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.

Respiratory syncytial virus (RSV) bronchiolitis: comparative study of RSV groups A and B infected children

The grouping characteristics of 29 respiratory syncitial virus (RSV) present in nasopharyngeal cells collectedfrom hospitalized children with bronchiolitis during the 1990RSVseason in Porto Alegre, RS, were analysed. Twenty-two were grouped as belonging to group A and 7 to group B. Cyanosis, oxigen therapy, cough, lenght of hospitalization and atelectasis were observed to be more frequently found within group B infected children. Other clinical signs and symptoms were similarly found in both groups.

Control of parasitic infections among school children in the peri-urban area of Botucatu, São Paulo, Brazil

Tide prevalence of intestinal parasitosis ivas investigated in a primaiy school located in Rubiâo Júnior, a peri-urban district of Botucatu, São Paulo slate, Brazil, in order to assess the effect of treatment and practical measures of prophylaxis in the control of parasitic infections among 7-to- 18-year-old school children of a low socio-economic status. The first series of parasitological examinations included 219 school children, ef which 123 (56.1 %) were found to be infected with one or more parasite species. Eighty- four children canying pathogenic parasites were submitted to various anti-parasitic treatment schedules. We re-evaluated 15 (89 %) students after 4 to 6 months post- chemotherapy. The results indicate that the combination of treatment with prophylactic measures has been successful in the control of parasitic infections, since reinfection rates were generally low (< 5-3 %), except for Giardia lamblia infections (18.6 %), and a marked reduction oti the prevalence rates was obsewed with a significant percentage of cure (> 73-1 %) in children infected with most parasite species. The reasons for the apparent failure in the control of infections caused by Hymenolepis nana and Strongyloides stercoralis are discussed.

Overexpressing BDNF in Neural Stem Cells using non-viral gene delivery strategies

Dissertação para obtenção do Grau de Mestre em Biotecnologia

Human parvovirus B19 infections among exanthematic diseases notified as measles

A total of 1397 sera collected from 1095 cases of exanthematic disease notified as measles in ES and RJ states during July 1992 to December 1994 were investigated. These sera were first tested for measles and rubella specific IgM. When they proved negative, they were tested for B19 specific IgM by an enzyme immunoassay. B19 infection was confirmed in 27 (2.5%) of these cases. Sera from 194 negative cases for measles and rubella IgM received from other Brazilian states were also investigated and B19 infection was confirmed for 11 of them. Sera from these 38 IgM positive cases for B19, were tested for anti-B19 IgG by an enzyme immunoassay and for B19 DNA by dot blot hybridization. Anti-B19 IgG antibodies were detected in most of the acute sera. B19 DNA was detected in the acute serum of one patient that had been splenectomized before. As the exanthem caused by human parvovirus infection may be clinically diagnosed as rubella, it could be important to diagnose B19 infection in Brazil since it is becoming prevalent as the cause of rash in countries where rubella is controlled by vaccination.

Hospital-acquired infections: a cost estimation for CLABSI in Portugal

A Work Project, presented as part of the requirements for the Award of a Masters Degree in Economics from the NOVA – School of Business and Economics

A longitudinal study on enteropathogenic infections of livestock in Trinidad

A longitudinal study was conducted on selected livestock farms to determine the prevalence of enteropathogens in diarrhoeic and non-diarrhoeic animals. The enteropathogens assayed from faecal samples and rectal swabs were bacteria (Escherichia coli, Campylobacter spp. Salmonella spp. and Yersinia enterocolitica), parasites (coccidia, gastrointestinal nematodes and Cryptosporidium spp.) and viruses (group A rotavirus and parvovirus). The prevalence of the enteropathogens in various animal species was related to age and month of the year. Generally, younger animals presented a higher prevalence of infection by enteropathogens than older animals while most infections occurred between the months of January and April.

Frequency of Mycoplasma hominis and Ureaplasma urealyticum infections in women with systemic lupus erythematosus

Ureaplasma urealyticum (UU) and Mycoplasma hominis (MH) have been detected in the urine of women with systemic lupus erythematosus (SLE). We evaluated the presence of these mycoplasma in the endocervix of women presenting SLE. A total of 40 SLE patients (mean age 40.2 years), and 51 healthy women (mean age 30.9 years), were studied. Endocervical swabs were cultured in specific liquid media for MH or UU, detected by a quantitative color assay, and considered positive at >10³ dilutions. Statistical analysis was performed using the two-tailed Fisher test. UU was detected in 52.5 % of patients and in 11.8% of controls (p= 0.000059). MH was detected in 20% of patients and 2% controls (p=0.003905). Both mycoplasmas were detected in 7.3% patients and 0% controls (p<0.000001). The results reported here corroborate the association of the mycoplasma infection and SLE. Thus, these agents may stimulate the production of autoreactive clones.

Entamoeba histolytica and Entamoeba dispar infections as detected by monoclonal antibody in an urban slum in Fortaleza, Northeastern Brazil

In this study the authors used the Elisa-based antigen detection tests that distinguish E. histolytica from E. dispar to examine the prevalence of E. histolytica infection in individuals from an urban slum in Fortaleza, Northeastern, Brazil. This test has a sensitivity and specificity that is comparable to PCR and isoenzyme analysis, which is the gold standard. Single stools samples were obtained from 735 individuals. The prevalence of E. histolytica infection was 14.9% (110/735) and 25.4%(187/735) for E. dispar-E. histolytica complex. The most affected age group for E. histolytica /E. histolytica-E. dispar infection was the 1-5 year olds but there was no remarkable decrease with age. There was no significant difference in colonization rates between males and females. The results from this survey demonstrate that E. histolytica is highly prevalent in the Community studied. Furthermore, it offers promise for the antigen detection test as a sensitive and technically simple tool for detecting E. histolytica infection in the field.

Impact of viral mmunomodulatory proteins

Dissertation presented to obtain the Ph.D degree in Biology

Trypanosoma cruzi in marsupial didelphids (Philander frenata and Didelhis marsupialis): differences in the humoral immune response in natural and experimental infections

Philander frenata and Didelphis marsupialis harbor parasitism by Trypanosoma cruzi without developing any apparent disease and on the contrary to D. marsupialis, P. frenata maintains parasitism by T. cruzi II subpopulations. Here we compared the humoral immune response of the two didelphids naturally and experimentally infected with T. cruzi II group, employing SDS-PAGE/Western blot techniques and by an Indirect immunofluorescence assay. We also studied the histopathological pattern of naturally and experimentally infected P. frenata with T. cruzi. P. frenata sera recognized more antigens than D. marsupialis, and the recognition pattern did not show any change over the course of the follow up of both didelphid species. Polypeptides of 66 and 90kDa were the most prominent antigens recognized by both species in the soluble and enriched membrane fractions. P. frenata recognized intensely also a 45kDa antigen. Our findings indicate that: 1) there were no quantitative or qualitative differences in the patent or subpatent phases in the recognition pattern of P. frenata; 2) the significant differences in the recognition pattern of parasitic antigens by P. frenata and D. marsupialis sera suggest that they probably "learned" to live in harmony with T. cruzi by different strategies; 3) although P. frenata do not display apparent disease, tissular lesions tended to be more severe than has been described in D. marsupialis; and 4) Both didelphids probably acquired infection by T. cruzi after their evolutionary divergence.

Supercomplexes of Prokaryotic Aerobic Respiratory Chains-Escherichia coli and Bacillus subtilis supramolecular assemblies

Dissertation presented to obtain the Ph.D degree in Biochemistry.