Development and Application of a Fast Method to Acquire the Accurate Whole-Genome Sequences of Human Adenoviruses

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.

Prevalence of Human Adenoviruses in Respiratory Tract-infected Patients in Anbar Governorate (West of Iraq): A Descriptive Cross-sectional Study

“…In the age group, 51+ years was also 3 (2.9%) using the IgM ELIZA test. These results were consistent with the results reported by Zhao et al (2021), 12 that HAdV ranges between 5 to 15% of patients with upper and lower hospitalized RTIs. Our study was consistent with previous studies that found that HAdV has a significant role in pediatric upper and lower RTIs, as its frequency range between 2 to 5% of the overall upper and lower RTIs and 4 to 10% of pneumonia, particularly in pediatric and immune-compromised patients.…”

“…2 Furthermore, some severe upper and lower RTIs duo to adenovirus may develop chronic difficulties, e.g., bronchiolitis obliterans and bronchiectasis cases. 12 The current study showed that 1(12.5%) was positive for HAdV using IgG ELIZA in 8 upper respiratory tract infected patients. Of 66 patients with pneumonia, 5(7.6%) were positive for HAdV using IgG ELIZA.…”

Isolation of novel simian adenoviruses from macaques for development of a vector for human gene therapy and vaccines

“…First, a novel adenovirus GZ3-12 was isolated from a fecal sample of a rhesus macaque as noted earlier; this was subsequently amplified in HEK293 cells. Viral genomic DNA was extracted from GZ3-12 using the modified extraction method described earlier by us ( 55 ). Separately, pBR322 was digested by Eco RI, with the linearized product serving as the PCR template.…”