| Measles is a kind of respiratory tract infectious diseases caused by measles virus (MV). It is still one of extremely serious infectious diseases to harms child life and health in the world. It is gradually concentrated in developing countries rather than all over the world after the use of measles vaccine.Although we have come to a great approach to controlling measles, incidence rate of measles was still relative higher in the remote province and outbreak in some regions. Suspecting arouses because of the increasing of measles spreading and prevail changing feature. Respecing the ascensus of cases reported recently in Jilin Province, this research studied the trait of measles spreading after scheme immunization for several years and timely intensify immunization, as well as the molecular etiological changes of measles wild virus.1. Study on the etiology of the wild-type measles virusCollect 105 measles borderline cases from 2005 to 2007, and succeeding separate 12 viruses with the Vero/SLAM cell and B95a cells lines. Confirmed completely 12 virus for the measles virus, by the typical CPE (cytomixis), the measles monoclonal antibody, and the N gene carboxyl terminal 450 nucleotides amplification. Viral RNA prepared from 12 strains wild-type measles viruses was used as a template for RT-PCR (reverse transcript polymerase chain reaction). 450 nucleotide pieces of the C- terminal region of the N gene, 537 nucleotide pieces of H gene and 143 nucleotide pieces of M gene were amplified and sequenced. Phylogenetic analysis and diversity analysis were done with DNAStar and the MEGA3.1. Their heredity distance of analyses were done: phylogenetic trees, the 22 measles WHO reference strains, H1 and H2 strains wild-type measles virus separated from 1993 to 1994, as well as the Chinese vaccine Changchun47 sequence to make phylogenetic analysis. The result showed that the 12 strains wild-type measles belong belong to the clade H and were one of big branch with H1 reference Hunan.China93-7. Further, it was discovered 12 wild-type measles viruses strains belonged to H1a subgenotype that represented strains were China93-2 and China93-4 of WHO reference strains ,and H1b and H1c subtypes were not found. The prevalent cases is H1a subgenotypes in Jilin Province druing 2005~2007, but it could not completely deny other subgenotypes existence, which coincided with the mainly popular genomes. This result showed that the outbreaking of measles during 2005 ~2007 come from native strains, not exogenous.The gene sequences of 12 measles wild virus were not completely same. Compared with the 12 strains wild-type measles viruses separated, N C-terminal 450 bp, 537 bp of H gene, and 143 bp of M gene, the nucleotides and amino acid homology are 97.6% and 96.7%, 98.3% and 97.2%, 98.6% and 95.8% above respectively. Compared themselves, the heredity distance are 0.002~0.032,0.002~0.017 and 0.002~0.021 respectively. These proved that the differene between measles wild viruses was not signifigant, and the difference between genomes altered in a micro extent in our province these 3 years. In homologic analyses showed that the nucleotide sequence the homology and the amino acid sequence homology always were not consistent, the nucleotide mutation possibly certainly did not cause the amino acid the variation, and could not affect the protein structure and the function. It is different with it, the minority position spot nucleotide mutation possibly could cause an amino acid bigger variation.Compared the 12 strains wild-type measles viruses with Edmonston standard strain, the difference in N gene C-terminal 450 nucleotides was 29~31bp (150 amino acid difference was 13); and in 537 nucleotide pieces of H gene was 30~32bp (175 amino acid difference was 9~11). The difference between the there gene pieces with Edmonston standard strains in the level of amino acid was 8.67% and 5.14%～6.29% respectively, which showed that wild-type measles wild viruses and Edmonston standard strains differed a lot on nucleotides homology and amino acid sequences.Compared with N and H gene, M gene is conservative. It is similar between earlier wild-type MV and Moraten about their M gene, but it has changed now under the vaccine immunity choice pressure. The main changing area is the 50th to 100th amino acids. Meanwhile, there was some reports that matrix protein has some relation with subacute sclerosing panencep halitis(SSPE). Our reseach cloned the most variation region of M gene 143 nucleotides, and the sequence analysis suggested that the difference with Edmonston standard strains altered between 2.80%~3.50%, that is, nucleotides altered between 4~5. There was only two strains of wild-type measles wild viruses(JL-5 and JL-12) in M gene differed 1 amino acid respectively (2.13%), coincided with which in other countries. Analysis on gene relationships of their sequence with MVi/Zhejiang.CHN/7.05/4 reference strains, SSPE wild-type measles wild viruses strains, Edmonston standard strains and vaccine strains showed that the 12 measles branch, and denied SSPE wild-type measles wild viruses strains with all the vaccine strains separated in other countries, which may be the molecular biology basement making native popular strains refuse SSPE.Glycosylation site of HA protein is a significant site of antigen neutralization. The 12 measles wild viruses in our research all lost a glycosylation site (S-N)on 38th site (nucleotides 113th G-A), and 5 glycosylation sites became 4. This change will lead to the molecular weight and biological activity change of H gene, cause the protein spatial structure change, and affect its immunity activeness. Whether the biological structure and the function changes affects the protection of measles vaccine, or the now-using vaccines could refuse the attack come from the wild virus still need to be discussed, which becomes the most concerned problem in our research.We balanced out the serum came from acute measles victim, healthy crowd and two share of primary immunity child blood with 3 strains of measles wild virus(1 strain of 2005,2006,2007 respectively) and vaccine strains. The findings showed that the serum comes from acute measles victim could not completely counteract the measles wild virus, hinting the lost of infection protection. The primary immunity child blood serum can completely neutralize wild-type measles virus strain, but the ability of neutralization wild-type measles virus was obviously lower than vaccine strain, the neutralizing antibody titer(NAT) differed as much as 4 times between vaccine strain and wilde-type MV. This hinted that the protection ability descended and time limit shortened. The healthy blood serum could neutralize wild-type measles virus strain (GMT is 1:14.42, 1:13.45, and 1:14.42 respectively) and vaccine strain (GMT is 1:26.91). Both of neutralization ability titer were not different more and statistical analysis did not have significant difference.This research analyzed the antigenicity of 3 from the 12 strains. The result of serum neutralization test of vaccine strains and 3 measles virus strains showed that their neutralization site were the same, and the now-using vaccines still play a part in the pretection. But it also showed that the ability of neutralization differed as much as 4 times above, coincided with primary immunity child blood serum test, which meant the descending of the vaccines'ability. The findings showed that cross neutralization test through measles virus strains with the corresponding immune serum was done. Antigen ratios between every each measles virus strains were calculated. Three wild-type measles virus antigen ratios only were 1.14, 1.06 and 1.02 respectively. It hinted these three wild poisonous had no non-antigenicity difference. (there being no difference when R equals 1-1.5, little difference when 1.5-2, the higher the R goes, the greater the difference is). But Antigen ratios between the three wild-type measles virus and measles vaccine were 3.26, 5.62 and 5.94 respectively. It cued that current wild-type measles virus had more obvious difference with present measles vaccine strains in the antigenicity. Antigen ratios between three wild-type measles virus and Edmonston were 4.16, 6.07 and 6.21 respectively. It simultaneously prompts wild-type measles virus have a bigger antigenicity difference with Edmonston strain than vaccine strain, which coincide with the gene sequence anslysis. The above serum research proved that the protection of measles vaccine descending at different degrees in the respect of neutralising capacity of measles wild virus and vaccine strains descended in different crowds'serum.2. The changes of Jilin Province measles popular characteristic around the intensive immunityThis article carries on the description epidemiology analysis and the data processing on the Jilin Province from 2004 to 2007 year measles epidemic disease material. and the measles epidemic situation monitor material. Carries on the descriptive epidemiology analysis and data statistics processing to our province measles popular changes, after the intensive immunity to 8 monthes ~ 14 years old children in April 2006. The analysis result is as follows:After the intensive immunity, the incidence of measles, and the mortality rate dropped obviously. The popular peak was reduced significantly. Fifty-three examples were reported in the whole Jilin Province in 2004 and measles incidence rate reported was 0.2 per hundred thousands. Measles incidence rate compared with 2003 dropped 78.33%. In 2005, the whole province measles arose 1 978 examples. Measles incidence rate was 7.27 per hundred thousands and compared with 2004 rose 3553.65%. The first overtopping was 3 per hundred thousands of disease incidence rates from 1985. In 2006, 4 278 examples were reported in the whole Jilin Province. Measles incidence rate was 17.41 per hundred thousands, compared with 2005 rose 138.42%. In 2005, the measles dropped to 390 examples. Measles incidence rate was 1.43 per hundred thousands and compared with 2006 dropped 1326.33%. Four years were no death cases of illness.After the intensive immunity, the patients age structure ratio had changed: the number of patients of yonger than 8 monthes and adult both rose. There were two peaks of age of onset every year in all age group: 0~2 years old group and 10~40 years old group respectively. But 1 year old of group was the measles rises peak age section. The cases of younger than one year old increased, the infants of less than 8 monthes was the highest, and the adults cases also rosed. The incidence presented polarization tendency, that is more than 15 year old to the adult case and the infants younger than 8 monthes.After the intensive immunity, the seasonal characteristic of the measles arose was not obvious. There was measles morbidity in each month during 2004~2007. Cases reported from January every year, started to have the big increased range from February, and the high stage from March to June every year. Case of illness number of these two months reported accounts for the whole year to report case of illness number 69.81%(2004), 69.41%(2005), 80.36%(2006) and 80.77%(2007) separately. Reported case of illness number both in April and May was the most and lowest in September. Although the morbidity has certain seasonal characteristic, already not really obvious.After the intensive immunity, the distribution of measles had moved to the city full of fluid population. There was only 8 districts (according to administrative division Jilin Province to be divided 9 areas) to report the measles case of illness in 2004, but in 2005 all 9 districts have the measles case reporting. Measles outbreak was in some districts. In 2006, the measles epidemic situation nearly affects all counties and city, the popular intensity surpassed in 2005 the same time 3~5 times. Tendency to assume partial exploded the situation to spread the entire province was. Measles incidence dropped rapidlys in 2007, after a strengthened immunity on April 2006 covered entire province. The measles case number of transient population occupied the total case about 30%, and was higher at the economic development quick big city. Some key city like Changchun, Jilinshi and Sipin, the incidence rates were higher than the far away city.Measles arises about the occupation distribution situation was in following four kinds of occupations that the measles arises the number were more. They were lives scattered the child, the student, the day care children and the farmer in turn. Occupies always arose the number were 70% above proportion. Looked from the immunity vaccination and the immunity history, not the immunity and the immunity history is unclear case of illness number occupies always sends case of illness number 82.82%, the crowd only has about 91% to the measles immunity, cannot prevent the measles virus circulation dissemination.After the intensive immunity, the popular measles intensity was weaken, and the popular cycle is broken. Before the intensive immunity, measles morbidity presented the scattered, small scope popular, and out break coexisting. There was no measles out break after the intensive immunity in April 2006 till the end of 2007. It was only scattered and small scope popular. Crowd immunity protection can be improved in short time by timely intensive immunity. The popular cycle will be broken by it. The measles popular cycle during 2004~2007 in our province was broken by the intensive immunity in April 2006.The epidemiology analysis result indicated that, the main reasons of measles erupts the popular in our province from 2005 to 2007 was about: (1) the crowd has not carried on a second dose of measles vaccine according to the stipulation related, has not specially carried on the regular strengthened immunity; (2) the individual place appears the immunity blind spot, the easy feeling crowd to increase related; (3) some children's immunity defeated, some others the immunity along with the age gradually to reduce after adds the primary immunity to grow, and all these persons had not carried on a second dose of measles vaccine. All these leads to the partial measles patient arises in the age to move concerns.This research analyzed the measles outbreak reasons from epidemiology, molecular biology and serology level in Jilin Province during 2005~2007, and the influence to our province measles epidemic situation after measles vaccine strengthening immunity in April 2006. Discussed the influence of the wild-type measles virus gene variation and the antigen drifting to the measles vaccine immunity protection effect. And confirmed the popular wild-tpye measles virus were belonged to Hla gene subgroup causing our province measles outbreak during 2005~2007.Above research prompt: for the molecular biology, it is necessary to continue to analyze measles virus other primary structure gene (F, P, and L gene); for serology monitoring, we should also take the measles virus antigen change dynamic observation; for epidemiological surveillance, we should observe the prevention effect of now-using vaccine to new appearing genotype measles virus. In order to prepare screening good immunity protect strain for researching new measles vaccine, and to prevent and control measles breakout provides the scientific value reference.
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