Identification of contamination in the American Type Culture Collection stock of human adenovirus type 8 by whole genome sequencing.
Identification of contamination in the American Type Culture Collection stock of human adenovirus type 8 by whole genome sequencing.
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通过全基因组测序鉴定美国典型培养物保藏中心库存中人类 8 型腺病毒的污染。
DOI：
10.1128/jvi.02875-12
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发表时间：
2013
期刊：
影响因子：
5.4
通讯作者：
Watanabe H.
中科院分区：
文献类型：
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作者：
Yamane S;Lee A;Hanaoka N;Gonzalez G;Kaneko H;Ishida S;Kitaichi N;Ohno S;Koyanagi K;Aoki K;Fujimoto T;Yawata N;Watanabe H.
人腺病毒感染全世界数十亿人，导致各种疾病，如结膜炎、呼吸系统疾病、胃肠炎和出血性膀胱炎(1)。HAdV-D型(HAdV-8)是流行性角膜结膜炎(EKC)的主要致病因子之一(2-9)，是流行性角膜结膜炎(EKC)的主要致病因子之一。在我们的研究过程中，通过对从ATCC获得的HAdV-8毒株(批号59323195)的基因组测序，发现基因组上存在广泛的多态性，表明HAdV-8股票受到了污染。对非HAdV-8病毒的Hexon蛋白编码区进行扩增和测序，结果表明，非HAdV-8病毒的Hexon序列与我们之前测得的HAdV-10原型(DDBJ登录号AB724351)的Hexon序列完全相同。然后，我们测定了ATCC HAdV-8批号59323195的两种类型，即HAdV-8和-10的全基因组序列。我们首先根据我们先前研究中已有的HAdV-8 TRIM(DDBJ登录号AB448767)、HAdV-8b(DDBJ登录号AB448768)、HAdV-8e(DDBJ登录号AB448769)和HAdV-10原型以及其他可用的HAdV基因组序列设计了类型特异性引物。然后我们对扩增产物进行测序、组装和分析(方法学见参考文献10)。ATCC HAdV-8“TRIM变异体”(DDBJ登录号AB746853)是HAdV-8的一个新变异体，全长34,978个碱基，与已有的HAdV-8 TRIM序列最接近，有5个基因的7个非同义突变，4个基因的5个同义突变，9个基因间的碱基替换，以及3个插入和5个缺失，包括CR1-α的单核苷酸插入(SNI)和CR1-γ基因的SNI和单核苷酸缺失。ATCC HAdV-8毒株的基因组序列(35,105bp；DDBJ登录号AB746854)与HAdV-10(DDBJ登录号AB724351)的基因组几乎完全相同，只是PTP基因发生了非同义改变，端粒区有12个碱基替换。我们还独立地对从ATCC购买的HAdV-8批次的基因组进行了测序，并提出了-
Human adenoviruses (HAdVs) infect billions of people world-wide, causing various diseases, such as conjunctivitis, respiratory disease, gastroenteritis, and hemorrhagic cystitis (1). HAdVs have been classified into six species and further classified into 51 types, available from the American Type Culture Collection (ATCC) as prototypes, based on serum neutralization and 16 additional type candidates (types 52 to 67) defined by the genome sequences.Type 8 of HAdV-D (HAdV-8) is one of the major causative agents of epidemic keratoconjunctivitis (EKC)(2–9), and as such, we have been studying modes of the pathogenesis with genome sequencing and other means. During the course of our studies, by sequencing the genome of the HAdV-8 strain available from ATCC (lot number 59323195), extensive polymorphisms were identified over the genome, indicating that the HAdV-8 stock was contaminated. Amplifying and sequencing the hexon protein-coding region, we found that the hexon sequences of the non-HAdV-8 viruses were identical to that of the HAdV-10 prototype (DDBJ accession number AB724351) that we had sequenced previously. We then proceeded to determine the whole-genome sequences of two types, ie, HAdV-8 and-10, in ATCC HAdV-8 lot number 59323195. We first amplified genome regions with type-specific primers designed based on preexisting sequences available from our previous study (10) on HAdV-8 Trim (DDBJ accession number AB448767), HAdV-8b (DDBJ accession number AB448768), HAdV-8e (DDBJ accession number AB448769), and the HAdV-10 prototype and from other HAdV genomes available. We then sequenced, assembled, and analyzed the amplicons (see reference 10 for the methodology). The genome of the ATCC HAdV-8 “Trim variant”(DDBJ accession number AB746853) is a new variant of HAdV-8 that is 34,978 bp in length and which is closest to the preexisting HAdV-8 Trim sequence, differing by seven nonsynonymous changes in five genes, five synonymous changes in four genes, and nine intergenic base substitutions, as well as three insertions and five deletions, including a single-nucleotide insertion (SNI) in CR1-α and an SNI and a single-nucleotide deletion in CR1-γ genes. The genome of the contaminant in the ATCC HAdV-8 stock (35,105 bp; DDBJ accession number AB746854) was almost identical to that of HAdV-10 (DDBJ accession number AB724351) except for a nonsynonymous change in the pTP gene and 12 base substitutions in the telomere regions. We also sequenced the genome of an HAdV-8 lot purchased from ATCC independently and propa-