Development and analysis of a tick-borne encephalitis virus infectious clone using a novel and rapid strategy

In less than 1 month we have constructed an infectious clone of attenuated tick-borne encephalitis virus (strain Vasilchenko) from 100 μl of unpurified virus suspension using long high fidelity PCR and a modified bacterial cloning system. Optimization of the 3′ antisense primer concentration was ess...

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Bibliographic Details
Published in:Journal of virological methods 1998-12, Vol.76 (1), p.109-120
Main Authors: Gritsun, T.S., Gould, E.A.
Format: Article
Language:English
Subjects:
Animals
Biological and medical sciences
Cells, Cultured
Cloning, Molecular
DNA Primers
DNA, Complementary
DNA, Viral - biosynthesis
Encephalitis Viruses, Tick-Borne - genetics
Encephalitis Viruses, Tick-Borne - physiology
Fundamental and applied biological sciences. Psychology
High fidelity
Infectious clone
Long RT-PCR
Mice
Microbiology
Molecular Sequence Data
Reverse Transcriptase Polymerase Chain Reaction
RNA, Viral - genetics
Techniques used in virology
Tick-borne encephalitis virus
Transfection
Viral Plaque Assay
Virology
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Summary:In less than 1 month we have constructed an infectious clone of attenuated tick-borne encephalitis virus (strain Vasilchenko) from 100 μl of unpurified virus suspension using long high fidelity PCR and a modified bacterial cloning system. Optimization of the 3′ antisense primer concentration was essential to achieve PCR synthesis of an 11 kb cDNA copy of RNA from infectious virus. A novel system utilising two antisense primers, a 14-mer for reverse transcription and a 35-mer for long PCR, produced high yields of genomic length cDNA. Use of low copy number Able™ K cells and an incubation temperature of 28°C increased the genetic stability of cloned cDNA. Clones containing 11 kb cDNA inserts produced colonies of reduced size, thus providing a positive selection system for full length clones. Sequencing of the infectious clone emphasised the improved fidelity of the method compared with conventional PCR and cloning methods. A simple and rapid strategy for genetic manipulation of the infectious clone is also described. These developments represent a significant advance in recombinant technology and should be applicable to positive stranded RNA viruses which cannot easily be purified or genetically manipulated.
ISSN:0166-0934
1879-0984
DOI:10.1016/S0166-0934(98)00130-X