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In this study, a simple one-step reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for rapid detection of Lassa virus (LASV) was established. The two primer sets were designed to detect LASV circulating in Sierra Leone and northeastern Nigeria. The RT-LAMP assay using these primer sets was able to detect 100 copies of the in vitro transcribed artificial LASV RNA within 25 min. The assay was also evaluated using intact viral RNA extracted from cell culture-propagated viruses and confirmed to be highly specific for LASV. The RT-LAMP assay developed in this study is rapid, simple, and highly specific for the detection of LASV, although its sensitivity is slightly lower than that of real-time RT-PCR. In addition, because the RT-LAMP assay does not require the use of sophisticated equipment, it would be advantageous for clinical diagnosis of LASV infection in developing countries. It might also be employed in cases of deliberate release during bioterrorism attacks or in epidemiological surveillance for disease outbreaks.

Human immunodeficiency virus (HIV) Gag precursor protein is cleaved by viral protease (PR) within GagPol precursor protein to produce the mature matrix (MA), capsid, nucleocapsid, and p6 domains. This processing is termed maturation and required for HIV infectivity. In order to understand the intracellular sites and mechanisms of HIV maturation, HIV molecular clones in which Gag and GagPol were tagged with FLAG and hemagglutinin epitope sequences at the C-termini, respectively were made. When coexpressed, both Gag and GagPol were incorporated into virus particles. Temporal analysis by confocal microscopy showed that Gag and GagPol were relocated from the cytoplasm to the plasma membrane. Mature cleaved MA was observed only at sites on the plasma membrane where both Gag and GagPol had accumulated, indicating that Gag processing occurs during Gag/GagPol assembly at the plasma membrane, but not during membrane trafficking. Fluorescence resonance energy transfer imaging suggested that these were the primary sites of GagPol dimerization. In contrast, with overexpression of GagPol alone an absence of particle release was observed, and this was associated with diffuse distribution of mature cleaved MA throughout the cytoplasm. Alteration of the Gag-to-GagPol ratio similarly impaired virus particle release with aberrant distributions of mature MA in the cytoplasm. However, when PR was inactive, it seemed that the Gag-to-GagPol ratio was not critical for virus particle release but virus particles encasing unusually large numbers of GagPol molecules were produced, these particles displaying aberrant virion morphology. Taken together, it was concluded that the Gag-to-GagPol ratio has significant impacts on either intracellular distributions of mature cleaved MA or the morphology of virus particles produced.

Human immunodeficiency virus (HIV) Gag precursor protein is cleaved by viral protease (PR) within GagPol precursor protein to produce the mature matrix (MA), capsid, nucleocapsid, and p6 domains. This processing is termed maturation and required for HIV infectivity. In order to understand the intracellular sites and mechanisms of HIV maturation, HIV molecular clones in which Gag and GagPol were tagged with FLAG and hemagglutinin epitope sequences at the C-termini, respectively were made. When coexpressed, both Gag and GagPol were incorporated into virus particles. Temporal analysis by confocal microscopy showed that Gag and GagPol were relocated from the cytoplasm to the plasma membrane. Mature cleaved MA was observed only at sites on the plasma membrane where both Gag and GagPol had accumulated, indicating that Gag processing occurs during Gag/GagPol assembly at the plasma membrane, but not during membrane trafficking. Fluorescence resonance energy transfer imaging suggested that these were the primary sites of GagPol dimerization. In contrast, with overexpression of GagPol alone an absence of particle release was observed, and this was associated with diffuse distribution of mature cleaved MA throughout the cytoplasm. Alteration of the Gag-to-GagPol ratio similarly impaired virus particle release with aberrant distributions of mature MA in the cytoplasm. However, when PR was inactive, it seemed that the Gag-to-GagPol ratio was not critical for virus particle release but virus particles encasing unusually large numbers of GagPol molecules were produced, these particles displaying aberrant virion morphology. Taken together, it was concluded that the Gag-to-GagPol ratio has significant impacts on either intracellular distributions of mature cleaved MA or the morphology of virus particles produced.

Neurovirulence of the Onderstepoort strain of canine distemper virus (CDV) adapted to human neural cell lines was determined by the intracerebral inoculation of DDD mice at 3 and 5 weeks of age. Intensity of neurovirulence was estimated by histopathological changes in the central nervous system and clinical symptoms. The original virus propagated in Vero cells induced leptomeningoencephalitis, whereas neuroblastoma-adapted virus induced nerve cell degeneration and mild encephalitis with relatively low morbidity and fatality. In contrast, the viruses adapted to glioblastoma and oligodendroglioma caused high morbidity and fatality. The latter two viruses induced necrotizing encephalopathy including edema and hyperemia. In addition, the glioblastoma-adapted virus induced formation of giant cells. The oligodendroglioma-adapted virus caused demyelination and spongy state associated with degeneration of glial cells and axons. These observations are discussed in regard to a possible correlation between the neurovirulence of CDV in mice and its tropism for neural cells in vitro.

The biochemical characteristics of canine distemper virus (CDV) adapted to three human neural cells (glioblastoma, oligodendroglioma, and neuroblastoma cells) were compared with those of the unadapted original virus. The specific gravity of the virions and nucleocapsids of the original and the three adapted viruses were not different. The molecular weights of genomic RNA and messenger RNAs encoding H, F, P, and NP proteins of the adapted viruses as estimated by Northern blot hybridization were similar to those of the original virus. By T1-resistant oligonucleotide analysis of the genomic RNA, the glioblastoma- and the neuroblastoma-adapted viruses gave two more spots than the original virus; the oligodendroglioma-adapted virus had a pattern identical to that of the original virus. By two-dimensional gel electrophoresis of virion proteins, we found a difference in the isoelectric point of the viral envelope proteins H and F between the original and the adapted viruses. These results suggest that viral genomic changes occurred during adaptation, resulting in the alteration of viral envelope proteins.

Interaction of the Onderstepoort strain of canine distemper virus (CDV) with three established human neural cells, i.e. IMR-32 neuroblastoma, 118-MGC glioma and KG-I oligodendroglioma, was examined, and adaptation of CDV to these cells was also attempted. The unadapted virus was found to grow at relatively low titers in the three neural cells inducing moderate to minimal cyto­ pathic effects (CPE). The virus was successfully grown at high titers in these cells after 8 to 10 passages. Biological characteristics such as growth rate, morphology of CPE and plaque size changed after adaptation. Analysis by SDS-polyacrylamide gel electrophoresis, however, failed to show any difference in the molecular weight of component proteins among the unadapted and three adapted viruses. Inbred DDD strain of mice developed clinical signs after intracerebral inocula­ tion with the unadapted virus but most of them survived with histological lesions of encephalitis. Neuroblastoma-adapted virus induced only transient clinical signs in some animals with mild encephalitic lesions in the gray matter. Increases in neurovirulence were found for viruses adapted to glioma and oligo­ dendroglioma cells. Almost all mice inoculated with these two viruses at 3 weeks of age died within 8 days with histological lesions consisting of hyperemia, edema, severe degeneration of nerve cells and a few giant cells. Demyelinating lesions in the absence of inflammatory changes were observed in the cerebellum, pons and medulla oblongata of animals inoculated with oligodendroglioma-adapted virus. Canine distemper virus (CDV) which belongs to the morbillivirus subgroup of paramyxoviridae is highly neurovirulent in dogs as indicated by acute distemper encephalitis at a high frequency and old dog encephalitis in rare cases. Because of some similarities of the former to measles encephalitis and of the latter to subacute sclerosing panencephalitis (SSPE), CDV infection is considered to serve as a model for the comparative study of neurovirulence of measles virus. Thus, several disease models of CDV-induced encephalitis have been established in experimental animals including rodents and nonhuman primates (11, 13, 17). Recently, the possible involvement of CDV as a causative agent in multiple sclerosis (MS) has been con­ troversial from the epidemiological standpoint (4, 5). Therefore, neurovirulence of CDV is now being investigated from the aspect of etiological agents for the human

The stability of neurovirulence and in vitro phenotypes of canine distemper viruses adapted to neural cells was examined. Neurovirulence was estimated by the morbidity, mortality, and histopathological changes in the central nervous system of mice. After a single passage of the adapted viruses in Vero cells in which the unadapted virus had been passed, the neurovirulence of glioblastoma-adapted and oligodendroglioma-adapted viruses reverted completely to that of the unadapted virus. However, the neurovirulence of a neuroblastoma-adapted virus reverted partially. In vitro phenotypes such as the two-dimensional electrophoretic patterns of viral proteins and the cross-neutralization patterns also reverted to those of the unadapted virus. However, plaque sizes remained similar to those of the viruses adapted to neural cells.