XAV939, a Wnt/ß-catenin pathway modulator, has inhibitory effects on LPS-induced inflammatory response.

Aim: In this study, we report the anti-inflammatory activity of XAV939, a modulator of the Wnt/ß-catenin pathway. Methods: WNT/ß-catenin pathway and NF-κB signaling pathway were examined in LPS-stimulated human bronchial epithelial cells and effects of XAV939 on these pathways were analyzed. The effect of XAV939 was confirmed in human umbilical vein endothelial cells. Results: LPS-induced expressions of pro-inflammatory genes IL-6, IL-8, TNF-α, IL-1ß, MCP-1, MMP-9, iNOS and COX-2 were significantly and dose-dependently suppressed by XAV939. LPS-induced NF-κB signaling, such as IκB phosphorylation and degradation as well as nuclear translocation of NF-κB, was also suppressed by XAV939. Target DNA binding of NF-κB was significantly and dose-dependently suppressed by XAV939 during LPS-induced inflammatory response. The suppressive effects of XAV939 on NF-κB signaling, target DNA binding of NF-κB and pro-inflammatory gene expression were all rescued by over expression of ß-catenin, which shows that the anti-inflammatory effect of XAV939 is mediated by the modulation of ß-catenin, a central component of the WNT/ß-catenin pathway. Conclusion: The findings of this study showed that XAV939 exerts anti-inflammatory effects through the modulation of the Wnt/ß-catenin pathway.

Berberine activates AMPK to suppress proteolytic processing, nuclear translocation and target DNA binding of SREBP-1c in 3T3-L1 adipocytes.

AMP-activated protein kinase (AMPK) and sterol regulatory element binding protein (SREBP)­1c are major therapeutic targets in the treatment of metabolic diseases. In the present study, the fat­reducing mechanisms of berberine (BBR), a natural isoquinoline, was investigated by examining the AMPK­mediated modulation of SREBP­1c in 3T3­L1 adipocytes. BBR activated AMPK in a dose­ and time­dependent manner, and increased the phosphorylation of the 125­kDa precursor form of SREBP­1c, which suppressed its proteolytic processing into the mature 68­kDa form and its subsequent nuclear translocation. The binding of nuclear SREBP­1c to its E­box motif­containing target DNA sequence was decreased following treatment with BBR, which led to a decrease in the expression of lipogenic genes and subsequently reduced intracellular fat accumulation. Transfection with AMPKα1 siRNA, and not control siRNA, inhibited BBR­induced phosphorylation of the 125­kDa SREBP­1c, which confirmed that AMPK was responsible for phosphorylating SREBP­1c. AMPKα1 siRNA transfection rescued the proteolytic processing, nuclear translocation and target DNA binding of SREBP­1c that had been suppressed by BBR. In addition, BBR­induced suppression of lipogenic gene expression and intracellular fat accumulation were rescued by AMPKα1 siRNA transfection. In conclusion, the results of the present study demonstrate that BBR activates AMPK to induce phosphorylation of SREBP­1c, thereby suppressing proteolytic processing, nuclear translocation and target DNA binding of SREBP­1c, which leads to a reduction in lipogenic gene expression and intracellular fat accumulation. The results of the present study indicate that BBR may be a potential candidate for the development of drugs to treat obesity.

WNT/ß-catenin pathway modulates the TNF-α-induced inflammatory response in bronchial epithelial cells.

In this study, TNF-α was found to activate the WNT/ß-catenin pathway in BEAS-2B human bronchial epithelial cells. Levels of phospho-LRP6, Dvl-2, and phospho-GSK-3ß were elevated, while that of Axin was reduced by TNF-α treatment. Nuclear translocation of ß-catenin and the reporter activity of a ß-catenin-responsive promoter were increased by TNF-α treatment. Under the same experimental conditions, TNF-α activated the NF-κB signaling, which includes the phosphorylation and degradation of IκB and nuclear translocation and target DNA binding of NF-κB, and it was found that an inhibitor of NF-κB activation, JSH-23, inhibited TNF-α-induced Wnt signaling as well as NF-κB signaling. It was also found that recombinant Wnt proteins induced NF-κB nuclear translocations and its target DNA binding, suggesting that Wnt signaling and NF-κB signaling were inter-connected. TNF-α-induced modulations of IκB and NF-κB as well as pro-inflammatory cytokine expression were significantly suppressed by the transfection of ß-catenin siRNA compared to that of control siRNA. Transfection of a ß-catenin expression plasmid augmented the TNF-α-induced modulations of IκB and NF-κB as well as pro-inflammatory cytokine expression. These results clearly demonstrated that the WNT/ß-catenin pathway modulates the inflammatory response induced by TNF-α, suggesting that this pathway may be a useful target for the effective treatment of bronchial inflammation.

AMPK and SREBP-1c mediate the anti-adipogenic effect of ß-hydroxyisovalerylshikonin.

ß-hydroxyisovalerylshikonin (ß-HIVS), which is a natural naphthoquinone compound, is one of the main chemicals isolated from a therapeutic plant, Lithospermum erythrorhizon. In the present study, we demonstrated that ß-HIVS inhibited the adipogenesis of 3T3-L1 cells through AMP-activated protein kinase (AMPK)-mediated modulation of sterol regulatory element binding protein (SREBP)­1c. The anti-adipogenic effect of ß-HIVS was accompanied by the increased phosphorylation of AMPK and precursor SREBP­1c. In ß-HIVS-treated 3T3-L1 cells, AMPK was activated and phosphorylated precursor SREBP­1c, preventing the cleavage of precursor SREBP­1c to mature SREBP­1c. Expression of the fat-forming enzymes, acetyl-CoA carboxylase (ACC)1, fatty acid synthase (FAS) and stearoyl-CoA desaturase (SCD)1, which are transcribed by mature SREBP­1c, were downregulated, resulting in reduced intracellular fat accumulation. The anti-adipogenic effect of ß-HIVS was significantly attenuated by AMPK knockdown. Knockdown of AMPK using siRNA decreased the phosphorylation of precursor SREBP­1c and increased the levels of mature SREBP. The levels of the fat-forming enzymes, ACC1, FAS and SCD1, as well as intracellular fat accumulation were also significantly increased by AMPK knockdown. These results suggest that ß-HIVS activated AMPK, which was followed by the downregulation of mature SREBP­1c and fat-forming enzymes, leading to the inhibition of adipogenesis.

Lipoteichoic acid upregulates NF-κB and proinflammatory cytokines by modulating ß-catenin in bronchial epithelial cells.

Lipoteichoic acid (LTA) is a major cell wall component and virulence factor of gram-positive bacteria. The present study investigated the LTA­induced inflammatory response of BEAS­2B human bronchial epithelial cells, and detected the expression levels of proinflammatory cytokines interleukin (IL)­6, IL­8, IL­1ß, tumour necrosis factor­α and monocyte chemotactic protein­1, the upregulation of NF­κB, and the phosphorylation and degradation of I­κB. During the LTA­induced inflammatory response of the BEAS­2B human bronchial epithelial cells, the activity levels of the ß­catenin­dependent promoter, and the protein expression levels of ß­catenin were significantly upregulated, whereas ß­catenin phosphorylation and the expression levels of AXIN were significantly downregulated. Following knockdown of ß­catenin by small interfering (si)RNA transfection, both the LTA-induced protein expression levels of NF­κB and the LTA-induced activity levels of the NF­κB­dependent promoter were significantly reduced. Similarly, a marked reduction in I­κB phosphorylation and degradation was observed following ß­catenin knockdown. The expression levels of the LTA­induced proinflammatory cytokines were also significantly reduced following ß­catenin siRNA. These results suggest that ß­catenin has a significant role in the regulation of NF­κB activity and proinflammatory cytokine expression during the LTA-induced inflammatory response of bronchial epithelial cells.

Β-catenin regulates NF-κB activity and inflammatory cytokine expression in bronchial epithelial cells treated with lipopolysaccharide.

In the present study, we demonstrate that lipopolysaccharide (LPS) induces the expression of inflammatory cytokines, including interleukin (IL)-6, IL-8, IL-1ß, tumor necrosis factor (TNF)-α and monocyte chemoattractant protein (MCP)-1 in BEAS-2B human bronchial epithelial cells in a dose- and time-dependent manner. This increase was accompanied by an increased activity of nuclear factor (NF)­κB. When the expression of ß-catenin was analyzed following treatment with LPS, the mRNA level was unaltered; however, the ß-catenin protein levels increased with a decrease in phosphorylation at the serine 33/37 residues. Nuclear ß-catenin protein levels also increased along with the reporter activity of a ß-catenin-responsive TOPFlash vector. To elucidate the regulatory role of ß-catenin in the LPS-induced inflammatory response of bronchial epithelial cells, ß-catenin production was knocked down using siRNA. Our results revealed that ß-catenin protein levels and TOPFlash vector reporter activity were reduced to basal levels by siRNA transfection. In this experimental condition, NF-κB activity, measured by enzyme-linked immunosorbent assay (ELISA), electrophoretic mobility shift assay (EMSA) and an NF-κB responsive reporter assay, was reduced to basal levels. Similarly, LPS-induced inflammatory cytokine expression was reduced almost to basal levels following transfection with ß-catenin siRNA. These results demonstrate that ß-catenin positively regulates NF-κB activity, as well as the expression of inflammatory cytokines in the inflammatory response of LPS-treated bronchial epithelial cells.

ß-catenin mediates the inflammatory cytokine expression induced by the Der p 1 house dust mite allergen.

The modulations of ß-catenin were analyzed during the inflammatory response induced by the Der p 1 house dust mite allergen. Der p 1 induced the dose-dependent expression of inflammatory cytokines, including interleukin (IL)-6, IL-8, monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α) in THP-1 human monocytic cells. The mRNA expression levels of ß-catenin were not altered, however protein levels increased following Der p 1 treatment, demonstrating that ß-catenin was modulated by post-transcriptional processes. It was also revealed that nuclear ß-catenin levels were significantly increased while cytoplasmic ß-catenin levels were reduced, which demonstrated the nuclear translocation of ß-catenin by the Der p 1 allergen. Glycogen synthase kinase 3ß (GSK3ß), a regulator of ß-catenin stability, was demonstrated to be phosphorylated following Der p 1 treatment. When ß-catenin was knocked down by the transfection of its small interfering RNA (siRNA), inflammatory cytokine expression as well as nuclear factor-κB (NF-κB) activity, which were induced by Der p 1 treatment, were all significantly reduced. The results demonstrated that Der p 1-induced inflammatory responses were mediated by ß-catenin.

Distribution of rotavirus G and P genotypes approximately two years following the introduction of rotavirus vaccines in South Korea.

Genotyping of human rotaviruses was performed on 299 (40.1%) rotavirus-positive samples obtained from 745 children with acute diarrhea in three provinces in South Korea between March 2008 and February 2010, approximately 2 years following the introduction of the RotaTeq (September 2007) and Rotarix (July 2008). The most prevalent G genotypes were G1 (51.5%), followed by G3 (24.0%), G4 (15.4%), G9 (6.4%), and G2 (4.7%). The predominant types of P genotypes were P[8] (72.6%), followed by P[6] (19.1%) and P[4] (6.0%). The phylogenetic analyses of the VP7 genes of G9 strains revealed they were highly identical and belonged in lineage III. This study highlights the consistency of the predominant G1 genotype and slightly higher predominance of the identical G9 strains over the G2 genotype.

Beta-catenin promoter polymorphism is associated with asthma risk in Korean subjects.

OBJECTIVES: The effects of ß-catenin promoter haplotypes on its mRNA expression levels and asthma risks were investigated in Korean subjects. DESIGN AND METHODS: The genotype analyses were conducted by a Taqman method for 684 Korean subjects, 400 controls and 284 with asthma. Measurement of mRNA expression levels in peripheral blood nucleated cells were conducted on subjects whose buffy coat fractions were available (n=185). Logistic regression analyses were conducted to test the associations of the ß-catenin promoter haplotypes with asthma risks. RESULTS: Four SNPs, -10,288C>T (rs7630377), -6,426C>G (rs9859392), -4,361G>C (rs9870255), and -765G>A (rs3864004), were identified in the promoter region of the ß-catenin gene, and three common haplotypes were constructed from them. Haplotype ht1[CCGG] was associated with decreased ß-catenin mRNA expression levels and a lower asthma risk with an odds ratio of 0.53, while ht2[TGCA] was associated with increased mRNA expression levels and a higher asthma risk with an odds ratio of 2.34. Ht3[TCGG] had no significant effects on both. CONCLUSIONS: Our findings show that ß-catenin promoter polymorphism affects its mRNA expression levels, and also is significantly associated with the asthma risk of Korean subjects.

Genomic characterization of a cell-culture-adapted Korean human G9P[8] rotavirus, CAU05-202.

The human rotavirus G9 strain is the fifth most common rotavirus worldwide. A human rotavirus G9P[8] strain CAU05-202 was isolated from a young child with diarrhea using a cell culture system, and its major gene sequences were determined. Phylogenetic analysis of the VP7 gene revealed that CAU05-202 clustered into genetic lineage III-d and was most closely related to G9 rotaviruses from Turkey (strain GUH13) and Sri Lanka (strain 05SLC056 and 05SLC057). VP4 and NSP4 gene analysis showed that CAU05-202 belongs to the P[8]-3 lineage and genotype B, respectively. In addition, CAU05-202 has a long RNA electropherotype, supported by VP6 gene analysis, which is clearly associated with subgroup II specificity. Analysis of the G9 rotavirus strain CAU05-202 provides information concerning the genetic relationships among global rotavirus G9 strains, suggesting that closely related G9 strains are persistent and widespread in Asian countries.

Molecular characterization of rare G12P[6] rotavirus isolates closely related to G12 strains from the United States, CAU 195 and CAU 214.

Two human G12 rotaviruses, CAU 195 and CAU 214, were isolated from South Korea using cell culture and characterized on the basis of sequence divergence in the VP7, VP4, and NSP4 genes. Phylogenetic analysis of the VP7 gene sequences indicated that these strains clustered into lineage III and were most closely related to G12 rotaviruses isolated in the United States. The VP4 and NSP4 gene sequences showed that two strains belonged to the P[6]-Ia lineage and genotype [B]. This finding provides information that can be used to evaluate G12 strains and aid in the development of effective vaccines in the future.

Shikonin inhibits adipogenesis by modulation of the WNT/ß-catenin pathway.

AIM: Our previous study showed for the first time that shikonin, a natural compound isolated from Lithospermun erythrorhizon Sieb. Et Zucc, inhibits adipogenesis and fat accumulation. This study was conducted to investigate the molecular mechanism of the anti-adipogenic effects of shikonin. MAIN METHODS: Gene knockdown experiments using small interfering RNA (siRNA) transfection were conducted to elucidate the crucial role of ß-catenin in the anti-adipogenic effects of shikonin. KEY FINDINGS: Shikonin prevented the down-regulation of ß-catenin and increased the level of its transcriptional product, cyclin D1, during adipogenesis of 3T3-L1 cells, preadipocytes originally derived from mouse embryo. ß-catenin was a crucial mediator of the anti-adipogenic effects of shikonin, as determined by siRNA-mediated knockdown. Shikonin-induced reductions of the major transcription factors of adipogenesis including peroxisome proliferator-activated receptor γ and CCAAT/enhancer binding protein α, and lipid metabolizing enzymes including fatty acid binding protein 4 and lipoprotein lipase, as well as intracellular fat accumulation, were all significantly recovered by siRNA-mediated knockdown of ß-catenin. Among the genes located in the WNT/ß-catenin pathway, the levels of WNT10B and DVL2 were significantly up-regulated, whereas the level of AXIN was down-regulated by shikonin treatment. SIGNIFICANCE: This study clearly shows that shikonin inhibits adipogenesis by the modulation of WNT/ß-catenin pathway in vitro, and also suggests that WNT/ß-catenin pathway can be used as a therapeutic target for obesity and related diseases using a natural compound like shikonin, even though the in vivo effects of shikonin and its clinical significance remain to be elucidated.

Beta-Catenin mediates the anti-adipogenic effect of baicalin.

beta-Catenin reportedly inhibits adipogenesis through the down-regulations of peroxisome proliferator-activated receptor (PPAR)gamma and CCAAT/enhancer binding protein (C/EBP)alpha. We report that baicalin, a natural flavonoid compound, inhibits adipogenesis by modulating beta-Catenin. During 3T3-L1 cell adipogenesis, beta-Catenin was down-regulated, but baicalin treatment maintained beta-Catenin expression. Anti-adipogenic effects of baicalin were significantly attenuated by beta-Catenin siRNA transfection. beta-Catenin siRNA rescued the reduced expressions of PPARgamma, C/EBPalpha, fatty acid binding protein 4 and lipoprotein lipase by baicalin. Furthermore, baicalin modulated members of the WNT/beta-Catenin pathway by maintaining the expressions of low-density lipoprotein receptor-related protein 6, disheveled (DVL)2 and DVL3. These findings suggest that beta-Catenin mediates the anti-adipogenic effects of baicalin.

Development and application of multiprobe real-time PCR method targeting the hsp65 gene for differentiation of Mycobacterium species from isolates and sputum specimens.

We developed a multiprobe real-time PCR assay targeting hsp65 (HMPRT-PCR) to detect and identify mycobacterial isolates and isolates directly from sputum specimens. Primers and probes for HMPRT-PCR were designed on the basis of the hsp65 gene sequence, enabling the recognition of seven pathogenic mycobacteria, including Mycobacterium tuberculosis, M. avium, M. intracellulare, M. kansasii, M. abscessus, M. massiliense, and M. fortuitum. This technique was applied to 24 reference and 133 clinical isolates and differentiated between all strains with 100% sensitivity and specificity. Furthermore, this method was applied to sputum specimens from 117 consecutive smear-positive patients with smear results of from a trace to 3+. These results were then compared to those obtained using the rpoB PCR-restriction analysis method with samples from cultures of the same sputum specimens. The HMPRT-PCR method correctly identified the mycobacteria in 89 samples (76.0%, 89/117), and moreover, the sensitivity level was increased to 94.3% (50/53) for sputa with an acid-fast bacillus score equal to or greater than 2+. Our data suggest that this novel HMPRT-PCR method could be a promising approach for detecting pathogenic mycobacterial species from sputum samples and culture isolates routinely in a clinical setting.

Genetic variation of prevalent G1P[8] human rotaviruses in South Korea.

The human rotavirus G1P[8] strain is one of the most common rotaviruses worldwide, including Korea. Six Korean G1P[8] human rotaviruses, isolated using cell culture techniques, were characterized on the basis of sequence differences in VP7, VP4, VP6, and NSP4 genes to elucidate the evolutionary relationships in the community. All strains had a long RNA electropherotype, supported by VP6 gene analysis, clearly associated with subgroup II specificity. The phylogenetic analysis of VP7 gene sequences showed that they all clustered into lineage I, as reported for G1 strains in Japan, China, Vietnam, and Thailand. In addition, phylogenetic analysis of the VP4 gene showed that they belong to two distinct lineages, P[8]-II and P[8]-III. With respect to the NSP4 gene, all strains belonged to genotype B. An understanding of the ecology and molecular evolution of rotaviruses circulating in the country is very important for the development of vaccines and vaccination strategies. This study provides new information concerning the genetic variability of the rotavirus strain G1P[8] occurring most commonly as a vaccine candidate.

A western Eurasian male is found in 2000-year-old elite Xiongnu cemetery in Northeast Mongolia.

We analyzed mitochondrial DNA (mtDNA), Y-chromosome single nucleotide polymorphisms (Y-SNP), and autosomal short tandem repeats (STR) of three skeletons found in a 2,000-year-old Xiongnu elite cemetery in Duurlig Nars of Northeast Mongolia. This study is one of the first reports of the detailed genetic analysis of ancient human remains using the three types of genetic markers. The DNA analyses revealed that one subject was an ancient male skeleton with maternal U2e1 and paternal R1a1 haplogroups. This is the first genetic evidence that a male of distinctive Indo-European lineages (R1a1) was present in the Xiongnu of Mongolia. This might indicate an Indo-European migration into Northeast Asia 2,000 years ago. Other specimens are a female with mtDNA haplogroup D4 and a male with Y-SNP haplogroup C3 and mtDNA haplogroup D4. Those haplogroups are common in Northeast Asia. There was no close kinship among them. The genetic evidence of U2e1 and R1a1 may help to clarify the migration patterns of Indo-Europeans and ancient East-West contacts of the Xiongnu Empire. Artifacts in the tombs suggested that the Xiongnu had a system of the social stratification. The West Eurasian male might show the racial tolerance of the Xiongnu Empire and some insight into the Xiongnu society.

Platycodin D inhibits adipogenesis of 3T3-L1 cells by modulating Kruppel-like factor 2 and peroxisome proliferator-activated receptor gamma.

In this study, platycodin D was found to inhibit intracellular triglyceride accumulation in 3T3-L1 cells with an IC(50) of 7.1 microM. The expression levels of genes involved in lipid metabolism such as fatty-acid-binding protein 4 and lipoprotein lipase were significantly downregulated following treatment with platycodin D. Treatment with platycodin D also resulted in a reduction of Peroxisome proliferator-activated receptor(PPAR)gamma expression and its binding to target DNA sequence. Among the various upstream regulators of PPARgamma, the expression of Kruppel-like factor(KLF)2, an anti-adipogenic factor, was significantly upregulated following platycodin D treatment. When the upregulation of KLF2 was inhibited by KLF2 siRNA, the expression and binding of PPARgamma to its target sequence were significantly recovered under these conditions. The results of this study suggested that anti-adipogenic effect of platycodin D involves the upregulation of KLF2 and subsequent downregulation of PPARgamma.

Antiobesity effect of baicalin involves the modulations of proadipogenic and antiadipogenic regulators of the adipogenesis pathway.

In this study, the antiobesity effects of baicalin, 5,6-dihydroxyflavone-7-glucuronic acid, were characterized using an in vitro system of adipogenesis, i.e. fat cell formation. Baicalin-treatment of 3T3-L1 preadipocytes was shown to inhibit triglyceride accumulation and lipid droplet formation during induced adipogenesis. Microarray analyses showed that baicalin modulated the expression of genes located in pathways such as adipogenesis, cholesterol biosynthesis, focal adhesion and others. In the adipogenesis pathway, treatment with baicalin significantly down-regulated terminal differentiation markers of adipocytes including fatty acid binding protein 4. The effects of baicalin on the core part of the adipogenesis pathway, however, were paradoxical; the expression levels of CCAAT/enhancer binding protein (C/EBP)beta and C/EBPdelta were up-regulated, while the expression levels of the peroxisome proliferator-activated receptor (PPAR)gamma and C/EBPalpha were down-regulated. The antiadipogenic mechanisms of baicalin can be explained by its effects on the upstream part of adipogenesis pathway; baicalin not only up-regulates the antiadipogenic regulators, C/EBPgamma, C/EBP homologous protein and Kruppel-like factor (KLF)2, but also down-regulates the proadipogenic regulator, KLF15. The overall effects of baicalin on these upstream regulators of adipogenesis were antiadipogenic, resulting in the down-regulation of downstream genes and the inhibition of cellular fat accumulation.

A UCP1-412A>C polymorphism is associated with abdominal fat area in Korean women.

We used genomic sequencing of Korean subjects to identify the uncoupling protein-1 (UCP1) polymorphism -412A>C (rs3811787 in the dbSNP database). This study is the first to associate this polymorphism with a human phenotype. The frequency of the major A allele was 0.53 and that of the minor C allele was 0.47. The -412A>C polymorphism was not linked to the well-known -3826A>G (rs1800592; mid R:D'mid R:=0.60 and r(2)=0.33), yielding four predicted haplotypes from these two polymorphisms. We associated -412A>C, -3826A>G and their haplotypes with computed tomography-measured body fat areas from 367 Korean female subjects. The G allele of -3826A>G and the C allele of -412A>C were significantly associated with larger areas of abdominal subcutaneous fat in a dominant model (p=0.001 and p=0.0004, respectively); combining them together (ht2[GC]) enhanced this significance (p=0.00005). In contrast, presence of the A allele in both polymorphisms (ht1[AA]) was significantly associated with smaller areas of abdominal subcutaneous fat (p=0.003). We observed no significant associations between these UCP1 genetic polymorphisms and thigh fat areas, visceral fat areas, or blood biochemical profiles, suggesting that this polymorphism might differentially affect fat accumulation in different parts of the human body, even though further study is needed to elucidate the mechanism of it.

Detection of unusual rotavirus genotypes G8P[8] and G12P[6] in South Korea.

Five hundred four fecal specimens, collected between 2004 and 2006 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.