Serial passage of a street rabies virus in mouse neuroblastoma cells resulted in attenuation: potential role of the additional N-glycosylation of a viral glycoprotein in the reduced pathogenicity of street rabies virus

Abstract

Street rabies viruses are field isolates known to be highly neurotropic. However, the viral elements related to their pathogenicity have yet to be identified at the nucleotide or amino acid level. Here, through 30 passages in mouse neuroblastoma NA cells, we have established an attenuated variant of street rabies virus strain 1088, originating from a rabid woodchuck followed by 2 passages in the brains of suckling mice. The variant, 1088-N30, was well adapted to NA cells and highly attenuated in adult mice after intramuscular (i.m.) but not intracerebral (i.c.) inoculations. 1088-N30 had seven nucleotide substitutions, and the R196S mutation of the G protein led to an additional N-glycosylation. Street viruses usually possess one or two N-glycosylation sites on the G protein, 1088 has two, while an additional N-glycosylation site is observed in laboratory-adapted strains. We also established a cloned variant 1088-N4#14 by limiting dilution. Apart from the R196S mutation, 1088-N4#14 possessed only one amino acid substitution in the P protein, which is found in several field isolates. 1088-N4#14 also efficiently replicated in NA cells and was attenuated in adult mice after i.m. inoculations, although it was more pathogenic than 1088-N30. The spread of 1088-N30 in the brain was highly restricted after i.m. inoculations, although the pattern of 1088-N4#14's spread was intermediate between that of the parental 1088 and 1088-N30. Meanwhile, both variants strongly induced humoral immune responses in mice compared to 1088. Our results indicate that the additional N-glycosylation is likely related to the reduced pathogenicity. Taken together, we propose that the number of N-glycosylation sites in the G protein is one of the determinants of the pathogenicity of street rabies viruses.