Representational difference analysis was used to search for pathogens in multiple sclerosis brains. We detected a 341-nucleotide fragment that was 99.4% identical to the major DNA binding protein gene of human herpesvirus 6 (HHV-6). Examination of 86 brain specimens by PCR demonstrated that HHV-6 was present in >70% of MS cases and controls and is thus a commensal virus of the human brain. By DNA sequencing, 36/37 viruses from MS cases and controls were typed as HHV-6 variant B group 2. Other herpesviruses, retroviruses, and measles virus were detected infrequently or not at all. HHV-6 expression was examined by immunocytochemistry with monoclonal antibodies against HHV-6 virion protein 101K and DNA binding protein p41. Nuclear staining of oligodendrocytes was observed in MS cases but not in controls, and in MS cases it was observed around plaques more frequently than in uninvolved white matter. MS cases showed prominent cytoplasmic staining of neurons in gray matter adjacent to plaques, although neurons expressing HHV-6 were also found in certain controls. Since destruction of oligodendrocytes is a hallmark of MS, these studies suggest an association of HHV-6 with the etiology or pathogenesis of MS.Multiple sclerosis (MS) is a disease of young adults that is characterized clinically by a variable relapsing and remitting course and pathologically by the progressive accumulation of plaques of demyelination within the white matter of the central nervous system. In normal white matter, the axons of neurons are surrounded by myelin sheaths, made from the cell membranes of oligodendrocytes. In MS plaques, the myelin sheaths are destroyed, leaving the naked axons intact but impaired in their conduction of action potentials. The currently held view is that an autoimmune inflammatory reaction against components of myelin results in destruction of oligodendrocytes. The demyelinating lesions in MS are found throughout the central nervous system, with a predilection for the periventricular white matter, optic nerve, brainstem, spinal cord, and cerebellum, resulting in a disease that is pleiomorphic in its clinical presentation.In spite of the substantial evidence that autoimmunemediated demyelination plays a major role in the progression of MS, epidemiologic studies suggest that an infectious agent may also be involved (1). Prior reports have suggested that viral infection of cells within the central nervous system may initiate the events leading to focal demyelination (2), and a number of viruses have been implicated in the pathogenesis of MS (3). Despite extensive investigation, however, none of these associations has been confirmed (4), and the issue of viral involvement in the pathogenesis of MS remains unresolved.To search for an MS-associated pathogen, we used representational difference analysis (RDA) (5). In RDA, successive rounds of subtractive hybridization and PCR amplification enriched for DNA sequences that were present in a DNA preparation from diseased tissue (MS brain) but absent from control DNA (n...

We demonstrated functional associations between mouse adenovirus type 1 (MAV-1) early region 1A (E1A) protein and both the mouse retinoblastoma protein (pRb) and the mouse pRb-related protein, p107. Interactions between MAV-1 E1A and mouse pRb or mouse p107 proteins were examined in infected cell lysates using a mouse embryonic fibroblast cell line infected with wild-type and mutant MAV-1 viruses. Using a polyclonal antibody to MAV-1 E1A, exogenously added mouse pRb or mouse p107 was coimmunoprecipitated from wild-type, dIE105 (CR1 delta)-, and dIE106 (CR3 delta)-infected cell lysates. No coimmunoprecipitation was seen with cell lysates from dIE102 (CR2 delta) or pmE109, a mutant virus that produces no detectable E1A protein due to an ATG to TTG point mutation in the initiator methionine. Introduction of mouse pRb into SAOS-2 cells resulted in a flat and enlarged cell phenotype, whereas cotransfection of mouse pRb and MAV-1 E1A resulted in a significant reduction of flat cells, presumably due to E1A binding pRb. CR1 delta and CR2 delta E1A proteins were less effective at reducing the number of flat, enlarged cells induced by pRb expression than were the CR3 delta or wild-type E1A proteins. The reduced ability of these mutants to inactivate pRb relative to wild-type E1A correlated with their reduced ability to bind pRb in the in vitro coimmunoprecipitation experiments. As a measure of p107/MAV-1 E1A complex formation in MAV-1-infected cells, we used mobility shift assays to examine cell extracts for the presence of p107-containing E2F protein-DNA complexes. Mock-, dIE102-, and pmE109-infected cell extracts formed a p107-containing complex, whereas wild-type-infected cell extracts did not. Thus the formation of a p107-E2F complex in wild-type- or these mutant-infected extracts inversely correlated with the presence of E1A-p107 complexes identified in the vitro coimmunoprecipitation experiments. This is consistent with E1A-p107 complexes forming in wild-type MAV-1-infected cells.

The attachment of Cryptosporidium sporozoites to Madin-Darby canine kidney (MDCK) cells was examined using transmission electron microscopy. As the anterior end of the sporozoite came into close proximity to the MDCK cell, the host cell membrane evaginated around the sporozoite, forming a parasitophorous vacuole. A dense band formed below the host cell membrane at the site nearest to the conoid. Variably electron-dense material was apparently released from the conoid and a large membrane-bound vacuole was formed in the anterior end of the sporozoite, displacing the typical anterior electron-dense organelles (rhoptries and micronemes). The outer membrane of the sporozoite pellicle then fused with the host cell membrane immediately adjacent to the conoid. The membrane surrounding the anterior vacuole was also fused with the common host-parasite membrane, forming Y-shaped membrane junctions where each limb was a unit membrane. A direct link was thereby established between the anterior vacuole of the sporozoite and the host cell cytoplasm. The anterior vacuole membrane separating the sporozoite and the host cell cytoplasm was the precursor of the feeder organelle.

Mouse adenovirus type 1 (MAV-1) early region 1A (E1A) viral mutants were used to determine the importance of this region in pathogenesis and establishment of a persistent infection in the natural host. Lethal dose analysis with adult male Swiss outbred mice revealed a significant reduction in virulence for all of the E1A mutants. During acute infections with 105 PFU of virus, an E1A null mutant,pmE109, was found in the same organs (brain, spleen, and spinal cord) and the same cell types (endothelial cells and mononuclear cells in lymphoid tissue) as wild-type virus. Another null mutant,pmE112, was detected in the same organs but in lower numbers. However, when mice were given a lower dose, 1 PFU,pmE109 and pmE112 reached none of the target organs analyzed by 14 days postinfection (p.i.). The absence of E1A did not hinder the ability of MAV-1 to establish a persistent infection. Viral nucleic acid was detected by PCR amplification or in situ hybridization in the kidneys, brains, spleens, and prefemoral lymph nodes of mice infected with wild-type or mutant virus up to 55 weeks p.i. The brain, spleen, and lymph node are recognized sites of acute viral infection but are previously unrecognized sites for MAV-1 persistence. Evidence for the potential reactivation of persistent MAV-1 infections is also presented.

Mouse adenovirus type 1 (MAV-1) mutants with deletions of conserved regions of early region 1A (E1A) or with point mutations that eliminate translation of E1A were used to determine the role of E1A in MAV-1 replication. MAV-1 E1A mutants expressing no E1A protein grew to titers comparable to wild-type MAV-1 titers on mouse fibroblasts (3T6 fibroblasts and fibroblasts derived from Rb+/+,Rb+/−, and Rb−/− transgenic embryos). To test the hypothesis that E1A could induce a quiescent cell to reenter the cell cycle, fibroblasts were serum starved to stop DNA replication and cellular replication and then infected with the E1A mutant and wild-type viruses. All grew to equivalent titers. Steady-state levels of MAV-1 early mRNAs (E1A, E1B, E2, E3, and E4) from 3T6 cells infected with wild-type or E1A mutant virus were examined by Northern analysis. Steady-state levels of mRNAs from the mutant-infected cells were comparable to or greater than the levels found in wild-type virus infections for most of the early regions and for two late genes. The E2 mRNA levels were slightly reduced in all mutant infections relative to wild-type infections. E1A mRNA was not detected from infections with the MAV-1 E1A null mutant, pmE109, or from infections with similar MAV-1 E1A null mutants, pmE112 andpmE113. The implications for the lack of a requirement of E1A in cell culture are discussed.

Mink lung epithelial (NBL-7) cells were shown to be permissive for human herpesvirus 6 (HHV-6) by four independent methods of analysis: detection of infectious virus, viral antigens, viral DNA sequences, and herpesvirus particles. Infection was serially passaged, with minimal cytopathology, for several months demonstrating for the first time that a cell of epithelial origin can support HHV-6 replication.

The tumor cells in the pleural effusions of a case of small cell undifferentiated carcinoma have been studied by light microscopy and by thin section and freeze-fracture electron microscopy. Evidence of exocytosis of neurosecretory granules from the tumor cells is presented. The tumor cells form clumps, with the cells joined by focal tight junctions and small desmosomes. These findings are discussed with reference to the neuroendocrine origin of these tumors and the role of the junctional complexes in the formation of tumor cell aggregates.