High Rates of Detection of Respiratory Viruses in Tonsillar Tissues from Children with Chronic Adenotonsillar Disease

Chronic tonsillar diseases are an important health problem, leading to large numbers of surgical procedures worldwide. Little is known about pathogenesis of these diseases. In order to investigate the role of respiratory viruses in chronic adenotonsillar diseases, we developed a cross-sectional study to determine the rates of viral detections of common respiratory viruses detected by TaqMan real time PCR (qPCR) in nasopharyngeal secretions, tonsillar tissues and peripheral blood from 121 children with chronic tonsillar diseases, without symptoms of acute respiratory infections. At least one respiratory virus was detected in 97.5% of patients. The viral co-infection rate was 69.5%. The most frequently detected viruses were human adenovirus in 47.1%, human enterovirus in 40.5%, human rhinovirus in 38%, human bocavirus in 29.8%, human metapneumovirus in 17.4% and human respiratory syncytial virus in 15.7%. Results of qPCR varied widely between sample sites: human adenovirus, human bocavirus and human enterovirus were predominantly detected in tissues, while human rhinovirus was more frequently detected in secretions. Rates of virus detection were remarkably high in tonsil tissues: over 85% in adenoids and close to 70% in palatine tonsils. In addition, overall virus detection rates were higher in more hypertrophic than in smaller adenoids (p = 0.05), and in the particular case of human enteroviruses, they were detected more frequently (p = 0.05) in larger palatine tonsils than in smaller ones. While persistence/latency of DNA viruses in tonsillar tissues has been documented, such is not the case of RNA viruses. Respiratory viruses are highly prevalent in adenoids and palatine tonsils of patients with chronic tonsillar diseases, and persistence of these viruses in tonsils may stimulate chronic inflammation and play a role in the pathogenesis of these diseases.

SEASONALITY OF VIRAL RESPIRATORY INFECTIONS IN SOUTHEAST OF BRAZIL: THE INFLUENCE OF TEMPERATURE AND AIR HUMIDITY

Viruses are the major cause of lower respiratory tract infections in childhood and the main viruses involved are Human Respiratory Syncytial Virus (HRSV), Human Metapneumovirus (HMPV), Influenzavirus A and B (FLUA and FLUB), Human Parainfluenza Virus 1, 2 and 3 (HPIV1, 2 and 3) and Human Rhinovirus (HRV). The purposes of this study were to detect respiratory viruses in hospitalized children younger than six years and identify the influence of temperature and relative air humidity on the detected viruses. Samples of nasopharyngeal washes were collected from hospitalized children between May/2004 and September/2005. Methods of viral detection were RT-PCR, PCR and HRV amplicons were confirmed by hybridization. Results showed 54% (148/272) of viral positivity. HRSV was detected in 29% (79/272) of the samples; HRV in 23.1% (63/272); HPIV3 in 5.1% (14/272); HMPV in 3.3% (9/272); HPIV1 in 2.9% (8/272); FLUB in 1.4% (4/272), FLUA in 1.1% (3/272), and HPIV2 in 0.3% (1/272). The highest detection rates occurred mainly in the spring 2004 and in the autumn 2005. It was observed that viral respiratory infections tend to increase as the relative air humidity decreases, showing significant association with monthly averages of minimal temperature and minimal relative air humidity. In conclusion, viral respiratory infections vary according to temperature and relative air humidity and viral respiratory infections present major incidences it coldest and driest periods.

Severe lower respiratory tract infection in infants and toddlers from a non-affluent population: viral etiology and co-detection as risk factors

Abstract Background Lower respiratory tract infection (LRTI) is a major cause of pediatric morbidity and mortality, especially among non-affluent communities. In this study we determine the impact of respiratory viruses and how viral co-detections/infections can affect clinical LRTI severity in children in a hospital setting. Methods Patients younger than 3 years of age admitted to a tertiary hospital in Brazil during the months of high prevalence of respiratory viruses had samples collected from nasopharyngeal aspiration. These samples were tested for 13 different respiratory viruses through real-time PCR (rt-PCR). Patients were followed during hospitalization, and clinical data and population characteristics were collected during that period and at discharge to evaluate severity markers, especially length of hospital stay and oxygen use. Univariate regression analyses identified potential risk factors and multivariate logistic regressions were used to determine the impact of specific viral detections as well as viral co-detections in relation to clinical outcomes. Results We analyzed 260 episodes of LRTI with a viral detection rate of 85% (n = 222). Co-detection was observed in 65% of all virus-positive episodes. The most prevalent virus was Respiratory Syncytial Virus (RSV) (54%), followed by Human Metapneumovirus (hMPV) (32%) and Human Rhinovirus (HRV) (21%). In the multivariate models, infants with co-detection of HRV + RSV stayed 4.5 extra days (p = 0.004), when compared to infants without the co-detection. The same trends were observed for the outcome of days of supplemental oxygen use. Conclusions Although RSV remains as the main cause of LRTI in infants our study indicates an increase in the length of hospital stay and oxygen use in infants with HRV detected by RT-PCR compared to those without HRV. Moreover, one can speculate that when HRV is detected simultaneously with RSV there is an additive effect that may be reflected in more severe clinical outcome. Also, our study identified a significant number of children infected by recently identified viruses, such as hMPV and Human Bocavirus (HBov), and this is a novel finding for poor communities from developing countries.

Seasonality of viral respiratory infections in Southeast of Brazil: the influence of temperature and air humidity

Viruses are the major cause of lower respiratory tract infections in childhood and the main viruses involved are Human Respiratory Syncytial Virus (HRSV), Human Metapneumovirus (HMPV), Influenzavirus A and B (FLUA and FLUB), Human Parainfluenza Virus 1, 2 and 3 (HPIV1, 2 and 3) and Human Rhinovirus (HRV). The purposes of this study were to detect respiratory viruses in hospitalized children younger than six years and identify the influence of temperature and relative air humidity on the detected viruses. Samples of nasopharyngeal washes were collected from hospitalized children between May/2004 and September/2005. Methods of viral detection were RT-PCR, PCR and HRV amplicons were confirmed by hybridization. Results showed 54% (148/272) of viral positivity. HRSV was detected in 29% (79/272) of the samples; HRV in 23.1% (63/272); HPIV3 in 5.1% (14/272); HMPV in 3.3% (9/272); HPIV1 in 2.9% (8/272); FLUB in 1.4% (4/272), FLUA in 1.1% (3/272), and HPIV2 in 0.3% (1/272). The highest detection rates occurred mainly in the spring 2004 and in the autumn 2005. It was observed that viral respiratory infections tend to increase as the relative air humidity decreases, showing significant association with monthly averages of minimal temperature and minimal relative air humidity. In conclusion, viral respiratory infections vary according to temperature and relative air humidity and viral respiratory infections present major incidences it coldest and driest periods.

In silico target fishing for rationalized ligand discovery exemplified on constituents of Ruta graveolens

The identification of targets whose interaction is likely to result in the successful treatment of a disease is of growing interest for natural product scientists. In the current study we performed an exemplary application of a virtual parallel screening approach to identify potential targets for 16 secondary metabolites isolated and identified from the aerial parts of the medicinal plant RUTA GRAVEOLENS L. Low energy conformers of the isolated constituents were simultaneously screened against a set of 2208 pharmacophore models generated in-house for the IN SILICO prediction of putative biological targets, i. e., target fishing. Based on the predicted ligand-target interactions, we focused on three biological targets, namely acetylcholinesterase (AChE), the human rhinovirus (HRV) coat protein and the cannabinoid receptor type-2 (CB (2)). For a critical evaluation of the applied parallel screening approach, virtual hits and non-hits were assayed on the respective targets. For AChE the highest scoring virtual hit, arborinine, showed the best inhibitory IN VITRO activity on AChE (IC (50) 34.7 muM). Determination of the anti-HRV-2 effect revealed 6,7,8-trimethoxycoumarin and arborinine to be the most active antiviral constituents with IC (50) values of 11.98 muM and 3.19 muM, respectively. Of these, arborinine was predicted virtually. Of all the molecules subjected to parallel screening, one virtual CB (2) ligand was obtained, i. e., rutamarin. Interestingly, in experimental studies only this compound showed a selective activity to the CB (2) receptor ( Ki of 7.4 muM) by using a radioligand displacement assay. The applied parallel screening paradigm with constituents of R. GRAVEOLENS on three different proteins has shown promise as an IN SILICO tool for rational target fishing and pharmacological profiling of extracts and single chemical entities in natural product research.

Antiviral agent blocks breathing of the common cold virus

A dynamic capsid is critical to the events that shape the viral life cycle; events such as cell attachment, cell entry, and nucleic acid release demand a highly mobile viral surface. Protein mass mapping of the common cold virus, human rhinovirus 14 (HRV14), revealed both viral structural dynamics and the inhibition of such dynamics with an antiviral agent, WIN 52084. Viral capsid digestion fragments resulting from proteolytic time-course experiments provided structural information in good agreement with the HRV14 three-dimensional crystal structure. As expected, initial digestion fragments included peptides from the capsid protein VP1. This observation was expected because VP1 is the most external viral protein. Initial digestion fragments also included peptides belonging to VP4, the most internal capsid protein. The mass spectral results together with x-ray crystallography data provide information consistent with a “breathing” model of the viral capsid. Whereas the crystal structure of HRV14 shows VP4 to be the most internal capsid protein, mass spectral results show VP4 fragments to be among the first digestion fragments observed. Taken together this information demonstrates that VP4 is transiently exposed to the viral surface via viral breathing. Comparative digests of HRV14 in the presence and absence of WIN 52084 revealed a dramatic inhibition of digestion. These results indicate that the binding of the antiviral agent not only causes local conformational changes in the drug binding pocket but actually stabilizes the entire viral capsid against enzymatic degradation. Viral capsid mass mapping provides a fast and sensitive method for probing viral structural dynamics as well as providing a means for investigating antiviral drug efficacy.

Viral capsid mobility: A dynamic conduit for inactivation

Mass spectrometry and fluorescent probes have provided direct evidence that alkylating agents permeate the protein capsid of naked viruses and chemically inactivate the nucleic acid. N-acetyl-aziridine and a fluorescent alkylating agent, dansyl sulfonate aziridine, inactivated three different viruses, flock house virus, human rhinovirus-14, and foot and mouth disease virus. Mass spectral studies as well as fluorescent probes showed that alkylation of the genome was the mechanism of inactivation. Because particle integrity was not affected by selective alkylation (as shown by electron microscopy and sucrose gradient experiments), it was reasoned that the dynamic nature of the viral capsid acts as a conduit to the interior of the particle. Potential applications include fluorescent labeling for imaging viral genomes in living cells, the sterilization of blood products, vaccine development, and viral inactivation in vivo.

Internal ribosomal entry site substitution eliminates neurovirulence in intergeneric poliovirus recombinants.

Neuropathogenicity of poliovirus can be attenuated by mutations in the internal ribosomal entry site (IRES) within the 5' nontranslated region of its genome. The Sabin vaccine strains used in prevention of poliomyelitis carry such mutations in their IRES elements. In addition, mutations within the structural and nonstructural proteins of Sabin strains may equally contribute to the attenuation phenotype. Despite their effectiveness as vaccines, the Sabin strains retain a neuropathogenic potential in animal models for poliomyelitis and, at a very low rate, they can cause poliomyelitis in vaccine recipients. The elimination of the neurocytopathic phenotype was achieved through the exchange of the entire poliovirus IRES with its counterpart from human rhinovirus type 2 without affecting growth properties in nonneuronal cells. The attenuating effect of the human rhinovirus type 2 IRES within the context of a poliovirus genome has been mapped to the 3' portion of this genetic element.

Programa Nacional de Vigilância da Gripe: relatório da época 2015/2016

O vírus da gripe é uma das maiores causas de morbilidade e mortalidade em todo o mundo, afetando um elevado número de indivíduos em cada ano. Em Portugal a vigilância epidemiológica da gripe é assegurada pelo Programa Nacional de Vigilância da Gripe (PNVG), através da integração da informação das componentes clínica e virológica, gerando informação detalhada relativamente à atividade gripal. A componente clínica é suportada pela Rede Médicos-Sentinela e tem um papel especialmente relevante por possibilitar o cálculo de taxas de incidência permitindo descrever a intensidade e evolução da epidemia de gripe. A componente virológica tem por base o diagnóstico laboratorial do vírus da gripe e tem como objetivos a deteção e caraterização dos vírus da gripe em circulação. Para o estudo mais completo da etiologia da síndrome gripal foi efectuado o diagnóstico diferencial de outros vírus respiratórios: vírus sincicial respiratório tipo A (RSV A) e B (RSV B), o rhinovírus humano (hRV), o vírus parainfluenza humano tipo 1 (PIV1), 2 (PIV2) e 3 (PIV3), o coronavírus humano (hCoV), o adenovírus (AdV) e o metapneumovirus humano (hMPV). Desde 2009 a vigilância da gripe conta também com a Rede Portuguesa de Laboratórios para o Diagnóstico da Gripe que atualmente é constituída por 15 hospitais onde se realiza o diagnóstico laboratorial da gripe. A informação obtida nesta Rede Laboratorial adiciona ao PNVG dados relativos a casos de doença respiratória mais severa com necessidade de internamento. Em 2011/2012, foi lançado um estudo piloto para vigiar os casos graves de gripe admitidos em Unidades de Cuidados Intensivos (UCI) que deu origem à atual Rede de vigilância da gripe em UCI constituída em 2015/2016 por 31 UCI (324 camas). Esta componente tem como objetivo a monitorização de novos casos de gripe confirmados laboratorialmente e admitidos em UCI, permitindo a avaliação da gravidade da doença associada à infeção pelo vírus da gripe. O Sistema da Vigilância Diária da Mortalidade constitui uma componente do PNVG que permite monitorizar a mortalidade semanal por “todas as causas” durante a época de gripe. É um sistema de vigilância epidemiológica que pretende detetar e estimar de forma rápida os impactos de eventos ambientais ou epidémicos relacionados com excessos de mortalidade. A notificação de casos de Síndrome Gripal (SG) e a colheita de amostras biológicas foi realizada em diferentes redes participantes do PNVG: Rede de Médicos-Sentinela, Rede de Serviços de Urgência/Obstetrícia, médicos do Projeto EuroEVA, Rede Portuguesa de Laboratórios para o Diagnóstico da Gripe e Rede vigilância da gripe em UCI. Na época de vigilância da gripe de 2015/2016 foram notificados 1.273 casos de SG, 87% dos quais acompanhados de um exsudado da nasofaringe para diagnóstico laboratorial. No inverno de 2015/2016 observou-se uma atividade gripal de baixa intensidade. O período epidémico ocorreu entre a semana 53/2015 e a semana 8/2016 e o valor mais elevado da taxa de incidência semanal de SG (72,0/100000) foi observado na semana 53/2015. De acordo com os casos notificados à Rede Médicos-Sentinela, o grupo etário dos 15 aos 64 anos foi o que apresentou uma incidência cumulativa mais elevada. O vírus da gripe foi detetado em 41,0% dos exsudados da nasofaringe recebidos tendo sido detetados outros vírus respiratórios em 24% destes. O vírus da gripe A(H1)pdm09 foi o predominantemente detetado em 90,4% dos casos de gripe. Foram também detetados outros vírus da gripe, o vírus B - linhagem Victoria (8%), o vírus A(H3) (1,3%) e o vírus B- linhagem Yamagata (0,5%). A análise antigénica dos vírus da gripe A(H1)pdm09 mostrou a sua semelhança com a estirpe vacinal 2015/2016 (A/California/7/2009), a maioria dos vírus pertencem ao novo grupo genético 6B.1, que foi o predominantemente detetado em circulação na Europa. Os vírus do tipo B apesar de detetados em número bastante mais reduzido comparativamente com o subtipo A(H1)pdm09, foram na sua maioria da linhagem Victoria que antigenicamente se distinguem da estirpe vacinal de 2015/2016 (B/Phuket/3073/2013). Esta situação foi igualmente verificada nos restantes países da Europa, Estados Unidos da América e Canadá. Os vírus do subtipo A(H3) assemelham-se antigenicamente à estirpe selecionada para a vacina de 2016/2017 (A/Hong Kong/4801/2014). Geneticamente a maioria dos vírus caraterizados pertencem ao grupo 3C.2a, e são semelhantes à estirpe vacinal para a época de 2016/2017. A avaliação da resistência aos antivirais inibidores da neuraminidase, não revelou a circulação de estirpes com diminuição da suscetibilidade aos inibidores da neuraminidase (oseltamivir e zanamivir). A situação verificada em Portugal é semelhante à observada a nível europeu. A percentagem mais elevada de casos de gripe foi verificada nos indivíduos com idade inferior a 45 anos. A febre, as cefaleias, o mal-estar geral, as mialgias, a tosse e os calafrios mostraram apresentar uma forte associação à confirmação laboratorial de um caso de gripe. Foi nos doentes com imunodeficiência congénita ou adquirida que a proporção de casos de gripe foi mais elevada, seguidos dos doentes com diabetes e obesidade. A percentagem total de casos de gripe em mulheres grávidas foi semelhante à observada nas mulheres em idade fértil não grávidas. No entanto, o vírus da gripe do tipo A(H1)pdm09 foi detetado em maior proporção nas mulheres grávidas quando comparado as mulheres não grávidas. A vacina como a principal forma de prevenção da gripe é especialmente recomendada em indivíduos com idade igual ou superior a 65 anos, doentes crónicos e imunodeprimidos, grávidas e profissionais de saúde. A vacinação antigripal foi referida em 13% dos casos notificados. A deteção do vírus da gripe ocorreu em 25% dos casos vacinados e sujeitos a diagnóstico laboratorial estando essencialmente associados ao vírus da gripe A(H1)pdm09, o predominante na época de 2015/2016. Esta situação foi mais frequentemente verificada em indivíduos com idade compreendida entre os 15 e 45 anos. A confirmação de gripe em indivíduos vacinados poderá estar relacionada com uma moderada efetividade da vacina antigripal na população em geral. A informação relativa à terapêutica antiviral foi indicada em 67% casos de SG notificados, proporção superior ao verificado em anos anteriores. Os antivirais foram prescritos a um número reduzido de doentes (9,0%) dos quais 45.0% referiam pelo menos a presença de uma doença crónica ou gravidez. O antiviral mais prescrito foi o oseltamivir. A pesquisa de outros vírus respiratórios nos casos de SG negativos para o vírus da gripe, veio revelar a circulação e o envolvimento de outros agentes virais respiratórios em casos de SG. Os vírus respiratórios foram detetados durante todo o período de vigilância da gripe, entre a semana 40/2015 e a semana 20/2016. O hRV, o hCoV e o RSV foram os agentes mais frequentemente detetados, para além do vírus da gripe, estando o RSV essencialmente associado a crianças com idade inferior a 4 anos de idade e o hRV e o hCoV aos adultos e população mais idosa (≥ 65 anos). A Rede Portuguesa de Laboratórios para o Diagnóstico da Gripe, efetuou o diagnóstico da gripe em 7443 casos de infeção respiratória sendo o vírus da gripe detetado em 1458 destes casos. Em 71% dos casos de gripe foi detetado o vírus da gripe A(H1)pdm09. Os vírus da gripe do tipo A(H3) foram detetados esporadicamente e em número muito reduzido (2%), e em 11% o vírus da gripe A (não subtipado). O vírus da gripe do tipo B foi detetado em 16% dos casos. A frequência de cada tipo e subtipo do vírus da gripe identificados na Rede Hospitalar assemelha-se ao observado nos cuidados de saúde primários (Rede Médicos-Sentinela e Serviços de Urgência). Foi nos indivíduos adultos, entre os 45-64 anos, que o vírus A(H1)pdm09 representou uma maior proporção dos casos de gripe incluindo igualmente a maior proporção de doentes que necessitaram de internamento hospitalar em unidades de cuidados intensivos. O vírus da gripe do tipo B esteve associado a casos de gripe confirmados nas crianças entre os 5 e 14 anos. Outros vírus respiratórios foram igualmente detetados sendo o RSV e os picornavírus (hRV, hEV e picornavírus) os mais frequentes e em co circulação com o vírus da gripe. Durante a época de vigilância da gripe, 2015/2016, não se observaram excessos de mortalidade semanais. Nas UCI verificou-se uma franca dominância do vírus da gripe A(H1)pdm09 (90%) e a circulação simultânea do vírus da gripe B (3%). A taxa de admissão em UCI oscilou entre 5,8% e 4,7% entre as semanas 53 e 12 tendo o valor máximo sido registado na semana 8 de 2016 (8,1%). Cerca de metade dos doentes tinha entre 45 e 64 anos. Os mais idosos (65+ anos) foram apenas 20% dos casos, o que não será de estranhar, considerando que o vírus da gripe A(H1)pdm09 circulou como vírus dominante. Aproximadamente 70% dos doentes tinham doença crónica subjacente, tendo a obesidade sido a mais frequente (37%). Comparativamente com a pandemia, em que circulou também o A(H1)pdm09, a obesidade, em 2015/2016, foi cerca de 4 vezes mais frequente (9,8%). Apenas 8% dos doentes tinha feito a vacina contra a gripe sazonal, apesar de mais de 70% ter doença crónica subjacente e de haver recomendações da DGS nesse sentido. A taxa de letalidade foi estimada em 29,3%, mais elevada do que na época anterior (23,7%). Cerca de 80% dos óbitos ocorreram em indivíduos com doença crónica subjacente que poderá ter agravado o quadro e contribuído para o óbito. Salienta-se a ausência de dados históricos publicados sobre letalidade em UCI, para comparação. Note-se que esta estimativa se refere a óbitos ocorridos apenas durante a hospitalização na UCI e que poderão ter ocorrido mais óbitos após a alta da UCI para outros serviços/enfermarias. Este sistema de vigilância da gripe sazonal em UCI poderá ser aperfeiçoado nas próximas épocas reduzindo a subnotificação e melhorando o preenchimento dos campos necessários ao estudo da doença. A época de vigilância da gripe 2015/2016 foi em muitas caraterísticas comparável ao descrito na maioria dos países europeus. A situação em Portugal destacou-se pela baixa intensidade da atividade gripal, pelo predomínio do vírus da gripe do subtipo A(H1)pdm09 acompanhada pela deteção de vírus do tipo B (linhagem Victoria) essencialmente no final da época gripal. A mortalidade por todas as causas durante a epidemia da gripe manteve-se dentro do esperado, não tendo sido observados excessos de mortalidade. Os vírus da gripe do subtipo predominante na época 2015/2016, A(H1)pdm09, revelaram-se antigénicamente semelhantes à estirpe vacinal. Os vírus da gripe do tipo B detetados distinguem-se da estirpe vacinal de 2015/2016. Este facto conduziu à atualização da composição da vacina antigripal para a época 2016/2017. A monitorização contínua da epidemia da gripe a nível nacional e mundial permite a cada inverno avaliar o impacto da gripe na saúde da população, monitorizar a evolução dos vírus da gripe e atuar de forma a prevenir e implementar medidas eficazes de tratamento da doença, especialmente quando esta se apresenta acompanhada de complicações graves.

Impaired type I and type III interferon induction and rhinovirus control in human cystic fibrosis airway epithelial cells

Rhinoviruses are important triggers of pulmonary exacerbations and possible contributors to long-term respiratory morbidity in cystic fibrosis (CF), but mechanisms leading to rhinovirus-induced CF exacerbations are poorly understood. It is hypothesised that there is a deficient innate immune response of the airway epithelium towards rhinovirus infection in CF.

The structure of the two amino-terminal domains of human ICAM-1 suggests how it functions as a rhinovirus receptor and as an LFA-1 integrin ligand

The normal function of human intercellular adhesion molecule-1 (ICAM-1) is to provide adhesion between endothelial cells and leukocytes after injury or stress. ICAM-1 binds to leukocyte function-associated antigen (LFA-1) or macrophage-1 antigen (Mac-1). However, ICAM-1 is also used as a receptor by the major group of human rhinoviruses and is a catalyst for the subsequent viral uncoating during cell entry. The three-dimensional atomic structure of the two amino-terminal domains (D1 and D2) of ICAM-1 has been determined to 2.2-Å resolution and fitted into a cryoelectron microscopy reconstruction of a rhinovirus–ICAM-1 complex. Rhinovirus attachment is confined to the BC, CD, DE, and FG loops of the amino-terminal Ig-like domain (D1) at the end distal to the cellular membrane. The loops are considerably different in structure to those of human ICAM-2 or murine ICAM-1, which do not bind rhinoviruses. There are extensive charge interactions between ICAM-1 and human rhinoviruses, which are mostly conserved in both major and minor receptor groups of rhinoviruses. The interaction of ICAMs with LFA-1 is known to be mediated by a divalent cation bound to the insertion (I)-domain on the α chain of LFA-1 and the carboxyl group of a conserved glutamic acid residue on ICAMs. Domain D1 has been docked with the known structure of the I-domain. The resultant model is consistent with mutational data and provides a structural framework for the adhesion between these molecules.

Bayesian inference of the number of factors in gene-expression analysis: application to human virus challenge studies.

BACKGROUND: Nonparametric Bayesian techniques have been developed recently to extend the sophistication of factor models, allowing one to infer the number of appropriate factors from the observed data. We consider such techniques for sparse factor analysis, with application to gene-expression data from three virus challenge studies. Particular attention is placed on employing the Beta Process (BP), the Indian Buffet Process (IBP), and related sparseness-promoting techniques to infer a proper number of factors. The posterior density function on the model parameters is computed using Gibbs sampling and variational Bayesian (VB) analysis. RESULTS: Time-evolving gene-expression data are considered for respiratory syncytial virus (RSV), Rhino virus, and influenza, using blood samples from healthy human subjects. These data were acquired in three challenge studies, each executed after receiving institutional review board (IRB) approval from Duke University. Comparisons are made between several alternative means of per-forming nonparametric factor analysis on these data, with comparisons as well to sparse-PCA and Penalized Matrix Decomposition (PMD), closely related non-Bayesian approaches. CONCLUSIONS: Applying the Beta Process to the factor scores, or to the singular values of a pseudo-SVD construction, the proposed algorithms infer the number of factors in gene-expression data. For real data the "true" number of factors is unknown; in our simulations we consider a range of noise variances, and the proposed Bayesian models inferred the number of factors accurately relative to other methods in the literature, such as sparse-PCA and PMD. We have also identified a "pan-viral" factor of importance for each of the three viruses considered in this study. We have identified a set of genes associated with this pan-viral factor, of interest for early detection of such viruses based upon the host response, as quantified via gene-expression data.

Comparison of fast-track diagnostics respiratory pathogens multiplex real-time RT-PCR assay with in-house singleplex assays for comprehensive detection of human respiratory viruses

Fast-track Diagnostics respiratory pathogens (FTDRP) multiplex real-time RT-PCR assay was compared with in-house singleplex real-time RT-PCR assays for detection of 16 common respiratory viruses. The FTDRP assay correctly identified 26 diverse respiratory virus strains, 35 of 41 (85%) external quality assessment samples spiked with cultured virus and 232 of 263 (88%) archived respiratory specimens that tested positive for respiratory viruses by in-house assays. Of 308 prospectively tested respiratory specimens selected from children hospitalized with acute respiratory illness, 270 (87.7%) and 265 (86%) were positive by FTDRP and in-house assays for one or more viruses, respectively, with combined test results showing good concordance (K=0.812, 95% CI = 0.786-0.838). Individual FTDRP assays for adenovirus, respiratory syncytial virus and rhinovirus showed the lowest comparative sensitivities with in-house assays, with most discrepancies occurring with specimens containing low virus loads and failed to detect some rhinovirus strains, even when abundant. The FTDRP enterovirus and human bocavirus assays appeared to be more sensitive than the in-house assays with some specimens. With the exceptions noted above, most FTDRP assays performed comparably with in-house assays for most viruses while offering enhanced throughput and easy integration by laboratories using conventional real-time PCR instrumentation. Published by Elsevier B.V.

Increased nuclear suppressor of cytokine signaling 1 in asthmatic bronchial epithelium suppresses rhinovirus induction of innate interferons.

BACKGROUND Rhinovirus infections are the dominant cause of asthma exacerbations, and deficient virus induction of IFN-α/β/λ in asthmatic patients is important in asthma exacerbation pathogenesis. Mechanisms causing this interferon deficiency in asthmatic patients are unknown. OBJECTIVE We sought to investigate the expression of suppressor of cytokine signaling (SOCS) 1 in tissues from asthmatic patients and its possible role in impaired virus-induced interferon induction in these patients. METHODS We assessed SOCS1 mRNA and protein levels in vitro, bronchial biopsy specimens, and mice. The role of SOCS1 was inferred by proof-of-concept studies using overexpression with reporter genes and SOCS1-deficient mice. A nuclear role of SOCS1 was shown by using bronchial biopsy staining, overexpression of mutant SOCS1 constructs, and confocal microscopy. SOCS1 levels were also correlated with asthma-related clinical outcomes. RESULTS We report induction of SOCS1 in bronchial epithelial cells (BECs) by asthma exacerbation-related cytokines and by rhinovirus infection in vitro. We found that SOCS1 was increased in vivo in bronchial epithelium and related to asthma severity. SOCS1 expression was also increased in primary BECs from asthmatic patients ex vivo and was related to interferon deficiency and increased viral replication. In primary human epithelium, mouse lung macrophages, and SOCS1-deficient mice, SOCS1 suppressed rhinovirus induction of interferons. Suppression of virus-induced interferon levels was dependent on SOCS1 nuclear translocation but independent of proteasomal degradation of transcription factors. Nuclear SOCS1 levels were also increased in BECs from asthmatic patients. CONCLUSION We describe a novel mechanism explaining interferon deficiency in asthmatic patients through a novel nuclear function of SOCS1 and identify SOCS1 as an important therapeutic target for asthma exacerbations.