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Generation of an Attenuated Tiantan Vaccinia Virus Strain by Deletion of Multiple Genes.

ABSTRACT: An attenuated vaccinia virus-MVTTEAB-was constructed by deletion of non-essential gene segments related to the immunomodulatory and virulence functions of the vaccinia virus Tiantan strain (VVTT). The shuttle plasmids pTC-EGFP, pTE-EGFP, pTA35-EGFP, pTB-EGFP, and pTA66-EGFP were constructed and combined with the early and late strong promoter pE/L and EGFP as an exogenous selectable marker. Then, through the homologous recombination technology and Cre/loxP system, the following gene fragments were gradually knocked out one by one: TC7L-TK2L, TE3L, TA35R, TB13R, and TA66R. Ultimately, the five-segment-deleted attenuated strain MVTTEAB was obtained. Knockout of these segments and genetic stability of MVTTEAB were confirmed, and it was also shown that knockout of these segments did not affect the replication ability of the virus. Further, a series of in vivo and in vitro experiments demonstrated that the virulence of MVTTEAB was attenuated significantly, but at same time, high immunogenicity was maintained. These results indicate that MVTTEAB has potential for clinical use as a safe viral vector or vaccine with good attenuation and immunogenicity.

SUBMITTER: Li Y

PROVIDER: S-EPMC5671601 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Generation of an Attenuated Tiantan Vaccinia Virus Strain by Deletion of Multiple Genes.

Li Yiquan Y Zhu Yilong Y Chen Shuang S Li Wenjie W Yin Xunzhe X Li Shanzhi S Xiao Pengpeng P Han Jicheng J Li Xiao X Sun Lili L Jin Ningyi N

Frontiers in cellular and infection microbiology 20171031

An attenuated vaccinia virus-MVTT<sub>EAB</sub>-was constructed by deletion of non-essential gene segments related to the immunomodulatory and virulence functions of the vaccinia virus Tiantan strain (VVTT). The shuttle plasmids pTC-EGFP, pTE-EGFP, pTA35-EGFP, pTB-EGFP, and pTA66-EGFP were constructed and combined with the early and late strong promoter pE/L and EGFP as an exogenous selectable marker. Then, through the homologous recombination technology and Cre/loxP system, the following gene f ...[more]

PMID: 29164070

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Project description:Unlike RNA viruses, most DNA viruses replicate their genomes with high-fidelity polymerases that rarely make base substitution errors. Nevertheless, experimental evolution studies have revealed rapid acquisition of adaptive mutations during serial passage of attenuated vaccinia virus (VACV). One way in which adaptation can occur is by an accordion mechanism in which the gene copy number increases followed by base substitutions and, finally, contraction of the gene copy number. Here, we show rapid acquisition of multiple adaptive mutations mediated by a gene-inactivating frameshift mechanism during passage of an attenuated VACV. Attenuation had been achieved by exchanging the VACV A8R intermediate transcription factor gene with the myxoma virus ortholog. A total of seven mutations in six different genes occurred in three parallel passages of the attenuated virus. The most frequent mutations were single-nucleotide insertions or deletions within runs of five to seven As or Ts, although a deletion of 11 nucleotides also occurred, leading to frameshifts and premature stop codons. During 10 passage rounds, the attenuated VACV was replaced by the mutant viruses. At the end of the experiment, virtually all remaining viruses had one fixed mutation and one or more additional mutations. Although nucleotide substitutions in the transcription apparatus accounted for two low-frequency mutations, frameshifts in genes encoding protein components of the mature virion, namely, A26L, G6R, and A14.5L, achieved 74% to 98% fixation. The adaptive role of the mutations was confirmed by making recombinant VACV with A26L or G6R or both deleted, which increased virus replication levels and decreased particle/PFU ratios.IMPORTANCE Gene inactivation is considered to be an important driver of orthopoxvirus evolution. Whereas cowpox virus contains intact orthologs of genes present in each orthopoxvirus species, numerous genes are inactivated in all other members of the genus. Inactivation of additional genes can occur upon extensive passaging of orthopoxviruses in cell culture leading to attenuation in vivo, a strategy for making vaccines. Whether inactivation of multiple viral genes enhances replication in the host cells or has a neutral effect is unknown in most cases. Using an experimental evolution protocol involving serial passages of an attenuated vaccinia virus, rapid acquisition of inactivating frameshift mutations occurred. After only 10 passage rounds, the starting attenuated vaccinia virus was displaced by viruses with one fixed mutation and one or more additional mutations. The high frequency of multiple inactivating mutations during experimental evolution simulates their acquisition during normal evolution and extensive virus passaging to make vaccine strains.

Dataset Information

Generation of an Attenuated Tiantan Vaccinia Virus Strain by Deletion of Multiple Genes.

Publications

Generation of an Attenuated Tiantan Vaccinia Virus Strain by Deletion of Multiple Genes.

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