Expressão e purificação da proteína E3 do vírus chikungunya (CHIKV)

RESUMO: O vírus chikungunya (CHIKV) é um vírus de RNA, com invólucro, da família Togaviridae, transmitido por mosquitos Aedes spp. Distribuído por largas regiões de África e Ásia, causa grandes epidemias de artrite grave. A semelhança de sintomas com outras doenças como a dengue e a malária e a persistência de IgM específicas, dificultam o diagnóstico da infeção por CHIKV. A deteção no sangue de E3, uma glicoproteína viral secretada, a incluir num ensaio imunoenzimático poderá melhorar o diagnóstico nos países onde as técnicas de biologia molecular são de difícil acesso. Para testar a utilidade de E3 num ensaio de diagnóstico, esta deverá ser expressa em quantidade, purificada e usada para produção de anticorpos específicos. Para expressar E3 numa forma solúvel, suscetível de ser purificada num único passo cromatográfico sem proteases, recorreu-se à estratégia da fusão com o domínio de ligação à quitina (CBD)-inteína (IMPACT™ System, NEB). A sequência codificadora de E3 foi amplificada a partir de RNA viral, clonada em pTYB21 e expressa em E. coli como uma proteína de fusão insolúvel de 64 kDa. A expressão a 12ºC induzida por IPTG 0,1 mM aumentou a solubilidade de CBD-inteína-E3. A aplicação de lisados celulares em colunas de quitina originou a retenção de CBD-inteína-E3 na matriz. Porém, a autoclivagem da inteína na coluna, induzida com reagentes tiol, foi pouco eficiente e mesmo a proteína E3 separada não eluiu da coluna. E3 foi ainda expressa em E. coli com uma cauda de seis histidinas (E3[His]6) por clonagem no vetor pET28b(+). Lisados celulares aplicados em colunas de níquel permitiram a eluição de uma proteína de 9 kDa, compatível com a massa molecular estimada para E3[His]6, ainda que com outros contaminantes proteicos. A identidade da proteína de 9 kDa será confirmada pela indução de anticorpos com esta preparação e reatividade daqueles com células infetadas com CHIKV.----------------ABSTRACT: Chikungunya virus (CHIKV) is an enveloped, positive strand RNA virus belonging to the family Togaviridae. Transmitted by Aedes spp mosquitoes, CHIKV causes large epidemics of severe arthritogenic disease in Africa and Asia and represents a serious threat in countries where vectors are present. Symptoms similarity with other diseases, e.g. dengue and malaria, along with CHIKV IgM persistence turns accurate CHIKV diagnosis a difficult task in low-income countries. Detection of E3, a small secreted viral glycoprotein, to be included in an immunoenzymatic test was envisaged as a possible improvement in CHIKV diagnosis. To test the diagnostic value of E3, recombinant E3 should be expressed and purified to generate antibodies. In order to express CHIKV E3 in a soluble form amenable to purification by a single step affinity chromatography, the chitin binding domain (CBD)-intein fusion strategy without proteases (IMPACT™ System, NEB) was employed. The E3 coding sequence was amplified from viral RNA, cloned in pTYB21 and expressed in E. coli ER2566 as an insoluble 64 kDa CBD-intein-E3 fusion protein. Solubility was partially achieved by lowering the expression temperature to 12ºC and the inducer (IPTG) concentration to 0.1 mM. Clarified cell lysate loaded onto a chitin column allowed ligation of the fusion protein but the intein-mediated cleavage efficiency was low and E3 failed to elute from the column as demonstrated by SDS-PAGE. E3 was further expressed with a six histidine tag, E3[His]6, employing the pET System (Novagen). E3[His]6 was expressed in E. coli Rosetta (30ºC, 0.4 mM IPTG) as a 9 kDa protein. Soluble cell extracts in 20-40 mM imidazole, applied onto a nickel column and eluted with 500 mM imidazole yielded a protein preparation enriched in the 9kDa protein. The 9 kDa will be used as antigen to generate antibodies that upon reaction with CHIKV infected cells will confirm its identity.

Modulation of α-synuclein aggregation and toxicity

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

Design of a tool to study the fate of Chlamydia trachomatis infected cells

Chlamydia trachomatis has a unique obligate intracellular developmental cycle that ends by the lysis of the cell and/or the extrusion of the bacteria in order to allow for re-infections. While Chlamydia trachomatis infections are often asymptomatic the diagnosis of Chlamydia trachomatis is usually late, occurring after manifestation of persistency. Investigations on the consequences of long-term infections and the molecular mechanisms behind it will reveal light to what extent bacteria can modulate host cell function and what the ultimate fate of host cells after clearance of an infection is. Such studies on the host cell fate could be greatly facilitated if the infected cells become permanently marked during and after the infection. Therefore, this project intends to develop a new genetic tool that would allow permanently labeling of Chlamydia trachomatis host cells. The plan was to generate a Chlamydia trachomatis strain that encodes a recombinant CRE recombinase, fused to a secretory effector function of the Chlamydia type 3 secretion system (T3SS). Upon translocation into the host cell, this recombinant CRE enzyme could then, owing to its site-specific recombination function, switch a reporter gene contained in the host cell genome. To this end, the reporter line carried a membrane-tagged tdTomato (mT) gene flanked by two LoxP sequences followed by a GFP gene. The translocation of the recombinant CRE recombinase into this cell line was designed to trigger the recombination of the LoxP sites whereby the cells would turn from red fluorescence to green as an irreversible label of the infected cells. Successful execution of this mechanism would allow to draw a direct link between Chlamydia trachomatis infection and the subsequent fate of the infected cell.

Functional and structural studies of a mini-ferritin protein

Dissertação para obtenção do Grau de Mestre em Bioquímica

Assessment of Notch1 ligands production - key protein targets in breast cancer

Cell-to-cell communication is required for many biological processes in development and adult life. One of the most common systems utilized by a wide range of eukaryotes is the Notch signalling pathway. Four Notch receptors and five ligands have been identified in mammals that interact via their extracellular domains leading to transcription activation. Studies have shown that the Notch ligands expression is undetectable in normal breast tissues, but moderate to high expression has been detected in breast cancer. Thus, any of the Notch1 ligands can be studied as possible therapeutic targets for breast cancer. To study Notch pathway proteins there is the need to obtain stable protein solutions. E. coli is the host of excellence for recombinant proteins for the ease of use, fast growth and high cell densities. However, the expression of mammalian proteins in such systems may overwhelm the bacterial cellular machinery, which does not possess the ability for post-translational modifications, or dedicated compartments for protein synthesis. Mammalian cells are therefore preferred, despite their technical and financial increased demands. We aim to determine the best expression and purification conditions for the different ligand protein constructs, to develop specific function-blocking antibodies using the Phage Display technology. Moreover, we propose to crystallize the Notch1 ligands alone and in complex with the phage display selected antibodies, unveiling molecular details. hJag2DE3 and hDll1DE6 proteins were purified from refolded inclusion bodies or mammalian cell culture supernatants, respectively, and purity was confirmed by SDS-PAGE (>95%). Protein produced in mammalian cells showed to be more stable, apparently with the physiological disulfide pattern, contrary to what was observed in the refolded protein. Several nano-scale crystallization experiments were set up in 96-well plates, but no positive result was obtained. We will continue to pursue for the best expression for the Notch ligand constructs in both expression systems.

Epidemiological and molecular characteristics of HIV-1 infection among female commercial sex workers, men who have sex with men and people living with AIDS in Paraguay

An HIV seroprevalence and molecular study was conducted among 935 subjects: 723 female commercial sex workers, 92 men who have sex with men and 120 HIV-positive volunteers. The reported injection drug use rates were 0.7% in female commercial sex workers and 3% in men who have sex with men. Sexually transmitted infections were reported in 265 (37%) of the female commercial sex workers and 38 (41%) of the men who have sex with men. A total of 20 (2.8%) female commercial sex workers and 12 (13%) men who have sex with men became HIV infected during the study period. A history of sexually transmitted infection increased the risk of subsequent HIV infection twofold (adjusted odds ratio of 2.5) among the female commercial sex workers, while cocaine use had an adjusted odds ratios of 6.61 among men who have sex with men. From 130 samples, and based on heteroduplex mobility assaying for the env gene, with sequencing of part of pol and/or full genomes, subtype B was the predominant subtype identified (66%); followed by subtype F (22%) and subtype C (4%). Recombinant CRF12-BF strains were identified in 6% and CRF17_BF was identified in 2%.

Genetic characterization of rabies virus isolated from bovines and equines between 2007 and 2008, in the States of São Paulo and Minas Gerais

INTRODUCTION: Rabies is an acute disease of the central nervous system and is responsible for the deaths of thousands of humans, wild animals and livestock, particularly cattle, as well as causing major economic losses. This study describes the genetic characterization of rabies virus variants that circulate in Desmodus rotundus populations and are transmitted to herbivores. METHODS: Fifty rabies virus isolates from bovines and equines in the States of São Paulo and Minas Gerais, Brazil, were genetically characterized and compared with sequences retrieved from GenBank. RESULTS: Two clusters (I and II) with mean nucleotide identities of 99.1 and 97.6% were found. The first of these contained nearly all the samples analyzed. Lineages from other Brazilian states grouped in cluster II. CONCLUSIONS: Analysis of the amino acid sequences of the N proteins revealed the existence of genetic markers that may indicate possible variations between geographic regions, although the biologically active regions are conserved within the species over space and time.

Serosurvey of hantavirus infection in humans in the border region between Brazil and Argentina

INTRODUCTION: According to reports by the Ministry of Health, in the far western region of the State of Santa Catarina, there have been no reports of hantavirus pulmonary syndrome, a zoonotic disease transmitted by feces of infected rodents. A seroepidemiological study of residents of this region, was conducted, with the aim of determining the presence of hantavirus infections. A total of 340 volunteers of both genus, from the towns of Belmonte and Paraíso, were studied. METHODS: The serum of these patients was collected and used to detect IgG antibodies against recombinant N protein of Araraquara hantavirus, by ELISA assay. The positive samples were then titrated and confirmed by immunofluorescence assay. RESULTS: This study demonstrated the presence of IgG antibodies against hantavirus N protein in 3.5% of the population. The most frequent occupation was farm worker, 81% had direct and indirect contact with rodents, 91.7% of positive cases were farm workers, indicating that the probable cause of infection occurred during barn cleaning. These antibodies are noteworthy, given that the levels of antibodies were verified in individuals whose contact with hantavirus may have occurred many years ago. CONCLUSIONS: This study shows the circulation of hantavirus in the region, a fact that until now, had not reported. All the serum reagents had contact with the pathogen, but did not develop pulmonary and cardiovascular syndrome. It is important to remain alert, because hantavirus is a serious and emerging disease of some relevance.

First report of rabies infection in bats, Molossus molossus, Molossops neglectus and Myotis riparius in the city of São Paulo, State of São Paulo, southeastern Brazil

INTRODUCTION: This paper presents the first report of rabies in three bat species, Molossus molossus, Molossops neglectus and Myotis riparius in the city of São Paulo, Brazil. METHODS: Bats were diagnosed as positive for rabies using the fluorescent antibody test and mouse inoculation test. The isolates were characterized antigenically using a panel of eight monoclonal antibodies. The samples were also genetically analyzed by partial sequencing of the portion of nucleoprotein gene between positions 1157 and 1445nt. RESULTS: Analysis of the results verified that the sample isolated from the species M. molossus presented antigenic variant 6, while the other two samples showed a different profile from that established in the panel, one not previously reported in the literature. The results of genetic analysis revealed that the M. molossus sample segregated with Lasiurus sp. isolates, M. neglectus segregated with a subgroup of Eptesicus furinalis isolates and the Myotis riparius sample segregated with Myotis sp. isolates. CONCLUSIONS: The cases reported in this paper emphasize the need for clarification of the circumstances in which cases of rabies in wildlife occur, principally in urban areas.

A retrospective serologic survey of hantavirus infections in the county of Cássia dos Coqueiros, State of São Paulo, Brazil

INTRODUCTION: In recent years, hantavirus infections producing severe diseases have obtained an increased attention from public health authorities from the countries of Eurasia to the Americas. Brazil has reported 1,300 cases of hantavirus cardiopulmonary syndrome (HCPS) from 1993 to 2010, with about 80 of them occurring in the northeast of the State of São Paulo, with 48% fatality rate. Araraquara virus was the causative agent of HCPS in the region. Considering that hantaviruses causing human disease in the Americas were unknown until 1993, we have looked for hantavirus infections in the population of Cássia dos Coqueiros county, northeast of the State of São Paulo, Brazil, before this time. This county has about 2,800 inhabitants and an economy based on agriculture, including cultivation of Brachiaria decumbens grass. The grass seeds are an important rodent attraction, facilitating transmission of hantavirus to man. Four HCPS cases were reported so far in the county. METHODS: In this study, 1,876 sera collected from 1987 to 1990 were tested for IgG to hantavirus by IgG-ELISA, using the N recombinant protein of Araraquara virus as antigen. RESULTS: Positive results were observed in 89 (4.7%) samples, which were all collected in 1987. The positivity among urban inhabitants was 5.3%, compared with 4.3% among those living in rural areas. CONCLUSIONS: Our results showed that hantavirus infections occurred in Cássia dos Coqueiros, completely unrecognized, even before hantaviruses were described in the Americas.

Serologic survey of hantavirus in a rural population from the northern State of Mato Grosso, Brazil

INTRODUCTION: Hantavirus is a genus of ribonucleic acid (RNA) viruses included in the family Bunyaviridae. Hantaviruses are rodent-borne zoonoses that, in the last 18 years, became an emergent public health problem in the Americas, causing a severe cardiopulmonary syndrome. This disease has no specific treatment and has a high case fatality. The transmission of hantavirus to man occurs by inhaling aerosols of rodent excreta. The aim of this study was to determine the prevalence of antibodies to hantavirus in the population of the rural settlement of Tupã in the county of Marcelândia, state of Mato Grosso, Brazil. METHODS: The participants of the serologic survey were visited at their homes and selected randomly among the settlement population. Blood samples of the participants were collected by venopuncture. The serum samples were tested by an IgG-ELISA using an N recombinant protein of Araraquara hantavirus as antigen, using the protocol previously established by Figueiredo et al. RESULTS: IgG antibodies to hantavirus were detected in 7 (13%) of the 54 participants. The positivity was higher among men. It was observed that there was an association of seropositivity to hantavirus within the participants born in the south of Brazil. CONCLUSIONS: The results suggest that, in this rural area, everyone is exposed to the same risk of becoming infected with hantavirus, and, therefore, there is a need to intensify surveillance activities and education of the local people to prevent this viral infection.

Prevalence and factors associated with HCV infection among elderly individuals in a southern Brazilian city

Introduction Few Latin American studies have assessed the prevalence of hepatitis C virus (HCV) infection in elderly individuals, in whom the highest rates are expected. We aimed to investigate the prevalence of and factors associated with HCV infection in elderly residents in the municipality of Tubarão, Santa Catarina. Methods This cross-sectional study included 820 individuals (aged ≥ 60 years) who were selected by simple random sampling. The presence of anti-HCV antibodies was tested by chemiluminescence, and HCV RNA detection was performed for the anti-HCV-reactive subjects. Those individuals who were anti-HCV reactive but had undetectable HCV RNA levels were tested using a third-generation recombinant immunoblot assay. The variables were compared using the chi-squared test or Fisher's exact test, and those variables with p < 0.05 were included in the logistic regression model. Results The mean patient age was 68.6 years (SD 7.0 years); 39% were men, and 92% were Caucasian. Eighteen subjects were anti-HCV positive. Among these individuals, 4 were characterized as false-positives, leaving 14 (1.7%) individuals with confirmed infections for analysis. HCV infection was associated with an age older than 65 years, households with 3 or more residents and the previous transfusion of blood products. In the logistic regression analysis, the following variables were independently associated with HCV infection: households with 3 or more residents (OR 7.9, 95% CI 1.7–35.9, p = 0.008) and previous blood transfusion (OR 6.2, 95% CI 2.1–18.6, p = 0.001). Conclusions The HCV prevalence in the elderly population in the municipality of Tubarão was higher than that found in previous studies of blood donors in the same region. Although exposure to contaminated blood products remained important, other transmission routes, such as household transmission, could play a role in HCV infection.

Indeterminate RIBA results were associated with the absence of hepatitis C virus RNA (HCV-RNA) in blood donors

Introduction: Hepatitis C virus (HCV) infection is diagnosed by the presence of antibodies and is supplemented by confirmatory testing methods, such as recombinant immunoblot assay (RIBA) and HCV-RNA detection. This study aimed to evaluate the efficacy of RIBA testing to diagnose HCV infection in blood donors positive for anti-HCV antibodies. Methods: A total of 102 subjects positive for anti-HCV determined by enzyme-linked immunosorbent assay (ELISA) at the Hematology and Hemotherapy Foundation of Bahia (HEMOBA) were later assessed with new samples using the Abbott Architect anti-HCV test (Abbott Diagnostics, Wiesbaden, Germany), the RIBA III test (Chiron RIBA HCV 3.0 SIA, Chiron Corp., Emeryville, CA, USA), the polymerase chain reaction (PCR; COBAS® AMPLICOR HCV Roche Diagnostics Corp., Indianapolis, IN, USA) and line probe assay (LiPA - Siemens, Tarrytown, NY, USA) genotyping for HCV diagnosis. Results: Of these new samples, 38.2% (39/102) were positive, 57.8% (59/102) were negative and 3.9% (4/102) were indeterminate for anti-HCV; HCV-RNA was detected in 22.5% (23/102) of the samples. RIBA results were positive in 58.1% (25/43), negative in 9.3% (4/43) and indeterminate in 32.6% (14/43) of the samples. The prevailing genotypes were 1 (78.3%, 18/23), 3 (17.4%, 4/23) and 2 (4.3%, 1/23). All 14 samples with indeterminate RIBA results had undetectable viral loads (detection limit ≤50 IU/mL). Of these samples, 71.4% (10/14) were reevaluated six months later. Eighty percent (8/10) of these samples remained indeterminate by RIBA, and 20% (2/10) were negative. Conclusions: In this study, individuals with indeterminate RIBA results had no detectable HCV-RNA.

A Saint Louis encephalitis and Rocio virus serosurvey in Brazilian horses

Introduction Arboviruses are an important public health problem in Brazil, in especially flaviviruses, including the Saint Louis encephalitis virus (SLEV) and the Rocio virus (ROCV), are especially problematic. These viruses are transmitted to humans or other vertebrates through arthropod bites and may cause diseases with clinical manifestations that range from asymptomatic infection, viral hemorrhagic fever to encephalitis. Methods A serological survey of horses from various regions of Brazil using an enzyme-linked immunosorbent assay (ELISA) with recombinant SLEV domain III peptides and ROCV E protein as antigens. Results Overall, 415 (55.1%) of the 753 horses that were screened were seropositive for flavivirus and, among them, monotypic reactions were observed to SLEV in 93 (12.3%) and to ROCV in 46 (6.1%). These results suggested that these viruses, or other closely related viruses, are infecting horses in Brazil. However, none of the studied horses presented central nervous system infection symptoms. Conclusions Our results suggest that SLEV and ROCV previously circulated among horses in northeast, west-central and southeast Brazil.

HIV-1 epidemiology and circulating subtypes in the countryside of South Brazil

INTRODUCTION: Human immunodeficiency virus type 1 (HIV-1) has spread worldwide, with several subtypes and circulating recombinant forms. Brazil has an incidence of 20.5 HIV-1/acquired immunodeficiency syndrome (AIDS) patients per 100,000 inhabitants; however, the Southernmost State of Rio Grande do Sul (RS) has more than twice the number of HIV-1-infected people (41.3/100,000 inhabitants) and a different pattern of subtype frequencies, as previously reported in studies conducted in the capital (Porto Alegre) and its metropolitan region. This study examined HIV-1/AIDS epidemiological and molecular aspects in the countryside of Rio Grande do Sul. METHODS: Socio-demographic, clinical and risk behavioral characteristics were obtained from HIV-1-positive adult patients using a structured questionnaire. HIV-1 subtypes were determined by nested-polymerase chain reaction (PCR) and sequencing of the pol and env genes. RESULTS: The study sample included 149 (55% women) patients with a mean age of 41.8 ± 11.9 years. Most (73.8%) patients had a low education level and reported heterosexual practices as the most (91.9%) probable transmission route. HIV-1 subtypes were detected in 26 patients: 18 (69.2%) infected with subtype C, six (23.1%) infected with subtype B and two (7.7%) infected with BC recombinant forms. CONCLUSIONS: These data highlight the increasing number of HIV-1 subtype C infections in the countryside of South Brazil.