Neuroadapted yellow fever virus 17D: genetic and biological characterization of a highly mouse-neurovirulent virus and its infectious molecular clone

A neuroadapted strain of yellow fever virus (YFV) 17D derived from a multiply mouse brain-passaged virus (Porterfield YF17D) was additionally passaged in SCID and normal mice. The virulence properties of this virus (SPYF) could be distinguished from nonneuroadapted virus (YF5.2iv, 17D infectious clone) by decreased average survival time in SCID mice after peripheral inoculation, decreased average survival time in normal adult mice after intracerebral inoculation, and occurrence of neuroinvasiveness in normal mice. SPYF exhibited more efficient growth in peripheral tissues of SCID mice than YF5.2iv, resulting in a more rapid accumulation of virus burden, but with low-titer viremia, at the time of fatal encephalitis. In cell culture, SPYF was less efficient in replication than YF5.2iv in all cell lines tested. The complete nucleotide sequence of SPYF revealed 29 nucleotide substitutions relative to YF5.2iv, and these were distributed throughout the genome. There were a total of 13 predicted amino acid substitutions, some of which correspond to known differences among the Asibi, French viscerotropic virus, French neurotropic vaccine, and YF17D vaccine strains. The envelope (E) protein contained five substitutions, within all three functional domains. Substitutions were also present in regions encoding the NS1, NS2A, NS4A, and NS5 proteins and in the 3' untranslated region (UTR). Construction of YFV harboring all of the identified coding nucleotide substitutions and those in the 3' UTR yielded a virus whose cell culture and pathogenic properties, particularly neurovirulence and neuroinvasiveness for SCID mice, generally resembled those of the original SPYF isolate. These findings implicate the E protein and possibly other regions of the genome as virulence determinants during pathogenesis of neuroadapted YF17D virus in mice. The determinants affect replication efficiency in both neural and extraneural tissues of the mouse and confer some limited host-range differences in cultured cells of nonmurine origin.     

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.     

Neuroadapted yellow fever virus 17D: determinants in the envelope protein govern neuroinvasiveness for SCID mice

A molecular clone of mouse-neuroadapted yellow fever 17D virus (SPYF-MN) was used to identify critical determinants of viral neuroinvasiveness in a SCID mouse model. Virus derived from this clone differs from nonneuroinvasive YF5.2iv virus at 29 nucleotide positions, encoding 13 predicted amino acid substitutions and 2 substitutions in the 3' untranslated region (UTR). The virulence determinants of SPYF-MN for SCID mice were identified by constructing and characterizing intratypic viruses in which the E protein of SPYF-MN was expressed in the YF5.2iv background (SPYF-E) or the E protein of YF5.2iv was expressed in the SPYF-MN background (YF5.2-E). SPYF-E caused lethal encephalitis in young adult SCID mice after intraperitoneal inoculation, with average survival times and tissue virus burdens resembling those of mice inoculated with the parental SPYF-MN virus. To define which domains of the E protein are involved in neuroinvasiveness, two viruses were tested in which the amino acid substitutions in domains I-II and III were segregated. This revealed that substitutions in domain III (residues 305, 326, and 380) were critical for the neuroinvasive phenotype, based on average survival times and tissue burdens of infectious virus. Comparison of growth properties of the various intratypic viruses in cell culture indicated that no inherent defects in replication efficiency were likely to account for the biological differences observed in these experiments. These findings demonstrate that the E protein is a critical factor for yellow fever virus neuropathogenesis in the SCID mouse model and that the neuroinvasive properties depend principally on functions contributed by domain III of this protein. To assess whether critical determinants for neuroinvasion of normal ICR mice by SPYF virus were also in the E protein, sequences of viruses recovered from brains of ICR mice succumbing to encephalitis with the parental SPYF virus were derived. No differences were found in the E protein; however, two substitutions were present in the 3' UTR compared to that of SPYF-MN, one of which is predicted to alter RNA secondary structure in this region. These findings suggest that the 3' UTR may also affect neuroinvasiveness of SPYF virus in the mouse model.     

Transcriptomic profile of host response in Japanese encephalitis virus infection

Background

The attenuated Yellow fever (YF) 17D vaccine virus is one of the safest and most effective viral vaccines administered to humans, in which it elicits a polyvalent immune response. Herein, we used the YF 17D backbone to express a Trypanosoma cruzi CD8⁺ T cell epitope from the Amastigote Surface Protein 2 (ASP-2) to provide further evidence for the potential of this virus to express foreign epitopes. The TEWETGQI CD8⁺ T cell epitope was cloned and expressed based on two different genomic insertion sites: in the fg loop of the viral Envelope protein and the protease cleavage site between the NS2B and NS3. We investigated whether the site of expression had any influence on immunogenicity of this model epitope.

Results

Recombinant viruses replicated similarly to vaccine virus YF 17D in cell culture and remained genetically stable after several serial passages in Vero cells. Immunogenicity studies revealed that both recombinant viruses elicited neutralizing antibodies to the YF virus as well as generated an antigen-specific gamma interferon mediated T-cell response in immunized mice. The recombinant viruses displayed a more attenuated phenotype than the YF 17DD vaccine counterpart in mice. Vaccination of a mouse lineage highly susceptible to infection by T. cruzi with a homologous prime-boost regimen of recombinant YF viruses elicited TEWETGQI specific CD8⁺ T cells which might be correlated with a delay in mouse mortality after a challenge with a lethal dose of T. cruzi.

Conclusions

We conclude that the YF 17D platform is useful to express T. cruzi (Protozoan) antigens at different functional regions of its genome with minimal reduction of vector fitness. In addition, the model T. cruzi epitope expressed at different regions of the YF 17D genome elicited a similar T cell-based immune response, suggesting that both expression sites are useful. However, the epitope as such is not protective and it remains to be seen whether expression of larger domains of ASP-2, which include the TEWETGQI epitope, will elicit better T-CD8+ responses to the latter. It is likely that additional antigens and recombinant virus formulations will be necessary to generate a protective response.     

The small nonstructural protein (NS2) of the parvovirus minute virus of mice is required for efficient DNA replication and infectious virus production in a cell-type-specific manner.

Seven mutations which affect only the small nonstructural protein NS2 were introduced into the infectious clone of the autonomous parvovirus, minute virus of mice (MVM). The majority of these mutants were severely defective for replication following transfection of normal host murine A9 fibroblasts; however, all were found to replicate more efficiently and produce infectious virus in certain other cell types, including human NB324K. The isolation of viral stocks from NB324K cells permitted a more detailed analysis of the mutant defect on A9 cells. NS2 mutant NS2-2018 was shown to be approximately 10-fold deficient for viral monomer replicative-form DNA production within a single-burst cycle in infected A9 cells and produced a reduced amount of progeny single strand. Mutant NS2-2018 generated wild-type levels of monomer replicative-form DNA on NB324K cells but made reduced levels of progeny single strand and small plaques on these cells. The accumulation of NS1 is reduced late in NS2-2018 infection of A9 cells, but NS1 accumulates to wild-type levels late in NB324K cell infections. NS1 nuclear localization is not dependent on NS2 in A9 or NB324K cells. These results indicate that NS2 participates in MVM DNA replication and is required for efficient viral growth. The requirement for NS2 during MVM replication is also host cell specific. This requirement is significantly more pronounced in the normal host murine A9 cells than in certain other cell types, including NB324K.     

Processing of the yellow fever virus nonstructural polyprotein: a catalytically active NS3 proteinase domain and NS2B are required for cleavages at dibasic sites.

The vaccinia virus-T7 transient expression system was used to further examine the role of the NS3 proteinase in processing of the yellow fever (YF) virus nonstructural polyprotein in BHK cells. YF virus-specific polyproteins and cleavage products were identified by immunoprecipitation with region-specific antisera, by size, and by comparison with authentic YF virus polypeptides. A YF virus polyprotein initiating with a signal sequence derived from the E protein fused to the N terminus of NS2A and extending through the N-terminal 356 amino acids of NS5 exhibited processing at the 2A-2B, 2B-3, 3-4A, 4A-4B, and 4B-5 cleavage sites. Similar results were obtained with polyproteins whose N termini began within NS2A (position 110) or with NS2B. When the NS3 proteinase domain was inactivated by replacing the proposed catalytic Ser-138 with Ala, processing at all sites was abolished. The results suggest that an active NS3 proteinase domain is necessary for cleavage at the diabasic nonstructural cleavage sites and that cleavage at the proposed 4A-4B signalase site requires prior cleavage at the 4B-5 site. Cleavages were not observed with a polyprotein whose N terminus began with NS3, but cleavage at the 4B-5 site could be restored by supplying the the NS2B protein in trans. Several experimental results suggested that trans cleavage at the 4B-5 site requires association of NS2B and the NS3 proteinase domain. Coexpression of different proteinases and catalytically inactive polyprotein substrates revealed that trans cleavage at the 2B-3 and 4B-5 sites was relatively efficient when compared with trans cleavage at the 2A-2B and 3-4A sites.     

Exchanging the yellow fever virus envelope proteins with Modoc virus prM and E proteins results in a chimeric virus that is neuroinvasive in SCID mice

A chimeric flavivirus infectious cDNA was constructed by exchanging the premembrane (prM) and envelope (E) genes of the yellow fever virus vaccine strain 17D (YF17D) with the corresponding genes of Modoc virus (MOD). This latter virus belongs to the cluster of the "not-known vector" flaviviruses and is, unlike YF17D, neuroinvasive in SCID mice. Replication of in vitro-transcribed RNA from this chimeric flavivirus was shown by [(3)H]uridine labeling and RNA analysis. Expression of the MOD prM and E proteins was monitored by radioimmunoprecipitation and revealed that the MOD proteins were correctly and efficiently produced from the chimeric precursor protein. The MOD E protein was shown to be N-linked glycosylated, whereas prM, as predicted from the genome sequence, did not contain N-linked carbohydrates. In Vero cells, the chimeric virus replicated with a similar efficiency as the parental viruses, although it formed smaller plaques than YF17D and MOD. In SCID mice that had been infected intraperitoneally with the chimeric virus, the viral load increased steadily until death. The MOD/YF virus, like MOD from which it had acquired the prM and E structural proteins, but unlike YF, proved neuroinvasive in SCID mice. Animals developed neurological symptoms about 15 days after inoculation and died shortly thereafter. The distribution of MOD/YF RNA in the brain of infected mice was similar to that observed in MOD-infected mice. The observations provide compelling evidence that the determinants of neuroinvasiveness of flaviviruses are entirely located in the envelope proteins prM and E.     

A tick-borne Langat virus mutant that is temperature sensitive and host range restricted in neuroblastoma cells and lacks neuroinvasiveness for immunodeficient mice

Langat virus (LGT), the naturally attenuated member of the tick-borne encephalitis virus (TBEV) complex, was tested extensively in clinical trials as a live TBEV vaccine and was found to induce a protective, durable immune response; however, it retained a low residual neuroinvasiveness in mice and humans. In order to ablate or reduce this property, LGT mutants that produced a small plaque size or temperature-sensitive (ts) phenotype in Vero cells were generated using 5-fluorouracil. One of these ts mutants, clone E5-104, exhibited a more than 10(3)-fold reduction in replication at the permissive temperature in both mouse and human neuroblastoma cells and lacked detectable neuroinvasiveness for highly sensitive immunodeficient mice. The E5-104 mutant possessed five amino acid substitutions in the structural protein E and one change in each of the nonstructural proteins NS3 and NS5. Using reverse genetics, we demonstrated that a Lys(46)-->Glu substitution in NS3 as well as a single Lys(315)-->Glu change in E significantly impaired the growth of LGT in neuroblastoma cells and reduced its peripheral neurovirulence for SCID mice. This study and our previous experience with chimeric flaviviruses indicated that a decrease in viral replication in neuroblastoma cells might serve as a predictor of in vivo attenuation of the neurotropic flaviviruses. The combination of seven mutations identified in the nonneuroinvasive E5-104 mutant provided a useful foundation for further development of a live attenuated TBEV vaccine. An evaluation of the complete sequence of virus recovered from brain of SCID mice inoculated with LGT mutants identified sites in the LGT genome that promoted neurovirulence/neuroinvasiveness.     

Clearance of an immunosuppressive virus from the CNS coincides with immune reanimation and diversification

Background

The yellow fever virus, a member of the genus Flavivirus, is an arthropod-borne pathogen causing severe disease in humans. The attenuated yellow fever 17D virus strain has been used for human vaccination for 70 years and has several characteristics that are desirable for the development of new, live attenuated vaccines. We described here a methodology to construct a viable, and immunogenic recombinant yellow fever 17D virus expressing a green fluorescent protein variant (EGFP). This approach took into account the presence of functional motifs and amino acid sequence conservation flanking the E and NS1 intergenic region to duplicate and fuse them to the exogenous gene and thereby allow the correct processing of the viral polyprotein precursor.

Results

YF 17D EGFP recombinant virus was grew in Vero cells and reached a peak titer of approximately 6.45 ± 0.4 log10 PFU/mL at 96 hours post-infection. Immunoprecipitation and confocal laser scanning microscopy demonstrated the expression of the EGFP, which was retained in the endoplasmic reticulum and not secreted from infected cells. The association with the ER compartment did not interfere with YF assembly, since the recombinant virus was fully competent to replicate and exit the cell. This virus was genetically stable up to the tenth serial passage in Vero cells. The recombinant virus was capable to elicit a neutralizing antibody response to YF and antibodies to EGFP as evidenced by an ELISA test. The applicability of this cloning strategy to clone gene foreign sequences in other flavivirus genomes was demonstrated by the construction of a chimeric recombinant YF 17D/DEN4 virus.

Conclusion

This system is likely to be useful for a broader live attenuated YF 17D virus-based vaccine development for human diseases. Moreover, insertion of foreign genes into the flavivirus genome may also allow in vivo studies on flavivirus cell and tissue tropism as well as cellular processes related to flavivirus infection.     

Recombinant Yellow Fever Viruses Elicit CD8+ T Cell Responses and Protective Immunity against Trypanosoma cruzi

Chagas’ disease is a major public health problem affecting nearly 10 million in Latin America. Despite several experimental vaccines have shown to be immunogenic and protective in mouse models, there is not a current vaccine being licensed for humans or in clinical trial against T. cruzi infection. Towards this goal, we used the backbone of Yellow Fever (YF) 17D virus, one of the most effective and well-established human vaccines, to express an immunogenic fragment derived from T. cruzi Amastigote Surface Protein 2 (ASP-2). The cDNA sequence of an ASP-2 fragment was inserted between E and NS1 genes of YF 17D virus through the construction of a recombinant heterologous cassette. The replication ability and genetic stability of recombinant YF virus (YF17D/ENS1/Tc) was confirmed for at least six passages in Vero cells. Immunogenicity studies showed that YF17D/ENS1/Tc virus elicited neutralizing antibodies and gamma interferon (IFN-γ) producing-cells against the YF virus. Also, it was able to prime a CD8⁺ T cell directed against the transgenic T. cruzi epitope (TEWETGQI) which expanded significantly as measured by T cell-specific production of IFN-γ before and after T. cruzi challenge. However, most important for the purposes of vaccine development was the fact that a more efficient protective response could be seen in mice challenged after vaccination with the YF viral formulation consisting of YF17D/ENS1/Tc and a YF17D recombinant virus expressing the TEWETGQI epitope at the NS2B-3 junction. The superior protective immunity observed might be due to an earlier priming of epitope-specific IFN-γ-producing T CD8⁺ cells induced by vaccination with this viral formulation. Our results suggest that the use of viral formulations consisting of a mixture of recombinant YF 17D viruses may be a promising strategy to elicit protective immune responses against pathogens, in general.     

Heterologous T cells can help restore function in dysfunctional hepatitis C virus nonstructural 3/4A-specific T cells during therapeutic vaccination

The hepatitis C virus (HCV)-specific T cell response in patients with chronic HCV is dysfunctional. In this study, we aimed at restoring immunological function through therapeutic vaccination in a transgenic mouse model with impaired HCV-specific T cell responses due to a persistent presence of hepatic HCV nonstructural (NS)3/4A Ags. The HCV-specific T cells have an actively maintained dysfunction reflected in reduced frequency, impaired cytokine production, and impaired effector function in vivo, which can be partially restored by blocking regulatory T cells or programmed cell death ligand 1. We hypothesized that the impairment could be corrected by including sequences that created a normal priming environment by recruiting "healthy" heterologous T cells and by activating innate signaling. Endogenously expressed hepatitis B core Ag (HBcAg) can recruit heterologous T cells and activate TLR (TLR7) signaling. Hence, by combining HCV NS3/4A with different forms of HBcAg we found that heterologous sequences somewhat improved activation and expansion of NS3/4A-specific T cells in a wild-type host. Importantly, the signals provided by HBcAg effectively restored the activation of HCV-specific T cells in a tolerant NS3/4A-transgenic mouse model. The adjuvant effect could also be transferred to the priming of dysfunctional HLA-A2-restricted NS3-specific T cells in vivo. Thus, recruiting healthy heterologous T cells to the site of priming may also help restore HCV-specific responses present in a chronically infected host.     

The acidic domain of hepatitis C virus NS4A contributes to RNA replication and virus particle assembly

Hepatitis C virus NS3-4A is a membrane-bound enzyme complex that exhibits serine protease, RNA helicase, and RNA-stimulated ATPase activities. This enzyme complex is essential for viral genome replication and has been recently implicated in virus particle assembly. To help clarify the role of NS4A in these processes, we conducted alanine scanning mutagenesis on the C-terminal acidic domain of NS4A in the context of a chimeric genotype 2a reporter virus. Of 13 mutants tested, two (Y45A and F48A) had severe defects in replication, while seven (K41A, L44A, D49A, E50A, M51A, E52A, and E53A) efficiently replicated but had severe defects in virus particle assembly. Multiple strategies were used to identify second-site mutations that suppressed these NS4A defects. The replication defect of NS4A F48A was partially suppressed by mutation of NS4B I7F, indicating that a genetic interaction between NS4A and NS4B contributes to RNA replication. Furthermore, the virus assembly defect of NS4A K41A was suppressed by NS3 Q221L, a mutation previously implicated in overcoming other virus assembly defects. We therefore examined the known enzymatic activities of wild-type or mutant forms of NS3-4A but did not detect specific defects in the mutants. Taken together, our data reveal interactions between NS4A and NS4B that control genome replication and between NS3 and NS4A that control virus assembly.     

Cell culture adaptation of hepatitis C virus and in vivo viability of an adapted variant

Production of infectious hepatitis C virus in cell culture has become possible because of the unique properties of the JFH1 isolate. However, virus titers are rather low, limiting the utility of this system. Here we describe the generation of cell culture-adapted JFH1 variants yielding higher titers of infectious particles and enhanced spread of infection in cultured cells. Sequence analysis of adapted genomes revealed a complex pattern of mutations that differed in two independent experiments. Adaptive mutations were observed both in the structural and in the nonstructural regions, with the latter having the highest impact on enhancement of virus titers. The major adaptive mutation was identified in NS5A, and it enhanced titers of three intergenotypic chimeras consisting of the structural region of a genotype 1a, 1b, or 3a isolate and the remainder of the JFH1 isolate. The mutation resides at the P3 position of the NS5A-B cleavage site and slows down processing, implying that subtle differences in replication complex formation appear to determine the efficiency of virus formation. Highly adapted JFH1 viruses carrying six mutations established a robust infection in uPA-transgenic SCID mice xenografted with human hepatocytes. However, the mutation in NS5A which enhanced virus titers in cell culture the most had reverted to wild type in nearly half of the viral genomes isolated from these animals at 15 weeks postinoculation. These results argue for some level of impaired fitness of this mutant in vivo.     

Mechanism of virulence attenuation of glycosaminoglycan-binding variants of Japanese encephalitis virus and Murray Valley encephalitis virus

The in vivo mechanism for virulence attenuation of laboratory-derived variants of two flaviviruses in the Japanese encephalitis virus (JEV) serocomplex is described. Host cell adaptation of JEV and Murray Valley encephalitis virus (MVE) by serial passage in adenocarcinoma cells selected for variants characterized by (i) a small plaque phenotype, (ii) increased affinity to heparin-Sepharose, (iii) enhanced susceptibility to inhibition of infectivity by heparin, and (iv) loss of neuroinvasiveness in a mouse model for flaviviral encephalitis. We previously suggested that virulence attenuation of the host cell-adapted variants of MVE is a consequence of their increased dependence on cell surface glycosaminoglycans (GAGs) for attachment and entry (E. Lee and M. Lobigs, J. Virol. 74:8867-8875, 2000). In support of this proposition, we find that GAG-binding variants of JEV and MVE were rapidly removed from the bloodstream and failed to spread from extraneural sites of replication into the brain. Thus, the enhanced affinity of the attenuated variants for GAGs ubiquitously present on cells and extracellular matrices most likely prevented viremia of sufficient magnitude and/or duration required for virus entry into the brain parenchyma. This mechanism may also account, in part, for the attenuation of the JEV SA14-14-2 vaccine, given the sensitivity of the virus to heparin inhibition. A pronounced loss of the capacity of the GAG-binding variants to produce disease was also noted in mice defective in the alpha/beta interferon response, a mouse strain shown here to be highly susceptible to infection with JEV serocomplex flaviviruses. Despite the close genetic relatedness of JEV and MVE, the variants selected for the two viruses were altered at different residues in the envelope (E) protein, viz., Glu(306) and Asp(390) for JEV and MVE, respectively. In both cases the substitutions gave the protein an increased net positive charge. The close spatial proximity of amino acids 306 and 390 in the predicted E protein structure strongly suggests that the two residues define a receptor-binding domain involved in virus attachment to sulfated proteoglycans.     

UV-Irradiation of related mouse hybrid cells: Similar increase in capacity to replicate intact minute-virus-of-mice but differential enhancement of survival of UV-irradiated virus

3 hybrid cell lines between mouse fibroblasts (A9) and mouse lymphoma cells (L5178YS) were compared with regard to the ability of UV-pre-irradiated cells to replicated intact (unirradiated) Minute-Virus-of-Mice (MVM) and to reactivate UV-irradiated MVM. UV irradiation of cells before virus infection enhanced their ability to plaque intact virus (Enhanced Capacity) to a similar extent in the 3 hybrid cell lines. However, pretreatment of cells with UV radiation enhanced the survival of UV-damaged virus (Enhanced Reactivation) in only 1 of these hybrids. The lack of detectable Enhanced Reactivation in the other hybrids without concomitant change in their Enhanced Capacity, suggets that these processes are at least partly independent. Virus survival in unirradiated cells was similar for all 3 hybrid cell lines, indicating that the absence of detectable Enhanced Reactivation in 2 of the hybrids was not due to the constitutive expression of this process, but might rather result from its loss or extinction. The expression of both Enhanced Capacity and Enhanced Reactivation requires synthesis of protein de novo shortly after cell irradiation.     

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.     

Only Pyrimidinoceptors Are Functionally Expressed in Mouse Neuroblastoma Cell Lines

The ability of UTP, UDP, ATP, and ADP to influence inositol phospholipid hydrolysis in neuroblastoma origin cell lines was assessed. The mouse neuroblastoma lines N1E 115, Neuro 2a, and NB4 1A3 and the rat glioma/mouse neuroblastoma hybrid line NG108-15 gave robust responses to both UTP and UDP, which were essentially equipotent. Thus a range of cell lines of mouse neuroblastoma origin express a pyrimidine-selective P2Y receptor. The NG108-15 cells were the only cell type tested at which ATP and ADP displayed activity with EC₅₀ values of greater than 100 μM, compared with values of 0.58 and 1.25 μM for UTP and UDP, respectively. In contrast to the cell lines derived from mouse neuroblastoma, the human neuroblastoma lines SH-SY5Y and SK-N-SH did not respond to any nucleotides, although both responded well to carbachol.     

CHARACTERIZATION OF BENZODIAZEPINE BINDING SITES IN CULTURED CELLS OF NEURAL ORIGIN

Abstract— The binding of [³H]diazepam to benzodiazepine receptors was investigated in cultured cell lines of neural origin. Two cell lines, the rat C6 glioma and mouse NB41A3 neuroblastoma possess large numbers of benzodiazepine binding sites, while the other neural cell lines examined had significantly fewer benzodiazepine binding sites. [³H]diazepam binding to membranes prepared from C6 or NB41A3 cells was saturable and of a relatively high affinity ( K _D± 12 and 20 n m , respectively) when compared with rat cerebral cortex ( K _D± 4.6 n m ). A single class of binding sites in both cell lines was demonstrated by Scatchard analysis. The maximum binding capacities ( B _max) in the C6 and NB41A3 cell lines were found to be 10 and 3.5 fold higher than in rat cerebral cortex, respectively. In contrast to the rat cerebral cortex, binding of [³H]diazepam in cultured cells was not displaced by the clinically active benzodiazepines clonazepam and oxazepam while the clinically inactive benzodiazepine Ro 5-4864 potently inhibited the binding of [³H]diazepam in both neural cell lines. In toto , this data suggests a change in the benzodiazepine binding sites in cultured cells of neural origin to that found in peripheral (kidney) tissue. The observation that cell lines derived from both neuronal and glial elements contain large numbers of benzodiazepine binding sites also suggests benzodiazepine receptors in the central nervous system may not be confined to a single cell type.     

Inhibition of vesicular stomatitis virus infection by nitric oxide.

Inhibitory effects of nitric oxide (NO) on vesicular stomatitis virus (VSV) infection were investigated by using a VSV-susceptible mouse neuroblastoma cell line, NB41A3. Productive VSV infection of NB41A3 cells was significantly inhibited by an organic NO donor, S-nitro-N-acetylpenicillamine (SNAP), while the control compound N-acetylpenicillamine (NAP) had no effect. Survival rate of VSV-infected cells was greatly increased by the treatment with SNAP, while the NAP treatment did not have any effect. Adding SNAP 30 min prior to infection resulted in complete inhibition of viral production when a low multiplicity of infection (MOI) was used. Substantial inhibition of viral production was also obtained with treating cells 6 h earlier before infection with a higher MOI. Activating the neuronal NO synthase by treating cells with N-methyl-D-aspartate (NMDA) led to significant inhibition of viral production by cells infected at the three doses of virus tested (MOIs of 0.1, 1, and 5). The inhibitory effect of NMDA on viral infection was totally blocked by the NO synthase inhibitor N-methyl-L-arginine. However, adding hemoglobin, a strong NO-binding protein and thus an inactivator of NO activity, did not reverse the NMDA-induced inhibition of viral production, suggesting that NO might exert its antiviral effects inside the NO-producing cells. Collectively, these data support the anti-VSV effects of NO, which might be one of the important factors of natural immunity in controlling the initial stages of VSV infection in the central nervous system.     

trans-Complementation of yellow fever virus NS1 reveals a role in early RNA replication.

Mutational analysis of the nonstructural protein 1 (NS1) of yellow fever virus (YF) has implicated it in viral RNA replication. To further explore this observation, we sought a method for uncoupling NS1 function from NS1 expression and processing as part of the large YF polyprotein. Here we describe a strategy for providing NS1 in trans, utilizing a noncytopathic Sindbis virus vector. Replication of a defective YF genome containing a large in-frame deletion of NS1 was dependent on functional expression of NS1. Recovered mutant virus was shown to contain the deletion and was neutralized by YF-specific antiserum. Complemented mutant virus increased in titer with kinetics similar to those of parental YF 17D but peaked at lower titers. trans-complementation has allowed us to derive high-titer, helper-free stocks of YF defective in NS1 with which to further characterize the role of this gene product in RNA replication. The first cycles of RNA replication were analyzed by using a sensitive strand-specific RNase protection assay. We document these events for mutant and wild-type viruses in the presence or absence of complementation. These data strongly suggest a role for NS1 prior to or at initial minus-strand synthesis.