Abstract
The whole-genome sequencing (WGS) of human adenoviruses (HAdVs) plays an important role in identifying, typing, and mutation analysis of HAdVs. Nowadays, three generations of sequencing have been developed. The accuracy of first-generation sequencing is up to 99.99%, whereas this technology relies on PCR and is time consuming; the next-generation sequencing (NGS) is expensive and not cost effective for determining a few special samples; and the third-generation sequencing technology has a higher error rate. In this study, first, we developed an efficient HAdV genomic DNA extraction method. Using the complete genomic DNA instead of the PCR amplicons as the direct sequencing template and a set of walking primers, we developed the HAdV WGS method based on first-generation sequencing. The HAdV whole genomes were effectively sequenced by a set of one-way sequencing primers designed, which reduced the sequencing time and cost. More importantly, high sequence accuracy is guaranteed. Four HAdV strains (GZ01, GZ02, HK35, and HK91) were isolated from children with acute respiratory diseases (ARDs), and the complete genomes were sequenced using this method. The accurate sequences of the whole inverted terminal repeats (ITRs) at both ends of the HAdV genomes were also acquired. The genome sequence of human adenovirus type 14 (HAdV-B14) strain GZ01 acquired by this method is identical to the sequence released in GenBank, which indicates that this novel sequencing method has high accuracy. The comparative genomic analysis identified that strain GZ02 isolated in September 2010 had the identical genomic sequence with the HAdV-B14 strain GZ01 (October 2010). Therefore, strain GZ02 is the first HAdV-B14 isolate emergent in China (September 2010; GenBank acc no. MW692349). The WGS of HAdV-C2 strain HK91 and HAdV-E4 strain HK35 isolated from children with acute respiratory disease in Hong Kong were also determined by this sequencing method. In conclusion, this WGS method is fast, accurate, and universal for common human adenovirus species B, C, and E. The sequencing strategy may also be applied to the WGS of the other DNA viruses.
Highlights
Human adenoviruses (HAdVs) have seven species, A (HAdVA) through G (HAdV-G), defined by various biological and morphological criteria, nucleic acid characteristics, and homologies (Zhang et al, 2017; Cheng et al, 2018)
We found that strain GZ02 isolated in September 2010 had the identical genomic sequence with strain GZ01 isolated in October 2010 (Zhang et al, 2012b, 2017), which suggests that both isolates derived from the same strain; human adenoviruses (HAdVs)-B14 strains GZ02, not strain GZ01, is the first HAdV-B14 isolate emergent in China
To verify if the viral genomic DNA obtained is correct or not, the genomes were digested with EcoRI restriction endonuclease
Summary
Human adenoviruses (HAdVs) have seven species, A (HAdVA) through G (HAdV-G), defined by various biological and morphological criteria, nucleic acid characteristics, and homologies (Zhang et al, 2017; Cheng et al, 2018). One-third of HAdVs are related to human diseases and are estimated to cause 8% of global clinically relevant viral diseases (Yu et al, 2016). Common adenoviral diseases include respiratory infections in children and military recruits, infantile gastroenteritis, and ocular infections among healthy individuals (Zhang et al, 2009, 2012b; Han et al, 2013). Less frequently, these pathogens can cause urinary tract infections, myocarditis, meningoencephalitis, and acute hemorrhagic cystitis. In neonates and immunocompromised individuals, HadVs have been reported to cause fulminant fatal pneumonia, hepatitis, and/or encephalitis (Adhikary et al, 2014; Scott et al, 2016; Cheng et al, 2018). In 2019, a local outbreak of adenovirus in southwest China killed more than 40 children
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