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Journal of Virological Methods Jan 2018For vaccine development, 3D-structure determination, direct fluorescent labelling, and numerous other studies, homogeneous virus preparations of high purity are...
For vaccine development, 3D-structure determination, direct fluorescent labelling, and numerous other studies, homogeneous virus preparations of high purity are essential. Working with human rhinoviruses (RVs), members of the picornavirus family and the main cause of generally mild respiratory infections, we noticed that our routine preparations appeared highly pure on analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), exclusively showing the four viral capsid proteins (VPs). However, the preparations turned out to contain substantial amounts of contaminating material when analyzed by orthogonal analytical methods including capillary zone electrophoresis, nano electrospray gas-phase electrophoretic mobility molecular analysis (nES GEMMA), and negative stain transmission electron microscopy (TEM). Because these latter analyses are not routine to many laboratories, the above contaminations might remain unnoticed and skew experimental results. By using human rhinovirus serotype A2 (RV-A2) as example we report monolithic anion-exchange chromatography (AEX) as a last polishing step in the purification and demonstrate that it yields infective, highly pure, virus (RV-A2 in the respective fractions was confirmed by peptide mass fingerprinting) devoid of foreign material as judged by the above criteria.
Topics: Chromatography, Ion Exchange; Rhinovirus; Virology
PubMed: 28966037
DOI: 10.1016/j.jviromet.2017.09.027 -
Respiratory Research Dec 2010Rhinoviruses (RV) are key triggers in acute asthma exacerbations. Previous studies suggest that men suffer from infectious diseases more frequently and with greater...
BACKGROUND
Rhinoviruses (RV) are key triggers in acute asthma exacerbations. Previous studies suggest that men suffer from infectious diseases more frequently and with greater severity than women. Additionally, the immune response to most infections and vaccinations decreases with age. Most immune function studies do not account for such differences, therefore the aim of this study was to determine if the immune response to rhinovirus varies with sex or age.
METHODS
Blood mononuclear cells were isolated from 63 healthy individuals and grouped by sex and age (≤50 years old and ≥52 years old). Cells were cultured with rhinovirus 16 at a multiplicity of infection of 1. The chemokine IP-10 was measured at 24 h as an index of innate immunity while IFNγ and IL-13 were measured at 5 days as an index of adaptive immunity.
RESULTS
Rhinovirus induced IFNγ and IL-13 was significantly higher in ≤50 year old women than in age matched men (p < 0.02 and p < 0.05) and ≥52 year old women (p < 0.02 and p > 0.005). There was no sex or age based difference in rhinovirus induced IP-10 expression. Both IFNγ and IL-13 were negatively correlated with age in women but not in men.
CONCLUSIONS
This study suggests that pre-menopausal women have a stronger adaptive immune response to rhinovirus infection than men and older people, though the mechanisms responsible for these differences remain to be determined. Our findings highlight the importance of gender and age balance in clinical studies and in the development of new treatments and vaccines.
Topics: Adaptive Immunity; Adolescent; Adult; Aged; Aged, 80 and over; Aging; Cells, Cultured; Female; Humans; Leukocytes, Mononuclear; Male; Middle Aged; Rhinovirus; Sex Factors; Young Adult
PubMed: 21194432
DOI: 10.1186/1465-9921-11-184 -
Mutations in VP1 and 3A proteins improve binding and replication of rhinovirus C15 in HeLa-E8 cells.Virology Dec 2016Viruses in the rhinovirus C species (RV-C) can cause severe respiratory illnesses in children including pneumonia and asthma exacerbations. A transduced cell line...
Viruses in the rhinovirus C species (RV-C) can cause severe respiratory illnesses in children including pneumonia and asthma exacerbations. A transduced cell line (HeLa-E8) stably expressing the CDHR3-Y receptor variant, supports propagation of RV-C after infection. C15 clinical or recombinant isolates replicate in HeLa-E8, however progeny yields are lower than those of related strains of RV-A and RV-B. Serial passaging of C15 in HeLa-E8 resulted in stronger cytopathic effects and increased (≥10-fold) virus binding to cells and progeny yields. The adaptation was acquired by two mutations which increased binding (VP1 TK) and replication (3A EK), respectively. A similar 3A mutation engineered into C2 and C41 cDNAs also improved viral replication (2-8 fold) in HeLa but the heparan sulfate mediated cell-binding enhancement by the VP1 change was C15-specific. The findings now enable large-scale cost-effective C15 production by infection and the testing of RV-C infectivity by plaque assay.
Topics: Amino Acid Motifs; HeLa Cells; Humans; Mutation, Missense; Picornaviridae Infections; Rhinovirus; Viral Proteins; Virus Attachment; Virus Replication
PubMed: 27743961
DOI: 10.1016/j.virol.2016.09.025 -
Antiviral Chemistry & Chemotherapy 2005Fluoro-substituted flavones and 2-styrylchromones, related to natural and synthetic flavonoids previously described, were prepared, characterized and tested for...
Fluoro-substituted flavones and 2-styrylchromones, related to natural and synthetic flavonoids previously described, were prepared, characterized and tested for anti-rhinovirus activity. Structural elucidation of the new compounds was performed by IR, NMR spectra and X-ray crystal structure analysis for 6-fluoro-3-hydroxy-2-styrylchromone. The antiviral potency was evaluated by a plaque reduction assay in HeLa cell cultures infected with rhinoviruses 1B and 14, selected as representative serotypes for viral groups B and A of human rhinoviruses, respectively. In comparison with results previously obtained, the introduction of the fluorine atom seems to exert a positive influence on the activity against serotype 14 while counteracting the effect against serotype 1B.
Topics: Antiviral Agents; Chromones; HeLa Cells; Humans; Hydrocarbons, Fluorinated; Inhibitory Concentration 50; Models, Chemical; Models, Molecular; Molecular Structure; Rhinovirus
PubMed: 16130524
DOI: 10.1177/095632020501600406 -
Cellular and Molecular Life Sciences :... Jan 2007Rhinoviruses, which cause common cold, belong to the Picornaviridae family, small non-enveloped viruses (diameter 15-30 nm) containing a single-stranded RNA genome... (Review)
Review
Rhinoviruses, which cause common cold, belong to the Picornaviridae family, small non-enveloped viruses (diameter 15-30 nm) containing a single-stranded RNA genome (about 7 kb). Over 100 different rhinoviral serotypes have been identified thus far, establishing rhinoviruses as the most diverse group of Picornaviridae. Based on receptor binding properties, rhinoviruses are divided into two classes: the major group binding to intracellular adhesion molecule-1 and the minor group binding to the very low density lipoprotein receptors. Interactions between virus and the receptor molecules cause a conformational change in the capsid, which is a prerequisite for viral uptake. Rhinoviruses trigger a chemokine response upon infection that may lead to exacerbation of the symptoms of common cold, i.e. asthma and inflammation. The following review aims to summarize the knowledge about rhinoviral infections and discusses therapeutical approaches against this almost perfectly adapted pathogen.
Topics: Antiviral Agents; Capsid; Capsid Proteins; Common Cold; Cytokines; Humans; Intercellular Adhesion Molecule-1; Receptors, LDL; Receptors, Virus; Rhinovirus; Signal Transduction
PubMed: 17131060
DOI: 10.1007/s00018-006-6266-5 -
Peptides Sep 2017Human rhinoviruses (HRVs) are the most common cause of viral respiratory tract infections, and are associated with significant morbidity and mortality in...
Human rhinoviruses (HRVs) are the most common cause of viral respiratory tract infections, and are associated with significant morbidity and mortality in immunocompromised individuals and patients with pre-existing pulmonary conditions. The therapeutic options available are extremely limited and therefore novel therapeutics for HRV infections are of significant interest. Cathelicidins have been shown to have potent antiviral activity against a range of pathogens and are known to be key immunomodulatory mediators during infection. We therefore assessed the antiviral potential of cathelicidins from humans and other mammalian species against HRV, together with the potential for the human cathelicidin to modulate apoptotic pathways and alter cell viability during HRV infection. We demonstrate that LL-37, the porcine cathelicidin Protegrin-1, and the ovine cathelicidin SMAP-29 display potent antiviral activity towards HRV and that this activity is visible when either the virus is exposed to the peptides prior to cell infection or after cells have been infected. We further demonstrate that, in contrast to established findings with bacterial infection models, LL-37 does not induce apoptosis or necrosis in HRV-infected lung epithelial cells at physiological or superphysiological concentrations, but does reduce the metabolic activity of infected cells compared to uninfected cells treated with similar peptide concentrations. Collectively, the findings from this study demonstrate that the mechanism of action of cathelicidins against rhinovirus is by directly affecting the virus and we propose that the delivery of exogenous cathelicidins, or novel synthetic analogues, represent an exciting and novel therapeutic strategy for rhinovirus infection.
Topics: Animals; Antimicrobial Cationic Peptides; Antiviral Agents; Blood Proteins; Cathelicidins; Epithelial Cells; Humans; Respiratory Tract Infections; Rhinovirus; Sheep; Swine
PubMed: 28764966
DOI: 10.1016/j.peptides.2017.07.013 -
Journal of Clinical Virology : the... Mar 2015human rhinovirus (HRV) is a major cause of influenza-like illness (ILI) in adults and children. Differences in disease severity by HRV species have been described among... (Observational Study)
Observational Study
BACKGROUND
human rhinovirus (HRV) is a major cause of influenza-like illness (ILI) in adults and children. Differences in disease severity by HRV species have been described among hospitalized patients with underlying illness. Less is known about the clinical and virologic characteristics of HRV infection among otherwise healthy populations, particularly adults.
OBJECTIVES
to characterize molecular epidemiology of HRV and association between HRV species and clinical presentation and viral shedding.
STUDY DESIGN
observational, prospective, facility-based study of ILI was conducted from February 2010 to April 2012. Collection of nasopharyngeal specimens, patient symptoms, and clinical information occurred on days 0, 3, 7, and 28. Patients recorded symptom severity daily for the first 7 days of illness in a symptom diary. HRV was identified by RT-PCR and genotyped for species determination. Cases who were co-infected with other viral respiratory pathogens were excluded from the analysis. We evaluated the associations between HRV species, clinical severity, and patterns of viral shedding.
RESULTS
eighty-four HRV cases were identified and their isolates genotyped. Of these, 62 (74%) were >18 years. Fifty-four were HRV-A, 11HRV-B, and 19HRV-C. HRV-C infection was more common among children than adults (59% vs. 10%, P<0.001). Among adults, HRV-A was associated with higher severity of upper respiratory symptoms compared to HRV-B (P=0.02), but no such association was found in children. In addition, adults shed HRV-A significantly longer than HRV-C (P trend=0.01).
CONCLUSIONS
among otherwise healthy adults with HRV infection, we observed species-specific differences in respiratory symptom severity and duration of viral shedding.
Topics: Adolescent; Adult; Aged; Child; Child, Preschool; Female; Genotype; Humans; Infant; Infant, Newborn; Male; Middle Aged; Molecular Epidemiology; Phylogeny; Picornaviridae Infections; Prospective Studies; Respiratory Tract Infections; Rhinovirus; Sequence Analysis, DNA; Virus Shedding; Young Adult
PubMed: 25728083
DOI: 10.1016/j.jcv.2015.01.007 -
Journal of Virology Dec 2016The human rhinovirus (HRV) 3C and 2A proteases (3C and 2A, respectively) are critical in HRV infection, as they are required for viral polyprotein processing as well as...
UNLABELLED
The human rhinovirus (HRV) 3C and 2A proteases (3C and 2A, respectively) are critical in HRV infection, as they are required for viral polyprotein processing as well as proteolysing key host factors to facilitate virus replication. Early in infection, 3C is present as its precursor 3CD, which, although the mechanism of subcellular targeting is unknown, is found in the nucleus as well as the cytoplasm. In this study, we use transfected and infected cell systems to show that 2A activity is required for 3CD nuclear localization. Using green fluorescent protein (GFP)-tagged forms of 3C, 3D, and mutant derivatives thereof, we show that 3C is located in the cytoplasm and the nucleus, whereas 3CD and 3D are localized predominantly in the cytoplasm, implying that 3D lacks nuclear targeting ability and that 3C activity within 3CD is not sufficient to allow the larger protein into the nucleus. Importantly, by coexpressing mCherry-2A fusion proteins, we demonstrate formally that 2A activity is required to allow HRV 3CD access to the nucleus. In contrast, mCherry-3C is insufficient to allow 3CD access to the nucleus. Finally, we confirm the relevance of these results to HRV infection by demonstrating that nuclear localization of 3CD correlates with 2A activity and not 3C activity, which is observed only later in infection. The results thus define the temporal activities of 2A and 3CD/3C activities in HRV serotype16 infection.
IMPORTANCE
The human rhinovirus genome encodes two proteases, 2A and 3C, as well as a precursor protease, 3CD. These proteases are essential for efficient virus replication. The 3CD protein is found in the nucleus early during infection, though the mechanism of subcellular localization is unknown. Here we show that 2A protease is required for this localization, the 3C protease activity of 3CD is not sufficient to allow 3CD entry into the nucleus, and 3D lacks nuclear targeting ability. This study demonstrates that both 2A and 3C proteases are required for the correct localization of proteins during infection and defines the temporal regulation of 2A and 3CD/3C protease activities during HRV16 infection.
Topics: 3C Viral Proteases; Cell Nucleus; Cysteine Endopeptidases; Cytoplasm; Gene Expression Regulation, Viral; Genes, Reporter; Green Fluorescent Proteins; HeLa Cells; Host-Pathogen Interactions; Humans; Luminescent Proteins; Protein Transport; Proteolysis; Rhinovirus; Serogroup; Viral Proteins; Virus Replication; Red Fluorescent Protein
PubMed: 27681132
DOI: 10.1128/JVI.00974-16 -
Journal of Virology Dec 2016Rhinovirus (RV) species A and C are the most frequent cause of respiratory viral illness worldwide, and RV-C has been linked to more severe exacerbations of asthma in...
UNLABELLED
Rhinovirus (RV) species A and C are the most frequent cause of respiratory viral illness worldwide, and RV-C has been linked to more severe exacerbations of asthma in young children. Little is known about the immune responses to the different RV species, although studies comparing IgG1 antibody titers found impaired antibody responses to RV-C. Therefore, the aim of this study was to assess whether T-cell immunity to RV-C is similarly impaired. We measured T-cell proliferation to overlapping synthetic peptides covering the entire VP1 capsid protein of an RV-A and RV-C genotype for 20 healthy adult donors. Human leukocyte antigen (HLA) was typed in all the donors in order to investigate possible associations between the HLA type and RV peptide recognition. Total and specific IgG1 antibody titers to the VP1 proteins of both RV-A and RV-C were also measured to examine associations between the antibody and T-cell responses. We identified T-cell epitopes that are specific to and representative of each RV-A and RV-C species. These epitopes stimulated CD4-specific T-cell proliferation, with similar magnitudes of response for both RV species. All the donors, independent of their HLA-DR or -DQ type, were able to recognize the immunodominant RV-A and -C regions of VP1. Furthermore, the presence or absence of specific antibody titers was not related to changes in T-cell recognition. Our results indicate a dissociation between the antibody and T-cell responses to rhinoviruses. The species-representative T-cell epitopes identified in this study are valuable tools for future studies investigating T-cell responses to the different RV species.
IMPORTANCE
Rhinoviruses (RVs) are mostly associated with the common cold and asthma exacerbations, although their contributions to most upper and lower respiratory tract diseases have increasingly been reported. Species C (RV-C) has been associated with more frequent and severe asthma exacerbations in young children and, along with RV-A, is the most clinically relevant species. Little is known about how our immune system responds to rhinoviruses, and there are limited tools to study specific adaptive immunity against each rhinovirus species. In this study, we identified immunodominant T-cell epitopes of the VP1 proteins of RV-A and RV-C, which are representative of each species. The study found that T-cell responses to RV-A and RV-C were of similar magnitudes, in contrast with previous findings showing RV-C-specific antibody responses were low. These findings will provide the basis for future studies on the immune response to rhinoviruses and can help elucidate the mechanisms of severity of rhinovirus-induced infections.
Topics: Adult; Amino Acid Sequence; Antibodies, Viral; Antibody Specificity; Asthma; Common Cold; Epitopes, T-Lymphocyte; Female; Healthy Volunteers; Histocompatibility Testing; Humans; Immunodominant Epitopes; Lymphocyte Activation; Male; Middle Aged; Rhinovirus; Sequence Homology, Amino Acid; Species Specificity; T-Lymphocytes; Viral Proteins; Young Adult
PubMed: 27630239
DOI: 10.1128/JVI.01701-16 -
PLoS Biology Jun 2019Rhino- and enteroviruses are important human pathogens, against which no antivirals are available. The best-studied inhibitors are "capsid binders" that fit in a...
Rhino- and enteroviruses are important human pathogens, against which no antivirals are available. The best-studied inhibitors are "capsid binders" that fit in a hydrophobic pocket of the viral capsid. Employing a new class of entero-/rhinovirus inhibitors and by means of cryo-electron microscopy (EM), followed by resistance selection and reverse genetics, we discovered a hitherto unknown druggable pocket that is formed by viral proteins VP1 and VP3 and that is conserved across entero-/rhinovirus species. We propose that these inhibitors stabilize a key region of the virion, thereby preventing the conformational expansion needed for viral RNA release. A medicinal chemistry effort resulted in the identification of analogues targeting this pocket with broad-spectrum activity against Coxsackieviruses B (CVBs) and compounds with activity against enteroviruses (EV) of groups C and D, and even rhinoviruses (RV). Our findings provide novel insights in the biology of the entry of entero-/rhinoviruses and open new avenues for the design of broad-spectrum antivirals against these pathogens.
Topics: Amino Acid Sequence; Amino Acids; Antigens, Viral; Antiviral Agents; Binding Sites; Capsid; Capsid Proteins; Cryoelectron Microscopy; Drug Development; Enterovirus; Humans; Models, Molecular; Molecular Conformation; Rhinovirus; Viral Proteins; Virion
PubMed: 31185007
DOI: 10.1371/journal.pbio.3000281