Kiyoshi Tanabayashi

Variations of nucleotide sequences and transcription of the SH gene among mumps virus strains

We have compared nucleotide sequences of the SH genes as well as their flanking regions of six mumps virus strains and found a high amino acid diversity (up to 23%) of the putative SH proteins among these strains. It was found, in addition, that one of these strains (Enders strain) contained a point mutation in putative polyadenylation signal for the F gene mRNA (TTTAGAAAAAAA to TTTAGAAGAAAA). Northern blot analysis showed that the Enders strain was also unique in that neither monocistronic SH nor bicistronic SH-HN mRNA could be detected in the cells infected with this particular strain.

Expression of mumps virus glycoproteins in mammalian cells from cloned cDNAs: Both F and HN proteins are required for cell fusion

Two recombinant plasmids were constructed by inserting the cDNAs of either the fusion (F) or the hemagglutinin-neuraminidase (HN) protein genes of mumps virus into the pcDL-SR alpha expression vector. Both the F and the HN proteins expressed in COS7 cells transfected with their respective recombinant plasmids were indistinguishable in terms of electrophoretic mobility from their counterparts synthesized in mumps virus-infected cells. The F protein was cleaved and expressed on the cell surface, but uncleaved forms were also detected. The expressed HN protein was transported to the cell surface and adsorbed guinea pig erythrocytes. Syncytium formation was induced when COS7 cells were transfected with both recombinant plasmid DNAs together, but not with the recombinant plasmid only carrying the F gene. This observation indicates that cell fusion mediated by mumps virus requires both the F and the HN glycoproteins.

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.

Recombinant Wild-Type and Edmonston Strain Measles Viruses Bearing Heterologous H Proteins: Role of H Protein in Cell Fusion and Host Cell Specificity

ABSTRACT Wild-type measles virus (MV) isolated from B95a cells has a restricted host cell specificity and hardly replicates in Vero cells, whereas the laboratory strain Edmonston (Ed) replicates in a variety of cell types including Vero cells. To investigate the role of H protein in the differential MV host cell specificity and cell fusion activity, H proteins of wild-type MV (IC-B) and Ed were coexpressed with the F protein in Vero cells. Cell-cell fusion occurred in Vero cells when Ed H protein, but not IC-B H protein, was expressed. To analyze the role of H protein in the context of viral infection, a recombinant IC-B virus bearing Ed H protein (IC/Ed-H) and a recombinant Ed virus bearing IC-B H protein (Ed/IC-H) were generated from cloned cDNAs. IC/Ed-H replicated efficiently in Vero cells and induced small syncytia in Vero cells, indicating that Ed H protein conferred replication ability in Vero cells on IC/Ed-H. On the other hand, Ed/IC-H also replicated well in Vero cells and induced small syncytia, although parental Ed induced large syncytia in Vero cells. These results indicated that an MV protein(s) other than H protein was likely involved in determining cell fusion and host cell specificity of MV in the case of our recombinants. SLAM (CDw150), a recently identified cellular receptor for wild-type MV, was not expressed in Vero cells, and a monoclonal antibody against CD46, a cellular receptor for Ed, did not block replication or syncytium formation of Ed/IC-H in Vero cells. It is therefore suggested that Ed/IC-H entered Vero cells through another cellular receptor.

Comparison of the entire nucleotide and deduced amino acid sequences of the attenuated hog cholera vaccine strain GPE− and the wild-type parental strain ALD

SummaryWe have determined the complete nucleotide sequences of a live attenuated hog cholera virus (HCV) and its progenitor strain. The viral RNA of each strain consisted of 12 298 nucleotides including untranslated regions of 373 and 228 bases at the 5′ and 3′ end, respectively. There was a single large open reading frame spanning 11 697 nucleotides which could encode a large protein of 3 899 amino acids with a calculated molecular weight of 438-kDa. We have found 225 nucleotide difference between the two strains, of which six were located in the untranslated region. Four-sixths of these differences resulted in amino acid substitutions.

Variation in Field Isolates of Measles Virus during an 8‐Year Period in Japan

Field isolates of measles virus (MV) during an 8‐year period in four areas of Japan, i.e., Osaka, Nagoya, Tokyo and Akita, were classified into three types in regard to the electrophoretic mobility of the hemagglutinin (HA) proteins: S type with small (78 K) HA, M type with intermediate (80 K) HA and L type with large (82K) HA. The type of field isolates was closely related with the geographical location and the year of virus isolation. The S type strain was isolated only in an outbreak from 1983 to 1984, whereas the M and L type strains were isolated between 1983 and 1990. The HA genes of the M and L type strains of MV were found to have a nucleotide substitution which introduces a new potential glycosylation site. In addition, the matrix proteins of all field strains isolated after 1977 showed slower electrophoretic mobility of 42 K than 39 K of the Edmonston and Toyoshima strains. These results indicate that MV strains of different HA types existed concomitantly and that major populations of MV currently circulating in Japan are changing from those prevalent in 1983‐1984.

Detection and characterization of mumps virus V protein

By cDNA cloning, DNA sequencing, and RNAse mapping analyses two distinct mRNA species were shown to be transcribed from the mumps virus P gene; one faithful and the other edited copies. The former was found to direct the synthesis of the V protein (25K), while the latter, after the addition of two nontemplated G residues, was found to direct the synthesis of the P protein (40K-42K). The carboxy terminal of the V protein contained a cysteine-rich region which was similar but not identical to the metal binding domain motif found in several nucleic acid binding proteins. The V protein was detected not only in mumps virus-infected cells but also in the virions by antiserum raised against a synthetic peptide.

Differentiation of the mumps vaccine strains from the wild viruses by the nucleotide sequences of the P gene

Nucleotide sequence analysis of a part of the P gene of mumps virus allowed the differentiation of live attenuated mumps vaccine strains from each other and from wild mumps viruses. Restriction enzyme analysis was found to serve as a convenient method of screening for one strain of mumps vaccine. Examination by the method of virus isolates obtained from the patients who developed mumps parotitis or meningitis following vaccination revealed that most of those viruses were related to the respective vaccine viruses used for immunization.

Nucleotide sequence of the leader and nucleocapsid protein gene of mumps virus and epitope mapping with the in vitro expressed nucleocapsid protein

The nucleotide sequence of the leader and the gene encoding the nucleocapsid protein (NP) of mumps virus Miyahara strain have been determined. The leader sequence is 55 nucleotides in length and the NP gene is 1845 nucleotides in length, exclusive of poly(A). The NP gene codes for a protein of 549 amino acids, with a calculated molecular weight of 61,365. For epitope mapping, a series of NPs from which C-termini were serially deleted were expressed in vitro from five mRNA constructs and were examined by radioimmunoprecipitation assay (RIPA) with eight nonoverlapping monoclonal antibodies (MoAbs) against the mumps virus NP. It was found that seven out of eight MoAbs reacted with the NP synthesized in vitro. Five recognized the epitopes located within the C-terminal 74 amino acids region and one within the adjacent 64 amino acids upstream. The epitope of the remaining one was in the N-terminal half of the NP.

Identification of the MHC class I B locus in cynomolgus monkeys

By determining the nucleotide sequences of more than 700 cDNA clones isolated from 16 cynomolgus monkeys, we identified 26 Mafa-B alleles. In addition, nine sequences with similarity to Mamu-I alleles were identified. Since multiple Mafa-B alleles were found in each individual, it was strongly suggested that the cynomolgus MHC class I B locus might be duplicated and that the Mafa-I locus was derived from the B locus by gene duplication, as in the case of the Mamu-I locus of rhesus monkeys.