Safety and immunogenicity of an attenuated Chinese pseudorabies variant by dual deletion of TK&gE genes

Jichun Wang,Mengwei Xu,Aimin Ge,Zengcai Song,Yongfeng Qiao,Yamei Liu,Hongjie Fan,Rongli Guo,Yating Zheng,Jibo Hou,Zhisheng Wang

doi:10.1186/s12917-018-1536-7

Abstract

BackgroundSince the outbreak of a new emerging virulent pseudorabies virus mutant in Chinese pig herds, intensive research has been focused on the construction of novel gene deletion vaccine based on the variant virulent viruses. An ideal vaccine candidate is expected to have a balanced safety and immunogenicity.ResultsFrom the infectious clone of PRV AH02LA strain, a TK deletion mutant was generated through two-step Red mutagenesis. After homologous recombination with a transfer vector, a TK&gE dual deficient mutant PRV (PRVΔTK&gE-AH02) was generated, and its structure verified by PCR, RFLP and sequencing. Growth kinetics test showed that PRVΔTK&gE-AH02 reached a titer of 107.5 TCID50 /mL on ST cells.The PRVΔTK&gE-AH02 at a dose of 106.0 TCID50 /animal was not virulent in mice or 1-day-old piglets with maternal PRV antibodies. No clinical signs or virus shedding were detected in 28~ 35-day-old piglets without maternal PRV antibodies after nasal or intramuscular administration with a dose of 106.0 TCID50, although it caused one death of four 1-day-old piglets without maternal PRV antibodies. In the efficiency test of PRVΔTK&gE-AH02, all four 28~ 35-day-old piglets without PRV antibody in the challenge control showed typical clinical symptoms and virus shedding, and two died at 4~ 5 days post challenge. All piglets in 105.0, 104.0 and 103.0 TCID50/dose PRVΔTK&gE-AH02 groups provided complete protection against challenge at only 7 days post intramuscular vaccination. More importantly, PRVΔTK&gE-AH02 stopped virus shedding in these piglets. In contrast, all four piglets in PRV Bartha K61 vaccine group developed high body temperature (≥40.5 °C) and viral shedding, despite they had mild or even no clinical symptoms.ConclusionsThe constructed TK&gE dual deletion mutant PRVΔTK&gE-AH02 can reach high titers on ST cells. The live vaccine of PRVΔTK&gE-AH02 is highly safe, and can not only provide clinical protection but also stops virus shedding. This study suggests that PRVΔTK&gE-AH02 might work as a promising vaccine candidate to combat the PRV variant emerging in Chinese herds since 2011.

Highlights

Since the outbreak of a new emerging virulent pseudorabies virus mutant in Chinese pig herds, intensive research has been focused on the construction of novel gene deletion vaccine based on the variant virulent viruses
Several colonies with resistance to chloramphenicol and kanamycin were obtained and verified through Polymerase chain reaction (PCR) with a pair of primers(PRV ΔTK check F/R)(data not shown) and through Restriction fragment length polymorphism (RFLP) after digestion with Kpn I(Fig. 2), which showed a band of 5975 bp missed and an additional band of 6639 bp present after the first recombination
Colonies with resistance to chloramphenicol, but sensitive to kanamycin were selected and one of them, named Bacterial artificial chromosome (BAC) Pseudorabies virus (PRV)△Thymidine kinase (TK)/glycoprotein E (gE)/Glycoprotein I (gI), was successfully confirmed through PCR with a pair of primers(PRV ΔTK check F/R)(data not shown) and through RFLP after digestion with Kpn I(Fig. 2), which showed a shows an additional band of 6639 bp and a 5975 bp band missed when compared to lane 3

Summary

Introduction

Since the outbreak of a new emerging virulent pseudorabies virus mutant in Chinese pig herds, intensive research has been focused on the construction of novel gene deletion vaccine based on the variant virulent viruses. Since 2011, a new emerging pseudorabies virus(PRV) variant has swept many Chinese pig herds, leading to infection or disease of variable severity [1,2,3,4]. A TK&gE dual deletion mutant of the wild-type PRV TNL strain, which was isolated from a commercial pig farm in southern Taiwan in 1976, has been generated and proposed potential vaccine candidate with safety, efficacy and DIVA capability [22]. No TK&gE deletion mutant from the new emerging Chinese variant has been reported so far

Methods

Results

Discussion

Conclusion