| Objective1. To study the variation and evolution characteristics of measles strains causing theepidemics that circulated in Zhejiang province during1999to2011, based on the analyze ofnucleotide, amino acid, glycosylation sites of each gene and whole genome.2. To analyze the differences among the measles vaccine strains, other genotypes and theZhejiang epidemic strains.3. To estimate the evolutionary rate and divergence time of N gene and H gene, as to explorethe evolutionary characteristics of measles virus clade H.MethodNine Zhejiang measles strains were proliferated in Vero/slam cell, identified with real-timefluorescent quantitative RT-PCR, amplified by RT-PCR assay. The obtained sequences wereanalyzed with other genotypes epidemic strains of foreign and vaccine strain S191, using variety ofbiological software. Finally, the rate of nucleotide substitution and the time to most recent commonancestor (TMRCA) in measles clade H, based on the nucleotide sequences of N gene and H gene,were analyzed by Bayesian Markov chain Monte Carlo (Bayesian-MCMC) and molecular clockmethods.Results1. The whole genome sequence length of nine measles strains prevalence in Zhejiangprovince is15894bp. Compared with the vaccine strain S191, the average nucleotide differencesare range from595to669, while the average amino acid variants are range from135to669inZhejiang strains. In nucleotide level, N gene variation is the largest (5.5%), while the largest one isP gene in amino acids (7.7%).2. The number of amino acid changes in N, H and F gene cell epitope regions, are14,6,7respectively. Other genes also have different numbers of amino acid variation. Some of aminoacids have the phenomenon of back mutation, entropy is also high, but the value of Ka/Ks issmaller than one. 3. There are five glycosylation sites change in Zhejiang measles strains. P gene and L generespectively added a glycosylation site, while a glycosylation site were reduced in the both F geneand H gene, and L gene1712glycosylation sites is not stable during1999to2012.4. All the Zhejiang measles virus strains separated in the year of1999to2011belonged toclade H, genotype H1. In addition to the strain isolated in1999, is subtype H1b, the rest of theepidemic strains are subtype H1a, and there is no obvious time and geographic course among theisolates.5. At the whole genome level, the genetic distance between Zhejiang epidemic strains andvaccine strains around the world is greater than the distances between epidemic strains of genotypeD4, B3and vaccine strains(t=-12.39，P<0.05；t=-9.76，P<0.05).6. The evolutionary average rate on N gene was8.802×10-4substitutions/site/year (95%HPD:5.688×10-4~1.184×10-3). The estimated dates for TMRCA of clade A and H of measles virus wasdated bake to1945(95%HPD:1920~1954), while the divergence times of clade H and genotypeH1were1977(95%HPD:1954~1987) and1986(95%HPD:1980~1990) respectively. Theevolutionary average rate on H gene was6.349×10-4substitutions/site/year (95%HPD:4.397×10-4~8.214×10-4). The estimated dates for TMRCA of clade A and H of measles virus was dated baketo1938(95%HPD:1908~1954), while the divergence times of clade H and genotype H1were1973(95%HPD:1959~1985) and1982(95%HPD:1973~1988) respectively.ConclusionThere were significant differences found in the each gene between vaccine strain S191andZhejiang epidemic strains, which belong to genotype H1. The differences between the currentvaccine strains and H genotype epidemic strains were much larger than the differences betweenvaccine and the foreign epidemic strains (genotype D4, B3) The most common ancestor of measleslineage A and H appears from the beginning20th century to the middle of20th century. Measleslineage H began to divergent around in1975, about ten years later genotype H1is also divided. Weshould pay much attention to these trends, strengthen the molecular epidemiology and evolutiondirection in measles virus, and develop candidate for vaccine as early as possible. |
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