Genetic and biological characterisation of Zika virus isolates from different Brazilian regions

BACKGROUND Zika virus (ZIKV) infections reported in recent epidemics have been linked to clinical complications that had never been associated with ZIKV before. Adaptive mutations could have contributed to the successful emergence of ZIKV as a global health threat to a nonimmune population. However, the causal relationships between the ZIKV genetic determinants, the pathogenesis and the rapid spread in Latin America and in the Caribbean remain widely unknown. OBJECTIVES The aim of this study was to characterise three ZIKV isolates obtained from patient samples during the 2015/2016 Brazilian epidemics. METHODS The ZIKV genomes of these strains were completely sequenced and in vitro infection kinetics experiments were carried out in cell lines and human primary cells. FINDINGS Eight nonsynonymous substitutions throughout the viral genome of the three Brazilian isolates were identified. Infection kinetics experiments were carried out with mammalian cell lines A549, Huh7.5, Vero E6 and human monocyte-derived dendritic cells (mdDCs) and insect cells (Aag2, C6/36 and AP61) and suggest that some of these mutations might be associated with distinct viral fitness. The clinical isolates also presented differences in their infectivity rates when compared to the well-established ZIKV strains (MR766 and PE243), especially in their abilities to infect mammalian cells. MAIN CONCLUSIONS Genomic analysis of three recent ZIKV isolates revealed some nonsynonymous substitutions, which could have an impact on the viral fitness in mammalian and insect cells.

Detection and clearance of a mosquito densovirus contaminant from laboratory stocks of Zika virus

BACKGROUND The Zika virus (ZIKV) epidemics that affected South America in 2016 raised several research questions and prompted an increase in studies in the field. The transient and low viraemia observed in the course of ZIKV infection is a challenge for viral isolation from patient serum, which leads to many laboratories around the world sharing viral strains for their studies. C6/36 cells derived from Aedes albopictus larvae are commonly used for arbovirus isolation from clinical samples and for the preparation of viral stocks. OBJECTIVES Here, we report the contamination of two widely used ZIKV strains by Brevidensovirus, here designated as mosquito densovirus (MDV). METHODS Molecular and immunological techniques were used to analyse the MDV contamination of ZIKV stocks. Also, virus passages in mammalian cell line and infecting susceptible mice were used to MDV clearance from ZIKV stocks. FINDINGS MDV contamination was confirmed by molecular and immunological techniques and likely originated from C6/36 cultures commonly used to grow viral stocks. We applied two protocols that successfully eliminated MDV contamination from ZIKV stocks, and these protocols can be widely applied in the field. As MDV does not infect vertebrate cells, we performed serial passages of contaminated stocks using a mammalian cell line and infecting susceptible mice prior to re-isolating ZIKV from the animals' blood serum. MDV elimination was confirmed with immunostaining, polymerase chain reaction (PCR), and analysis of the mosquitoes that were allowed to feed on the infected mice. MAIN CONCLUSIONS Since the putative impact of viral contaminants in ZIKV strains generally used for research purposes is unknown, researchers working in the field must be aware of potential contaminants and test viral stocks to certify sample purity.

Organoid as a culture system for viral vaccine strains

Organoid is an in vitro multicellular form mimicking in vivo organ. Its similarity to human organ including cellular organization, molecular expression patterns, as well as genetic signatures enables to study the characteristics of infectious agents and host-pathogen interaction. For the features of organoid, this system also can be potentially used to cultivate currently uncultivable viruses of vaccine candidates. This paper will briefly describe problems in the current culture system for virus production and the possibility of organoid as culture system for viral vaccine and their current limitations that should be solved to meet the goal.

Three-dimensional Culture of Human Airway Epithelium in Matrigel for Evaluation of Human Rhinovirus C and Bocavirus Infections / 生物医学与环境科学（英文）

OBJECTIVE@#Newly identified human rhinovirus C (HRV-C) and human bocavirus (HBoV) cannot propagate in vitro in traditional cell culture models; thus obtaining knowledge about these viruses and developing related vaccines are difficult. Therefore, it is necessary to develop a novel platform for the propagation of these types of viruses.@*METHODS@#A platform for culturing human airway epithelia in a three-dimensional (3D) pattern using Matrigel as scaffold was developed. The features of 3D culture were identified by immunochemical staining and transmission electron microscopy. Nucleic acid levels of HRV-C and HBoV in 3D cells at designated time points were quantitated by real-time polymerase chain reaction (PCR). Levels of cytokines, whose secretion was induced by the viruses, were measured by ELISA.@*RESULTS@#Properties of bronchial-like tissues, such as the expression of biomarkers CK5, ZO-1, and PCK, and the development of cilium-like protuberances indicative of the human respiration tract, were observed in 3D-cultured human airway epithelial (HAE) cultures, but not in monolayer-cultured cells. Nucleic acid levels of HRV-C and HBoV and levels of virus-induced cytokines were also measured using the 3D culture system.@*CONCLUSION@#Our data provide a preliminary indication that the 3D culture model of primary epithelia using a Matrigel scaffold in vitro can be used to propagate HRV-C and HBoV.

Frecuencia de escherichia coli betalactamasa de espectro extendido (BLEE), en pacientes con infeccion de vías urinarias. Hospital José Carrasco Arteaga / Frequency of escherichia coli beta lactamase extended spectrum (ESBL), in patients with urinary tract infection. Hopital José Carrasco Arteaga

OBJETIVOS: Determinar la Frecuencia de Eschericia coli Beta Lactamasa de Espectro Extendido en Infecciones de Vías Urinarias, en el hospital José Carrasco Arteaga 2012-2013. MÉTODOLOGÍA: Observacional de tipo descriptivo prospectivo transversal. La población estudiada estuvo conformada por 605 muestras de urocultivos de pacien-tes de las áreas de: consulta externa, emergencia, hos-pitalización y cuidados intensivos. Se efectuó la prueba del método de confirmación apropiado, basado en la inhibición de la enzima ß-Lactamasas de confirmación productora de ß-Lactamasa según las normas Stan-dard Institute Clinical Laboratory. Se realizó el análisis estadístico descriptivo para comprensión e interpreta-ción de datos. RESULTADOS: Se cultivó 605 muestras, se reportó E. coli en 455 muestras de las cuales 82 correspondieron a la cepa productora de ß-Lactamasas de Expectro Ex-tendido un 18%. Según las variables consideradas, de acuerdo al sexo las mujeres representaron el mayor por-centaje con un 87,8%, el grupo etario con mayor repor-te fue el de 51-60 años con el 20,7%, seguido del grupo de 61-70 con el 17,1%, según la procedencia, el área urbana representó 69,5%, de acuerdo a los servicios en consulta externa se reportó 37,8% y en emergencia el 34,1%. CONCLUSIÓN: en el hospital José Carrasco Arteaga en el periodo septiembre 2012-enero 2013 se reportó una prevalencia del 18% de E. coli productora de ß-Lacata-masa en muestras de urocultivo de pacientes de los ser-vicios de consulta externa, emergencia, hospitalización y cuidados intensivos. Las mujeres fueron las más afec-tadas, según procedencia el mayor porcentaje fue del área urbana, el grupo de adultos representó el mayor porcentaje y la consulta externa fue el servicio con mayor frecuencia. (Impacto de los resultados

OBJECTIVES: To determine the frequency of Escherichia coli extended spectrum lactamasa in urinary tract in-fections, at José Carrasco Arteaga Hospital, 2012-2013. METHODOLOGY: It is an observational descriptive cross-sectional study. The population consisted of 605 samples of urine cultures of patients from the areas of: external consultation, emergency, hospitalization and intensive care. The appropriate confirmatory method was tested, it was based on the inhibition of ß-Lacta-masa-producing confirmation enzyme according to the Standard Institute Clinical Laboratory. A descriptive sta-tistical analysis was performed for data comprehension and interpretation. RESULTS: A total of 605 samples were cultured, E. coli was reported in 455 samples, and only 82 corresponded to the extended Expectrum ß-lactamase producing strain with 18%. According to the variables considered, regar-ding sex, women represented the highest percentage with 87.8%, the highest age group was 51-60 years with 20.7%, followed by the group of 61-70 with 17.1%, de-pending on the source, the urban area accounted for 69.5%, according to the services in external consultation 37.8% were reported, and 34.1% were emergency ones. CONCLUSION: A prevalence of 18% of E. coli producing ß-Lactamasa was reported at the José Carrasco Artea-ga Hospital in the period September 2012-January 2013 in urine samples of patients from external consultation, emergency, hospitalization and Intensive care servi-ces. The women were the most affected, according to provenance, the highest percentage was of the urban area, the group of adults represented the highest per-centage and the external consultation service was the most frequent. (Impact of results).

Desempenho da cultura líquida MGIT após implementação em uma rede de laboratórios públicos do estado de São Paulo / Performance of liquid culture MGIT after implementation in a network of public laboratories of Sao Paulo state

A OMS, em 2007, recomendou a implementação da cultura líquida para o diagnóstico da tuberculose (TB) e teste de sensibilidade para países de baixa e média renda. Neste estudo foi avaliado o desempenho da cultura líquida MGIT em condição de rotina após dois anosde implantação em uma rede de laboratórios públicos. Foi efetuada análise retrospectiva de dados da cultura líquida, realizadas em dez laboratórios regionais do Instituto Adolfo Lutz, de janeiro a março de 2010. Foram incluídas amostras submetidas a baciloscopia, cultura líquida MGIT automatizada ou manual e identificação presuntiva do complexo Mycobacterium tuberculosis (CMTB). Foram detectadas 1.159 culturas positivas. Destas, 113 (9,7%) contaminaram, e 1.046 foram analisadas, sendo 850 (81,3%) CMTB, 116 (11,1%) micobactérias não tuberculosas e 6 (0,6%) Nocardia sp A taxa de contaminação foi de 2,2% e o acréscimo da cultura para o diagnóstico da TB foi de 29,9%. A média do tempo de detecção da cultura foi de 14,7 dias (DP+/- 11,7 dias). A acurácia da identificação presuntiva foi de 91,3%. A cultura líquida MGIT demonstrou ser excelente alternativa para efetuar diagnóstico da TB e das micobacterioses, em razão da rapidez possibilitando uma intervenção rápida e eficaz no tratamento.

In 2007, WHO recommended the implementation of liquid culture for tuberculosis (TB) diagnosis and drug-susceptibility test in low and middle-income countries. This study evaluated the performance of MGIT culture in routine condition after two years of its implementation in a public laboratories network.This is a retrospective study, which analyzed the data on the liquid culture performed in ten regional laboratories of the Institute Adolfo Lutz, from January to March 2010. The data included clinical samples submitted to microscopy, automated or manual MGIT culture and presumptive M. tuberculosis complex (MTBC) identification by analyzing the cord formation. Culture was positive in 1,159 samples. Of these, 113 (9.7%) contaminated, and 1,046 were analyzed, of which 850 (81.3%) were identified as MTBC, 116 (11.1%) as non-tuberculous mycobacteria and 6 (0.6%) as Nocardia sp. Contamination rate was 2.2% and the contribution of culture to the TB diagnosis was 29.9%. The detection mean time was 14.7 days (SD+/-11.7 days). The accuracy of the presumptive identification of MTBC was 91.3%. MGIT liquid culture demonstrated to be an excellent alternative for diagnosing TB and mycobacterioses, because of the rapidity of diagnosis, thus allowing an immediate and effective treatment.

Repercusión de los dispositivos móviles en la atención de enfermería a usuarios en estado crítico / Repercussion of mobile devices on nursing care for users in critical state

Introducción: actualmente en los hospitales de México, especialmente en las áreas de cuidados críticos, se ha incrementado el uso de dispositivos móviles de comunicación, repercutiendo en el cuidado del paciente; esto pudiera representar no solamente un distractor, sino una fuente portadora de gérmenes. Objetivo: evaluar la repercusión de los dispositivos móviles en la atención de enfermería a usuarios en estado crítico. Métodos: estudio descriptivo, trasversal; donde fueron medidos los tiempos de interrupción del cuidado de enfermería en el uso de dispositivos móviles de comunicación; se describió la exposición de estos artefactos con los equipos biomédicos por medio de una guía observacional, además se tomó muestra de los dispositivos móviles para su cultivo en agar nutritivo. Resultados: el 75,00 por ciento de los enfermeros estudiados hacían uso de los dispositivos móviles dentro de su jornada laboral; el 68,00 por ciento hizo uso de algún dispositivo móvil mientras realizaba alguna actividad con el paciente; el 64,00 por ciento tenía contacto con equipo biomédico; el 100,00 por ciento no se lavaba las manos antes y después de usarlos; en el 100,00 por ciento de las muestras tomadas y cultivadas hubo crecimiento Unidades Formadoras de Colonias a las 48 horas. Conclusiones: los dispositivos móviles son distractores, adictivos y cuentan con carga bacteriológica, esto afecta en la atención directa al paciente, su uso aún no está regulado; por esta razón sería importante considerar limitar el uso en las unidades de cuidados críticos, esto ayudara a brindar una mejor atención viéndose reflejado en la seguridad del paciente(AU)

Introduction: In Mexico hospitals today, especially in critical care areas, the use of mobile devices of communication has increased, which has had a repercussion on the care for the patient; this could represent not only a distracting aspect, but a germ-bearing source. Objective: Assess the repercussion of mobile devices on nursing care for user in critical state. Methods: cross-sectional, descriptive study in which we measured the interruption times for nursing care in the use of mobile devices of communication; we described the exposition of this artifacts with biomedical equipment by means of an observational guide, we also took sample of mobile devices for their culture in a nutrient agar. Results: 75.00 percentof the studied nurses used mobile devices within their working day; 68.00 percent used any mobile device while doing any activity with the patient; 64.00 percent had contact with biomedical equipment; 100.00 percent did not wash their hands before or after using them; in the 100.00 percent of the samples taken and cultured there were colonies growing after 48 hours. Conclusions: Mobile devices are distracting, addictive and have bacteriologic charge, which affects the direct care for the patient, their use is not regulated; therefore, it would be important to consider limiting their use in critical care units, which will help provide better attention reflected on the patient's safety(AU)

Tuberculose pulmonar paucibacilar em Centros de Detenção Provisória / Paucibacillary pulmonary tuberculosis in Provisional Detention Centers

Cultura de micobactérias proporciona o crescimento de bacilos viáveis, mesmo presentes em escassa quantidade e não detectados pela baciloscopia. Neste estudo foram analisadas as amostras de escarro que apresentaram baciloscopia negativa e cultura positiva. As amostras foram coletadas de 2008 a 2013, de indivíduos detidos em Centros de Detenção Provisória de Santo André, Mauá e Diadema, Estado de São Paulo. As metodologias utilizadas foram baciloscopia por coloração Ziehl-Neelsen e cultura pelo Sistema BACTEC MGIT 960 e Ogawa-Kudoh. Dos 11.529 exames realizados, 221 (1,9 %) apresentaram baciloscopias negativas e culturas positivas. Dos 221 isolados, 166 (75,1 %) pertenciam ao Complexo Mycobacterium tuberculosis, 21 (9,5 %) micobactérias não membros do Complexo Mycobacterium tuberculosis (MNT), 33 (14,9 %) Mycobaterium sp e uma cultura mista do Complexo M. tuberculosis e M. avium. MNT mais frequentes foram M. avium (23,8 %) e M. fortuitum (19,0 %). A maioria dos isolados do Complexo M. tuberculosis (155/166 - 93,4 %) foi sensível aos antimicrobianos. Sete amostras apresentaram resistência à isoniazida e uma apresentou multirresistência à isoniazida e rifampicina. Este estudo mostra a importância da realização da cultura em escarros que apresentam baciloscopia negativa no diagnóstico da TB e micobacteriose. O tratamento tardio causa a continuidade da transmissão da doença e agravamento do quadro clínico.

Culture of mycobacteria induces the growth of viable bacillus occurring in small quantity, which are no detectable by bacilloscopy. This study aimed at identifying the mycobacteria isolates from sputum presenting negative bacilloscopy and positive culture. The samples were collected from 2008 to 2013 from criminals of Provisional Detention Centers in Santo André, Mauá and Diadema/SP. Smears were stained by Ziehl-Neelsen staining and the cultures were performed by the BACTEC MGIT 960 system and Ogawa-Kudoh culture medium. Of 11,529 isolates, 221 (1.9 %) showed negative bacilloscopy and positive cultures. Of 221 isolates, 166 (75.1 %) belonged to Mycobacterium tuberculosis complex, 21 (9.5 %) were nontuberculous mycobacteria (NTM), 33 (14.9 %) Mycobacterium sp, and one identified as a mixed culture of M. tuberculosis and M. avium complex. The most common NTM species were M. avium (23.8 %) and M. fortuitum (19.0 %). Most of the isolates (155/166-93.4 %) were susceptible to antimicrobial agents. Seven samples were resistant to isoniazid, and one presented multiresistance to isoniazid and rifampicin. This study shows the importance in performing sputum culture, when these samples are negative on bacilloscopy in diagnosing TB and mycobacteriosis. The treatment delay results in the maintenance of disease transmission and worsening of clinical symptoms.

Enhancement of Virus Replication in An Influenza A Virus NS1-Expresssing 293 Cell Line / 生物医学与环境科学（英文）

The nonstructural protein 1 (NS1) of influenza A virus, which is absent from the viral particle, but highly expressed in infected cells, strongly antagonizes the interferon (IFN)-mediated antiviral response. We engineered an NS1-expressing 293 (293-NS1) cell line with no response to IFN stimulation. Compared with the parental 293 cells, the IFN-nonresponsive 293-NS1 cells improved the growth capacity of various viruses, but the introduction of NS1 barely enhanced the propagation of Tahyna virus, a negative-strand RNA virus. In particular, fastidious enteric adenovirus that replicates poorly in 293 cells may grow more efficiently in 293-NS1 cells; thus, IFN-nonresponsive 293-NS1 cells might be of great value in diagnostic laboratories for the cultivation and isolation of human enteric adenoviruses.

Effects of temperature and pH on the growth of H1N1 subtype of influenza A virus by surface-enhanced Raman spectroscopy / 生物工程学报

Surface enhanced Raman spectroscopy technology (SERS), using gold nanoparticles as a base, was developed for rapid and sensitive detection of virus strains. SERS can be used as a rapid and reliable method to distinguish the titers of viral replication. In the present study, we characterized H1N1 subtypes of influenza A virus strains in different conditions of pH or temperatures, while we analyzed data from SERS technology using gold nanoparticles as a base and cell cultures were employed to further confirm the data from virus strains. Origin8.0 was used to collect Raman spectra, smooth and homogenize data, and to contrast spectra. Our results indicated that the peaks of different virus strains in optimal environmental conditions (T=37 ℃/pH=7.2) reached ≥3 000. This criterion was verified by subsequent virological method. The present data indicate that the established SERS protocol can be used as a rapid and reliable method to distinguish the replication rate of virus, which can be further used in clinical samples.

Agentes bacterianos en la sepsis neonatal. cuidados intensivos neonatales, Hospital Mario Catarino Rivas / Bacterial agents in neonatal sepsis. Neonatal Intensive Care unit Mario Catarino Rivas National Hospital

Antecedentes: Las infecciones nosocomiales son aquellas que se adquieren y se mani estan luego de 48 horas de hospitalización Objetivo: Determinar los gérmenes aislados por cultivos de los recién nacidos diagnosticados como sepsis nosocomial en la unidad de cuidados intensivos neonatales (UCIN), Hospital Nacio- nal Mario Catarino Rivas (HNMCR), en los meses de julio a septiembre del 2015. Pacientes y métodos: Estudio transversal, de los 443 pacientes ingresados a UCIN, 221 neonatos que desarrollaron infección posterior a 48 horas de internamiento. La información se obtuvo del expediente clínico y se procesó en el software estadístico Epi Info 3.02 Resulta- dos: De los cultivos obtenidos; (165) 75% resultaron positivos para algún germen especí- co. Los gérmenes aislados fueron; Pseudomo- na spp 71 (43%) y Pseudomona aeruginosa 58 (35%), haciendo un total de 78% de sepsis nosocomial por Pseudomona. Conclusión: La sepsis intrahospitalaria es un problema frecuente en UCIN, por lo tanto es necesario el cumplimiento de las normas de vigilancia y control de este tipo de infecciones...(AU)

Bovine Herpesvirus 4 in Parana State, Brazil: case report, viral isolation, and molecular identification

Bovine Herpesvirus 4 (BoHV-4) is a member of Gammaherpesvirinae sub-family and belongs to genus Rhadinovirus. This virus has been associated with different clinical manifestations and research activity has put forward a strong correlation among virus infection, postpartum metritis, and abortion. The goal of this work was to characterize a virus strain isolate from a cow’s uterine outflow. From swabs drawn of uterine secretion, a virus strain was isolated and characterized by its cytopathology, morphology, and molecular biology approaches. In culture there was CPE development, characterized mainly by long strands with several small balloons along them, radiated from infected cells. Electron microscopy analysis revealed virus particles that had icosahedrical capsid symmetry surrounded by a loose envelope, typical of a herpesvirus. A 2,571 bp PCR product after HindIII digestion generated four fragments, whose base pair composition were 403, 420, 535, and 1,125 bp. Restriction enzymes HindIII and BamHI generated the expected diagnostic bands as well as a 2,350 bp hypermolar fragment as a result of BamHI treatment to demonstrate that agent was a bovine herpesvirus 4, appertaining to DN-599 group.

Selection and Identification of the Biological Characteristics of a Cold-adapted Genotype G1P8 ZTR-68 Rotavirus by Serial Cold-adapted Passaging / 病毒学报

We wished to select a cold-adapted genotype G1P[8] ZTR-68 rotavirus (China southwest strain) in MA104 cells for possible use as a live vaccine. ZTR-68 was recovered originally from children with diarrhea. The virus was cultivated at 37 degrees C at the first passage. Then, the cultivation temperature was decreased stepwise by 3 degrees C per eight passages. In total, the virus was passaged 32 times, and cultivation was terminated at 28 degrees C. Biological characteristics of the virus were analyzed during serial passages. There was no difference between the migration patterns of genomic dsRNA segments according to polyacrylamide gel electrophoresis of original and cold-adapted viruses. Infectious and red cell-agglutination titers of cold-adapted virus were lower than those of the parent virus. Also, the virus formed small-size plaques with irregular shapes at 31 degrees C and 28 degrees C. These results suggested that a genetically stable attenuated virus can be obtained through serial cold-adapted passages. Thus, an alternative strategy is provided by cold-adaption for development of attenuated live rotavirus vaccines.

Rescuing Bombyx mori bidensovirus in BmN cells in vitro / 生物工程学报

Bombyx mori bidensovirus (BmBDV) has been identified as causing chronic densonucleosis in Bombyx mori specifically. The replication mechanism of BmBDV remains unknown. Its genome comprises two single stands DNA (VD1 and VD2). In order to rescue infectious virions in vitro, we obtained the total viral DNA extracted from the BmBDV-infected larvae midguts, subsequently cloned the full-length sequence of BmBDV genome fragments by PCR and constructed recombinant plasmids pMD18T-VD1 and pUC-VD2. The linear genome fragments were obtained by digesting recombinant plasmids with corresponding restriction enzymes, and then collectively transfected BmN cells by the method of liposome-embedding. We determined the replication of the virus gene by PCR with the template of demethylated total DNA extracted from the post-transfect BmN cells. Meanwhile, we collected the total proteins from the post-transfect BmN cells and the larvae midgut of feeding the post-transfect BmN cells to perform Western blotting analysis, and detected the expression of viral genes. Here we firstly confirm that infectious virions can be rescued in BmN cells by linear co-transfect method.

Efficient assembly of full-length infectious clone of Brazilian IBDV isolate by homologous recombination in yeast

The Infectious Bursal Disease Virus (IBDV) causes immunosuppression in young chickens. Advances in molecular virology and vaccines for IBDV have been achieved by viral reverse genetics (VRG). VRG for IBDV has undergone changes over time, however all strategies used to generate particles of IBDV involves multiple rounds of amplification and need of in vitro ligation and restriction sites. The aim of this research was to build the world's first VRG for IBDV by yeast-based homologous recombination; a more efficient, robust and simple process than cloning by in vitro ligation. The wild type IBDV (Wt-IBDV-Br) was isolated in Brazil and had its genome cloned in pJG-CMV-HDR vector by yeast-based homologous recombination. The clones were transfected into chicken embryo fibroblasts and the recovered virus (IC-IBDV-Br) showed genetic stability and similar phenotype to Wt-IBDV-Br, which were observed by nucleotide sequence, focus size/morphology and replication kinetics, respectively. Thus, IBDV reverse genetics by yeast-based homologous recombination provides tools to IBDV understanding and vaccines/viral vectors development.

Evaluación del cultivo liofilizado de Candida albicans utilizado en esquemas de certificaciones de calidad / Evaluation of the lyophilized culture of Candida albicans used in quality certification schemes

Introducción: el laboratorio de control microbiológico de la UEB Laboratorios Liorad dispone de una colección de cultivos microbianos para la conservación de microorganismos, donde se encuentra depositada la levadura Candida albicans que se emplea en esquemas de certificaciones de calidad establecidos para la evaluación de ensayos como: promoción de crecimiento de los medios de cultivos, validación de técnicas microbiológicas, entre otros. Objetivo: evaluar los resultados de la conservación de esta cepa por el método de liofilización durante un periodo de ocho años. Métodos: para el crecimiento de la cepa se utilizó el medio de cultivo Caldo Saboraud y variantes de sustancias lioprotectoras puras como: (leche descremada al 20 por ciento, glicerol 20 por ciento, sacarosa al 10 por ciento y peptona 5 por ciento) así como la mezcla de lioprotectores (leche 10 por ciento, sacarosa 5 por ciento, glicerol 5 por ciento). Se evaluó viabilidad, pureza y estabilidad genética de esta cepa durante el tiempo objeto de estudio. Resultados: las características propias de la especie estudiada se mantuvieron inalterables con un elevado grado de pureza en todas las variantes estudiadas. En cuanto a la supervivencia, cuando se emplearon las sustancias lioprotectoras puras se evidenció una marcada disminución de la viabilidad. No así al emplear la mezcla de lioprotectores que mantuvo niveles de viabilidad por encima del límite establecido durante todo el tiempo objeto de estudio. Conclusiones: los valores obtenidos en cuanto a la supervivencia de este microorganismo permiten inferir que para la conservación por largos periodos de tiempo la variante donde se empleó mezclas de lioprotectores resultó una buena opción para la conservación de C. albicans(AU)

Introduction: the microbiological control laboratory at the Basic Enterprise Unit Liorad Laboratories stores a collection of microbial cultures for the preservation of microorganisms, including the Candida albicans yeast used in the quality certification schemes established for the evaluation of assays such as fostering of culture medium growth and validation of microbiological techniques, among others. Objective: evaluate the results obtained in the preservation of this strain by the lyophilization method during a period of eight years. Methods: for strain growth, use was made of Saboraud broth culture medium as well as variants of pure lyoprotective substances such as 20 percent skimmed milk, 20 percent glycerol, 10 percent saccharose and 5 percent peptone, and the mixture of lyoprotectors (10 percent milk, 5 percent saccharose, 5 percent glycerol). An evaluation was conducted of the viability, purity and genetic stability of the strain during the study period. Results: characteristics typical of the study species remained unchanged with a high degree of purity in all the variants examined. As to survival, a marked reduction in viability was observed when pure lyoprotective substances were used, but not with the mixture of lyoprotectors, in which case viability levels exceeded the established limit during the entire study period. Conclusions: the survival values obtained for this microorganism indicate that preservation for long periods with mixtures of lyoprotectors was a good option for the preservation of C. albicans(AU)

Evaluation of a rainbow trout (Oncorhynchus mikyss) culture water recirculating system / Evaluación de un sistema de recirculación de agua para levante de trucha arcoiris (Oncorhynchus mikyss)

Objective. To evaluate a water recirculation system of rainbow trout fish culture at the recirculating laboratory of the Aquaculture Engineering Production Program of the Universidad of Nariño. Materials and Methods. There were cultured 324 rainbow trout (Oncorhynchus mikyss) fries in 12 plastic tanks of 250 L capacity in a recirculation aquaculture system which treatment system was made up by a conventional sedimentation tank, a fixed bead up flow biofilter with recycled PVC tube pieces used as carrier, and a natural degassing system; the sedimentation unit effluent was pumped to a reservoir tank by a centrifugal 2 HP after passed by gravity through the biofilter and was distributed to the 12 culture units in which there were injected a constant amount of air from a blower. Results. The waste water treatment system removes 31% of the Total Suspended Solids; 9.5% of total ammonia nitrogen, and increased the dissolved oxygen to the final effluent in a 6.5%. It was calculated a biomass increase of 305% on the 75 days, the mortality percentage registered during the research period was of 4.9%. Conclusions. The wastewater treatment system maintained the physic chemical water quality parameters in the recommended values for the specie. The values of weight and size gain, food conversion, mortality and biomass production reported were between the normal values of rainbow trout fish culture in recirculating systems.

Objetivo. Evaluar un sistema de recirculación de agua para cultivo de trucha arcoiris en el laboratorio de recirculación del Programa Ingeniería en Producción Acuícola de la Universidad de Nariño. Materiales y métodos. Se cultivaron 324 alevinos de trucha arco íris (Oncorhynchus mikyss) en 12 tanques plásticos de 250 L de capacidad en un sistema de recirculación para acuacultura cuyo sistema de tratamiento estuvo constituido por un sedimentador convencional, un biofiltro de flujo ascendente con medio soporte fijo conformado por segmentos reciclados de tubos PVC, y un sistema de desgasificación natural; el efluente del sedimentador fue elevado a un tanque reservorio por medio de una bomba centrífuga de 2 HP para después pasar por gravedad a través del biofiltro y posteriormente ser distribuido a las 12 unidades de cultivo a las que de manera permanente se inyectó aire proveniente de un blówer. Resultados. El sistema de tratamiento del agua removió 31% de los sólidos suspendidos totales; 9.5% del nitrógeno amoniacal total, e incrementó el oxígeno disuelto al efluente final en un 6.5%. Se calculó un incremento de la biomasa del 345% en los 75 días, el porcentaje de mortalidad registrado durante todo el periodo de estudio fue del 4.9%. Conclusiones. El sistema de tratamiento mantuvo los parámetros físico-químicos de la calidad de agua dentro de los rangos requeridos por la especie. El incremento de peso y talla, la conversión alimenticia, la mortalidad y la producción de biomasa reportaron valores normales para producción de trucha en sistemas de recirculación.

Selección de variantes de virus dengue sensibles a la heparina en células BHK-21 / Selection of heparin-sensitive dengue virus variants in BHK-21 cells

Estudios previos han demostrado que la adaptación de diversos virus a crecer en líneas celulares de vertebrados, conduce a la selección de variantes virales que unen al heparán sulfato (HS) con alta afinidad. En el presente trabajo se determinó la susceptibilidad de cepas del virus dengue (DENV) a la heparina hipersulfatada un análogo al HS, después de pases seriados en células BHK-21. A aislados de campo de los cuatro serotipos de DENV, se les realizaron ocho pases seriados en células BHK-21. La adaptación de los DENV al cultivo celular seleccionó variantes virales con una aumentada capacidad replicativa en células BHK-21 y una incrementada susceptibilidad a la heparina, en relación a las respectivas cepas no adaptadas, obteniéndose una inhibición de la infectividad más significativa en DENV-2 y DENV-4. Las cepas de DENV adaptadas presentaron cambios en la secuencia de aminoácidos de la proteína de envoltura (E), en particular una substitución K204R para DENV-1, N67K para DENV-2, K308R y V452A para DENV-3 y E327G para DENV-4. Estas sustituciones implicaron ganancia de residuos básicos que incrementaron la carga neta positiva de la proteína. Los resultados sugieren, que la adaptación de cepas de DENV a células BHK-21 selecciona variantes virales sensibles a la heparina y que la efectividad de este compuesto varía dependiendo de la cepa viral. Además sugieren que el HS puede jugar un papel importante en la infectividad de las cepas de DENV adaptadas al cultivo celular, a diferencia de los aislados de DENV no adaptados.

Several studies have shown that adaptation of various viruses to grow in certain cell lines of vertebrates, leads to the selection of virus variants that bind heparan sulfate (HS) with high affinity. In this study we investigated the susceptibility of strains of dengue virus (DENV) to oversulfated heparin an analogue of HS after passages in BHK-21 cells. Field isolates of the four serotypes of DENV with a limited number of passes in mosquito cells C6/36HT were serially passaged eight times in BHK-21 cells. The adaptation of the DENV to the cell culture selected viral variants with an increased replicative capacity in BHK-21 cells and an increased susceptibility to heparin compared with the original not adapted strains, with a more significant inhibition of the infectivity in DENV-2 and DENV-4.The E protein of the adapted strains showed changes in the amino acid sequence, particularly at the position K204R to DENV-1, N67K to DENV-2, K308R and V452A for DENV-3 and E327G to DENV-4. These substitutions implicated a gain of basic residues that increased the net positive charge of the protein. These results suggest that adaptation of DENV strains to BHK-21 cells implies changes in the envelope protein, changes associated to the protein reactivity with heparin, the inhibitory effectiveness of this compound varying depending on the viral strain. In addition, these results suggest that the HS can play an important role in the infectivity of the DENV strains adapted to vertebrate cell culture, but not in the infectivity of non-adapted DENV isolates.

Construction of cDNA infectious clones of EV71 highly-pathogenic and cell-culture-adapted strains / 病毒学报

The highly-pathogenic EV71 strain is the primary cause of mortality in hand-foot-and-mouth disease (HFMD) associated with neurological symptoms, for which no clinically effective drugs or vaccines exist. This study aimed to construct infectious cDNA clones of the EV71 highly-pathogenic strain and the cell-culture adapted strain using a reverse genetics approach. The genomic RNAs of EV71 parent strains were used as the templates for RT-PCR amplification, and then the PCR products that overlapped the full-length genome were connected into the pBR322 vector to produce infectious clones of pEV71 (HP) and pEV71 (CCA), respectively. The results showed that the HP strain propagated much more quickly than the CCA strain. The rescued viruses derived from the infectious clones not only maintained their consistency with their parent strains in terms of genomic sequences, but also retained their respective biological phenotypes. This research will contribute to our understanding of EV71 pathogenesis and the development of novel vaccines against HFMD.

Producing Ad-IFN gamma by suspension culture of HEK293 cells in a disposable bioreactor / 生物工程学报

Adenovirus vectors are promising delivery systems for gene therapy. We established a new process for clinic trial of recombinant adenovirus vectors using a novel disposable bioreactor. The suspension HEK293 cells were inoculated into a 5 L disposable bioreactor with parameters control of pH, DO, agitation and temperature. After 6 days of a fed-batch culture, the final cell density reached 2.0 x 10(6) cells/mL. The culture was infected with Ad-IFNγ at an MOI of 30. The harvest was performed at approximately 48 h post-infection and crude viral lysate was obtained after 3 freeze/thaw cycles and centrifugation. The maximum titers of crude viral lysate was 1.49 x 10(13) Infectious units (IFU) and the bulk product specific was 3,800 IFU/cell. Purified Ad-IFNγ by anion-exchange chromatography and the final recovery of infectious unit reached 35.9%. The result demonstrates that an efficient and stable process of producing Ad-IFNγ using a disposable fed-batch bioreactor is established.