We reported earlier that B95a, an Epstein-Barr virus-transformed marmoset B lymphoblastoid cell line, is more susceptible to infection with measles virus than other cells. The cell line also was found to be susceptible to infection with the lapinized Nakamura III (L) strain of rinderpest virus and various strains derived from it. The B95a cell line was therefore the only host cell system available for the propagation and quantification of the L strain. In contrast to the adaptation of the L strain to Vero cells which results in a diminution of virulence in rabbits, the propagation of the virus in B95a cells preserved the virulence and some other properties in rabbits. Furthermore, when Vero cell-adapted variants of the L strain with diminished virulence were serially passaged in B95a cells, virulence in rabbits was gradually regained.
B95-8, an Epstein-Barr virus-transformed marmoset B-lymphoblastoid cell line, and its derivative B95a, capable of attachment to a substrate surface, were 10,000-fold more sensitive to measles virus present in clinical specimens than were Vero cells. B95-8 and B95a cells were thus thought to be useful host cells for the isolation of measles virus. Quantitation of measles virus present in clinical specimens showed that a large quantity of virus, exceeding 10(6) 50% tissue culture infective doses per ml of a nasal-swab eluate, is shed into secretions by patients with acute measles, consistent with the contagiousness of the disease. Measles viruses isolated in B95a cells differed in some biological properties from those adapted to Vero cells. First, the viruses isolated in B95a cells did replicate in Vero cells, but release into the fluid phase was less efficient than that of Vero cell-adapted viruses. Second, minor antigenic differences were found between virus strains isolated in B95a cells and those isolated in Vero cells from the same clinical specimens. Third, the viruses isolated and propagated in B95a cells caused clinical signs in experimentally infected monkeys resembling those of human measles. It was suspected that measles virus is subject to host cell-mediated selection and that the viruses grown in B95a cells are more representative of measles virus circulating among humans than are the viruses selected in Vero cells.
ABSTRACT Reverse genetics technology so far established for measles virus (MeV) is based on the Edmonston strain, which was isolated several decades ago, has been passaged in nonlymphoid cell lines, and is no longer pathogenic in monkey models. On the other hand, MeVs isolated and passaged in the Epstein-Barr virus-transformed marmoset B-lymphoblastoid cell line B95a would retain their original pathogenicity (F. Kobune et al., J. Virol. 64:700–705, 1990). Here we have developed MeV reverse genetics systems based on the highly pathogenic IC-B strain isolated in B95a cells. Infectious viruses were successfully recovered from the cloned cDNA of IC-B strain by two different approaches. One was simple cotransfection of B95a cells, with three plasmids each encoding the nucleocapsid (N), phospho (P), or large (L) protein, respectively, and their expression was driven by the bacteriophage T7 RNA polymerase supplied by coinfecting recombinant vaccinia virus vTF7-3. The second approach was transfection with the L-encoding plasmid of a helper cell line constitutively expressing the MeV N and P proteins and the T7 polymerase (F. Radecke et al., EMBO J. 14:5773–5784, 1995) on which B95a cells were overlaid. Virus clones recovered by both methods possessed RNA genomes identical to that of the parental IC-B strain and were indistinguishable from the IC-B strain with respect to growth phenotypes in vitro and the clinical course and histopathology of experimentally infected cynomolgus monkeys. Thus, the systems developed here could be useful for studying viral gene functions in the context of the natural course of MeV pathogenesis.
The course of natural infection with measles virus was examined serologically and histopathologically in monkeys which were kept in routine quarantine. Giant cells of the Warthin-Finkeldey type were consistently detected in the lymph nodes biopsied one week after the serologically estimated time of natural infection, and they were considered to be caused by natural infection.Histopathological responses to subcutaneous inoculations of various strains of measles virus were then compared in monkeys which were kept in strict isolation from natural infection. The wild virus consisting of either throat washings from measles patients or infected monkey tissue extract produced a large number of giant cells widely distributed in the lymphoid tissues of all the monkeys inoculated. On the contrary, attenuated vaccines and other laboratory strains did not produce giant cells in most of the monkeys inoculated except in few monkeys which had small number of giant cells only in a limited distribution. In spite of such a marked difference of pathological changes between the wild virus and the vaccine or laboratory strains, no definite correlation of the pathological changes with the virulence of measles virus could be elucidated since the several laboratory strains which were supposed to be unattenuated behaved apparently in a similar way as the attenuated vaccines.
Cynomolgus monkeys with or without measles antibody were intracerebrally inoculated with measles or canine distemper viruses, and antibody responses in the cerebrospinal fluid (CSF) were investigated. In measles antibody-free monkeys, natural infection with wild measles virus or intracerebral inoculations with two attenuated measles vaccines evoked primary antibody responses to measles virus in the sera but not in the CSF. In measles-immune monkeys, intracerebral inoculation with the TYCSA strain of measles virus produced a significantly high titer of measles antibody in the CSF with a minimal rise in the serum antibody and resulted in a significant decrease in serum/CSF antibody ratios. Intracerebral inoculation of a neurotropic canine distemper virus, the Onderstepoort strain, into measles-immune monkeys caused production of both measles and distemper antibodies in the CSF. Inoculation of measles-immune monkeys intravenously with measles virus or intracerebrally with rubella virus, which has no antigenic relation to measles virus, failed to evoke a measles antibody response in the CSF. These results indicated that local production of measles antibody in the CSF was caused by a stimulus within the central nervous system of measles virus antigen or canine distemper virus antigen that partially cross-reacted with measles virus antigen as a secondary antibody response.
The lymphoid tissues of monkeys infected with wild measles virus were examined by light microscopy and the fluorescent antibody technique. There was a good correlation between distribution of viral antigen and giant cells, most of which were of the reticular type. A large amount of viral antigen was always detected in giant cells, which suggested that these cells might be formed as a result of growth of measles virus in situ. Major growth sites of measles virus were found to be as the spleen, lymph nodes, and tonsils. It also appeared that the thymus and cecum might be involved to a lesser extent than the other lymphoid organs. Evidence of growth of virus in the bone marrow was not obtained. The possibility that the reticular cells are a main target of growth of measles virus was considered.
A strain of canine distemper virus was shown to be highly neurovirulent in non-human primates. Intracerebral inoculation induced in monkeys histological lesions of encephalomyelitis, i.e., degenerative changes consisting mainly of neuronal damage and inflammatory changes such as perivascular cuffings and glial proliferation, in wide areas in the brain and spinal cord. In one monkey observed for 70 days, lesions with a tendency of subacute sclerosing were also noticed. Immunosuppression with cyclophosphamide or antithymocyte serum was found to aggravate the clinical course and to modify the histological lesions in the central nervous system as well as the level of antibody response to the virus in cerebrospinal fluid. Possible application of distemper encephalomyelitis in monkeys as a primate model for analysis of the immune mechanism involved in paramyxovirus-induced encephalomyelitis was discussed.
