Randomly primed,strand-switching,MinION-based sequencing for the detection and characterization of cultured RNA viruses

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.     

头骨形态指标鉴别水貂来源有效性的评估

人工驯养野生动物是获取野生动物产品的重要手段,但是非法盗猎的野生动物产品以人工饲养的名义进入市场,就会对野生动物的保护形成挑战。因此,准确鉴别市场上的动物产品是来自养殖场还是野外,是保护野外种群,维持养殖业正常秩序的关键。本研究以北美水貂(Neovison vison)为例,基于颧骨内侧空间(ZAS)、颧宽(ZAA和ZBA)、颅基长(BSL)、下颌长(MDL)、眶后收缩(POC)和最大面部宽度(GFB)等量度,建立了9个头骨形态计量学指标:POC/GFB、POC/BSL、POC/MDL、ZAS SQRT/BSL、ZA SSQRT/MDL、ZBA×BSL/2ZAS、ZBA×MDL/2ZAS、ZBB×BSL/2ZAS和ZBB×MDL/2ZAS。野生组(n=32,9雄23雌)和饲养组(n=45,35雄10雌)的比较表明,除POC/GFB外,所有指标对于在两组样品之间均有显著差异,整体判别正确率为73.2%—85.5%。在性别已知的情况下,雄性的整体正确率为82.4%—93.8%,雌性为41.7%—85.7%。为此,应针对不同性别、不同来源的动物建立相应的参考数据,利用似然比进行鉴别。本研究的结果不仅可用于北美水貂的鉴别,也为其他小型食肉动物的鉴别提供经验。