Characterization of a neurologic disease induced by a polytropic murine retrovirus: evidence for differential targeting of ecotropic and polytropic viruses in the brain.

A variety of ecotropic murine leukemia viruses cause neurodegenerative disease. We describe here the clinical and histopathological features of a neurologic disease induced by a polytropic murine leukemia virus, FMCF98. Clinical disease was dominated by hyperexcitability and ataxia, and the histopathology was characterized primarily by astrocytosis and astrocytic degeneration. The viral envelope gene harbored the determinants of neurovirulence, since the chimeric virus Fr98E, which contained the envelope gene of FMCF98 on a background of the nonneurovirulent virus FB29, caused a similar disease. The disease caused by Fr98E differed from that induced by the coisogenic neurovirulent ecotropic virus FrCasE in clinical presentation, histopathology, and distribution of virus in the central nervous system. Since Fr98E contains a polytropic envelope gene and FrCasE contains an ecotropic envelope gene, these phenotypic differences appeared to be determined by envelope sequences and may reflect differences in virus receptor usage in the central nervous system.     

Neurologic disease induced by polytropic murine retroviruses: neurovirulence determined by efficiency of spread to microglial cells.

Several murine leukemia viruses (MuLV) induce neurologic disease in susceptible mice. To identify features of central nervous system (CNS) infection that correlate with neurovirulence, we compared two neurovirulent MuLV, Fr98 and Fr98/SE, with a nonneurovirulent MuLV, Fr54. All three viruses utilize the polytropic receptor and are coisogenic, each containing a different envelope gene within a common genetic background. Both Fr98 and Fr98/SE induce a clinical neurologic disease characterized by hyperexcitability and ataxia yet differ in incubation period, 16 to 30 and 30 to 60 days, respectively. Fr54 infects the CNS but fails to induce clinical signs of neurologic disease. In this study, we compared the histopathology, regional virus distribution, and cell tropism in the brain, as well as the relative CNS viral burdens. All three viruses induced similar histopathologic effects, characterized by intense reactive astrogliosis and microglial activation associated with minimal vacuolar degeneration. The infected target cells for each virus consisted primarily of endothelial and microglial cells, with rare oligodendrocytes. Infection localized predominantly in white matter tracts of the cerebellum, internal capsule, and corpus callosum. The only feature that correlated with relative neurovirulence was viral burden as measured by both viral CA protein expression in cerebellar homogenates and quantification of infected cells. Interestingly, Fr54 (nonneurovirulent) and Fr98/SE (slow disease) had similar viral burdens at 3 weeks postinoculation, suggesting that they entered the brain with comparable efficiencies. However, spread of Fr98/SE within the brain thereafter exceeded that of Fr54, reaching levels of viral burden comparable to that seen for Fr98 (rapid disease) at 3 weeks. These results suggest that the determinants of neurovirulence in the envelope gene may influence the efficiency of virus spread within the brain and that a critical number of infected cells may be required for induction of clinical neurologic disease.     

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.     

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.     

Assembly of Theiler's virus recombinants used in mapping determinants of neurovirulence.

A major determinant of neurovirulence for the GDVII strain of Theiler's virus, a murine picornavirus, was mapped to the P1 capsid protein region. Chimeric viruses were constructed by using sequences from the 5' noncoding and P1 regions of the virulent GDVII strain to replace equivalent regions of the less virulent BeAn strain. Neurovirulence in mice progressively increased as larger regions of BeAn capsid protein-encoding sequences were replaced. The in vitro growth characteristics of the chimeras showed that some chimeras were growth delayed in BHK-21 cells even though the viral constructs exhibited larger plaque sizes, were less temperature sensitive, and were more thermally stable than BeAn. Examination of assembly intermediates revealed an altered pentamer conformation and delayed empty capsid formation for the growth-compromised viruses. For these constructs, their chimeric nature inadvertently resulted in virion assembly defects that complicated finer-scale mapping of the determinants of virulence within the capsid region. These results demonstrate the importance of determining in vitro growth characteristics of chimeras to correctly decipher the significance of their phenotypes. VP1 does not contain a complete determinate for virulence because a chimera with VP1-encoding sequences from GDVII in an otherwise BeAn virus has an attenuated phenotype but is not growth compromised in vitro. The source of sequences, BeAn or GDVII, in the 5' noncoding region had only slight effects on the virulence of recombinant constructs.     

Neurovirulence of polytropic murine retrovirus is influenced by two separate regions on opposite sides of the envelope protein receptor binding domain

Changes in the envelope proteins of retroviruses can alter the ability of these viruses to infect the central nervous system (CNS) and induce neurological disease. In the present study, nine envelope residues were found to influence neurovirulence of the Friend murine polytropic retrovirus Fr98. When projected on a three-dimensional model, these residues were clustered in two spatially separated groups, one in variable region B of the receptor binding site and the other on the opposite side of the envelope. Further studies indicated a role for these residues in virus replication in the CNS, although the residues did not affect viral entry.     

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.     

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.     

Neuropeptide Y Has a Protective Role during Murine Retrovirus-Induced Neurological Disease

Viral infections in the central nervous system (CNS) can lead to neurological disease either directly by infection of neurons or indirectly through activation of glial cells and production of neurotoxic molecules. Understanding the effects of virus-mediated insults on neuronal responses and neurotrophic support is important in elucidating the underlying mechanisms of viral diseases of the CNS. In the current study, we examined the expression of neurotrophin- and neurotransmitter-related genes during infection of mice with neurovirulent polytropic retrovirus. In this model, virus-induced neuropathogenesis is indirect, as the virus predominantly infects macrophages and microglia and does not productively infect neurons or astrocytes. Virus infection is associated with glial cell activation and the production of proinflammatory cytokines in the CNS. In the current study, we identified increased expression of neuropeptide Y (NPY), a pleiotropic growth factor which can regulate both immune cells and neuronal cells, as a correlate with neurovirulent virus infection. Increased levels of Npy mRNA were consistently associated with neurological disease in multiple strains of mice and were induced only by neurovirulent, not avirulent, virus infection. NPY protein expression was primarily detected in neurons near areas of virus-infected cells. Interestingly, mice deficient in NPY developed neurological disease at a faster rate than wild-type mice, indicating a protective role for NPY. Analysis of NPY-deficient mice indicated that NPY may have multiple mechanisms by which it influences virus-induced neurological disease, including regulating the entry of virus-infected cells into the CNS.The early innate immune response to virus infection in the central nervous system (CNS) plays an important regulatory role in controlling both viral infection and pathogenesis. The neuroinflammatory response can limit virus replication through production of type I interferons and recruitment of virus-specific T cells to the CNS (5, 9, 12, 15, 19). However, the neuroinflammatory response can also lead to chronic gliosis, the production of cytokines that are toxic to neurons, and the recruitment of virus-infected cells to the CNS (6, 8, 18, 35). Understanding the relationship between the innate immune response and viral disease is essential in order to manipulate this response to control virus infection in the CNS.To better understand the role of the innate immune responses in viral pathogenesis in the CNS, we have utilized a mouse model of polytropic retrovirus infection. In this model, neuropathogenesis is indirect, since the polytropic retroviruses do not productively infect neurons. Instead, the viruses predominantly infect macrophages and microglia in the CNS (32). Despite severe neurological disease development following polytropic retrovirus infection, the only histologic changes observed in the brain are the activations of microglia and astrocytes (31). In addition, we have found high levels of proinflammatory cytokines and chemokines in brain tissue from infected mice, including tumor necrosis factor (TNF); interleukin 1 alpha (IL-1α), IL-1β, and IL-6; and the chemokines chemokine ligand 2 (CCL2/MCP-1), CCL3 (MIP-1α), CCL4 (MIP-1β), CCL5 (RANTES), and chemokine (C-X-C motif) ligand 10 (CXCL10/IP-10) (28). Studies with different chemokine receptors and cytokine-deficient mice demonstrated that at least two of these proinflammatory cytokines, CCL2 and TNF, can contribute to retrovirus-induced neurological disease (26, 27). However, neither of these molecules was necessary for neurological disease for all of the neurovirulent polytropic retroviruses studied, suggesting that other host factors contribute to retroviral pathogenesis.Analysis of the envelope protein of the neurovirulent polytropic retrovirus identified key residues in the envelope protein that influence the ability of the virus to induce neurological disease (28). In this study, we utilized neurovirulent and nonneurovirulent chimeric viruses that differ by only a few amino acid residues in these envelope regions to identify host response factors whose expression correlated with neurovirulence. We also utilized two different mouse strains, Inbred Rocky Mountain White (IRW) and 129S6, to confirm that expression of these host response factors is consistently induced or suppressed during neurovirulent virus infection. We determined that, although a number of host response genes are induced by polytropic retrovirus infection of the CNS, the expression of several of these factors correlated only with neuroinvasion and was not strongly correlative of neurovirulence. However, we have identified a neurotrophin, neuropeptide Y (NPY), whose expression strongly correlates with neurovirulence. We found that NPY had a protective influence on retroviral neuropathogenesis and examined the mechanisms by which NPY influences retrovirus infection of the CNS.     

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.     

Molecular and biological characterization of a neurovirulent molecular clone of simian immunodeficiency virus.

To identify the molecular determinants of neurovirulence, we constructed an infectious simian immunodeficiency virus (SIV) molecular clone, SIV/17E-Fr, that contained the 3' end of a neurovirulent strain of SIV, SIV/17E-Br, derived by in vivo virus passage. SIV/17E-Fr is macrophage tropic in vitro and neurovirulent in macaques. In contrast, a molecular clone, SIV/17E-Cl, that contains the SU and a portion of the TM sequences of SIV/17E-Br is macrophage tropic but not neurovirulent. To identify the amino acids that accounted for the replication differences between SIV/17E-Fr and SIV/17E-Cl in primary macaque cells in vitro, additional infectious molecular clones were constructed. Analysis of these recombinant viruses revealed that changes in the TM portion of the envelope protein were required for the highest level of replication in primary macaque macrophages and brain cells derived from the microvessel endothelium. In addition, a full-length Nef protein is necessary for optimum virus replication in both of these cell types. Finally, viruses expressing a full-length Nef protein in conjunction with the changes in the TM had the highest specific infectivity in a sMAGI assay. Thus, changes in the TM and nef genes between SIV/17E-Cl and SIV/17E-Fr account for replication differences in vitro and correlate with replication in the central nervous system in vivo.     

Molecular analysis of the envelope gene and long terminal repeat of Friend mink cell focus-inducing virus: implications for the functions of these sequences.

We sequenced the envelope (env) gene and 3' long terminal repeat of a Friend mink cell focus-inducing virus (F-MCFV). We also sequenced the gp70 coding regions for two cDNA clones of another F-MCFV. The deduced amino acid sequence of the env gene products of both F-MCFVs were compared to the corresponding sequences of other MCFVs and of ecotropic viruses. The env polypeptides of the different viruses showed long stretches of homology in the carboxy-terminal half of gp70 and in p15env ("constant region"). The amino-terminal half of gp70 was very similar in all MCFVs, but showed extensive variations relative to the ecotropic viruses ("differential region"). This differential region in all MCFVs is of endogeneous origin. We show evidence that this region carries determinants for ecotropic or polytropic host range. No indication could be found that the env gene products determine the histological type of disease caused by particular MCFVs. When the long terminal repeats of F-MCFV and Friend murine leukemia virus were compared with those of other viruses causing either lymphatic leukemia or erythroleukemia, several nucleotides were localized which might determine the histological type of disease caused by these viruses.     

Brain infection by neuroinvasive but avirulent murine oncornaviruses

The chimeric murine oncornavirus FrCas(E) causes a rapidly progressive noninflammatory spongiform encephalomyelopathy after neonatal inoculation. The virus was constructed by the introduction of pol-env sequences from the wild mouse virus CasBrE into the genome of a neuroinvasive but nonneurovirulent strain of Friend murine leukemia virus (FMuLV), FB29. Although the brain infection by FrCas(E) as well as that by other neurovirulent murine retroviruses has been described in detail, little attention has been paid to the neuroinvasive but nonneurovirulent viruses. The purpose of the present study was to compare brain infection by FrCas(E) with that by FB29 and another nonneurovirulent virus, F43, which contains pol-env sequences from FMuLV 57. Both FB29 and F43 infected the same spectrum of cell types in the brain as that infected by FrCas(E), including endothelial cells, microglia, and populations of neurons which divide postnatally. Viral burdens achieved by the two nonneurovirulent viruses in the brain were actually higher than that of FrCas(E). The widespread infection of microglia by the two nonneurovirulent viruses is notable because it is infection of these cells by FrCas(E) which is thought to be a critical determinant of its neuropathogenicity. These results indicate that although the sequence of the envelope gene determines neurovirulence, this effect appears to operate through a mechanism which does not influence either viral tropism or viral burden in the brain. Although all three viruses exhibited similar tropism for granule neurons in the cerebellar cortex, there was a striking difference in the distribution of envelope proteins in those cells in vivo. The FrCas(E) envelope protein accumulated in terminal axons, whereas those of FB29 and F43 remained predominantly in the cell bodies. These observations suggest that differences in the intracellular sorting of these proteins may exist and that these differences appear to correlate with neurovirulence.     

Modification of the untranslated regions of human enterovirus 71 impairs growth in a cell-specific manner

Human enterovirus 71 (HEV71) is the causative agent of hand, foot, and mouth disease and associated acute neurological disease. At present, little is known about the genetic determinants of HEV71 neurovirulence. Studies of related enteroviruses have indicated that the untranslated regions (UTRs), which control virus-directed translation and replication, also exert significant influence on neurovirulence. We used an infectious cDNA clone of a subgenogroup B3 strain to construct and characterize chimeras with 5'- and 3'-UTR modifications. Replacement of the entire HEV71 5' UTR with that of human rhinovirus 2 (HRV2) resulted in a small reduction in growth efficiency in cells of both nonneuronal (rhabdomyosarcoma) and neuronal (SH-SY5Y) origin due to reduced translational efficiency. However, the introduction of a 17-nucleotide deletion into the proximal region of the 3' UTR significantly decreased the growth of HEV71-HRV2 in SH-SY5Y cells. This observation is similar to that made with stem-loop domain Z (SLD Z)-deleted coxsackievirus B3-HRV2 5'-UTR chimeras reported previously and provides the first evidence of a potentially functional SLD Z in the 3' UTR in human enterovirus A species viruses. We further showed that the cell-specific growth impairment was caused by the synergistic effects of cis-acting UTR control elements on different stages of the virus life cycle. These chimeras will further improve our understanding of the control of HEV71 replication and its relationship to neurovirulence.     

Yellow fever/Japanese encephalitis chimeric viruses: construction and biological properties

A system has been developed for generating chimeric yellow fever/Japanese encephalitis (YF/JE) viruses from cDNA templates encoding the structural proteins prM and E of JE virus within the backbone of a molecular clone of the YF17D strain. Chimeric viruses incorporating the proteins of two JE strains, SA14-14-2 (human vaccine strain) and JE Nakayama (JE-N [virulent mouse brain-passaged strain]), were studied in cell culture and laboratory mice. The JE envelope protein (E) retained antigenic and biological properties when expressed with its prM protein together with the YF capsid; however, viable chimeric viruses incorporating the entire JE structural region (C-prM-E) could not be obtained. YF/JE(prM-E) chimeric viruses grew efficiently in cells of vertebrate or mosquito origin compared to the parental viruses. The YF/JE SA14-14-2 virus was unable to kill young adult mice by intracerebral challenge, even at doses of 10(6) PFU. In contrast, the YF/JE-N virus was neurovirulent, but the phenotype resembled parental YF virus rather than JE-N. Ten predicted amino acid differences distinguish the JE E proteins of the two chimeric viruses, therefore implicating one or more residues as virus-specific determinants of mouse neurovirulence in this chimeric system. This study indicates the feasibility of expressing protective antigens of JE virus in the context of a live, attenuated flavivirus vaccine strain (YF17D) and also establishes a genetic system for investigating the molecular basis for neurovirulence determinants encoded within the JE E protein.     

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.     

Pathogenesis of early and late disease in mice infected with Theiler''s virus, using intratypic recombinant GDVII/DA viruses.

Intratypic recombinant Theiler's viruses prepared between GDVII and DA strains were used to identify genomic sequences important in neurovirulence, virus persistence, and demyelination and to clarify the mechanisms involved in disease induction. The coding region between 1B and 2C of the highly virulent GDVII strain contains a determinant partly responsible for neurovirulence (early paralysis and death) which correlates with elevated levels of infectious virus and the presence of virus antigen within neurons of the brain stem and gray matter of the spinal cord. Both the GDVII and the DA strains of virus contain genetic determinants for late demyelination in spinal cord. However, quantitative analysis of demyelination produced by recombinant GDVII/DA viruses suggest that multiple gene segments influence the number and extent of demyelinating lesions.     

Comparison of the Neurovirulence of a Vaccine and a Wild-Type Mumps Virus Strain in the Developing Rat Brain

Prior to the adoption of widespread vaccination programs, mumps virus was the leading cause of virus-induced central nervous system (CNS) disease. Mumps virus-associated CNS complications in vaccinees continue to be reported; outside the United States, some of these complications have been attributed to vaccination with insufficiently attenuated neurovirulent vaccine strains. The development of potentially neurovirulent, live, attenuated mumps virus vaccines stems largely from the lack of an animal model that can reliably predict the neurovirulence of mumps virus vaccine candidates in humans. The lack of an effective safety test with which to measure mumps virus neurovirulence has also hindered analysis of the neuropathogenesis of mumps virus infection and the identification of molecular determinants of neurovirulence. In this report we show, for the first time, that mumps virus infection of the neonatal rat leads to developmental abnormalities in the cerebellum due to cerebellar granule cell migration defects. The incidence of the cerebellar abnormalities and other neuropathological and clinical outcomes of mumps virus infection of the neonatal rat brain demonstrated the ability of this model to distinguish neurovirulent (Kilham) from nonneurovirulent (Jeryl Lynn) mumps virus strains. Thus, this neonatal rat model may prove useful in evaluating the neurovirulence potential of new live, attenuated vaccine strains and may also be of value in elucidating the molecular basis of mumps virus neurovirulence.     

Identification of two determinants that attenuate vaccine-related type 2 poliovirus.

The poliovirus P2/P712 strain is an attenuated virus that is closely related to the type 2 Sabin vaccine strain. By using a mouse model for poliomyelitis, sequences responsible for attenuation of the P2/P712 strain were previously mapped to the 5' noncoding region of the genome and a central region encoding VP1, 2Apro, 2B, and part of 2C. To identify specific determinants that attenuate the P2/P712 strain, recombinants between this virus and the mouse-adapted P2/Lansing were constructed and their neurovirulence in mice was determined. By using this approach, the attenuation determinant in the central region was mapped to capsid protein VP1. Candidate attenuating sequences in VP1 and the 5' noncoding region were identified by comparing the P2/P712 sequence with that of vaccine-associated isolate P2/P117, and the P2/117 sequences were introduced into the P2/Lansing-P2/P712 recombinants by site-directed mutagenesis. Results of neurovirulence assays in mice indicate that an A at nucleotide 481 in the 5' noncoding region and isoleucine (Ile) at position 143 of capsid protein VP1 are the major determinants of attenuation of P2/P712. These determinants also attenuated neurovirulence in transgenic mice expressing human poliovirus receptors, a new model for poliomyelitis in which virulent viruses are not host restricted. These results demonstrate that A-481 and Ile-143 are general determinants of attenuation.     

Yellow fever virus/dengue-2 virus and yellow fever virus/dengue-4 virus chimeras: biological characterization,immunogenicity, and protection against dengue encephalitis in the mouse model

Two yellow fever virus (YFV)/dengue virus chimeras which encode the prM and E proteins of either dengue virus serotype 2 (dengue-2 virus) or dengue-4 virus within the genome of the YFV 17D strain (YF5.2iv infectious clone) were constructed and characterized for their properties in cell culture and as experimental vaccines in mice. The prM and E proteins appeared to be properly processed and glycosylated, and in plaque reduction neutralization tests and other assays of antigenic specificity, the E proteins exhibited profiles which resembled those of the homologous dengue virus serotypes. Both chimeric viruses replicated in cell lines of vertebrate and mosquito origin to levels comparable to those of homologous dengue viruses but less efficiently than the YF5.2iv parent. YFV/dengue-4 virus, but not YFV/dengue-2 virus, was neurovirulent for 3-week-old mice by intracerebral inoculation; however, both viruses were attenuated when administered by the intraperitoneal route in mice of that age. Single-dose inoculation of either chimeric virus at a dose of 10(5) PFU by the intraperitoneal route induced detectable levels of neutralizing antibodies against the homologous dengue virus strains. Mice which had been immunized in this manner were fully protected from challenge with homologous neurovirulent dengue viruses by intracerebral inoculation compared to unimmunized mice. Protection was associated with significant increases in geometric mean titers of neutralizing antibody compared to those for unimmunized mice. These data indicate that YFV/dengue virus chimeras elicit antibodies which represent protective memory responses in the mouse model of dengue encephalitis. The levels of neurovirulence and immunogenicity of the chimeric viruses in mice correlate with the degree of adaptation of the dengue virus strain to mice. This study supports ongoing investigations concerning the use of this technology for development of a live attenuated viral vaccine against dengue viruses.