Desenvolvimento de uma plataforma de microarranjo de DNA para detecção de vírus transmitidos por pequenos mamíferos e artrópodes

Human activities have being responsible for the global environmental changes, resulting in an increase number of incident of vector- and rodent-borne diseases worldwide. Rodents and arthropods-borne viruses are important globally emerging and re-emerging viruses and most of them are RNA viruses. Efficient and early diagnosis of these infections are very important to prevent their spread, to improve clinical management of the patients, as wells to protect livestock and domestic animals. Currently, available diagnostic methods such as immunoassays, polymerase chain reaction and virus isolation can detect only one or few viruses in a single assay. The DNA microarray platform has emerged as diagnostic tool suitable for high throughput screening of pathogenic agents. The aim of this study was to develop a DNA microarray platform (RoboArboVirusChip) for detecting rodent- and arthropod-borne viruses, which belong to seven families: Bunyaviridae (genera Orthobunyavirus, Nairovirus and Phlebovirus), Flaviviridae (genus Flavivirus), Togaviridae (genus Alphavirus), Reoviridae (genera Orbivirus, Seadornavirus and Coltvivirus), Rhabdoviridae (genera Vesiculovirus and Ephemerovirus), and Asfarviridae (genus Asfarvirus). Specific oligonucleotide probes of 60-mer (n=4209) targeting 412 virus species and generic probes of 25-35-mer (n=87) targeting viruses at the genus level were designed. A total of 17 reference viruses belonging to the Bunyaviridae, Flaviviridae, Rhabdoviridae and Togaviridae families were used to standardize RoboArboVirusChip. All reference viruses were specifically detected without any cross hybridization; however, the generic probes were not able to identify the viruses at the genus level. The RoboArboVirusChip was able to specifically identify four viruses contained in three different mixtures: i) virus of different families, ii) virus of the Flavivirus genus, and iii) the Dengue virus (DENV) serotypes. The four DENV serotypes were use to evaluate the sensitivity of the RoboArboVirusChip, which was able to detect a minimum of 25 RNA copies/mL of the viruses, confirming its high sensitivity. The applicability of the RoboArboVirusChip to detect viruses in clinical samples was tested with serum samples obtained from dengue suspected cases (four positive cases and 40 negative cases). DENV was detected in the four positive serum samples, while in the 40 negative serum samples, it was not detected any virus. The results obtained in this study suggest that the RoboArboVirusChip platform could be a useful tool for early diagnosis of robovirus and arbovirus infections during epidemic outbreaks, helping in the rapid implementation of disease containment strategiesAs atividades humanas têm sido responsável por mudanças ambientais globais, resultando num aumento do número de casos de doenças transmitidas por vetores e roedores em todo o mundo. Os vírus transmitidos por roedores e artrópodes são vírus emergentes e re-emergentes de importância global, sendo que a maioria deles são vírus de RNA. O diagnóstico eficiente e precoce dessas infecções são muito importantes para evitar a sua propagação, para melhorar o manejo clínico dos pacientes e também, para proteger o gado e os animais domésticos. Atualmente, os métodos de diagnóstico disponíveis, tais como os imunoensaios, a reação em cadeia da polimerase e o isolamento viral podem detectar apenas um ou poucos vírus em um único ensaio. A plataforma de microarranjo de DNA tem surgido como uma ferramenta de diagnóstico apropriada para o monitoramento em larga escala de agentes patogênicos. O objetivo deste estudo foi desenvolver uma plataforma de microarranjo de DNA (RoboArboVirusChip) para a detecção de vírus transmitidos por roedores e artrópodes, os quais pertencem a sete famílias: Bunyaviridae (gêneros Orthobunyavirus, Nairovirus e Phlebovírus), Flaviviridae (gênero Flavivirus), Togaviridae (gênero Alphavirus), Reoviridae (gênero Orbivirus, Seadornavirus e Coltvivirus), Rhabdoviridae (géneros Vesiculovirus e Ephemerovirus), e Asfarviridae (gênero Asfarvirus). Sondas oligonucleotídicas de 60-mer (n=4209) específicas contra 412 espécies virais, e sondas genéricas de 25-35-mer (n=87) para detecção de vírus a nível do gênero foram desenhados. Um total de 17 vírus de referência, pertencentes às famílias Bunyaviridae, Flaviviridae, Rhabdoviridae e Togaviridae foram utilizados para padronizar o RoboArboVirusChip. Todos os vírus de referência foram detectados especificamente sem apresentação de hibridação cruzada, porem as sondas genéricas não foram capazes de detectar os vírus a nível do gênero. O RoboArboVirusChip foi capaz de identificar especificamente quatro vírus contidos em diferentes misturas: i) vírus de diferentes famílias, ii) vírus pertencentes ao gênero a Flavivirus, e iii) os sorotipos do vírus da dengue (DENV). Os quatro sorotipos do DENV foram utilizados para determinar a sensibilidade do RoboArboVirusChip, o qual foi capaz de detectar um mínimo de 25 copias de RNA/mL. A aplicabilidade do RoboArboVirusChip para detectar vírus em amostras clínicas foi avaliada testando amostras de soro de pacientes com suspeita de dengue (quatro casos positivos e 40 casos negativos). Os resultados obtidos neste estudo sugerem que o RoboArboVirusChip poderá ser uma ferramenta útil para o diagnóstico precoce da infecção causada por robovírus e arbovírus, auxiliando na rápida implementação de estratégias de contenção das doenças causadas por esses víru

Desenvolvimento de uma plataforma de microarranjo de DNA para detecção de vírus transmitidos por pequenos mamíferos e artrópodes

Human activities have being responsible for the global environmental changes, resulting in an increase number of incident of vector- and rodent-borne diseases worldwide. Rodents and arthropods-borne viruses are important globally emerging and re-emerging viruses and most of them are RNA viruses. Efficient and early diagnosis of these infections are very important to prevent their spread, to improve clinical management of the patients, as wells to protect livestock and domestic animals. Currently, available diagnostic methods such as immunoassays, polymerase chain reaction and virus isolation can detect only one or few viruses in a single assay. The DNA microarray platform has emerged as diagnostic tool suitable for high throughput screening of pathogenic agents. The aim of this study was to develop a DNA microarray platform (RoboArboVirusChip) for detecting rodent- and arthropod-borne viruses, which belong to seven families: Bunyaviridae (genera Orthobunyavirus, Nairovirus and Phlebovirus), Flaviviridae (genus Flavivirus), Togaviridae (genus Alphavirus), Reoviridae (genera Orbivirus, Seadornavirus and Coltvivirus), Rhabdoviridae (genera Vesiculovirus and Ephemerovirus), and Asfarviridae (genus Asfarvirus). Specific oligonucleotide probes of 60-mer (n=4209) targeting 412 virus species and generic probes of 25-35-mer (n=87) targeting viruses at the genus level were designed. A total of 17 reference viruses belonging to the Bunyaviridae, Flaviviridae, Rhabdoviridae and Togaviridae families were used to standardize RoboArboVirusChip. All reference viruses were specifically detected without any cross hybridization; however, the generic probes were not able to identify the viruses at the genus level. The RoboArboVirusChip was able to specifically identify four viruses contained in three different mixtures: i) virus of different families, ii) virus of the Flavivirus genus, and iii) the Dengue virus (DENV) serotypes. The four DENV serotypes were use to evaluate the sensitivity of the RoboArboVirusChip, which was able to detect a minimum of 25 RNA copies/mL of the viruses, confirming its high sensitivity. The applicability of the RoboArboVirusChip to detect viruses in clinical samples was tested with serum samples obtained from dengue suspected cases (four positive cases and 40 negative cases). DENV was detected in the four positive serum samples, while in the 40 negative serum samples, it was not detected any virus. The results obtained in this study suggest that the RoboArboVirusChip platform could be a useful tool for early diagnosis of robovirus and arbovirus infections during epidemic outbreaks, helping in the rapid implementation of disease containment strategiesAs atividades humanas têm sido responsável por mudanças ambientais globais, resultando num aumento do número de casos de doenças transmitidas por vetores e roedores em todo o mundo. Os vírus transmitidos por roedores e artrópodes são vírus emergentes e re-emergentes de importância global, sendo que a maioria deles são vírus de RNA. O diagnóstico eficiente e precoce dessas infecções são muito importantes para evitar a sua propagação, para melhorar o manejo clínico dos pacientes e também, para proteger o gado e os animais domésticos. Atualmente, os métodos de diagnóstico disponíveis, tais como os imunoensaios, a reação em cadeia da polimerase e o isolamento viral podem detectar apenas um ou poucos vírus em um único ensaio. A plataforma de microarranjo de DNA tem surgido como uma ferramenta de diagnóstico apropriada para o monitoramento em larga escala de agentes patogênicos. O objetivo deste estudo foi desenvolver uma plataforma de microarranjo de DNA (RoboArboVirusChip) para a detecção de vírus transmitidos por roedores e artrópodes, os quais pertencem a sete famílias: Bunyaviridae (gêneros Orthobunyavirus, Nairovirus e Phlebovírus), Flaviviridae (gênero Flavivirus), Togaviridae (gênero Alphavirus), Reoviridae (gênero Orbivirus, Seadornavirus e Coltvivirus), Rhabdoviridae (géneros Vesiculovirus e Ephemerovirus), e Asfarviridae (gênero Asfarvirus). Sondas oligonucleotídicas de 60-mer (n=4209) específicas contra 412 espécies virais, e sondas genéricas de 25-35-mer (n=87) para detecção de vírus a nível do gênero foram desenhados. Um total de 17 vírus de referência, pertencentes às famílias Bunyaviridae, Flaviviridae, Rhabdoviridae e Togaviridae foram utilizados para padronizar o RoboArboVirusChip. Todos os vírus de referência foram detectados especificamente sem apresentação de hibridação cruzada, porem as sondas genéricas não foram capazes de detectar os vírus a nível do gênero. O RoboArboVirusChip foi capaz de identificar especificamente quatro vírus contidos em diferentes misturas: i) vírus de diferentes famílias, ii) vírus pertencentes ao gênero a Flavivirus, e iii) os sorotipos do vírus da dengue (DENV). Os quatro sorotipos do DENV foram utilizados para determinar a sensibilidade do RoboArboVirusChip, o qual foi capaz de detectar um mínimo de 25 copias de RNA/mL. A aplicabilidade do RoboArboVirusChip para detectar vírus em amostras clínicas foi avaliada testando amostras de soro de pacientes com suspeita de dengue (quatro casos positivos e 40 casos negativos). Os resultados obtidos neste estudo sugerem que o RoboArboVirusChip poderá ser uma ferramenta útil para o diagnóstico precoce da infecção causada por robovírus e arbovírus, auxiliando na rápida implementação de estratégias de contenção das doenças causadas por esses víru

DNA Microarray Platform for Detection and Surveillance of Viruses Transmitted by Small Mammals and Arthropods

<div><p>Viruses transmitted by small mammals and arthropods serve as global threats to humans. Most emergent and re-emergent viral agents are transmitted by these groups; therefore, the development of high-throughput screening methods for the detection and surveillance of such viruses is of great interest. In this study, we describe a DNA microarray platform that can be used for screening all viruses transmitted by small mammals and arthropods (SMAvirusChip) with nucleotide sequences that have been deposited in the GenBank. SMAvirusChip was designed with more than 15,000 oligonucleotide probes (60-mers), including viral and control probes. Two SMAvirusChip versions were designed: SMAvirusChip v1 contains 4209 viral probes for the detection of 409 viruses, while SMAvirusChip v2 contains 4943 probes for the detection of 416 viruses. SMAvirusChip was evaluated with 20 laboratory reference-strain viruses. These viruses could be specifically detected when alone in a sample or when artificially mixed within a single sample. The sensitivity of SMAvirusChip was evaluated using 10-fold serial dilutions of dengue virus (DENV). The results showed a detection limit as low as 2.6E3 RNA copies/mL. Additionally, the sensitivity was one log₁₀ lower (2.6E2 RNA copies/mL) than quantitative real-time RT-PCR and sufficient to detect viral genomes in clinical samples. The detection of DENV in serum samples of DENV-infected patients (n = 6) and in a whole blood sample spiked with DENV confirmed the applicability of SMAvirusChip for the detection of viruses in clinical samples. In addition, in a pool of mosquito samples spiked with DENV, the virus was also detectable. SMAvirusChip was able to specifically detect viruses in cell cultures, serum samples, total blood samples and a pool of mosquitoes, confirming that cellular RNA/DNA did not interfere with the assay. Therefore, SMAvirusChip may represent an innovative surveillance method for the rapid identification of viruses transmitted by small mammals and arthropods.</p></div

Results of statistical analyses of reference viruses and control samples tested with the SMAvirusChip platform.

<p>Results of statistical analyses of reference viruses and control samples tested with the SMAvirusChip platform.</p

Detection of DENV-2 spiked in a whole blood sample and a pool of mosquitoes with SMAvirusChip v1.

<p>Detection of DENV-2 spiked in a whole blood sample and a pool of mosquitoes with SMAvirusChip v1.</p

Laboratory viruses used in this study.

<p>Laboratory viruses used in this study.</p

Screening of febrile patients with suspected malaria from the Brazilian Amazon for virus infection

Sao Paulo Research Foundation (FAPESP), Sao Paulo, Brazil, Grant no. 2017/09194-3 and no. 2015/04882-3. Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES)— Finance Code 001. Brazilian National Council for Scientific and Technological Development—Brazil (CNPq)—PQ fellowship (Grant no. 306471/2017-5).University of Sao Paulo. School of Pharmaceutical Sciences of Ribeirao Preto. Department of Clinical Analyses, Toxicology and Food Sciences. Laboratory of Virology. Ribeirao Preto, SP, Brazil.The University of Tennessee Health Science Center. Department of Microbiology, Immunology, and Biochemistry. Memphis, TN, USA.The University of Tennessee Health Science Center. Department of Microbiology, Immunology, and Biochemistry. Memphis, TN, USA.University of Sao Paulo. School of Pharmaceutical Sciences of Ribeirao Preto. Department of Clinical Analyses, Toxicology and Food Sciences. Laboratory of Virology. Ribeirao Preto, SP, Brazil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.University of Sao Paulo. Ribeirao Preto Medical School. Virology Research Center. Ribeirao Preto, SP, Brazil.Fundação de Medicina Tropical Dr Heitor Vieira Dourado. Manaus, AM, Brazil.Fundação de Medicina Tropical Dr Heitor Vieira Dourado. Manaus, AM, Brazil / Universidade do Estado do Amazonas. Manaus, AM, Brazil.Fundação de Medicina Tropical Dr Heitor Vieira Dourado. Manaus, AM, Brazil / Fundação Oswaldo Cruz. Instituto Leônidas and Maria Deane. Manaus, AM, Brazil.University of Sao Paulo. School of Pharmaceutical Sciences of Ribeirao Preto. Department of Clinical Analyses, Toxicology and Food Sciences. Laboratory of Virology. Ribeirao Preto, SP, Brazil.Arthropod-borne viruses (arboviruses) are a significant public health threat, especially in tropical and subtropical regions. More than 150 arboviruses can cause febrile illness following infection in humans. The Brazilian Amazon region has the highest number of arboviruses detected worldwide. In addition to arboviruses, malaria, caused by Plasmodium vivax, is endemic in the Amazon. Patients with malaria and arboviral disease frequently show similar clinical presentation and laboratory findings, making the diagnosis of the cause of the infection challenging. The aim of this study was to evaluate the potential for viral infections in patients with suspected malaria but without Plasmodium infection in the Brazilian Amazon. We recruited 200 subjects with suspected malaria in Manaus, Brazil. First, we tested for arboviruses in serum samples from 124 of the 200 participants using an arbovirus DNA microarray platform, which did not detect any virus. Then, we mixed the serum samples of the other 76 participants in 10 pools and subjected them to next-generation sequencing. Analysis of the sequencing data revealed the presence of only one arbovirus (Zika virus) in one sample pool. This analysis also detected the presence of primate erythroparvovirus 1 and pegivirus C. These results suggest that arboviruses are not the most frequent viral infections in patients with suspected malaria but without Plasmodium infection in the metropolitan region of Manaus. Implementation of specific viral surveillance tests will help in the early detection of viruses with epidemic potential

SMAvirusChip v2 form (RAF) showing the results of statistical analysis of an array hybridized with ZIKV.

<p>SMAvirusChip v2 form (RAF) showing the results of statistical analysis of an array hybridized with ZIKV.</p

Results of statistical analyses of pools of viruses analyzed with SMAvirusChip.

<p>Results of statistical analyses of pools of viruses analyzed with SMAvirusChip.</p