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Doctoral Thesis
DOI
https://doi.org/10.11606/T.17.2005.tde-04092020-140921
Document
Author
Full name
Marcos Lazaro Moreli
Institute/School/College
Knowledge Area
Date of Defense
Published
Ribeirão Preto, 2005
Supervisor
Committee
Figueiredo, Luiz Tadeu Moraes (President)
Fonseca, Benedito Antonio Lopes da
Martinez, Roberto
Zanchin, Nilson Ivo Tonin
Zanotto, Paolo Marinho de Andrade
Title in Portuguese
Diagnóstico de Hantavírus por detecção genômica com estudo filogenético e produção de uma proteína N recombinante
Keywords in Portuguese
ELISA
Estudos filogenéticos
Expressão da proteína N
Hantavírus
IgG
RT-PCR
Seqüenciamento de N Gl G2
Abstract in Portuguese
Hantaviroses são zoonoses distribuídas mundialmente, associadas a roedores (família Muridae) e transmitidas ao homem pela inalação de partículas virais contidas nas excretas destes roedores. Os Hantavírus são causadores de duas doenças principais: a Febre Hemorrágica com Síndrome Renal, de baixa letalidade, que ocorre na Ásia e Europa e a Síndrome Pulmonar e Cardiovascular por Hantavírus (SPCVH) que ocorre nas Américas, associada a roedores da subfamília Sigmodontinae. Hantavirus são um gênero na família Bunyaviridae. São envelopados, com 80 a l10 nm, possuindo RNA de fita simples e polaridade negativa, divididos em 3 segmentos, o grande (L), o médio (M) e o pequeno (S), que codificam respectivamente a polimerase viral, as glicoproteínas de superfíce (G1 e G2) e a proteína do nucleocapsídeo (N). Vários Hantavírus tem sido identificados em países da América do sul, incluindo o Brasil, onde são conhecidas as espécies, Juquitiba (JUQ), Araraquara (ARA), Castelos dos Sonhos (CAS), Anajatuba (ANJ) ou Rio Mearim (RIME). A SPCVH é problema de saúde pública na região de Ribeirão Preto, Estado de São Paulo, onde já se notificou 37 casos desde 1998, com alta letalidade. O diagnóstico de infecções por Hantavírus no país tem sido feito por ELISA, detectando anticorpos contra a proteína N dos vírus Sin Nombre e Andes. Um método alternativo ao ELISA é a RT-PCR. Como parte deste trabalho, selecionou-se primers de alta homologia entre distintos Hantavírus, para utilização em RT-PCR. Por esta técnica, o gene N de Hantavírus foi detectado em 8 de 9 amostras séricas de pacientes com SPCVH (88,9%) e em todas as 9 amostras por uma nested-PCR (100%). Seqüenciou-se nucleotídios dos amplicons da RT-PCR e da nested-PCR evidenciando alta similaridade destas seqüências com o gene N do Hantavírus ARA (94,8% a 99,1 %). A RT-PCR e a nested-PCR mostraram-se métodos úteis ao diagnóstico de infecções por Hantavirus, prestando-se seus ... produtos a estudos filogenéticos. Também, selecionou-se primers para amplificação por RT-PCR dos genes G1 e G2. De 10 pacientes que tiveram amplicons de G1, G2 e N seqüenciados, fez-se análise filogenética. Para tanto, seqüências foram primeiramente alinhadas pelo programa ClustalW e editadas com o BioEdit e os cladogramas feitos com POY 3.1.1, por otimização direta, utilizando algoritmo de máxima-parsimônia e análise simultânea de N, Gl e G2. A edição de árvores foi feita no programa WinClada e mostrou 3 grupos distintos de vírus da América do Sul. O do vírus Andes (AND), o de outros vírus Argentinos, Pergamino (PRG) , Maciel (MAC), Lechiguanas, Hu39694 e o do vírus Araraquara (ARA), que se agrupou com as seqüências de fragmentos genômicos obtidos dos pacientes. A proximidade filogenética mostra que o Hantavirus causador de SPCVH na região de Ribeirão Preto foi o ARA. Também, seqüenciou-se todo o segmento S do ARA, e com este buscou-se expressar o gene da proteína N. Para tanto, selecionou-se vários primers e com eles, produziu-se amplicons de 'DA ORDEM DE' 500, 'DA ORDEM DE' 1400, 'DA ORDEM DE' 1500 e 'DA ORDEM DE' 1700 pb, que foram clonados e seqüenciados. O segmento S inteiro de ARA possui 1858 nucleotídios (nt) e a sua região codificadora de N, 1287 nt. Comparou-se filogeneticamente o gene de N com o de outros Hantavírus, tendo este se mostrado-se mais similar com MAC e PERG. Expressou-se o gene de N com novos primers e um amplicon com tamanho esperado foi diretamente clonado no vetor de expressão pET 200D, que transformou colônia de Escherichia coli BL21. Esta bactéria, induzida com IPTG produziu altos teores de uma proteína detectável em WesternBlot por anticorpos de soros de pacientes convalescentes de SPCVH. Avaliou-se o grau de solubilidade e purificou-se esta proteína N por coluna de afinidade Ni-NTA, quantificou-se o produto e utilizou-se o mesmo em ELISA juntamente com controle ... negativo (Escherishia coli, contendo plasmídeo sem gene de N). O ELISA foi testado em 24 amostras de soro utilizando a proteína N recombinante de ARA como antígeno, sendo que 17 destas foi detectado anticorpos IgG contra Hantavírus e pode ser utilizado rotineiramente no diagnóstico da SPCVH.
Title in English
Diagnosis of Hantavirus for genomic detection with phylogenetic study and production of recombinant N protein
Keywords in English
ELISA
Hantavirus
IgG
Phylogenetic studies
Protein N expression
RT-PCR
Sequencing N Gl G2
Abstract in English
Hantaviruses, family Bunyaviridae, have been implicated as etiologic agents for two acute diseases: The Hemorrhagic Fever with Renal Syndrome (HFRS), occurring in the Europe and Asia and Hantavirus Cardio-Pulmonary Syndrome (HCPS), occurring in the Américas. Both diseases are carried by rodent vectors and they are transmitted to humans by contact or through inhalation of aerosols contaminated with saliva, feces or urine of the infected rodent host. In the case of American hantaviruses which cause HCPS, these hosts belong to the order Rodentia, family Muridae, subfamily Sigmodontinae. The genome of these viruses consist of three single-stranded RNA segments, S, M and L, which encode the nucleocapsid (N), glycoproteins (G1 and G2), and the polymerase protein, respectively. Presently, HCPS is more common in countries of South America than in North America. In Brazil more than 540 cases of HCPS have ever been reported. Among to the total reported cases, 37 cases occurred in the Ribeirão Preto region with a case-fatality rate of 50%. This is an emerging infectious disease with profound impact on public health and therefore it demands studies involving laboratory diagnosis and genomic characterization of strains circulating of hantaviruses. Previous studies have characterized 4 different hantavirus genetic lineages in Brazil, which were named according to the site of detection: Juquitiba (JUQ), Castelo dos Sonhos (CAS), Araraquara (ARA) and Anajatuba (ANJ) or Rio Mearim (RIME). The diagnosis of hantavirus infections in Brazil is done by an enzyme-linked immunosorbent assay based on detection of Immunoglobulin G (IgG) and M (IgM) responses to non-homologous recombinant nucleocapsid (N) proteins of Sin Nombre (SN) and Andes (AND) viruses. An altemative molecular approach used to detect gene regions of hantaviruses would be the RT-PCR and nested-PCR. In this study, in order to determine the genotypes and distribution of hantavirus causing HCPS, selected primers anneling to regions of high similarity were used initially to amplify partial N regions of hantavirus in a RT-PCR. Partial sequences of the N gene of hantavirus were detected in 8 out of 9 serum samples obtained from patients with HCPS (88,9%) and in 9 samples by nested-PCR (100%). Nucleotides sequences of the N genes of hantavirus obtained from all amplicons showed high similarity (94,8% to 99,1%) when compared with Araraquara (ARA) virus partial sequences of the N gene. The RT-PCR and Nested-PCR demonstrated to be a useful tool for the molecular diagnosis of hantavirus infection also becoming possible the molecular analysis of genomic characterization of strains circulating of hantaviruses. Additionaly, for a more refined diagnostic and robust phylogenetic analysis two pairs of primers were selected in high homology regions of G1 and G2 glycoprotein genes after multiple alignment among nucleotide sequences of the M segment of hantavirus. These selected primers were used together with previous selected primers for the N gene. RT-PCR detected in 16 blood serum samples obtained from patients with HCPS the production of three amplicons (N, G1 and G2). Three amplicons of each 10 blood serum samples out of 16 were cloned, sequenced and their obtained sequences compared with different hantaviruses. For phylogenetic analysis, after multiple alignment performed with ClustalW software, the sequences obtained from blood serum samples of patients together sequences of different hantavirus were splited into short fragments with BioEdit software to construct the cladograms by POY 3.1.1 software by optimization direct method (DO), using the principie of maximum parsimony with jackknife support. Four cladograms were constructed for each N, G1 and G2 genes, including one of them by simultaneously analysis (N+ G1 + G2). To visualize the trees, WinClada software was used showing in a similar way in four trees ARA virus agrouped with patients sequences, demonstrating that HCPS cases were caused by ARA. Also, in this study we report the entire nucleotide sequence of the S segment of a Brazilian hantavirus aiming the expression of homologous recombinant antigens with N protein of a local virus as well as the phylogenetic analysis with complete S segment. This sequence was obtained by RT-PCR using the combination of new primers selected from terminal nucleotide sequence after multiple aligment of South American Hantavirus with others previously described for diagnosis based on N gene. The S segment of ARA consists 1858 nucleotide (nt) along with a coding region of 1287 nt, similar to others South American Hantavirus. For expression of N protein, new selected primers flanking the coding region of N gene were used in a RT-PCR. This amplicons was directly cloned in pET 200D expression vector and transformed in Escherichia coli, BL21 to induce the production ofthe N recombinant protein with IPTG. High levei of expression of histidine-tagged fusion N protein with a molecular mass of 52 kDa was determined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) analysis. The N protein was also recognized by sera of patients that they had had HCPS by Western Blotting assay. By chromatography on nickel-agarose column, the expressed N protein was purified and used together with a negative control (E. coli BL21 containing plasmid without N gene) in a ELISA for detection of IgG. Finally, ELISA were tested for 24 serum samples, showing reaction with 17 HCPS patients serum samples. At last, this assay can be used for studies of seroprevalence and serological diagnosis for HCPS.
 
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