Recovery Infectious Enterovirus 71 by Bac-to-Bac Expression System in vitro and in vivo.

Baojing Lu,Zeng Li,Xuejuan Liu,Hui Peng,Bao Li,Qi Tang,Binbin Zhu,Li Xie,Hanzhong Wang,Linding Wang,Zhenhua Zheng,Qianyun Wang

doi:10.3389/fmicb.2022.825111

Abstract

Enterovirus 71 (EV71) is one of the most important etiological agents for hand–foot–mouth disease. Compared with coxsackievirus A16 infection, EV71 infection is often associated with severe central nervous system complications, such as encephalitis, encephalomyelitis, and acute flaccid paralysis in infants and young children. In this study, we constructed a recombinant baculovirus with T7 ribonucleic acid polymerase under the control of a cytomegalovirus promoter and simultaneously engineered the T7 promoter upstream of a full-length EV71 complementary deoxyribonucleic acid. After transduction into mammalian cells, typical cytopathic effects (CPEs) and VP1 signals were detected in cells transfected with recombinant baculovirus. Additionally, viral particles located in the cytoplasm of human rhabdomyosarcoma cells (Rd) and Vero cells were observed by electron microscope, indicating that EV71 was recovered using a Bac-to-Bac expression system in vitro. After four passages, the rescued virus had a growth curve and plaque morphology similar to those of the parental virus. Furthermore, the Vp1 gene and the protein from the mouse brain were detected by reverse transcription polymerase chain reaction and immunohistochemistry after intracerebral injection of purified recombinant baculovirus. Typical CPEs were observed after inoculation of the supernatant from mouse brain to Rd cells, revealing a reconstruction of EV71 in vivo. Thus, we established a new approach to rescue EV71 based on a baculovirus expression system in vitro and in vivo, which may provide a safe and convenient platform for fundamental research and a strategy to rescue viruses that currently lack suitable cell culture and animal models.

Highlights

Enterovirus 71 (EV71) belongs to the Enterovirus genus of the Picornaviridae family, which is a non-enveloped, positive, single-stranded ribonucleic acid virus, that is a common cause of hand– foot–mouth disease (HFMD) in young children (Mao et al, 2016), but occasionally it can lead to severe diseases such as aseptic meningitis, poliomyelitis-like paralysis, and possibly fatalProducing Infectious EV71 by Bac-to-Bac System encephalitis (Solomon et al, 2010)
Recombinant bacmid DNA was transfected into Sf9 cells to produce recombinant baculovirus virus, and the virus Ac- T7BrCr, which can express T7 RNA polymerase in mammalian cells and contains the T7 promoter before the genome of BrCr was constructed using a Bac-to-Bac expression system
Rescued EV71induced cytopathic effects (CPEs) and fluorescence appeared 18 h posttransduction, and approximately 80% of the cells were rounded and lost normal cell morphology 48 h posttransduction compared with the controls

Summary

Introduction

Enterovirus 71 (EV71) belongs to the Enterovirus genus of the Picornaviridae family, which is a non-enveloped, positive, single-stranded ribonucleic acid virus, that is a common cause of hand– foot–mouth disease (HFMD) in young children (Mao et al, 2016), but occasionally it can lead to severe diseases such as aseptic meningitis, poliomyelitis-like paralysis, and possibly fatalProducing Infectious EV71 by Bac-to-Bac System encephalitis (Solomon et al, 2010). Reverse genetics systems are one of the most important and powerful tools to study the molecular biology of viruses (Yu et al, 2019). Most positive-strand RNA viruses, such as hepatitis A virus, coxsackievirus B6, and norovirus, have been recovered based on the reverse genetics system (Tellier et al, 1996; Martino et al, 1999; Todd and Tripp, 2019). Arita et al (2005) constructed an infectious clone of BrCr to explore the neurovirulence site of EV71, which was the first study of EV71 rescue to the best of our knowledge. Various groups constructed infectious clones of EV71 using different strategies that showed potential advantages for gene mutation, animal model application, and vaccine production of rescue virus (Han et al, 2010; Meng et al, 2012; Zhang et al, 2020)

Methods

Results

Conclusion