AIMS: To evaluate the stability of coxsackievirus B (CVB) genome integrated with the enhanced green fluorescent protein gene (egfp) and provide valuable information for the use of the recombinant CVB variant. METHODS: A CVB3 variant expressing eGFP was constructed by insertion of the egfp open-reading frame (ORF) at the 5' end of CVB3 ORF. The recombinant virus CVB3-eGFP was serially passaged in HeLa cells. The deletions in the CVB3-eGFP genome around egfp were examined by reverse transcription polymerase chain reaction and sequencing. RESULTS: Genomic deletions of CVB3-eGFP could be observed as early as the 2nd passage. Sequencing showed that the genomic deletions caused either viral ORF shifts or partial deletions of the viral VP4 coding sequence. The 6th passage of CVB3-eGFP was checked by plaque assay for eGFP expression. All plaque-like foci showed eGFP expression. eGFP expression was also viewed in HeLa cells infected with plaque-forming viruses. CONCLUSIONS: The insertion of egfp destabilized the CVB3 genome. The genomic deletions led to lethal mutations because of the termination of viral protein synthesis due to viral ORF shift and loss of partial viral gene. These findings imply that experimental data based on CVB integrated with the reporter gene should be interpreted with caution.
AIMS: To evaluate the stability of coxsackievirus B (CVB) genome integrated with the enhanced green fluorescent protein gene (egfp) and provide valuable information for the use of the recombinant CVB variant. METHODS: A CVB3 variant expressing eGFP was constructed by insertion of the egfp open-reading frame (ORF) at the 5' end of CVB3 ORF. The recombinant virus CVB3-eGFP was serially passaged in HeLa cells. The deletions in the CVB3-eGFP genome around egfp were examined by reverse transcription polymerase chain reaction and sequencing. RESULTS: Genomic deletions of CVB3-eGFP could be observed as early as the 2nd passage. Sequencing showed that the genomic deletions caused either viral ORF shifts or partial deletions of the viral VP4 coding sequence. The 6th passage of CVB3-eGFP was checked by plaque assay for eGFP expression. All plaque-like foci showed eGFP expression. eGFP expression was also viewed in HeLa cells infected with plaque-forming viruses. CONCLUSIONS: The insertion of egfp destabilized the CVB3 genome. The genomic deletions led to lethal mutations because of the termination of viral protein synthesis due to viral ORF shift and loss of partial viral gene. These findings imply that experimental data based on CVB integrated with the reporter gene should be interpreted with caution.
Authors: Irena Corbic Ramljak; Julia Stanger; Antonio Real-Hohn; Dominik Dreier; Laurin Wimmer; Monika Redlberger-Fritz; Wolfgang Fischl; Karin Klingel; Marko D Mihovilovic; Dieter Blaas; Heinrich Kowalski Journal: PLoS Pathog Date: 2018-08-06 Impact factor: 6.823