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To describe the presence of mutations and the ART resistance profile in PLHIV in Belém, Pará. Methods: This is a cross-sectional, descriptive and retrospective study, where 189 genotyping reports of individuals were included, regardless of age or sex and who were seen in the period from 2013 to 2015. Results: Most individuals had a high viral load (93%), low TCD4+ lymphocyte levels (97%) and the most prevalent HIV subtype was B (78%). We observed in this study a high presence of mutations in all classes of drugs used in ART with emphasis on M184V (ITRNN), K103N (ITRNN) and L63A/P/T/Q (PI), demonstrating that many mutations can contribute to the profile of viral resistance. Conclusion: The results presented show the importance of genotyping testing not only for choosing therapeutic and surveillance regimens, but also to contribute to patient adherence to antiretroviral treatment.

Background The forkhead box protein 3 (FOXP3) transcription factor is one of the main markers of immunological suppression in different pathological profiles, and the presence of polymorphic variants may alter the gene expression of this factor. Despite descriptions of an association between the presence of the rs2232365 polymorphism and chronic diseases, the role of the sex variant in this context has not yet been elucidated, as the FOXP3 gene is located on the human sex chromosome X. Results To contribute to this topic, 323 women and 373 men were enrolled in the study, of which 101 were diagnosed with chronic viral liver diseases (39 women and 62 men), 67 with HTLV-1 infection (44 women and 23 men), 230 with coronary artery disease (91 women and 139 men) and 298 healthy and uninfected blood donors (149 women and men). They were genotyped for the rs2232365 polymorphism. The rs2232365 polymorphism was associated with clinical and pathological aspects and biomarkers of viral infections only in men, with functional differences between different infections. Conclusions A relationship is suggested between sex and FOXP3 rs2232365 polymorphism, resulting in different biological repercussions.

A inflamação das meninges pode ser causada por diversos agentes infecciosos e sua prevalência está associada a fatores como o estado imunitário do indivíduo. Pessoas que vivem com HIV/AIDS (PVHIV) apresentam maior risco de adoecimento, podendo evoluir com complicações severas devido a gravidade desta doença. Este foi um estudo retrospectivo, descritivo e observacional em PVHIV adultos com notificação compulsória de meningite, no período de janeiro de 2015 a dezembro de 2017. Dos 130 casos analisados, 65,3% eram do sexo masculino, 96,5% eram pardos e 79,2% dos casos residiam na região metropolitana de Belém. A idade da população variou de 19 a 61 anos com média de 33,9 anos sendo 55,4% analfabetos ou tinham apenas o ensino fundamental, 13,1% apresentavam carga viral com o valor de 105 a 106 cópias/mL e 46,2% apresentaram LTCD4+ menor que 250 células/mm³. Quanto ao tipo de meningite, 42,3% dos casos eram de meningite tuberculosa (TBM), 40,8% meningite criptocócica (MC) e 16,9% era meningite por outras causas. Os sinais e sintomas mais frequentes na população estudada foram cefaleia (93,1%), febre (86,0%) e vômito (61,5%). A síndrome da imunodeficiência adquirida (SIDA) foi responsável por 26,1% das causas de óbito, seguida de meningite com 22,8% de casos. Os pacientes com MC acumularam o maior número de óbitos (46,0%). A contagem de LTCD4 e LTCD8 foi significativamente mais alta em pacientes com TBM quando comparada a pacientes com MC. A maioria dos casos de óbito apresentava contagem de linfócitos TCD4+ abaixo de 200 células/mm³ e contagem de LTCD8+ abaixo de 1.000 células/mm³. A população analisada em nosso estudo apresentou um padrão clínico e epidemiológico com predomínio de homens, com idade média de 34 anos, pardos, com baixo nível de escolaridade, pertencentes à região metropolitana de Belém e com alto grau de comprometimento imunológico. Demonstrando também que embora a maioria dos casos fosse de meningite tuberculosa o maior índice de óbitos foi associado aos casos de meningite criptocócica.

Background The HIV-1 epidemic is still considered a global public health problem, but great advances have been made in fighting it by antiretroviral therapy (ART). ART has a considerable impact on viral replication and host immunity. The production of type I interferon (IFN) is key to the innate immune response to viral infections. The STING and cGAS proteins have proven roles in the antiviral cascade. The present study aimed to evaluate the impact of ART on innate immunity, which was represented by STING and cGAS gene expression and plasma IFN-α level. Methods This cohort study evaluated a group of 33 individuals who were initially naïve to therapy and who were treated at a reference center and reassessed 12 months after starting ART. Gene expression levels and viral load were evaluated by real-time PCR, CD4+ and CD8+ T lymphocyte counts by flow cytometry, and IFN-α level by enzyme-linked immunosorbent assay. Results From before to after ART, the CD4+ T cell count and the CD4+/CD8+ ratio significantly increased (p + T cell count slightly decreased, and viral load decreased to undetectable levels in most of the group (84.85%). The expression of STING and cGAS significantly decreased (p = 0.0034 and p = 0.0001, respectively) after the use of ART, but IFN-α did not (p = 0.1558). Among the markers evaluated, the only markers that showed a correlation with each other were STING and CD4+ T at the time of the first collection. Conclusions ART provided immune recovery and viral suppression to the studied group and indirectly downregulated the STING and cGAS genes. In contrast, ART did not influence IFN-α. The expression of STING and cGAS was not correlated with the plasma level of IFN-α, which suggests that there is another pathway regulating this cytokine in addition to the STING–cGAS pathway.

Background: The forkhead box protein 3 (FOXP3) transcription factor is one of the main markers of immunological suppression in different pathological profiles, and the presence of polymorphic variants may alter the gene expression of this factor. Despite descriptions of an association between the presence of the rs2232365 polymorphism and chronic diseases, the role of the sex variant in this context has not yet been elucidated, as the FOXP3 gene is located on the human sex chromosome X. Results: To contribute to this topic, 323 women and 373 men were enrolled in the study, of which 101 were diagnosed with chronic viral liver diseases (39 women and 62 men), 67 with HTLV-1 infection (44 women and 23 men), 230 with coronary artery disease (91 women and 139 men) and 298 healthy and uninfected blood donors (149 women and men). They were genotyped for the rs2232365 polymorphism. The rs2232365 polymorphism was associated with clinical and pathological aspects and biomarkers of viral infections only in men, with functional differences between different infections. Conclusions: A relationship is suggested between sex and FOXP3 rs2232365 polymorphism, resulting in different biological repercussions.

Background: Human papillomavirus (HPV) is the most common sexually transmitted disease 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.Methods: 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 the commercial kit.Results: 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 used 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.Conclusions: 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.

National Council for Scientific and Technological Development (Conselho Nacional de Desenvolvimento Cient?fico and Tecnol?gico - CNPQ) and the Institutional Support Program for Qualified Production (Programa de Apoio a Produ??o Qualificada - PAPQ/2019) of the Universidade Federal do Par?. Universidade Federal do Par?. Instituto de Ci?ncias Biol?gicas. Laborat?rio de Virologia. Bel?m, PA, Brazil / Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil. Universidade Federal do Par?. Instituto de Ci?ncias Biol?gicas. Laborat?rio de Virologia. Bel?m, PA, Brazil. Universidade Federal do Par?. Instituto de Ci?ncias Biol?gicas. Laborat?rio de Virologia. Bel?m, PA, Brazil. Universidade Federal do Par?. Instituto de Ci?ncias Biol?gicas. Laborat?rio de Virologia. Bel?m, PA, Brazil. Universidade Federal do Par?. Instituto de Ci?ncias Biol?gicas. Laborat?rio de Virologia. Bel?m, PA, Brazil. Universidade Federal do Par?. Instituto de Ci?ncias Biol?gicas. Laborat?rio de Virologia. Bel?m, PA, Brazil. Universidade Federal do Par?. Instituto de Ci?ncias Biol?gicas. Laborat?rio de Virologia. Bel?m, 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 Par? 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 Par? 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 Salin?polis, Augusto Corr?a, S?o Caetano de Odivelas and Curu??, respectively. Notably, the sample pool from the municipality of Bragan?a 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 Par? 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 HPyV2

Yellow fever (YF) is a viral disease transmitted by mosquitoes and endemic mostly in South America and Africa with 20–50% fatality. All current licensed YF vaccines, including YF-Vax® (Sanofi-Pasteur, Lyon, France) and 17DD-YFV (Bio-Manguinhos, Rio de Janeiro, Brazil), are based on live attenuated virus produced in hens’ eggs and have been widely used. The YF vaccines are considered safe and highly effective. However, a recent increase in demand for YF vaccines and reports of rare cases of YF vaccine-associated fatal adverse events have provoked interest in developing a safer YF vaccine that can be easily scaled up to meet this increased global demand. To this point, we have engineered the YF virus envelope protein (YFE) and transiently expressed it in Nicotiana benthamiana as a stand-alone protein (YFE) or as fusion to the bacterial enzyme lichenase (YFE-LicKM). Immunogenicity and challenge studies in mice demonstrated that both YFE and YFE-LicKM elicited virus neutralizing (VN) antibodies and protected over 70% of mice from lethal challenge infection. Furthermore, these two YFE-based vaccine candidates induced VN antibody responses with high serum avidity in nonhuman primates and these VN antibody responses were further enhanced after challenge infection with the 17DD strain of YF virus. These results demonstrate partial protective efficacy in mice of YFE-based subunit vaccines expressed in N. benthamiana. However, their efficacy is inferior to that of the live attenuated 17DD vaccine, indicating that formulation development, such as incorporating a more suitable adjuvant, may be required for product development.

O presente estudo objetivou caracterizar o perfil clinico-epidemiológico de pessoas vivendo com HIV (PVHIV) residentes no estado do Pará. Foram incluídos todos os indivíduos, independente de idade ou sexo, cujas amostras, de 2013 a 2015, foram encaminhadas ao Laboratório Central do Pará (LACEN-PA) para a realização do teste de genotipagem do HIV. Os dados foram obtidos por meio da coleta de informações nos laudos do teste de genotipagem guardados no LACEN. Foram coletadas informações como sexo, idade, nível de escolaridade, raça, ano de diagnóstico, motivos relacionados a mudança de esquema terapêutico e presença de mutações. O gênero masculino foi predominante, com faixa etária entre 40 e 50 anos ou mais, pardos e com ensino fundamental. A maioria teve o diagnóstico para o HIV entre os anos 2000 e 2009, tendo a maior parte apresentado mudanças de esquema terapêutico. Tal fato se deu em grande parte pela presença de falha terapêutica e intolerância, corroborando assim com a presença de mutações. No presente estudo observamos um perfil epidemiológico de PVHIV condizente com a literatura, caracterizado pela prevalência do gênero masculino, com faixa etária entre 40 e 50 anos ou mais, pardo e escolaridade em nível fundamental. Foi possível observar também um alto índice de mudanças nos regimes terapêuticos, de falha terapêutica, além de elevadas taxas de mutações. Demonstrando assim, a importância do exame de genotipagem não só para a escolha da terapia mais adequada como também para a adoção de estratégias que busquem melhorar a adesão de PVHIV ao tratamento com antirretrovirais. Novos estudos que possam contribuir com a compreensão e identificação das mudanças dos regimes terapêuticos assim como a contribuição destes para o aparecimento de mutações e consequente falha terapêutica são necessários.

GB virus C (GBV-C) is a lymphotropic virus with a low level or non-existent replication in the liver. The interaction between HIV-1 and GBV-C apparently reduces the progression of HIV-1 infection to AIDS and improves the quality of life of HIV-1 infected individuals. A cross-sectional study was established to determine the possible effect of HIV-1/GBV-C coinfection on HIV-1 viral load and CD4+ T lymphocyte counts. Samples from 313 HIV-1 infected persons from the Virus Laboratory of the Federal University of Para as well as demographic and clinical information were obtained from medical records. This study used a nested PCR method to determine GBV-C viremia. The prevalence of HIV-1/GBV-C coinfection was 17%. There were no significant differences in the distribution according to age, sex or ethnicity between the groups. The differences in HIV-1 viral load and CD4+ T lymphocyte count between the HIV-1 and HIV-1/GBV-C groups were highly significant, indicating that coinfection results in lower viral loads and higher CD4+ T lymphocyte counts compared to HIV-1 mono-infection. The results indicate a protective effect among coinfected individuals.

Background HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is an infectious chronic-inflammatory disease, which can lead to lower limb motions. Methods The study evaluated the effects of serial Pilates exercises on the clinical and immunological profiles of patients with HAM/TSP. Eight patients with ages ranging from 39 to 70 years old (2 males and 6 females), 2 wheelchair users and 6 with compromised gait, were evaluated. The patients were submitted to 20 Pilates sessions for 10 weeks. Data were collected at 3 time points (beginning of the study, after Pilates sessions and after 10 weeks without Pilates) and consisted of evaluations of the pain level, spasticity, motor strength, balance, mobility, functional capacity, quality of life and quantification of IFN-γ, IL-10 and IL-9 cytokines levels. Results After the Pilates sessions, significant improvements in pain level, static and dynamic balance, trunk control, mobility and quality of life were observed, with simultaneous and significant reductions in the serum levels of the cytokines IFN-γ and IL-10. However, after 10 weeks without Pilates, there were significant changes in terms of increasing pain and regression of mobility, with no changes in strength, spasticity, functional capacity in any of the periods of the study. Conclusions The results suggest that Pilates may be a promising auxiliary physical therapy for patients with HAM/TSP.

Background Studies have shown that the human T-lymphotropic virus 2 (HTLV-2) is endemic in several indigenous populations of the Brazilian Amazon and molecular analyses have shown the exclusive presence of HTLV-2 subtype 2c among the indigenous groups of this geographical region. Methods The present study characterizes the prevalence of HTLV-2 infection in three new villages of the Xikrin tribe, in the Kayapo group, according to their distribution by sex and age. The study included 263 samples from individuals from the Kateté, Djujeko and Oodjã villages. Plasma samples were tested for the presence of anti-HTLV-1/2 antibodies using enzyme-linked immunosorbent assays (ELISA). Seropositive samples were confirmed using real-time PCR, nested PCR and sequencing. Results The serological and molecular results confirmed the sole presence of HTLV-2 in 77 (29%) samples, with a prevalence of 38% among women and 18% among men. In these communities, it was found that the prevalence of HTLV-2 infection increased with age. Nucleotide sequences (642 bp, 5’LTR) from eight samples were subjected to phylogenetic analysis by the neighbor-joining method to determine the viral subtype, which confirmed the presence of HTLV-2c. Conclusions The results of the present study establish the presence of HTLV-2 infection in three new villages of the Xikrin tribe and confirm the high endemicity of the infection in the Kayapo indigenous group of the Brazilian Amazon.

This study was financed in part by the Coordenac??o de Aperfeic?oamento de Pessoal de N??vel Superior - Brasil (CAPES) - Finance Code 001. Universidade Federal do Par?. Instituto de Ci?ncias Biol?gicas. Programa de P?s-gradua??o em Biologia de Agentes Infecciosos e Parasit?rios. Bel?m, PA, Brazil. Universidade Federal do Par?. Instituto de Ci?ncias Biol?gicas. Programa de P?s-gradua??o em Biologia de Agentes Infecciosos e Parasit?rios. Bel?m, PA, Brazil. Universidade Federal do Par?. Instituto de Ci?ncias Biol?gicas. Programa de P?s-gradua??o em Biologia de Agentes Infecciosos e Parasit?rios. Bel?m, PA, Brazil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Laborat?rio de Virologia. Ananindeua, PA, Brasil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Laborat?rio de Virologia. Ananindeua, PA, Brasil. Universidade Federal do Par?. Instituto de Ci?ncias Biol?gicas. Laborat?rio de Virologia. Bel?m, PA, Brazil. Universidade Federal do Par?. Instituto de Ci?ncias Biol?gicas. Laborat?rio de Virologia. Bel?m, PA, Brazil. Universidade Federal do Par?. Instituto de Ci?ncias Biol?gicas. Laborat?rio de Virologia. Bel?m, PA, Brazil. Universidade Federal do Par?. Instituto de Ci?ncias Biol?gicas. Laborat?rio de Virologia. Bel?m, PA, Brazil. Universidade Federal do Par?. Instituto de Ci?ncias Biol?gicas. Laborat?rio de Virologia. Bel?m, PA, Brazil. Universidade Federal do Par?. Instituto de Estudos Costeiros. Grupo de Estudo e Pesquisa em Popula??es Vulner?veis. Bragan?a, PA, Brazil. Universidade Federal do Par?. Instituto de Ci?ncias Biol?gicas. Laborat?rio de Virologia. Bel?m, PA, Brazil. Universidade Federal do Par?. Instituto de Ci?ncias Biol?gicas. Programa de P?s-gradua??o em Biologia de Agentes Infecciosos e Parasit?rios. Bel?m, PA, Brazil / Universidade Federal do Par?. Instituto de Ci?ncias Biol?gicas. Laborat?rio de Virologia. Bel?m, PA, Brazi Introduction - Human pegivirus 1 (HPgV-1) is a single-stranded, positive-sense RNA virus belonging to the Flaviviridae family with limited cause-effect evidence of the causation of human diseases. However, studies have shown a potential beneficial impact of HPgV-1 coinfection in HIV disease progression. Human T lymphotropic virus-1 (HTLV-1) is a retrovirus known for causing diseases, especially in muscle and white blood cells, in approximately 5% of patients. Thus, this study aimed to investigate the potential effects of an HPgV-1 infection in patients carrying HTLV-1 in the state of Par? in the North Region of Brazil. Methods - A group of HTLV-1 carriers was compared to healthy controls. Blood samples were collected, data from medical regards were collected, and a questionnaire was administered. HPgV-1 and HTLV-1 positivity was determined by quantitative polymerase chain reaction (qRT-PCR). The data were analyzed to correlate the effects of HPgV-1 coinfection in HTLV-1 carriers. Results - A total of 158 samples were included in the study: 74 HTLV-1-positive patients (46,8%) and 84 healthy controls (53,2%). The overall HPgV-1 positivity rate was 7.6% (12/158), resulting in a prevalence of 5.4% (4/74) and 9.5% (8/84) in HTLV-1 carriers and healthy controls, respectively. No significant differences were found when comparing any clinical or demographic data between groups. Conclusion - This study indicated that the prevalence of HPgV-1 infection is low in HTLV-1 carriers in Bel?m, Par?, and probably does not alter the clinical course of HTLV-1 infection, however, further studies are still needed.

The transcription factor FOXP3 is an essential marker of the development and activation of regulatory T cells (Tregs), which are cells specialized in the regulation and normal tolerance of the immune response. In the context of chronic viral liver diseases, Tregs participate in the maintenance of infections by promoting histopathological control and favor the immune escape of viral agents by suppressing the antiviral response. Single nucleotide polymorphisms (SNPs) may influence the function of FOXP3 in a number of pathological conditions. The present study sought to evaluate the influence of SNPs in the FOXP3 gene promoter region in patients with chronic viral liver diseases. Three SNPs (−3279C>A, −2383C>T, and −924A>G) were analyzed in groups of patients with chronic hepatitis C (CHC), active chronic hepatitis B (CHB-A), inactive chronic hepatitis B (CHB-I), and a healthy control group (CG) using real-time PCR. The frequencies of the polymorphic variants were compared between groups and correlated with liver histopathological characteristics and enzyme levels [i.e., alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma-glutamyl transpeptidase (GGT)] obtained via biopsy and from the clinical records of the participating patients, respectively. For the −2338C>T SNP, no significant differences were found in the frequencies of variants between groups or in the histological findings. Significant associations between the polymorphisms and the CHB-I group were not established. The −3279C>A SNP was associated with altered viral loads (log10) and GGT levels in CHC patients with advanced stages of inflammatory activity and liver fibrosis. The −924A>G SNP was associated with altered viral loads (log10) and liver enzyme levels among CHB-A patients with milder inflammation and fibrosis. However, the frequencies of the −3279C>A and −924A>G polymorphisms were not directly associated with the histopathological profiles of the analyzed patients. These polymorphic variants may influence hepatic function in patients with chronic viral liver diseases but are not directly associated with the establishment of the degree of inflammatory activity and liver fibrosis.

A terapia genica tem como principio a transferencia de material genetico para dentro das celulas de um individuo, com o intuito de corrigir genes responsaveis por caracteristicas patologicas e, assim, tratar doencas. Desse modo, a entrega eficiente e a expressao apropriada dos genes terapeuticos sao requisitos fundamentais para a eficiencia da terapia genica. Diante disso, os virus se apresentam como importantes sistemas para a transferencia de genes, sendo utilizados como uma excelente estrategia para o tratamento de doencas monogenicas e nao monogenicas. Os vetores virais baseados em retrovirus, adenovirus, virus adenoassociados, herpesvirus e poxvirus tem sido estudados para o tratamento de doencas monogenicas, incluindo a doenca granulomatosa cronica, a imunodeficiencia combinada grave ligada ao X e a deficiencia da enzima adenosina desaminase, assim como para as doencas nao monogenicas como o câncer. A presente revisao objetiva explorar os aspectos mais relevantes do uso de vetores virais na terapia genica. Embora sejam eficientes na entrega do material genetico, os vetores virais apresentam risco de infeccao, ativam a resposta imune do hospedeiro e podem apresentar um potencial oncogenico. Assim, para que a terapia genica possa ser aplicada na rotina clinica, muitas pesquisas ainda sao necessarias para o refinamento e aperfeicoamento da tecnica, incluindo o desenvolvimento dos vetores virais.

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.

Human T-lymphotropic virus 1 (HTLV-1) infection has been associated with ATL and inflammatory diseases but remains a neglected health problem. HTLV-1 associated diseases were originally described as sporadic entities, but family aggregations have been reported. Viral, genetic, immunological and behavioral factors were used to explain family clusters, but until now a clear explanation remains uncertain. In the present study we report, for the first time, a family cluster of diseased persons presenting the infection across three generations associated with FAS -670A/G polymorphism.

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.

Yellow fever (YF) is an endemic disease in some tropical areas of South America and Africa that presents lethality rate between 20 and 50%. There is no specific treatment and to control this disease a highly effective live-attenuated egg based vaccine is widely used for travelers and residents of areas where YF is endemic. However, recent reports of rare, sometimes fatal, adverse events post-vaccination have raised concerns. In order to increase safety records, alternative strategies should be considered, such as developing a new inactivated vaccine using a cell culture based technology, capable of meeting the demands in cases of epidemic. With this goal, the production of YF virus in Vero cells grown on microcarriers and its subsequent purification by chromatographic techniques was studied. In this work we investigate the capture step of the purification process of the YF virus. At first, virus stability was studied over a wide pH range, showing best results for the alkaline region. Considering this result and the pI of the envelope protein previously determined in silico, a strong anion exchanger was considered most suitable. Due to the easy scalability, simplicity to handle, absence of diffusional limitations and suitability for virus handling of membrane adsorbers, a Q membrane was evaluated. The amount of antigen adsorbed onto the membrane was investigated within the pH range for virus stability, and the best pH for virus adsorption was considered to be 8.5. Finally, studies on gradient and step elution allowed to determine the most adequate salt concentration for washing (0.15 M) and virus elution (0.30 M). Under these operating conditions, it was shown that this capture step is quite efficient, showing high product recovery (93.2 ± 30.3%) and efficient DNA clearance (0.9 ± 0.3 ng/dose).

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. Made available in DSpace on 2018-01-26T23:42:11Z (GMT). No. of bitstreams: 1 final7824.pdf: 751184 bytes, checksum: 8a69b369ac5eac46f9115317b9ab5c75 (MD5) Previous issue date: 2015-10-15

Yellow fever is an acute infectious disease caused by prototype virus of the genus Flavivirus. It is endemic in Africa and South America where it represents a serious public health problem causing epidemics of hemorrhagic fever with mortality rates ranging from 20% to 50%. There is no available antiviral therapy and vaccination is the primary method of disease control. Although the attenuated vaccines for yellow fever show safety and efficacy it became necessary to develop a new yellow fever vaccine due to the occurrence of rare serious adverse events, which include visceral and neurotropic diseases. The new inactivated vaccine should be safer and effective as the existing attenuated one. In the present study, the immunogenicity of an inactivated 17DD vaccine in C57BL/6 mice was evaluated. The yellow fever virus was produced by cultivation of Vero cells in bioreactors, inactivated with β-propiolactone, and adsorbed to aluminum hydroxide (alum). Mice were inoculated with inactivated 17DD vaccine containing alum adjuvant and followed by intracerebral challenge with 17DD virus. The results showed that animals receiving 3 doses of the inactivated vaccine (2 μg/dose) with alum adjuvant had neutralizing antibody titers above the cut-off of PRNT50 (Plaque Reduction Neutralization Test). In addition, animals immunized with inactivated vaccine showed survival rate of 100% after the challenge as well as animals immunized with commercial attenuated 17DD vaccine.

RNA replicon derived from Flavivirus genome is a valuable tool for studying viral replication independent of virion assembly and maturation, besides being a great potencial for heterologous gene expression. In this study we described the construction of subgenomic replicons of yellow fever virus by yeast-based homologous recombination technique. The plasmid containing the yellow fever 17D strain replicon (pBSC-repYFV-17D), previously characterized, was handled to heterologous expression of the green fluorescent protein (repYFV-17D-GFP) and firefly luciferase (repYFV-17D-Luc) reporter genes. Both replicons were constructed by homologous recombination between the linearized vector pBSC-repYFV-17D and the PCR product containing homologous 25 nucleotides ends incorporated into PCR primers. The genomic organization of these constructs is similar to repYFV-17D, but with insertion of the reporter gene between the remaining 63 N-terminal nucleotides of the capsid protein and 72 C-terminal nucleotides of the E protein. The replicons repYFV-17D-GFP and repYFV-17D-Luc showed efficient replication and expression of the reporter genes. The yeast-based homologous recombination technique used in this study proved to be applicable for manipulation of the yellow fever virus genome in order to construct subgenomic replicons. O replicon de RNA derivado do genoma de Flavivirus é uma ferramenta valiosa para o estudo de replicação viral independente da montagem e da maturação do virion, além de possuir um grande potencial para expressão de genes heterólogos. Neste estudo nós descrevemos a construção de replicons subgenômicos do vírus da febre amarela utilizando a técnica de recombinação homóloga em levedura. O plasmídeo contendo o replicon do vírus febre amarela cepa 17D (pBSC-repYFV-17D), caracterizado anteriormente, foi manipulado para a expressão heteróloga dos genes repórteres green fluorescent protein (repYFV-17D-GFP) e firelly luciferase (repYFV-17D-Luc). Ambos os replicons foram construídos por recombinação homóloga entre o vetor pBSC-repYFV-17D linearizado e o produto de PCR contendo 25 nucleotídeos terminais homólogos incorporados aos oligonucleotídeos iniciadores. A organização genômica dos construídos é semelhante ao repYFV-17D, com inserção de um gene repórter entre os restantes 63 nucleotídeos N-terminais da proteína do capsídeo e 72 nucleotídeos C-terminais da proteína E. Os replicons repYFV-17D-GFP e repYFV-17D-Luc mostraram uma eficiente replicação e expressão dos genes repórteres. A técnica de recombinação homóloga em levedura usada neste estudo demonstrou ser aplicável à manipulação do genoma do vírus da febre amarela para a construção de replicons subgenômicos.