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Resumo: O Curso de Medicina da Universidade Estadual de Londrina Implementou em 1998 uma profunda mudança do currículo, adotando como estratégia a Aprendizagem Baseada em Problemas. Esta demanda o desempenho de novos e complexos papéis por parte dos docentes. Para isto, foram oferecidos cursos de capacitação ao longo dos anos de implementação. Este estudo analisa as perspectivas dos docentes que já atuaram como tutores acerca da capacitação recebida e das dificuldades e necessidades percebidas. Destacam-se como resultados a dificuldade em aplicar os instrumentos de avaliação dos alunos, na proposta formativa e participativa, vem como desafio percebido de mobilizar alunos indiferentes e lidar com conteúdos de várias disciplinas ao longo dos módulos tutoriais. Conclui-se que é necessário ampliar a capacitação num processo de educação permanente que garanta a plena implantação e a sustentabilidade do novo currículo. Abstract: In 1998 the Medical School at the State University in Londrina, Paraná, Brazil, implanted radical curricular changes, adopting Problem-Based Learning (PBL), among other innovations. The strategy has demanded the development of complex new roles for the faculty. In order to act as tutors, faculty received specific training throughout the implementation years. This study analyzes tutors´ perspectives about the training, their difficulties, and observed needs for improvement. Results show that tutors find it difficult to apply new instruments for assessing students´ learning as well as for encouraging less engaged students to participate. Dealing with contents from several different disciplines through the development of teaching units is also identified as a challenge. We conclude that it is necessary to broken faculty training in a process of continuing education that assures full implementation and sustainability of the new curriculum.

Resumo: Objetivo: Conhecer a visão dos docentes do curso de Medicina da UEL a respeito do processo de implantação e do momento atual do novo currículo em ABP. Método: Aplicação de um questionário aos 268 docentes envolvidos com o curso e avaliação das respostas pela escala Likert. Resultados: Responderam o questionário 220 docentes, 31,49% são doutores e 40,5% são mestres. Somentem31,6% dos docentes avaliaram a qualidade do currículo anterior de primeira a quarta séries como boa, mas 83,3% consideram o internato bom. Sentem-se bem informados sobre as mudanças curriculares 55,5% dos docentes, porém somente 28,9% consideram que elas foram suficientemente discutidas. Apenas 32,7% tiveram boa participação na fase de planejamento e implantação. A maioria dos docentes (63,6%) já foi tutor. A ABP é considerada um avanço em relação ao método anterior por 64,8% dos docentes, 69,8% acham que ela facilita o aprendizado dos alunos e 70,9% consideram que eles ficam mais motivados. Conclusão: Embora não tenha participado adequadamente da fase inicial de discussão e execução dos trabalhos, a maioria dos docentes considera a ABP vantajosa em relação ao método anterior e vem aumentado seu grau de inserção no processo, apontando para a consolidação das mudanças propostas. Abstract: Objective: To know the view of medical faculty in UEL regarding the process of implantation and the current status of the new PBL curriculum. Method: Application of a questionnaire to 268 teachers involved with the course and evaluation of their answers using the Likert scale. Results: 220 teachers have answered the questionnaire, 31.4% have a Doctoral degree and 40.5% have a Master´s degree. Only 31.6% of the faculty considered the previous curriculum for the 1st through 4th years as good; nevertheless, 83.3% considered is Internship good. 55% of the teachers consider themselves well informed about the curricular changes undertaken; however, only 28.9% consider those changes discusses thoroughly enough within the. Only 32.7% affirmed having had an intensive participation in the planning and implantation phase. Most teachers (63,6%) have been tutors. The PBL is considered an advance in relation to the previous method by 64,8% of the faculty; 69,8% believe that PBL fosters students´ learning process and 70.9% consider it as more motivating. Conclusion: Although most teachers have not been engaged since the earlier phases of discussion and curriculum implementation, they consider PBL advantageous in relation to the previous method and observe that the new curriculum has increased its acceptability among the academic community, in a consolidation of the proposed changes.

Azadirachta indica leaf is used by Indian population for the healing of various diseases including viral infection. Herein, we analyzed the antiherpetic (HSV-1) activity of two polysaccharides (P1 and P2) isolated from the leaf of A. indica and their chemically sulfated derivatives (P1S and P2S). The molecular weights of P1S and P2S are 41 and 11 kDa, respectively. Sulfate groups are located at positions C3 of the Araf and C6 of both Galp and Glcp residues of the most active polysaccharide (P1S). These compounds were not cytotoxic in HEp-2 cells, up to 1000 μg/mL. Both P1S and P2S exhibited antiviral activity when used simultaneously to HSV-1, with 50% inhibitory concentration/selectivity index, respectively, of 31.1 μg/mL/>51.4 and 80.5 μg/mL/>19.8. P1S showed better inhibitory effect (91.8%) compared to P1 (50%), P2 (71.1%) and P2S (70%) at 200 μg/mL. Synthesis of viral protein showed a dose–dependent response and the nucleic acid synthesis was inhibited up to 25 μg/mL, by P1 and P1S and up to 50 μg/mL, by P2 and P2S. The antiviral effect is probably due to the interference of polysaccharides at the early stages of HSV-1 replication, including adsorption. Further studies are under way to get insight into the mechanism of action of the substances.

Adenanthera pavonina is a native tree of Africa and Asia, introduced in Brazil for reforestation and wood industry. Several pharmacological activities have described scientifically, including antiviral activity. This study evaluated the antiviral effect of sulfated polysaccharide of Adenanthera pavonina (SPAp) against acyclovir (ACV)—resistant (AR-29) and sensitive (KOS) herpes simplex virus strains. The 50% cytotoxic concentration (CC50) was determined by MTT method and the 50% inhibitory concentration (IC50) was evaluated by plaque reduction assay. The in vivo SPAp antiviral activity was performed in Balb/c mice infected by skin scarification and treated with topical 0.5% (w/w) SPAp formulations. SPAp showed a CC50 of 47.81 μg/mL and the IC50 were 0.49 μg/mL (SI = 97.5) and 0.54 μg/mL (SI = 88.5) for the strains KOS and AR-29, respectively. Our results demonstrated that mice treated with SPAp presented a delay in the development and progression of skin lesions compared with the control group.

Background: Herpes simplex virus (HSV) and poliovirus (PV) are both agents of major concern in the public health system. It has been shown that Dimorphandra gardneriana galactomannans can be used as solubilizer vehicles in the manufacturing of medicine. Mangiferin is the major constituent of Mangifera indica and presents multiple medicinal and biological activities. Objective: This study assayed the effect of D. gardneriana galactomannan combined with mangiferin (DgGmM) against HSV-1 and PV-1. Methods: The DgGmM cytotoxicity was evaluated by the colorimetric MTT method and the antiviral activity by plaque reduction assay, immunofluorescence and polymerase chain reaction (PCR), in HEp-2 cells. Results: The DgGmM showed a 50% cytotoxic concentration (CC50) > 2000 µg/mL. The 50% inhibitory concentrations (IC50) for HSV-1 and PV-1 were, respectively, 287.5 µg/mL and 206.2 µg/mL, with selectivity indexes (SI) > 6.95 for the former and > 9.69 for the latter. The DgGmM time-ofaddition protocol for HSV-1 showed a maximum inhibition at 500 µg/mL, when added concomitantly to infection and at the time 1 h post-infection (pi). While for PV-1, for the same protocol, the greatest inhibition, was also observed concomitantly to infection at 500 μg/mL and at the times 4 h and 8 h pi. The inhibition was also demonstrated by the decrease of fluorescent cells and/or the inhibition of specific viral genome. Conclusion: These results suggested that the DgGmM inhibited HSV-1 and PV-1 replication, with low cytotoxicity and high selectivity and, therefore, represents a potential candidate for further studies on the control of herpes and polio infections.

The herpes simplex virus, also known as HSV, is an important human pathogen. Acyclovir (ACV) is the first-line antiviral for the treatment of HSV infections; nevertheless HSV resistance to ACV has been increasingly reported and, therefore, search for alternative drugs have been encouraged. Herein, the effect of Cucumis melo sulfated pectin (SPCm) was evaluated in the HSV-1 infection. Pectin cytotoxicity and its antiherpetic action were determined by assays of MTT and plaque reduction, respectively. The SPCm concentration that reduced the cell viability by 50% (CC50) was 1440 μg/mL, while the concentration that reduced PFU in 50% (IC50) was 6 μg/mL against ACV-sensitive (KOS) strain and 12 μg/mL for ACV-resistant (AR-29) strain. The pectin showed high selectivity index (SI) for both viral strains. Therefore, we suggest that SPCm has been effective for HSV-1, strenghten by viral protein and DNA syntheses inhibition. In conclusion, we have found that SPCm is a promising alternative compound to control HSV infection.

Poliovirus (PV),an enterovirus of the Picornaviridae family, is the agent of poliomyelitis, a devastating neurologic disease that may result in paralysis and even death.Mangiferin is the major constituent of Mangifera indica and has multiple pharmacological activities, including antiviral. This study evaluated the activity of mangiferin against poliovirus type-1 (PV-1). The compound exhibited the 50% cytotoxic concentration (CC50) > 2000 μg/mL in HEp-2 cell cultures, by the dimethylthiazolyl-diphenyltetrazolium bromide method (MTT). The 50% inhibitory concentration (IC50) was of 53.5 μg/mL, determined by the plaque reduction assay (PRA) with selectivity index (SI) > 37.4. We investigate the possible mechanism of action of mangiferin and a maximum inhibition of PV was found for virucidal and inhibition of adsorption assays, at all tested concentrations. The inhibition of viral protein synthesis was also demonstrated by immunofluorescence assay (IFA).These results suggested that mangiferin is an attractive candidate for the control of PV infection.

The herpes simplex virus (HSV) diseases represent a relevant medical and social problem due to their communicability and recurrence following latency. The green algae are rich source of polysaccharides referred to as ulvans, reported as being biologically and pharmacologically active. In this work, we analyzed the activity of seven chemically modified polysaccharides from Enteromorpha compressa (Chlorophyta, Ulvaceae), against HSV. Only the derivative named SU1F1 showed satisfactory viral inhibition activity, with a high selectivity index, and, therefore, it was submitted to analysis of the probable mechanism of action and structure. SU1F1 is a sulphated (22% w/w) heteroglycuronan with an apparent molecular mass of 34kDa. The antiviral activity was assayed by plaque reduction assay under the protocols of the time-of-addition (from 3h before infection to 16h after infection), the inhibition of virus adsorption and penetration, and the virucidal effects. SU1F1 showed a high viral activity at the time 0h. We demonstrated that its inhibitory effect was maintained until 4h post-treatment with 100% of viral inhibition at 100μg/ml. No effect was observed in additional protocols (the pre-treatment, the inhibition of adsorption and penetration and virucidal assays). Reverse Transcriptase associated PCR (RT-PCR) results were in accordance with plaque reduction assay and demonstrated the activity of SU1F1 at the initial stages of HSV replication.

Propolis is a rich source of bioactive molecules made by bees by collecting an infinite variety of secondary metabolites from plants. This study determined the chromatographic and mass spectrometry profile along with the biological activity of propolis produced by the native Brazilian bee Melipona quadrifasciata. A hydroalcoholic crude extract was prepared and partitioned in solvents of different polarities, generating aqueous, dichloromethane, ethyl acetate, butanol and insoluble fractions. The phenolic and flavonoid content was also determined in crude aqueous and methanolic extracts as well. The antioxidant activity was determined by three different methods and correlated with the phenolic and flavonoid content. Extracts and fractions were tested against the herpes simplex virus type 1, and the cytotoxicity was evaluated in fibroblast L929 cells. The chromatographic and mass spectrometry analysis revealed the presence of catechin, epicatechin, aromadendrin, naringenin, pinocembrin and p-coumaric acid. The methanolic extract, ethyl acetate fraction and insoluble fraction possess higher phenolic and flavonoid content along with better antioxidant activities. The ethyl acetate and butanol fractions, along with the FM14 and FM45 subfractions demonstrated better viral inhibition, with 50% inhibitory concentrations of 90.5, 294, 188 and 58.5 μg ml−1, respectively. Among these, the ones presenting the best selectivity index were the ethyl acetate and FM45 fractions, with a pronounced virucidal effect. The methanolic extract stood out in the antioxidant activities and showed no cytotoxicity bellow 125 μg ml−1, exhibiting the most promising biological activity. Although this propolis seems to act in the early stages of herpetic infections, it is still difficult to correlate this activity with a single constituent of this complex mixture, suggesting a synergism in the extract components. To our knowledge, this is the first study of its kind with this propolis type. Keywords: Antioxidant, Antiviral, Cytotoxicity, Herpes simplex, Propolis, Stingless bee

Adenanthera pavonina is a native tree of Africa and Asia, introduced in Brazil for reforestation and wood industry. Several pharmacological activities have described scientifically, including antiviral activity. This study evaluated the antiviral effect of sulfated polysaccharide of Adenanthera pavonina (SPAp) against acyclovir (ACV)—resistant (AR-29) and sensitive (KOS) herpes simplex virus strains. The 50% cytotoxic concentration (CC(50)) was determined by MTT method and the 50% inhibitory concentration (IC(50)) was evaluated by plaque reduction assay. The in vivo SPAp antiviral activity was performed in Balb/c mice infected by skin scarification and treated with topical 0.5% (w/w) SPAp formulations. SPAp showed a CC(50) of 47.81 μg/mL and the IC(50) were 0.49 μg/mL (SI = 97.5) and 0.54 μg/mL (SI = 88.5) for the strains KOS and AR-29, respectively. Our results demonstrated that mice treated with SPAp presented a delay in the development and progression of skin lesions compared with the control group.

Mangiferin is found in many plant species as the mango tree (Mangifera indica) with ethnopharmacological applications and scientific evidence. The emergence of resistant herpes simplex virus (HSV) strains to Acyclovir (ACV) has encouraged the search for new drugs. We investigated the in vitro and in vivo activity of mangiferin obtained from M. indica against ACV-resistant HSV-1 (AR-29) and sensitive (KOS) strains. The in vitro activity was performed under varying treatment protocols. The substance showed a CC50 > 500 μg/mL and IC50 of 2.9 μg/mL and 3.5 μg/mL, respectively, for the AR-29 and KOS strains. The in vivo activity was performed in Balb/c mice treated with 0.7% topical mangiferin formulation. This formulation inhibited most effectively the AR-29 strain, attenuated the lesions, postponed their appearance or enhanced healing, in comparison to control group. We demonstrated the potentiality of mangiferin from M. indica to control HSV replication with emphasis to ACV-resistant infection.

The herpes simplex virus (HSV) is a widespread human pathogen and for many reasons the development of anti-herpetic drugs from natural products has been encouraged. Adenanthera pavonina (Ap) is a medicinal plant widely used in Brazil, among other uses. Herein, a native Ap seed polysaccharide (PLSAp) and its chemically sulfated derivate (SPLSAp) were studied by Fourier transform IR spectra (FT-IR), gel permeation chromatography (GPC) for molar mass determination and their intrinsic viscosity [η]. Biologically, the compounds were evaluated for anti-HSV activity, in HEp2 cell cultures. The cytotoxic concentrations (CC50) and the inhibitory concentrations (IC(50)) of the polysaccharides were determined by the colorimetric assay (dimethyl-thiazolyl-diphenyltetrazolium bromide) and plaque reduction assay (PRA), respectively. The SPLSAp showed a better antiviral activity when compared to the PLSAp with a CC(50) of 500 μg/ml, the IC(50) equal to 15 μg/ml and the selectivity index (SI) of 33.3. The time-of-addition and the time-of-removal assays demonstrated the highest inhibitory activity between 8-16h after the infection. The inhibition of viral DNA and protein syntheses by SPLSAp monitored by PCR and immunofluorescence assay (IFA), respectively, has also demonstrated. These findings demonstrated that the SPLSAp inhibited HSV-1 infection in different steps of the replication and, therefore, represents a valuable compound for preclinical studies in anti-herpetic therapy.

Herpesvirus and poliovirus are responsible for important diseases in human and animal. Trichilia catigua a Brazilian native plant known as catigua has several medicinal properties among them antimicrobial for bacteria and protozoa, however, no antiviral activity has been reported yet. This study evaluated the antiviral activity of the crude extract (CE) and aqueous and ethyl acetate fractions (AF, EAF) obtained from T. catigua in the replication of the Herpes simplex virus (HSV-1), bovine herpesvirus (BoHV-1) and poliovirus (PV-1). The cytotoxicity was analyzed by MTT assay and the antiviral effect was determined by the addition of extracts (0.25 to 100.0 μg/ml), before (-2h and -1h), during (Oh) and after (1h and 2h) the viral infection, by plaque reduction assay, in HEp-2 cell culture. The virucidal activity and inhibition of viral adsorption were also evaluated. In addition, the combination index (CI) with Acyclovir (ACV - reference drug) was determined for HSV-1. CE, AF and EAF showed a low toxicity (CC(50) >400 µg/ml) and low inhibitory concentration (IC50), ranging from 2.44-34.25 µg/ml for herpesvirus and 0.67 to 1.8 µg/ml for PV-1, associated with high selectivity index. The tested compounds showed high virucidal effect and high ability to inhibit viral adsorption, for all virus. The CI demonstrated a synergic effect (CI

Adenanthera pavonina, popularly known as red-bead tree, carolina, pigeon’s eye, and dragon’s eye, is a plant traditionally used in Brazil for the treatment of several diseases. The present study aimed at evaluating the activity of sulfated polysaccharide from theAdenanthera pavonina(SPLSAp) seeds against poliovirus type 1 (PV-1) in HEp-2 cell cultures. The SPLSAppresented a cytotoxic concentration (CC50) of 500 μg/mL in HEp-2 cell cultures, evaluated by the dimethylthiazolyl-diphenyltetrazolium bromide method (MTT). The SPLSApexhibited a significant antiviral activity, with a 50% inhibitory concentration (IC50) of 1.18 µg/mL, determined by plaque reduction assay and a high selectivity index (SI) of 423. The maximum inhibition (100%) of PV replication was found when the SPLSAptreatment was concomitant with viral infection (time 0 h), at all tested concentrations. The maximal inhibition was also found when the SPLSApwas used 1 h and 2 h postinfection, albeit at 50 μg/mL and 100 μg/mL. Therefore, we demonstrated that the SPLSApinhibited PV growth. We also suggested that SPLSApinhibited PV in more than one step of the replication, as the mechanism of antiviral action. We, therefore, selected the compound as a potential candidate for further development towards the control of the infection.

Herpes simplex virus (HSV) is one of the most regular human pathogens, being a public health problem, and causal agent of several diseases. Poliovirus (PV) is an enteric virus and about 1% of infected individuals develop paralytic poliomyelitis due to viral invasion of the central nervous system and destruction of motor neurons. This work evaluated the activity of a sulfated polysaccharide of Caesalpinia ferrea (SPLCf) in HSV and PV replication. The antiviral effect of SPLCf at varying concentrations was tested by plaque assay under several protocols, such as time-of-addition, adsorption and penetration inhibition and virucidal. Syntheses of viral protein and nucleic acid were also monitored by the immunofluorescence assay and PCR. The SPLCf inhibited virus adsorption and steps after penetration, and inhibited the synthesis of viral protein. Virucidal effect was also shown and nucleic acid synthesis was concurrent with positive results. Our findings suggested that the substance with low toxicity represent a potential viral inhibitor.

Agaricus brasiliensis is an edible mushroom, traditionally used for the treatment of several diseases. In this paper, a polysaccharide (PLS) from A. brasiliensis, its carboxymethylated (CPLS) and sulfated (SPLS) derivatives, as well as, fractions (F1–F3) obtained from the PLS were investigated for their effect in the replication of herpes simplex virus and bovine herpes virus in HEp-2 cell cultures. The PLS, SPLS and F3 inhibited both virus strains similarly, in a dose-dependent curve. F1, F2 and CPLS did not show significant effect even at higher concentrations. All the compounds showed neither virucidal or viral adsorption inhibition activities nor effect when cells were treated prior to infection. Our study demonstrated that the extracts of A. brasiliensis, can be promising for future antiviral drug design and its biotechnological production is economically feasible.

Herpes simplex virus (HSV) infections are common and widespread; nevertheless, their outcome can be of unpredictable prognosis in neonates and in immunosuppressed patients. Anti-HSV therapy is effective, but the emergence of drug-resistant strains or the drug toxicity that hamper the treatment is of great concern. Vaccine has not yet shown relevant benefit; therefore, palliative prophylactic measures have been adopted to prevent diseases. This short review proposes to present concisely the history of HSV, its taxonomy, physical structure, and replication and to explore the pathogenesis of the infection, clinical manifestations, laboratory diagnosis, treatment, prophylaxis and epidemiology of the diseases.

Ethnopharmacological relevance Azadirachta indica A. Juss, popularly known as neem, has been extensively used in Ayurvedic medicine by Indian population for over 2000 years. It is used traditionally for the healing of various diseases. Natural products and their derivatives provide an excellent source for new anti-viral drugs. Aim of the study The present study aims at evaluating the activity of two polysaccharides (P1 and P2) isolated from the leaves of Azadirachta indica and their chemical sulfated derivatives (P1S and P2S) against poliovirus type 1 (PV-1). Materials and methods The cytotoxicity of the compounds was analyzed by MTT and the antiviral effect was determined by plaque reduction assay in different protocols. Results The polysaccharides did not show any cytotoxic effects on HEp-2 cells at the highest tested concentration (200 μg/ml) and exhibited significant antiviral activity with inhibitory concentrations (IC50) of 80 μg/ml, 37.5 μg/ml, 77.5 μg/ml, and 12.1 μg/ml for P1, P1S, P2 and P2S, respectively, and the selectivity indexes (SI) ranged from 18 to 131.9. The compounds demonstrated better inhibitory effect when added concomitantly with the virus infection with a dose-dependent curve inhibition. Lesser effect was observed when the compounds were added after viral infection and the least effect at pre-treatment. Conclusions We suggested that the polysaccharides obtained from Azadirachta indica act against PV-1 by inhibiting the initial stage of viral replication. Importantly, original polysaccharides showed better virucidal effect than their sulfated derivatives at all tested concentrations. This study provides a scientific basis for the past and present ethnomedical uses of this plant.

Aims: Chlorophyllin (CHLN), a synthetic derivative of chlorophyll, was assayed in the replication of poliovirus (PV-1) and bovine herpesvirus (BoHV-1) in HEp-2 cell cultures. Methods and Results: Virucidal activity of CHLN was evaluated and the time-of-addition assay was performed as follows: before the infection (−1 and −2 h), at the time of the infection (0 h) and after the infection (1 and 2 h). Plaque reduction assay (PRA) showed that CHLN inhibited BoHV-1 and PV-1 infection and the 50% inhibitory concentrations (IC50) against BoHV-1 and PV-1 infection were 8·6 and 19·8 μg ml−1, respectively. The time-of-addition study demonstrated that the CHLN was effective inhibiting viral replication in 51% and 66·5% for PV-1 and BoHV-1, respectively, at the highest concentration of 20·0 μg ml−1, when added during the infection. The directed effect of CHLN on viral strains demonstrated an inhibition of 62% and 66·4% for PV-1 and BoHV-1, respectively, by PRA. Conclusions: These results demonstrated that CHLN could be used as an antiviral suggesting directed activity on virus particles and on virus-receptor sites to BoHV. For poliovirus, CHLN also demonstrated virucide activity, moreover, showed to inhibit early steps of the replication cycle. Significance and Impact of the Study: CHLN demonstrated promising selectivity index for both virus strains; therefore, it can be used for the development of an antiviral agent.

Made available in DSpace on 2013-09-27T14:52:48Z (GMT). No. of bitstreams: 1 WOS000254531400003.pdf: 130501 bytes, checksum: 369c383ba2c72794d74caa40832d33f0 (MD5) Previous issue date: 2008-01-01 Made available in DSpace on 2013-09-30T19:24:11Z (GMT). No. of bitstreams: 1 WOS000254531400003.pdf: 130501 bytes, checksum: 369c383ba2c72794d74caa40832d33f0 (MD5) Previous issue date: 2008-01-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T15:34:30Z No. of bitstreams: 1 WOS000254531400003.pdf: 130501 bytes, checksum: 369c383ba2c72794d74caa40832d33f0 (MD5) Made available in DSpace on 2014-05-20T15:34:30Z (GMT). No. of bitstreams: 1 WOS000254531400003.pdf: 130501 bytes, checksum: 369c383ba2c72794d74caa40832d33f0 (MD5) Previous issue date: 2008-01-01 We investigated the antiviral activity of an aliphatic nitro compound (NC) isolated from Heteropteris aphrodisiaca O. Mach. (Malpighiaceae), a Brazilian medicinal ptant. The NC was tested for its antiviral activity against poliovirus type 1 (PV-1) and bovine herpes virus type 1 (BHV-1) by plaque reduction assay in cell culture. The NC showed a moderate antiviral activity against PV-1 and BHV-1 in HEp-2 celts, and the 50% inhibitory concentration (IC50) were 22.01 mu g/ml (selectivity index (SI) = 2.83) and 21.10 mu g/mi (SI = 2.95), respectivety. At the highest concentration of the drug (40 mu g/ml) a reduction of approximately 80% in plaque assay was observed for both viruses. The treatment of cells or virus prior to infection did not inhibit the replication of virus strains. (C) 2006 Elsevier GmbH. All rights reserved. Universidade Estadual de Londrina (UEL), Dept Microbiol CCB, BR-86051990 Londrina, Parana, Brazil Univ Estadual Paulista, PPG Ciencias Farmaceut, Araraquara, SP, Brazil Universidade Estadual de Maringá (UEM), Dept Farm & Farmacol, Maringa, Parana, Brazil Univ Estadual Paulista, PPG Ciencias Farmaceut, Araraquara, SP, Brazil

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% to 7.8%. The dose-response relationship was determined by simple linear regression with a coefficient of correlation (R²) equal to 96.4%. 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% a 7.8%. A relação dose-resposta foi determinada por regressão linear simples com o estabelecimento do coeficiente de correlação (R²) igual a 96.4%. O ensaio possibilitou a detecção mínima de 2.3 µg/ml de antígeno de poliovírus.

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% at the concentrations of 50 and 100 µg/mL, respectively moreover, reduction in 47% 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% o número de plaques e em cerca de 47% o número de células apresentando fluorescência específica, nas concentrações de 50 e 100 µg/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.

Crude extract (CE) and aqueous (AqF) and ethyl acetate (EtOAcF) fractions of Guazuma ulmifolia LAM., Sterculiaceae and the corresponding AqF, EtOAcF of Stryphnodendron adstringens (MART.) COVILLE, Leguminosae were tested for their antiviral activity against poliovirus 1 (P-1) and bovine herpesvirus 1 (BHV-1) in HEp-2 cultured cells. The antiviral activity was monitored by plaque assay and immunofluorescence assay (IFA) under virucidal and therapeutic protocols. The therapeutic protocol demonstrated statistically significant positive results with both plants and for both virus strains. The highest percentages of viral inhibition were found for G. ulmifolia EtOAcF which inhibited BHV-1 and P-1 replication by 100% and 99%, respectively (p

Between May and August in 2003, a total of 251 fecal samples were collected from children and adults with diarrhea (5 inpatients and 246 outpatients) at a private hospital in the city of Ponta Grossa, the state of Paraná, Brazil. Group A rotavirus was detected in 71 of 251 (28.3%) specimens: 55 (77.5%) from children under 5 years of age and 16 (22.5%) from individuals aged 6-72 years. All 71 strains exhibited a "long" RNA pattern when analyzed by PAGE. Sixty-one positive samples that yielded enough RNA were submitted to PCR genotyping. The most frequent G/P genotype combination detected was G1P[8] (86.9%; 53/61) followed by G9P[8] (3.3%; 2/61) and G12P[9] (1.6%; 1/61). Rotaviruses with G2, G3, G4, P[4], or P[6] specificity were not detected. For three strains (4.9%) bearing G1 genotype, the VP4 specificity could no be determined, and two specimens (3.3%) remained G/P non-typeable. One rotavirus strain (HC91) bearing G12P[9] genotype with a "long" electropherotype was isolated from an 11-month-old boy with diarrhea for the first time in Brazil. The cell-culture grown HC91 strain was shown to belong to serotype G12 by neutralization.

Aims: Chlorophyllin (CHLN) is a synthetic derivative of chlorophyll that possesses antimutagenic activity against several environmental contaminants. In the present study, CHLN was assayed for its capacity to prevent nuclear fragmentation (NF) in HEp-2 cells infected with poliovirus. Methods and Results: CHLN was assayed at concentrations of 0·5 and 2·5 μg ml−1, and NF was monitored using the comet assay and acridine orange staining. We demonstrated that CHLN reduced the percentage of NF in poliovirus-infected HEp-2 cells, when cells were treated with drug before infection or exposed continuously to drug. However, the highest degree of protection was achieved when the virus was exposed to CHLN before infection followed by protocol where infected cultures were continuously exposed to the drug after infection. Conclusions: It is suggested that CHLN primarily binds to the virus which inhibits cell penetration, thereby maintaining nuclear integrity. Significance and Impact of the Study: Considering that CHLN has several beneficial properties and no significant toxic effects in humans and animals, it would be an ideal candidate drug to test for antiviral activity.

Single-stranded conformation polymorphism (SSCP) analysis and heteroduplex mobility assays (HMAs) were used to identify and genotype enteric adenoviruses (EAd). The results were compared to those of restriction endonuclease assays, species-specific PCRs, and direct nucleotide sequence analyses. Of the 31 stool samples tested, 15 isolates were identified as EAd and 7 were identified as nonenteric Ad by all methods. An agreement of 100% was found between the SSCP and HMA results.

Rotaviruses are the major cause of viral diarrhea in humans and animals. Actinomycin D (Act D) is an antibiotic that intercalates DNA and therefore inhibits DNA-dependent transcription. The current study was carried out to assess the influence of Act D on the replication of simian rotavirus (SA11) in cell culture. Virus-infected MA-104 cell cultures were studied in the presence of Act D at concentrations of 1.25 and 2.5 microg/ml. Treatment of rotavirus-infected cells with 2.5 microg/ml Act D 48 h post-infection reduced the cytoplasmic metachromasia after staining with acridine orange by 25%. Viral RNA labeled with 3H-uridine in the presence of the drug was separated by polyacrylamide gel electrophoresis. Viral RNA replication was not affected by Act D, but increased 3H-uridine uptake was demonstrable by infected cells in the presence of the drug. This possibly was due to the inhibition of cellular RNA synthesis by Act D, which thus enhances incorporation of the radionuclide into the viral RNA. Act D reduced the number of infected cells presenting virus-specific fluorescence 48 h post-infection by more than 50%. These data suggest that Act D may have complexed with viral RNA and prevented newly synthesized mRNA from being translated, but may not have prevented early replication.

Arbovirus (Arthropod-borne virus) sao assim designados pelo fato de parte de seu ciclo de replicacao ocorrer nos insetos, podendo ser transmitidos aos seres humanos e outros animais pela picada de artropodes hematofagos. Dos mais de 545 especies de arbovirus conhecidos, cerca de 150 causam doencas em humanos. As arboviroses tem representado um grande desafio a saude publica, devido as mudancas climaticas e ambientais e aos desmatamentos que favorecem a amplificacao, a transmissao viral, alem da transposicao da barreira entre especies. Neste trabalho de revisao discorremos sobre as caracteristicas gerais, patogenia e epidemiologia dos arbovirus e das infeccoes resultantes, que tem emergido ou reemergido no Brasil nas ultimas decadas. Pela potencial ameaca que significam a saude humana no Brasil, os arbovirus mencionados neste trabalho merecem especial atencao no que refere ao estabelecimento/consolidacao de programas compulsorios de controle e combate eficazes das doencas em humanos e animais domesticos.

Rotaviruses (RV) are the single most important cause of severe diarrheal illness in infants and young children in both developed and developing countries worldwide. They affect mainly children less than 24 months of age. Presently, we adapted human rotavirus strains from field materials in cell culture, as well as, studied their properties by the analyses of the cytopathic effect, immunofluorescence features, plaque properties and virus genome stability. The study was carried out with 53 rotavirus-positive fecal specimens, collected in the city of Ponta Grossa, Parana, before the introduction of vaccination program in Brazil. The samples were submitted to at least 3 blind passages in MA-104 cell culture before discarded as non-cultivable. The adaptation process was monitored by the development of the cytopathic effect, immunofluorescence assay , plaque assay and genome stability by reverse transcriptase-polymerase chain reactions amplification. Fourteen human rotavirus strains were adapted to grow in cell culture. The isolation was demonstrated by the development of typical cytopathic effect. Immunofluorescence assay revealed granular inclusions with specific fluorescence mainly at the perinuclear area of the infected cells. Plaque assay allowed the demonstration of plaques with varying diameter depending upon the strains. Reverse transcriptase-polymerase chain reactions resulted in amplification and genotyping, demonstrating the stability of the genome through cell culture adaptation. DOI: http://dx.doi.org/10.17525/vrr.v19i1.97

Rotaviruses are common pathogens and the causal agents of acute diarrhea among children and young animals. The involvement of rotavirus in human diarrheal disease among population of urban and rural areas of the city of Londrina, Parana was evaluated. Nine hundred and five fecal specimens from persons with diarrhea were studied, being 686 and 219 from urban and rural areas, respectively. Thirty-eight samples (4,2%) were positive for rotavirus by polyacrylamide gel electrophoresis of viral RNA and latex agglutination test of which 36 were from urban and two from rural areas. Out of the positive specimens, 17 strains were further characterized by RT-PCR typing assay, resulting in 16 strains of G1 genotype while one sample was found to be a mixture of G1 and G3 genotypes.Os rotavírus são patógenos comuns e causam diarréia aguda em crianças e animais jovens. Neste trabalho avaliamos a participação do vírus na diarréia de populações humanas das áreas urbana e rural da cidade de Londrina, Paraná. Foram analisadas 905 amostras fecais de indivíduos com diarréia aguda, sendo 686 e 219 amostras das zonas urbana e rural, respectivamente. Trinta e oito amostras (4,2%) foram consideradas positivas pelas técnicas de eletroforese em gel de poliacrilamida do RNA viral e aglutinação 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 considerada mistura dos genótipos G1 e G3.

A citopatologia in vitro de uma cepa de rotavirus porcino adaptado em cultura de celulas foi comparada a estirpe-prototipo simia (SA-11). O efeito citopatico (ECP) produzido pelos virus foi semelhante embora a estirpe porcina tivesse apresentado algumas alteracoes diferentes, como o acentuado estreitamento do citoplasma, com grande perda do volume citoplasmatico. O virus porcino apresentou menor numero de plaques de ECP porem com diâmetro maior em relacao ao virus simio, demonstrando maior capacidade de disseminacao celula-celula, quase oito vezes mais, a julgar pelo diâmetro dos plaques de ECP. Os elementos do citoesqueleto das celulas infectadas revelaram uma reorganizacao semelhante para ambas as estirpes, nao sendo possivel observar nenhuma diferenca, embora o ECP do virus porcino tenha sido mais acentuado.

A total of 919 Escherichia coli isolates from 125 children with diarrhoea (cases) and 98 controls were assayed for adherence to HEp-2 cells. Localised adherence was found only in isolates from cases. Diffuse, aggregative (AA), chain-like adherence (CLA) and variants of the AA pattern were found in both cases and controls. The AA isolates were tested for gene sequences associated with enteroaggregative E. coli (EAEC). Only 25% of the isolates hybridised with the EAEC probe, and the aafA, astA and pet gene sequences were found in 7.9%, 44.7% and 7.9% of the isolates, respectively. The aggA gene was not found, although 7.9% were positive for aggC. The CLA isolates reacted with the EAEC probe (55.6%), and the aggC, astA and pet gene sequences were found in 66.7%, 33.3% and 11.1%, respectively. The aggR (55.6%), aspU (55.6%), shf (33.3%) and she (22.2%) genes were also found in CLA isolates.

Rotaviruses are known as major causal agents of diarrhea in humans and animals. They affect young animals in intensive rearing and cause great economic losses. This study evaluated the infectivity of porcine rotavirus maintained for 32 months at approximately 10 degrees C in the original stool specimens. Thirty stool specimens of 1-4-week-old piglets from breeding farms located in the southwest of the State of Parana were selected for this study. They were randomly chosen from stool samples positive for rotavirus RNA by polyacrylamide gel electrophoresis (PAGE) at the time of collection. The thirty stool samples maintained for 32 months were re-tested by PAGE and 11 out of 30 were still positive showing physical integrity of the eleven segments of viral RNA. In order to demonstrate the maintenance of viral infectivity processed fecal homogenates were inoculated in MA-104 cell cultures. After an average of three blind passages 5 out of 11 samples demonstrated cytopathic effect similar to that of a simian rotavirus (SA-11) used as positive control. To confirm these findings an immunofluorescence test was performed and typical cytoplasmatic granular fluorescence was observed. Electron microscopy of stool samples showed that most of the virus particles were single-shelled and some were found to be in advanced state of degradation. The viral nucleic acid extracted from six fecal specimens out of those that showed physical integrity of rotavirus RNA by PAGE were also amplified when submitted to RT-PCR demonstrating stability of viral RNA. We therefore concluded that porcine rotavirus infectivity is maintained for a long period of time in stool specimens at low temperature.

Thirty one Escherichia coli strains isolated from pigs with urinary tract infections were investigated for presence of virulence factors and plasmid DNA profile. The most frequent virulence factors presented by these strains were mannose-resistant fimbriae, including P. fimbriae (54.8%) and aerobactin production (45.2%). The pap) operon, detected by PCR, was found in 54.8% of the strains, which is similar to its frequency in human strains. Other characteristics such as the presence of mannose-sensitive hemagglutinin (16.1%), indicative of type 1 pili, and production of hemolysin (25.8%), colicin (38.7%) and toxins (22.6% for LT and for VT) were less frequent. No strains were positive for STa production. Plasmid profiles were variable among isolates from either the same or different farms.

The fourth gene of a porcine (S8) and eight human rotavirus isolates possessing the major human VP4 specificity (P1A serotype and/or P[8] genotype) were partially sequenced and compared to other available P[8] sequences from rotaviruses types G1, G3, G5 and G9 specificities which had been originally recovered from children with diarrhea in Japan, Brazil and the USA. Brazilian rotavirus S8 represented the single known porcine rotavirus with this P specificity. Phylogenetic analysis revealed two lineages or subgenotypes within P[8] strains: the F45-like P subgenotype comprised most of the strains, including all the human G5 isolates analyzed, whereas the Wa- or S8-like subgenotype consisted of only a human isolate obtained in the same geographic region as S8 and an American strain with atypical RNA profile besides the prototypes Wa and S8 viruses. A conserved basic amino acid residue at position 131 in VP4 seemed characteristic of the F45-like P[8] subgenotype.

The viral contamination of water for human consumption is a common cause of diseases outbreaks, therefore, a matter of great relevance for public health, especially in developing countries. In most countries, legislation recommends the study of coliforms to indicate the potability of water, however, this is not correlated with the presence of virus. The present review sought to characterize the epidemiological importance of waterborne viral diseases and the limitations of laboratorial detection and elimination of these pathogens. Besides, the state of art of virus as a tool to assess the quality of the water, as well as, the use of a single virus as the indicator of environmental contamination by virus itself are presented. Reports have demonstrated that some viruses are resistant to conventional water treatment process. Changes in water treatment policy should be achieved, and, should involve viral detection and resistance to physical and chemical agents, amongst other methods. However, we have to bear in mind that the decrease of waterborne infectious diseases is greatly dependent upon the improvement of sanitation, water treatment, together with preventive measures. All those measures together could ensure appropriate conditions for the promotion of human health and the reduction of environmental impacts. DOI: http://dx.doi.org/10.17525/vrrjournal.v18i1-2.65

BACKGROUND: Herpes simplex virus (HSV) infection is an endemic disease and it is estimated that 60- 95% of the adult population are infected with symptoms that are usually self-limiting, though they can be seri- ous, extensive and prolonged in immunocompromised individuals, highlighted by the emergence of drug-resist- ant strains. The study of the wild-type HSV strains based on the cytopathogenic features and its antiviral sensi- tivity 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 per- formed 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.

Submitted by ROBERTO PAULO CORREIA DE ARAÚJO (ppgorgsistem@ufba.br) on 2017-06-20T15:18:30Z No. of bitstreams: 1 16_v.14_2.pdf: 678061 bytes, checksum: 061e409a2950096d03202bf70557f815 (MD5) Made available in DSpace on 2017-06-20T15:18:30Z (GMT). No. of bitstreams: 1 16_v.14_2.pdf: 678061 bytes, checksum: 061e409a2950096d03202bf70557f815 (MD5) Previous issue date: 2015-05 Introdução: a poliomielite é uma doença infecciosa aguda causada pelo poliovírus, afeta o sistema nervoso central e leva à destruição de neurônios motores, resultando em paralisia flácida. O vírus pertence à família Picornaviridae e é constituído por RNA de fita simples de polaridade positiva e capsídeo icosaédrico. Objetivo: esta revisão propõe contextualizar as informações atuais da doença, do agente etiológico, vacinas e suas intercorrências. Metodologia: foram selecionados artigos de pesquisa originais e de revisão disponíveis nas bases de dados PubMed, Portal Periódicos CAPES e ScienceDirect, utilizando os seguintes descritores: erradicação, poliomielite, poliovírus e vacinas. Resultados: as consultas realizadas nos bancos de dados resultaram em 120 artigos, dos quais 20 foram utilizados para escrita dessa revisão. Conclusão: a doença está sob controle na maior parte do mundo, devido ao uso da vacina a partir de 1955 e a consolidação das medidas imunoprofiláticas, através do programa de imunização massiva iniciada na década de setenta. Entretanto, a ameaça de sua re-emergência persiste. Apesar dos intensos esforços para a erradicação da doença, prevista pela OMS para o ano de 2000, a poliomielite permanece endêmica na Nigéria, Paquistão e Afeganistão. Até que a erradicação do poliovírus seja consolidada, as demais regiões do mundo continuam em risco de importação da doença. Além disso, a poliomielite paralítica associada às cepas derivadas dos vírus vacinais (VDPV) impõe um novo e complexo desafio à erradicação da doença. Salvador

Background Lentinula edodes, known as shiitake, has been utilized as food, as well as, in popular medicine, moreover, compounds isolated from its mycelium and fruiting body have shown several therapeutic properties. The aim of this study was to determine the antiviral activity of aqueous (AqE) and ethanol (EtOHE) extracts and polysaccharide (LeP) from Lentinula edodes in the replication of poliovirus type 1 (PV-1) and bovine herpes virus type 1 (BoHV-1). Methods The time-of-addition assay was performed at the times -2, -1, 0, 1 and 2 h of the infection. The virucidal activity and the inhibition of viral adsorption were also evaluated. Plaque assay was used to monitor antiviral activity throughout. Results The AqE and LeP were more effective when added at 0 h of infection, however, EtOHE was more effective at the times 1 h and 2 h of the infection. AqE, EtOHE and LeP showed low virucidal activity, and the inhibition of viral adsorption was not significant. Conclusions The results allowed us to conclude that AqE, EtOHE and LeP act on the initial processes of the replication of both strains of virus.