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

The Whole Genome Sequence of Enterovirus 71 Isolated in Henan Porvince in 2008

Author ZhangShaoLi
Tutor XuZuoLi
School Zhengzhou University
Course Epidemiology and Biostatistics,
Keywords HFMD EV71 Whole genome Sequence analysis
CLC R373.2
Type Master's thesis
Year 2010
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ObjectiveTo learn the epidemiological Characteristics of pathogens of hand, foot and mouth disease (HFMD) and to explore the hereditary characteristics of the whole genome sequence and sub-type of one Enterovirus 71 isolated in Henan Province in 2008.Materials and methodsSpecimens such as Serum and/or faeces (throat swab, herpes fluid, CSF) from HFMD case diagnosed by Clinic were identified by molecular biology recommended by the National CDC. Some selected samples were also inoculated rhabdomyosarcoma cells (RD cells) for virus isolation. Designed eight pairs of primers,using reverse transcription-polymerase chain reaction (RT-PCR) to extract the RNA from one EV71. Products were purified and then be bi-directional sequenced. Nucleotide was finished, stitched assembly and proofreaded by a variety of software such as Clustal (1.8) X, GeneDoc, DNAStar and MEGA3. Comparison of the nucleotide and amino acid of EV71 strains, representatives of the various genetic subtypes of strains from GenBank to construct of phylogenetic tree and homology analysis and shape strain of the strain.Results1 Test results435 specimens of clinically diagnosed cases were detected. EV71 detection rate was 16.78% while Cox.A16 was 1.38%. The difference is significant. Of the positive cases, the male and female ratio was 2.3:1 (54/23), but there is no significant difference (P>0.05).69 specimens were carried on cell culture and virus isolation, and 7 specimens appeared cytopathic effect (CPE). The isolating rate was 10.15%. EV71 accounted for 85.7%(6/7) and Cox.A16,14.3%. Using RT-PCR, isolates were tested again and the positive rate was 100%.2 Whole genome sequence analysis of one EV71 isolatesThe length of the EV71 isolates (not including the polyA tail) was 7405 bp, including 27.01% A,24.00% G,24.92% T and 24.07% C. In which, A and T are rich. From the genome 5’end, there was a 742 bases 5’non-coding region, that wasn’t the same as SHZH98 strain, BrCr strain, Zhejiang08 strain and TW2086 strain, whose was 743. The later was a coding region including 6579bp bases, encoding 2193 amino acid-containing poly-protein. After that was one 84 bp 3’non-coding region (3’UTR). Compared HENAN08 strain with other EV71 strains, there was no nucleotide deletion and insertion in the coding region, but in 5’UTR and 3’UTR districts deletion and insertion exists.Nucleotide homology:In the whole genome, including the 5’UTR area, coding region (P1, P2 and P3) and the 3’UTR area, compared HENAN08 with AnhuiFY08 strain and Zhejiang08 strain, the homology was both higher than 95%. In the P1 area, compared HENAN08 with AnhuiFY08, SHZH98, Zhejiang08 and SHZH03, the homology was all higher than 90%. With BrCr and TW2086, the homology was all higher than 80%. With Cox.A16, the homology was lower than 70%. In the 3’UTR area, the homology was the lost with BrCr and TW2086.Amino acid homology: In the entire coding region, compared with other domestic and foreign EV71 strains, there had a high homology, but the lowest homology with the Cox.A16. In the P2 and P3 areas, compared HENAN08 with the other domestic EV71 strains, the homology was higher. While compared HENAN08 with the standard strains, BrCr and TW2086, the homology was lower than the Cox.A16. In the VP1, part of the structural protein area, compared HENAN08 strain with AnhuiFY08, SHZH98, BrCr, Zhejiang08, SHZH03 and TW2086, the homology was higher than 95%, but 72.7%with the Cox.A16, the lowest one. In the VP4, another part of the structural protein area, the homology was 100%.Conclusions1 EV71 was the advantage of pathogen strains in HENAN Province in 2008.2 It’s the first time to extract all of the EV71 virus gene fragments successfully in HENAN Province. The sequence number was GU366191.3 Whether genetic kinship relations or popular time, there had a closest relationship between the HENAN08 strain and AnhuiFY08 strain. The two EV71 isolates belonged to the same genotype-C genotype C4 subtypes.

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