Genetic Diversity Does Not Contribute to Attenuation of HeLa Passaged Wild-Type Yellow Fever Virus Strain French Viscerotropic Virus.

Alan D T Barrett,Jill K Thompson,Ashley E Strother,Steven G Widen

doi:10.3390/v14030527

Abstract

The disease yellow fever was prevented by two live attenuated vaccines, strains 17D and French neurotropic vaccine (FNV), derived by serial passage of wild-type (WT) strains Asibi and French Viscerotropic virus (FVV), respectively. Both 17D and FNV displayed decreased genetic diversity and resistance to the antiviral Ribavirin compared to their WT parental strains, which are thought to contribute to their attenuated phenotypes. Subsequent studies found that only a few passages of WT strain FVV in HeLa cells resulted in an attenuated virus. In the current study, the genome sequence of FVV following five passages in HeLa cells (FVV HeLa p5) was determined through Next Generation Sequencing (NGS) with the aim to investigate the molecular basis of viral attenuation. It was found that WT FVV and FVV HeLa p5 virus differed by five amino acid substitutions: E-D155A, E-K331R, E-I412V, NS2A-T105A, and NS4B-V98I. Surprisingly, the genetic diversity and Ribavirin resistance of the FVV HeLa p5 virus were not statistically different to WT parent FVV. These findings suggest that while FVV HeLa p5 is attenuated, this is not dependent on a high-fidelity replication complex, characterized by reduced genetic diversity or increased Ribavirin stability, as seen with FNV and 17D vaccines.

Highlights

Yellow fever virus (YFV) is the prototypical member of the genus Flavivirus [1]
The French Viscerotropic virus (FVV) virus used in the study was passaged minimally in Vero cells and the genome was used as a reference, along with previously published FVV and French neurotropic vaccine (FNV) genomes to align the reads using the Bowtie2 program, and PCR duplicates were removed using Picard
The consensus sequence for the FVV HeLa p5 (Genbank MZ285906) virus was aligned to the published WT FVV and FNV genome sequences as well as the newly sequenced FVV

Summary

Introduction

Yellow fever virus (YFV) is the prototypical member of the genus Flavivirus [1]. The viscerotropic disease, yellow fever (YF), caused by wild-type YFV is characterized by infection of the liver, resulting in the onset of jaundice in severe cases for which the disease and virus are named [2–5]. The vaccine currently used today, strain 17D, was derived by 176 serial passages of wild-type (WT) strain Asibi in mouse tissue and chicken embryo tissue both with and without neurological tissue [6]. This resulted in an attenuated phenotype characterized by a loss of viscerotropism, loss of neurotropism, and loss of mosquito competence.

Methods

Results

Conclusion