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

Haibin Pan,Jaya Rajaiya,Shan Zhao,Ashrafali Ismail,Chengsong Wan,Na Cao,Min Li,Yuqian Yan,Jing Zhang,Wei Zhao,Junxian Ou,James Chodosh,Qiwei Zhang,Liqiang Feng

doi:10.3390/v10100568

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.

Highlights

Human adenoviruses (HAdVs) are non-enveloped and double-strand DNA viruses which have a diameter of 70–90 nanometers and a genome of 35–40 kb
The infectious clones of plasmid pBRAdV14 were generated by the ligation of the PCR product of linearized pBR322 and HAdV-B14 genomic DNA at 50 ◦ C for 1 h (Figure 1)
Plasmid pBRAdV14 was linearized with AsiSI and the HAdV-B14 genome was released, which was used to transfect the A549 cells

Summary

Introduction

Human adenoviruses (HAdVs) are non-enveloped and double-strand DNA viruses which have a diameter of 70–90 nanometers and a genome of 35–40 kb. Viruses 2018, 10, 568 and classified into seven species (A to G), in which types 1–51 were initially characterized according to serum neutralization and hemagglutination-inhibition tests, whereas types 52 and the other following types were identified using the whole-genome sequence as the typing standard [1,2,3,4,5,6,7]. Species B HAdVs, as well as HAdV-E4 cause epidemics of a similar syndrome, acute respiratory disease (ARD) [8]. HAdV-B3, -B7, -B16 and -B21 in HAdV-B1 are respiratory pathogens and can infect the respiratory tract; HAdV-B50, another member in HAdV-B1, does not cause a specific disease. The other two members in HAdV-B2, HAdV-B14 and B55, are identified as respiratory pathogens [8,10,11,12]

Methods

Results

Conclusion