Rapid Construction of a Replication-Competent Infectious Clone of Human Adenovirus Type 14 by Gibson Assembly

Abstract

In 1955, Human adenovirus type 14 (HAdV-B14p) was firstly identified in a military trainee diagnosed as acute respiratory disease (ARD) in the Netherlands. Fifty years later, a genomic variant, HAdV-B14p1, re-emerged in the U.S. and caused large and fatal ARD outbreaks. Subsequently, more and more ARD outbreaks occurred in Canada, the UK, Ireland, and China, in both military and civil settings. To generate a tool for the efficient characterization of this new genomic variant, a full-length infectious genomic clone of HAdV-B14 was successfully constructed using one-step Gibson Assembly method in this study. Firstly, the full genome of HAdV-B14p1 strain GZ01, the first HAdV-B14 isolate in China, was assembled into pBR322 plasmid by Gibson Assembly. The pBRAdV14 plasmid, generated by Gibson Assembly, was analyzed and verified by PCR, restriction enzymes digestion and the sequencing. Secondly, viruses were rescued from pBRAdV14-transfected A549 cells. The integrity of the rescued viruses was identified by restriction enzyme analysis. The complete sequence of the infectious clone was further sequenced. No mutation was found in the infectious clone during the construction when compared with the parental virus and pBR322 sequences. The direct immunofluorescence assay indicated the expression of the hexon protein. Finally, typical virions were observed; the one-step growth curves further showed that the DNA replication and viral reproduction efficiency of pBRAd14 derived viruses was similar with that of wild-type HAdV-B14 strain. The successful construction of the replication-competent infectious clone of pBRAdV14 facilitates the development of vaccine and antiviral drugs against HAdV-B14, as well as provides a novel strategy for rapid construction of infectious viral clones for other large-genome DNA viruses.

Construction and Characterization of a Novel Recombinant Attenuated and Replication-Deficient Candidate Human Adenovirus Type 3 Vaccine: “Adenovirus Vaccine Within an Adenovirus Vector”

“…Prior to sequencing, as a rapid check, restriction endonuclease analysis (REA) was performed to characterize the recombinant plasmid rapidly. Restriction maps generated with five restriction endonucleases were consistent with the in silico restriction maps predicted by the Vector NTI 11.5.1 software (Invitrogen Corp; San Diego, CA, USA) (Zhao et al 2014;Yu et al 2016;Zhang et al 2017;Pan et al 2018) (Fig. 2).…”

High-Throughput Cloning and Characterization of Emerging Adenovirus Types 70, 73, 74, and 75

“…However, the further exploration of the more than 100 human adenovirus types was hindered for a long time by lack of convenient genetic modification methods. To convert adenoviruses to vectors, several strategies have been devised, such as cosmid-based methods, traditional cut- and-paste based molecular cloning, homologous recombination in bacteria or in eukaryotic cells (summarized in review [ 18 ]), and most recently described Gibson gene assembly technique [ 19 , 20 ]. Those methods were either time consuming or complicated.…”

A one-step construction of adenovirus (OSCA) system using the Gibson DNA Assembly technology

“… 1 , 2 Interestingly, several recent studies explored the use of the GDA technology for the construction of special Ad vectors. 17 , 18 , 19 Freedman et al. used the GDA technology to generate a modified oncolytic group B Ad EnAdenotucirev (EnAd) to express a bispecific single-chain antibody, which was controlled by the virus major late promoter.…”

“…Interestingly, several recent studies explored the use of the GDA technology to construct Ad. 17 , 18 , 19 However, those reports were mainly involved in the construction of either specific oncolytic Ad or rarely used Ad viruses without broad utilities for conventional Ad generation. For the OSCA system, we first engineered novel adenoviral recipient vectors that contain two 20-bp unique sequences, namely modified one-step site 1 (MOS1) and MOS2, which serve as the universal overlapping sites for Gibson Assembly reactions at the ΔE1 region of the Ad genome.…”