Simultaneous Packaging of Two Different RNA Segments into an Influenza C Virus-like Particle Occurs Inefficiently

Reverse genetics systems for influenza C virus encounter challenges due to the inefficient production of infectious virus particles. In the present study, we explored the underlying cause by assessing the efficiency of generating influenza C virus-like particles (C-VLPs) containing specific virus RNA (vRNA) segments. Using 293T cells transfected with plasmids encoding GFP- and DsRed2-vRNAs (each flanked by the non-coding regions of Segments 5 and 6, respectively), along with plasmids expressing virus proteins, we observed that C-VLPs containing a single vRNA segment were generated efficiently. However, the simultaneous packaging of two vRNA segments into a single C-VLP was less frequent, as demonstrated by flow cytometry and reverse-transcription PCR analyses. Statistical evaluations revealed a decreased efficiency of incorporating multiple vRNA segments into single particles, which likely contributes to the reduced production of infectious virus particles in reverse genetics systems. These findings highlight a critical limitation in the vRNA incorporation mechanism of influenza C virus, contrasting with that of influenza A virus. Hence, further studies are required to elucidate specific vRNA packaging signals and optimize vRNA expression levels to improve the production of infectious influenza C virus particles.