Viral coexistence and insertional mutations in the ORF8 region of SARS-CoV-2: A possible mechanism of nucleotide insertion

•Virus from swab sample S66 in hiroshima had T-base insertions in the ORF8 region.•Cloning, and TIDE analysis confirmed mixed viruses with different T-base insertions.•Virus with one t (T1+) was 70–75 %; others included T0, T2+, T3+, T4+, and T5+.•C27911T point mutation led to a sequence of seven or...

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Published in:	Virus research Vol. 350; p. 199478
Main Authors:	Kurose, Miuko, Yamamoto, Akima, Elsayed, Abeer Mohamed Abdelfattah, Lawal-Ayinde, Basirat Mojisola, Nomura, Toshihito, Higashiura, Akifumi, Irie, Takashi, Fukushi, Masaya, Kanda, Miyuki, Tahara, Hidetoshi, Morita, Daichi, Kuroda, Teruo, Ko, Ko, Takahashi, Kazuaki, Tanaka, Junko, Sakaguchi, Takemasa
Format:	Journal Article
Language:	English
Published:	Netherlands Elsevier B.V 01-12-2024 Elsevier
Subjects:	Covid-19 NGS analysis Polymerase stuttering T-base insertions TIDE analysis Covid-19 MOI TIDE analysis T-base insertions TCID50 DMEM TIDE Polymerase stuttering RT-PCR NGS analysis CPE BSL NGS COVID-19 polymerase stuttering
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Summary:	•Virus from swab sample S66 in hiroshima had T-base insertions in the ORF8 region.•Cloning, and TIDE analysis confirmed mixed viruses with different T-base insertions.•Virus with one t (T1+) was 70–75 %; others included T0, T2+, T3+, T4+, and T5+.•C27911T point mutation led to a sequence of seven or more consecutive t bases.•Study highlights a potential mechanism for insertion mutations in SARS-CoV-2. The virus obtained from a swab sample ID: S66 in Hiroshima was reported to have a single T-base insertion in the ORF8 coding region. However, no T insertion was observed when we determined the genomic sequence using another method. We then extracted RNA from the S66 swab sample and sequenced the insertion site using the Sanger method. The resulting waveform was disrupted beyond the insertion site, suggesting the presence of a mixed population of viruses with different sequences. Through plasmid cloning of RT-PCR amplification fragments and virus cloning by limiting dilution, along with TIDE analysis to determine the ratio of components from the Sanger sequencing waveform, it was confirmed that the sample contained a mixture of viruses with varying numbers of T-base insertions. The virus with one T insertion (T1+) was predominant in 70–75 % of the genomes, and genomes with T0, T2+, T3+, T4+, and T5+ were also detected. No T-base insertion mutations were observed in the ORF8 region in three other SARS-CoV-2 samples. In the S66 sample, a C27911T point mutation near the insertion site in the ORF8 region resulted in a sequence of seven or more consecutive T bases, which was the cause of the T-base insertion. When the cloned S66 virus (T1+) was passaged in cultured cells, there was a tendency for viruses with more insertion bases to become dominant with successive generations, suggesting that the T-base insertion was due to polymerase stuttering. The insertion of T bases resulted in synthesis of deletion mutants of the ORF8 protein, but no significant change was observed in the proliferation of the viruses in cultured cells. A search of the GenBank database using NCBI BLAST for viruses similar to S66 with T-base insertion mutations revealed hundreds of viruses widely distributed on the molecular phylogenetic tree. These base insertion viruses were thought to have occasionally arisen during the virus infection process. This study suggests one mechanism of insertion mutations in SARS-CoV-2, and it is important to consider the emergence of future mutant strains.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0168-1702 1872-7492 1872-7492
DOI:	10.1016/j.virusres.2024.199478