The rate at which emergent Alphavirus epidemics occur globally is increasing. Of the alphaviruses, Chikungunya Virus (CHIKV) is the most geographically and medically prominent, causing number of recent epidemics and increasing in geographic range – due to a range of factors including changing land use, international travel, climate change and mutations associated with changing vector competence. CHIKV’s closest genetic relative, O’nyong-nyong Virus (ONNV), has been identified as the causative pathogen of a serious epidemic that affected approximately 2 million people in Sub-Saharan Africa in 1959. However, ONNV has not been well studied, in part due to a paucity of molecular tools and an optimised reverse genetic system.
In the current study replication of three alternative recombinant ONNV infectious clones, expressing different molecular markers of replication, was assessed and optimised across a range of mammalian and mosquito cell lines. We demonstrated that BHK-21, Huh7, RD, C6/36 and U4.4 cells were all permissible to ONNV infection, producing titres ranging from 105 PFU/mL to 108 PFU/mL. Notably, infectious clones replicating using nsP3-mCherry fusion proteins replicated producing lower titres in C6/36 relative to the other clones over 48 hours. The efficiency of antibodies raised against CHIKV non-structural proteins was demonstrated against ONNV expressed proteins, using both western blot and Immunofluorescence using confocal microscopy.
In order to further validate the reverse genetic systems, for the first time ONNV virions were imaged using negative staining and transmission electron microscopy, and a 3D structure of ONNV virions was generated by image averaging. Comparisons to published CHIKV virus-like particles showed differences in size and icosahedral structure. In summary, for the first time this study optimised the reverse genetic system for ONNV replication, across alternative physiologically relevant cell lines and molecular tools for further investigation of ONNV replication and structure.
