Efficient N-glycosylation at position 37, but not at position 146, in the street rabies virus glycoprotein reduces pathogenicity

•Most street RABVs have two N-glycosylation sites in the G protein.•The core-glycosylation at position 37 is usually inefficiently.•We characterized two N-glycosylation-modified variants of a street RABV.•The efficient glycosylation at position 37 led to cell adaptation and reduced pathogenicity.•The additional N-glycosylation at 146 led to the cell adaptation, but not reduced pathogenicity.

Most street rabies viruses have two N-glycosylation sites in their glycoproteins (G proteins), i.e., at Asn37 and Asn319, but Asn37 is usually not core-glycosylated in an efficient manner. Previously, we reported the possible roles of single additional N-glycosylations at Asn194 or Asn247 in the cell adaptation and reduced pathogenicity of a street rabies virus, which suggest that N-glycosylation is closely related to the evolution of rabies viruses. In this study, we characterized two novel N-glycosylation-modified variants, N5C#7 and N5C#8, which were cloned using the limiting dilution method after serial passaging of the street rabies virus strain 1088 in mouse neuroblastoma-derived NA cells. N5C#7 had an L38R mutation in the G protein, which led to efficient core glycosylation at Asn37. On the other hand, N5C#8 had a D146 N mutation in the G protein, which led to an additional N-glycosylation at position 146. Both variants replicated highly efficiently in NA cells compared with the parental strain. Like the parental strain, both variants caused lethal infections in adult mice after intracerebral inoculation. However, N5C#7 exhibited reduced pathogenicity after intramuscular inoculation, whereas N5C#8 displayed the same level of pathogenicity as the parental strain. In summary, the efficient core glycosylation at position 37 was related to cell adaptation and the reduced pathogenicity of the street rabies virus. By contrast, despite of being related to cell adaptation, the additional N-glycosylation at position 146 did not affect the pathogenicity, which is consistent with a report that street rabies virus strains with N-glycosylation sites at positions 37, 146, and 319 have been isolated from rabid animals. Thus, the results of the present study provide additional evidence that supports the relationship between G protein N-glycosylation and rabies virus evolution.