Luận Án Ph.D. degree của Ts Lê văn Phan với đầu đề:

Molecular Characterization of Human Group A Rotavirus Infections. Expression of Human Rotavirus Genes and Rotavirus like Particle Production
Microbiology Major
Department of Medical Science, Graduate School, Chung-Ang University
December 2008

ABSTRACT
Rotaviruses are the most common etiological agent of severe diarrhea in infants and young children and are responsible, worldwide, for an estimated 454,000–705,000 death annually. In this study, five hundred four fecal specimens, collected between 2004 and 2006 in Seoul, South Korea from young children with acute diarrhea, were screened for rotavirus by ELISA with VP6-specific antibody. Of these samples, 394 (78.2%) were confirmed as group A rotavirus and they underwent G- and P typing using a combination of ELISA, RT-PCR, and sequence analysis methods. The dominant circulating G serotype was G1 (35.6%) followed by G3 (26.4%), G4 (14.7%), and G2 (11.9%). There was a low prevalence of G9 (1.0%) and of unusual G type rotavirus, in particular, G12 (0.5%) and G8 (0.3%). Of the P genotype rotavirus in circulation, P[8] (53.0%) was most common followed by P[6] (15.5%), P[4] (15.2%), and P[9] (2.3%). Determination of G- and P type combinations revealed that G1P[8] strains were most prevalent (25.4%), amid G3P[8] (16.8%), G2P[4] (6.3%), and G4P[6] (6.1%) strains.
Unusual or rare combinations such as G2P[6], G2P[8], G3P[4], G2P[9], G1P[9], G3P[9], G12P[6], G1P[4], G3P[6], and G8P[8] were also found. Owing to the recent emergence of G8 and G12 rotavirus, the findings from this study are important since they provide new information concerning the local and global spread of rotavirus genotypes.

Previous epidemiological studies reported that G1 to G4 serotypes were common, particularly G1 as the predominant strain during the past years. Recently, increasing numbers of studies have documented that G9 and G12 strains may represent the new epidemiologically important G serotypes. Several reports have revealed considerable genetic diversity within the VP7 gene of predominant G1 strains and G9 strains as well. In this work Korean G1P[8], G9P[8], and unusual G12P[6] human rotavirus strains have also been isolated and characterized for VP7, VP4, VP6, and NSP4 gene.
For G1P[8] human rotavirus strains, all strains showed a “long” RNA pattern and VP6 subgroup II specificity. The phylogenetic analysis of VP7 gene sequences showed that they all clustered into lineage I, the same as reported G1 strains in Japan, China, Vietnam, and Thailand except that one old Korean strain (Kor-64) belonged to lineage IV. Also, phylogenetic analysis of VP4 genes revealed that they were of P[8] genotype with two distinct lineages (P[8]-3 and P[8]-2). With respect to the NSP4 gene, all G1 strains fell into the genotype B. For G9P[8] human rotavirus strain (CAU 202), CAU 202 strain showed a “long” RNA pattern and VP6 subgroup II specificity. The phylogenetic analysis of VP7 gene sequences showed that CAU 202 and other Korean G9 strains deposited in GenBank databases clustered into genetic lineage III. Also, phylogenetic analysis of VP4 genes revealed that CAU 202 was of P[8] genotype with lineages P[8]-3. With respect to the NSP4 gene, CAU 202 fell into the genotype B.
Also for G12 human rotavirus strains, CAU 195 and CAU 214 were isolated and characterized. Both CAU 195 and CAU 214 strain showed a “long” RNA pattern and subgroup II specificity. VP7 gene of CAU 195 and CAU 214 strains exhibited 89.8% to 99.7% at the nucleotide similarities and 92.3% to 100% at the amino acid identities respectively, to those of rotaviruses with G12 specificity in GenBank. VP4 gene sequences of CAU 195 and CAU 214 showed the highest identity with P[6] specificity to porcine strain Gottfried with homologies of 83.2 to 83.3% at the nucleotide and 89.5 to 89.7% at the amino acid levels. In addition, the NSP4 gene of these strains were most closely related to the human and porcine branch within the Wa lineage, and revealed that they belong to genotype B. Finally, four structural proteins of VP2, VP4, VP6, and VP7 of human rotavirus have been cloned and expressed successfully using baculovirus expression system, respectively. The double-layered rotavirus like particles (2/6 VLPs) and triple-layered rotavirus like particles (2/4/6/7 VLPs) were then generated and characterized, respectively.

Keywords: Human rotavirus; Phylogenetic analysis; Virus like particles.

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