Mumps virus neurovirulence assessment-impact of viral doses, animal sex and age on results dispersion

Mumps epidemics highlight the necessity for new live mumps vaccines. For vaccine candidate neurosafety assessment, the most commonly used test is the rat-based neurovirulence test (RNVT). Its main experimental and ethical deficiency is the results dispersion which requires large number of animals to obtain statistically relevant results. To additionally characterize the RNVT the impact of two experimental animal characteristics, age for inoculation and sex, as well as range of infectious viral doses were analysed. Results obtained from two neurovirulent mumps viruses, SHdeopti-MRV2 and 9218/Zg98, demonstrated no corelation between animal sex and RNVT results (p = 0.9638 and p = 0.3337). Analysis of animal inoculation age found that hydrocephalus formation is impaired in neonatal rats older than 3 days. The inoculation period could be extended from first to the second postanatal day without the impact on RNVT results (p = 0.5865). RNVT results demonstrated robustness to infectious doses in a range from 100 to 1000 PFU, but using the non-standard infectious doses,10 PFU or 100 CCID50, poses a risk of underestimating the neurovirulence potential of the MuV. However, none of the analysed parameters influenced the high dispersion of results. In conclusion, although our finding will facilitate the implementation of the RNVT, the dispersion of the results indicates the need for a better mumps neurovirulence test that would align more closely with the 3R principles.