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Isolation and Characterization of a Variant Psedorabies Virus HNXY and Construction of rHNXY-?TK/?gE.

ABSTRACT: The outbreak of pseudorabies in China, caused by more virulent pseudorabies virus (PRV) than the classical strains, has led to considerable economic losses. In this study, PRV strain HNXY was isolated from the Henan province of China in 2015 from the pig farm with severe reproductive failure in sows and a high mortality in piglets. The 50% tissue culture infectious doses (TCID50) of HNXY in Vero cells were examined to be 106.5/mL, and the neutralisation titer against Bartha-K61 was significantly higher than against HNXY when tested with the serum from Bartha-K61 vaccinated pigs. The 50% lethal doses (LD50) of HNXY to six-week-old BALB/c mice and two-month-old PRV-free pigs were both 102.3 TCID50. HNXY was classified as genotype II, and numerous amino acid variations were found in gB, gE, gC, gD, TK, and RR1 proteins, compared with PRV from other countries or those prevalent in China before 2012. The attenuated rHNXY-?TK/?gE was further constructed, which presented significantly smaller plaques than HNXY, as well as the similar growth kinetics. rHNXY-?TK/?gE was confirmed to be non-pathogenic to six-week-old BALB/c mice and zero-day-old piglets. This study isolated updated PRV promising to develop into a new vaccine candidate.

SUBMITTER: Wu F

PROVIDER: S-EPMC7600349 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Isolation and Characterization of a Variant Psedorabies Virus HNXY and Construction of rHNXY-∆TK/∆gE.

Wu Fengsun F Lv Yujin Y Zhang Shijun S Liu Lingling L Wu Yuchen Y Zhao Pandeng P Peng Zhifeng Z Liu Shengli S Zhang Zhonghua Z Li Wengang W

Animals : an open access journal from MDPI 20201004 10

The outbreak of pseudorabies in China, caused by more virulent pseudorabies virus (PRV) than the classical strains, has led to considerable economic losses. In this study, PRV strain HNXY was isolated from the Henan province of China in 2015 from the pig farm with severe reproductive failure in sows and a high mortality in piglets. The 50% tissue culture infectious doses (TCID50) of HNXY in Vero cells were examined to be 106.5/mL, and the neutralisation titer against Bartha ...[more]

PMID: 33020441

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Project description:Since 2011, pseudorabies based on the pseudorabies virus (PRV) variant has emerged as a serious health issue in pig farms in China. The PRV gE/TK or gE/gI/TK deletion strains protect against emerging PRV variants. However, these variants may cause lethal infections in newborn piglets without PRV antibodies. Previous studies have shown that codon deoptimization of a virulence gene causes virus attenuation. Accordingly, we deoptimized US3-S (US3 gene encoding a short isoform that represents approximately 95% of the total US3 transcription) and UL56 genes (first 10 or all codons) of PRV gE/TK deletion strain (PRVΔTK&gE-AH02) to generate six recombinant PRVs through bacterial artificial chromosome technology. In swine testicular cells, recombinant PRVs with all codon deoptimization of US3-S or UL56 genes were grown to lower titers than the parental virus. Notably, US3-S or UL56 with all codon deoptimization reduced mRNA and protein expressions. Subsequently, the safety and immunogenicity of recombinant PRVs with codon deoptimization of US3-S or UL56 are evaluated as vaccine candidates in mice and piglets. The mice inoculated with recombinant PRVs with codon deoptimization of US3-S or UL56 showed exceptional survival ability without severe clinical signs. All codons deoptimized (US3-S and UL56) significantly decreased virus load and attenuated pathological changes in the brains of the mice. Moreover, the protection efficiency offered by recombinant PRVs with codon deoptimization of US3-S or UL56 showed similar effects to PRVΔTK&gE-AH02. Remarkably, the 1-day-old PRV antibody-negative piglets inoculated with PRVΔTK&gE-US3-ST-CD (a recombinant PRV with all codon deoptimization of US3-S) presented no abnormal clinical symptoms, including fever. The piglets inoculated with PRVΔTK&gE-US3-ST-CD showed a high serum neutralization index against the PRV variant. In conclusion, these results suggest using codon deoptimization to generate innovative live attenuated PRV vaccine candidates.

Project description: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?TK&gE-AH02) was generated, and its structure verified by PCR, RFLP and sequencing. Growth kinetics test showed that PRV?TK&gE-AH02 reached a titer of 107.5 TCID50 /mL on ST cells. The PRV?TK&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 PRV?TK&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 PRV?TK&gE-AH02 groups provided complete protection against challenge at only 7 days post intramuscular vaccination. More importantly, PRV?TK&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?TK&gE-AH02 can reach high titers on ST cells. The live vaccine of PRV?TK&gE-AH02 is highly safe, and can not only provide clinical protection but also stops virus shedding. This study suggests that PRV?TK&gE-AH02 might work as a promising vaccine candidate to combat the PRV variant emerging in Chinese herds since 2011.

Dataset Information

Isolation and Characterization of a Variant Psedorabies Virus HNXY and Construction of rHNXY-?TK/?gE.

Publications

Isolation and Characterization of a Variant Psedorabies Virus HNXY and Construction of rHNXY-∆<i>TK</i>/∆<i>gE</i>.

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