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

Abstract

Background: 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. An ideal vaccine candidate is expected to have a balanced safety and immunogenicity.

Results: From 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 (PRV^ΔTK&gE-AH02) was generated, and its structure verified by PCR, RFLP and sequencing. Growth kinetics test showed that PRV^ΔTK&gE-AH02 reached a titer of 10^7.5 TCID₅₀ /mL on ST cells. The PRV^ΔTK&gE-AH02 at a dose of 10^6.0 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 10^6.0 TCID₅₀, although it caused one death of four 1-day-old piglets without maternal PRV antibodies. In the efficiency test of PRV^ΔTK&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 10^5.0, 10^4.0 and 10^3.0 TCID₅₀/dose PRV^ΔTK&gE-AH02 groups provided complete protection against challenge at only 7 days post intramuscular vaccination. More importantly, PRV^ΔTK&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.

Conclusions: The constructed TK&gE dual deletion mutant PRV^ΔTK&gE-AH02 can reach high titers on ST cells. The live vaccine of PRV^ΔTK&gE-AH02 is highly safe, and can not only provide clinical protection but also stops virus shedding. This study suggests that PRV^ΔTK&gE-AH02 might work as a promising vaccine candidate to combat the PRV variant emerging in Chinese herds since 2011.

Keywords: Attenuation; Immunogenicity; Live vaccine; Pseudorabies virus emerging variant; Safety; TK&gE dual deletion.

Fig. 1

Construction of TK&gE dual deletion mutant virus (PRV^ΔTK&gE-AH02 strain). a A fragment with selection mark(sm) was amplified to target part of UL23(TK) gene in the genome of PRV. b Homologous recombination was conducted through En Passant protocol to delete part of TK gene. c The sm of kanamycin resistance gene was removed in the 2nd Red recombination. d Another homologous recombination was performed to recover the whole gI gene and part of gE gene during virus rescuing. e Schematic presentation of the TK&gE dual deletion mutant was shown. Scales in bp or kbp are provided

Fig. 2

Restriction fragment length polymorphism (RFLP) of BAC^PRV-G and its TK deletion mutants. a RFLP pattern of BAC ^{PRV△TK/gE/gI}, BAC^{PRV△TK/gE/gI&K+} and BAC^PRV-G. Lane 1, 2 and 3 are BAC ^{PRV△TK/gE/gI}, BAC^{PRV△TK/gE/gI&K+} and BAC^PRV-G respectively after digestion with Kpn I. The arrow (lane 2) shows an additional band of 6639 bp and a 5975 bp band missed when compared to lane 3. The arrow(lane 1) showed an additional band of 5628 bp and a missing band of 6639 bp when compared to lane 2. M is 1Kb DNA marker. b Predicted RFLP pattern with PRV ZJ01 strain (GenBank:KM061380.1) as a reference. Lane 1, 2 and 3 are prediction of BAC ^{PRV△TK/gE/gI}, BAC^{PRV△TK/gE/gI&K+} and BAC^PRV-G respectively digested with Kpn I. c Kpn I sites were cited in lane 1, 2 and 3 for BAC ^{PRV△TK/gE/gI}, BAC^{PRV△TK/gE/gI&K+} and BAC^PRV-G respectively. Sites underlined with red lines indicate the changed position of Kpn I restriction site leading to the bands changing accordingly

Fig. 3

Plaques of TK&gE dual deletion mutant virus. Plaques are shown under UV excitation(left) or phase control(right). Arrow shows the plaque of rescued virus from BAC ^{PRV△TK/gE/gI}. Arrowhead shows the plaque of TK&gE dual deletion mutant virus (PRV^ΔTK&gE-AH02) after replacement of mini-F sequences through another homologous recombination. Individual panels present views of 600 × 600 μm

Fig. 4

Multi-step growth kinetics of AH02LA, LA-A^B and PRV^ΔTK&gE-AH02 on ST cells Titers of infected-cell supernatants (a) and cell-associated virus(b) of AH02LA, LA-A^B and PRV^ΔTK&gE-AH02 were tested at 0, 6, 12, 23, 36, 48 and 72 h post infection with an MOI of 0.01. Shown are means of titers in three independent experiments. Standard deviation are shown with the error bar

Fig. 5

Safety and efficiency of TK&gE dual deletion mutant virus for piglets a Safety for 1-day-old PRV gB antibody negative piglets were tested. b Safety for 1-day-old PRV gB antibody positive piglets were tested. c A total of 24 28~ 35-day-old PRV gB antibody negative piglets were randomly divided into six groups of A-F. One week post vaccination, groups A, B, C, D and E were challenged intranasally with 2LD₅₀ PRV AH02LA per piglet. Group F piglets were not challenged. The survival rates of these groups over the 14 days post challenge are shown. I.N.: intranasally; I.M.: intramuscularly