Viral determinants of age-dependent virulence of Sindbis virus for mice.

Many alphaviruses cause more severe disease in young animals than in older animals. The age-dependent resistance to severe disease is determined primarily by maturation of the host, but strains of virus can be selected that overcome the increased resistance of mature animals. Sindbis virus (SV) strain AR339 causes fatal encephalitis in newborn mice and nonfatal encephalitis in weanling mice, whereas NSV, a neuroadapted strain of SV, causes fatal encephalitis in weanling as well as newborn mice. We have previously shown that the E2 glycoprotein of NSV contained His-55, whereas AR339 E2 had Gln-55 (S. Lustig, A. C. Jackson, C. S. Hahn, D. E. Griffin, E. G. Strauss, and J. H. Strauss, J. Virol. 62:2329-2336, 1988) and that SV with E2 containing Gly-172 was more virulent for newborn mice than SV with E2 containing Arg-172 (P. C. Tucker and D. E. Griffin, J. Virol. 65:1551-1557, 1991). Here we tested the virulence for both newborn and older mice of SV containing a number of different amino acids at E2 position 55 (His, Gln, Lys, Arg, Glu, Gly) in combination with both Gly-172 and Arg-172. All the viruses were virulent for newborn mice, but the residues at both 55 and 172 influenced the virulence of the virus, and there were differences in virulence observed among the various viruses. However, only viruses with His-55 were fully virulent for 14-day-old mice, and this virulence was independent of the residue at position 172. Virus with Lys-55 was virulent for 7-day-old mice, although slightly attenuated relative to His-55. Viruses with His-55 grew more rapidly and to higher titer in the brains of 7- and 14-day-old mice, in N18 neuroblastoma cells, and in BHK cells. Our data suggest that His-55 is important for neurovirulence in older mice and acts by increasing the efficiency of virus replication.     

Molecular analysis of neurovirulent strains of Sindbis virus that evolve during persistent infection of scid mice.

To understand the role of tissue-specific adaptation and antibody-induced selectional pressures in the evolution of neurovirulent viruses, we analyzed three strains of Sindbis virus isolated from the brains of persistently infected scid mice and four strains of Sindbis virus isolated from the brains of scid mice with viral reactivation following immune serum treatment. For each viral isolate, we tested neurovirulence in weanling BALB/c mice and sequenced regions of the E2 and E1 envelope glycoprotein genes that are known to contain important determinants of Sindbis virus neurovirulence. One strain isolated from a persistently infected scid mouse and two strains isolated from scid mice with viral reactivation were neurovirulent, resulting in mortality in 80 to 100% of weanling BALB/c mice. All three neurovirulent strains contained an A-->U change at nucleotide 8795, which predicts a Gln-->His substitution at E2 amino acid position 55. No nucleotide changes were detected in the other sequenced regions of the E2 and E1 envelope glycoprotein genes or in the avirulent isolates. Our findings indicate that tissue-specific adaptations, rather than antibody-induced selectional pressures, are a critical determinant of the evolution of neurovirulent strains of Sindbis virus and provide evidence that E2 His-55 is an important neuroadaptive mutation that confers neurovirulence properties on Sindbis virus.     

Alphavirus neurovirulence: monoclonal antibodies discriminating wild-type from neuroadapted Sindbis virus.

Wild-type Sindbis virus strain AR339 (SV) and a neurovirulent mutant (NSV), derived by neonatal and weanling mouse brain passage, both cause acute fatal encephalitis in neonatal mice, but NSV alone kills adult mice. NSV cannot be distinguished from SV by immune sera or simple biochemical tests. To localize the molecular changes associated with neuroadaptation, we used a new array of 30 anti-SV monoclonal antibodies to probe for differences between SV and NSV in four tests: immunoprecipitation, enzyme-linked immunosorbent assay binding, neutralization, and hemagglutination inhibition. Seventeen monoclonal antibodies detected differences. Both E1 and E2 glycoprotein gene products were altered during neuroadaptation, but the preponderance of changes was clustered on E2. The capsid protein C was not measurably altered. Mapping of both viruses with these monoclonal antibodies showed that during neuroadaptation SV topography substantially shifted, masking and unmasking biologically important neutralization and hemagglutination inhibition sites. These conformational rearrangements, predominantly on E2, coincided with the acquisition of increased neurovirulence and new lethality for adult mice.     

Identification of adult mouse neurovirulence determinants of the Sindbis virus strain AR86

Sindbis virus infection of mice has provided valuable insight into viral and host factors that contribute to virus-induced neurologic disease. In an effort to further define the viral genetic elements that contribute to adult mouse neurovirulence, the neurovirulent Sindbis virus strain AR86 was compared to the closely related (22 single amino acid coding changes and the presence or absence of an 18-amino-acid sequence in nsP3 [positions 386 to 403]) but avirulent Girdwood strain. Initial studies using chimeric viruses demonstrated that genetic elements within the nonstructural and structural coding regions contributed to AR86 neurovirulence. Detailed mapping studies identified three major determinants in the nonstructural region, at nsP1 538 (Ile to Thr; avirulent to virulent), an 18-amino-acid deletion in nsP3 (positions 386 to 403), and nsP3 537 (opal to Cys; avirulent to virulent), as well as a single determinant in the structural genes at E2 243 (Leu to Ser; avirulent to virulent), which were essential for AR86 adult mouse neurovirulence. Replacing these codons in AR86 with those found in Girdwood resulted in the attenuation of AR86, while the four corresponding AR86 changes in the Girdwood genetic background increased virulence to the level of wild-type AR86. The attenuating mutations did not adversely affect viral replication in vitro, and the attenuated viruses established infection in the brain and spinal cord as efficiently as the virulent viruses. However, the virus containing the four virulence determinants grew to higher levels in the spinal cord at late times postinfection, suggesting that the virus containing the four attenuating determinants either failed to spread or was cleared more efficiently than the wild-type virus.     

Monoclonal antibody cure and prophylaxis of lethal Sindbis virus encephalitis in mice

Neuroadapted Sindbis virus (NSV) causes acute encephalitis and paralyzes and kills adult mice unless they are treated with primary immune serum after infection. To study the nature and specificity of curative antibodies, we gave mice 30 different monoclonal antibodies (MAbs) against Sindbis virus (SV) 24 h after lethal intracerebral inoculation of NSV. By the time of MAb treatment, NSV replication in the brain had been well established (7.5 X 10(7) PFU/g). Seventeen MAbs directed against multiple biological domains on the NSV E1 and E2 envelope glycoproteins prevented paralysis and death. Anticapsid MAbs failed to protect. Altogether, 15 of 17 curative MAbs either neutralized NSV infectivity or lysed NSV-infected cells with complement, but neither ability was necessary or sufficient to guarantee recovery. All 5 protective anti-E2 MAbs neutralized NSV infectivity; 6 of 10 protective anti-E1 MAbs neutralized NSV; 4 did not. Plaque assay or immunohistochemical staining showed that neutralizing and nonneutralizing curative MAbs decreased NSV in the brain, brainstem, and spinal cord. Despite high neutralization titers, hyperimmune anti-SV and anti-NSV mouse sera prevented only 6 and 30% of deaths, respectively, while primary immune sera prevented 50 (SV) and 90% (NSV) of deaths. Secondary intravenous immunization with a live virus apparently diminished, obscured, or failed to boost a class of protective antibodies. When separate mouse groups were given these 30 MAbs 24 h before lethal intracerebral inoculation of NSV, a slightly different set of 17 neutralizing or nonneutralizing anti-E1 and anti-E2 antibodies protected. Two nonneutralizing MAbs and hyperimmune anti-SV serum, which had failed to promote recovery, prophylactically protected 100% of the mice. The antibody requirements or mechanisms of prophylaxis and recovery may differ.     

Pathogenesis of encephalitis induced in newborn mice by virulent and avirulent strains of Sindbis virus.

Strains of Sindbis virus differ in their virulence for mice of different ages; this variation is related in large part to variations in the amino acid compositions of E1 and E2, the surface glycoproteins. The comparative pathogenesis of Sindbis virus strains which are virulent or avirulent for newborn mice has not been previously examined. We have studied the diseases caused by a virulent wild-type strain, AR339, and two less virulent laboratory strains, Toto1101 and HRSP (HR small plaque). After peripheral inoculation of 1,000 PFU, AR339 causes 100% mortality within 5 days (50% lethal dose [LD50] = 3 PFU) while Toto1101 causes 70% mortality (LD50 = 10(2.4) PFU) and HRSP causes 50 to 60% mortality (LD50 = 10(5.1) PFU) with most deaths occurring 7 to 11 days after infection. However, after intracerebral inoculation of 1,000 PFU, Toto1101 is virulent (100% mortality within 5 days; LD50 = 4 PFU) while HRSP is not (75% mortality; LD50 = 10(4.2) PFU). After intracerebral inoculation, all three strains initiate new virus formation within 4 h, but HRSP reaches a plateau of 10(6) PFU/g of brain while Toto1101 and AR339 replicate to a level of 10(8) to 10(9) PFU/g of brain within 24 h. Interferon induction parallels virus growth. Mice infected with HRSP develop persistent central nervous system infection (10(6) PFU/g of brain) until the initiation of a virus-specific immune response 7 to 8 days after infection when virus clearance begins. The distribution of virus in the brains of mice was similar, but the virus was more abundant in the case of AR339. HRSP continued to spread until day 9. Clearance from the brain was complete by day 17. We conclude that the decreased virulence of HRSP is due to an intrinsic decreased ability of this strain of Sindbis virus to grow in neural cells of the mouse. We also conclude that CD-1 mice do not respond to the antigens of Sindbis virus until approximately 1 week of age. This lack of response does not lead to tolerance and persistent infection but rather to late virus clearance whenever the immune response is initiated.     

Effect of E2 envelope glycoprotein cytoplasmic domain mutations on Sindbis virus pathogenesis.

The cytoplasmic domain of the E2 envelope glycoprotein is important in Sindbis virus assembly, but little is known about its role in the pathogenesis of Sindbis virus encephalitis. To investigate its role in viral pathogenesis, we constructed six recombinant viruses containing site mutations in the E2 cytoplasmic domain, using the neurovirulent background strain, TE12. Our findings demonstrate that the E2 cytoplasmic domain is a determinant of Sindbis virus growth and neurovirulence in suckling mice as well as persistent infection in weanling scid mice. They also suggest that the tyrosine, serine, or threonine residues are not essential for replication in mouse brain or anti-E2 monoclonal antibody-mediated restriction of Sindbis virus replication.     

Luciferase imaging of a neurotropic viral infection in intact animals

The identification of viral determinants of virulence and host determinants of susceptibility to virus-induced disease is essential for understanding the pathogenesis of infection. Obtaining this information requires infecting large numbers of animals to assay amounts of virus in a variety of organs and to observe the onset and progression of disease. As an alternative approach, we have used a murine model of viral encephalitis and an in vivo imaging system that can detect light generated by luciferase to monitor over time the extent and location of virus replication in intact, living mice. Sindbis virus causes encephalomyelitis in mice, and the outcome of infection is determined both by the strain of virus used for infection and by the strain of mouse infected. The mode of entry into the nervous system is not known. Virulent and avirulent strains of Sindbis virus were engineered to express firefly luciferase, and the Xenogen IVIS system was used to monitor the location and extent of virus replication in susceptible and resistant mice. The amount of light generated directly reflected the amount of infectious virus in the brain. This system could distinguish virulent and avirulent strains of virus and susceptible and resistant strains of mice and suggested that virus entry into the nervous system could occur by retrograde axonal transport either from neurons innervating the initial site of replication or from the olfactory epithelium after viremic spread.     

Neuroattenuation of an avirulent bunyavirus variant maps to the L RNA segment.

The derivation and characterization of a neuroattenuated reassortant clone (RFC 25/B.5) of California serogroup bunyavirus was described previously (M. J. Endres, A. Valsamakis, F. Gonzalez-Scarano, and N. Nathanson, J. Virol. 64:1927-1933, 1990). To map the RNA segment responsible for this attenuation, a panel of reassortants was constructed between the attenuated clone B.5 (genotype TLL) and a virulent clone (B1-1a) of reciprocal genotype (LTT). Parent viruses and clones representing all of the six possible reassortants were examined for neurovirulence by intracerebral injection in adult mice. Reassortants bearing the large RNA segment from the virulent parent were almost as virulent as the virulent parent virus, while reassortants bearing the large RNA segment from the avirulent parent virus exhibited low or intermediate virulence. These results indicate that the large RNA segment is the major determinant of neuroattenuation of clone B.5. In addition to its neuroattenuation, clone B.5 was temperature sensitive and exhibited an altered plaque morphology. These phenotypes also segregated with the large RNA segment. The importance of the large RNA segment (which encodes the viral polymerase) in neurovirulence contrasts with prior studies which indicate that the ability to cause lethal encephalitis after peripheral injection of suckling mice (neuroinvasiveness) is primarily determined by the middle-sized RNA segment, which encodes the viral glycoproteins.     

Induction of apoptosis by Sindbis virus occurs at cell entry and does not require virus replication

Sindbis virus (SV) is an alphavirus that causes encephalitis in mice and can lead to the apoptotic death of infected cells. To determine the step in virus replication during which apoptosis is triggered, we used UV-inactivated SV, chemicals that block virus fusion or protein synthesis, and cells that do and do not express heparan sulfate, the initial binding molecule for SV infection of many cells. In initial experiments, UV-inactivated neuroadapted SV (NSV) induced apoptosis in Chinese hamster ovary (CHO) cells lacking heparan sulfate in the presence of cycloheximide. When fusion of prebound UV-inactivated NSV was rapidly induced at the plasma membrane by exposure to acidic pH, apoptosis was induced in CHO cells with or without heparan sulfate in the presence or absence of cycloheximide in a virus dose-dependent manner. In N18 neuroblastoma cells, the relative virulence of the virus strain was an important determinant of apoptosis induced by UV-inactivated SV. Treatment of N18 cells with monensin to prevent endosomal acidification an hour before, but not 2 h after, exposure to live NSV blocked the induction of cell death, as did treatment with NH(4)Cl or bafilomycin A1. These studies indicate that SV can induce apoptosis at the time of fusion with the cell membrane and that virus replication is not required.     

Identification of genes involved in the host response to neurovirulent alphavirus infection

Single-amino-acid mutations in Sindbis virus proteins can convert clinically silent encephalitis into uniformly lethal disease. However, little is known about the host gene response during avirulent and virulent central nervous system (CNS) infections. To identify candidate host genes that modulate alphavirus neurovirulence, we utilized GeneChip Expression analysis to compare CNS gene expression in mice infected with two strains of Sindbis virus that differ by one amino acid in the E2 envelope glycoprotein. Infection with Sindbis virus, dsTE12H (E2-55 HIS), resulted in 100% mortality in 10-day-old mice, whereas no disease was observed in mice infected with dsTE12Q (E2-55 GLN). dsTE12H, compared with dsTE12Q, replicated to higher titers in mouse brain and induced more CNS apoptosis. Infection with the neurovirulent dsTE12H strain was associated with both a greater number of host genes with increased expression and greater changes in levels of host gene expression than was infection with the nonvirulent dsTE12Q strain. In particular, dsTE12H infection resulted in greater increases in the levels of mRNAs encoding chemokines, proteins involved in antigen presentation and protein degradation, complement proteins, interferon-regulated proteins, and mitochondrial proteins. At least some of these increases may be beneficial for the host, as evidenced by the demonstration that enforced expression of the antiapoptotic mitochondrial protein peripheral benzodiazepine receptor (PBR) protects neonatal mice against lethal Sindbis virus infection. Thus, our findings identify specific host genes that may play a role in the host protective or pathologic response to neurovirulent Sindbis virus infection.     

Structural and nonstructural protein genome regions of eastern equine encephalitis virus are determinants of interferon sensitivity and murine virulence

Eastern equine encephalitis virus (EEEV) causes sporadic epidemics of human and equine disease in North America, but South American strains have seldom been associated with human neurologic disease or mortality, despite serological evidence of infection. In mice, most North American and South American strains of EEEV produce neurologic disease that resembles that associated with human and equine infections. We identified a South American strain that is unable to replicate efficiently in the brain or cause fatal disease in mice yet produces 10-fold higher viremia than virulent EEEV strains. The avirulent South American strain was also sensitive to human interferon (IFN)-alpha, -beta, and -gamma, like most South American strains, in contrast to North American strains that were highly resistant. To identify genes associated with IFN sensitivity and virulence, infectious cDNA clones of a virulent North American strain and the avirulent South American strain were constructed. Two reciprocal chimeric viruses containing swapped structural and nonstructural protein gene regions of the North American and South American strains were also constructed and found to replicate efficiently in vitro. Both chimeras produced fatal disease in mice, similar to that caused by the virulent North American strain. Both chimeric viruses also exhibited intermediate sensitivity to human IFN-alpha, -beta, and -gamma compared to that of the North American and South American strains. Virulence 50% lethal dose assays and serial sacrifice experiments further demonstrated that both structural and nonstructural proteins are important contributors to neurovirulence and viral tissue tropism. Together, the results of this study emphasize the complex and important influences of structural and nonstructural protein gene regions on EEEV virulence.     

Recombinant sindbis/Venezuelan equine encephalitis virus is highly attenuated and immunogenic

Venezuelan equine encephalitis virus (VEEV) is an important, naturally emerging zoonotic virus. VEEV was a significant human and equine pathogen for much of the past century, and recent outbreaks in Venezuela and Colombia (1995), with about 100,000 human cases, indicate that this virus still poses a serious public health threat. The live attenuated TC-83 vaccine strain of VEEV was developed in the 1960s using a traditional approach of serial passaging in tissue culture of the virulent Trinidad donkey (TrD) strain. This vaccine presents several problems, including adverse, sometimes severe reactions in many human vaccinees. The TC-83 strain also retains residual murine virulence and is lethal for suckling mice after intracerebral (i.c.) or subcutaneous (s.c.) inoculation. To overcome these negative effects, we developed a recombinant, chimeric Sindbis/VEE virus (SIN-83) that is more highly attenuated. The genome of this virus encoded the replicative enzymes and the cis-acting RNA elements derived from Sindbis virus (SINV), one of the least human-pathogenic alphaviruses. The structural proteins were derived from VEEV TC-83. The SIN-83 virus, which contained an additional adaptive mutation in the nsP2 gene, replicated efficiently in common cell lines and did not cause detectable disease in adult or suckling mice after either i.c. or s.c. inoculation. However, SIN-83-vaccinated mice were efficiently protected against challenge with pathogenic strains of VEEV. Our findings suggest that the use of the SINV genome as a vector for expression of structural proteins derived from more pathogenic, encephalitic alphaviruses is a promising strategy for alphavirus vaccine development.     

Inhibition of nitric oxide synthesis increases mortality in Sindbis virus encephalitis.

Sindbis virus (SV) is an alphavirus that causes acute encephalomyelitis in mice. The outcome is determined by the strain of virus and by the age and genetic background of the host. The mortality rates after infection with NSV, a neurovirulent strain of SV, were as follows v: 81% (17 of 21) in BALB/cJ mice; 20% (4 of 20) in BALB/cByJ mice (P < 0.001); 100% in A/J, C57BL/6J, SJL, and DBA mice; and 79% (11 of 14) in immunodeficient scid/CB17 mice. Treatment with Nomega-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthetase (NOS) inhibitor, increased mortality to 100% (P < 0.05) in NSV-infected BALB/cJ mice, to 95% (P < 0.001) in BALB/cByJ mice, and to 100% in scid/CB17 mice. BALB/cJ and BALB/cByJ mice had similar levels of inducible NOS mRNA in their brains, which were not affected by L-NAME or NSV infection. Brain NOS activity was similar in BALB/cJ and BALB/cByJ mice before and after infection and was markedly inhibited by L-NAME. NSV replication in the brains of BALB/cJ mice, BALB/cByJ mice, and mice treated with L-NAME was similar. Treatment of N18 neuroblastoma cells with NO donors S-nitroso-N-acetylpenicillamine or sodium nitroprusside in vitro before infection increased cell viability at 42 to 48 h compared with untreated NSV-infected N18 cells with little effect on virus replication. These data suggest that NO protects mice from fatal encephalitis by a mechanism that does not directly involve the immune response or inhibition of virus growth but rather may enhance survival of the infected neuron until the immune response can control virus replication.     

Mechanism of altered Sindbis virus neurovirulence associated with a single-amino-acid change in the E2 Glycoprotein.

The mechanism by which amino acid changes in the E1 and E2 surface glycoproteins of Sindbis virus affect neurovirulence is unknown. We have studied two recombinant viruses which differ in virulence. One (TE) contains Gly and the other (TES) contains Arg at position 172 in E2. TE causes more rapid death than TES in newborn mice. Both viruses replicate similarly in nonneuronal cells, but TE replicates more rapidly in the brains of newborn mice and in neuroblastoma cells. TE also induces earlier viral RNA synthesis in neuroblastoma cells. 35S-labeled TE binds more efficiently to brain and neuroblastoma cells, but not to nonneuronal cells, than TES. We propose that a region of the E2 glycoprotein affected by the amino acid occupying position 172 is important for binding to an alphavirus receptor on neurons and influences neurovirulence by this mechanism.     

Safety testing for neurovirulence of novel live, attenuated flavivirus vaccines: infant mice provide an accurate surrogate for the test in monkeys.

Current requirements for control of live viral vaccines, including yellow fever 17D, produced from potentially neurotropic wild-type viruses include tests for neurovirulence in nonhuman primates. We have used yellow fever 17D virus as a live vector for novel flavivirus vaccines (designated ChimeriVax) against dengue, Japanese encephalitis (JE), and West Nile (WN) viruses. For control of these vaccines, it would be preferable to substitute a test in mice for the test in a higher species (monkeys). In this study, we compare the neurovirulence of ChimeriVax vaccine candidates in suckling mice inoculated by the intracerebral (IC) route with graded doses of the test article or yellow fever 17D vaccine as a reference control. Mortality ratio and survival distribution are the outcome measures. The monkey safety test is performed as described for control of yellow fever vaccines. In both mice and monkeys, all chimeric vaccines were significantly less neurovirulent than yellow fever 17D vaccine. The test in suckling mice discriminated between strains of two different vaccines (ChimeriVax-JE and ChimeriVax-DEN1) differing by a single amino acid change, and was more sensitive for detecting virulence differences than the test in monkeys. The results indicate that the suckling mouse test is simple to perform, highly sensitive and, with appropriate validation, could complement or possibly even replace the neurovirulence component of the monkey safety test. The test in infant mice is particularly useful as a means of demonstrating biological consistency across seed virus and vaccine lots.     

Contributions of the viral genetic background and a single amino acid substitution in an immunodominant CD8+ T-cell epitope to murine coronavirus neurovirulence

The immunodominant CD8+ T-cell epitope of a highly neurovirulent strain of mouse hepatitis virus (MHV), JHM, is thought to be essential for protection against virus persistence within the central nervous system. To test whether abrogation of this H-2Db-restricted epitope, located within the spike glycoprotein at residues S510 to 518 (S510), resulted in delayed virus clearance and/or virus persistence we selected isogenic recombinants which express either the wild-type JHM spike protein (RJHM) or spike containing the N514S mutation (RJHM(N514S)), which abrogates the response to S510. In contrast to observations in suckling mice in which viruses encoding inactivating mutations within the S510 epitope (epitope escape mutants) were associated with persistent virus and increased neurovirulence (Pewe et al., J Virol. 72:5912-5918, 1998), RJHM(N514S) was not more virulent than the parental, RJHM, in 4-week-old C57BL/6 (H-2b) mice after intracranial injection. Recombinant viruses expressing the JHM spike, wild type or encoding the N514S substitution, were also selected in which background genes were derived from the neuroattenuated A59 strain of MHV. Whereas recombinants expressing the wild-type JHM spike (SJHM/RA59) were highly neurovirulent, A59 recombinants containing the N514S mutation (SJHM(N514S)/RA59) were attenuated, replicated less efficiently, and exhibited reduced virus spread in the brain at 5 days postinfection (peak of infectious virus titers in the central nervous system) compared to parental virus encoding wild-type spike. Virulence assays in BALB/c mice (H-2d), which do not recognize the S510 epitope, revealed that attenuation of the epitope escape mutants was not due to the loss of a pathogenic immune response directed against the S510 epitope. Thus, an intact immunodominant S510 epitope is not essential for virus clearance from the CNS, the S510 inactivating mutation results in decreased virulence in weanling mice but not in suckling mice, suggesting that specific host conditions are required for epitope escape mutants to display increased virulence, and the N514S mutation causes increased attenuation in the context of A59 background genes, demonstrating that genes other than that for the spike are also important in determining neurovirulence.     

The Neurovirulence of the DA and GDVII Strains of Theiler’s Virus Correlates with Their Ability To Infect Cultured Neurons

The strains of Theiler’s murine encephalomyelitis virus, a picornavirus, are divided into two groups according to their neurovirulence after intracerebral inoculation. The highly virulent GDVII strain causes an acute, fatal encephalomyelitis, whereas the DA strain causes a mild encephalomyelitis followed by a chronic inflammatory demyelinating disease associated with viral persistence. Studies with recombinant viruses showed that the capsid plays the major role in determining these phenotypes. However, the molecular basis for the effect of the capsid on neurovirulence is still unknown. In this paper, we describe a large difference in the patterns of infection of primary neuron cultures by the GDVII and DA strains. Close to 90% of the neurons were infected 12 h after inoculation with the GDVII strain, and the cytopathic effect was complete 24 h postinoculation. In contrast, with the DA strain, viral antigens were not detected in neurons until 24 h postinoculation. Infected neurons accounted for only 2% of the total number of neurons, even 6 days after inoculation. No cytopathic effect was visible, and the cultures could be kept for the same length of time as the noninfected controls. Because the neurovirulence of the GDVII strain has been mapped to the capsid, we examined the role of the capsid in this difference of phenotype. We showed, using recombinant viruses, that the capsid was indeed responsible for the pattern of infection observed in vitro, most likely through its role in viral entry. Thus, the levels of neurovirulence of the GDVII and DA strains correlate with their abilities to infect cultured neurons, and this ability is controlled by the capsid.     

Immunization with nonstructural proteins promotes functional recovery of alphavirus-infected neurons.

The encephalitic alphaviruses are useful models for understanding virus-neuron interactions. A neurovirulent strain of Sindbis virus (NSV) causes fatal paralysis in mice by infecting motor neurons and inducing apoptosis of these nonrenewable cells. Antibodies to the surface glycoproteins suppress virus replication, but other recovery-promoting components of the immune response have not been recognized. We assessed the effect on the outcome of NSV-induced encephalomyelitis of immunization of mice with nonstructural proteins (nsPs) by using recombinant vaccinia viruses. Mice immunized with vaccinia virus expressing nsPs and challenged with NSV initially developed paralysis similar to unimmunized mice but then recovered neurologic function. Mice preimmunized with vaccinia virus expressing structural proteins were completely protected from paralysis. Mice immunized with vaccinia virus alone showed paralysis with little evidence of recovery. Vaccinia virus expressing only nsP2 was as effective as vaccinia virus expressing all the nsPs. Protection provided by immunity to nsPs was not associated with a reduction in virus replication or with improved antibody responses to structural proteins. Protection could not be passively transferred with nsP immune serum. The depletion of T cells at the time of NSV infection decreased protection. The data show that antiviral immune responses can improve the ability of neurons to survive infection and to recover function without altering virus replication.