The mumps virus SH protein is a membrane protein and not essential for virus growth.

By immunoprecipitation analysis using antisera against oligo peptides synthesized based on the deduced N-terminal and C-terminal amino acid sequences of the SH proteins of the mumps virus, the SH protein was detected in mumps virus-infected cells. The SH protein expressed from cDNA by the vaccinia-T7 expression system was recovered in the membrane fraction. Association of the SH protein with the membrane was resistant to high salt, EDTA, and alkaline treatment but sensitive to detergents. Indirect immunofluorescence experiments showed that the SH protein is involved in the exocytotic pathway. These data indicate that the SH protein is a membrane protein. Treatment of microsomes with TPCK-trypsin suggested that the SH protein is oriented in the membrane with its C-terminal facing the cytoplasm. Furthermore the SH protein was not detected in a particular strain (Enders strain) of mumps virus, indicating that the mumps virus SH protein is not essential for virus replication.

Adverse events associated with MMR vaccines in Japan.

The largest nationwide active surveillance of four Measles-Mumps-Rubella (MMR) vaccines was conducted in Japan. A total of 1255 pediatricians actively participated in the study, which comprised 8.6% of all members of the Japanese Pediatric Society. The total number of registered recipients of MMR vaccines was 38 203. They were arbitrarily given one of the MMR vaccines produced by three makers (Takeda, Osaka city, Kitasato Minato-ku. Tokyo and Biken Suita city, Japan) or the standard MMR vaccine made of designated strains (Kitasato's measles-AIK-C, Biken's mumps-Urabe Am9 and Takeda's rubella-To336) produced by Takeda, Kitasato and Biken and were observed for 35 days. The rates of virologically confirmed aseptic meningitis per 10,000 recipients were 16.6, 11.6, 3.2 and 0 for the standard MMR, Takeda MMR, Kitasato MMR and Biken MMR vaccines, respectively. The incidence of convulsions between 15 and 35 days was the highest with the standard MMR vaccine and the incidence of fever associated with vomiting occurring between 15 and 35 days (symptoms relevant to aseptic meningitis) were also the highest with the standard MMR vaccine. The incidence of parotid swelling was the lowest with Takeda MMR vaccine. This surveillance revealed that incidences of aseptic meningitis after administration of the standard MMR vaccine and of Biken MMR vaccine were different. This posed questions about the manufacturing consistency of the Urabe Am9 mumps virus vaccines. On the other hand, the National Institute of Health found that the biological characteristics of the Urabe Am9 mumps virus contained in the standard MMR vaccine and in the Biken MMR vaccine were different. The Biken Company reported that the mumps vaccine in the standard MMR vaccine was a mixture of two Urabe Am9 mumps vaccine bulks; one identical to that contained in the Biken MMR vaccine and the other produced by a different manufacturing process.

Establishment of cell lines stably expressing mumps virus glycoproteins.

We established two cell lines that stably express hemagglutinin-neuraminidase (HeLa-HN) and fusion proteins (HeLa-F) of a fusogenic strain of mumps virus. Infection of HeLa-F cells with a nonfusogenic strain resulted in induction of extensive cell fusion. On the other hand, HeLa-HN cells appeared resistant to cell fusion induced by mumps virus infection.

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.

We 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.

Effect on fusion induction of point mutations introduced into the F protein of mumps virus.

We have tested the effect of substitution of an amino acid at position 195 of the F protein of mumps virus on cell-to-cell fusion caused by the virus. Introduction of amino acids with aromatic side chains into this position resulted in reduction of fusion induction. Furthermore the F protein was not cleaved when the amino acid at this position was substituted by several amino acids, suggesting that the amino acid at this position was essential to keep the tertiary structure of the protein that might be required not only for proper folding of the protein but also for induction of membrane fusion.

Identification of an amino acid that defines the fusogenicity of mumps virus.

Recombinant cDNA clones representing the fusion (F) and hemagglutinin-neuraminidase (HN) proteins of two mumps virus strains different in fusogenicity were constructed. Upon transfection of COS7 cells, extensive cell fusion was observed only when cells expressed the F protein of the fusing strain together with the HN protein derived from either strain. Mutational analyses further showed that the amino acid at position 195 of the F protein plays a critical role in determining the extent of cell fusion induced by mumps virus, since replacement of Ser-195 by Tyr significantly reduced the fusion inducibility of otherwise fusion-competent F protein.

Differentiation of mumps vaccine strains from wild viruses by single-strand conformation polymorphism of the P gene.

The DNA fragments amplified through the polymerase chain reaction from part of the P gene of four mumps vaccine strains (Urabe, Torii, Hoshino and Miyahara) were subjected to single-strand conformation polymorphism (SSCP) analysis. These four vaccine strains were differentiated from each other. Furthermore, twelve wild viruses and a laboratory strain (Enders strain) were also distinguished by this method. Viruses isolated from patients who developed aseptic meningitis 4 to 6 weeks after measles-mumps-rubella vaccination showed identical SSCP patterns with the vaccine strain used for immunization. These results were well correlated with sequence analysis of P-gene segments, indicating high applicability of the SSCP analysis for differentiation of mumps vaccine strains not only from each other but from wild viruses.

Molecular cloning and sequence analysis of the mumps virus gene encoding the L protein and the trailer sequence.

We have cloned and determined the nucleotide sequences of the seventh gene of the Miyahara strain of mumps virus (MuV) encoding the L protein. The L gene is 6925 nucleotides in length and contains a single long open reading frame which is capable of coding for a protein of 2261 amino acids with a calculated molecular weight of 256,571 Da. The deduced amino acid sequence of the L protein of MuV showed significant homology with those of six other paramyxoviruses, human parainfluenza type 2 virus, Newcastle disease virus, Sendai virus, measles virus, human parainfluenza type 3 virus, and human respiratory syncytial virus. The predicted MuV L protein contained distinct elements thought to be essential for RNA polymerase activity. A noncoding sequence of 24 nucleotides downstream of the presumed polyadenylation site of the L gene showed significant complementarity with the leader sequence composed of 55 nucleotide at the 3' end of the genomic RNA.

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.

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.

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.

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.

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.

Monoclonal antibodies against the nucleoprotein of mumps virus: their binding characteristics and cross-reactivity with other paramyxoviruses.

Twelve monoclonal antibodies (MoAbs) directed against the nucleoprotein (NP) of mumps virus were analysed for their binding characteristics. Competitive binding in enzyme-linked immunosorbent assay divided them into eight groups. Two of the MoAbs recognized exclusively 66 kD polypeptide of NP, two recognized 66 kD and 60 kD, and one recognized 66 kD (and 60 kD to a lesser extent) in Western blot assays under either denaturing or partially denaturing conditions. Under partially denaturing condition, another five MoAbs reacted faintly but the remaining two did not react at all. Under denaturing condition, on the other hand, these seven MoAbs showed little reactivity with any polypeptide. Furthermore, denaturation resulted in formation of other polypeptides 55 kD, 50 kD, and 43 kD which all were detected by MoAbs reacting with 66 kD and/or 60 kD. Previously demonstrated antigenic cross-reactivity among the NPs of mumps virus and those of human parainfluenza viruses type 2 and type 4 in radioimmunoprecipitation assay using polyclonal antisera was confirmed by an anti-NP MoAb which showed little reactivity in denaturing Western blot assay.

Nucleotide sequence of the gene encoding the matrix protein of mumps virus (Miyahara strain).

The nucleotide sequence of the gene encoding the matrix (M) protein of mumps virus (MuV), Miyahara strain, has been determined from several overlapping cDNA clones. The M protein mRNA is 1248 nucleotides in length, exclusive of the poly(A) tail, and codes for a protein of 375 amino acids (Mr41,556). Comparison of the deduced amino acid sequence of the M protein of the Miyahara strain with that of the SBL-1 strain revealed that the M proteins of both strains are highly conserved. A significantly lower rate of nucleotide differences conducive to amino acid differences in the M gene compared with other genes appeared to indicate the importance of the conserved primary structure of the M protein for its function.

Improvement in potency assay of measles-mumps-rubella trivalent vaccine: interference between components and measures for its elimination.

In the potency assay of trivalent measles-mumps-rubella (MMR) vaccine by the immunocytochemical focus assay reported previously (Fukuda et al., 1987), development of rubella foci in RK13 cells was inhibited in the presence of a large excess of mumps component, resulting in an underestimation of the titre of the rubella component. When RK13 cells are infected with the mixture of mumps and rubella viruses, mumps virus interfered with the growth of rubella virus. Interference was mediated most likely by interferon induced by mumps virus. The interference was eliminated by a partial neutralization of mumps component by the addition of anti-mumps serum to the inoculum to RK13 cells. Improved method of potency assay of MMR vaccine incorporating the above measures and other modifications are described.

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.

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.