Mapping the viral determinants of neuroattenuation for a unique California serogroup bunyavirus

Many members of the California serogroup of bunyaviruses are significant human pathogens. For example, La Crosse virus, the prototype of the California serogroup, is the second leading cause of arboviral encephalitis in the United States. At the onset of this project, however, little was known about the determinants of neurovirulence among these viruses. Therefore, the goal of this study has been to map the viral determinants of neurovirulence using a genetic approach. In the course of these studies, the first highly attenuated California serogroup virus (clone B.5) was isolated and characterized. B.5 virus was more than 1,000,000-fold more attenuated than the prototypic La Crosse virus and the study showed that the decreased virulence of B.5 virus correlated with its inability to grow in adult mouse brain. The segmented nature of the bunyavirus genome allowed for reassortant viruses to be produced by coinfections with the neuroattenuated, B.5, and the neurovirulent, B1-1a, viruses. The genotyping of progeny viruses with respect to the origin of their three RNA genome segments was greatly facilitated by the development of a simple yet rapid dot-blot hybridization protocol. This study describes the virulence characteristics of a complete panel of reassortant viruses. Examination of these reassortants in a mouse model system enabled us to map the major determinant of neuroattenuation to the L RNA segment. We have demonstrated that this RNA segment codes for the large viral protein which is believed to be the viral polymerase and have endonuclease activity. In addition to the L RNA, the S RNA segment also was observed to have a modulating affect on neuroattenuation suggesting that the interaction between these two segments or their products is important in determining the neurovirulence phenotype among members of the California serogroup