Cloning and sequencing of the fusion protein gene of mumps virus (Miyahara strain).
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Amino Acid Sequence↗
Biomedical subjects
M Hishiyama
Publications and source records attributed to M Hishiyama.
Cloning and sequencing of the haemagglutinin-neuraminidase gene of mumps virus (Miyahara strain).
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Amino Acid Sequence↗
Sequence variation of the P gene among mumps virus strains.
We have determined nucleotide sequences of a 183-nucleotide long region of the P gene of 10 mumps virus strains after gene amplification mediated by DNA polymerase catalyzed chain reaction (PCR) and have compared them with those of two strains which had been reported earlier (K. Takeuchi et al., J. Gen. Virol., 69, 2043-2049 (1988]. It was shown that mutation is generally noncumulative, i.e., most nucleotide substitutions in earlier strains do not appear in later strains. Viruses of different lineages appeared to cocirculate, but the comparison of American and Japanese strains suggested that those isolated in one country are more closely related to each other than to those isolated in the other country.
Base Sequence↗
Virus-specific polypeptides of human parainfluenza virus type 4 and their synthesis in infected cells.
We have studied the structural components of human parainfluenza virus type 4A (PIV-4A) and identified some virus-specific polypeptides by immunoprecipitation with polyclonal and monoclonal antibodies followed by one- or two-dimensional SDS-PAGE. HN polypeptides existed as monomer, disulfide-linked dimer, and disulfide-linked larger oligomer in cells infected with PIV-4A. Interestingly, the nonreduced NP, the nonreduced fusion, and the reduced F1 proteins migrated as doublets. Two F1 polypeptides were derived from different F1 + 2 proteins which migrated separately under nonreducing condition. In Vero cells infected with two strains of PIV-4A, two lower-molecular-weight proteins related to NP were detected. Oligopeptide patterns of the lower-molecular-weight protein were similar to those of NP protein synthesized in primary monkey kidney cells. The NP-related low-molecular-weight protein(s) was immunoprecipitated by 1 of 11 monoclonal antibodies against mumps virus NP protein. The MAb also reacted with NP proteins of PIV-2 and SV5. Thus, the epitope recognized by the MAb was common among PIV-2, PIV-4, mumps virus, and SV5, suggesting that the epitope might have an important biological function. However, the MAb did not react with the intact NP protein from cells infected with PIV-4, indicating that the epitope of PIV-4A was presented only when NP was cleaved. Phosphorylation was demonstrated for NP and P proteins.
Animals↗
Intracellular processing of mumps virus glycoproteins.
Using newly isolated monoclonal antibodies (MoAb) directed to the hemagglutinin-neuraminidase (HN) and the fusion (F) glycoproteins of mumps virus, we have analyzed post-translational modification of both glycoproteins. The HN glycoprotein synthesized as a monomer slowly acquires immunoreactivity with a monoclonal antibody (MoAb) to the HN in conjunction with the formation of oligomers. The oligomerization of the HN protein appeared to take place during transport of the protein through the Golgi apparatus. The immunoreactivity of the F protein evolved quickly in the rough endoplasmic reticulum, and the precursor of the F protein appeared to be proteolytically cleaved into F1 and F2 at the trans Golgi cisternae.
Animals↗
Complement-mediated neutralization test for determination of mumps vaccine-induced antibody.
When assessed by a conventional plaque neutralization (NT) test, about one third of recipients of live mumps vaccine were found to have failed to seroconvert, although the majority of them became seropositive by the complement-mediated neutralization (CNT) test. The discrepancy between the conventional NT and CNT was found to result from two factors. First, the predominant production of antibodies to the fusion (F) protein during the early phase of antibody response, and second, a low efficiency of NT of the early antibodies in the absence of complement. These features of antibody response were also seen in natural mumps infection and in experimental infection of monkeys, but were particularly prominent in vaccinees because of the limited extent of antibody response in the latter. The discrepancy between conventional NT and CNT diminished with time after vaccination, after natural infection, and also after experimental infection of monkeys. The CNT test was therefore considered better suited than the conventional NT test for assessment of the outcome of vaccination, at least until 6 to 9 weeks after vaccination. The protective role of the antibody in vivo, which requires the addition of complement for neutralization, was inferred from the observation that the antibody incorporated in the agar overlay significantly reduced the size of plaques formed by mumps virus.
Animals↗
Molecular cloning and sequence analysis of the mumps virus gene encoding the P protein: mumps virus P gene is monocistronic.
The nucleotide sequence of the P (phosphoprotein) gene of two strains of mumps virus has been determined from overlapping cDNA clones. The P gene contained a single open reading frame coding for a protein of 391 amino acids with a calculated Mr of 41,587, in good agreement with the value (40K to 45K) estimated from electrophoretic mobility on SDS-polyacrylamide gels. No open reading frame analogous to the C gene of other paramyxoviruses existed in the mumps virus P gene region. Comparison of the amino acid sequence of the mumps virus P protein with that of Newcastle disease virus showed a limited sequence homology.
Amino Acid Sequence↗
The polypeptides of human parainfluenza type 2 virus and their synthesis in infected cells.
We have studied the structural components of three strains of human parainfluenza virus type 2 (HPI-2) and identified the virus-specific polypeptides. Molecular weight of P and F1 polypeptides determined by us, when compared with that reported previously, was in reversed order. HN polypeptide existed chiefly as disulfide-linked dimers in cells infected with Toshiba strain, while as monomers and dimers in nearly equal proportion in cells infected with two other strains. Similar disulfide-linked NP oligomers were found in cells infected with all three strains. F1 and F2, cleaved forms of F protein, could be detected in cells infected with all three strains, but the ratio of cleaved (F1 and F2) to uncleaved (F0) forms was markedly lower in 62-M 786- and 63-M 1-infected than in Toshiba strain-infected cells. However, there was no difference of oligopeptide mapping patterns and isoelectric point of F polypeptide between Toshiba and 62-M 786 strains. By contrast, oligopeptide mapping patterns of HN protein of Toshiba strain differed from those of the two other strains. Furthermore, the HN polypeptide of Toshiba strain was phosphorylated in the infected Vero cells, but that of the other two strains was not.
Animals↗
Replication of mumps virus in mouse: transient replication in lung and potential of systemic infection.
A mumps virus strain, which replicated in mouse lung after aerosol inhalation, was obtained by selective replication of a wild strain in L929 cells and by further passaging in mice by intraperitoneal inoculation. All of infected mice survived and rechallenge of the survived mice with the same virus resulted in no virus growth in the lung. Treatment of infected mice with antiserum against interferon (IFN) or asialo GM1 delayed virus clearance from lung. Mice at 5 weeks of age were also sensitive to the virus as well as those at 1 week. When injected intravenously, the virus could grow not only in lung but also in salivary glands, heart and spleen. Furthermore, the virus replicated in liver, spleen, pancreas and testis after intraperitoneal inoculation. Antibody response of mice infected by aerosol inhalation was slower than that of intraperitoneally infected ones in either IgG or IgM production. These results indicated that the adapted virus replicated in mouse lung by a natural route of infection and had a potential to cause systemic infection in mouse.
Animals↗
Immunocytochemical focus assay for potency determination of measles-mumps-rubella trivalent vaccine.
The immunocytochemically stained focus assay for the determination of potency of individual components in measles-mumps-rubella trivalent vaccine is described. The method involves the reaction of infected cultures maintained under the agar overlay medium sequentially with rabbit antiserum specific to each component, biotinylated anti-rabbit IgG serum, avidin-biotinylated-peroxidase complex, and substrate mixture. The potency of one component determined by the method was not influenced by the presence of two other components and was comparable to that determined by either dilution end point titration or plaque assay. The method offers a number of advantages over the current method based on neutralization of components other than the one to be titrated.
Antibodies, Viral↗
Induction of cell fusion in Newcastle disease virus-infected L929 cells by anti-L929 cell antisera.
Infection of L929 cells by Newcastle disease virus (NDV) did not induce cell fusion, whereas addition of anti-L929 cell antiserum to the culture medium did result in cell fusion. When the antiserum was added to the culture medium of NDV-infected cells at the start of incubation, polykaryocytes appeared after 18 h post-infection (p.i.). On the other hand, when the antiserum was added to the culture medium at 18 h p.i., no polykaryocytes appeared, suggesting that the presence of the antiserum at the stage of NDV replication was necessary for the antiserum-induced cell fusion. Since anti-F polypeptide antiserum inhibited the cell fusion, functional F protein was essential for the antiserum-induced cell fusion. All the anti-L929 cell xenogenic sera tested were able to induce the cell fusion, whereas anti-L929 cell allogenic sera did not induce cell fusion. Anti-MCA cell serum also induced the cell fusion. Little replication of NDV was observed in the presence of anti-L929 cell serum and synthesis of virus-specific polypeptides, especially of M protein, was suppressed. However, there was no difference in expression of virus-specific glycoproteins on the surface membranes between cells treated with normal rabbit serum and those treated with anti-L929 cell serum. On the basis of the above results, the mechanism of induction of cell fusion by anti-host cell serum is discussed.
Animals↗
Immunological interrelationships among human and non-human paramyxoviruses revealed by immunoprecipitation.
Antigenic interrelationships among paramyxoviruses were examined by immunoprecipitation of isotope-labelled virus-infected cell lysates with specific antisera against virions or virus components. Sendai virus, human parainfluenza virus type 1 (Pa-1) and human parainfluenza virus type 3 (Pa-3) belonged to one antigenic group, and human parainfluenza virus type 2 (Pa-2), human parainfluenza virus type 4 (Pa-4), mumps virus (MuV) and simian virus 5 to a second group. Furthermore, the human paramyxoviruses Pa-1, Pa-2, Pa-3, Pa-4 and MuV formed a single antigenic group which overlapped the above groups. Although Newcastle disease virus (NDV) belonged to a separate group it showed some cross-reaction with the human paramyxoviruses. In particular, certain batches of anti-NDV antisera reacted with the fusion (F) polypeptide of Pa-2 and the reciprocal reaction of anti-Pa-2 antiserum with NDV F was also found. The nucleoprotein showed the broadest cross-reactions among these paramyxoviruses, whereas the matrix polypeptide exhibited antigenic individuality. The nucleoprotein of MuV was most cross-reactive. Pa-2 haemagglutinin-neuraminidase (HN) and anti-MuV HN serum showed cross-reactivity with various antisera and antigens.
Antibodies, Viral↗
[Infection defense antigens against pathogenic viruses to human. Infection defense antigens and genetic code--trend and progress of study on biomolecular analysis. i. Mumps virus].
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Antibodies, Monoclonal↗
Effects of antibodies and interferon on mumps virus persistently infected L929 cells. Generation of variant viruses in the cells during incubation with monoclonal antibodies and interferon.
We studied the effect of antibodies and interferon (IFN) on L929 cells persistently infected with mumps virus (MuV). The persistent infection was maintained by horizontal transmission of the virus within the culture but was regulated by endogenously produced IFN (8). The maintenance of the persistently infected cells in the continuous presence of anti-MuV serum suppressed the production of infectious virus. No virus antigen-positive cells were detected beyond 14 passages. Once antiserum was removed from the seemingly "cured cultures", however, a small number of antigen-positive cells appeared and the release of infectious virus into the culture fluid resumed. Even after 67 passages (200 days) of culture in the presence of anti-serum, the virus reappeared in the culture. At least 0.01 per cent of L929 cells in cultures maintained under antiserum harbored the virus without expression of virus antigen. The virus recovered from such cells was temperature sensitive. The infection of fresh L929 cells with the variant led to noncytocidal persistent infection which was maintained by propagation of virus-infected cells. MuV carrier cultures could be cured by serial cultivation in medium containing the mixture of antiserum and IFN. When L-MuV cells were subcultured with medium containing neutralizing monoclonal antibodies (MoAbs), antibody-resistant variant viruses were rapidly generated and selected. In the presence of IFN variant viruses resistant to IFN were generated. In view of the small amount of virus produced from persistently infected cells, variant viruses appeared to be generated in an unusually high frequency in carrier cultures. Thus, this experimental system may offer a useful in vitro model for studying antigenic variation and generation of various variant viruses.
Animals↗
Monoclonal antibodies against the glycoproteins of mumps virus: fusion inhibition by anti-HN monoclonal antibody.
Six monoclonal antibodies (MAbs) against the haemagglutinin-neuraminidase (HN) protein and three against the fusion (F) protein of mumps virus were obtained. Anti-HN MAbs were classified into three antigen-specific groups in competitive binding assays. All of the anti-HN MAbs showed virus-neutralizing and haemolysis-inhibiting activities and inhibited fusion from without (FFWO). Three of them inhibited fusion from within (FFWI) and the cell-to-cell spread of infection. The most effective MAb in inhibiting FFWI possessed neuraminidase-inhibiting activity and little haemagglutination-inhibiting activity. All anti-F MAbs inhibited virus-induced haemolysis but none of them inhibited FFWO, FFWI or the cell-to-cell spread of infection.
Animals↗
Enzyme-linked immunosorbent assay for mumps IgM antibody: comparison of IgM capture and indirect IgM assay.
We have established two enzyme-linked immunosorbent assays (ELISAs) for detection of mumps IgM antibody, i.e., indirect IgM ELISA and IgM capture ELISA, for serodiagnosis of recent mumps infection. In the latter method, peroxidase-conjugated monoclonal antibody to mumps virus was employed. Both methods detected mumps antibody of IgM class only in serum fractions separated by centrifugation through a sucrose density gradient. Optical density values given by both ELISAs were correlated for most sera examined. Indirect IgM ELISA, however, gave a false positive reaction for sera containing both rheumatoid factor and mumps IgG antibody, while giving a false negative reaction for sera containing high titers of mumps IgG antibody. This technique was, therefore, less reliable than IgM capture ELISA. IgM antibody detectable by IgM capture ELISA was present in all patients with mumps by the fifth day of illness and persisted for up to 3 mth in most and up to 5 mth in same cases.
Antibodies, Monoclonal↗
Persistence of mumps virus in mouse L929 cells.
The characteristics of a persistent infection of L929 cells with mumps virus (MuV) is presented. The persistent infection (L-MuV cells) was regulated by interferon (IFN) produced endogenously and almost all the properties showed that the carrier culture was maintained by horizontal transmission of the virus. Small-plaque mutants, but not temperature-sensitive variants, were selected during the persistent infection. MuV released from L-MuV cells (MuV-pi) replicated efficiently in L929 cells, while infection of L929 cells with the original MuV-o resulted in an abortive infection. The efficient replication of MuV-pi in L929 cells can be explained by the findings that MuV-pi induced IFN more slowly and had lower susceptibility to IFN in L929 cells than MuV-o did. M protein was synthesized to a considerable degree in MuV-pi-infected cells, while it could not be detected in MuV-o-infected cells. By contrast, MuV-pi formed small plaques in Vero cell monolayers and the yield of MuV-pi in Vero cells was lower than that of MuV-o. M protein induced by MuV-pi decayed easily in Vero cells. M protein was considered to be a limiting factor for MuV replication in both cell lines.
Animals↗
Replication of mumps virus in murine cells.
Mumps virus replication was examined in various culture cells derived from mice. Eight of 16 lymphoid cell lines and 4 of 13 non-lymphoid cell lines supported the replication of Vero cell-adapted Enders strain (EY) of mumps virus. EY strain replicated more efficiently in lymphoid cell lines than in non-lymphoid ones. T cell preference, however, was not observed in this study. The growth kinetics of EY strain in high yield cell lines such as EL-4, L1210 and NS-18 cells were similar to that in Vero cells, while in low yield cell lines such as DK, C243 and 203 GL cells the growth patterns varied respectively. Nineteen kinds of primary culture cells of murine origin all proved not to be susceptible for EY strain, even when spleen cells were stimulated with lectins or allogeneic cells. Seven other strains of mumps virus were examined for their ability to replicate in EL-4, L1210 and L929 cells. Four and 6 strains replicated in EL-4 cells and L1210 cells respectively, although the growth patterns and yields varied in each virus-cell combinations. On the other hand, none of 7 strains showed sufficient replication in L929 cells.
Animals↗