patogenicidad , Respirovirus/fisiologíaRESUMEN
Our laboratory provided the first proof-of-concept that double-stranded short interfering RNA (ds-siRNA) can act as potent and specific antiviral agents. Designed against specific mRNAs of nonsegmented negative-stranded RNA (NNR) viruses, siRNAs abrogated expression of the corresponding viral proteins, and generated the predicted viral phenotypes. Knockdown was demonstrated across different genera: respiratory syncytial virus (RSV), a pneumovirus; vesicular stomatitis virus (VSV), a rhabdovirus; and human parainfluenza virus (HPIV), a paramyxovirus. The targeted genes could have a wide range of functions, thus documenting the versatility of the technique. Interestingly, antisense single-stranded siRNA (ss-siRNA) was also effective, albeit at a higher concentration. NNR viral genomic and antigenomic RNA, which are encapsidated by nucleocapsid protein and serve as templates for viral RNA-dependent RNA polymerase, were resistant to siRNA. Together, siRNAs offer complementary advantages over traditional mutational analyses that are difficult to perform in NNR viruses, and are also an important new tool to dissect host-virus interactive pathways.