Utilisation of ISA Reverse Genetics and Large-Scale Random Codon Re-Encoding to Produce Attenuated Strains of Tick-Borne Encephalitis Virus within Days.

Lauriane De Fabritus,Fabien Aubry,Antoine Nougairède,Xavier De Lamballerie,Ernest A Gould,Alessandro Marcello

doi:10.1371/journal.pone.0159564

Abstract

Large-scale codon re-encoding is a new method of attenuating RNA viruses. However, the use of infectious clones to generate attenuated viruses has inherent technical problems. We previously developed a bacterium-free reverse genetics protocol, designated ISA, and now combined it with large-scale random codon-re-encoding method to produce attenuated tick-borne encephalitis virus (TBEV), a pathogenic flavivirus which causes febrile illness and encephalitis in humans. We produced wild-type (WT) and two re-encoded TBEVs, containing 273 or 273+284 synonymous mutations in the NS5 and NS5+NS3 coding regions respectively. Both re-encoded viruses were attenuated when compared with WT virus using a laboratory mouse model and the relative level of attenuation increased with the degree of re-encoding. Moreover, all infected animals produced neutralizing antibodies. This novel, rapid and efficient approach to engineering attenuated viruses could potentially expedite the development of safe and effective new-generation live attenuated vaccines.

Highlights

Many emerging infectious diseases are caused by arthropod-borne viruses of which pathogenic flaviviruses, such as yellow fever virus (YFV), dengue virus, Japanese encephalitis virus (JEV), West Nile virus and tick-borne encephalitis virus (TBEV) are major human pathogens [1,2,3]
We demonstrate the feasibility of producing attenuated viruses using the ISA method combined with random codon re-encoding
Using the Oshima 5–10 strain and the ISA protocol, we engineered the WT (WT_ISA virus) and two randomly re-encoded viruses in days

Summary

Introduction

Many emerging infectious diseases are caused by arthropod-borne viruses (arboviruses) of which pathogenic flaviviruses, such as yellow fever virus (YFV), dengue virus, Japanese encephalitis virus (JEV), West Nile virus and tick-borne encephalitis virus (TBEV) are major human pathogens [1,2,3]. The large-scale codon re-encoding procedure is a recently developed attenuating technique that modifies the nucleic acid composition of large coding regions without modifying the encoded proteins, by introducing a large number of slightly deleterious synonymous mutations. This method of attenuation takes generic advantage of live attenuated vaccines and enables precise modulation of the attenuated phenotype. A new bacterium-free approach, called ISA (Infectious subgenomic amplicons), was described to generate infectious single-stranded positive-sense RNA viruses [26] This method avoids the need for cDNA cloning procedures and shortens the time to produce engineered virus. We demonstrate the feasibility of producing attenuated viruses using the ISA method combined with random codon re-encoding

Materials and Methods

Ethics statement

Results and Discussion