| Measles is a kind of respiratory infectious diseases caused by the measles virus, and it is one of limited kinds of infectious diseases that could be effectively controlled via vaccination. Although the morbidity and mortality caused by measles were significantly reduced by the widely use of measles vaccines, measles is still a problem of public health and still threats to the children's health and safety all over the world. Measles virus is single serotype, and its heredity has been considered relatively stable. However, measles strains isolated in recent years had a great genetic variation compared to the ones which were isolated in the 60s of last century. Whether the elevation of prevalence of measles has significant correlations with the variations of measles? Whether the variations of wild measles strains will result in reduction in protection of measles vaccine? Is there a need of developing new measles vaccines to achieve the ultimate goal of measles elimination in the world? All of these topics above have attracted wide attention from scholars around the world.To investigate the variations of wild strains of measles, the effects of current live attenuated measles vaccine and the mechanisms of live attenuated measles vaccine and other issues, this research carried out the following works:1. Variations of wild measles strains:40 samples of suspected cases of measles from 2008 to 2009 (32 of throat swabs and 8 of urine samples) were collected and 4 virus strains were successfully isolated by Vero / SLAM cells. According to results of cross-neutralization assays and the sequences of the 450 nucleotides carboxyl(-COOH) terminus of N gene, all of 4 strains of the virus were identified as measles virus.The 450 nucleotides fragments of carboxyl end of N gene on 16 wild measles strains(4 wild strains isolated in this research and 12 wild strains stored in our laboratory which were isolated in Jilin Province from 2005 to 2007) were amplified with RT-PCR. Software MEGA4.0 was applied to analyze the genetic affinity of corresponding sequences between test results and international standard strain Edmonston strain, Shanghai-191 and Changchun-47 vaccine strains. The results showed that the sequence homologies of 450 nucleotides and 150 amino acid in carboxyl-terminal of N gene were respectively 99.11% and 99.33%, the group genetic distance is less than 0.009; The genetic distance among 16 wild strains and Edmonston standard strain was 0.069 ~ 0.074, and the differences in nucleotide and amino acid were 6.00% ~ 7.55% and 8.00%~8.67%, respectively; The genetic distance among 16 wild strains and Shanghai-191 vaccine strain was 0.084 ~ 0.091, and the differences in nucleotide and amino acid were 6.67% ~ 8.22% and 10.00% ~ 10.67%, respectively; The genetic distance among 16 wild strains and Changchun-47 vaccine strain was 0.071 ~ 0.076 and the differences in nucleotide and amino acid were 6.22%~ 6.67% and 8.67%~9.33%, respectively.The results showed that the 16 strains of measles virus had neither significant genome sequences differences nor evident group genetic distance differences each other. But the 16 strains of measles virus had evident differences in group genetic distance and great variations in nucleotide and amino acid levels compared with Edmonston standard strain, Shanghai -191 and Changchun-47 vaccine strains.These experiments above showed that great variations in genetic level existed in the wild type strains compared with Edmonston strain and measles vaccine strain The variations accumulated gradually and might affect the effects of the current measles vaccine.2. Research of the mechanism of measles vaccine attenuation.Wild type measles strains were purified via the method of virus plaque experiments, and we observed the morphological characteristics of the virus plaque and the spot rates, then picked the spot and amplified the clone of the virus. Consequently we observed the characteristics of produced cytopathic and determined their virulence. In this research, we simulated the attenuated measles vaccine cultivation technology, cultured the attenuated measles wild strains which was separated from the specimens of the patients and the purified measles cloning strains to see the attenuation result.The results showed that the plaques generated from the same wild strains of measles had different size, shape, center and edge features as well as the spot rate. After picking out from the spot of each clone strains, different strains had different virulence ranged, which is 6.17lgTCID50/0.1ml~7.40lgTCID50/0.1ml. The characteristics and appears time of cell lesions were different. These results showed that the virulence of wild strains of measles had declined, while the clone strains had no significant change in virulence, which revealed that there were differences between clone strains and wild-type strains.Reduction of the virulence of wild strains occurred in the process of adaptation of virus from Primary human amnion cells(HAM) to Primary chick embryo cells(CEC). But there were no obvious changing neither in the process of virus adapts primary human embryonic kidney cells(HK) to HAM nor in the process of continuous reproduction on CEC. The result showed that one wild type measles strain may be a hybrid, which contained a large number of virulent strains and a small amount of attenuated strains, CEC played a selection role of the virus, the attenuated strains which were adapted to CEC would be retained while virulent strain which were not well adapted to CEC were eliminated. The reproduction of attenuated strain on CEC had amplified the attenuated strain to a large number.3. Vaccination and Epidemiological studies of measlesWe collected the epidemiological data of measles since 1988 in China and then analysed the circumstances of the rate of measles vaccination and the changing of national incidence rates in 1990~2007 and the differences of incidence in 31 provinces in China in 2003~2009 by the way of descriptive epidemiology. And then, we analysed the characteristics of distributions of measles'seasonal incidence of nationwide in 2004~2010 and the incidence of measles after immunization in Jilin Province.The results indicated that although the rate of measles vaccination had remained high level in China, however measles was still one of the leading legal infectious diseases in China. There would be a small-scale outbreak every 2 to 3 years, and there were significant differences in the incidence of measles in different provinces. The peak time of the measles disease was delayed obviously and seasonal incidence of the measles disease had changed greatly. Strengthening the vaccination might soon receive nice preventive effect; however the epidemic cycle of measles had not been broken through the Strengthened vaccination, long-term effects of vaccination needed further observation and research.This study suggests that good basis of immunization through vaccination can't effectively prevent the outbreaks of measles. To achieve the goal of the elimination of measles, we should continue strengthening the epidemiological surveillance of the serological and molecular epidemiology of measles virus, as well as carry out the basic works of children's immunization and vaccination. More over, checking and adjusting the current strategies and immunization programs in our country, developing new measles vaccines in time are needed to improve the efficacy of the measles vaccine.
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