Identification of Contamination in the American Type Culture Collection Stock of Human Adenovirus Type 8 by Whole-Genome Sequencing

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Human adenoviruses (HAdVs) infect billions of people worldwide, 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, i.e., 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 propagated thereafter at the National Institute of Infectious Diseases of Japan (NIID) and found that the sequence (HAdV-10 NIID-1; DDBJ accession number AB695621) was entirely identical to that of the contaminant HAdV-10 (DDBJ accession number AB746854). In the phylogenetic tree of these newly sequenced genomes and those of 27 other HAdV types (Fig. 1), the genomes deposited under DDBJ accession numbers AB746853 and AB746854 were placed in the HAdV-8 and the HAdV-10 clades, respectively, with no ambiguity.

Fig 1.

Molecular phylogenetic tree of the two genomes from the ATCC HAdV-8 stock (in bold) together with 29 complete genome sequences of HAdV-D viruses in the International Nucleotide Sequence Database (INSD). The multiple alignment of these sequences was constructed using MAFFT version 6 (11), the evolutionary distances were estimated using Kimura's two-parameter model (12) based on 33,884 gap-free sites in the alignment, and then an unrooted tree was constructed using the neighbor-joining method (13). The bootstrap probability calculated for 1,000 replicates is shown on each branch. The genomes that were newly sequenced in this study are indicated by an asterisk.

Here we report on the whole-genome sequences and phylogenetic analyses of the HAdV-8 and HAdV-10 reference virus stocks distributed by the ATCC and conclude that HAdV-8 lot number 59323195, provided by the ATCC, contained both HAdV-8 and HAdV-10 types. Due to HAdV-8 being one of the major causative agents of EKC, we would like to draw attention of the adenovirus research community to this contamination issue so as to prevent misinterpretation.

Nucleotide sequence accession numbers.

The whole-genome sequences of the HAdV-8 Trim variant, the HAdV-10 contaminant in the ATCC HAdV-8 stock, HAdV-10 NIID-1, and the HAdV-10 prototype have been deposited at DDBJ under accession numbers AB746853, AB746854, AB695621, and AB724351, respectively.