Direct or DNA Extraction-Free Amplification and Quantification of Foodborne Pathogens.

The use of direct nucleic acid amplification of pathogens from food matrices has the potential to reduce time to results over DNA extraction-based approaches as well as traditional culture-based approaches. Here we describe protocols for assay design and experiments for direct amplification of foodborne pathogens in food sample matrices using loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR). The examples provided include the detection of Escherichia coli in milk samples and Salmonella in pork meat samples. This protocol includes relevant reagents and methods including obtaining target sequences, assay design, sample processing, and amplification. These methods, though used for specific example matrices, could be applied to many other foodborne pathogens and sample types.

Role of RNA polymerase III transcription and regulation in ischaemic stroke.

Ischaemic stroke is a leading cause of death and life-long disability due to neuronal cell death resulting from interruption of glucose and oxygen supplies. RNA polymerase III (Pol III)-dependent transcription plays a central role in protein synthesis that is necessary for normal cerebral neuronal functions, and the survival and recovery under pathological conditions. Notably, Pol III transcription is highly sensitive to ischaemic stress that is known to rapidly shut down Pol III transcriptional activity. However, its precise role in ischaemic stroke, especially during the acute and recovery phases, remains poorly understood. The microenvironment within the ischaemic brain undergoes dynamic changes in different phases after stroke. Emerging evidence highlights the distinct roles of Pol III transcription in neuroprotection during the acute phase and repair during the recovery phase of stroke. Additionally, investigations into the mTOR-MAF1 signalling pathway, a conserved regulator of Pol-III transcription, reveal its therapeutic potential in enhancing acute phase neuroprotection and recovery phase repair.

AAV2 can replicate its DNA by a rolling hairpin or rolling circle mechanism, depending on the helper virus.

Adeno-associated virus type 2 (AAV2) is a small, non-pathogenic, helper virus-dependent parvovirus with a single-stranded (ss) DNA genome of approximately 4.7 kb. AAV2 DNA replication requires the presence of a helper virus such as adenovirus type 5 (AdV5) or herpes simplex virus type 1 (HSV-1) and is generally assumed to occur as a strand-displacement rolling hairpin (RHR) mechanism initiated at the AAV2 3' inverted terminal repeat (ITR). We have recently shown that AAV2 replication supported by HSV-1 leads to the formation of double-stranded head-to-tail concatemers, which provides evidence for a rolling circle replication (RCR) mechanism. We have revisited AAV2 DNA replication and specifically compared the formation of AAV2 replication intermediates in the presence of either HSV-1 or AdV5 as the helper virus. The results confirmed that the AAV2 DNA replication mechanism is helper virus-dependent and follows a strand-displacement RHR mechanism when AdV5 is the helper virus and primarily an RCR mechanism when HSV-1 is the helper virus. We also demonstrate that recombination plays a negligible role in AAV2 genome replication. Interestingly, the formation of high-molecular-weight AAV2 DNA concatemers in the presence of HSV-1 as the helper virus was dependent on an intact HSV-1 DNA polymerase. IMPORTANCE: AAV is a small helper virus-dependent, non-pathogenic parvovirus. The AAV genome replication mechanism was extensively studied in the presence of AdV as the helper virus and described to proceed using RHR. Surprisingly, HSV-1 co-infection facilitates RCR of the AAV2 DNA. We directly compared AdV5 and HSV-1 supported AAV2 DNA replication and showed that AAV2 can adapt its replication mechanism to the helper virus. A detailed understanding of the AAV replication mechanism expands our knowledge of virus biology and can contribute to increase gene therapy vector production.

Strand-specific PCR-competitive replication and adduct bypass assay for assessing how DNA adducts perturb DNA replication in mammalian cells.

Human genomes are susceptible to damage by a variety of endogenous and exogenous agents. If not repaired, the resulting DNA lesions can potentially lead to mutations, genome instability, and cell death. While existing in vitro experiments allow for characterizing replication outcomes from the use of purified translesion synthesis (TLS) DNA polymerases, such studies often lack the sophistication and dynamic nature of cellular contexts. Here, we present a strand-specific PCR-based Competitive Replication and Adduct Bypass (ssPCR-CRAB) assay designed to investigate quantitatively the impact of DNA lesions on replication efficiency and fidelity in mammalian cells. Combined with genetic manipulation, this approach facilitates the revelation of diverse functions of TLS polymerases in replication across DNA lesions.

Direct, ensemble FRET approaches to monitor transient state kinetics of human DNA polymerase δ holoenzyme assembly and initiation of DNA synthesis.

In humans, DNA polymerase δ (pol δ) holoenzymes, comprised of pol δ and the processivity sliding clamp, proliferating cell nuclear antigen (PCNA), carry out DNA synthesis during lagging strand replication, the initiation of leading strand DNA replication as well as most of the major DNA damage repair pathways. In each of these contexts, pol δ holoenzymes are assembled at primer/template (P/T) junctions and initiate DNA synthesis in a stepwise process that involves the PCNA clamp loader, replication factor C and, depending on the DNA synthesis pathway, the major single strand DNA-binding protein complex, replication protein A (RPA). In a recent report from our laboratory, we designed and utilized direct, ensemble Förster Resonance Energy Transfer approaches to monitor the transient state kinetics of pol δ holoenzyme assembly and initiation of DNA synthesis on P/T junctions engaged by RPA. In this chapter, we detail the original approaches and discuss adaptations that can be utilized to monitor fast kinetic reactions in the millisecond (ms) timescale. All approaches described in this chapter utilize a commercially-available fluorescence spectrophotometer, can be readily evolved for alternative DNA polymerases and P/T DNA substrates, and permit incorporation of protein posttranslational modifications, accessory factors, DNA covalent modifications, accessory factors, enzymes, etc. Hence, these approaches are widely accessible and broadly applicable for characterizing DNA polymerase holoenzyme assembly and initiation of DNA synthesis during any PCNA-dependent DNA synthesis pathway.

Studying the intersection of nucleoside modifications and SARS-CoV-2 RNA-dependent RNA transcription using an in vitro reconstituted system.

There is growing recognition that viral RNA genomes possess enzymatically incorporated modified nucleosides. These small chemical changes are analogous to epigenomic modifications in DNA and have the potential to be similarly important modulators of viral transcription and evolution. However, the molecular level consequences of individual sites of modification remain to be broadly explored. Here we describe an in vitro assay to examine the impact of nucleoside modifications on the rate and fidelity of SARS-CoV-2 RNA transcription. Establishing the role of modified nucleotides in SARS-CoV-2 is of interest both for advancing fundamental knowledge of RNA modifications in viruses, and because modulating the modification-landscape of SARS-CoV-2 may represent a therapeutic strategy to interfere with viral RNA replication. Our approach can be used to assess the influence both of modifications present in a template RNA, as well nucleotide analog inhibitors. These methods provide a reproducible guide for generating active SARS-CoV-2 replication/transcription complexes capable of establishing how RNA modifications influence the pre-steady state rate constants of nucleotide addition by RNA-dependent RNA polymerases.

A model for transcription-dependent R-loop formation at double-stranded DNA breaks: Implications for their detection and biological effects.

R-loops are structures containing an RNA-DNA duplex and an unpaired DNA strand. During R-loop formation an RNA strand invades the DNA duplex, displacing the homologous DNA strand and binding the complementary DNA strand. Here we analyze a model for transcription-dependent R-loop formation at double-stranded DNA breaks (DSBs). In this model, R-loop formation is preceded by detachment of the non-template DNA strand from the RNA polymerase (RNAP). Then, strand exchange is initiated between the nascent RNA and the non-template DNA strand. During that strand exchange the length of the R-loop could either increase, or decrease in a biased random-walk fashion, in which the bias would depend upon the DNA sequence. Eventually, the restoration of the DNA duplex would completely displace the RNA. However, as long as the RNAP remains bound to the template DNA strand it prevents that displacement. Thus, according to the model, RNAPs stalled at DSBs can increase the lifespan of R-loops, increasing their detectability in experiments, and perhaps enhancing their biological effects.

Rapid genome-wide profiling of DNA methylation and genetic variation using guide positioning sequencing (GPS).

Whole-genome bisulfite sequencing (WGBS) has been extensively utilized for DNA methylation profiling over the past decade. However, it has shown limitations in terms of high costs and inefficiencies. The productivity and accuracy of DNA methylation detection rely critically on the optimization of methodologies and the continuous refinements of related sequencing platforms. Here, we describe a detailed protocol of guide positioning sequencing (GPS), a bisulfite-based, location-specific sequencing technology designed for comprehensive DNA methylation characterization across the genome. The fundamental principle of GPS lies in the substitution of dCTP with 5-methyl-dCTP (5 mC) at the 3'-end of DNA fragments by T4 DNA polymerase, which protects cytosines from bisulfite conversion to preserve the integrity of the base composition. This alteration allows the 3'-end to independently facilitate genetic variation profiling and guides the 5'-end, enriched with methylation information, to align more rapidly to the reference genome. Hence, GPS enables the concurrent detection of both genetic and epigenetic variations. Additionally, we provide an accessible description of the data processing, specifically involving certain software and scripts. Overall, the entire GPS procedure can be completed within a maximum of 15 days, starting with a low initial DNA input of 100-500 ng, followed by 4-5 days for library construction, 8-10 days for high-throughput sequencing (HTS) and data analysis, which can greatly facilitate the promotion and application of DNA methylation detection, especially for the rapid clinical diagnosis of diverse disease pathologies associated with concurrent genetic and epigenetic variations.

HIV-1 reverse transcriptase error rates and transcriptional thresholds based on single-strand consensus sequencing of target RNA derived from in vitro-transcription and HIV-infected cells.

Nucleotide incorporation and lacZ-based forward mutation assays have been widely used to determine the accuracy of reverse transcriptases (RTs) in RNA-dependent DNA polymerization reactions. However, they involve quite complex and laborious procedures, and cannot provide accurate error rates. Recently, NGS-based methods using barcodes opened the possibility of detecting all errors introduced by the RT, although their widespread use is limited by cost, due to the large size of libraries to be sequenced. In this study, we describe a novel and relatively simple NGS assay based on single-strand consensus sequencing that provides robust results with a relatively small number of raw sequences (around 60 Mb). The method has been validated by determining the error rate of HIV-1 (BH10 strain) RT using the HIV-1 protease-coding sequence as target. HIV-1 reverse transcription error rates in standard conditions (37°C/3 mM Mg2+) using an in vitro-transcribed RNA were around 7.3×10-5. In agreement with previous reports, an 8-fold increase in RT's accuracy was observed after reducing Mg2+ concentration to 0.5 mM. The fidelity of HIV-1 RT was also higher at 50°C than at 37°C (error rate 1.5×10-5). Interestingly, error rates obtained with HIV-1 RNA from infected cells as template of the reverse transcription at 3 mM Mg2+ (7.4×10-5) were similar to those determined with the in vitro-transcribed RNA, and were reduced to 1.8×10-5 in the presence of 0.5 mM Mg2+. Values obtained at low magnesium concentrations were modestly higher than the transcription error rates calculated for human cells, thereby suggesting a realistic transcriptional threshold for our NGS-based error rate determinations.

Attenuation of the neuropathogenic equine herpesvirus type 1 strain Ab4p in hamsters by a single amino acid mutation (D752N) in viral DNA polymerase ORF30.

Equine herpesvirus type 1 (EHV-1) causes abortion, respiratory infection, and neurological diseases (equine herpesvirus myeloencephalopathy) in horses. A single nucleotide polymorphism (SNP) associated with a single amino acid in the DNA polymerase gene (ORF30, in which D752 is neuropathogenic and N752 is non-neuropathogenic) of EHV-1 has been associated with neuropathogenicity in horses. We constructed an EHV-1 Ab4p ORF30 N752 mutant and a repair virus to examine the effect of a D752N mutation on the neuropathogenicity of the virus in Syrian hamsters. The N752 mutation did not affect viral growth in cultured cells but it did attenuate the neuropathogenicity of Ab4p in the hamsters. The results suggest that D752N is involved in neuropathogenicity not only in horses but also in hamsters.

LINC01116-dependent upregulation of RNA polymerase I transcription drives oncogenic phenotypes in lung adenocarcinoma.

BACKGROUND: Hyperactive RNA Polymerase I (Pol I) transcription is canonical in cancer, associated with malignant proliferation, poor prognosis, epithelial-mesenchymal transition, and chemotherapy resistance. Despite its significance, the molecular mechanisms underlying Pol I hyperactivity remain unclear. This study aims to elucidate the role of long noncoding RNAs (lncRNAs) in regulating Pol I transcription in lung adenocarcinoma (LUAD). METHODS: Bioinformatics analyses were applied to identify lncRNAs interacting with Pol I transcriptional machinery. Fluorescence in situ hybridization was employed to examine the nucleolar localization of candidate lncRNA in LUAD cells. RNA immunoprecipitation assay validated the interaction between candidate lncRNA and Pol I components. Chromatin isolation by RNA purification and Chromatin Immunoprecipitation (ChIP) were utilized to confirm the interactions of candidate lncRNA with Pol I transcriptional machinery and the rDNA core promoter. Functional analyses, including lncRNA knock-in and knockdown, inhibition of Pol I transcription, quantitative PCR, cell proliferation, clonogenicity, apoptosis, cell cycle, wound-healing, and invasion assays, were performed to determine the effect of candidate lncRNA on Pol I transcription and associated malignant phenotypes in LUAD cells. ChIP assays and luminometry were used to investigate the transcriptional regulation of the candidate lncRNA. RESULTS: We demonstrate that oncogenic LINC01116 scaffolds essential Pol I transcription factors TAF1A and TAF1D, to the ribosomal DNA promoter, and upregulate Pol I transcription. Crucially, LINC01116-driven Pol I transcription activation is essential for its oncogenic activities. Inhibition of Pol I transcription abrogated LINC01116-induced oncogenic phenotypes, including increased proliferation, cell cycle progression, clonogenicity, reduced apoptosis, increased migration and invasion, and drug sensitivity. Conversely, LINC01116 knockdown reversed these effects. Additionally, we show that LINC01116 upregulation in LUAD is driven by the oncogene c-Myc, a known Pol I transcription activator, indicating a functional regulatory feedback loop within the c-Myc-LINC01116-Pol I transcription axis. CONCLUSION: Collectively, our findings reveal, for the first time, that LINC01116 enhances Pol I transcription by scaffolding essential transcription factors to the ribosomal DNA promoter, thereby driving oncogenic activities in LUAD. We propose the c-Myc-LINC01116-Pol I axis as a critical oncogenic pathway and a potential therapeutic target for modulating Pol I transcription in LUAD.

Complete genome sequence of a novel mitovirus identified in the phytopathogenic fungus Alternaria tenuissima.

In this study, a novel positive-sense single-stranded RNA (+ ssRNA) mycovirus, Alternaria tenuissima mitovirus 1 (AtMV1), was identified in Alternaria tenuissima strain YQ-2-1, a phytopathogenic fungus causing leaf blight on muskmelon. The genome of AtMV1 is a single RNA molecule that is 3013 nt in length with an A + U content of 66.58% and contains a single open reading frame (ORF) using the fungal mitochondrial genetic code. The ORF was predicted to encode a 313-amino-acid RNA-dependent RNA polymerase (RdRp) with a molecular mass of 35.48 kDa, which contains six conserved motifs with the highly conserved GDD tripeptide in motif IV. The 5' and 3' untranslated regions were predicted to fold into stem-loop and panhandle secondary structures. The results of a BLASTp search revealed that the amino acid (aa) sequence of RdRp of AtMV1 shared the highest sequence similarity (51.04% identity) with that of Sichuan mito-like virus 30, a member of the genus Duamitovirus within the family Mitoviridae. Phylogenetic analysis based on the aa sequence of the RdRp suggested that AtMV1 is a novel member of the genus Duamitovirus. To our knowledge, this is the first report of the complete genome sequence of a new mitovirus infecting A. tenuissima.

A case of immunocompetent intracranial cryptococcoma in which intraoperative rapid pathological diagnosis and polymerase chain reaction led to early treatment: What to know to avoid misdiagnosis as brain tumor.

Background: Cryptococcal infections of the central nervous system are infrequent in immunocompetent hosts and usually present as meningitis. However, a fungal mass called a cryptococcoma may form, requiring caution in therapeutic intervention. Here, we report a rare case in which treatment of intraventricular cryptococcoma in an immunocompetent patient was facilitated by rapid pathological diagnosis. Case Description: A 58-year-old previously healthy man was admitted to our hospital with fever, headache, and gradually worsening hearing loss over 1 month. Cerebrospinal fluid analysis showed moderately elevated levels of protein and lymphocytic cells and decreased glucose. In addition, ß2-microglobulin was highly elevated. Magnetic resonance imaging showed homogeneously enhanced lesions in lateral ventricles of the left and right hemispheres and the subarachnoid space, and 18F-fluorodeoxyglucose positron emission tomography revealed abnormal uptake corresponding to the lesion. A surgical excision was performed to achieve a definitive diagnosis. Intraoperative rapid pathology, including immunohistochemistry (IHC), yielded negative results for malignant tumor, suggesting the possibility of inflammatory granuloma. Additional targeted pathological diagnosis was immediately performed. Paraffin-embedded histopathological examination showed fibrocaseous granuloma and numerous fungal spores. Cryptococcus neoformans within the granuloma were suggested by Fontana-Masson and Grocott staining and confirmed by polymerase chain reaction (PCR), leading to a diagnosis of cryptococcoma. Antifungal agents were started 3 days postoperatively. The patient has since been doing well, with no recurrence. Conclusion: This pathology can be difficult to distinguish from a brain tumor, so early pathological diagnosis, including rapid pathology with IHC and PCR, may be crucial.

NusG-Spt5 Transcription Factors: Universal, Dynamic Modulators of Gene Expression.

The accurate and efficient biogenesis of RNA by cellular RNA polymerase (RNAP) requires accessory factors that regulate the initiation, elongation, and termination of transcription. Of the many discovered to date, the elongation regulator NusG-Spt5 is the only universally conserved transcription factor. With orthologs and paralogs found in all three domains of life, this ubiquity underscores their ancient and essential regulatory functions. NusG-Spt5 proteins evolved to maintain a similar binding interface to RNAP through contacts of the NusG N-terminal domain (NGN) that bridge the main DNA-binding cleft. We propose that varying strength of these contacts, modulated by tethering interactions, either decrease transcriptional pausing by smoothing the rugged thermodynamic landscape of transcript elongation or enhance pausing, depending on which conformation of RNAP is stabilized by NGN contacts. NusG-Spt5 contains one (in bacteria and archaea) or more (in eukaryotes) C-terminal domains that use a KOW fold to contact diverse targets, tether the NGN, and control RNA biogenesis. Recent work highlights these diverse functions in different organisms. Some bacteria contain multiple specialized NusG paralogs that regulate subsets of operons via sequence-specific targeting, controlling production of antibiotics, toxins, or capsule proteins. Despite their common origin, NusG orthologs can differ in their target selection, interacting partners, and effects on RNA synthesis. We describe the current understanding of NusG-Spt5 structure, interactions with RNAP and other regulators, and cellular functions including significant recent progress from genome-wide analyses, single-molecule visualization, and cryo-EM. The recent findings highlight the remarkable diversity of function among these structurally conserved proteins.

Description of the complete rDNA repeat unit structure of Coturnixjaponica Temminck et Schlegel, 1849 (Aves).

Ribosomal RNA (18S, 5.8S, 28S) gene clusters in genomes form regions that consist of multiple tandem repeats. They are located on a single or several pairs of chromosomes and play an important role in the formation of the nucleolus responsible for the assembly of ribosome subunits. The rRNA gene cluster sequences are widely used for taxonomic studies, however at present, complete information on the avian rDNA repeat unit structure including intergenic spacer sequence is available only for the chicken (Gallusgallusdomesticus Linnaeus, 1758). The GC enrichment and high-order repeats peculiarities within the intergenic spacer described for the chicken rDNA cluster may be responsible for these failures. The karyotype of the Japanese quail (Coturnixjaponica Temminck et Schlegel, 1849) deserves close attention because, unlike most birds, it has three pairs of nucleolar organizer bearing chromosomes, two of which are microchromosomes enriched in repeating elements and heterochromatin that carry translocated terminal nucleolar organizers. Here we assembled and annotated the complete Japanese quail ribosomal gene cluster sequence of 21166 base pairs (GenBank under the registration tag BankIt2509210 CoturnixOK523374). This is the second deciphered avian rDNA cluster after the chicken. Despite the revealed high similarity with the chicken corresponding sequence, it has a number of specific features, which include a slightly lower degree of GC content and the presence of bendable elements in the content of both the transcribed spacer I and the non-transcribed intergenic spacer.

Engineering Phi29-DNAP Variants for Customized DNA Hydrogel Materials.

DNA hydrogels, which hold potential for use in medicine, biosensors, and tissue engineering, can be produced through enzymatic rolling circle amplification (RCA) using phi29 DNA polymerase (DNAP). This paper introduces new DNAP variants designed for RCA-based DNA hydrogel production, featuring enzymes with modified DNA binding, enhanced thermostability, reduced exonuclease activity, and protein tags for fluorescence detection or specific immobilization. We evaluated these enzymes by quantifying DNA output via quantitative PCR (qPCR) and assessing hydrogel mechanical properties through micromechanical indentation. The results showed that most variants generated similar DNA amounts and hydrogels with comparable mechanical properties. Additionally, all variants successfully incorporated non-natural nucleotides, such as base-modified dGTP derivatives and 2'-fluoro-dGTP, during RCA. This study's robust analytical approach offers a strong foundation for selecting new enzymes and producing DNA hydrogels with tailored material properties.

[Regulation of Transcription by RNA Polymerase III Promotors in the Norm and Pathology].

RNA polymerase III synthesizes a wide range of noncoding RNAs shorter than 400 nucleotides in length. These RNAs are involved in protein synthesis (tRNA, 5S rRNA, and 7SL RNA), maturation, and splicing of different types of RNA (RPR, MRP RNA, and U6 snRNA), regulation of transcription (7SK RNA), replication (Y RNA), and intracellular transport (vault RNA). BC200 and BC1 RNA genes are transcribed by RNA polymerase III in neurons only where these RNAs regulate protein synthesis. Mutations in the regulatory elements of the genes transcribed by RNA polymerase III as well as in transcription factors of this RNA polymerase are associated with the development of a number of diseases, primarily oncological and neurological. In this regard, the mechanisms of regulation of the expression of the genes containing various RNA polymerase III promoters were actively studied. This review describes the structural and functional classification of polymerase III promoters, as well as the factors involved in the regulation of promoters of different types. A number of examples demonstrate the role of the described factors in the pathogenesis of human diseases.

A gold nanoparticle-enhanced dCas9-mediated fluorescence resonance energy transfer for nucleic acid detection.

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas proteins coupled with pre-amplification have shown great potential in molecular diagnoses. However, the current CRISPR-based methods require additional reporters and time-consuming process. Herein, a gold nanoparticle (AuNP)-enhanced CRISPR/dCas9-mediated fluorescence resonance energy transfer (FRET) termed Au-CFRET platform was proposed for rapid, sensitive, and specific detection of nucleic acid for the first time. In the Au-CFRET sensing platform, AuNP was functionalized with dCas9 and used as nanoprobe. Target DNA was amplified with FAM-labeled primers and then precisely bound with AuNP-dCas9. The formed complex rendered the distance between AuNP acceptor and FAM donor to be short enough for the occurrence of FRET, thus resulting in fluorescence quenching. Moreover, AuNPs were demonstrated to enhance binding efficiency of dCas9 to target DNA in Au-CFRET system. The key factors regarding the FRET efficiency were analyzed and characterized in detail, including the length of donor/acceptor and the size of AuNPs. Under the optimal conditions, Au-CFRET could determinate CaMV35S promoter of genetically modified rice as low as 21 copies µL-1. Moreover, Au-CFRET sensing system coupled with one-step extraction and recombinase polymerase amplification can identify the genuine plant seeds within 30 min from sampling to results at room/body temperature without expensive equipment or technical expertise, and requires no additional exogenous reporters. Therefore, the proposed sensing platform significantly simplified the system and shortened the assay time for nucleic acid diagnoses.

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

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.