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Detection and Genotyping of Noroviruses in Children with Acute Gastroenteritis in Jiangmen

Author: SongCanLei
Tutor: NieJun；DaiYingChun
School: Southern Medical University,
Course: Epidemiology and Biostatistics,
Keywords: Norovirus Recombinant RdRp Capsid ORF
CLC: R440
Type: Master's thesis
Year: 2010
Downloads: 56
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Abstract

Objective:1.To study the genotypes of NoVs after sequence analysis and further determine the predominant strain of NoVs among the children with non-bacterial diarrhea in Jiang men.2. To identify the recombinant NoV strain and analyse nucleotide sequences of recombinant region and RNA secondary structures on the recombinant strain and the reference strains, investigating the possible information of the recombinant strain.Methods:1.15 days every month., fecal speciments were collected from non-bacterial dirrhea children in Jiangmen Maternity & Child Health Care Hospital and the period last for two whole years.Additionally,test conclutions were reported to the patients by communicating with telephone.2. Rotavirus were detected with Rotavirus Group A Diagnostic Kit (Colloidal Gold Device),and all specimens were detected for NoV.RNA extraction and cDNA synthesis, The fractional RdRp region and the N/S region of Capsid of NoV were respectively amplified by primer JV12Y/JV13I and typing primers GⅠSKF/GⅠSKR、COG2F/GⅡSKR using PCR, The PCR products were purified, sequenced,comparing sequences of the all PCR products in GenBank using on line server BLAST and the sequences aligned viral sequences by using Clustal W,employing MEGA 4.1 program package to construct the phylogenetic trees.3.After the Suspected recombinant NoV strains were found, the regions of NoV on norovirus genome were amplified by primers JV12Y/G I SKR or JV12Y/GⅡSKR using RT-PCR, comparing sequences of the all PCR products in GenBank using on line server BLAST and employing SimPlot3.5.1 program package to indentify the recombinant NoV strain,analysing nucleotide sequences of recombinant region and RNA secondary structures on the recombinant strain and the reference strains by BioEdit and DNAMAN.Result:1.881 fecal specimens were collected from non-bacterial diarrhea children in Jiangmen, and NoV positive rate was 13.1%(115/881).67 random sequenced strains belonged to NoV, in which 57 strains were GⅡ.4,6 strains were GⅡ.3,2 strains were GⅡ.6,2 strains were recombination strains,and the GⅡ.4 Genotype of the NoV was predominant strain.2.The NoV/Jiangmen056/2006/china strain was identified as a recombinant of RdRp region of Lordsdal strain and capsid region of Mexico strain, and NoV/Jiangmen380/2006/china strain was a recombinant of RdRp region of NLV/VannesL169/2000 strain and capsid region of SaKaeo-53 strain.3.As a result of analysing nucleotide sequences of ORF1/2 overlapping region and RNA secondary structures on the NoV/Jiangmen 056/2006/China strain, NoV/Jiangmen 380/2006/China strain and the reference strains, there were 28 highly conserved nucleotide sites and similar hairpin like structures. Conclusion1.NoVs are important etiological agent of children under five years old in Jiangmen. There were different genotypes circulated among children, NoV GⅡ-4 genetic cluster was the predominant strain.2.NoV/Jiangmen056/2006/china strain and NoV/Jiangmen380/2006/china strain were two different norovirus recombinant strains which were first found in the southen of china, and NoV/Jiangmen380/2006/china strain was a novel GⅡ.b norovirus recombinant strain,which may supply important information to study the norovirus recombination in china. The highly conserved nucleotide sequence, along with RNA secondary structure-hairpin like structure on overlapping region of ORF1/2 may play an important part in norovirus recombination.

Detection and Genotyping of Noroviruses in Children with Acute Gastroenteritis in Jiangmen

Abstract

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