Cheng-Ming Lee

Molecular epidemiology of HCV genotypes among injection drug users in Taiwan: Full-length sequences of two new subtype 6w strains and a recombinant form_2b6w.

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. J. Med. Virol. 82:57–68, 2010.

Molecular Epidemiology of HIV-1 Infection and Full-Length Genomic Analysis of Circulating Recombinant Form 07_BC Strains from Injection Drug Users in Taiwan

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.

Molecular epidemiology and trends of HIV-1 subtypes in Taiwan.

To understand the trends of distribution and risk factors associated with different HIV-1 subtypes in different populations in Taiwan, blood samples and questionnaires were collected from 267 male and 21 female HIV-1-infected people in a multicenter survey from 1993 to 1996. This group represented about one quarter of the total registered HIV-1 cases in Taiwan. The HIV-1 subtypes were determined using V3-based peptide-enzyme immunoassays complemented by heteroduplex mobility assay and phylogenetic tree analysis. The results showed that in Taiwan, men were primarily infected with HIV-1B (68.2%) and HIV-1E (27.3%), whereas women were mainly infected with non-B subtypes (4.8% A, 4.8% C, 71.4% E, and 9.5% G). In addition, 71.4% of men with HIV-1B were homosexual or bisexual, whereas 56.2% of men with HIV-1E were heterosexual (p < .001). Although HIV-1E subtype came to Taiwan later than HIV-1B, it has become a major subtype in the heterosexual population.

Molecular epidemiology of HIV-1 infection in Taiwan from 2005 to 2008: further spread of CRF07_BC and emergence of CRF07_BC/subtype B dual infection.

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.

Molecular Epidemiology of Severe Acute Respiratory Syndrome–Associated Coronavirus Infections in Taiwan

Abstract 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

Characterization of the 5' regulatory region of the human Glycine N-methyltransferase gene

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.

Community-based molecular epidemiology of HTLV type I in Taiwan and Kinmen: implication of the origin of the cosmopolitan subtype in northeast Asia.

To understand the possible origin and dissemination of HTLV-I infection in northeast Asia, community-based molecular epidemiological studies were conducted on the Kinmen Islands (off the coast of Fukien Province, China) and in Taiwan. A total of 3831 Taiwanese from 3 townships (Pu-Li, Chu-Dung, and Pu-Tze) and 993 aborigines from 4 tribes in Taiwan participated in this study. The prevalence rates of HTLV-I infection in adult residents from Pu-Li, Chu-Dung, and Pu-Tze were 0.82, 1.72, and 1.63%, respectively. None of the aborigines had HTLV-I infection. Previously, 0.73% of the adult population of Kin-Hu, Kinmen were found to have HTLV-I infection. Peripheral blood mononuclear cells were collected from HTLV-I carriers identified both in Taiwan and Kinmen and the HTLV-I LTR sequences were PCR amplified, subcloned, and sequenced for phylogenetic tree analysis. The results showed that all 6 HTLV-I isolates from Kinmen and 13 of 18 (72.2%) isolates from Taiwan were group a (transcontinental) of Cosmopolitan subtype, while 5 of 18 (27.8%) isolates from Taiwan were group b (Japanese) of Cosmopolitan subtype. Since all of the HTLV-I-infected persons were descendants of immigrants from mainland China, the origin of the Cosmopolitan subtype in Taiwan and Kinmen may not have been Japan, as previously theorized, but China, possibly the result of the migration of an infected population in the past several centuries.

Detection of hepatitis C virus subtypes 6a, 6n, 6w and mixed infections using a modified multiplex real-time polymerase chain reaction protocol

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.

Virological investigation of four outbreaks of influenza B reassortants in the northern region of Taiwan from October 2006 to February 2007

BackgroundFrom 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.FindingsPhylogenetic 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.ConclusionThe 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.