Molecular basis of Sindbis virus neurovirulence in mice.

We examined a variety of strains of Sindbis virus for the genetic changes responsible for differences in neurovirulence in mice. SV1A (a low passage of the AR339 strain of Sindbis virus), a neuroadapted Sindbis virus (NSV), and two laboratory strains of Sindbis virus (HRSP and Toto1101) were examined. NSV causes severe encephalomyelitis with hind-limb paralysis and high mortality after intracerebral inoculation in weanling mice. In contrast, SV1A causes only mild, nonfatal disease in weanling mice; however, in suckling mice, SV1A causes a fatal encephalomyelitis after either intracerebral or subcutaneous inoculation. The two laboratory strains used have a greatly reduced neurovirulence for suckling mice and are avirulent for weanling mice. The nucleotide sequences and encoded amino acid sequences of the structural glycoproteins of these four strains were compared. Hybrid genomes were constructed by replacing restriction fragments in a full-length cDNA clone of Sindbis virus, from which infectious RNA can be transcribed in vitro, with fragments from cDNA clones of the various strains. These recombinant viruses allowed us to test the importance of each amino acid difference between the various strains for neurovirulence in weanling and suckling mice. Glycoproteins E2 and E1 were of paramount importance for neurovirulence in adult mice. Recombinant viruses containing the nonstructural protein region and the capsid protein region from an avirulent strain and the E1 and E2 glycoprotein regions from NSV were virulent, although they were less virulent than NSV. Furthermore, changes in either E2 (His-55 in NSV to Gln in SV1A) or E1 (Ala-72 in NSV to Val in SV1A and Asp-313 in NSV to Gly in SV1A) reduced virulence. For virulence in suckling mice, we found that a number of changes in E2 and E1 can lead to decreased virulence and that in fact, a gradient of virulence exists.     

A deletion at the UL/IR junction reduces pseudorabies virus neurovirulence.

A recombinant pseudorabies virus (PRV), designated LLT beta delta 2, which contains a 3-kbp deletion spanning the junction of the unique long and internal repeat sequences was constructed. Compared with the parental strain and a virus rescued for the deleted sequences, LLT beta delta 2 exhibited similar replication characteristics in tissue culture. When inoculated intranasally in swine, LLT beta delta 2 was significantly reduced in virulence and did not produce neurological signs characteristic of PRV infection. LLT beta delta 2 replicated efficiently at the site of inoculation and in peripheral nervous tissues, but replication was restricted in the central nervous system. These results indicate the presence of a PRV neurovirulence determinant in the vicinity of the junction.     

Influence of pseudorabies virus proteins on neuroinvasion and neurovirulence in mice

Neurotropism is a distinctive feature of members of the Alphaherpesvirinae. However, its molecular basis remains enigmatic. In the past, research has been focused mainly on the role of viral envelope proteins in modulating herpesvirus neuroinvasion and neurovirulence (T. C. Mettenleiter, Virus Res. 92:192-206, 2003). To further analyze the molecular requirements for neuroinvasion of the alphaherpesvirus pseudorabies virus (PrV), adult mice were infected intranasally with a set of single- or multiple-deletion mutants lacking the UL3, UL4, UL7, UL11, UL13, UL16, UL17, UL21, UL31, UL34, UL37, UL41, UL43, UL46, UL47, UL48, UL51, US3, US9, glycoprotein E (gE), gM, UL11/US9, UL11/UL16, UL16/UL21, UL11/UL16/UL21, UL11/gE, UL11/gM, UL43/gK, UL43/gM, or UL43/gK/gM genes. Neurovirulence was evaluated by measuring mean survival times compared to that after wild-type virus infection. Furthermore, by immunohistochemical detection of infected neurons, the kinetics of viral spread in the murine central nervous system was investigated.     

Genetic determinants of dengue type 4 virus neurovirulence for mice.

Mouse-adapted dengue type 4 virus (DEN4) strain H241 is highly neurovirulent for mice, whereas its non-mouse-adapted parent is rarely neurovirulent. The genetic basis for the neurovirulence of the mouse-adapted mutant was studied by comparing intratypic chimeric viruses that contained the three structural protein genes from the parental virus or the neurovirulent mutant in the background sequence of nonneurovirulent DEN4 strain 814669. The chimera that contained the three structural protein genes from mouse neurovirulent DEN4 strain H241 proved to be highly neurovirulent in mice, whereas the chimera that contained the corresponding genes from its non-mouse-adapted parent was not neurovirulent. This finding indicates that most of the genetic loci for the neurovirulence of the DEN4 mutant lie within the structural protein genes. A comparison of the amino acid sequences of the parent and its mouse neurovirulent mutant proteins revealed that there were only five amino acid differences in the structural protein region, and three of these were located in the envelope (E) glycoprotein. Analysis of chimeras which contained one or two of the variant amino acids of the mutant E sequence substituting for the corresponding sequence of the parental virus identified two of these amino acid changes as important determinants of mouse neurovirulence. First, the single substitution of Ile for Thr-155 which ablated one of the two conserved glycosylation sites in parental E yielded a virus that was almost as neurovirulent as the mouse-adapted mutant. Thus, the loss of an E glycosylation site appears to play a role in DEN4 neurovirulence. Second, the substitution of Leu for Phe-401 also yielded a neurovirulent virus, but it was less neurovirulent than the glycosylation mutant. These findings indicate that at least two of the genetic loci responsible for DEN4 mouse neurovirulence map within the structural protein genes.     

Biological basis of rabies virus neurovirulence in mice: comparative pathogenesis study using the immunoperoxidase technique.

The CVS strain of fixed rabies virus causes acute, fatal encephalomyelitis in young adult ICR mice. Variant RV194-2, which was selected from CVS virus in cell culture with a neutralizing antiglycoprotein monoclonal antibody, has a single amino acid change in the glycoprotein. The infections caused by CVS virus and RV194-2 virus were compared in mice for 14 days postinoculation of 5 x 10(7) PFU into the right masseter muscle. All CVS virus-infected mice died (mean time to death, 7.9 days), compared with a mortality rate of 8.5% for RV194-2 virus-infected mice. RV194-2 virus spread to the ipsilateral trigeminal ganglion during the first 2 days postinoculation, and both viruses spread to the ipsilateral motor nucleus of the trigeminal nerve in the pons. Both viruses spread centrifugally and caused infection of bilateral trigeminal ganglia on day 3. The viruses spread throughout the central nervous system (CNS) at similar rates, but CVS virus infected many more neurons than did RV194-2 virus. Rabies virus antigen was observed in only occasional CNS neurons after day 6 of RV194-2 virus infection. By this time, CVS virus had caused severe widespread infection. In this model, virulence depends on improved efficiency of viral spread between CNS neurons rather than the rate of spread or topographical distribution of the infection.     

Molecular basis for interference of defective interfering particles of pseudorabies virus with replication of standard virus.

Serial passage of pseudorabies virus (PrV) at high multiplicity yields defective interfering particles (DIPs), but the sharp cyclical increases and decreases in titer of infectious virus that are observed upon continued passage at high multiplicity of most DIPs of other viruses are not observed with DIPs of PrV (T. Ben-Porat and A. S. Kaplan, Virology 72:471-479). We have studied the dynamics of the interactions of the virions present in a population of DIPs to assess the cis functions for which the genomes of the DIPs are enriched. The defective genomes present in one population of DIPs, [PrV(1)42], replicate preferentially over the nondefective genomes present in that virion population at early stages of infection, indicating that the DIP DNA is enriched for sequences that can serve as origins of replication at early stages of infection. This replicative advantage of the DIP DNA is transient and disappears at later stages of infection. The defective DNA does not appear to be encapsidated preferentially over the nondefective DNA present in this virion population, which might indicate that it is not enriched for cleavage-encapsidation sites. However, the nondefective DNA in the DIP virion population has become modified and has acquired reiterations of sequences originating from the end of the unique long (UL) region of the genome. Furthermore, both the infectious and defective genomes present in the DIP population compete for encapsidation more effectively than do the genomes of standard PrV. These results indicate that the defective genomes in the population of virions studied are enriched not only for an origin of replication but probably also for sequences necessary for efficient cleavage-encapsidation. Furthermore, the nondefective genomes present in this population of DIPs have also been modified and have acquired the ability to compete with the defective genomes for cleavage-encapsidation.     

Genetic and biological analyses of a herpes simplex virus intertypic recombinant reduced specifically for neurovirulence.

RS6 is a herpes simplex virus intertypic recombinant derived from type 1 strain 17 syn+ and type 2 strain HG52. With a 50% lethal dose of about 10(5) PFU after intracerebral inoculation of mice, RS6 was approximately 100,000 times less neurovirulent than either of its wild-type parental viruses were. When compared with strains 17 syn+ and HG52, RS6 replicated intermediately in primary mouse embryo fibroblasts in vitro at 38.5 degrees C (mouse temperature) and to wild-type peak titers in mouse feet in vivo. In contrast, following intracranial inoculation of mice, RS6 replicated significantly less well than did either of its parental viruses in brains. The genetic defect(s) responsible for the reduced neurovirulence of RS6 was stable after in vitro and in vivo serial passage, was not manifested as temperature-sensitive plaquing in vitro, and did not affect thymidine kinase expression. These data indicate that RS6 has a genetic defect(s) specifically affecting its ability to replicate in the mouse brain. Using marker rescue technologies, we increased the neurovirulence of RS6 and localized one genetic determinant(s) involved with the reduced neurovirulence of this agent to 0.72 to 0.87 map units (and, tentatively, to 0.79 to 0.83 map units) of the herpes simplex virus genome. When coupled with the work suggesting that thymidine kinase expression is essential for efficient replication in nerve tissues and earlier reports from this laboratory and others, the results presented in this study indicate that more than one herpes simplex virus gene is involved with neurovirulence.     

Characterization of the envelope proteins of pseudorabies virus.

Previously we have reported that among the proteins of purified pseudorabies virions there are four major glycoproteins (T. Ben-Porat and A. S. Kaplan, Virology 41:265-273, 1970). Several minor glycoproteins can also be identified by two-dimensional gel electrophoresis. Removal of the viral envelope with Triton X-100 selectively removes from the virions all of the glycoproteins as well as several non-glycosylated proteins. Sedimentation analysis or chromatography of these proteins reveals that several are complexed with one another, some being covalently linked via disulfide bridges. Analysis of the proteins by immunoprecipitation with monoclonal antibodies reactive with the membrane proteins showed also that three of the four major virus glycoproteins (125K, 74K, and 58K; gIIa, gIIb, and gIIc, respectively) are linked covalently by disulfide bridges. Furthermore, all three share extensive sequence homology as indicated by the identity of their antigenic determinants and by partial peptide mapping; they probably originate from a single protein precursor. The fourth major glycoprotein (98K; gIII) is not complexed to any other protein. Three minor glycoproteins (130K [gI], 98K [gIV], and 62K [gV]), which form a noncovalently linked complex with a 115K nonglycosylated protein, have also been identified. Of the monoclonal antibodies used in this study, only those reactive with the major 98K glycoprotein (gIII) inhibit virus adsorption and neutralize virus infectivity in the absence of complement. However, all react with surface components of the virion, indicating that the proteins with which they react are exposed on the surface of the virions. A nomenclature for the pseudorabies virus glycoproteins is proposed.     

Processing of pseudorabies virus glycoprotein gII.

The glycoprotein complex gII of pseudorabies virus was isolated by immunoprecipitation with the monoclonal antibody M5, which was covalently linked to protein A-Sepharose. After sodium dodecyl sulfate-polyarylamide gel electrophoresis under reducing conditions and blotting onto poly(vinylidene difluoride) membrane, its subunits, gIIa, gIIb, and gIIc, were subjected to N-terminal sequencing. gIIa and gIIb start at position 59 and gIIc starts at position 503 according to the amino acid sequence deduced from the gene, indicating that there is one major protein (gIIa) which is cleaved into the two protein fragments gIIb and gIIc. Protein labeling with 14C-amino acids gave no indication that the three proteins (gIIa, gIIb, and gIIc) of the complex are present in equimolar ratios. It seems that gIIa is only a minor component of the complex, whereas gIIb and gIIc are contained in equimolar amounts.     

Inverted repetition in the chromosome of pseudorabies virus.

An electron microscope examination of pseudorabies virus DNA single strands after self-annealing shows a loop of single-stranded DNA at one end of the molecule contiguous to a double-strand region. The molecule then terminates in a further single-stranded region that does not form a loop. It is suggested that the DNA contains a sequence of 13.3 x 106 daltons at one end, which is repeated internally with opposite polarity. The segment of the genome separating the repeats has a double-strand molecular weight of 5.4 x 106. The whole native DNA has a molecular weight of 90 x 106 to 95 x 106.     

Measles virus neurovirulence and host immunity

Measles virus is highly neuroinvasive, yet host immune responses are highly effective at limiting neurovirulence in humans. We know that neurons are an important target of infection and that both IFN-γ and -β expression are observed in the measles virus-infected human brain. Rodent models can be used to understand how this response is orchestrated. Constitutive expression of the major inducible 70-kDa heat-shock protein is a feature of primate tissues that is lacking in mice. This article examines the importance of addressing this difference when modeling outcomes of brain infection in mice, particularly in terms of understanding how infected neurons may activate uninfected brain macrophages to produce IFN-β and support T-cell production of IFN-γ, a mediator of noncytolytic viral clearance. New and historical data suggest that the virus heat-shock protein 70 relationship is key to a protective host immune response and has potential broad relevance.     

Antigenic determinants of measles virus hemagglutinin associated with neurovirulence.

The biological activity of monoclonal antibodies specific for the hemagglutinin protein of measles virus strain CAM recognizing six epitope groups according to their binding properties to measles virus strain CAM/R401 was investigated in vivo in our rat model of measles encephalitis. When injected intraperitoneally into measles virus-infected suckling rats, some monoclonal antibodies modified the disease process and prevented the necrotizing encephalopathy seen in untreated animals. The analysis of measles virus brain isolates revealed emergence of variants that resisted neutralization with the passively transferred selecting monoclonal antibody but not with other monoclonal antibodies. Monoclonal antibody escape mutants were also isolated in vitro, and their neurovirulence varied in the animal model. Sequence data from the hemagglutinin gene of measles virus localize a major antigenic surface determinant of the hemagglutinin protein between amino acid residues 368 and 396, which may be functionally important for neurovirulence. The data indicate that the interaction of antibodies with the measles virus H protein plays an important role in the selection of neurovirulent variants. These variants have biological properties different from those of the parent CAM virus.     

Chimeric cDNA studies of Theiler's murine encephalomyelitis virus neurovirulence.

Strain GDVII and other members of the GDVII subgroup of Theiler's murine encephalomyelitis virus are highly neurovirulent and rapidly fatal, while strain DA and other members of the TO subgroup produce a chronic, demyelinating disease. GDVII/DA chimeric cDNA studies suggest that a major neurovirulence determinant is within the GDVII 1B through 1D capsid protein coding region, although the additional presence of upstream GDVII sequences, including the 5' untranslated region, contributes to full neurovirulence. Our studies indicate that there are limitations in precisely delineating neurovirulence determinants with chimeric cDNAs between evolutionarily diverged viruses, such as GDVII and DA.     

Glycoprotein gIII of pseudorabies virus is multifunctional.

One of the major glycoproteins of pseudorabies virus, gIII, is nonessential for growth in cell culture. Mutants defective in gIII, however, consistently yield lower titers of infectious virus (3- to 20-fold) than does wild-type virus. The interactions of gIII- mutants with their host cells were compared with those of wild-type virus in an attempt to uncover the functions of gIII. We show that gIII plays a major role in the stable adsorption of the virus to its host cell; in the absence of gIII, the rate of adsorption is reduced and adsorption is easily reversed by washing. Thus, adsorption of pseudorabies virus can be said to occur in at least the following two ways: (i) a gIII-mediated rapid adsorption or (ii) a slower and more labile adsorption that is independent of gIII. After virions have been complexed with monoclonal antibodies against gIII (but not some monoclonal antibodies against other glycoproteins), both modes of adsorption were inhibited. Glycoprotein gIII affects virus stability and virus release, as well as adsorption. The effect on virus release is marked when the virus is defective in additional functions. Thus, although we found no obvious difference in the release of virus from gIII- or wild-type virus-infected rabbit kidney cells, release of a gIII-/gI- double mutant from the cells occurred less readily than did release of a gI- mutant. The gIII-/gI- and gIII- mutants, however, adsorbed to cells at a similar rate, indicating that the effects of gIII on adsorption and virus release constitute separate functions. The Bartha vaccine strain of pseudorabies virus has a defective gIII gene and is released poorly from rabbit kidney cells. After the resident Bartha gIII gene was replaced by the gIII gene of wild-type virus, virus release was enhanced considerably. Since inactivation of gIII in wild-type pseudorabies virus did not significantly affect virus release, the Bartha strain must be defective in another function which, in conjunction with gIII, significantly affects virus release. These results indicate again that gIII affects virus release in conjunction with other functions. Also, although the Bartha strain was functionally defective in virus release, it adsorbed to cells as well as wild-type virus did, showing that the effects of gIII on virus adsorption and release constitute separate functions. We conclude that gIII is a multifunctional glycoprotein.     

Identification and characterization of pseudorabies virus dUTPase.

Sequence analysis within the long segment of the pseudorabies virus (PrV) genome identified an open reading frame of 804 bp whose deduced protein product of 268 amino acids exhibited homology to dUTPases of other herpesviruses. The gene was designated UL50 because of its colinearity with the homologous gene of herpes simplex virus type 1. An antiserum raised against a bacterially expressed fragment of PrV UL50 specifically detected a 33-kDa protein in lysates of infected cells, which is in agreement with the predicted molecular mass of the PrV UL50 protein. A UL50-negative PrV mutant (PrV UL50-) was constructed by the insertion of a beta-galactosidase expression cassette into the UL50 coding sequence. A corresponding rescuant (PrV UL50resc) was also isolated. The interruption of the UL50 gene led to the disappearance of the 33-kDa protein, whereas restoration of UL50 gene expression restored detection of the 33-kDa protein. Enzyme activity assays confirmed that UL50 of PrV codes for a dUTPase which copurifies with nuclei of infected cells. PrV UL50- replicated with an only slightly reduced efficiency in epithelial cells in culture compared with that of its parental wild-type virus strain. Our results thus demonstrate that UL50 of PrV encodes a protein of 33 kDa with dUTPase activity which copurifies with nuclei of infected cells and is dispensable for replication in cultured epithelial cells.     

Genetic determinants of neurovirulence and attenuation of poliovirus

The results of mapping of genetic determinants for poliovirus attenuation are reviewed. Possible mechanisms of the phenotypic expression of attenuating mutations located in different regions of the viral genome are discussed.     

Disease duration determines canine distemper virus neurovirulence

The Morbillivirus hemagglutinin (H) protein mediates attachment to the target cell. To evaluate its contribution to canine distemper virus neurovirulence, we exchanged the H proteins of the wild-type strains 5804P and A75 and assessed the pathogenesis of the chimeric viruses in ferrets. Both strains are lethal to ferrets; however, 5804P causes a 2-week disease without neurological signs, whereas A75 is associated with a longer disease course and neurological involvement. We observed that both H proteins supported neuroinvasion and the subsequent development of clinical neurological signs if given enough time, demonstrating that disease duration is the main neurovirulence determinant.     

Localization of a herpes simplex virus neurovirulence gene dissociated from high-titer virus replication in the brain.

Previous studies with the herpes simplex virus type 1 X type 2 intertypic recombinant RS6 suggested that the genomic region from 0.11 to 0.14 map units is involved in neurovirulence (R. T. Javier, R. L. Thompson, and J. G. Stevens, J. Virol. 61:1978-1984, 1987). To study this further, we isolated an RS6-derived herpes simplex virus intertypic recombinant (R13-1) which has a genetic defect within this area. After inoculation into mouse brains, R13-1 was found to be approximately 10,000-fold less neurovirulent than either the wild-type type 1 or type 2 parental virus. However, R13-1 replicated in the mouse brain to titers resembling those of the wild-type parents. Further comparisons with wild-type counterparts indicated that R13-1 expressed equivalent levels of the enzyme thymidine kinase and replicated to intermediate levels in primary mouse embryo fibroblasts maintained at the normal body temperature for mice. Using marker rescue techniques combined with in vivo selection, we found that recombination between unit-length R13-1 DNA and a cloned type 1 DNA fragment spanning the region from 0.11 to 0.14 map units (EcoRI-d, 0.079 to 0.192 map units) generated viruses with a wild-type neurovirulence phenotype. To further refine the genomic region of interest, we performed marker rescue experiments using two EcoRI-d subclones, EcoRI/BamHI dc (0.079 to 0.143 map units) and BamHI/EcoRI and (0.143 to 0.192 map units), representing the left and right halves of the EcoRI d fragment, respectively. In these experiments the EcoRI/BamHI dc clone, but not the BamHI/EcoRI ad clone, yielded recombinant viruses exhibiting wild-type neurovirulence. These results show that at least one herpes simplex virus gene function associated with neurovirulence is located within a 9.1-kilobase region at 0.079 to 0.143 map units of the viral genome. Perhaps more significantly, the results indicate that this neurovirulence property functions independently of high-titer virus replication in the brain.     

Pathogenesis of simian immunodeficiency virus encephalitis: viral determinants of neurovirulence.

To examine the relationship between macrophage tropism and neurovirulence, macaques were inoculated with two recombinant hybrid viruses derived from the parent viruses SIVmac239, a lymphocyte-tropic, non-neurovirulent clone, and SIV/17E-Br, a macrophage-tropic, neurovirulent virus strain. The first recombinant, SIV/17E-Cl, contained the portion of the env gene that encodes the surface glycoprotein and a short segment of the transmembrane glycoprotein of SIV/17E-Br in the backbone of SIVmac239. Unlike SIVmac239, SIV/17E-Cl replicated productively in macrophages, demonstrating that sequences in the surface portion of env determine macrophage tropism. None of five macaques inoculated with SIV/17E-Cl developed simian immunodeficiency virus (SIV) encephalitis. The second recombinant, SIV/17E-Fr, which contained the entire env and nef genes and the 3' long terminal repeat of SIV/17E-Br in the SIVmac239 backbone, was also macrophage tropic. Six of nine macaques inoculated with SIV/17E-Fr developed SIV encephalitis ranging from mild to moderate in severity, indicating a significant (P = 0.031) difference in the neurovirulence of the two recombinants. In both groups of macaques, CD4+ cell counts declined gradually during infection and there was no significant difference in the rate of the decline between the two groups of macaques. This study demonstrated that macrophage tropism alone is not sufficient for the development of neurological disease. In addition, it showed that while sequences in the surface portion of the envelope gene determine macrophage tropism, additional sequences derived from the transmembrane portion of envelope and/or nef confer neurovirulence.