Identification of B-cell epitopes in the envelope glycoprotein of dengue virus type 3

Made available in DSpace on 2012-05-07T14:43:57Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) 000032.pdf: 4330648 bytes, checksum: bd80b9612f7455644b3a3466bb6b9b7e (MD5) Previous issue date: 2007Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Recife, PE, Brasil.As infecções pelo vírus dengue têm se tornado um problema crescente de Saúde Pública em regiões tropicais e subtropicais do mundo. O vírus pertence à família Flaviviridae com quatro sorotipos antigenicamente distintos (DENV-1 a DENV-4). Uma possível estratégia para evitar a patogenia associada com uma vacina para o dengue (que deve ser tetravalente), seria a construção de uma vacina quimérica composta de epítopos críticos selecionados dos quatro sorotipos. A maioria dos epítopos envolvidos na neutralização do vírus está presente na glicoproteína E do envelope, que é a maior proteína de superfície da partícula viral. O objetivo deste trabalho foi identificar epítopos de célula B na glicoproteína E do vírus dengue sorotipo 3. Para o mapeamento de epítopos imunodominantes, noventa e cinco peptídeos (15-mers cada, sobreposição de 10) foram sintetizados (Synpep, California-USA), a partir da sequência de 490 aminoácidos da glicoproteína E do envelope do DENV-3, de cepa circulante no Brasil. Estes peptídeos foram testados por ELISA contra um pool de soros de pacientes positivos e negativos para dengue, coletados durante a fase de convalescença da infecção por DENV-3. Os resultados mostraram que os soros de humanos reagiram com onze, dos noventa e cinco peptídeos testados, distribuídos em 5 regiões com aminoácidos na posições 51-65 (peptídeo 11), 71-90 (peptídeos 15 e 16), 131-170 (peptídeos 27, 28, 29, 30, 31 e 32), 196-210 (peptídeo 40) e 246-260 (peptídeo 50). A análise da curva ROC mostrou que, dentre os peptídeos identificados, nove seriam capazes de diferenciar entre pacientes com DENV-3 de pacientes não-dengue e três capazes de diferenciar a infecção por DENV-3 daquelas por outros sorotipos virais (DENV-1 e DENV-2). Os epítopos imunodominantes aqui descritos, junto com outros epítopos bem documentados, são potencialmente relevantes para o desenho de uma vacina para o vírus dengue e para o desenvolvimento de kits de diagnóstico específico

Development and evaluation of two new vaccine strategies against dengue virus

Made available in DSpace on 2015-06-01T19:28:11Z (GMT). No. of bitstreams: 2 536.pdf: 12060845 bytes, checksum: 5af1c56abad31cc9c82bc99e14652b56 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2011Fundação Oswaldo Cruz. Centro de Pesquisas Aggeu Magalhães. Recife, PE, BrasilA dengue é um problema de Saúde Pública em termos de morbidade e mortalidade, sendo reconhecida em mais de 100 países. No entanto, o desenvolvimento de uma vacina encontra sua dificuldade na imunopatogênese da doença, fazendo-se necessária a construção de uma vacina tetravalente que seja capaz de imunizar contra os quatro sorotipos do vírus, em diferentes faixas etárias, sem elicitar o efeito deletério da febre hemorrágica do dengue. Para isto, novas tecnologias têm sido utilizadas no lugar dos sistemas de atenuação e inativação viral. Diante disto, o presente trabalho teve como objetivo desenvolver duas novas estratégias vacinais contra o vírus dengue, utilizando as seguintes abordagens: a primeira consistiu no desenvolvimento de uma vacina de DNA que expressava epítopos definidos de células B e T, do DENV-3, associados ao sinal de tráfego celular da proteína de membrana do lisossomo- LAMP. A segunda consistiu na expressão in tandem dos domínios III da proteína do envelope dos quatro sorotipos, fusionados ao replicon do vírus da febre amarela 17D. Foram utilizadas técnicas padrão de clonagem e de recombinação homóloga em levedura para a construção das diferentes abordagens. A expressão da vacina de DNA e a replicação autônoma dos replicons foram confirmadas pelo ensaio de imunofluorescência indireta de células transfectadas. Os resultados para a vacina de DNA mostraram com sucesso a expressão do LAMP-1 humano fusionado aos epítopos, no entanto, a construção não foi capaz de gerar resposta imune por anticorpos neutralizantes. A vacina baseada em replicons mostrou que é possível utilizar com sucesso os replicons como vetores vacinais e que estes permitem a expressão de genes heterólogos alvos. Nossos estudos representam uma etapa inicial para o desenvolvimento de novas formulações vacinais que podem constituir um importante avanço para o desenvolvimento de vacinas de última geração para o dengu

Full-length infectious clone of a low passage dengue virus serotype 2 from Brazil

Full-length dengue virus (DENV) cDNA clones are an invaluable tool for many studies, including those on the development of attenuated or chimeric vaccines and on host-virus interactions. Furthermore, the importance of low passage DENV infectious clones should be highlighted, as these may harbour critical and unique strain-specific viral components from field-circulating isolates. The successful construction of a functional Brazilian low passage DENV serotype 2 full-length clone through homologous recombination reported here supports the use of a strategy that has been shown to be highly useful by our group for the development of flavivirus infectious clones and replicons

Detection of human polyomavirus 2 (HPyV2) in oyster samples in northern Brazil

National Council for Scientific and Technological Development (Conselho Nacional de Desenvolvimento Científico and Tecnológico - CNPQ) and the Institutional Support Program for Qualified Production (Programa de Apoio a Produção Qualificada - PAPQ/2019) of the Universidade Federal do Pará.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Virologia. Belém, PA, Brazil / Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Virologia. Belém, PA, Brazil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Virologia. Belém, PA, Brazil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Virologia. Belém, PA, Brazil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Virologia. Belém, PA, Brazil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Virologia. Belém, PA, Brazil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Virologia. Belém, PA, Brazil.Background: Human polyomavirus 2 (HPyV2 or JCPyV) is persistent in the environment due to its excretion in urine and feces; it is detected in samples of wastewater, surface water and drinking water. A lack of basic sanitation and sewage collection results in the presence of this virus in food, especially in oysters, since they are bioaccumulators and are consumed in their natural form, thus posing a risk to human health. Methods: This study investigated the frequency of HPyV2 in samples of oysters marketed in northeastern Pará State, Brazil, and optimized a real-time PCR (qPCR) protocol for the detection of an endogenous oyster control. A total of 217 oysters in 22 pools from five municipalities in the state of Pará were analyzed. Samples underwent dissection and total maceration of oyster tissue using a viral concentration technique, followed by DNA extraction with phenol-chloroform and amplification of the VP1 region for molecular detection via qPCR. Results: HPyV2 was detected in 18.2% (4/22) of the pooled samples, with frequencies of 25, 20, 20 and 16% in the municipalities of Salinópolis, Augusto Corrêa, São Caetano de Odivelas and Curuçá, respectively. Notably, the sample pool from the municipality of Bragança did not have detectable HPyV2 and this was the only sampled location with a water treatment station. In this study, Crassostrea genus-specific primers (AFL52 ribosomal RNA gene) of oyster were developed for use as an endogenous control in the qPCR analysis, which will be useful for future studies. Conclusions: The detection of HPyV2 in oyster samples commercialized in the state of Pará shows the circulation of this virus in the studied municipalities. Thus, it is necessary to implement measures for improving sewage collection and basic sanitation to avoid contamination of water and food with HPyV

Development and characterization of a packaging cell line for pseudo-infectious yellow fever virus particle generation

Abstract INTRODUCTION: Pseudo-infectious yellow fever viral particles (YFV-PIVs) have been used to study vaccines and viral packaging. Here, we report the development of a packaging cell line, which expresses the YFV prM/E proteins. METHODS: HEK293 cells were transfected with YFV prM/E and C (84 nt) genes to generate HEK293-YFV-PrM/E-opt. The cells were evaluated for their ability to express the heterologous proteins and to package the replicon repYFV-17D-LucIRES, generating YFV-PIVs. RESULTS: The expression of prM/E proteins was confirmed, and the cell line trans-packaged the replicon for recovery of a reporter for the YFV-PIVs. CONCLUSIONS: HEK293-YFV-prM/E-opt trans-packaging capacity demonstrates its possible biotechnology application

Prevalence of high risk HPV in HIV-Infected women from Belem, Para, Amazon Region of Brazil: a cross-sectional study

Federal University of Pará. Biology of Infectious and Parasitic Agents Post-Graduate Program. Belém, PA, Brazil / Federal University of Pará. Institute of Biological Sciences. Virology Laboratory. Belém, PA, Brazil.Federal University of Pará. Biology of Infectious and Parasitic Agents Post-Graduate Program. Belém, PA, Brazil / Federal University of Pará. Institute of Biological Sciences. Virology Laboratory. Belém, PA, Brazil.Federal University of Pará. Biology of Infectious and Parasitic Agents Post-Graduate Program. Belém, PA, Brazil / Federal University of Pará. Institute of Biological Sciences. Virology Laboratory. Belém, PA, Brazil.Federal University of Pará. Biology of Infectious and Parasitic Agents Post-Graduate Program. Belém, PA, Brazil / Federal University of Pará. Institute of Biological Sciences. Virology Laboratory. Belém, PA, Brazil.Federal University of Pará. Biology of Infectious and Parasitic Agents Post-Graduate Program. Belém, PA, Brazil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Laboratório de Papilomavírus. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Laboratório de Papilomavírus. Ananindeua, PA, Brasil.Federal University of Pará. Institute of Biological Sciences. Virology Laboratory. Belém, PA, Brazil.Reference Unit Specialized in Infectious and Parasitic Diseases. Belém, PA, BrazilFederal University of Pará. Biology of Infectious and Parasitic Agents Post-Graduate Program. Belém, PA, Brazil / Federal University of Pará. Institute of Biological Sciences. Virology Laboratory. Belém, PA, Brazil.Federal University of Pará. Biology of Infectious and Parasitic Agents Post-Graduate Program. Belém, PA, Brazil / Federal University of Pará. Institute of Biological Sciences. Virology Laboratory. Belém, PA, Brazil.Federal University of Pará. Biology of Infectious and Parasitic Agents Post-Graduate Program. Belém, PA, Brazil / Federal University of Pará. Institute of Biological Sciences. Virology Laboratory. Belém, PA, Brazil.Human papillomavirus (HPV) is the most common sexually transmitted infection in the world. Several studies have shown a higher prevalence of HPV infection in HIV-infected women. The aim of this study was to determine the prevalence and the genotype diversity of HPV infection in HIV-infected women. From April 2010 to December 2012 cervical specimens were collected from 169 HIV-infected women who screening for cervical cancer at Reference Unit in Belém. The detection of HPV infection was performed by nested PCR and HPV type was performed using a commercial system. The prevalence of HPV infection was 63.3%. Of the 47 genotyped samples, 40.4% was found positive for high risk-HPV 16 and 12.8% for high risk-HPV 52. HPV infection was predominant in the group of women with no incidence of cytological abnormalities and more prevalent in women of reproductive age, unmarried, low education level, and who reported use condoms during sexual intercourse. It was observed an association between HPV infection and independent variables, such as condom use, multiple sexual partners, and history of sexually transmitted diseases. High-risk types of HPV infection were prevalent in our study. Infection with multiple high-risk HPV genotypes may potentiate the development of cervical cancer in HIV-infected women

Antibody Response to the SARS-CoV-2 Spike and Nucleocapsid Proteins in Patients with Different COVID-19 Clinical Profiles

The first case of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in Brazil was diagnosed on February 26, 2020. Due to the important epidemiological impact of COVID-19, the present study aimed to analyze the specificity of IgG antibody responses to the S1, S2 and N proteins of SARS-CoV-2 in different COVID-19 clinical profiles. This study enrolled 136 individuals who were diagnosed with or without COVID-19 based on clinical findings and laboratory results and classified as asymptomatic or as having mild, moderate or severe disease. Data collection was performed through a semistructured questionnaire to obtain demographic information and main clinical manifestations. IgG antibody responses to the S1 and S2 subunits of the spike (S) protein and the nucleocapsid (N) protein were evaluated using an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions. The results showed that among the participants, 87.5% (119/136) exhibited IgG responses to the S1 subunit and 88.25% (120/136) to N. Conversely, only 14.44% of the subjects (21/136) displayed S2 subunit responses. When analyzing the IgG antibody response while considering the different proteins of the virus, patients with severe disease had significantly higher antibody responses to N and S1 than asymptomatic individuals (p ≤ 0.0001), whereas most of the participants had low antibody titers against the S2 subunit. In addition, individuals with long COVID-19 showed a greater IgG response profile than those with symptomatology of a short duration. Based on the results of this study, it is concluded that levels of IgG antibodies may be related to the clinical evolution of COVID-19, with high levels of IgG antibodies against S1 and N in severe cases and in individuals with long COVID-19

Antibody response to the SARS-CoV-2 spike and nucleocapsid proteins in patients with different COVID-19 clinical profiles

National Council for Scientific and Technological Development (CNPQ #401235/2020-3; #302935/2021-5), Fundação Amazônia de Amparo a Estudos e Pesquisa do Pará (FAPESPA #005/2020 and #006/2020) and Secretaria de Ciência, Tecnologia e Educação Superior, Profissional e Tecnológica (SECTET #09/2021)Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Virologia. Belém, PA, Brazil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Virologia. Belém, PA, Brazil / Universidade Federal do Pará. Programa de Pós-Graduação em Biologia de Agentes Infecciosos e Parasitários. Belém, PA, Brazil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Virologia. Belém, PA, Brazil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Genética de Doenças Complexas. Belém, PA, Brazil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Genética de Doenças Complexas. Belém, PA, Brazil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Genética de Doenças Complexas. Belém, PA, Brazil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Genética de Doenças Complexas. Belém, PA, Brazil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Genética de Doenças Complexas. Belém, PA, Brazil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Genética de Doenças Complexas. Belém, PA, Brazil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Genética de Doenças Complexas. Belém, PA, Brazil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Genética de Doenças Complexas. Belém, PA, Brazil.Hospital Adventista de Belém. Belém, PA, Brazil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Virologia. Belém, PA, Brazil / Universidade Federal do Pará. Programa de Pós-Graduação em Biologia de Agentes Infecciosos e Parasitários. Belém, PA, Brazil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Virologia. Belém, PA, Brazil / Universidade Federal do Pará. Programa de Pós-Graduação em Biologia de Agentes Infecciosos e Parasitários. Belém, PA, Brazil.Universidade Federal do Pará. Programa de Pós-Graduação em Biologia de Agentes Infecciosos e Parasitários. Belém, PA, Brazil / Universidade do Estado do Pará. Centro de Ciências Biológicas e da Saúde. Belém, PA, Brazil.Ministério da Saúde. Secretaria de Vigilância em Saúde e Ambiente. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Universidade Federal do Pará. Programa de Pós-Graduação em Biologia de Agentes Infecciosos e Parasitários. Belém, PA, Brazil / Ministério da Saúde. Secretaria de Vigilância em Saúde e Ambiente. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Universidade do Estado do Pará. Centro de Ciências Biológicas e da Saúde. Belém, PA, Brazil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Genética de Doenças Complexas. Belém, PA, Brazil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Virologia. Belém, PA, Brazil.Universidade Federal do Pará. Instituto de Ciências Biológicas. Laboratório de Virologia. Belém, PA, Brazil / Universidade Federal do Pará. Programa de Pós-Graduação em Biologia de Agentes Infecciosos e Parasitários. Belém, PA, Brazil.The first case of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in Brazil was diagnosed on February 26, 2020. Due to the important epidemiological impact of COVID-19, the present study aimed to analyze the specificity of IgG antibody responses to the S1, S2 and N proteins of SARS-CoV-2 in different COVID-19 clinical profiles. This study enrolled 136 individuals who were diagnosed with or without COVID-19 based on clinical findings and laboratory results and classified as asymptomatic or as having mild, moderate or severe disease. Data collection was performed through a semistructured questionnaire to obtain demographic information and main clinical manifestations. IgG antibody responses to the S1 and S2 subunits of the spike (S) protein and the nucleocapsid (N) protein were evaluated using an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions. The results showed that among the participants, 87.5% (119/136) exhibited IgG responses to the S1 subunit and 88.25% (120/136) to N. Conversely, only 14.44% of the subjects (21/136) displayed S2 subunit responses. When analyzing the IgG antibody response while considering the different proteins of the virus, patients with severe disease had significantly higher antibody responses to N and S1 than asymptomatic individuals (p ≤ 0.0001), whereas most of the participants had low antibody titers against the S2 subunit. In addition, individuals with long COVID-19 showed a greater IgG response profile than those with symptomatology of a short duration. Based on the results of this study, it is concluded that levels of IgG antibodies may be related to the clinical evolution of COVID-19, with high levels of IgG antibodies against S1 and N in severe cases and in individuals with long COVID-19