Passage in Vero cells alters the characteristics of measles AIK-C vaccine strain.

Objective of this study is to evaluate the feasibility of measles vaccine production in Vero cell culture. We constructed the full-length cDNA, pIC-MVAIK-F278Leu (small plaque-type) and pIC-MVAIK-F278Phe (large plaque-type) from the AIK-C measles vaccine strain attenuated from the Edmonston wild-type. MVAIK-S/B2 was rescued from pIC-MVAIK-F278Leu after two passages in B95a cells and MVAIK-SL/B2V1 was obtained through large plaque cloning in Vero cells. MVAIK-SL/B2V8 was obtained after eight passages in Vero cells. It produced large plaques in Vero cells, grew well at 39 degrees C, and thus the characteristics of the AIK-C vaccine strain were lost. Thirteen amino acid changes were observed; one in the N, two in the P, one in the C, three in the F, one in the H, and five in the L protein regions. Twelve of these changes excluding one in the L gene were back mutated to the Edmonston strain. Change from Leu to Phe at position 278 of the F protein was an early event during adaptation to Vero cells and the P gene was back-mutated to the Edmonston wild-type. As for the control, MVAIK-L/B9 strain was obtained after passages in B95a cells from pIC-MVAIK-F278Phe (large plaque-type). It maintained the same temperature sensitivity as the AIK-C vaccine strain and only four amino acid changes, one in the N and three in the L protein region, were observed without any mutations in the P, C, M, F, and H genes. The passage of the measles vaccine AIK-C strain in Vero cells lost the characteristics of small plaque inducibility and temperature sensitivity (ts) phenotype.

The phosphoprotein of attenuated measles AIK-C vaccine strain contributes to its temperature-sensitive phenotype.

Measles AIK-C vaccine strain exhibits temperature-sensitivity (ts). To identify the structural proteins, which contribute to the ts property of AIK-C virus, reverse genetics was used. MV-minigenome RNA was replicated at 32.5, 37, and 39 degrees C when the plasmids expressing N, P, and L proteins of the Edmonston strain (the parental strain of AIK-C) were used, whereas the minigenome RNA replicated only at 32.5 degrees C but did not at 37 degrees C and higher temperature when N, P, and L protein expression plasmids of the AIK-C strain were used. A series of minigenome experiments revealed that the amino acid substitution of leucine at position 439 of the P protein by proline (P439-Pro) contributes to the ts phenotype of AIK-C. Four recombinant viruses having various P genes were rescued from the modified AIK-C genome cDNA and ts-characteristics were compared in Vero cells by plaque formation assay. The results showed that the P439-Pro of AIK-C virus played a key role in the ts phenotype, but the other substitutions in the P gene might have an accessory function in the expression of the phenotype.

Molecular epidemiology of mumps virus in Japan and proposal of two new genotypes.

We isolated 872 strains of mumps virus from naso-pharyngeal secretions in seven different districts of Japan from January 2000 to July 2001. Among them, 57 strains were geno-typed by nucleotide sequencing in part of the hemagglutinin-neuraminidase (HN) and small hydrophobic (SH) protein regions. Four different genotypes (B, G, K, and L) of mumps virus were co-circulating in Japan and the distribution of genotypes varied in geographically different districts. Two new clusters designated as genotypes K and L had more than 7% nucleotide variation in the SH gene. Among the 57 strains, 11 were classified as B, 35 as G, three as K, and eight as L, which was mainly isolated in Tokyo. We also examined 104 stains isolated in a clinic in Mie prefecture from 1993 to 2003. Genotype B was the indigenous strain and genotype K was introduced in 1994. Genotypes B and K co-circulated in the 1990s and were replaced by genotype G in 2000. There was no significant change in neutralizing test antibody titers against genotypes B, G, K, and L using seven post-vaccination sera with Hoshino strain (genotype B) and these four genotypes had a different antigenicity from genotype A. We should continue to watch on mumps virus molecular epidemiology.