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Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors
Published on: September 13, 2018
Infection study of Bombyx mori macula-like virus (BmMLV) using a BmMLV-negative cell line and an infectious cDNA
Masashi Iwanaga1, Tomoyuki Hitotsuyama, Susumu Katsuma
1Department of Bioproductive Science, Faculty of Agriculture, Utsunomiya University, Utsunomiya-shi, Tochigi 321-8505, Japan. iwanaga@cc.utsunomiya-u.ac.jp
Abstract:
Previously, a novel macula-like virus was identified from Bombyx mori cultured cell line BmN and termed B. mori macula-like virus (BmMLV). BmMLV encodes a 6.5-kb-long positive, single-strand RNA genome, which contains putative RNA-dependent RNA polymerase (RdRp), coat protein (cp) and p15 genes. In this study, CP expression in several B. mori-derived cell lines was examined by using the CP antibody. Surprisingly, Western blot analysis revealed that all of the cell lines tested have already been infected with BmMLV. To perform reverse genetic studies in BmMLV, a new BmMLV-negative cell line, designated as BmVF from the embryos of B. mori was established. Infection studies showed that BmVF cells were permissive to BmMLV persistent infection. In addition, a full-length infectious cDNA clone of BmMLV, termed pHMLV was developed. Upon transfection of pHMLV into BmMLV-negative BmVF cells, viral CP was detected in both cells and conditioned medium. When the cDNA-derived virus in conditioned medium was inoculated onto BmVF cells, efficient propagation of BmMLV was observed. Collectively, these results indicate that the new BmMLV-negative cell line and the infectious cDNA clone of BmMLV will be useful for elucidation of the mechanism of BmMLV replication and the functional roles of BmMLV genes.
Insights
A novel Bombyx mori macula-like virus (BmMLV) was studied. Researchers developed a BmMLV-negative cell line and an infectious clone, enabling further research into BmMLV replication and gene function.
Area of Science:
- Virology
- Molecular Biology
- Insect Pathology
Background:
- A novel macula-like virus, Bombyx mori macula-like virus (BmMLV), was previously identified in silkworm cells.
- BmMLV possesses a positive-sense single-stranded RNA genome encoding RNA-dependent RNA polymerase (RdRp), coat protein (CP), and p15.
- Initial investigations revealed widespread BmMLV infection across various Bombyx mori cell lines.
Purpose of the Study:
- To establish a BmMLV-negative cell line for reverse genetics studies.
- To develop a full-length infectious cDNA clone of BmMLV.
- To investigate BmMLV replication and gene function.
Main Methods:
- Western blot analysis using a CP antibody to detect BmMLV infection.
- Establishment of a new BmMLV-negative cell line (BmVF) from Bombyx mori embryos.
- Development of a full-length infectious cDNA clone (pHMLV) of BmMLV.
- Transfection of pHMLV into BmVF cells and subsequent inoculation with cDNA-derived virus.
Main Results:
- All tested Bombyx mori cell lines were found to be infected with BmMLV.
- The newly established BmVF cell line supported persistent BmMLV infection.
- Transfection of the pHMLV clone into BmVF cells resulted in CP expression and virus production.
- Inoculation of BmVF cells with the cDNA-derived virus led to efficient BmMLV propagation.
Conclusions:
- The BmVF cell line serves as a valuable tool for studying BmMLV.
- The infectious cDNA clone pHMLV facilitates reverse genetic analysis of BmMLV.
- These resources will aid in elucidating BmMLV replication mechanisms and gene functions.
