A versatile and stable reporter platform for human rhinovirus and other enteroviruses
- 1Laboratory of Virus Control, Center for Infectious Disease Education and Research (CiDER), Research Institute for Microbial Diseases (RIMD), and Center for Adva
Abstract
Human rhinovirus (HRV), a major cause of upper respiratory infections, has increasingly been associated with lower respiratory tract disease. Although tools to investigate HRV pathogenesis are needed, they remain limited. Reporter viruses expressing fluorescent or luminescent proteins are powerful tools for analyzing viral distribution and replication; however, insertion of a reporter gene often impairs viral replication, and the inserted gene is readily lost during serial passage. To address these problems, we established a versatile and stable strategy for generating reporter HRV and other enteroviruses. The gene encoding mScarlet3, a red fluorescent protein, was inserted between the 5'UTR and VP4 gene, a site previously used in other enteroviruses but not in HRV. Optimizing the 2A cleavage sequence between mScarlet3 and VP4 yielded a reporter HRV with growth kinetics comparable to those of the wild-type virus. To improve the genetic stability of the reporter insert, we evaluated GC-content optimization of the reporter gene and relocation of the cis-acting replication element (CRE). GC-content optimization substantially improved reporter stability in both HRV-B14 and EV-A71, whereas CRE relocation provided additional stabilization in EV-A71 but not in HRV-B14, demonstrating the potential applicability of this strategy to other enteroviruses. This strategy for constructing stable reporter HRV and other enteroviruses will facilitate studies of enterovirus pathogenesis.
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BibTeX
@misc{okada2026versatile,
title = {{A versatile and stable reporter platform for human rhinovirus and other enteroviruses}},
author = {K. Okada and J. Hirano and T. Adachi and T. Noguchi and K. Uemura and C. Ono and S. Nagata and Y. Yamamoto and T. Nagamoto and S. Taguwa and M. T. Yeh and Y. Matsuura},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.02.756346},
url = {https://www.biorxiv.org/content/10.64898/2026.10.02.756346v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - A versatile and stable reporter platform for human rhinovirus and other enteroviruses
AU - K. Okada
AU - J. Hirano
AU - T. Adachi
AU - T. Noguchi
AU - K. Uemura
AU - C. Ono
AU - S. Nagata
AU - Y. Yamamoto
AU - T. Nagamoto
AU - S. Taguwa
AU - M. T. Yeh
AU - Y. Matsuura
PY - 2026
DA - 2026/10/02
PB - bioRxiv
DO - 10.64898/2026.10.02.756346
UR - https://www.biorxiv.org/content/10.64898/2026.10.02.756346v1
AB - Human rhinovirus (HRV), a major cause of upper respiratory infections, has increasingly been associated with lower respiratory tract disease. Although tools to investigate HRV pathogenesis are needed, they remain limited. Reporter viruses expressing fluorescent or luminescent proteins are powerful tools for analyzing viral distribution and replication; however, insertion of a reporter gene often impairs viral replication, and the inserted gene is readily lost during serial passage. To address these problems, we established a versatile and stable strategy for generating reporter HRV and other enteroviruses. The gene encoding mScarlet3, a red fluorescent protein, was inserted between the 5'UTR and VP4 gene, a site previously used in other enteroviruses but not in HRV. Optimizing the 2A cleavage sequence between mScarlet3 and VP4 yielded a reporter HRV with growth kinetics comparable to those of the wild-type virus. To improve the genetic stability of the reporter insert, we evaluated GC-content optimization of the reporter gene and relocation of the cis-acting replication element (CRE). GC-content optimization substantially improved reporter stability in both HRV-B14 and EV-A71, whereas CRE relocation provided additional stabilization in EV-A71 but not in HRV-B14, demonstrating the potential applicability of this strategy to other enteroviruses. This strategy for constructing stable reporter HRV and other enteroviruses will facilitate studies of enterovirus pathogenesis.
ER -