Flavivirus cDNA clones frequently demonstrate genetic instability in transformed bacteria, which hampers the construction and manipulation of cDNAs for infectious flaviviruses. In this study, we developed a stable, full-length cDNA clone, pJEHEN, of a GI JEV strain HEN0701 using a medium-copy-number pBR322 vector and propagating cDNA clones at room temperature. The virus vJEHEN recovered from the infectious clone was indistinguishable from the parent virus HEN0701 with respect to plaque morphology, growth kinetics, and virulence characteristics. A T-to-A silent mutation of nucleotide 24 of the NS2a gene was introduced into the infectious cDNA clone to eliminate frameshifting. The rescued mutant virus vJETA did not express NS1′ in infected cells and showed reduced growth and neurovirulence in mice. This convenient method for the construction and manipulation of infectious JEV cDNA clones may be of use in further studies to improve our understanding of the molecular mechanisms responsible for JEV replication and pathogenesis.
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This study was supported by the International Scientific and Technological Cooperation Projects of China (Grant Number: 2014FE30140) and the Natural Science Foundation of China (Grant Number: 31201917).
Conflict of interest
All individual participants approve to submit and declare no conflict of interests.
The animal experiments in this study were approved by the Animal Care and Ethics Committee of Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, and were carried out in accordance with conventional animal-welfare regulations and standards.
Hao Zheng and Xuchen Zheng have contributed equally to the study.
Edited by Simon D. Scott.
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Zheng, H., Zheng, X., Tong, W. et al. A simple method for developing an infectious cDNA clone of Japanese encephalitis virus. Virus Genes 53, 4–14 (2017). https://doi.org/10.1007/s11262-016-1387-x
- Japanese encephalitis virus
- Infectious clone
- NS1′ protein