Dissertation
Dissertation > Medicine, health > Basic Medical > Medical Microbiology ( pathogenic bacteriology,pathogenic microbiology ) > Human Virology ( pathogenic virus) > Enteroviruses and hepatitis virus

Studies on Genotype and Serology of Measles Virus Strains

Author LiuYang
Tutor ChenJinYing
School Tianjin Medical University
Course Pathogen Biology
Keywords Meaasles virus Genotype Antibody Attenuated live Measles vaccine Reverse transcription-polymerase chain reaction(RT-PCR) Restriction fragment length polymorphism(RFLP) Sequence analysis
CLC R373.2
Type Master's thesis
Year 2010
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Objective:To study the genovariation of wild-type measles virus from the patiens in Tianjin and evaluate the immuno-effect of the attenuated live measles vaccine after measles vaccine immunization.Methods:1. Throat swab and urine specimens were collected from patients with suspected measles from 2002 to 2008 and measles virus were isolated on Vero-SLAM cells.2. RNA of measles virus were extracted from the infected cell lysates, and C terminal 594 nucleotide fragment of nucleoprotein (N) gene was amplified by one step RT-PCR. PCR amplified products were sequenced and compared with the international standard strain to analyze sequence.3. Restriction enzyme sites of C terminal 594 nucleotide fragment of nucleoprotein (N) gene was selected by Genruner software,Bcn I was choosed to establish a method of RT-PCR-RFLP to identify genotype of measles virus. Measles virus vaccine strains from wild strains was identified by the method.4. Level of antibody was analyzed before measles vaccine immunization on the children under 8 months of age to understanding of the decay process of immunoprotective antibody.5. Antibody titers were detected by enzyme linked immunosorbent assay and Neutralizing antibodies assay before and after current attenuated live vaccine immunization on health human to evaluate the immuno-effect of measles vaccine.6. Specific IgM antibodies were detected in the serums of the patients with suspected measles in 2010 by enzyme linked immunosorbent assay to understangding measles cases trends in Tianjin and evaluating the Measles planned immunity and the immuno-effect of measles vaccine.Results:1. A total of 189 specimens were collected from patients with suspected measles of sporadic or outbreak cases from 2002 to 2008 in Tianjin.57 strains of measles virus were isolated from 132 urine specimens with a positive rate of 43.18%, and 23 strains of measles virus were isolated from 57 throat swabs with a positive rate of 40.35%.2. In the total 80 strains of measles virus,11 strains were positive both in throat swabs and urine specimens at the same time. In this case, one of them was sequenced, so the total number of sequencing was 69. Sequence analysis of nucleoprotein (N) gene C terminal 450 bp demonstrated all of the 69 strains of measles virus were genotype H1, and no vaccine-associated strains (A genotype) was isolated. All of the 69 strains were subgenotype H1a except one strain isolated in 2002, which was subgenotype Hlb. The data showed subgenotype H1a was predominant epidemic strain.3. The phylogenetic analysis of C terminal 594 nucleotide fragment of nucleoprotein (N) gene by randomly sampled 24 measles virus showed the subgenotype Hla viral strains have a variation range from 0.002 to 0.038 (1-17 nucleotide variation) from 2002 to 2008. The sequence data indicated the presence of multiple transmission chains caused by different measles virus in Tianjin.4. In this paper, a method of RT-PCR-RFLP was established to identify measles virus subgenotype Hla, subgenotype Hlb from genotype A,the results of this method were identical to those of sequence analysis.The method is simple, efficient and unique, economic and suitable for a lot of epidemiological investigations.5. Comparing the specific IgG anibodies before and after vaccine immunization on 922 of different age groups of health the crowd, the data showed the positive rates have no significant gender difference, but the age in august, High school junior and 20-25 are three levels lower age group with analysis of variance, the differences have statistical significance (P<0.0001)6. Immunoprotective level of IgG antibody was compared between vaccine stains and epidemic strains by neutralizing antibody assay after vaccine immunization on 13 august age children.No significant gender difference between the two, the result indicated the effectiveness of the attenuated live measles vaccine prevent.7. Protective levels of antibody analysis on 160 august children under age,the positive rate of IgG antibody of newborn only,36.8%, By 12.5% lower the age of july.The positive rate were gradual waning with age increasing month.8. The age group on august and than 14 group were a total of 509 people on 565 patients with suspected measles(90.09%),far more than august age or more to 14 group(9.91%). Laboratory treated 329 examples of the 509 patients with suspected measles,the positive rate as high as 64.64%, this indicated intensify immune of the measles vaccine is effective in 2008.But the current measles vaccine program needs further adjustment.Conclusion:1. Subgenotype H1a was predominant epidemic strain of measles virus in Tianjin.. Nucleotide analysis of C terminal 450 nucleotide fragment of nucleoprotein (N) gene showed the viral strains circulating have a 1-17 nucleotide variation. The sequence data indicated the presence of multiple transmission chains caused by different measles virus in Tianjin, Such variation is the antigens drift.2. Neutralizing antibody after vaccine immunization was detected to evaluate the immunoprotective level of measles vaccine on august age children. There were no significant difference between vaccine strains and epidemic strains, the results indicated the effectiveness of the attenuated live measles vaccine prevent.3. GMC of immunoprotective antibody on august children under age are gradual waning with age increasing month, therefore lead to more susceptible children. The age in august, High school junior and 20-25 are three levels lower age group by comparing GMT of the specific IgG anibodies before and after vaccine immunization on different age groups of health the crowd with analysis of variance, the differences have statistical significance, combinating epidemiological analysis of the Measles rebound cases, the results prompted the current measles vaccine program needs further adjustment.4. In this research a method of RT-PCR-RFLP was established to identify measles virus subgenotype H1a, subgenotype Hlb from genotype A,the results of this method were identical to those of sequence analysis.The method is simple, efficient and unique, economic and suitable for a lot of epidemiological investigations.

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