A versatile and stable reporter platform for human rhinovirus and other enteroviruses

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. IMPORTANCE Human rhinovirus (HRV) has lacked a reporter system expressing widely used reporter proteins, such as GFP, that enables visualization of viral dynamics in infected cells and tissues. The only reporter HRV reported to date loses fluorescence upon fixation and tolerates only small inserts, limiting its utility. Meanwhile, the clinical importance of HRV has become increasingly recognized, as reflected by its high detection rate among hospitalized children and recent outbreaks associated with fatal cases. A versatile and stable strategy for constructing reporter HRV is therefore urgently needed. Here, we overcome the two major obstacles to generating stable reporter HRV - impaired viral replication and loss of the inserted gene - and demonstrate that this strategy is also applicable to other enteroviruses. By providing a broadly applicable framework for constructing stable reporter viruses, this work establishes a foundation for visualizing infection dynamics and studying the pathogenesis of HRV and other enteroviruses.