RESUMEN
Androgen plays an important role in the pathogenesis of PCa (prostate cancer). Previously, we identified GNMT (glycine N-methyltransferase) as a tumour susceptibility gene and characterized its promoter region. Besides, its enzymatic product-sarcosine has been recognized as a marker for prognosis of PCa. The goals of this study were to determine whether GNMT is regulated by androgen and to map its AREs (androgen response elements). Real-time PCR analyses showed that R1881, a synthetic AR (androgen receptor) agonist induced GNMT expression in AR-positive LNCaP cells, but not in AR-negative DU145 cells. In silico prediction showed that there are four putative AREs in GNMT-ARE1, ARE2 and ARE3 are located in the intron 1 and ARE4 is in the intron 2. Consensus ARE motif deduced from published AREs was used to identify the fifth ARE-ARE5 in the coding region of exon 1. Luciferase reporter assay found that only ARE5 mediated the transcriptional activation of R1881. ARE3 overlaps with a YY1 [Yin and Yang 1 (motif (CaCCATGTT, +1118/+1126)] that was further confirmed by antibody supershift and ChIP (chromatin immunoprecipitation) assays. EMSA (electrophoretic mobility shift assay) and ChIP assay confirmed that AR interacts with ARE5 in vitro and in vivo. In summary, GNMT is an AR-targeted gene with its functional ARE located at +19/+33 of the first exon. These results are valuable for the study of the influence of androgen on the gene expression of GNMT especially in the pathogenesis of cancer.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Glicina N-Metiltransferasa/genética , Elementos de Respuesta , Antagonistas de Receptores Androgénicos/farmacología , Anilidas/farmacología , Secuencia de Bases , Sitios de Unión , Línea Celular Tumoral , Exones , Humanos , Masculino , Metribolona/farmacología , Nitrilos/farmacología , Sistemas de Lectura Abierta , Neoplasias de la Próstata , Unión Proteica , Receptores Androgénicos/metabolismo , Análisis de Secuencia de ADN , Congéneres de la Testosterona/farmacología , Compuestos de Tosilo/farmacología , Transcripción GenéticaRESUMEN
BACKGROUND: In 2004, HIV-1 infection among Taiwanese injection drug users (IDUs) started to surge. In 2007, a resurgence of HIV-1 epidemic among men having sex with men (MSM) occurred. We conducted a molecular epidemiological study of HIV-1 among different risk groups in Taiwan from 2005 to 2008. METHODS: In total 1133 HIV-1-infected adults including 576 IDUs, 464 MSM, and 93 heterosexuals were recruited. HIV-1 subtypes were determined using nested multiplex polymerase chain reaction and phylogenetic analysis. Dual infection was confirmed using cloning, sequencing, and heteroduplex mobility assay. RESULTS: Among HIV-1/AIDS subjects, 96.1% MSM and 62.5% heterosexual males were infected with subtype B, whereas 66.7% female heterosexuals were infected with CRF01_AE. Most IDUs (84.5%) were infected with CRF07_BC. Four heterosexual males, 2 females and 2 MSM who were not IDUs had CRF07_BC. Forty-nine patients had CRF07_BC/subtype B dual infection and 44 (89.8%) were IDUs. Multivariate logistic regression showed that the odds ratio for dual infection among IDUs who shared syringes >5 times per month was 4.7 (95% confidence interval = 1.3 to 17.7). Phylogenetic analyses revealed that there were 2 main groups of CRF07_BC strains with sporadic transmission between different risk groups. Among 10 IDUs infected with CRF01_AE, 7 cases were clustered with an outbreak happened in 2005 and 3 cases were clustered with other strains from heterosexual population. CONCLUSIONS: In Taiwan, 7.8% of HIV-1-infected IDUs had dual infection. It may have important impact to their clinical management. Although CRF07_BC was still remained in IDUs, it has spread to MSM and heterosexual populations.
Asunto(s)
Infecciones por VIH/epidemiología , Infecciones por VIH/virología , VIH-1/genética , Adulto , ADN Viral/análisis , Brotes de Enfermedades , Femenino , VIH-1/clasificación , VIH-1/aislamiento & purificación , Homosexualidad Masculina , Humanos , Masculino , Persona de Mediana Edad , Epidemiología Molecular , Datos de Secuencia Molecular , Análisis Multivariante , Abuso de Sustancias por Vía Intravenosa , Taiwán/epidemiologíaRESUMEN
Glycine N-methyltransferase (GNMT) is a tumor suppressor for hepatocellular carcinoma (HCC). High rates of Gnmt knockout mice developed HCC. Epigenetic alteration and dysregulation of several pathways including wingless-type MMTV integration site (Wnt), mitogen-activated protein kinase (MAPK) and Janus kinase and signal transducer and activator of transcription (JAK-STAT) are associated with HCC development in Gnmt knockout mice. We hypothesized that GNMT may regulate signal transduction through interacting with other proteins directly. In this report, we identified a mammalian target of rapamycin (mTOR) inhibitor (DEP domain containing MTOR-interacting protein [DEPDC6/DEPTOR]) as a GNMT-binding protein by using yeast two-hybrid screening. Fluorescence resonance energy transfer assay demonstrated that the C-terminal half of GNMT interact with the PSD-95/Dlg1/ZO-1 (PDZ) domain of DEPDC6/DEPTOR. Immunohistochemical staining showed that 27.5% (14/51) of HCC patients had higher expression levels of DEPDC6/DEPTOR in the tumorous tissues than in tumor-adjacent tissues, especially among HCC patients with hepatitis B viral infection (odds ratio 10.3, 95% confidence interval [CI] 1.05-11.3) or patients with poor prognosis (death hazard ratio 4.51, 95% CI 1.60-12.7). In terms of molecular mechanism, knockdown of DEPDC6/DEPTOR expression in HuH-7 cells caused S6K and 4E-BP activation, but suppressed Akt. Overexpression of DEPDC6/DEPTOR activated Akt and increased survival of HCC cells. Overexpression of GNMT caused activation of mTOR/raptor downstream signaling and delayed G2/M cell cycle progression, which altogether resulted in cellular senescence. Furthermore, GNMT reduced proliferation of HuH-7 cells and sensitized them to rapamycin treatment both in vitro and in vivo. In conclusion, GNMT regulates HCC growth in part through interacting with DEPDC6/DEPTOR and modulating mTOR/raptor signaling pathway. Both GNMT and DEPDC6/DEPTOR are potential targets for developing therapeutics for HCC.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Glicina N-Metiltransferasa/metabolismo , Neoplasias Hepáticas/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Adulto , Anciano , Animales , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Everolimus , Femenino , Células HEK293 , Hepatitis B/complicaciones , Hepatitis B/metabolismo , Hepatitis C/complicaciones , Hepatitis C/metabolismo , Humanos , Inmunosupresores/uso terapéutico , Péptidos y Proteínas de Señalización Intracelular , Masculino , Ratones , Ratones SCID , Persona de Mediana Edad , Sirolimus/análogos & derivados , Sirolimus/uso terapéutico , Técnicas del Sistema de Dos Híbridos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND/PURPOSE: In the past few years, many new subtypes in hepatitis C virus (HCV) genotype 6 have been identified. The aim of this study was to modify the multiplex real-time polymerase chain reaction (RT-PCR) protocol and use it to determine the HCV subtypes of a group of Taiwanese injection drug users (IDUs). METHODS: We used 76 serum specimens collected in northern Taiwan in 2008. Multiplex RT-PCR was used for HCV subtyping among those serum samples having anti-HCV antibodies. Twenty cases were randomly selected for comparison with subtyping results from Inno-LiPa II tests and phylogenetic tree analysis using NS5B sequences. RESULTS: Multiplex RT-PCR assays showed that 60.5% (46/76) of IDUs had single HCV infection. Three out of 76 (3.9%) had double HCV infection (1b/6a, 2a/2b and 2b/6a). Besides this, 27.6% (21/76) had no HCV signal. One IDU had subtype 6n and two had subtype 6w infection. Inno-LiPa II tests misclassified all 6n and 6w cases as 1b subtype. CONCLUSION: Our modified multiplex RT-PCR protocol can be used to support molecular epidemiological studies and laboratory diagnoses of different HCV subtypes including genotype 6.
Asunto(s)
Hepacivirus/clasificación , Hepatitis C/diagnóstico , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Técnicas de Genotipaje , Hepacivirus/genética , Humanos , FilogeniaRESUMEN
In both humans and rodents, males are known to be more susceptible than females to hepatocarcinogenesis. We have previously reported that glycine N-methyltransferase (GNMT) interacts with aflatoxin B(1) (AFB(1)) and reduces both AFB(1)-DNA adduct formation and hepatocellular carcinoma (HCC) in mice. We also reported that 50% of the males and 100% of the females in a small group of Gnmt null (Gnmt-/-) mice developed HCC, with first dysplastic hepatocellular nodules detected at mean ages of 17 and 16.5 months, respectively. In our study, we tested our hypothesis that male and female Gnmt-/- mice are susceptible to AFB(1) carcinogenesis, and that the absence of Gnmt expression may accelerate AFB(1)-induced liver tumorigenesis. We inoculated Gnmt-/- and wild-type mice intraperitoneally with AFB(1) at 7 days and 9 weeks of age and periodically examined them using ultrasound. Dysplastic hepatocellular nodules were detected in six of eight males and five of five females at 12.7 and 12 months of ages, respectively. Dysplastic hepatocellular nodules from 5/8 (62.5%) male and 4/5 (80%) female Gnmt-/- mice were diagnosed as having HCC, â¼6 months earlier than AFB(1)-treated wild-type mice. Results from microarray and real-time PCR analyses indicate that five detoxification pathway-related genes were downregulated in AFB(1)-treated Gnmt-/- mice: Cyp1a2, Cyp3a44, Cyp2d22, Gsta4 and Abca8a. In summary, we observed overall higher susceptibility to AFB(1)-related HCC in Gnmt-/- mice, further evidence that GNMT overexpression is an important contributing factor to liver cancer resistance.
Asunto(s)
Aflatoxina B1/toxicidad , Carcinoma Hepatocelular/inducido químicamente , Carcinoma Hepatocelular/genética , Glicina N-Metiltransferasa/deficiencia , Transportadoras de Casetes de Unión a ATP/genética , Animales , Autopsia , Carcinoma Hepatocelular/enzimología , Cruzamientos Genéticos , Citocromo P-450 CYP1A2/deficiencia , Citocromo P-450 CYP1A2/genética , Sistema Enzimático del Citocromo P-450/deficiencia , Sistema Enzimático del Citocromo P-450/genética , Familia 2 del Citocromo P450 , Cartilla de ADN , Femenino , Tamización de Portadores Genéticos , Predisposición Genética a la Enfermedad , Glutatión Transferasa/deficiencia , Glutatión Transferasa/genética , Hígado/patología , Masculino , Ratones , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa/métodos , Transcripción GenéticaRESUMEN
Human immunodeficiency virus type 1 (HIV-1) circulating recombinant form (CRF) 07_BC strain has caused serious outbreaks among injection drug users in Taiwan since 2004. The objective of this study was to conduct a molecular epidemiological study of HCV genotypes in intravenous drug users in Taiwan. Blood samples and questionnaires from 591 intravenous drug users infected with HIV-1 were collected nationwide. In total, 180 samples were selected for HCV genotyping using multiplex PCR and phylogenetic analysis of the core, E1 and NS5B regions. The Inno-Lipa assay was used to confirm multiple infections with different genotypes. Eighty percent had a single infection with subtype 1b being the most common subtype (24%), 12% had double infections and two had triple infections. In addition, three recombinant forms (RFs)-2a1a, 3a1b, and 2b6w were identified. Phylogenetic analyses showed that the 3a, 6a, and 6n strains were clustered with strains present in Thailand and mainland China. Full-length sequence analysis showed that two 6w strains shared 89.4-90.2% sequence homology with the 6(r) strain from the Guangdong Province, China. Bootscan analysis revealed that the recombination breakpoint of RF_2b6w was located at the NS2-NS3 junction. In summary, the distribution of HCV genotypes among Taiwanese intravenous drug users was complex and more than 12% of the drug users were infected with more than one genotype of HCV.
Asunto(s)
Hepacivirus/clasificación , Hepacivirus/genética , Genotipo , Infecciones por VIH/complicaciones , Infecciones por VIH/epidemiología , Infecciones por VIH/virología , VIH-1 , Hepacivirus/aislamiento & purificación , Hepatitis C/complicaciones , Hepatitis C/epidemiología , Hepatitis C/virología , Humanos , Epidemiología Molecular , Hibridación de Ácido Nucleico/métodos , Filogenia , Reacción en Cadena de la Polimerasa , Recombinación Genética , Abuso de Sustancias por Vía Intravenosa/complicaciones , Abuso de Sustancias por Vía Intravenosa/epidemiología , Encuestas y Cuestionarios , Taiwán/epidemiologíaRESUMEN
BACKGROUND: From October 2006 to February 2007, clinical specimens from 452 patients with symptoms related to respiratory tract infection in the northern region of Taiwan were collected. Real-time PCR and direct immunofluorescent antibody tests showed that 145 (32%) patients had influenza B virus infections. Subsequently, nucleotide sequence analyses of both hemagglutinin (HA) and neuraminidase (NA) genes of 39 isolates were performed. Isolated viruses were antigenically characterized using hemagglutinin inhibition (HI) test. FINDINGS: Phylogenetic tree analysis showed that all the isolates belonged to the B reassortant lineage with HA gene belonged to the B/Victoria/2/87 lineage and the NA gene belonged to the B/Yamagata/16/88 lineage. In addition, a group of children aged between 6 to 8 years old resided in Yilan county were infected with a variant strain. Hemagglutinin inhibition (HI) tests confirmed that all the reassortant influenza B viruses were B/Malaysia/2506/04-like viruses. Pre- and post-immunized serum samples from 4 normal volunteers inoculated with 2007 influenza vaccine were evaluated for their HI activity on 6 reassortant B isolates including two variants that we found in the Yilan county. The results demonstrated that after vaccination, all four vaccinees had at least 4-fold increases of their HI titers. CONCLUSION: The results indicate that the 2006-2007 seasonal influenza vaccine was effective in stimulating protective immunity against the influenza B variants identified in Yilan county. Continuous surveillance of emerging influenza B variants in the northern region of Taiwan is important for the selection of proper vaccine candidate in the future.
RESUMEN
Glycine N-methyltransferase (GNMT) is a tumor susceptibility gene for both hepatocellular carcinoma and prostate cancer. We have previously characterized GNMT genomic structure and mapped its chromosomal localization to 6p12. For this study we identified a GNMT transcriptional start site at the 14th position upstream of the ATG codon. Electrophoretic mobility shift assay results indicate binding of the nuclear factor-Y (NF-Y) transcription factor to the CCAAT box (-71/-67) of the GNMT gene. Mutation assay results suggest that the nucleotide sequence in the -56/-47 region is a binding site for a putative transcriptional factor. The TATA-less core promoter (-133/+14) contains three major elements: an Sp1 site, CCAAT box, and a novel box within the CTGTCGGCTG sequence. One functional xenobiotic response element (XRE) located at the -104/-82 region is inducable by benzo[a]pyrene treatment. We believe our results have value for the study of GNMT transcriptional regulation.
Asunto(s)
Glicina N-Metiltransferasa/genética , Secuencias Reguladoras de Ácidos Nucleicos , Secuencia de Bases , Factor de Unión a CCAAT/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Humanos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Sitio de Iniciación de la TranscripciónRESUMEN
Hepatocellular carcinoma (HCC) is the fifth common cancer in the world and it mainly occurs in men. Glycine N-methyltransferase (GNMT) participates in one-carbon metabolism and affects DNA methylation by regulating the ratio of S-adenosylmethionine to S-adenosylhomocystine. Previously, we described that the expression of GNMT was diminished in human HCC. Here, we showed that 50% (3/6) male and 100% (7/7) female Gnmt-/- mice developed HCC, and their mean ages of HCC development were 17 and 16.5 months, respectively. In addition, 42.9% (3/7) of female Gnmt-/- mice had hemangioma. Wnt reporter assay demonstrated that Gnmt is a negative regulator for canonical Wnt signaling pathway. Beta-catenin, cyclin D1 and c-Myc, genes related to Wnt pathway, were upregulated in the liver tissues from both 11 weeks and HCC stage of Gnmt-/- mice. Furthermore, global DNA hypomethylation and aberrant expression of DNA methyltransferases 1 and 3b were found in the early and late stages of HCC development. Hierarchical cluster analysis of 6,023 transcripts from microarray data found that gene expression patterns of HCC tumors from male and female Gnmt-/- mice were distinctively different. Real-time PCR confirmed that Gadd45a, Pak1, Mapk3 and Dsup3 genes of mitogen-activated protein kinase (MAPK) pathway were activated in Gnmt-/- mice, especially in the female mice. Therefore, GNMT is a tumor suppressor gene for liver cancer, and it is associated with gender disparity in liver cancer susceptibility.
Asunto(s)
Carcinoma Hepatocelular/enzimología , Glicina N-Metiltransferasa/genética , Neoplasias Hepáticas/enzimología , Animales , Carcinoma Hepatocelular/genética , Metilación de ADN , Salud de la Familia , Femenino , Predisposición Genética a la Enfermedad , Humanos , Neoplasias Hepáticas/genética , Masculino , Ratones , Ratones Noqueados , Mutación Missense , Factores Sexuales , Proteínas Wnt/metabolismoRESUMEN
BACKGROUND: Previously, we reported that there was an outbreak of human immunodeficiency virus type 1 (HIV-1) circulating recombinant form (CRF) 07_BC among injection drug users (IDUs) in Taiwan in 2004. The objectives of the present study were to conduct a molecular epidemiological analysis and to characterize the full-length genome of the Taiwanese CRF07_BC. METHODS: Three hundred and fifty-eight patients with HIV-1/AIDS from hospitals and 133 HIV-1-infected inmates from detention centers were recruited. DNA sequencing and phylogenetic analysis were conducted to determine subtypes and evolutionary relationships. Recombination breakpoints of 2 full-length CRF07_BC strains were elucidated using a bootscanning method. RESULTS: Of 206 HIV-1-infected patients who received a diagnosis in 2004, 44.7% were infected with subtype B, 53.4% with CRF07_BC, and 1.5% with CRF01_AE. Ninety-eight percent (109/111) of IDUs were infected with CRF07_BC. Deletions of 7-11 amino acids in both p6(gag) and p6(pol) proteins were noted among the Taiwanese CRF07_BC strains. The CRF_07BC strains belonged to 2 phylogenetic clusters, and the first cluster contained only CRF07_BC strains from the southern part of Taiwan. CONCLUSIONS: The Taiwanese CRF07_BC strains had 97% full-length sequence homology with the prototype from mainland China. CRF07_BC was first introduced into the southern region in 2002 and then spread to other regions in Taiwan in 2004.
Asunto(s)
Brotes de Enfermedades , Infecciones por VIH/epidemiología , Infecciones por VIH/virología , VIH-1/genética , Abuso de Sustancias por Vía Intravenosa , Secuencia de Aminoácidos , ADN Viral/análisis , Femenino , Genes env/genética , Genes gag/genética , Genes pol/genética , Infecciones por VIH/etiología , VIH-1/clasificación , VIH-1/aislamiento & purificación , Humanos , Masculino , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Encuestas y Cuestionarios , Taiwán/epidemiologíaRESUMEN
PURPOSE: Glycine N-methyltransferase (GNMT) affects genetic stability by regulating DNA methylation and interacting with environmental carcinogens. In a previous study, we showed that GNMT acts as a susceptibility gene for hepatocellular carcinoma. Here, we report on our efforts to characterize the haplotypes, loss of heterozygosity (LOH), and expression levels of the GNMT in prostate cancer. EXPERIMENTAL DESIGN: Peripheral blood mononuclear cell DNA collected from 326 prostate cancer patients and 327 age-matched controls was used to determine GNMT haplotypes. Luciferase reporter constructs were used to compare the promoter activity of different GNMT haplotypes. GNMT LOH rates in tumorous specimens were investigated via a comparison with peripheral blood mononuclear cell genotypes. Immunohistochemical staining was used to analyze GNMT expression in tissue specimens collected from 5 normal individuals, 33 benign prostatic hyperplasia patients, and 45 prostate cancer patients. RESULTS: Three major GNMT haplotypes were identified in 92% of the participants: A, 16GAs/DEL/C (58%); B, 10GAs/INS/C (19.9%); and C, 10GAs/INS/T (14.5%). Haplotype C carriers had significantly lower risk for prostate cancer compared with individuals with haplotype A (odds ratio, 0.68; 95% confidence interval, 0.48-0.95). Results from a phenotypic analysis showed that haplotype C exhibited the highest promoter activity (P < 0.05, ANOVA test). In addition, 36.4% (8 of 22) of the prostatic tumor tissues had LOH of the GNMT gene. Immunohistochemical staining results showed abundant GNMT expression in normal prostatic and benign prostatic hyperplasia tissues, whereas it was diminished in 82.2% (37 of 45) of the prostate cancer tissues. CONCLUSIONS: Our findings suggest that GNMT is a tumor susceptibility gene for prostate cancer.
Asunto(s)
Predisposición Genética a la Enfermedad , Glicina N-Metiltransferasa/genética , Haplotipos , Pérdida de Heterocigocidad , Neoplasias de la Próstata/genética , Anciano , Estudios de Casos y Controles , Humanos , Inmunohistoquímica , Masculino , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Hiperplasia Prostática/genéticaRESUMEN
Benzo[a]pyrene (BaP) is one of many polycyclic aromatic hydrocarbons that have been identified as major risk factors for developing various cancers. We previously demonstrated that the liver cancer susceptibility gene glycine N-methyltransferase (GNMT) is capable of binding with BaP and protecting cells from BaP-7,8-diol 9,10-epoxide-DNA adduct formation. In this study, we used a cytotoxicity assay to demonstrate that the higher expression level of GNMT, the lower cytotoxicity occurred in the cells treated with BaP. In addition, a cDNA microarray containing 7,597 human genes was used to examine gene expression patterns in BaP-treated HepG2 (a liver cancer cell line that expresses very low levels of GNMT) and SCG2-1-1 (a stable HepG2 clone that expresses high levels of GNMT) cells. The results showed that among 6,018 readable HepG2 genes, 359 (6.0%) were up-regulated more than 1.5-fold and 768 (12.8%) were down-regulated. Overexpression of GNMT in SCG2-1-1 cells resulted in the down-regulation of genes related to the detoxification, kinase/phosphatase pathways, and oncogenes. Furthermore, real-time PCR was used to validate microarray data from 21 genes belonging to the detoxification pathway. Combining both microarray and real-time PCR data, the results showed that among 89 detoxification pathway genes analyzed, 22 (24.7%) were up-regulated and 6 (6.7%) were down-regulated in BaP-treated HepG2 cells, while in the BaP-treated SCG2-1-1 cells, 12 (13.5%) were up-regulated and 26 (29.2%) were down-regulated (P < 0.001). Therefore, GNMT sequesters BaP, diminishes BaP's effects to the liver detoxification pathway and prevents subsequent cytotoxicity.
Asunto(s)
Benzo(a)pireno/farmacología , Perfilación de la Expresión Génica , Glicina N-Metiltransferasa/genética , Inactivación Metabólica/genética , Hígado/efectos de los fármacos , Benzo(a)pireno/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Análisis por Conglomerados , Regulación hacia Abajo/genética , Glicina N-Metiltransferasa/metabolismo , Humanos , Hígado/metabolismo , Hígado/patología , Receptores X del Hígado , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Receptores Nucleares Huérfanos , Reacción en Cadena de la Polimerasa/métodos , Unión Proteica , Receptores Citoplasmáticos y Nucleares/genética , Regulación hacia Arriba/genéticaRESUMEN
BACKGROUND: In 2003, Taiwan experienced a series of outbreaks of severe acute respiratory syndrome (SARS) and 1 laboratory-contamination accident. Here we describe a new phylogenetic analytical method to study the sources and dissemination paths of SARS-associated coronavirus (SARS-CoV) infections in Taiwan. METHODS: A phylogenetic analytical tool for combining nucleotide sequences from 6 variable regions of a SARS-CoV genome was developed by use of 20 published SARS-CoV sequences; and this method was validated by use of 80 published SARS-CoV sequences. Subsequently, this new tool was applied to provide a better understanding of the entire complement of Taiwanese SARS-CoV isolates, including 20 previously published and 19 identified in this study. The epidemiological data were integrated with the results from the phylogenetic tree and from the nucleotide-signature pattern. RESULTS: The topologies of phylogenetic trees generated by the new and the conventional strategies were similar, with the former having better robustness than the latter, especially in comparison with the maximum-likelihood trees: the new strategy revealed that during 2003 there were 5 waves of epidemic SARS-CoV infection, which belonged to 3 phylogenetic clusters in Taiwan. CONCLUSIONS: The new strategy is more efficient than its conventional counterparts. The outbreaks of SARS in Taiwan originated from multiple sources.