Serial passage of neurotropic-neurovirulent viruses in nonneuronal substrates to achieve neuroattenuation for the preparation of vaccines has been successfully performed for a number of viruses, including polio virus and mumps virus (9
). This process apparently selects for mutant viruses with an increased ability to replicate under specific conditions, with a concomitant reduction in the ability to infect the natural target tissue. Neuroattenuation is evaluated on an empirical basis, with passage continued until sufficient attenuation is achieved. The reverse process, that of neuroadaptation, is conceptually similar. Prolonged successive passages of virus in neuronal tissues or cells are used to select for mutant viruses with an increased tropism for those substrates. This technique has been employed for research purposes to generate neuroadapted variants of several viruses, including Sindbis virus, Newcastle disease virus, respiratory syncytial virus, yellow fever virus, influenza virus, and measles virus (7
). In this study, we exploited the neuroadaptation and neuroattenuation processes in combination with virus genome sequencing to identify specific genomic changes that might influence mumps virus neurotropism (the ability of the virus to infect and replicate in the brain) and neurovirulence (the ability of the presence of the virus to lead to damage in the brain).
Neuroadaptation of the JL parental strain, a nonneurotropic, nonneurovirulent mumps vaccine strain (5
), was achieved by serial passage on human neuronal cells (SY5Y). The resulting variant virus strain, JL-SY5Y, appeared to have a slight replication advantage in rat brain (neurotropism) and, more significantly, was more neurovirulent (judged on the basis of an increased incidence and severity of hydrocephalus) than the JL parental strain. Despite an increased ability of JL-SY5Y to replicate and cause damage in brain, the gross tissue distributions of the JL parental and JL-SY5Y strains appeared to be similar (both being limited to the ventricular ependymal cells). Of note, the increased ability of strain JL-SY5Y to grow in brain is not likely a simple reflection of the fact that JL-SY5Y is mammalian cell adapted whereas the JL parental strain is chick cell adapted, since no differences in virus growth were observed between rats inoculated with the JL parental strain versus those inoculated with strain JL-Vero, another mammalian-cell-adapted variant of the JL parental strain.
In comparing the genomic sequence between the JL parental and JL-SY5Y strains, seven nucleotide changes were found, all in coding regions. Three of the changes were predicted to result in amino acid substitutions: one each in the NP (Phe→ Pro468
), matrix protein (Val→Ala85
), and polymerase (Glu→Asp1165
). As the NP and the polymerase are involved in replication and transcription and the matrix protein is believed to be involved in the virion assembly process (10
), it is quite plausible that changes in these proteins are responsible for the observed in vitro and in vivo findings of enhanced growth and virulence of strain JL-SY5Y. Notably, amino acid changes in the NP, matrix protein, and polymerase in other viruses have been associated with changes in neurovirulence phenotype (40
). While in some cases, changes in protein sequence have been predicted to result in structural-functional changes, e.g., elimination of a phosphorylation site in the rabies virus NP (59
) or disruption of the pH-dependent association-disassociation between the matrix protein and the ribonucleoprotein complex in influenza virus (47
), the precise mechanism by which these changes affect neurovirulence of these and other viruses is not clear. In this study, neither of the amino acid changes in the NP and matrix protein of JL-SY5Y was predicted to result in structural-functional changes. However, not all structural-functional motifs have been identified for these proteins; therefore the possibility of the NP and matrix protein amino acid changes affecting virus phenotype cannot be ruled out. Unlike the NP and matrix protein amino acid changes, evidence does exist for a functional role of the Glu→Asp1165
substitution in the polymerase. This particular amino acid change is located in the highly conserved V domain of the protein (14
). Nearby mutations in this domain in Sendai virus, a close relative of mumps virus, have been shown to significantly affect RNA synthesis in vitro and in vivo (14
). Thus, it is possible that the Glu→Asp1165
substitution in the JL polymerase can affect virus growth. Confirmation of an effect on replication, transcription, or virulence caused by the NP, matrix protein, and polymerase amino acid changes identified here will ultimately require application of a reverse genetics system.
An alternate possible explanation for the observed differences in JL parental and JL-SY5Y strain replication in vitro and replication and virulence in vivo might lie in the relative proportions of JL1 and JL2, the two substrains known to comprise the JL strain (1
). Since it has been demonstrated that the JL1:JL2 ratio is host substrate dependent (4
), we measured the JL1:JL2 ratios in the three JL-based viruses used here. Our results showed that JL2 was comprised of 15% JL parental strain and 10% strain JL-Vero, whereas JL-SY5Y was entirely comprised of JL1. Thus, it is tempting to speculate that relative to JL2, JL1 is of increased replication competence, which may manifest as heightened neurotropism and neurovirulence in the rat assay. At this point, however, potential effects of JL1:JL2 ratio changes cannot be separated from potential effects of the NP, matrix protein, and polymerase amino acid changes.
To examine genomic differences following neuroattenuation, the 88-1961 parental strain, a highly neurotropic and neurovirulent strain (44
), was passaged on an avian fibroblast cell line (CEF), mimicking classical techniques used to produce live, attenuated vaccine from wild-type mumps virus (9
). As predicted, relative to the 88-1961 parental virus, the 88-1961-CEF variant showed a reduced ability to replicate in rat brain, a reduced incidence and severity of hydrocephalus, and a lower incidence of clinical disease. Consistent with these in vivo findings, strain 88-1961-CEF also showed a reduced ability to replicate on Vero cells, suggesting that reduced tropism for mammalian cells contributed to the observed decrease in infectivity in rats. Again, however, simple differences in replication competence in mammalian cells cannot fully explain the neurovirulence outcomes, since strains 88-1961-CEF and JL-SY5Y replicated to similar titers in mammalian cells in vitro yet had vastly different neurovirulence outcomes in vivo.
Unlike the JL-based virus stains, replication of the strain 88-1961-based viruses was not restricted to the ependymal cells. Numerous virus-infected neurons were also detected in the surrounding brain parenchyma. These data suggest that infection of cell types other than the ependymal cells can contribute to the heightened incidence and severity of hydrocephalus and clinical disease. However, while differences in neuroanatomic or cellular sites of replication may account for some of the variations in hydrocephalus severity between JL and 88-1961 strains, those differences may not explain the observed variations in neurovirulence between 88-1961 parental and 88-1961-CEF, since no obvious variations were observed in the neuroinvasiveness or distribution of virus among rats infected with these viruses. Certainly, a better understanding of the precise mechanism(s) by which mumps virus leads to hydrocephalus will ultimately be required for a better appreciation of the role of virus-infected subventricular neurons in hydrocephalus development. To date, the preponderance of data suggests that hydrocephalus is the result of accumulation of virus-infected cell debris in the aqueduct of Sylvius, blocking cerebrospinal fluid egress (27
). A similar mechanism is believed to be responsible for mumps virus hydrocephalus in humans (21
). However, hydrocephalus has also been observed prior to, or in the total absence of, aqueductal stenosis (26
), suggesting that stenosis of the aqueduct can be a secondary consequence of external compression by surrounding edematous tissue in already hydrocephalic animals and not causally related to the pathogenesis of hydrocephalus.
In comparing the genomic sequences of 88-1961 parental and 88-1961-CEF strains, four nucleotide substitutions were found, three of which were predicted to result in amino acid substitutions: one each in the polymerase (Ile→Val736), fusion (Pro/Thr→Thr91), and HN (Ser→Asn466) proteins.
All three of the predicted amino acid substitutions, individually or in combination, are good candidates for affecting virus replication and virulence. The polymerase Ile→Val736
substitution is located within a putative functional motif in the highly conserved domain III region of the polymerase protein (42
). This motif, identified by Poch et al. as motif B, is believed to represent an important element of the active site for template recognition and/or phosphodiester bond formation (49
). If such a function exists for this region, changes in its amino acid sequence could lead to changes in virus growth and, by extension, virulence.
The predicted amino acid substitutions in the fusion and HN glycoproteins are also located in known functional regions. The Pro/Thr→Thr91 change in the fusion protein, a protein responsible for fusion of viral and cellular membranes, is located within a region involved in the disulfide bond linkage between the F1 and F2 active subunits of the F0 precursor protein (61). In addition to possibly affecting the stability of the fusion protein, this change also impacts the polarity-hydrophobicity at this position and gives rise to a N-glycosylation site at amino acids 89 to 92. While this amino acid population change therefore has the potential to affect protein function, no differences were observed between levels of fusogenicity of the 88-1961 parental strain and 88-1961-CEF strain in vitro. Other more sensitive measurements of fusion protein function are planned.
Of the strain 88-1961 amino acid changes, the Ser→Asn466
substitution in the HN glycoprotein, the protein that promotes viral attachment to cellular receptors, is most promising. The HN protein has been shown to play a role in mumps virus neurovirulence. For example, a change at position 360 in the highly neurovirulent hamster brain-adapted Kilham mumps virus strain was associated with a decreased ability to infect neurons in hamsters and attenuated disease (32
), and amino acid changes at positions 335, 464, and 498 of the Urabe-AM9 mumps virus vaccine strain have been associated with meningitis in vaccinees (2
). The HN protein has two apparently opposing activities, adsorption to sialic acid-containing cell surface molecules (hemagglutination) and enzymatic cleavage of sialic acid (neuraminidase activity). Recent studies have indicated that both functions are located in a single sialic acid recognition site (13
). A recent crystal structure study of the HN glycoprotein of a closely related paramyxovirus, Newcastle disease virus, indicates that amino acid position 466 may be at or near this active site (15
). Thus, the Ser→Asn466
substitution in the HN glycoprotein of strain 88-1961-CEF, which is predicted to result in a loss of an N-glycosylation site (amino acids 464 to 467), has the potential to affect virus tropism and virulence. Whether or not the loss the N-glycosylation site translates to a change in secondary structure is not clear. While the Garnier, Osguthorpe, and Robson algorithm predicted a significant change in the secondary structure (turns and β-sheets) of this protein, the Chou-Fasman algorithm predicts no structural changes. Nonetheless, loss of N-glycosylation sites, regardless of effects on secondary structure, have been linked to changes in the neurovirulence in other viruses, including influenza virus (34
), lactate dehydrogenase-elevating virus (19
) and yellow fever virus (39
Considering that JL and 88-1961 strains represent different genotypes (25
), it is not surprising that none of the amino acids associated with JL neuroattenuation were identified in the attenuated 88-1961-CEF virus strain and none of the amino acids associated with the enhanced neurovirulence of the JL-SY5Y virus were identified in the neurovirulent 88-1961 parental virus strain. In a more expansive intergenotypic comparison of several mumps virus strains representing a wide range of neuropathogenicity for humans, no attenuation or neurovirulence-specific clusters were identified (unpublished data).
The results presented here demonstrate that the affinity of mumps virus for the brain can be increased or decreased by passage on neuronal or nonneuronal-nonmammalian cells, respectively. The relative abilities of these viruses to replicate in rat brains could not completely account for the relative levels of neurovirulence of the viruses, since both strain JL-SY5Y and strain 88-1961-CEF replicated to similar titers and yet strain 88-1961-CEF was significantly more neurovirulent than strain JL-SY5Y. Remarkably, despite multiple passages in vitro on cells of differing classes (avian versus mammalian), a small number of genetic changes, individually or in combination, appeared to account for these observed differences. Further studies, including reverse genetics, are required for confirmation of the specific role each of these protein changes in neurovirulence.