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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  TCID 50  /mL on ST cells.  The PRV ΔTK&gE-AH02  at a dose of 10 6.0  TCID 50  /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  TCID 50 , 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  TCID 50 /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.

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