A Chimeric Virus Approach Reveals the Matrix (M) Gene as a Critical Modulator of Mumps Virus Neurovirulence

Background/Objectives: Live attenuated mumps virus (MuV) vaccine strains have significantly reduced disease incidence since their introduction in the 1960s; however, a recent resurgence of outbreaks has led to calls for the development of new vaccines to overcome what appears to be reduced effectiveness linked to waning immunity and the emergence of strains antigenically mismatched to vaccine strains. A major obstacle to the development of newer live, attenuated MuV vaccines is ensuring their safety, particularly given the virus's neurotropic properties. Indeed, several vaccine strains licensed for use outside the US have proven to be insufficiently attenuated (such as the Urabe AM9 vaccine strain) and have caused aseptic meningitis in recipients, despite efforts to test these strains for neurotoxicity pre-licensure. Historically, attenuation was achieved empirically, and pre-clinical testing for neurovirulence safety has proven unreliable. Despite efforts in recent years, the genetic basis of attenuation of MuV vaccines remains inadequately understood. The objective of this study was to elucidate the genetic basis of neurovirulence of the Urabe AM9 vaccine strain. Methods: To this end, we generated a series of chimeric viruses in which genes of the highly attenuated Jeryl Lynn (JL) vaccine strain were exchanged with corresponding genes from the Urabe AM9 vaccine strain. The resulting chimeric viruses were tested for neurovirulence in a rat model and characterized for replication in vitro and in vivo. Using a multi-tiered approach consisting of independent rescue and analysis of two to three viruses per cDNA construct, possible off-target effects of identified single-nucleotide heterogeneities were mitigated. Results: All viruses were shown to be replication-competent in Vero cells, but differences in replication efficiencies and virus-induced neurotoxicity were observed in the in vivo model. In a rat neuronal cell line, the Urabe AM9 M and HN genes had opposite effects on the growth of chimeric JL- and Urabe AM9-based viruses. Conclusions: The results presented herein indicate that MuV neuroattenuation is mediated by the concerted action of multiple genes, with the matrix (M) gene exerting a dominant effect. These findings contribute to a growing body of evidence that informs the rational design of next-generation live-attenuated mumps virus vaccines.