Biology subjects

Matsugo, H.

Publications and source records attributed to Matsugo, H..

7 recordsLinked to original sources

bioRxiv2026

Distinct virus-specific regulation of RNA synthesis across genome segments by thogotovirus polymerases: insights from Oz virus and Dhori virus

Thogotoviruses are a group of tick-borne, six-segmented, negative-sense single-stranded RNA viruses. These viruses encode an RNA-dependent RNA polymerase that recognizes promoter sequences located at the genomic termini to initiate RNA synthesis. The 5' and 3' ends of the genome bind to the polymerase and function as a promoter. Outside the catalytic center, they base-pair with each other to form a double-stranded RNA structure. This structure is referred to as the distal duplex and plays an important role in RNA synthesis. In this study, we investigated how the RNA sequence of the distal duplex influences polymerase activity using minigenome systems of two thogotoviruses, Oz virus (OZV) and Dhori virus (DHOV). Each virus exhibits distinct activities among its six segments. In OZV, one determinant of these differences is the base pair at positions 5'12 and 3'11 within the distal duplex, where promoter activity varies depending on whether the base pair is G:C or A:U. In contrast, the DHOV polymerase is not affected by this difference. These results indicate that, even within the genus Thogotovirus, viruses differ in whether they possess a mechanism that modulates promoter activity based on subtle sequence differences within the distal duplex. Furthermore, phylogenetic analysis and comparison of promoter sequences suggest that thogotoviruses can be divided into groups that do or do not regulate intersegment promoter activity via the base pair at positions 5'12 and 3'11. HighlightsO_LIMinigenome systems of Oz virus and Dhori virus reveal segment-specific differences in promoter activity C_LIO_LIThe distal duplex sequence modulates RNA synthesis in a virus-dependent manner C_LIO_LIThe base pair at positions 5'12/3'11 determines promoter activity in Oz virus but not in Dhori virus C_LIO_LIThogotoviruses can be divided into groups that do or do not regulate promoter activity via distal duplex sequence variation at positions 5'12/3'11 C_LI

microbiology↗

bioRxiv2025

Recovery of infectious Oz virus from cloned cDNA

Oz virus (OZV) is a tick-borne, six-segmented, negative-strand RNA virus in the genus Thogotovirus, family Orthomyxoviridae. A fatal human infection was reported in Japan in 2023. In this study, we established a reverse genetics system to generate infectious recombinant OZV. Six plasmids encoding the full-length OZV genome segments under a murine RNA polymerase I promoter, together with four plasmids expressing viral proteins essential for polymerase activity, were co-transfected into murine cells. This approach enabled efficient recovery of infectious OZV. The recovered recombinant virus exhibited replication kinetics comparable to wild-type OZV. This system provides a platform for molecular studies of OZV.

microbiology↗

bioRxiv2024

Segment-specific promoter activity for RNA synthesis in the genome of Oz virus, genus Thogotovirus

Oz virus (OZV), a tick-borne, six-segmented negative-strand RNA virus in the genus Thogotovirus, caused a fatal human infection in Japan in 2023. To investigate mechanisms of viral RNA synthesis, we developed an OZV minigenome assay used in mammalian cells. Comparisons of promoter activities across six segments revealed that Segment 5 exhibited markedly lower promoter activity. Unlike the other segments forming a "distal duplex", a double-strand RNA beginning at the 11th nucleotide on the 5 end and the 10th on the 3, Segment 5 partially lacks this feature. Introducing a mutation in Segment 5 distal duplex resulted in a substantial increase in promoter activity. Further, we examined the promoter structures of other viruses in the genus Thogotovirus using public database. Thogoto virus Segment 1 also partially lacks a base pair in the distal duplex. In six-segmented RNA viruses, promoter activities are varied, with notable differences in activities likely existing among segments. HighlightsO_LIA minigenome assay was developed to elucidate the RNA synthesis mechanism of Oz virus. C_LIO_LISegment 5 of the Oz virus exhibits significantly lower promoter activity compared to other segments. C_LIO_LIThe distal duplex region formed by double-strand RNA at the genomic ends is essential for Oz virus RNA synthesis. C_LIO_LIPromoter activities in six-segmented RNA viruses are inherently variable among segments. C_LI

microbiology↗

bioRxiv2024

A single amino acid substitution in the Borna disease virus glycoprotein enhances the infectivity titer of vesicular stomatitis virus pseudotyped virus by altering membrane fusion activity

Borna disease virus 1 (BoDV-1) causes acute fatal encephalitis in mammals, including humans. Despite its importance, research on BoDV-1 cell entry has been hindered by low viral particle production in cells and the lack of cytopathic effects. To address these issues, we developed a method to efficiently produce vesicular stomatitis virus (VSV) pseudotyped with glycoprotein (G) of the genus Orthobornavirus, including BoDV-1. We discovered that optimal G expression is required to obtain a high infectivity titer of the VSV pseudotyped virus. Remarkably, the infectivity of the VSV pseudotyped virus with G from the BoDV-1 strain huP2br was significantly higher than that of the VSV pseudotyped virus with G from the He/80 strain. Mutational analysis demonstrated that the BoDV-1-G residue 307 determines the infectivity titer of VSV pseudotyped with BoDV-1-G (VSV-BoDV-1-G). A cell-cell fusion assay indicated that this residue plays a pivotal role in membrane fusion, thus suggesting that high membrane fusion activity and a broad pH range for membrane fusion are crucial for achieving a high infectivity titer of VSV-BoDV-1-G. This finding may be extended to increase the infectivity titer of VSV pseudotyped virus with other orthobornavirus G. Our study also contributes to identifying functional domains of BoDV-1-G and provides insight into G-mediated cell entry.

microbiology↗

bioRxiv2022

Isolation of bat sarbecoviruses of SARS-CoV-2 clade, Japan

Betacoronaviruses have caused 3 outbreaks in the past 2 decades. SARS-CoV-2, in particular, has caused a serious pandemic. As the betacoronaviruses are considered to originate from bats, surveillance of bat betacoronaviruses is crucial for understanding the mechanism of cross-species transition and potential for future outbreaks. We previously detected and characterized a SARS-CoV-2-related sarbecovirus, Rc-o319, from Rhinolophus cornutus in Japan. Here, we detected several bat sarbecoviruses of the SARS-CoV-2 clade from R. cornutus in multiple locations in Japan, and successfully isolated them using Vero/TMPRSS2 cells stably expressing R. cornutus ACE2 (Vero-RcACE2). The coding sequences of S1 region varied among isolates, whereas other genetic regions were highly conserved. Isolates were efficiently grown in Vero-RcACE2 cells, but did not replicate in Vero/TMPRSS2 cells stably expressing human ACE2, suggesting a narrow host range. Further long-term epidemiological studies of sarbecoviruses in wildlife are expected to facilitate the assessment of the risk of their spillover potential.

microbiology↗

bioRxiv2022

Influenza A virus agnostic receptor tropism revealed using a novel biological system with terminal sialic acid-knockout cells

Avian or human influenza A viruses bind preferentially to avian- or human-type sialic acid receptors, respectively, indicating that receptor tropism is an important factor for determining the viral host range. However, there are currently no reliable methods for analyzing receptor tropism biologically under physiological conditions. Here, we established a novel system using MDCK cells with avian- or human-type sialic acid receptors and with both sialic acid receptors knocked out (KO). When we examined the replication of human and avian influenza viruses in these KO cells, we observed unique viral receptor tropism that could not be detected using a conventional solid-phase sialylglycan binding assay, which directly assesses physical binding between the virus and sialic acids. Furthermore, we serially passaged an engineered avian-derived H4N5 influenza virus, whose PB2 gene was deleted, in avian-type receptor-KO cells stably expressing PB2 to select a mutant with enhanced replication in KO cells; however, its binding to human-type sialylglycan was undetectable using the solid-phase binding assay. These data indicate that a panel of sialic acid receptor-KO cells could be a useful tool for determining the biological receptor tropism of influenza A viruses. Moreover, the PB2-KO virus experimental system could help to safely and efficiently identify the mutations required for avian influenza viruses to adapt to human cells that could trigger a new influenza pandemic. Author summaryInfluenza A virus initiates infection via hemagglutinin by binding to avian- or human-type receptors. The acquisition of mutations that allow avian virus hemagglutinins (HAs) to recognize human-type receptors is mandatory for the transmission of avian influenza viruses to humans, which could lead to a pandemic. Therefore, it is important to detect such mutation(s) in animal influenza viruses for pandemic surveillance and risk assessment. In this study, we established a novel system using a set of genetically engineered MDCK cells with knocked out sialic acid receptors to biologically evaluate the receptor tropism for influenza A viruses. Using this system, we observed unique receptor tropism in several virus strains that was undetectable using conventional solid-phase binding assays that measure physical binding between the virus and artificially synthesized sialylglycans. This study makes a significant contribution to the literature because our findings suggest the pitfall of conventional receptor binding assay and the existence of a sialic acid-independent pathway for viral infection. In addition, our system could be safely used to identify mutations that could acquire human-type receptor tropism. Thus, this system could contribute not only toward basic analyses, such as elucidating the mechanism of influenza virus host range determination, but also the surveillance of viruses of animal origin that could be capable of infecting via human-type receptors, triggering a new influenza pandemic.

microbiology↗

bioRxiv2019

Establishment of a Reverse Genetics System for Influenza D Virus

Influenza D virus (IDV) was initially isolated in the USA in 2011. IDV is distributed worldwide and is one of the causative agents of bovine respiratory disease complex (BRDC), which exhibits high morbidity and mortality in feedlot cattle. Molecular mechanisms of IDV pathogenicity are still unknown. Reverse genetics systems are vital tools not only for studying the biology of viruses, but also for use in applications such as recombinant vaccine viruses. Here, we report the establishment of a plasmid-based reverse genetics system for IDV. We first verified that the 3'-terminal nucleotide of each 7-segmented genomic RNA contained uracil in contrary to the previous report, and were then able to successfully generate recombinant IDV by co-transfecting 7 plasmids containing these genomic RNAs along with 4 plasmids expressing polymerase proteins and NP into HRT-18G cells. The recombinant virus had a growth deficit compared to the wild-type virus, and we determined the reason for this growth difference by examining the genomic RNA content of the viral particles. We found that recombinant virus incorporated an unbalanced ratio of viral RNA segments into particles as compared to the wild-type virus, and thus we adjusted the amount of each plasmid used in transfection to obtain recombinant virus with the same replicative capacity as wild-type virus. Our work here in establishing a reverse genetics system for IDV will have a broad range of applications, including uses in studies focused on better understanding IDV replication and pathogenicity as well as those contributing to the development of BRDC countermeasures.\n\nIMPORTANCEBovine respiratory disease complex (BRDC) exhibits high mortality and morbidity in cattle, causing economic losses worldwide. Influenza D virus (IDV) is considered to be a causative agent of BRDC. Here, we developed a reverse genetics system that allows for the generation of IDV from cloned cDNAs, and the introduction of mutations into the IDV genome. This reverse genetics system will become a powerful tool for use in studies related to understanding the molecular mechanisms of viral replication and pathogenicity, and will also lead to the development of new countermeasures against BRDC.

microbiology↗