A cDNA for the prion protein (PrP) derived from scrapie-infected mouse brain was expressed in C127 mouse cells in vitro under the control of the mouse metallothionein promoter. PrP synthesis was detected by immunoprecipitation using a rabbit antibody specific for a 15 amino acid PrP peptide. Homogenates of cells expressing the cloned PrP cDNA inoculated into weanling mice failed to induce clinical scrapie during 190 days of observation. We conclude that either PrP is not the transmissible agent of scrapie or the PrP is not processed appropriately in this cell system to create the infectious agent.

A group of brain diseases, including BSE (or mad cow disease) and Creutzfeldt-Jacob disease of humans, are transmitted by prions. In his Perspective, Cheseboro outlines the evidence that prions are composed only of protein (an idea advanced by recent Nobel Prize winner Stanley Prusiner) and the evidence against that hypothesis. He cautions against complacency regarding the obstacles remaining in the field, since, he believes, the central core of the problem remains unresolved.

Most individuals infected with human immunodeficiency virus type 1 (HIV-1) initially harbor macrophage-tropic, non-syncytium-inducing (M-tropic, NSI) viruses that may evolve into T-cell-tropic, syncytium-inducing viruses (T-tropic, SI) after several years. The reasons for the more efficient transmission of M-tropic, NSI viruses and the slow evolution ofT-tropic, SI viruses remain unclear, although they may be linked to expression of appropriate chemokine coreceptors for virus entry. We have examined plasma viral RNA levels and the extent of CD4+ T-cell depletion in SCID mice reconstituted with human peripheral blood leukocytes following infection with M-tropic, dual-tropic, or T-tropic HIV-1 isolates. The cell tropism was found to determine the course of viremia, with M-tropic viruses producing sustained high viral RNA levels and sparing some CD4+ T cells, dual-tropic viruses producing a transient and lower viral RNA spike and extremely rapid depletion of CD4+ T cells, and T-tropic viruses causing similarly lower viral RNA levels and rapid-intermediate rates of CD4+ T-cell depletion. A single amino acid change in the V3 region of gp120 was sufficient to cause one isolate to switch from M-tropic to dual-tropic and acquire the ability to rapidly deplete all CD4+ T cells.

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) infection of the brain results in viral replication primarily in macrophages and microglia. Despite frequent detection of viral genome and proteins in the brains of AIDS patients with and without HIV dementia, only 20% of AIDS patients become demented. To investigate the role of viral envelope gene variation in the occurrence of dementia, we examined regions of variability in the viral envelope gene isolated from brains of AIDS patients. Brain-derived HIV-1 V1-V2 envelope sequences from seven demented and six nondemented AIDS patients displayed significant sequence differences between clinical groups, and by phylogenetic analysis, sequences from the demented group showed clustering. Infectious recombinant viruses containing brain-derived V3 sequences from both clinical groups were macrophagetropic, and viruses containing brain-derived V1, V2, and V3 sequences from both clinical groups spread efficiently in macrophages. In an indirect in vitro neurotoxicity assay using supernatant fluid from HIV-1-infected macrophages, recombinant viruses from demented patients induced greater neuronal death than viruses from nondemented patients. Thus, the HIV-1 envelope diversity observed in these patient groups appeared to influence the release of neurotoxic molecules from macrophages and might account in part for the variability in occurrence of dementia in AIDS patients.

Antigen capture enzyme-linked immunosorbent assay (ELISA) for quantitation of the p24 gag protein of human immunodeficiency virus type-1 (HIV-1) is currently the most common method used to demonstrate virus replication both in vivo and in vitro. The present paper describes an ELISA employing readily available inexpensive reagents and gives detailed suggestions for optimizing the variables for specific purposes. The assay is as sensitive as commercial kits (25 pg/ml) and has a linear dose response over a wide range of p24 concentrations (25-1000 pg/ml). For these reasons, as well as its low cost, this assay has proven useful in a variety of research applications. This assay also has been found to be effective in detecting the gag protein of human immunodeficiency virus type-2 and simian immunodeficiency virus.

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.

Transmissible spongiform encephalopathies are characterized by spongiosis, astrocytosis and accumulation of PrPSc, an isoform of the normal host protein PrPC. The exact cell types responsible for agent propagation and pathogenesis are still uncertain. To determine the possible role of astrocytes, we generated mice devoid of murine PrP but expressing hamster PrP transgenes driven by the astrocyte‐specific GFAP promoter. After inoculation with hamster scrapie, these mice accumulated infectivity and PrPSc to high levels, developed severe disease after 227 ± 5 days and died 7 ± 4 days later. Therefore, astrocytes could play an important role in scrapie pathogenesis, possibly by an indirect toxic effect on neurons. Interestingly, mice expressing the same transgenes but also endogenous murine PrP genes propagated infectivity without developing disease.

The V3 region of the human immunodeficiency virus type 1 (HIV-1) envelope protein is known to have a major influence on macrophage tropism as well as the ability to cause syncytium formation or fusion in CD4-positive lymphocyte cultures. Using infectious molecular HIV-1 clones, a series of mutant clones was created which allowed detailed mapping of V3 amino acid positions involved in these properties. In these experiments the non-syncytium-inducing phenotype in T cells did not always correlate with macrophage tropism. Macrophage tropism appeared to depend on the presence of certain combinations of amino acids at five specific positions within and just outside of the V3 loop itself, whereas syncytium formation in lymphocytes was influenced by substitution of particular residues at two to four positions within V3. In most cases, different V3 amino acid positions were found to independently influence macrophage tropism and syncytium formation in T cells and position 13 was the only V3 location which appeared to simultaneously influence both macrophage tropism and syncytium formation in lymphocytes.

Two infectious molecular clones of human immunodeficiency virus type 1, NL4-3 and JR-CSF, differ in their abilities to productively infect human brain capillary endothelial (HBCE) cells. The phenotypes of recombinants between these two molecular strains were examined to identify viral sequences responsible for the difference in HBCE cell tropism between the two parental strains. Our results indicate that HBCE cell tropism maps to a region that encompasses the C1 region of env and includes overlapping reading frames for the accessory genes vpr, vpu, tat, and rev. This region was unique for HBCE cell tropism and did not cosegregate with either macrophage or T-cell line tropism. However, several recombinant clones displayed dual tropism for both HBCE cells and macrophages. These endothelial cell- and macrophage-tropic strains may have a unique pathogenic advantage by entering the brain via HBCE cells and subsequently infecting microglial cells with high efficiency, leading to the induction of human immunodeficiency virus dementia.

The major determinants involved in neurological disease induction by polytropic murine leukemia virus FMCF98 are encoded by the envelope gene. To map these determinants further, we produced four chimeras which contained neurovirulent FMCF98 envelope sequences combined with envelope sequences from the closely related nonneurovirulent polytropic virus FMCF54. Surprisingly, two chimeric viruses containing completely separate envelope regions from FMCF98 could both induce neurological disease. Clinical signs caused by both neurovirulent chimeras appeared to be indistinguishable from those caused by FMCF98, although the incubation periods were longer. One neurovirulence determinant mapped to the N-terminal portion of gp7O, which contains the VRA and VRB receptor-binding regions, while the other determinant mapped downstream of both of the variable regions. Western blot (immunoblot) analyses and immunohistochemical staining of tissue sections indicated that the variations in neurovirulence of these viruses could not be explained by differences in either the quantitative level or the location of virus expression in the brain.

Friend murine leukemia virus is a retrovirus complex that induces rapid erythroleukemia and immunosuppression in susceptible strains of adult mice. Using this model, we directly examined the T-cell subsets required for a protective retrovirus vaccine. Paradoxically, recovery in mice immunized with a chimeric envelope containing only T-helper (TH) and B-cell epitopes was dependent on CD8+ T cells as well as CD4+ T cells despite the fact that the vaccine contained no CD8+ cytolytic T-lymphocyte (CTL) epitopes. However, the requirement for CD8+ T cells was overcome by inclusion of additional TH and B-cell epitopes in the immunizing protein. These additional epitopes primed for more rapid production of virus-neutralizing antibody which appeared to limit virus spread sufficiently to protect even in the absence of CD8+ T cells. Inclusion of an immunodominant CTL epitope in the vaccine was not sufficient to overcome dependence on CD4+ T cells. These data suggest that TH priming is more critical for retrovirus immunity than CTL priming.

Administration of virus-specific antibodies is known to be an effective early treatment for some viral infections. Such immunotherapy probably acts by antibody-mediated neutralization of viral infectivity and is often thought to function independently of T-cell-mediated immune responses. In the present experiments, we studied passive antibody therapy using Friend murine leukemia virus complex as a model for an immunosuppressive retroviral disease in adult mice. The results showed that antibody therapy could induce recovery from a well-established retroviral infection. However, the success of therapy was dependent on the presence of both CD4(+) and CD8(+) T lymphocytes. Thus, cell-mediated responses were required for recovery from infection even in the presence of therapeutic levels of antibody. The major histocompatibility type of the mice was also an important factor determining the relative success of antibody therapy in this system, but it was less critical for low-dose than for high-dose infections. Our results imply that limited T-cell responsiveness as dictated by major histocompatibility genes and/or stage of disease may have contributed to previous immunotherapy failures in AIDS patients. Possible strategies to improve the efficacy of future therapies are discussed.