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

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. Refer to individual Series

由小型哺乳动物和节肢动物传播的病毒，是威胁人类健康的全球性公共卫生威胁。当前绝大多数新发与再发病毒病原体均经由此类媒介传播，因此开发用于这类病毒检测与监测的高通量筛选方法具有重要研究价值。本研究介绍了一款DNA微阵列平台（SMAvirusChip），可用于筛查所有由小型哺乳动物和节肢动物传播的病毒，其探针核苷酸序列均取自基因序列数据库（GenBank）。SMAvirusChip搭载超过15000条寡核苷酸探针（60-mers），涵盖病毒特异性探针与阴性对照探针。该平台共推出两个版本：SMAvirusChip v1包含4209条病毒探针，可检测409种病毒；SMAvirusChip v2则包含4943条病毒探针，可检测416种病毒。研究采用20株实验室标准病毒毒株对SMAvirusChip开展性能验证。结果显示，无论是单一病毒样本还是人工混合的多病毒样本，该平台均可实现特异性检测。针对登革病毒（Dengue virus, DENV）的10倍系列稀释液开展灵敏度测试，结果表明其检测下限低至2.6×10³ RNA拷贝/毫升。此外，该平台的灵敏度较定量实时RT-PCR（quantitative real-time RT-PCR）低一个对数级，检测下限可达2.6×10² RNA拷贝/毫升，仍足以满足临床样本中病毒基因组的检测需求。通过对6份登革病毒感染患者的血清样本、1份人工掺入登革病毒的全血样本进行检测，证实了SMAvirusChip可用于临床样本中的病毒检测。此外，在登革病毒人工掺入的蚊虫混合样本池中，该平台同样可成功检出目标病毒。SMAvirusChip可在细胞培养物、血清样本、全血样本以及蚊虫混合样本中特异性识别病毒，证实了细胞来源的RNA/DNA不会对检测体系造成干扰。综上，SMAvirusChip可作为一种创新性的监测手段，用于快速鉴定小型哺乳动物和节肢动物传播的病毒。详见各对应数据集系列。