Akira Sugiura

Further isolation and characterization of temperature-sensitive mutants of influenza virus

Abstract We have assigned 34 temperature-sensitive ( ts ) mutants of the WSN strain of influenza virus into seven nonoverlapping recombination-complementation groups by conducting pairwise crosses. A single gene of the viral genome was affected in 27 mutants while six were double mutants and one was a triple mutant. The synthesis of virion RNA in mutant-infected cells at the nonpermissive temperature was studied by the incorporation of [ 3 H]uridine in the presence of actinomycin D into an acid-insoluble product. This method separated mutants into two classes. Mutants belonging to Groups I, II, III, and V were RNA − , while those belonging to Groups IV, VI, and VII were RNA + . The results of temperature shift-up experiments suggested that all four groups of RNA − mutants were defective in replicating virion RNA. Production of serologically or functionally active subviral components (ribonucleoprotein, hemagglutinin, and neuraminidase) by RNA − mutants at the nonpermissive temperature was variable. RNA + mutants produced a normal amount of these components, except Group IV mutants in which the production of functional hemagglutinin and neuraminidase was greatly depressed.

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.

GENETIC STUDIES OF INFLUENZA VIRUSES. II. PLAQUE FORMATION BY INFLUENZA VIRUSES IN A CLONE OF A VARIANT HUMAN HETEROPLOID CELL LINE.

Abstract Plaque formation with representative strains of all major influenza A virus subtypes and with influenza B (Lee) virus has been obtained in a clone (clone 1-5C-4) of a human cell line of apparently unique susceptibility. The efficiency of plaque formation relative to egg infective dose (EIU) varies with the strain of virus, ranging from 2 to 70%. The clone (1-5C-4) has remained stable with respect to viral susceptibility for at least 36 transfers in mass culture during a period of 9 months. Certain strains of virus formed plaques of characteristic and distinctive type of potential value in genetic studies. Kinetic studies of the virus-cell interaction with NWS indicated that adsorption proceeded rapidly. However, about 40% of inoculated virus was unadsorbed. Adsorption not reversed with antiserum was a slower process and required 3–4 hours. Single-cycle infection with a high input multiplicity of virus required 20 hours for completion and resulted in a yield of 4 PFU/cell.

B95A, A MARMOSET LYMPHOBLASTOID CELL LINE, AS A SENSITIVE HOST FOR RINDERPEST VIRUS

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.

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.

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.

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.

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.