Generation of an Attenuated Tiantan Vaccinia Virus Strain by Deletion of Multiple Genes.

Yiquan Li,Wenjie Li,Lili Sun,Ningyi Jin,Xunzhe Yin,Yilong Zhu,Shuang Chen,Pengpeng Xiao,Jicheng Han,Xiao Li,Shanzhi Li

doi:10.3389/fcimb.2017.00462

Abstract

An attenuated vaccinia virus-MVTTEAB-was constructed by deletion of non-essential gene segments related to the immunomodulatory and virulence functions of the vaccinia virus Tiantan strain (VVTT). The shuttle plasmids pTC-EGFP, pTE-EGFP, pTA35-EGFP, pTB-EGFP, and pTA66-EGFP were constructed and combined with the early and late strong promoter pE/L and EGFP as an exogenous selectable marker. Then, through the homologous recombination technology and Cre/loxP system, the following gene fragments were gradually knocked out one by one: TC7L-TK2L, TE3L, TA35R, TB13R, and TA66R. Ultimately, the five-segment-deleted attenuated strain MVTTEAB was obtained. Knockout of these segments and genetic stability of MVTTEAB were confirmed, and it was also shown that knockout of these segments did not affect the replication ability of the virus. Further, a series of in vivo and in vitro experiments demonstrated that the virulence of MVTTEAB was attenuated significantly, but at same time, high immunogenicity was maintained. These results indicate that MVTTEAB has potential for clinical use as a safe viral vector or vaccine with good attenuation and immunogenicity.

Highlights

The vaccinia virus (VV), which is used as a smallpox vaccine, belongs to the family Poxviridae and genus Orthopoxvirus
The Enhanced green fluorescent protein (EGFP) gene of rVVTT-CEGFP+ was knocked out with the Cre-loxP system, and the recombinant vaccinia virus rVVTT-C− that did not contain the TC7L–TK2L genes was obtained by 10 rounds of fluorescence plaque screening (Figures 1, 2)
The same screening method was used to knock out the other deletion fragments and construct a multi-gene-deleted attenuated strain of vaccinia virus Tiantan strain (VVTT) (MVTTEAB) in which five gene fragments and an exogenous selectable marker gene were deleted (Figure 2)

Summary

Introduction

The vaccinia virus (VV), which is used as a smallpox vaccine, belongs to the family Poxviridae and genus Orthopoxvirus. It is a complex double-stranded DNA virus that replicates and forms peculiar viral particles in host cells. The genes present at both ends of the genome are commonly used to identify species or host specificities and to encode proteins that regulate the host immune system and virulence factors (Liu and McFadden, 2015). Research findings have indicated that the genes in this virus are likely to have come from their eukaryotic host genes via horizontal gene transfer, and that these slow and sustained processes have contributed to the evolution of VV.

Methods

Results

Conclusion