RESUMEN
BACKGROUND: It has long been known that nasal inoculation with influenza A virus produces asymptomatic to febrile infections. Uncertainty persists about whether these infections are sufficiently similar to natural infections for studying human-to-human transmission. METHODS: We compared influenza A viral aerosol shedding from volunteers nasally inoculated with A/Wisconsin/2005 (H3N2) and college community adults naturally infected with influenza A/H3N2 (2012-2013), selected for influenza-like illness with objectively measured fever or a positive Quidel QuickVue A&B test. Propensity scores were used to control for differences in symptom presentation observed between experimentally and naturally infected groups. RESULTS: Eleven (28%) experimental and 71 (86%) natural cases shed into fine particle aerosols (P < .001). The geometric mean (geometric standard deviation) for viral positive fine aerosol samples from experimental and natural cases was 5.1E + 3 (4.72) and 3.9E + 4 (15.12) RNA copies/half hour, respectively. The 95th percentile shedding rate was 2.4 log10 greater for naturally infected cases (1.4E + 07 vs 7.4E + 04). Certain influenza-like illness-related symptoms were associated with viral aerosol shedding. The almost complete lack of symptom severity distributional overlap between groups did not support propensity score-adjusted shedding comparisons. CONCLUSIONS: Due to selection bias, the natural and experimental infections had limited symptom severity distributional overlap precluding valid, propensity score-adjusted comparison. Relative to the symptomatic naturally infected cases, where high aerosol shedders were found, experimental cases did not produce high aerosol shedders. Studying the frequency of aerosol shedding at the highest observed levels in natural infections without selection on symptoms or fever would support helpful comparisons.
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Virus de la Influenza A , Gripe Humana , Adulto , Aerosoles , Humanos , Subtipo H3N2 del Virus de la Influenza A , Esparcimiento de VirusRESUMEN
Uncertainty about the importance of influenza transmission by airborne droplet nuclei generates controversy for infection control. Human challenge-transmission studies have been supported as the most promising approach to fill this knowledge gap. Healthy, seronegative volunteer 'Donors' (n = 52) were randomly selected for intranasal challenge with influenza A/Wisconsin/67/2005 (H3N2). 'Recipients' randomized to Intervention (IR, n = 40) or Control (CR, n = 35) groups were exposed to Donors for four days. IRs wore face shields and hand sanitized frequently to limit large droplet and contact transmission. One transmitted infection was confirmed by serology in a CR, yielding a secondary attack rate of 2.9% among CR, 0% in IR (p = 0.47 for group difference), and 1.3% overall, significantly less than 16% (p<0.001) expected based on a proof-of-concept study secondary attack rate and considering that there were twice as many Donors and days of exposure. The main difference between these studies was mechanical building ventilation in the follow-on study, suggesting a possible role for aerosols.
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Gripe Humana/transmisión , Aerosoles , Femenino , Humanos , Subtipo H3N2 del Virus de la Influenza A , MasculinoRESUMEN
An authentic, hands-on experience in the laboratory is an important part of any undergraduate biology course. However, there are a limited number of mammalian virus systems that students can work with safely in an undergraduate teaching laboratory. For many systems, the risk to the students is too high. The influenza A virus M2 protein trans-complementation system bridges this gap. This system consists of a virus with mutations that prevent the expression of the essential M2 protein; therefore this virus can only replicate in a cell line that provides M2 in trans. Here, we describe the use of this system to carry out hemagglutination, real-time reverse transcriptase PCR, 50% tissue culture infectious dose, and plaque assays in an undergraduate lab setting.
RESUMEN
The influenza A virus M1 and M2 proteins play important roles in virus assembly and in the morphology of virus particles. Mutations in the distal cytoplasmic tail region of M2, and in particular a tyrosine-to-alanine mutation at residue 76 (Y76A), were essential for infectious virus production and filament formation while having limited effects on total virus particle budding. Using a novel selection method, mutations at seven different M1 amino acids (residue 73, 94, 135, 136, or 138 or a double mutation, 93/244) that are not found in circulating influenza virus strains or have not been previously identified to play a role in influenza A virus assembly were found to complement the lethal M2Y76A mutation. These M1 suppressor mutations restored infectious virus production in the presence of M2Y76A and mediated increased budding and filament formation even in the absence of M2. However, the efficiency of infectious virus replication was still dependent on the presence of the distal region of the M2 cytoplasmic tail. The data suggest that influenza A virus budding and genome incorporation can occur independently and provide further support for complementary roles of the M1 and M2 proteins in virus assembly.IMPORTANCE Influenza virus particle assembly involves the careful coordination of various viral and host factors to optimally produce infectious virus particles. We have previously identified a mutation at position 76 of the influenza A virus M2 protein that drastically reduces infectious virus production and filament formation with minimal effects on virus budding. In this work, we identified suppressor mutations in the M1 protein which complement this lethal M2 mutation by increasing the efficiency with which virus particles bud from infected cells and restoring filament formation at the infected-cell surface. M2 distal cytoplasmic domain sequences were still required for optimal infectivity. This indicates that M1 and M2 can functionally replace each other in some, but not all, aspects of virus particle assembly.
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Virus de la Influenza A/fisiología , Mutación , Proteínas de la Matriz Viral/genética , Liberación del Virus/genética , Animales , Citoplasma/metabolismo , Perros , Genoma Viral , Células HEK293 , Humanos , Virus de la Influenza A/genética , Virus de la Influenza A/crecimiento & desarrollo , Células de Riñón Canino Madin Darby , ARN Viral , Supresión Genética , Ensamble de Virus/genética , Replicación ViralRESUMEN
The CDC recommends that healthcare settings provide influenza patients with facemasks as a means of reducing transmission to staff and other patients, and a recent report suggested that surgical masks can capture influenza virus in large droplet spray. However, there is minimal data on influenza virus aerosol shedding, the infectiousness of exhaled aerosols, and none on the impact of facemasks on viral aerosol shedding from patients with seasonal influenza. We collected samples of exhaled particles (one with and one without a facemask) in two size fractions ("coarse">5 µm, "fine"≤5 µm) from 37 volunteers within 5 days of seasonal influenza onset, measured viral copy number using quantitative RT-PCR, and tested the fine-particle fraction for culturable virus. Fine particles contained 8.8 (95% CI 4.1 to 19) fold more viral copies than did coarse particles. Surgical masks reduced viral copy numbers in the fine fraction by 2.8 fold (95% CI 1.5 to 5.2) and in the coarse fraction by 25 fold (95% CI 3.5 to 180). Overall, masks produced a 3.4 fold (95% CI 1.8 to 6.3) reduction in viral aerosol shedding. Correlations between nasopharyngeal swab and the aerosol fraction copy numbers were weak (râ=â0.17, coarse; râ=â0.29, fine fraction). Copy numbers in exhaled breath declined rapidly with day after onset of illness. Two subjects with the highest copy numbers gave culture positive fine particle samples. Surgical masks worn by patients reduce aerosols shedding of virus. The abundance of viral copies in fine particle aerosols and evidence for their infectiousness suggests an important role in seasonal influenza transmission. Monitoring exhaled virus aerosols will be important for validation of experimental transmission studies in humans.
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Infección Hospitalaria/prevención & control , Gripe Humana/transmisión , Máscaras , Orthomyxoviridae , Aerosoles , Microbiología del Aire , Tos/virología , Infección Hospitalaria/virología , Espiración , Humanos , Orthomyxoviridae/fisiología , Tamaño de la Partícula , ARN Viral , Respiración , Esparcimiento de VirusRESUMEN
Influenza remains a serious public health threat throughout the world. Vaccines and antivirals are available that can provide protection from infection. However, new viral strains emerge continuously because of the plasticity of the influenza genome, which necessitates annual reformulation of vaccine antigens, and resistance to antivirals can appear rapidly and become entrenched in circulating virus populations. In addition, the spread of new pandemic strains is difficult to contain because of the time required to engineer and manufacture effective vaccines. Monoclonal antibodies that target highly conserved viral epitopes might offer an alternative protection paradigm. Herein we describe the isolation of a panel of monoclonal antibodies derived from the IgG(+) memory B cells of healthy, human subjects that recognize a previously unknown conformational epitope within the ectodomain of the influenza matrix 2 protein, M2e. This antibody binding region is highly conserved in influenza A viruses, being present in nearly all strains detected to date, including highly pathogenic viruses that infect primarily birds and swine, and the current 2009 swine-origin H1N1 pandemic strain (S-OIV). Furthermore, these human anti-M2e monoclonal antibodies protect mice from lethal challenges with either H5N1 or H1N1 influenza viruses. These results suggest that viral M2e can elicit broadly cross-reactive and protective antibodies in humans. Accordingly, recombinant forms of these human antibodies may provide useful therapeutic agents to protect against infection from a broad spectrum of influenza A strains.
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Epítopos/inmunología , Subtipo H5N1 del Virus de la Influenza A/inmunología , Virus de la Influenza A/genética , Virus de la Influenza A/inmunología , Gripe Aviar/inmunología , Animales , Anticuerpos/genética , Anticuerpos/inmunología , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Aves , Reacciones Cruzadas/genética , Reacciones Cruzadas/inmunología , Brotes de Enfermedades , Epítopos/genética , Humanos , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H5N1 del Virus de la Influenza A/genética , Vacunas contra la Influenza/genética , Vacunas contra la Influenza/inmunología , Gripe Aviar/genética , Gripe Humana/genética , Gripe Humana/inmunología , Gripe Humana/prevención & control , Ratones , Datos de Secuencia MolecularRESUMEN
The cytoplasmic tail of the influenza A virus M2 protein is required for the production of infectious virions. In this study, critical residues in the M2 cytoplasmic tail were identified by single-alanine scanning mutagenesis. The tyrosine residue at position 76, which is conserved in >99% of influenza virus strains sequenced to date, was identified as being critical for the formation of infectious virus particles using both reverse genetics and a protein trans-complementation assay. Recombinant viruses encoding M2 with the Y76A mutation demonstrated replication defects in MDCK cells as well as in primary differentiated airway epithelial cell cultures, defects in the formation of filamentous virus particles, and reduced packaging of nucleoprotein into virus particles. These defects could all be overcome by a mutation of serine to tyrosine at position 71 of the M2 cytoplasmic tail, which emerged after blind passage of viruses containing the Y76A mutation. These data confirm and extend our understanding of the significance of the M2 protein for infectious virus particle assembly.
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Virus de la Influenza A/fisiología , Tirosina/metabolismo , Proteínas de la Matriz Viral/química , Proteínas de la Matriz Viral/metabolismo , Virión/fisiología , Replicación Viral , Secuencias de Aminoácidos , Animales , Línea Celular , Cricetinae , Humanos , Virus de la Influenza A/química , Virus de la Influenza A/genética , Gripe Humana/virología , Mutación , Tirosina/química , Tirosina/genética , Proteínas de la Matriz Viral/genética , Virión/química , Virión/genética , Ensamble de VirusRESUMEN
The influenza A virus M2 protein has important roles during virus entry and in the assembly of infectious virus particles. The cytoplasmic tail of the protein can be palmitoylated at a cysteine residue, but this residue is not conserved in a number of human influenza A virus isolates. Recombinant viruses encoding M2 proteins with a serine substituted for the cysteine at position 50 were generated in the A/WSN/33 (H1N1) and A/Udorn/72 (H3N2) genetic backgrounds. The recombinant viruses were not attenuated for replication in MDCK cells, Calu-3 cells, or in primary differentiated murine trachea epithelial cell cultures, indicating there was no significant contribution of M2 palmitoylation to virus replication in vitro. The A/WSN/33 M2C50S virus displayed a slightly reduced virulence after infection of mice, suggesting that there may be novel functions for M2 palmitoylation during in vivo infection.
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Subtipo H1N1 del Virus de la Influenza A/fisiología , Subtipo H3N2 del Virus de la Influenza A/fisiología , Proteínas de la Matriz Viral/metabolismo , Factores de Virulencia/metabolismo , Replicación Viral , Sustitución de Aminoácidos , Animales , Peso Corporal , Línea Celular , Células Cultivadas , Perros , Humanos , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Subtipo H3N2 del Virus de la Influenza A/patogenicidad , Lipoilación , Pulmón/virología , Ratones , Mutagénesis Sitio-Dirigida , Infecciones por Orthomyxoviridae/patología , Infecciones por Orthomyxoviridae/virología , Análisis de Supervivencia , VirulenciaRESUMEN
Small polykaryocytes resulting from cell fusion are found in herpes simplex virus (HSV) lesions in patients, but their significance for viral spread and pathogenesis is unclear. Although syncytial variants causing extensive fusion in tissue culture can be readily isolated from laboratory strains, they are rarely found in clinical isolates, suggesting that extensive cell fusion may be deleterious in vivo. Syncytial mutations have previously been identified for several laboratory strains, but not for clinical isolates of HSV type 2. To address this deficiency, we studied a recent syncytial clinical isolate, finding it to be a mixture of two syncytial and one nonsyncytial strain. The two syncytial strains have novel mutations in glycoprotein B, and in vitro cell fusion assays confirmed that they are responsible for syncytium formation. This panel of clinical strains may be ideal for examining the effect of increased cell fusion on pathogenesis.
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Herpesvirus Humano 2/genética , Proteínas Virales de Fusión/genética , Secuencia de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Ciclosporina/farmacología , Células Gigantes/virología , Herpes Genital/virología , Herpesvirus Humano 2/efectos de los fármacos , Herpesvirus Humano 2/aislamiento & purificación , Humanos , Datos de Secuencia Molecular , Mutación , Alineación de Secuencia , Ensayo de Placa ViralRESUMEN
Formation of small polykaryons by cell-cell fusion is characteristic of herpes simplex virus (HSV) lesions, but the great majority of viruses isolated from such lesions produce only limited cell fusion in tissue culture. Because of this, HSV laboratory strains that produce extensive cell fusion (syncytium formation) in culture are regarded as variants or mutants. Furthermore, the rarity of clinical isolates able to produce syncytia in culture suggests that extensive cell fusion is deleterious in vivo. Mutations that confer a syncytial phenotype can then be regarded as bypassing a mechanism that normally limits cell fusion. Determination of how these mutations, some of which are in the cytoplasmic tail of glycoprotein B (gB), lead to syncytium formation will likely reveal how fusion is controlled. Here we show the following. (i) Truncation of the cytoplasmic tail of HSV type 2 gB (gB-2) by a minimum of 25 residues or a maximum of 49 residues produces a syncytial phenotype. (ii) Truncation by 20 to 49 residues increases cell fusion when gB-2 is coexpressed with only gD-2, gH-2, and gL-2. (iii) Truncation by 25 or more residues removes a potential endocytosis motif and increases gB-2 cell surface expression. (iv) Mutation of this motif increases gB-2 cell surface expression but does not increase fusogenic activity, whereas mutation of another potential endocytosis motif does not increase surface expression but does increase fusogenic activity. Therefore, syncytial mutations in the cytoplasmic tail of gB-2 do not act by increasing cell surface levels of the protein.