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1.
Curr Microbiol ; 76(12): 1477-1486, 2019 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-31612259

RESUMEN

Expression and secretion of recombinant proteins in the endotoxin-free bacterium, Bacillus subtilis, has been thoroughly studied, but overexpression in the cytoplasm has been limited to only a few proteins. Here, we used the robust IPTG-inducible promoter, Pgrac212, to overexpress human rhinovirus 3C protease (HRV3C) in the cytoplasm of B. subtilis cells. A novel solubility tag, the N-terminal domain of the lysS gene of B. subtilis coding for a lysyl-tRNA synthetase was placed at the N terminus with a cleavage site for the endoprotease HRV3C, followed by His-HRV3C or His-GST-HRV3C. The recombinant protease was purified by using a Ni-NTA column. In this study, the His-HRV3C and His-GST-HRV3C proteases were overexpressed in the cytoplasm of B. subtilis at 11% and 16% of the total cellular proteins, respectively. The specific protease activities were 8065 U/mg for His-HRV3C and 3623 U/mg for His-GST-HRV3C. The purified enzymes were used to cleave two different substrates followed by purification of the two different protein targets, the green fluorescent protein and the beta-galactosidase. In conclusion, the combination of an inducible promoter Pgrac212 and a solubility tag allowed the overexpression of the HRV3C protease in the cytoplasm of B. subtilis. The resulting fusion protein was purified using a nickel column and was active in cleaving target proteins to remove the fusion tags. This study offers an effective method for producing recombinant proteins in the cytoplasm of endotoxin-free bacteria.


Asunto(s)
Bacillus subtilis/genética , Cisteína Endopeptidasas/genética , Citoplasma/metabolismo , Microbiología Industrial/métodos , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes de Fusión/genética , Rhinovirus/enzimología , Proteínas Virales/genética , Bacillus subtilis/metabolismo , Clonación Molecular , Cisteína Endopeptidasas/aislamiento & purificación , Expresión Génica/efectos de los fármacos , Proteínas Fluorescentes Verdes/genética , Isopropil Tiogalactósido/farmacología , Lisina-ARNt Ligasa/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Rhinovirus/genética , Solubilidad , Proteínas Virales/aislamiento & purificación , beta-Galactosidasa/genética
2.
Zhonghua Liu Xing Bing Xue Za Zhi ; 40(8): 904-910, 2019 Aug 10.
Artículo en Chino | MEDLINE | ID: mdl-31484252

RESUMEN

Objective: To analyze the etiologic and epidemiological characteristics of adult acute respiratory infections in Shanghai during 2015-2017. Methods: Data was collected from outpatients with acute respiratory infections who visited the Fever Clinics in three hospitals of different levels in three administrative regions of Shanghai, from 2015 to 2017. Basic information and nasopharyngeal swabs were collected from cases in line with the inclusion criteria. Multiplex RT-PCR and bacterial cultures were performed to detect the respiratory pathogens. Results: A total of 806 individuals were enrolled from 2015 to 2017. Respiratory pathogens were identified in 73.45% (592/806) of the cases, with the virus detection rate as 66.75% (538/806). It was found that the major respiratory pathogens for virus detection were influenza A in 326 (40.45%), influenza B in 116 (14.39%), rhinovirus/enterovirus in 39 (4.84%) of the cases. The overall detection rate of bacteria was 16.13% (130/806), including Klebsiella pneumoniae in 90 (11.17%) cases, Staphylococcus Aureus in 46 (5.71%) cases. Other kind of bacteria were not detected in our study. The detection rates on Mycoplasma pneumoniae was 5.33% (43/806) and on Chlamydia pneumonia was 0.37% (3/806). Co-infection with multiple pathogens was detected in 18.61% (150/806) of the cases, including 135 with double infection (accounting for 90.00%), 14 with triple infection and 1 with quadruple infection (accounted for 9.33% and 0.67%, respectively). Among the 150 cases with co-infections, the main identified pathogens were influenza A, Klebsiella pneumoniae, Staphylococcus aureus, and Mycoplasma pneumoniae. Pathogens of acute respiratory infections that identified among the outpatients from the Fever Clinics at different time, region or population, the characteristics were different (P<0.001). Conclusions: In 2015-2017, outpatients with acute respiratory infections in Shanghai were mainly caused by influenza virus or other viruses, however dynamically with its composition, time, region and characteristics of the population. It is necessary to strengthen and combine related medical and preventive services and to develop the appropriate strategies regarding clinical diagnosis and treatment.


Asunto(s)
Bacterias/aislamiento & purificación , Infecciones Bacterianas/diagnóstico , Gripe Humana/diagnóstico , Reacción en Cadena de la Polimerasa Multiplex/métodos , Nasofaringe , Infecciones del Sistema Respiratorio/epidemiología , Infecciones del Sistema Respiratorio/etiología , Virosis/diagnóstico , Virus/aislamiento & purificación , Enfermedad Aguda , Adulto , Bacterias/genética , Infecciones Bacterianas/epidemiología , Infecciones Bacterianas/microbiología , China/epidemiología , Coinfección/diagnóstico , Enterovirus/genética , Enterovirus/aislamiento & purificación , Monitoreo Epidemiológico , Humanos , Incidencia , Virus de la Influenza A/genética , Virus de la Influenza A/aislamiento & purificación , Virus de la Influenza B/genética , Virus de la Influenza B/aislamiento & purificación , Gripe Humana/epidemiología , Gripe Humana/virología , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/aislamiento & purificación , Mycoplasma pneumoniae , Nasofaringe/microbiología , Nasofaringe/virología , Vigilancia de la Población , Infecciones del Sistema Respiratorio/diagnóstico , Rhinovirus/genética , Rhinovirus/aislamiento & purificación , Staphylococcus aureus/genética , Staphylococcus aureus/aislamiento & purificación , Virosis/epidemiología , Virosis/virología , Virus/genética
3.
J Microbiol Immunol Infect ; 52(2): 233-241, 2019 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-30201131

RESUMEN

BACKGROUND: Human rhinovirus (HRV) can cause severe illnesses in hospitalized patients. However, there are no studies regarding the prevalence of HRV infection, particularly the recently identified HRV-C, in hospitalized patients reported from Taiwan. METHODS: Respiratory specimens collected from 487 hospitalized patients in designated wards between 2013 and 2014 in a medical center in northern Taiwan were retrospectively detected for HRV. Positive specimens were further determined for genotyping. Medical charts of the HRV-positive patients were reviewed retrospectively. RESULTS: Totally, 76 patients (15.6%) were HRV positive, of which 60 were pediatric patients. HRV-A was identified in 41 (54%) patients, HRV-B in 6 patients (7.9%) and HRV-C in 29 patients (38%). A total of 47 different genotypes were identified. HRV infections were predominant during fall and winter seasons. 21.1% were affected by HRV alone and 78.9% were found to be co-infected with other microorganisms. The detection rate of HRV in children (18.6%) was significantly higher than in adults (9.6%). Compared with pediatric patients, adult patients were significantly associated with underlying disease, Pneumocystis jirovesii pneumonia co-infection, a diagnosis of pneumonia, fatal outcome, hospital acquisition of HRV, antibiotics administration and requiring intensive care, while pediatric patients were significantly associated with viral co-infection. CONCLUSIONS: HRV was a common cause of respiratory tract infection in Taiwan, particularly in pediatric patients. Eighty percent of HRV-infected inpatients had other microorganisms co-infection. Adult patients were more likely to be associated with a severe respiratory disease entity.


Asunto(s)
Coinfección/epidemiología , Epidemiología Molecular , Infecciones por Picornaviridae/epidemiología , Infecciones del Sistema Respiratorio/epidemiología , Rhinovirus/clasificación , Rhinovirus/patogenicidad , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Antibacterianos/uso terapéutico , Niño , Preescolar , Coinfección/tratamiento farmacológico , Coinfección/microbiología , Enterovirus/genética , Enterovirus/patogenicidad , Femenino , Genotipo , Hospitales , Humanos , Lactante , Masculino , Persona de Mediana Edad , Tipificación Molecular , Infecciones por Picornaviridae/complicaciones , Neumonía por Pneumocystis/complicaciones , Neumonía por Pneumocystis/tratamiento farmacológico , Prevalencia , Infecciones del Sistema Respiratorio/tratamiento farmacológico , Infecciones del Sistema Respiratorio/microbiología , Infecciones del Sistema Respiratorio/virología , Estudios Retrospectivos , Rhinovirus/genética , Taiwán/epidemiología , Adulto Joven
4.
Virology ; 526: 72-80, 2019 01 02.
Artículo en Inglés | MEDLINE | ID: mdl-30366300

RESUMEN

Recombination is a driving force for the emergence, evolution and virulence/epidemics of viruses, comprising the Enterovirus genus of the Picornaviridae family, important for human and animal health. By analyzing 2949 complete genomes/coding sequences, we provide a thorough and up-to-date overview of the genome-wide patterns and hotspots of intertypic recombination between the genogroups of this genus. Two prominent recombination hotspots are identified/verified, at the 5'UTR-capsid region junction, and at the beginning of the P2 region. In general, P2 was enriched in recombination events. Key phylogenetic groups implicated in recombination events are E71 and CVA6 in Enterovirus A species, E30 and E6 in Enterovirus B species, polioviruses 1 and 2 in Enterovirus C species. In addition, many events involve recombination partners that have not been sequenced yet, thus strongly suggesting a large environmental reservoir of genetic variation with a high potential for the emergence of new modified pathogens by recombination.


Asunto(s)
Enterovirus/genética , Evolución Molecular , Genoma Viral/genética , Recombinación Genética , Regiones no Traducidas 5'/genética , Proteínas de la Cápside/genética , Bases de Datos Genéticas , Enterovirus/clasificación , Genotipo , Humanos , Filogenia , Rhinovirus/clasificación , Rhinovirus/genética , Proteínas no Estructurales Virales/genética
5.
Future Microbiol ; 13: 1565-1573, 2018 11.
Artículo en Inglés | MEDLINE | ID: mdl-30417657

RESUMEN

AIM: To describe the genetic diversity of rhinovirus (RV) from patients attended at a tertiary hospital in Barcelona (Spain) from October 2014 to May 2017. METHODS: RV detection was performed by real-time multiplex RT-PCR. A specific real-time quantitive retrotranscription PCR (qRT-PCR) was carried out to select those samples (Ct < 35) for molecular characterization based on partial VP4/2 protein. RESULTS: Phylogenetic characterization revealed proportions of 63% RV-A, 6% RV-B and 31% RV-C (119 different types). RV-A circulated throughout all the study period, with a minor circulation during winter, just when RV-C prevailed. Differences between age medians by RV-specie were reported. CONCLUSION: The large genetic diversity of RV detected in our area is described here. The variable cocirculation of multiple RV types is also reported, showing differences by age.


Asunto(s)
Variación Genética/genética , Infecciones por Picornaviridae/virología , Infecciones del Sistema Respiratorio/virología , Rhinovirus/genética , Adolescente , Adulto , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Oportunidad Relativa , Filogenia , Infecciones por Picornaviridae/epidemiología , Infecciones del Sistema Respiratorio/epidemiología , Estudios Retrospectivos , Rhinovirus/aislamiento & purificación , Estaciones del Año , España/epidemiología , Estadísticas no Paramétricas , Centros de Atención Terciaria , Proteínas de la Matriz Viral/genética
6.
Virol J ; 15(1): 167, 2018 10 30.
Artículo en Inglés | MEDLINE | ID: mdl-30376870

RESUMEN

BACKGROUND: Respiratory syncytial virus (RSV), human Rhinovirus (HRV) and human Metapneumo Virus (HMPV) are important viral pathogens causing acute respiratory tract infections in the hospitalized patients. Sensitive and accurate detection of RSV, HRV and HMPV is necessary for clinical diagnosis and treatment. RESULTS: A locked nucleic acid (LNA)-based multiplex closed one-tube nested real-time RT-PCR (mOTNRT-PCR) assay was developed for simultaneous detection of RSV, HRV and HMPV. The sensitivity, specificity, reproducibility and clinical performance of mOTNRT-PCR were evaluated and compared with individual real time PCR (RT-qPCR) assay using clinical samples. The analytical sensitivity of mOTNRT-PCR assay was 5 copies/reaction for RSV, HRV and HMPV, respectively, and no cross-reaction with other common respiratory viruses was observed. The coefficients of variation (CV) of intra-assay and inter-assay were between 0.51 to 3.67%. Of 398 nasopharyngeal aspirates samples tested, 109 (27.39%), 150 (37.69%) and 44 (11.06%) were positive for RSV, HRV and HMPV, respectively, whereas 95 (23.87%), 137 (34.42%) and 38 (9.55%) were positive for RSV, HRV and HMPV, respectively, by individual RT-qPCR assay. Thirty three samples that were positive by mOTNRT-PCR but negative by RT-qPCR were confirmed as true positives by sequencing using reported traditional two-step nested PCR assay. CONCLUSION: mOTNRT-PCR assay reveals extremely higher sensitivity than that of RT-qPCR assay for detecting RSV, HRV and HMPV in clinical settings.


Asunto(s)
Metapneumovirus/aislamiento & purificación , Infecciones por Paramyxoviridae/diagnóstico , Infecciones por Picornaviridae/diagnóstico , Infecciones por Virus Sincitial Respiratorio/diagnóstico , Virus Sincitial Respiratorio Humano/aislamiento & purificación , Rhinovirus/aislamiento & purificación , Enfermedad Aguda , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Metapneumovirus/genética , Reacción en Cadena de la Polimerasa Multiplex , Nasofaringe/virología , Reproducibilidad de los Resultados , Virus Sincitial Respiratorio Humano/genética , Rhinovirus/genética , Sensibilidad y Especificidad
8.
Am J Trop Med Hyg ; 99(4): 1089-1095, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-30182916

RESUMEN

Military recruits are at high risk of respiratory infections. However, limited data exist on military populations in tropical settings, where the epidemiology of respiratory infections differs substantially from temperate settings. We enrolled recruits undertaking a 10-week military training at two Royal Thai Army barracks between May 2014 and July 2015. We used a multiplex respiratory panel to analyze nose and throat swabs collected at the start and end of the training period, and from participants experiencing respiratory symptoms during follow-up. Paired sera were tested for influenza seroconversion using a hemagglutinin inhibition assay. Overall rates of upper respiratory illness and influenza-like illness were 3.1 and 2.0 episodes per 100 person-weeks, respectively. A pathogen was detected in 96% of samples. The most commonly detected microbes were Haemophilus influenzae type B (62.7%) or non-type B (58.2%) and rhinovirus (22.4%). At baseline, bacterial colonization was high and included H. influenzae type B (82.3%), H. influenzae non-type B (31.5%), Klebsiella pneumoniae (14.6%), Staphylococcus aureus (8.5%), and Streptococcus pneumoniae (8.5%). At the end of follow-up, colonization with H. influenzae non-type B had increased to 74.1%, and S. pneumoniae to 33.6%. In the serology subset, the rate of influenza infection was 3.4 per 100 person-months; 58% of influenza infections resulted in clinical disease. Our study provides key data on the epidemiology and transmission of respiratory pathogens in tropical settings. Our results emphasize the need for improved infection prevention and control in military environments, given the high burden of illness and potential for intense transmission of respiratory pathogens.


Asunto(s)
Infecciones por Haemophilus/epidemiología , Gripe Humana/epidemiología , Infecciones por Klebsiella/epidemiología , Infecciones por Picornaviridae/epidemiología , Neumonía Neumocócica/epidemiología , Infecciones del Sistema Respiratorio/epidemiología , Infecciones Estafilocócicas/epidemiología , Adolescente , Adulto , Infecciones por Haemophilus/transmisión , Haemophilus influenzae tipo b/genética , Haemophilus influenzae tipo b/aislamiento & purificación , Humanos , Incidencia , Gripe Humana/transmisión , Infecciones por Klebsiella/transmisión , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/aislamiento & purificación , Masculino , Personal Militar , Orthomyxoviridae/genética , Orthomyxoviridae/aislamiento & purificación , Infecciones por Picornaviridae/transmisión , Neumonía Neumocócica/transmisión , Reacción en Cadena de la Polimerasa , Estudios Prospectivos , Infecciones del Sistema Respiratorio/transmisión , Rhinovirus/genética , Rhinovirus/aislamiento & purificación , Infecciones Estafilocócicas/transmisión , Staphylococcus aureus/genética , Staphylococcus aureus/aislamiento & purificación , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/aislamiento & purificación , Tailandia/epidemiología
9.
BMC Infect Dis ; 18(1): 459, 2018 Sep 10.
Artículo en Inglés | MEDLINE | ID: mdl-30200897

RESUMEN

BACKGROUND: Acute Respiratory Infections (ARI) are common causes of febrile illnesses in many settings in Senegal. These infections are usually managed presumptively due to lack of appropriate diagnostic tools. This situation, can lead to poor management of febrile illness or antibiotic misuse. In addition, there are limited data on the spectrum of pathogens commonly responsible for these ARI. This study was conducted to explore the pathogens community among patients with acute respiratory infection in a rural area in Senegal. METHODS: A cross sectional study was conducted from August to December 2015. Children and adult patients attending Keur Socé health post for signs suggestive of acute respiratory infection were enrolled after providing inform consent. Eligible participants were recruited using a consecutive sampling method. Paired nose and throat swabs were collected for pathogen detection. Samples were processed using a multiplex PCR designed to identify 21 pathogens including both virus and bacteria. RESULTS: Two hundred and fifty patients participated in the study. Samples positivity rate was evaluated at 95.2% (238/250). Streptococcus pneumoniae was the predominant pathogen (74%) and was present in all months and all age-groups, followed by Staphylococcus aureus (28,8%) and rhinovirus (28,4%). Respiratory syncytial virus (RSV) was detected only among children under 5 years old in August and September while coronavirus was present in all age groups, during the months of October and December. CONCLUSION: This pilot study revealed a diversity of pathogens over the time and across all age groups, highlighting the need for further exploration. A pathogen community approach including both virus and bacteria at a larger scale becomes crucial for a better understanding of transmission dynamics at population level in order to help shape ARI control strategies.


Asunto(s)
Malaria/complicaciones , Virus Sincitial Respiratorio Humano/aislamiento & purificación , Infecciones del Sistema Respiratorio/microbiología , Infecciones del Sistema Respiratorio/virología , Rhinovirus/aislamiento & purificación , Staphylococcus aureus/aislamiento & purificación , Streptococcus pneumoniae/aislamiento & purificación , Adolescente , Niño , Preescolar , Estudios Transversales , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Femenino , Humanos , Lactante , Malaria/diagnóstico , Malaria/transmisión , Masculino , Reacción en Cadena de la Polimerasa Multiplex , ARN Viral/genética , ARN Viral/metabolismo , Virus Sincitial Respiratorio Humano/genética , Infecciones del Sistema Respiratorio/complicaciones , Infecciones del Sistema Respiratorio/diagnóstico , Rhinovirus/genética , Población Rural , Estaciones del Año , Senegal , Staphylococcus aureus/genética , Streptococcus pneumoniae/genética
10.
J Virol ; 92(23)2018 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-30209171

RESUMEN

Rhinoviruses (RVs) replicate on cytoplasmic membranes derived from the Golgi apparatus. They encode membrane-targeted proteins 2B, 2C, and 3A, which control trafficking and lipid composition of the replication membrane. The virus recruits host factors for replication, such as phosphatidylinositol 4 (PI4)-kinase 3beta (PI4K3b), which boosts PI4-phosphate (PI4P) levels and drives lipid countercurrent exchange of PI4P against cholesterol at endoplasmic reticulum-Golgi membrane contact sites through the lipid shuttling protein oxysterol binding protein 1 (OSBP1). We identified a PI4K3b inhibitor-resistant RV-A16 variant with a single point mutation in the conserved 2B protein near the cytosolic carboxy terminus, isoleucine 92 to threonine (termed 2B[I92T]). The mutation did not confer resistance to cholesterol-sequestering compounds or OSBP1 inhibition, suggesting invariant dependency on the PI4P/cholesterol lipid countercurrents. In the presence of PI4K3b inhibitor, Golgi reorganization and PI4P lipid induction occurred in RV-A16 2B[I92] but not in wild-type infection. The knockout of PI4K3b abolished the replication of both the 2B[I92T] mutant and the wild type. Doxycycline-inducible expression of PI4K3b in PI4K3b knockout cells efficiently rescued the 2B[I92T] mutant and, less effectively, wild-type virus infection. Ectopic expression of 2B[I92T] or 2B was less efficient than that of 3A in recruiting PI4K3b to perinuclear membranes, suggesting a supportive rather than decisive role of 2B in recruiting PI4K3b. The data suggest that 2B tunes the recruitment of PI4K3b to the replication membrane and allows the virus to adapt to cells with low levels of PI4K3b while still maintaining the PI4P/cholesterol countercurrent for establishing Golgi-derived RV replication membranes.IMPORTANCE Human rhinoviruses (RVs) are the major cause of the common cold worldwide. They cause asthmatic exacerbations and chronic obstructive pulmonary disease. Despite recent advances, the development of antivirals and vaccines has proven difficult due to the high number and variability of RV types. The identification of critical host factors and their interactions with viral proteins and membrane lipids for the establishment of viral replication is a basis for drug development strategies. Our findings here shed new light on the interactions between nonstructural viral membrane proteins and class III phosphatidylinositol 4 kinases from the host and highlight the importance of phosphatidylinositol 4 phosphate for RV replication.


Asunto(s)
Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Infecciones por Picornaviridae/virología , Mutación Puntual , Rhinovirus/genética , Proteínas no Estructurales Virales/genética , Replicación Viral , Membrana Celular/metabolismo , Membrana Celular/virología , Colesterol/metabolismo , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/virología , Aparato de Golgi/metabolismo , Aparato de Golgi/virología , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Infecciones por Picornaviridae/genética , Infecciones por Picornaviridae/metabolismo , Transporte de Proteínas
11.
BMC Infect Dis ; 18(1): 462, 2018 Sep 14.
Artículo en Inglés | MEDLINE | ID: mdl-30217168

RESUMEN

BACKGROUND: Multiplex real-time polymerase chain reaction assays have improved diagnostic sensitivity for a wide range of pathogens. However, co-detection of multiple agents and bacterial colonization make it difficult to distinguish between asymptomatic infection or illness aetiology. We assessed whether semi-quantitative microbial load data can differentiate between symptomatic and asymptomatic states for common respiratory pathogens. METHODS: We obtained throat and nasal swab samples from military trainees at two Thai Army barracks. Specimens were collected at the start and end of 10-week training periods (non-acute samples), and from individuals who developed upper respiratory tract infection during training (acute samples). We analysed the samples using a commercial multiplex respiratory panel comprising 33 bacterial, viral and fungal targets. We used random effects tobit models to compare cycle threshold (Ct) value distributions from non-acute and acute samples. RESULTS: We analysed 341 non-acute and 145 acute swab samples from 274 participants. Haemophilus influenzae type B was the most commonly detected microbe (77.4% of non-acute and 64.8% of acute samples). In acute samples, nine specific microbe pairs were detected more frequently than expected by chance. Regression models indicated significantly lower microbial load in non-acute relative to acute samples for H. influenzae non-type B, Streptococcus pneumoniae and rhinovirus, although it was not possible to identify a Ct-value threshold indicating causal etiology for any of these organisms. CONCLUSIONS: Semi-quantitative measures of microbial concentration did not reliably differentiate between illness and asymptomatic colonization, suggesting that clinical symptoms may not always be directly related to microbial load for common respiratory infections.


Asunto(s)
Reacción en Cadena de la Polimerasa Multiplex/métodos , Infecciones del Sistema Respiratorio/diagnóstico , Enfermedad Aguda , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Femenino , Haemophilus influenzae tipo b/genética , Haemophilus influenzae tipo b/aislamiento & purificación , Humanos , Masculino , Personal Militar , Cavidad Nasal/microbiología , Faringe/microbiología , Estudios Prospectivos , ARN Viral/genética , ARN Viral/metabolismo , Infecciones del Sistema Respiratorio/microbiología , Infecciones del Sistema Respiratorio/virología , Rhinovirus/genética , Rhinovirus/aislamiento & purificación , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/aislamiento & purificación , Tailandia
13.
Influenza Other Respir Viruses ; 12(6): 717-727, 2018 11.
Artículo en Inglés | MEDLINE | ID: mdl-30120824

RESUMEN

BACKGROUND: Rhinovirus (RV) causes the common cold and asthma exacerbations. The RV genome is a 7.3 kb single-strand positive-sense RNA. OBJECTIVE: Using minor group RV1A as a backbone, we sought to design and generate a recombinant RV1A accommodating fluorescent marker expression, thereby allowing tracking of viral infection. METHOD: Recombinant RV1A infectious cDNA clones harboring the coding sequence of green fluorescent protein (GFP), Renilla luciferase, or iLOV (for light, oxygen, or voltage sensing) were engineered and constructed. RV-infected cells were determined by flow cytometry, immunohistochemistry, and immunofluorescence microscopy. RESULTS: RV1A-GFP showed a cytopathic effect in HeLa cells but failed to express GFP or Renilla luciferase due to deletion. The smaller fluorescent protein construct, RV1A-iLOV, was stably expressed in infected cells. RV1A-iLOV expression was used to examine the antiviral effect of bafilomycin in HeLa cells. Compared to parental virus, RV1A-iLOV infection of BALB/c mice yielded a similar viral load and level of cytokine mRNA expression. However, imaging of fixed lung tissue failed to reveal a fluorescent signal, likely due to the oxidation and bleaching of iLOV-bound flavin mononucleotide. We therefore employed an anti-iLOV antibody for immunohistochemical and immunofluorescence imaging. The iLOV signal was identified in airway epithelial cells and CD45+ CD11b+ lung macrophages. CONCLUSIONS: These results suggest that RV1A-iLOV is a useful molecular tool for studying RV pathogenesis. The construction strategy for RV1A-iLOV could be applied to other RV serotypes. However, the detection of iLOV-expressing RV in fixed tissue required the use of an anti-iLOV antibody, limiting the value of this construct.


Asunto(s)
Proteínas Luminiscentes/análisis , Infecciones por Picornaviridae/virología , Rhinovirus/crecimiento & desarrollo , Coloración y Etiquetado/métodos , Animales , Efecto Citopatogénico Viral , Citometría de Flujo , Expresión Génica , Inestabilidad Genómica , Células HeLa , Humanos , Inmunohistoquímica , Proteínas Luminiscentes/genética , Ratones Endogámicos BALB C , Microscopía Fluorescente , Infecciones por Picornaviridae/patología , Proteínas Recombinantes/análisis , Proteínas Recombinantes/genética , Rhinovirus/genética , Carga Viral
14.
J Med Chem ; 61(18): 8402-8416, 2018 09 27.
Artículo en Inglés | MEDLINE | ID: mdl-30153009

RESUMEN

Rhinoviruses (RVs) have been linked to exacerbations of many pulmonary diseases, thus increasing morbidity and/or mortality in subjects at risk. Unfortunately, the wide variety of RV genotypes constitutes a major hindrance for the development of Rhinovirus replication inhibitors. In the current investigation, we have developed a novel series of pyrazole derivatives that potently inhibit the Rhinovirus replication. Compounds 10e and 10h behave as early stage inhibitors of Rhinovirus infection with a broad-spectrum activity against RV-A and RV-B species (EC50 < 0.1 µM). We also evaluate the dynamics of the emerging resistance of these promising compounds and their in vitro genotoxicity. Molecular docking experiments shed light on the pharmacophoric elements interacting with residues of the drug-binding pocket.


Asunto(s)
Antivirales/química , Antivirales/farmacología , Diseño de Drogas , Infecciones por Enterovirus/tratamiento farmacológico , Pirazoles/química , Rhinovirus/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Animales , Infecciones por Enterovirus/virología , Células HeLa , Humanos , Masculino , Pruebas de Micronúcleos , Modelos Moleculares , Estructura Molecular , Conformación Proteica , Ratas , Ratas Sprague-Dawley , Rhinovirus/genética , Relación Estructura-Actividad
15.
Epidemiol Mikrobiol Imunol ; 67(1): 18-23, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30157660

RESUMEN

  Background: Acute respiratory infection result in high mortality and morbidity worldwide. There are several viral factors that originate respiratory diseases among them Enteroviruses(EVs) and Human Rhinoviruses(HRVs) can be mentioned. HRVs and EVs belong to Picornaviridae family and they have been recently classified under Enteroviruses. The pattern of respiratory infections generating organisms varies according to geographical locations. Therefore, it seems necessary to organize an appropriate plan to manage common viral diseases exclusively about Rhinoviruses and Enteroviruses. PATIENT AND METHODS: A total of 100 samples were collected from patients with acute respiratory infections (ARIs) who were hospitalized in Ahvaz city hospitals during December 2012 to November 2013 (one year longitude). Semi-Nested PCR was done on samples for detection of HRVs and EVs using region gene of VP4/VP2. Phylogenetic and molecular evolutionary analyses performed with MEGA version 5 software find out the sequence homology among the detected HRV and EV serotype. RESULTS: The results of this study revealed that from of 100 cases of ARIs 19 patients (19%) were HRV positive and 3 (3%) patients positive for EVs. Most positive cases of HRVs were observed in the autumn season while 3 positive cases of EVs were equally found in spring, summer and autumn. Phylogenetic analyses showed that the HRV strains were HRV-A9, HRV-A49, HRV-B14 and EV strains were Echo3 and 9. CONCLUSION: The results of this study revealed that high prevalence of 19% HRVs, HRV-A9, HRV-A49, HRV-B14 serotypes and low frequency of 3% Echo Viruses, Echo3 and Echo 9 serotypes have been detected in patients with ARI.


Asunto(s)
Enterovirus Humano B , Infecciones por Picornaviridae , Infecciones del Sistema Respiratorio , Rhinovirus , Infecciones por Echovirus/epidemiología , Infecciones por Echovirus/patología , Infecciones por Echovirus/virología , Enterovirus Humano B/clasificación , Enterovirus Humano B/genética , Enterovirus Humano B/fisiología , Humanos , Filogenia , Infecciones por Picornaviridae/epidemiología , Infecciones por Picornaviridae/patología , Infecciones por Picornaviridae/virología , Infecciones del Sistema Respiratorio/epidemiología , Infecciones del Sistema Respiratorio/virología , Rhinovirus/clasificación , Rhinovirus/genética , Rhinovirus/fisiología , Estaciones del Año , Serogrupo
16.
BMC Infect Dis ; 18(1): 437, 2018 Aug 29.
Artículo en Inglés | MEDLINE | ID: mdl-30157776

RESUMEN

BACKGROUND: International and national travelling has made the rapid spread of infectious diseases possible. Little information is available on the role of major traffic hubs, such as airports, in the transmission of respiratory infections, including seasonal influenza and a pandemic threat. We investigated the presence of respiratory viruses in the passenger environment of a major airport in order to identify risk points and guide measures to minimize transmission. METHODS: Surface and air samples were collected weekly at three different time points during the peak period of seasonal influenza in 2015-16 in Finland. Swabs from surface samples, and air samples were tested by real-time PCR for influenza A and B viruses, respiratory syncytial virus, adenovirus, rhinovirus and coronaviruses (229E, HKU1, NL63 and OC43). RESULTS: Nucleic acid of at least one respiratory virus was detected in 9 out of 90 (10%) surface samples, including: a plastic toy dog in the children's playground (2/3 swabs, 67%); hand-carried luggage trays at the security check area (4/8, 50%); the buttons of the payment terminal at the pharmacy (1/2, 50%); the handrails of stairs (1/7, 14%); and the passenger side desk and divider glass at a passport control point (1/3, 33%). Among the 10 respiratory virus findings at various sites, the viruses identified were: rhinovirus (4/10, 40%, from surfaces); coronavirus (3/10, 30%, from surfaces); adenovirus (2/10, 20%, 1 air sample, 1 surface sample); influenza A (1/10, 10%, surface sample). CONCLUSIONS: Detection of pathogen viral nucleic acids indicates respiratory viral surface contamination at multiple sites associated with high touch rates, and suggests a potential risk in the identified airport sites. Of the surfaces tested, plastic security screening trays appeared to pose the highest potential risk, and handling these is almost inevitable for all embarking passengers.


Asunto(s)
Aeropuertos , Contaminación de Equipos/estadística & datos numéricos , Infecciones del Sistema Respiratorio/virología , Virus/aislamiento & purificación , Adenoviridae/genética , Adenoviridae/aislamiento & purificación , Aeropuertos/normas , Aeropuertos/estadística & datos numéricos , Coronavirus/genética , Coronavirus/aislamiento & purificación , Infecciones por Coronavirus/transmisión , Infecciones por Coronavirus/virología , Finlandia/epidemiología , Humanos , Gripe Humana/transmisión , Gripe Humana/virología , Reacción en Cadena en Tiempo Real de la Polimerasa , Virus Sincitial Respiratorio Humano/genética , Virus Sincitial Respiratorio Humano/aislamiento & purificación , Infecciones del Sistema Respiratorio/transmisión , Rhinovirus/genética , Rhinovirus/aislamiento & purificación , Tacto , Viaje/estadística & datos numéricos , Enfermedad Relacionada con los Viajes , Virus/genética
17.
J Theor Biol ; 456: 34-40, 2018 11 07.
Artículo en Inglés | MEDLINE | ID: mdl-30059661

RESUMEN

Comparing DNA and protein sequence groups plays an important role in biological evolutionary relationship research. Despite many methods available for sequence comparison, only a few can be used for group comparison. In this study, we propose a novel approach using convex hulls. We use statistical information contained within the sequences to represent each sequence as a point in high dimensional space. We find that the points belonging to one biological group are located in a different region of space than points belonging to other biological groups. To be more precise, the convex hull of the points from one group are disjoint from the convex hulls of points from other groups. This finding allows us to do phylogenetic analysis for groups in an efficient way. Five different theorems are presented for checking whether two convex hulls intersect or are disjoint. Test results for datasets related to HRV, HPV, Ebolavirus, PKC and protein phosphatase domains demonstrate that our method performs well and provides a new tool for studying group phylogeny. More significantly, the convex analysis presents a new way to search for sequences belonging to a biological group by examining points within the group's convex hull.


Asunto(s)
Evolución Biológica , Rhinovirus/genética , Análisis de Secuencia de ADN/métodos , Análisis de Secuencia de Proteína/métodos , Algoritmos , Ebolavirus/genética , Genoma Viral/genética , Humanos , Análisis Numérico Asistido por Computador , Papillomaviridae/genética , Filogenia , Proteína Quinasa C/genética
18.
J Clin Virol ; 106: 13-17, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-30007137

RESUMEN

BACKGROUND: Respiratory infections are common reasons for hospital admission, and are associated with enormous economic burden due to significant morbidity and mortality. The wide spectrum of microbial agents underlying the pathology renders the diagnosis of respiratory infections challenging. Molecular diagnostics offer an advantage to the current serological and culture-based methods in terms of sensitivity, coverage, hands-on time, and time to results. OBJECTIVES: This study aimed to compare the clinical performance of three commercial kits for respiratory viral detection. STUDY DESIGN: The performance of FilmArray Respiratory Panel, AnyplexII RV16, and Argene was compared using clinical respiratory samples (n = 224, comprising 189 nasopharyngeal swabs in Universal Transport Medium (UTM) and 35 endotracheal aspirates), based on common overlapping targets across the platforms. Influenza A "equivocal" and "no-subtype" samples by FilmArray were further compared to a laboratory-developed Influenza A/B test. RESULTS AND CONCLUSIONS: The overall performance of all three platforms appeared to be comparable with regards to sensitivities (95.8-97.9%) and specificities (96.1-98.0%), detection of coinfections, and distinguishment of influenza from non-influenza cases. "Equivocal" and "no-subtype" samples by FilmArray mostly represented weak Influenza A by laboratory-developed test. Lower respiratory tract samples had comparable final-run success-rates and discordant-rates as compared to UTM. Coronavirus HKU1, which was not targeted by AnyplexII RV16, were detected as OC43. The expected test volume would be the main determinant for the selection of platform. Among the platforms, the FilmArray is the most automated but is of the lowest-throughput and has the highest reagent cost.


Asunto(s)
Técnicas de Diagnóstico Molecular , Juego de Reactivos para Diagnóstico/normas , Infecciones del Sistema Respiratorio/diagnóstico , Infecciones del Sistema Respiratorio/virología , Virus/genética , Coinfección/diagnóstico , Coinfección/virología , Enterovirus/genética , Enterovirus/aislamiento & purificación , Hospitalización , Humanos , Gripe Humana/diagnóstico , Reacción en Cadena de la Polimerasa Multiplex , Nasofaringe/virología , Juego de Reactivos para Diagnóstico/economía , Rhinovirus/genética , Rhinovirus/aislamiento & purificación , Sensibilidad y Especificidad , Virus/clasificación , Virus/aislamiento & purificación
19.
PLoS One ; 13(7): e0198624, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29969445

RESUMEN

The clinical profile of human rhinovirus (HRV) with regard to lower respiratory infections remains unclear. We analyzed the clinical features and cytokine responses of HRV isolates in children with respiratory infections. Quantitative analysis and genotyping of the HRV-positive samples from 601 nasopharyngeal aspirates (NPAs) were performed using VP4/VP2 sequencing. To compare T-helper1 (Th1) type (IFN-γ, TNF-α) and Th2 type (IL-4, IL-10) cytokine responses between HRV-A, B and C, the levels of the four cytokines were measured. The HRV-positive children had shorter fever duration (P = 0.018), and higher frequencies of chest retraction (P = 0.002) and wheezing (P = 0.022) than did the HRV-negative group. HRV-A was identified in 55 cases (58.5%), HRV-B in 8 (8.5%), and HRV-C in 31 (33.0%). There were no significant differences in the clinical data or NPA cytokines levels between patients with HRV-A and HRV-C infections. HRV is an important pathogen of the lower respiratory tract in young children. HRV-A and HRV-C are the dominant species that cause respiratory difficulty in young children.


Asunto(s)
Fiebre/diagnóstico , Infecciones por Picornaviridae/diagnóstico , Ruidos Respiratorios/diagnóstico , Infecciones del Sistema Respiratorio/diagnóstico , Rhinovirus/genética , Balance Th1 - Th2/genética , Enfermedad Aguda , Preescolar , Femenino , Fiebre/inmunología , Fiebre/fisiopatología , Fiebre/virología , Expresión Génica , Genotipo , Humanos , Lactante , Recién Nacido , Interferón gamma/genética , Interferón gamma/inmunología , Interleucina-10/genética , Interleucina-10/inmunología , Interleucina-4/genética , Interleucina-4/inmunología , Masculino , Infecciones por Picornaviridae/inmunología , Infecciones por Picornaviridae/fisiopatología , Ruidos Respiratorios/inmunología , Ruidos Respiratorios/fisiopatología , Infecciones del Sistema Respiratorio/inmunología , Infecciones del Sistema Respiratorio/fisiopatología , Infecciones del Sistema Respiratorio/virología , Estudios Retrospectivos , Rhinovirus/clasificación , Rhinovirus/aislamiento & purificación , Células TH1/inmunología , Células TH1/virología , Células Th2/inmunología , Células Th2/virología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología
20.
Epidemiol Infect ; 146(11): 1384-1388, 2018 08.
Artículo en Inglés | MEDLINE | ID: mdl-29970200

RESUMEN

To investigate the impact of viral and bacterial co-infection in hospitalised children with Mycoplasma pneumoniae pneumonia (RMPP). Retrospective analysis of 396 children with RMPP in our hospital admitted between 1 January 2011 and 31 December 2016 was performed. Nasal aspirate samples were collected for pathogen detection and clinical data were collected. We analysed clinical characteristics, lung imaging characteristics and pathogenic species among these children. Of the 396 RMPP cases, 107 (27.02%) had co-infection with other pathogen, with Streptococcus pneumoniae, Haemophilus influenzae and Staphylococcus aureus being the most common bacteria of infection and human bocavirus (HBoV), human rhinovirus, respiratory syncytial virus being the most common viruses of infection. Children with co-infection were younger than that with single infection (P = 0.010). Children with both virus and bacteria co-infection had been the youngest (P = 0.040). Children with co-infection had a longer fever process, higher leukocyte count, higher C-reactive protein compared with single infection (P < 0.05). Children with co-infection had a higher percentage of pnemothorax and diffuse large area of inflammation in chest X-ray manifestation compared with children with single infection (P < 0.05). S. pneumonia and HBoV was the leading cause of co-infection in RMPP. Co-infections led to more disease severity in children with RMPP compared with single infections.


Asunto(s)
Infecciones Bacterianas/complicaciones , Coinfección , Neumonía por Mycoplasma/complicaciones , Virosis/complicaciones , Distribución por Edad , Anticuerpos Antibacterianos/aislamiento & purificación , Infecciones Bacterianas/microbiología , Niño , Preescolar , Femenino , Haemophilus influenzae/aislamiento & purificación , Bocavirus Humano/genética , Bocavirus Humano/aislamiento & purificación , Humanos , Inmunoglobulina M/aislamiento & purificación , Lactante , Pacientes Internos , Masculino , Metapneumovirus/genética , Metapneumovirus/aislamiento & purificación , Nasofaringe/virología , ARN Ribosómico 16S/genética , Radiografía Torácica , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Estudios Retrospectivos , Rhinovirus/genética , Rhinovirus/aislamiento & purificación , Estaciones del Año , Staphylococcus aureus/aislamiento & purificación , Streptococcus pneumoniae/aislamiento & purificación , Virosis/virología
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