Herpes simplex virus: isolation, cytopathological characterization and antiviral sensitivity

BACKGROUND: Herpes simplex virus (HSV) infection is an endemic disease and it is estimated that 6095% of the adult population are infected with symptoms that are usually self-limiting, though they can be serious, extensive and prolonged in immunocompromised individuals, highlighted by the emergence of drug-resistant strains. The study of the wild-type HSV strains based on the cytopathogenic features and its antiviral sensitivity are important in the establishment of an antivirogram for controlling the infection. OBJECTIVE: This study sought to isolate and examine the cytopathological characteristics of circulating strains of the Herpes simplex virus, from clinical specimens and their sensitivity to commercially available antiherpesvirus drugs, acyclovir, phosphonophormic acid and trifluridine. METHODS: Herpes simplex virus isolation, cytopathological features and antiviral sensitivity assays were performed in cell culture by tissue culture infectious dose or plaque forming unit assay. RESULTS: From twenty-two clinical specimens, we isolated and adapted nine strains. Overall, the cytopathic effect was detected 24 h post-infection (p.i.) and the presence of syncytia was remarkable 48 h p.i., observed after cell staining. Out of eight isolates, four developed plaques of varying sizes. All the isolates were sensitive to acyclovir, phosphonophormic and trifluridine, with the percentage of virus inhibition (%VI) ranging from 49.7-100%. CONCLUSIONS: The methodology for HSV isolation and characterization is a straightforward approach, but the drug sensitivity test, regarded as being of great practical importance, needs to be better understood. .

Detection and characterization of human rotavirus in hospitalized patients in the cities of Ponta Grossa, Londrina and Assai - PR, Brazil

Acute diarrheal disease is still one of the major public health problems worldwide. Rotaviruses (RV) are the most important viral etiologic agents and children under five years of age are the target population. OBJECTIVE: To investigate the rate of RV infection in hospitalized patients due to acute diarrhea in the cities of Ponta Grossa, Londrina and Assai - Paraná. METHODS: Latex agglutination (LA); immunochromatography (ICG); polyacrylamide gel electrophoresis (PAGE) and negative staining electron microscopy (ME) tests were used to detect the virus. For the genotyping, RT-PCR and RT-PCR-ELISA were used, respectively, for NSP4 and VP4/VP7. RESULT: Out of 124 samples there were 69 positive stool samples for RV, for at least one of the used tests, 67 of them being RV group A (RV-A). Overall, most of the RV positive stool samples came from children under thirteen years of age. However, 12 positive cases occurred in patients aged 13 years or above, including an 81-year old patient. CONCLUSION: The data showed similar electropherotypes and genotypes G, P and NSP4 of the inland wild circulating strains of RV.

Inserção das ciências básicas no currículo integrado do curso de Medicina da Universidade Estadual de Londrina / The participation of basic sciences in the integrated curriculum of the Londrina State University medical school

O curso de Medicina da Universidade Estadual de Londrina (UEL) implementou um currículo integrado que utiliza a metodologia da Aprendizagem Baseada em Problemas (ABP) nos módulos temáticos. Embora dados da literatura apontem que o uso dessa metodologia aumenta o entendimento e a retenção de conceitos das ciências básicas, alguns autores demonstram preocupação com o fato de estas não receberem suficiente ênfase nesta metodologia. Esse trabalho teve por objetivo avaliar a inserção das ciências básicas no currículo integrado do curso de Medicina da UEL. Para isto, foi elaborado um questionário semiestruturado sobre o processo de inclusão dos docentes das áreas básicas nas várias atividades desenvolvidas no atual currículo. Houve aumento de 36 por cento na participação dos docentes em relação ao currículo anterior. Foi verificado que ocorreu uma distribuição dos docentes em atividades do currículo integrado nas quatro primeiras séries, embora com maior concentração nas duas primeiras. Devem ser feitos esforços para que a integração básico-clínico possa ocorrer com maior intensidade em todas as séries.

The medical school of the State University of Londrina established an integrated curriculum using Problem Based Learning (PBL) as teaching strategy in the thematic modules of the medical course. Although according to the literature Problem Based Learning enhances the understanding and retention of basic sciences concepts, some authors fear that these basic concepts are not sufficiently emphasized in this methodology. The objective of this study is to evaluate the participation of basic sciences in the integrated curriculum of the Londrina State University medical school. To this purpose a semi-structured questionnaire was elaborated for evaluating the participation of teachers of the basic areas in the different activities making part of the current curriculum. There was an increase of 36 percent in the participation of basic science teachers in relation to the previous curriculum. It was verified that basic sciences teachers participated in activities of the integrated curriculum during the first four but mainly during the first two years. Efforts should be made for achieving a more intense integration of basic sciences and clinical practice throughout the course.

Escrever bons problemas / Writing good problems

Bons problemas são fundamentais em um currículo que utiliza o Aprendizado Baseado em Problemas. Escrevê-los requer conhecimento sobre os elementos básicos que devem fazer parte da estrutura do problema e também os fatores fundamentais que devem ser levados em consideração para que os objetivos sejam alcançados. Neste trabalho foram revistos alguns artigos da literatura com sugestões sobre a estrutura de problemas para uso em atividades tutoriais. São também apresentados princípios que devem ser respeitados e o modelo de estrutura de um problema, tal como têm sido sugeridos para os grupos de planejamento dos módulos temáticos e tutoriais do curso de Medicina da Universidade Estadual de Londrina além de exemplos de erros, observados em nossa experiência de 10 anos com este modelo de currículo, e que devem ser evitados.

Good problems are crucial for the development of a Problem-Based Learning curriculum. Writing good problems requires discipline and knowledge of the basic principle of the structure and fundamental features that have been considered to achieve the goals. Herein, the authors reviewed some of the rules suggested by the medical literature, and presented a model of written problems that are suggested for the tutorial planning groups of the Medical Course at the State University of Londrina, Brazil. Examples of errors observed in our 10-year experience in Problem-Based Learning are commented.

Clorofilina inibe a multiplicação de poliovírus em cultura de células HEp-2 / Chlorophyllin inhibits the multiplication of poliovirus in HEp-2 cell cultures

A clorofilina (CHLN), derivado sintético da clorofila com atividade antimutagênica, foi analisada quanto a sua atividade antiviral para o poliovírus em culturas de células HEp-2. A droga foi avaliada quanto às atividades virucida, profilática e terapêutica na multiplicação do poliovirus em culturas de célulasHEp-2, nas concentrações de 0,5 e 2,5 µg/mL. A inibição do título viral e a reação de imunofluorescência foram utilizadas para avaliação. A CHLN inibiu a multiplicação do poliovírus nos três protocolos de tratamento, porém foi mais efetiva no tratamento virucida inibindo em 95,7 por cento a multiplicação viral na concentração de 2,5 µg/mL. A CHLN reduziu o número de células infectadas com fluorescência específica, nos tratamentos virucida, e terapêutico, 8h e 10h pós-infecção, nas duas concentrações (0,5e 2,5 µg/mL) . Sugere-se que a CHLN tem uma ação direta na partícula viral ou provavelmente age nos estágios iniciais da multiplicação do poliovirus.

Chlorophyllin (CHLN), a semisynthetic derivative of chlorophyll with antimutagenic properties, wasassayed for its antiviral effect on the replication of poliovirus in cell culture. The drug was tested forthe virucide, prophylactic and therapeutic activities on the replication of the poliovirus in HEp-2 cellscultures, at concentrations of 0.5 and 2.5 µg/mL. Virus titration and an indirect immunofluorescencetest were used for the evaluation. The CHLN inhibited poliovirus replication in all treatment protocols;however, it was more effective on virucide treatment, with a 95.7 percent reduction in viral multiplication atconcentration of 2.5 µg/mL. CHLN reduced the number of specifically fluorescent infected cells in bothvirucide and therapeutic treatments, 8h and 10h post-infection, at both concentrations (0.5 and 2.5 µg/mL). It is suggested that CHLN either has a direct action on the virus particles or acts on the initial stage of the polivirus replication.

Development of an enzyme immunoassay for poliovirus antigens

An indirect solid-phase enzyme immunoassay (EIA) was developed for the detection of poliovirus antigen. Virus antigen was obtained in LLC-MK2 cell cultures and used to prepare antibodies in rabbit and guinea pig. Antibodies were evaluated by double immunodiffusion and neutralization test. Optimal concentrations of guinea pig and rabbit immunoglobulins were determined by checkerboard titration. Microtitre plates were coated with 15.0 µg/ml guinea pig anti-polio immunoglobulin and rabbit anti-polio immunoglobulin at the concentration of 7.94 µg/ml was used as detecting antibody. The standard curve with eight different antigen concentrations in eight replicates resulted in a coefficient of variation (CV) between 2.1 percent to 7.8 percent. The dose-response relationship was determined by simple linear regression with a coefficient of correlation (R²) equal to 96.4 percent. The assay detected a minimum of 2.3 µg/ml poliovirus antigen.

O trabalho apresenta o desenvolvimento de um ensaio imunoenzimático indireto para a detecção de antígeno de poliovírus. O antígeno viral foi obtido em cultura de células LLC-MK2 e usado para imunização de coelho e cobaia. Os soros hiperimunes foram avaliados por imunodifusão dupla e teste de neutralização. Após padronização, o soro de captura, produzido em cobaia, foi usado na concentração protéica de 15.0 µg/ml para sensibilizar microplacas de poliestireno e o soro de coelho (detector) foi usado na concentração de 7.94 µg/ml. A curva padrão resultante da utilização de oito diferentes concentrações do antígeno padrão definiu um coeficiente de variação de 2.1 por cento a 7.8 por cento. A relação dose-resposta foi determinada por regressão linear simples com o estabelecimento do coeficiente de correlação (R²) igual a 96.4 por cento. O ensaio possibilitou a detecção mínima de 2.3 µg/ml de antígeno de poliovírus.

Antiviral activity of Agaricus blazei Murrill ss. Heinem extract against human and bovine herpesviruses in cell culture

The aqueous extract of Agaricus blazei Murill ss. Heinem, a basidiomycete native from Brazil, frequently used by popular medicine, mainly in the form of tea, was assessed to its antiviral action against herpes simplex type 1 (HSV-1) and bovine herpes type 1 (BoHV-1) in HEp-2 cell culture. Viral replication inhibition was evaluated by plaque assay and immunofluorescence test. The extract demonstrated virucide action for both viruses, being more effective against HSV-1, inhibiting its infectivity in 78.4 and 73.9 percent at the concentrations of 50 and 100 æg/mL, respectively moreover, reduction in 47 percent the number of fluorescent cells was observed for both concentrations. The extract also showed discrete therapeutic activity. These results suggest that A. blazei extract acts mainly in the viral particle, however, the effect during virus replication can not be ruled out.

O extrato aquoso de Agaricus blazei Murill ss. Heinem, um basidiomiceto nativo do Brasil, usado na medicina popular, na forma de chá, foi avaliado quanto suas propriedades antivirais contra herpes simplex tipo 1 (HSV-1) e herpes bovino tipo 1 (BoHV-1) em cultura de células HEp-2. A inibição da replicação viral foi monitorada pelos ensaio de placa e reação de imunofluorescência. O extrato apresentou atividade virucida mais efetiva do que terapêutica para ambos os vírus, sendo mais efetivo portanto para HSV-1, inibindo em mais de 70 por cento o número de plaques e em cerca de 47 por cento o número de células apresentando fluorescência específica, nas concentrações de 50 e 100 æg/mL, nas duas técnicas utilizadas. Os resultados obtidos sugerem que o extrato aquoso de A. blazei deve agir principalmente sobre a partícula viral, embora a inibição durante o ciclo replicativo do vírus não deva ser excluída.

The in vitro cytopathology of a porcine and the simian (SA-11) strains of rotavirus

Rotaviruses have been implicated as the major causal agents of acute diarrhoea in mammals and fowls. Experimental rotavirus infection have been associated to a series of sub-cellular pathologic alterations leading to cell lysis which may represent key functions in the pathogenesis of the diarrhoeic disease. The current work describes the cytopathic changes in cultured MA-104 cells infected by a simian (SA-11) and a porcine (1154) rotavirus strains. Trypan blue exclusion staining showed increased cell permeability after infection by both strains, as demonstrated by cell viability. This effect was confirmed by the leakage of infected cells evaluated by chromium release. Nuclear fragmentation was observed by acridine orange and Wright staining but specific DNA cleavage was not detected. Ultrastructural changes, such as chromatin condensation, cytoplasm vacuolisation, and loss of intercellular contact were shown in infected cells for both strains. In situ terminal deoxynucleotidyl transferase (Tunel) assay did not show positive result. In conclusion, we demonstrated that both strains of rotavirus induced necrosis as the major degenerative effect.

Incidence of group A rotavirus in urban and rural areas of the city of Londrina-Brazil, from 1995 to 1997

Os rotavírus säo patógenos comuns e causam diarréia aguda em crianças e animais jovens. Neste trabalho avaliamos a participaçäo do vírus na diarréia de populaçäo humanas das áreas urbana e rural da cidade de Londrina, Paraná. Foram analisadas 905 amostras fecais de indivíduos com diarréia aguda, senso 686 e 219 amostras das zonas urbanas e rural, respectivamente. Trinta eoito amostras (4,2 por cento) foram consideradas positivas pelas técnicas de eletroforese em gel de poliacrilamida do RNA viral e aglutinaçäo passiva de látex, das quais 36 da área urbana e dois da área rural. Das amostras positivas, 17 foram genotipadas por RT-PCR tendo sido caracterizadas 16 cepas G1 e uma consideradas mistura dos gfenótipos G1 e G3