Desenvolvimento de um controle externo positivo do tipo viral - like - particle para utilização em um teste diagnóstico de arboviroses emergentes

Orientador: Prof. Dr. Marco Aurélio KriegerCoorientadores: Drª. Rita de Cássia Pontello Rampazzo, Profª. Drª. Vanete ThomazSoccolTese (doutorado) - Universidade Federal do Paraná, Setor de Tecnologia, Programa de Pós-Graduação em Engenharia de Bioprocessos e Biotecnologia. Defesa : Curitiba, 22/06/2018Inclui referênciasResumo: Os arbovírus como dengue, Zika e chikungunya têm se proliferado principalmente em regiões tropicais e subtropicais por conta de condições climáticas, ambientais e falta de saneamento. Os sinais e sintomas das arboviroses citadas podem ser bastante semelhantes e representam um problema para o diagnóstico clínico. Assim, o diagnóstico laboratorial é imprescindível para definição das condutas terapêuticas a serem adotadas no tratamento dos pacientes suspeitos. Os testes mais usados no diagnóstico laboratorial são os sorológicos e moleculares, sendo que os testes moleculares apresentam a vantagem de serem mais sensíveis e específicos, enquanto que os sorológicos têm a desvantagem de apresentar riscos de reações cruzadas, uma vez que esses vírus tem genomas bastante semelhantes. O objetivo deste trabalho foi construir um controle externo positivo único contendo sequencias dos arbovírus dengue, Zika e chikungunya e de um transcrito humano específico para validar as reações do kit BIOMOL ZDC. A construção do controle foi feita utilizando técnicas de engenharia genética, foi desenhada uma sequência sintética única contendo fragmentos de todos os alvos do kit, e esta foi sintetizada e clonada em um sistema de expressão conhecido, contendo o promotor T7 e o fago MS2 modificado. Após a expressão e purificação foi feita a extração do RNA das partículas e a amplificação na RT- qPCR. Na análise verificou-se que todos os alvos do kit kit BIOMOL ZDC presentes no controle construído foram amplificados, validando todo o processo desde a extração, a transcrição reversa até a reação de PCR. Portanto, o controle construído atendeu aos objetivos deste projeto e desta forma o kit BIOMOL ZDC que já estava sendo usado para pesquisa no Brasil sem a presença do controle positivo, passou a ser utilizado com o módulo completo pelos mesmos laboratórios que obtiveram resultados semelhantes. Portanto, este produto (kit com o controle), desenvolvido com tecnologia nacional é biosseguro, tendo custo menor e alto valor agregado, e potencial para ser utilizado a fim de atender as necessidades da saúde pública brasileira. Palavras-chave: arbovírus, controle externo, diagnóstico molecular, RTqPCR.Abstract: The arboviruses such as dengue, Zika and chikungunya have proliferated mainly in tropical and subtropical regions due to climatic and environmental conditions and lack of sanitation. The signs and symptoms of the aforementioned arboviruses can be very similar and represent a problem for the clinical diagnosis. Thus, the laboratory diagnosis is essential for defining the therapeutic behaviors to be adopted in the treatment of suspected patients. The most commonly used tests in laboratory diagnosis are serological and molecular tests; molecular tests have the advantage of being more sensitive and specific, whereas serological tests have the disadvantage of presenting cross-reactivity risks, since these viruses have very similar genomes. The objective of this work was to construct a single positive external control containing dengue, Zika and chikungunya arbovirus sequences and a specific human transcript to validate the reactions of the BIOMOL ZDC kit. Construction of the control was done using genetic engineering techniques, a single synthetic sequence containing fragments of all kit targets was drawn and synthesized and cloned into a known expression system containing the T7 promoter and the modified MS2 phage. After the expression and purification, the RNA extraction from the particles and the amplification in the RT- qPCR were made. In the analysis it was verified that all the targets of the BIOMOL ZDC kit present in the constructed control were amplified validating the whole process from the extraction, the reverse transcription until the PCR reaction. Therefore, the constructed control met the objectives of this project and in this way the BIOMOL ZDC kit that was already being used for research in Brazil without the presence of the positive control was used with the complete module by the same laboratories that obtained similar results. Therefore, this product (kit with the control), developed with national technology is biosecure, having a lower cost and high added value, having potential to be used to meet the needs of Brazilian public health. Keywords: arbovirus, external control, molecular diagnosis, RT- qPCR

Epidemiological profile of Zika, Dengue and Chikungunya virus infections identified by medical and molecular evaluations in Rondonia, Brazil

Several arboviruses have emerged and/or re-emerged in North, Central and SouthAmerican countries. Viruses from some regions of Africa and Asia, such as the Zika and Chikungunya virus have been introduced in new continents causing major public health problems. The aim of this study was to investigate the presence of RNA from Zika, Dengue and Chikungunya viruses in symptomatic patients from Rondonia, where the epidemiological profile is still little known, by one-step real-time RT-PCR. The main clinical signs and symtoms were fever (51.2%), headache (78%), chills (6.1%), pruritus (12.2%), exanthema (20.1%), arthralgia (35.3%), myalgia (26.8%) and retro-orbital pain (19.5%). Serum from 164 symptomatic patients were collected and tested for RNA of Zika, Dengue types 1 to 4 and Chikungunya viruses, in addition to antibodies against Dengue NS1 antigen. Direct microscopy for Malaria was also performed. Only ZIKV RNA was detected in 4.3% of the patients, and in the remaining 95.7% of the patients RNA for Zika, Dengue and Chikungunya viruses were not detected. This finding is intriguing as the region has been endemic for Dengue for a long time and more recently for Chikungunya virus as well. The results indicated that medical and molecular parameters obtained were suitable to describe the first report of symptomatic Zika infections in this region. Furthermore, the low rate of detection, compared to clinical signs and symptoms as the solely diagnosis criteria, suggests that molecular assays for detection of viruses or other pathogens that cause similar symptoms should be used and the corresponding diseases could be included in the compulsory notification list

Desenvolvimento de um controle externo positivo do tipo viral - like - particle para utilização em um teste diagnóstico de arboviroses emergentes

Orientador: Prof. Dr. Marco Aurélio KriegerCoorientadores: Drª. Rita de Cássia Pontello Rampazzo, Profª. Drª. Vanete ThomazSoccolTese (doutorado) - Universidade Federal do Paraná, Setor de Tecnologia, Programa de Pós-Graduação em Engenharia de Bioprocessos e Biotecnologia. Defesa : Curitiba, 22/06/2018Inclui referênciasResumo: Os arbovírus como dengue, Zika e chikungunya têm se proliferado principalmente em regiões tropicais e subtropicais por conta de condições climáticas, ambientais e falta de saneamento. Os sinais e sintomas das arboviroses citadas podem ser bastante semelhantes e representam um problema para o diagnóstico clínico. Assim, o diagnóstico laboratorial é imprescindível para definição das condutas terapêuticas a serem adotadas no tratamento dos pacientes suspeitos. Os testes mais usados no diagnóstico laboratorial são os sorológicos e moleculares, sendo que os testes moleculares apresentam a vantagem de serem mais sensíveis e específicos, enquanto que os sorológicos têm a desvantagem de apresentar riscos de reações cruzadas, uma vez que esses vírus tem genomas bastante semelhantes. O objetivo deste trabalho foi construir um controle externo positivo único contendo sequencias dos arbovírus dengue, Zika e chikungunya e de um transcrito humano específico para validar as reações do kit BIOMOL ZDC. A construção do controle foi feita utilizando técnicas de engenharia genética, foi desenhada uma sequência sintética única contendo fragmentos de todos os alvos do kit, e esta foi sintetizada e clonada em um sistema de expressão conhecido, contendo o promotor T7 e o fago MS2 modificado. Após a expressão e purificação foi feita a extração do RNA das partículas e a amplificação na RT- qPCR. Na análise verificou-se que todos os alvos do kit kit BIOMOL ZDC presentes no controle construído foram amplificados, validando todo o processo desde a extração, a transcrição reversa até a reação de PCR. Portanto, o controle construído atendeu aos objetivos deste projeto e desta forma o kit BIOMOL ZDC que já estava sendo usado para pesquisa no Brasil sem a presença do controle positivo, passou a ser utilizado com o módulo completo pelos mesmos laboratórios que obtiveram resultados semelhantes. Portanto, este produto (kit com o controle), desenvolvido com tecnologia nacional é biosseguro, tendo custo menor e alto valor agregado, e potencial para ser utilizado a fim de atender as necessidades da saúde pública brasileira. Palavras-chave: arbovírus, controle externo, diagnóstico molecular, RTqPCR.Abstract: The arboviruses such as dengue, Zika and chikungunya have proliferated mainly in tropical and subtropical regions due to climatic and environmental conditions and lack of sanitation. The signs and symptoms of the aforementioned arboviruses can be very similar and represent a problem for the clinical diagnosis. Thus, the laboratory diagnosis is essential for defining the therapeutic behaviors to be adopted in the treatment of suspected patients. The most commonly used tests in laboratory diagnosis are serological and molecular tests; molecular tests have the advantage of being more sensitive and specific, whereas serological tests have the disadvantage of presenting cross-reactivity risks, since these viruses have very similar genomes. The objective of this work was to construct a single positive external control containing dengue, Zika and chikungunya arbovirus sequences and a specific human transcript to validate the reactions of the BIOMOL ZDC kit. Construction of the control was done using genetic engineering techniques, a single synthetic sequence containing fragments of all kit targets was drawn and synthesized and cloned into a known expression system containing the T7 promoter and the modified MS2 phage. After the expression and purification, the RNA extraction from the particles and the amplification in the RT- qPCR were made. In the analysis it was verified that all the targets of the BIOMOL ZDC kit present in the constructed control were amplified validating the whole process from the extraction, the reverse transcription until the PCR reaction. Therefore, the constructed control met the objectives of this project and in this way the BIOMOL ZDC kit that was already being used for research in Brazil without the presence of the positive control was used with the complete module by the same laboratories that obtained similar results. Therefore, this product (kit with the control), developed with national technology is biosecure, having a lower cost and high added value, having potential to be used to meet the needs of Brazilian public health. Keywords: arbovirus, external control, molecular diagnosis, RT- qPCR

External control viral-like particle construction for detection of emergent Arboviruses by real-time reverse-transcription PCR

Submitted by Manoel Barata ([email protected]) on 2019-12-04T17:17:15Z No. of bitstreams: 1 BMRI2019-25604ok.pdf: 1048630 bytes, checksum: a8f1a3ae8fad241e2707cfd63c41f3c7 (MD5)Approved for entry into archive by Manoel Barata ([email protected]) on 2020-02-12T14:23:15Z (GMT) No. of bitstreams: 1 BMRI2019-25604ok.pdf: 1048630 bytes, checksum: a8f1a3ae8fad241e2707cfd63c41f3c7 (MD5)Made available in DSpace on 2020-02-12T14:23:15Z (GMT). No. of bitstreams: 1 BMRI2019-25604ok.pdf: 1048630 bytes, checksum: a8f1a3ae8fad241e2707cfd63c41f3c7 (MD5) Previous issue date: 2019Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil / Universidade Federal do Paraná. Programa de Pós-Graduação em Engenharia de Bioprocessos e Biotecnologia. Curitiba, PR, Brasil.Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil.Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Escritório Rondônia. Porto Velho, Brasil.Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil / Universidade Federal do Paraná. Programa de Pós-Graduação em Engenharia de Bioprocessos e Biotecnologia. Curitiba, PR, Brasil / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil.Arboviruses have been emerging and reemerging worldwide, predominantly in tropical and subtropical areas. As many arbovirus infections, including dengue (DENV), Zika (ZIKV), and chikungunya (CHIKV), have similar signs and symptoms, clinical diagnosis of arbovirus infections is challenging. Therefore, reliable laboratory tests are necessary to improve the clinical management of patients with suspected arbovirus infections. Real-time reverse-transcription PCR (RT-qPCR) is among the more effective methods to distinguish these viruses. The aim of this study was to construct a unique positive external control derived from a unique plasmid using genetic engineering for specific use in RT-qPCR assays to detect Zika, dengue (1-4), and chikungunya. An external control derived from the MS2 bacteriophage was constructed using sequences from arbovirus and human genomes. Laboratories were asked to test the control in the ZDC Biomol kit, a RT-qPCR kit which is able to detect Zika, dengue serotypes 1-4, chikungunya, and an internal human control. RNA extracted from the external control was able to be amplified and detected in RT-qPCR assays for each virus detected by using the ZDC Biomol kit. The external control, samples from viral culture, and infected patient samples display similar amplification using this assay. The pET47b(+)MS2-ZDC vector is a viable expression system for the production of external control viral-like particles (MS2-ZDC). The RNA from the recombinant particles can be easily extracted and can function as a tool to validate all steps of process from the extraction to the amplification of all targets in specific reaction. Thus, the MS2-ZDC particles are laboratory-safe in order to avoid risk for operators, and the phages are effective as positive control for use in the ZDC Biomol kit amplifying all kit targets making them effective for commercial profile