Optimized expression of duck tembusu virus e gene delivered by a vectored duck enteritis virus in

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Molecular Biotechnology

https://doi.org/10.1007/s12033-019-00206-1

ORIGINAL PAPER

Optimized Expression of Duck Tembusu Virus E Gene Delivered

by a Vectored Duck Enteritis Virus In Vitro

Liu Chen1  · Bin Yu1

 · Jonggang Hua1

 · Zheng Ni1

 · Weicheng Ye1

 · Tao Yun1

 · Cun Zhang1

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Abstract

In our previous study, a recombinant duck enteritis virus (DEV) delivering codon-optimized E gene (named as E-ch) of duck

Tembusu virus (DTMUV) optimized referring to chicken’s codon bias has been obtained based on the infectious bacterial

artifcial chromosome (BAC) clone of duck enteritis virus vaccine strain pDEV-EF1, but the expression level of E-ch in

recombinant virus rDEV-E-ch-infected cells was very low. To optimize DTMUV E gene expression delivered by the vectored

DEV, diferent forms of E gene (collectively called EG) including origin E gene (E-ori), truncated E451-ori gene, codon￾optimized E-dk gene optimized referring to duck’s codon bias, as well as the truncated E451-ch and E451-dk, Etpa-ori and

Etpa-451-ori, which contain prefxing chick TPA signal peptide genes, were cloned into transfer vector pEP-BGH-end, and

several recombinant plasmids pEP-BGH-EG were constructed. Then the expression cassettes pCMV-EG-polyABGH ampli￾fed from pEP-BGH-EG by PCR were inserted into US7/US8 gene intergenic region of pDEV-EF1 by two-step Red/ET

recombination, 7 strain recombinant mutated BAC clones pDEV-EG carrying diferent E genes were constructed. Next, the

recombinant viruses rDEV-EG were reconstituted from chicken embryo fbroblasts (CEFs) by calcium phosphate precipita￾tion. Western blot analysis showed that E or E451 protein is expressed in rDEV-E-ori, rDEV-E-ch, rDEV-Etpa-ori, rDEV￾E451-ori, rDEV-E451-dk, and rDEV-E451-ch-infected CEFs, and protein expression level in rDEV-E451-dk-infected CEFs

is the highest. These studies have laid a foundation for developing bivalent vaccine controlling DEV and DTMUV infection.

Keywords Duck Tembusu virus · Duck enteritis virus · Vectored vaccine · Optimized expression · E antigen

Introduction

In April 2010, a novel infectious disease has spread around

the main duck-producing regions of egg-laying and breeder

ducks in China. This disease was characterized by retarded

growth, high fever, loss of appetite, decline in egg produc￾tion, and death emerged in ducks, and the main pathologic

features of the disease were ovarian hemorrhage, follicle

atresia, and rupture [1, 2].

By pathogen isolation and identifcation, as well as full￾genome sequencing, this pathogen is considered a new

member of Tembusu virus in ducks, which exhibits the

closest genetic evolutionary relationship with Tembusu

virus, a mosquitoborne favivirus of the Ntaya virus group,

thus tentatively named as duck Tembusu virus (DTMUV).

This pathogen can infect diferent breeds of egg-laying

and breeder ducks, with varying degrees of morbidity.

The incidence rate in the group-infected DTMUV is up

to 100%, and total mortality ranges from 5 to 15% while

occasionally increasing up to 30% due to secondary

Supplementary information Supplementary information

accompanies this paper at https://doi.org/10.1007/s12033-019-

00206-1.

* Cun Zhang

[email protected]

Liu Chen

[email protected]

Bin Yu

[email protected]

Jonggang Hua

[email protected]

Zheng Ni

[email protected]

Weicheng Ye

[email protected]

Tao Yun

[email protected]

1 Institute of Animal Husbandry and Veterinary

Sciences, Zhejiang Academy of Agriculture Sciences,

Hangzhou 310021, China