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Around 15% of our anti-glycoprotein monoclonal antibodies (MAbs) failed to neutralize the infectivity of the rabies virus during a 1-hr incubation at room temperature. In previous studies, we have demonstrated that it is possible to induce a massive conformational change of the glycoprotein population by incubating the virus at acidic pH. The conformational change is reversible and consequently viral infectivity is not affected by transient exposure at acidic pH. The proportion of glycoproteins in acidic or neutral configuration depends on the pH which means that even at neutral pH some glycoproteins transiently adopt the acidic configuration and vice versa. Here we report that some of our nonneutralizing MAbs recognize the acidic form of the glycoprotein at the virion surface. After incubation of the virus at pH 6.4, most glycoproteins are in the acidic configuration. Further 1-hr incubation with these MAbs at the same pH resulted in more immunoglobulins being attached to the virus and consequently neutralization was induced. It was also possible to induce neutralization with the same MAbs by incubation at neutral pH for a longer period or at a higher temperature. Mutants resistant to neutralization by these MAbs could be selected. Mutations confering resistance to neutralization were not localized in previously described antigenic sites and did not modify these sites at distance. They had no effect on the pathogenic power of the virus. Either they are situated in the epitope or they modify the epitope, so that it is no longer recognized by the antibody on the acidic configuration of the protein. Alternatively, these mutations may stabilize the protein in its neutral configuration. In addition, these experiments confirm our previous finding that neutralization requires the fixation of a large number of immunoglobulins on the virus, irrespective of the region of the protein recognized by the antibody.

The sequence of the glycoprotein gene of a street rabies virus was determined directly using fragments of a rabid dog brain after PCR amplification. Compared with that of the prototype strain CVS, this sequence displayed 10% divergence in overall amino acid composition. However only 6% divergence was noted in the ectodomain suggesting that structural constraints are exerted on this portion of the glycoprotein. A human strain isolated on cell culture from the saliva of a patient with clinical rabies had only five amino acid differences with the canine isolate, an indication of their close relatedness. These differences could have originated during transmission from dog to dog, or from dog to man, or during isolation on cell culture; they are nonetheless indicative of a genetic evolution of street rabies virus. This evolution was further evidenced by the selection of cell-adapted variants which displayed new amino acid substitutions in the glycoprotein. One of them concerned antigenic site III where arginine at position 333 was replaced by glutamine. As expected this substitution conferred resistance to a site IIIa monoclonal antibody (MAb), but surprisingly did not abolish neurovirulence for adult mice. However, a decrease in the neurovirulence of the cell-adapted variant in the presence of a site IIIa specific MAb was noted, suggesting that neurovirulence was due to a subpopulation neutralizable by the MAb. Simultaneous presence of both the parental and variant sequences was indeed evidenced in the brain of a mouse inoculated with the cell-adapted variant; during multiplication in the mouse brain, the frequency of the parental sequence rose from less than 10% to nearly 50%, indicating the selective advantage conferred by arginine 333 in nervous tissue. Altogether these results were suggestive of an intrinsic heterogeneity of street rabies virus. This heterogeneity was further demonstrated by the sequencing of molecular clones of the glycoprotein gene, which revealed that only one-third of the viral genomes present in the brain of a rabid dog had the consensus sequence. Two-thirds of the clones analyzed displayed from one to three amino acid substitutions. Such heterogeneous populations have been referred to as quasispecies, a concept which implies heterogeneous populations kept together in a dynamic equilibrium. This equilibrium could be rapidly displaced, giving the virus the capacity to adapt easily to new environmental conditions.

After intranasal instillation in the mouse, rabies virus (CVS strain) selectively infected olfactory receptor cells. In the main olfactory bulb (MOB), infection was observed in periglomerular, tufted, and mitral cells and in interneurons located in the internal plexiform layer. Beyond the MOB, CVS spread into the brain along the olfactory pathways. This infection is specific to chains of functionally related neurons but at the death of the animal some nuclei remain uninfected. CVS also penetrated the trigeminal system. The avirulent mutant AvO1, carrying a mutation in position 333 of the glycoprotein, infected the olfactory epithelium and the trigeminal nerve as efficiently as CVS. During the second cycle of infection, the mutant was able to infect efficiently periglomerular cells in the MOB and neurons of the horizontal limb of the diagonal band, which indicates that maturation of infective particles is not affected in primarily infected neuronal cells. On the other hand, other neuronal cells permissive for CVS, such as mitral cells or the anterior olfactory nucleus, are completely free of infection with the mutant, indicating that restriction is related to the ability of AvO1 to penetrate several categories of neurons. From these observations, we concluded that CVS should be able to bind several different receptors to penetrate neurons, while the mutant would be unable to recognize some of them.

Fixed rabies virus strains (ERA and CVS) produce a fatal paralytic disease in mice after intracerebral or intramuscular injection. Some antigenic mutants of both CVS and ERA viruses with a substitution in position 333 of the glycoprotein (arginine is replaced either by a glutamine, a glycine, or an isoleucine) are totally avirulent for adult mice whatever the dose and the route of inoculation. Here we report an exhaustive investigation of the effect of amino acid 333 on viral virulence. New antigenic mutants were isolated from either CVS, CVS derivatives, or SADBern having arginine in position 333 encoded by CGG, AGG, CGU, or AGA respectively. This study shows that when arginine is replaced by either a leucine, an isoleucine, a methionine, a cysteine, or a serine, the antigenic mutant is also totally avirulent. But when arginine is replaced by a lysine it is still pathogenic although the LD50 by the intracerebral route is higher. Furthermore 41 independent virulent revertants were isolated from four avirulent mutants (with a glycine, a glutamine, a methionine, or a serine in position 333 of the glycoprotein). Thirty-nine regained an arginine at position 333 and 2 had a lysine. From this analysis it appears that the presence of a positively charged amino acid (arginine or lysine) in position 333 of the glycoprotein is necessary for viral virulence.