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A Novel Strategy of US3 Codon De-Optimization for Construction of an Attenuated Pseudorabies Virus against High Virulent Chinese Pseudorabies Virus Variant.

ABSTRACT: In this study, we applied bacterial artificial chromosome (BAC) technology with PRV^ΔTK/gE/gI as the base material to replace the first, central, and terminal segments of the US3 gene with codon-deoptimized fragments via two-step Red-mediated recombination in E. coli GS1783 cells. The three constructed BACs were co-transfected with gI and part of gE fragments carrying homologous sequences (gI+gE'), respectively, in swine testicular cells. These three recombinant viruses with US3 codon de-optimization ((PRV^{ΔTK&gE-US3deop-1}, PRV^{ΔTK&gE-US3deop-2}, and PRV^{ΔTK&gE-US3deop-3}) were obtained and purified. These three recombinant viruses exhibited similar growth kinetics to the parental AH02LA strain, stably retained the deletion of TK and gE gene fragments, and stably inherited the recoded US3. Mice were inoculated intraperitoneally with the three recombinant viruses or control virus PRV^ΔTK&gEAH02 at a 10^7.0 TCID₅₀ dose. Mice immunized with PRV^{ΔTK&gE-US3deop-1} did not develop clinical signs and had a decreased virus load and attenuated pathological changes in the lungs and brain compared to the control group. Moreover, immunized mice were challenged with 100 LD₅₀ of the AH02LA strain, and PRV^{ΔTK&gE-US3deop-1} provided similar protection to that of the control virus PRV^ΔTK&gEAH02. Finally, PRV^{ΔTK&gE-US3deop-1} was injected intramuscularly into 1-day-old PRV-negative piglets at a dose of 10^6.0 TCID₅₀. Immunized piglets showed only slight temperature reactions and mild clinical signs. However, high levels of seroneutralizing antibody were produced at 14 and 21 days post-immunization. In addition, the immunization of PRV^{ΔTK&gE-US3deop-1} at a dose of 10^5.0 TCID₅₀ provided complete clinical protection and prevented virus shedding in piglets challenged by 10^6.5 TCID₅₀ of the PRV AH02LA variant at 1 week post immunization. Together, these findings suggest that PRV^{ΔTK&gE-US3deop-1} displays great potential as a vaccine candidate.

SUBMITTER: Xu M

PROVIDER: S-EPMC10458909 | biostudies-literature | 2023 Jul

REPOSITORIES: biostudies-literature

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A Novel Strategy of US3 Codon De-Optimization for Construction of an Attenuated Pseudorabies Virus against High Virulent Chinese Pseudorabies Virus Variant.

Xu Mengwei M Wang Yiwei Y Liu Yamei Y Chen Saisai S Zhu Laixu L Tong Ling L Zheng Yating Y Osterrieder Nikolaus N Zhang Chuanjian C Wang Jichun J

Vaccines 20230727 8

In this study, we applied bacterial artificial chromosome (BAC) technology with PRV<sup>ΔTK/gE/gI</sup> as the base material to replace the first, central, and terminal segments of the US3 gene with codon-deoptimized fragments via two-step Red-mediated recombination in <i>E. coli</i> GS1783 cells. The three constructed BACs were co-transfected with gI and part of gE fragments carrying homologous sequences (gI+gE'), respectively, in swine testicular cells. These three recombinant viruses with US3 ...[more]

PMID: 37631856

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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:Tunneling nanotubes (TNTs) are long bridge-like structures that connect eukaryotic cells and mediate intercellular communication. We found earlier that the conserved alphaherpesvirus US3 protein kinase induces long cell projections that contact distant cells and promote intercellular virus spread. In this report, we show that the US3-induced cell projections constitute TNTs. In addition, we report that US3-induced TNTs mediate intercellular transport of information (e.g., green fluorescent protein [GFP]) in the absence of other viral proteins. US3-induced TNTs are remarkably stable compared to most TNTs described in the literature. In line with this, US3-induced TNTs were found to contain stabilized (acetylated and detyrosinated) microtubules. Transmission electron microscopy showed that virus particles are individually transported in membrane-bound vesicles in US3-induced TNTs and are released along the TNT and at the contact area between a TNT and the adjacent cell. Contact between US3-induced TNTs and acceptor cells is very stable, which correlated with a marked enrichment in adherens junction components beta-catenin and E-cadherin at the contact area. These data provide new structural insights into US3-induced TNTs and how they may contribute to intercellular communication and alphaherpesvirus spread.IMPORTANCE Tunneling nanotubes (TNT) represent an important and yet still poorly understood mode of long-distance intercellular communication. We and others reported earlier that the conserved alphaherpesvirus US3 protein kinase induces long cellular protrusions in infected and transfected cells. Here, we show that US3-induced cell projections constitute TNTs, based on structural properties and transport of biomolecules. In addition, we report on different particular characteristics of US3-induced TNTs that help to explain their remarkable stability compared to physiological TNTs. In addition, transmission electron microscopy assays indicate that, in infected cells, virions travel in the US3-induced TNTs in membranous transport vesicles and leave the TNT via exocytosis. These data generate new fundamental insights into the biology of (US3-induced) TNTs and into how they may contribute to intercellular virus spread and communication.

Dataset Information

A Novel Strategy of US3 Codon De-Optimization for Construction of an Attenuated Pseudorabies Virus against High Virulent Chinese Pseudorabies Virus Variant.

Publications

A Novel Strategy of US3 Codon De-Optimization for Construction of an Attenuated Pseudorabies Virus against High Virulent Chinese Pseudorabies Virus Variant.

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