Ye Fuqiang, Han Yifang, Zhu Juanjuan, Li Peng, Zhang Qi, Lin Yanfeng, Wang Taiwu, Lv Heng, Wang Changjun, Wang Chunhui, Zhang Jinhai
Department of Disease Control and Prevention, Center for Disease Control and Prevention of Eastern Theater Command, Nanjing, China.
School of Life Science and Technology, China Pharmaceutical University, Nanjing, China.
Front Genet. 2020 Apr 7;11:285. doi: 10.3389/fgene.2020.00285. eCollection 2020.
Human adenoviruses (HAdVs) have been demonstrated to cause a diversity of diseases among children and adults. The circulation of human adenovirus type 21 (HAdV21) has been mainly documented within closed environments in several countries. Nonetheless, respiratory infections or outbreaks due to HAdV21 have never been reported in China. MinION and Illumina platforms were employed to identify the potential pathogen from a throat swab. Discrepancies between MinION and Illumina sequencing were validated and corrected via polymerase chain reaction (PCR). Genomic characterization and recombinant event detection were then performed. Among the 35,466 high-quality MinION reads, a total of 5,999 reads (16.91%) could be aligned to HAdV21 reference genomes (genome sizes ≈35.3 kb), among which 20 had a length of >30 kb. A genome sequence assembled from MinION reads was further classified as HAdV subtype 21a. Random downsampling revealed as few as 500 nanopore reads could cover ≥96.49% of current genome. Illumina sequencing displayed good consistency (pairwise nucleotide identity = 99.91%) with MinION sequencing but with 31 discrepancies that were further validated and confirmed by PCR coupled with Sanger sequencing. Restriction enzymes such as HI and I were able to distinguish the present genome from HAdV21 prototype and HAdV21b. Phylogenetic analysis employing whole-genome sequences placed our genome with members only from subtype 21a. Common features among HAdV21a strains were identified, including polymorphisms discovered in penton and 100 kDa hexon assembly-associated proteins and a recombinant event in the E4 gene. Using MinION and Illumina sequencers, we identified the first HAdV21a strain from China, which could provide key genomic data for disease control and epidemiological investigations.
人类腺病毒（HAdVs）已被证明可在儿童和成人中引发多种疾病。21型人类腺病毒（HAdV21）的传播主要记录在几个国家的封闭环境中。然而，中国从未报告过由HAdV21引起的呼吸道感染或疫情。采用MinION和Illumina平台从咽喉拭子中鉴定潜在病原体。通过聚合酶链反应（PCR）验证并校正了MinION和Illumina测序之间的差异。然后进行基因组特征分析和重组事件检测。在35466条高质量的MinION reads中，共有5999条reads（16.91%）可与HAdV21参考基因组（基因组大小≈35.3 kb）比对，其中20条长度大于30 kb。从MinION reads组装的基因组序列进一步被分类为HAdV亚型21a。随机下采样显示，仅500条纳米孔reads就能覆盖当前基因组的≥96.49%。Illumina测序与MinION测序显示出良好的一致性（成对核苷酸同一性=99.91%），但存在31处差异，通过PCR结合桑格测序进一步验证和确认。诸如HI和I等限制酶能够将当前基因组与HAdV21原型和HAdV21b区分开来。采用全基因组序列的系统发育分析将我们的基因组与仅来自21a亚型的成员归为一类。确定了HAdV21a菌株的共同特征，包括在五聚体和100 kDa六聚体组装相关蛋白中发现的多态性以及E4基因中的重组事件。通过使用MinION和Illumina测序仪，我们从中国鉴定出了首例HAdV21a菌株，这可为疾病控制和流行病学调查提供关键的基因组数据。
