RNA viruses rapidly mutate, which can result in increased virulence, increased escape from vaccine protection, and false-negative detection results. Targeted detection methods have a limited ability to detect unknown viruses and often provide insufficient data to detect coinfections or identify antigenic variants. Random, deep sequencing is a method that can more fully detect and characterize RNA viruses and is often coupled with molecular techniques or culture methods for viral enrichment. We tested viral culture coupled with third-generation sequencing for the ability to detect and characterize RNA viruses. Cultures of bovine viral diarrhea virus, canine distemper virus (CDV), epizootic hemorrhagic disease virus, infectious bronchitis virus, 2 influenza A viruses, and porcine respiratory and reproductive syndrome virus were sequenced on the MinION platform using a random, reverse primer in a strand-switching reaction, coupled with PCR-based barcoding. Reads were taxonomically classified and used for reference-based sequence building using a stock personal computer. This method accurately detected and identified complete coding sequence genomes with a minimum of 20× coverage depth for all 7 viruses, including a sample containing 2 viruses. Each lineage-typing region had at least 26× coverage depth for all viruses. Furthermore, analyzing the CDV sample through a pipeline devoid of CDV reference sequences modeled the ability of this protocol to detect unknown viruses. Our results show the ability of this technique to detect and characterize dsRNA, negative- and positive-sense ssRNA, and nonsegmented and segmented RNA viruses.

RNA 病毒迅速变异，这会导致毒力增加、疫苗保护的逃逸增加和假阴性检测结果。靶向检测方法检测未知病毒的能力有限，并且通常无法提供足够的数据来检测合并感染或识别抗原变异。随机、深度测序是一种可以更全面地检测和表征 RNA 病毒的方法，通常与分子技术或培养方法相结合以进行病毒富集。我们测试了病毒培养与第三代测序相结合的检测和表征 RNA 病毒的能力。牛病毒性腹泻病毒、犬瘟热病毒 (CDV)、流行性出血性疾病病毒、传染性支气管炎病毒、2 种甲型流感病毒的培养物，和猪呼吸和生殖综合征病毒在 MinION 平台上使用随机反向引物在链转换反应中进行测序，并结合基于 PCR 的条形码。读数进行分类分类，并使用库存个人计算机用于基于参考的序列构建。该方法准确检测并鉴定了完整的编码序列基因组，所有 7 种病毒的覆盖深度至少为 20 倍，包括含有 2 种病毒的样本。每个谱系分型区域对所有病毒至少有 26 倍的覆盖深度。此外，通过没有 CDV 参考序列的管道分析 CDV 样本模拟了该协议检测未知病毒的能力。我们的结果显示了该技术检测和表征 dsRNA、负义和正义 ssRNA、