Khan Mohd Jaseem, Trabuco Amanda Cristina, Alfonso Helda Liz, Figueiredo Mario Luis, Batista Weber Cheli, Badra Soraya Jabur, Figueiredo Luiz Tadeu, Lavrador Marco Aurélio, Aquino Victor Hugo
Laboratory of Virology, Department of Clinical Analyses, Toxicology and Food Sciences, Faculty of Pharmaceutical Sciences of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Sao Paulo, Brazil.
Laboratory of Virology, Research Center of Tropical Medicine, Porto Velho, Rondonia, Brazil.
PLoS Negl Trop Dis. 2016 Sep 21;10(9):e0005017. doi: 10.1371/journal.pntd.0005017. eCollection 2016 Sep.
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.
由小型哺乳动物和节肢动物传播的病毒对人类构成全球威胁。大多数新出现和重新出现的病毒病原体都是由这些群体传播的；因此，开发用于检测和监测此类病毒的高通量筛选方法备受关注。在本研究中，我们描述了一种DNA微阵列平台（SMAvirusChip），可用于筛选所有由小型哺乳动物和节肢动物传播且核苷酸序列已存入GenBank的病毒。SMAvirusChip设计有超过15,000个寡核苷酸探针（60聚体），包括病毒探针和对照探针。设计了两个SMAvirusChip版本：SMAvirusChip v1包含4209个病毒探针，用于检测409种病毒，而SMAvirusChip v2包含4943个探针，用于检测416种病毒。使用20种实验室参考菌株病毒对SMAvirusChip进行了评估。这些病毒在单独存在于样品中或在单个样品中人工混合时都能被特异性检测到。使用登革热病毒（DENV）的10倍系列稀释液评估了SMAvirusChip的灵敏度。结果显示检测限低至2.6E3 RNA拷贝/毫升。此外，其灵敏度比定量实时RT-PCR低一个对数10（2.6E2 RNA拷贝/毫升），足以检测临床样品中的病毒基因组。在登革热病毒感染患者的血清样品（n = 6）和加有登革热病毒的全血样品中检测登革热病毒，证实了SMAvirusChip在检测临床样品中病毒方面的适用性。此外，在加有登革热病毒的一组蚊子样品中也可检测到该病毒。SMAvirusChip能够特异性检测细胞培养物、血清样品、全血样品和一组蚊子中的病毒，证实细胞RNA/DNA不会干扰该检测。因此，SMAvirusChip可能代表一种用于快速鉴定由小型哺乳动物和节肢动物传播的病毒的创新监测方法。
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