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Antimicrobial Agents and Chemotherapy, November 2007, p. 3924-3931, Vol. 51, No. 11
0066-4804/07/$08.00+0     doi:10.1128/AAC.00408-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Novel Small-Molecule Inhibitors of Transmissible Gastroenteritis Virus

Cheng-Wei Yang,¹ Yung-Ning Yang,¹ Po-Huang Liang,^2, Chi-Min Chen,^3, Wei-Liang Chen,¹ Hwan-You Chang,⁴ Yu-Sheng Chao,¹ and Shiow-Ju Lee^*

Division of Biotechnology and Pharmaceutical Research, National Health Research Institutes,¹ Animal Technology Institute Taiwan, Miaoli,³ Institute of Biological Chemistry, Academia Sinica, Taipei,² Institute of Molecular Medicine, National Tsing Hua University, Hsin Chu, Taiwan, Republic of China⁴

Received 25 March 2007/ Returned for modification 3 May 2007/ Accepted 14 August 2007

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We used swine testicle (ST) cells infected with transmissible gastroenteritis virus (TGEV) and an indirect immunofluorescent assay with antibodies against TGEV spike and nucleocapsid proteins to screen small-molecule compounds that inhibit TGEV replication. Analogues of initial hits were collected and subjected to a 3CL protease (3CL^pro) inhibition assay with recombinant 3CL^pro and a fluorogenic peptide substrate. A series of benzothiazolium compounds were found to have inhibitory activity against TGEV 3CL^pro and to exert anti-TGEV activities in terms of viral protein and RNA replication in TGEV-infected ST cells, with consequent protection of TGEV-infected ST cells from cytopathic effect by blocking the activation of caspase-3.

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Porcine transmissible gastroenteritis virus (TGEV), an enteropathogenic coronavirus (CoV), causes diarrhea in pigs, and piglets under the age of 3 weeks usually die from the infection. Priming of piglets with a respiratory variant (porcine respiratory CoV [PRCV]) has been found beneficial in preventing TGEV infection (20, 21). PRCV and TGEV share 96% homology in the genome sequence; the spike (S) protein of PRCV is amino-terminally truncated (224 to 227 amino acids) relative to that of TGEV. This deletion changes the infectivity of the virus. Thus, PRCV is no longer enteropathogenic and acts like a naturally occurring vaccine against TGEV.

Among many virally encoded components, viral proteases play vital roles in virus replication and transcription and have become the key targets in the search for antiviral agents, for example, against human immunodeficiency virus or hepatitis C virus (10, 17-19, 22, 24). To our knowledge, no inhibitor of TGEV 3CL protease (3CL^pro) has been identified or investigated. The development of inhibitors of TGEV 3CL^pro will provide an alternative means besides PRCV for treating swine gastroenteritis caused by TGEV. Currently, farmers would not use an antiviral drug as a means of prophylaxis, because an efficacious TGEV vaccine (PRCV) is available. However, when new virulent TGEV variants evolve to evade immune protection, as severe acute respiratory syndrome (SARS)-associated CoV (SARS CoV) evolved from human CoV, the antiviral product will be most useful not only to control disease but also to reduce transmission. In addition, the inhibitors identified could be used to probe the biology and pathogenesis of the virus.

Due to the laboratory constraints of biosafety levels 3 and 4, swine testes (ST) cells infected with TGEV and MRC5 or Vero E6 cells infected with human CoV 229E have been used as surrogate systems for screening agents that inhibit the activity of SARS CoV. The SARS CoV 3CL^pro has gained much attention in the development of anti-SARS CoV agents (3, 7, 13, 19, 24), since to date no effective therapeutic methods or vaccines are available. The substrate specificity and structure of CoV 3CL^pros are highly conserved (2, 11, 26). The TGEV 3CL^pro has been suggested to be the most homologous to the SARS CoV 3CL^pro, based on results of sequence alignment analysis of CoV 3CL^pros (25).

TGEV is the first CoV reported to trigger apoptosis in infected cells (8). Subsequently, other CoVs, including infectious bronchitis virus (IBV) (14), murine hepatitis virus (MHV) (15), and, lately, SARS CoV (16), were found to have similar apoptotic effects. In addition, apoptosis-associated caspase activation has been documented among numerous CoVs, including TGEV, SARS CoV, MHV, and IBV (4, 5, 8, 14-16). Therefore, if inhibitors of 3CL^pro block virus replication, virus-induced caspase activation and subsequent apoptosis should be effectively prevented as well.

Here we identified a series of benzothiazolium compounds that inhibit TGEV 3CL^pro activity and exert anti-TGEV activity, including prevention of TGEV replication and TGEV-induced apoptosis, in cultured TGEV-infected ST cells.

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Cells, virus, and compounds. The ST epithelial cells were grown as monolayers in a growth medium consisting of minimum essential medium (MEM; Invitrogen) and 10% fetal bovine serum (HyClone Co.). The Taiwan field-isolated (TFI) virulent strain of TGEV (6) was propagated in ST cells cultured with MEM and 2% fetal bovine serum. TGEV strain TFI infected ST cells but not RPTG (pig kidney) cells, whereas TGEV strains TLM-83 (PRCV; Belgium), TO-163 (TGEV; Japan), and Purdue-115 (TGEV; United States) infected both cell lines (6). ZVAD-fmk was purchased from Promega (Madison, WI). The 20,000 compounds (CSV0A000001 to -10000 and CSV0C000001 to -10000; part of the compound collection of the Division of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Taiwan) used in the primary screening of anti-TGEV agents and benzothiazolium compounds were purchased from Chemical Diversity Lab (San Diego, CA) and had >95% purity.

IFA, cytopathic effect (CPE) assay, and cytotoxicity assay. The ST cells in 96-well plates, with or without a 2-h pretreatment with test compounds, were infected with TGEV at a multiplicity of infection (MOI) of 10. After 6 h of TGEV infection, ST cells were fixed with 80% acetone and subjected to an indirect immunofluorescent assay (IFA) with antibodies against the S and nucleocapsid (N) proteins of TGEV (monoclonal antibodies generated by C.-M. Chen's lab; unpublished data). After three washes with phosphate-buffered saline, cells were incubated with fluorescein isothiocyanate-conjugated anti-mouse immunoglobulin (Capell Inc.) for 60 min at room temperature. The cells were washed three times with phosphate-buffered saline, and the fluorescence intensities were either measured by use of the Wallac Victor II system (Packard, Inc.) (excitation and emission wavelengths, 485 and 535 nm, respectively) to determine the 50% effective concentrations (EC₅₀) for inhibiting S and N protein expression or viewed by fluorescence microscopy. The images were captured by a charge-coupled device linked to a Leica IM50 Image Manager. The cells were treated with eight different concentrations of test compounds. The results of these assays were used to obtain the dose-response curves from which EC₅₀s were determined.

For the CPE assay, TGEV was inoculated into a monolayer of ST cells in 6-well plates at an MOI of 5. The plates were incubated at 37°C under 5% CO₂; the CPE in each well was observed at 18 to 24 h postinfection (hpi); and images were recorded as described above. TGEV-induced CPE is characterized by rounding and enlargement of cells, formation of syncytia, and detachment of cells into the medium.

For the cytotoxicity assay, ST cells cultured in MEM (Invitrogen) and 10% fetal bovine serum (HyClone Co.) in 96-well plates were treated with eight different concentrations of test compounds for 18 h. The results of these assays were used to obtain the dose-response curves from which the concentrations at which 50% growth inhibition occurred (GI₅₀) were determined.

Cloning, expression, and purification of TGEV 3CL^pro. The 3CL^pro gene of TGEV strain TFI (6) was amplified as a cDNA fragment of 906 bp by reverse transcription-PCR (RT-PCR) with primers 5'-CCGCTCGAGAAGATTTACACCATACATTTGCCTT-3' and 5'-CGGGATCCACCATGTCAGGTTTGCGGAAAATGGCACAGCC-3' containing XhoI and BamHI, respectively, for subsequent subcloning. The cDNA fragment of TGEV 3CL^pro digested with XhoI and BamHI at each end was ligated into the pGEX-6p vector (Amersham Biosciences) and expressed as a glutathione S-transferase (GST) tag fusion. Escherichia coli strain BL21(DE3) was transformed with the resulting plasmid, pGEX-6p-TGEV-3CL^pro, and then cultured in Luria-Bertani broth medium in the presence of carbenicillin. Once the cultures reached an absorbance at 600 nm of 0.5 to 0.6, they were harvested by centrifugation at 10,000 rpm for 15 min at 4°C and resuspended in lysis buffer (20 mM Tris-HCl [pH 7.5], 200 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol [DTT]) for sonication. The filtered supernatant was applied to a GST Sepharose 4B column and washed with lysis buffer. The GST-tagged protease was eluted with 20 mM Tris-HCl (pH 7.5), 200 mM NaCl, 1 mM EDTA, 1 mM DTT, and 30 mM glutathione. The eluted protein was then digested with PreScission protease to remove the GST tag, and the mixture was reloaded onto a GST Sepharose 4B column. The flowthrough fraction was collected for an enzyme activity assay. The concentration of the purified protein was determined by use ofthe Bio-Rad protein assay reagent with a standard curve plotted against bovine serum albumin.

Inhibition assay of SARS CoV 3CL^pro and TGEV 3CL^pro. SARS CoV 3CL^pro was prepared, and the assay was set up, as described elsewhere (12). The concentrations for 50% inhibitory activity (IC₅₀) of the tested compounds were determined in a reaction mixture containing 50 nM SARS CoV 3CL^pro, 6 µM fluorogenic substrate (Dabcyl-KTSAVLQSGFRKME-Edans), 20 mM Tris-HCl (pH 7.5), 4 mM NaCl, 3.3 µM EDTA, 33 µM DTT, and 25 µM ß-mercaptoethanol in the presence of 0 to 50 µM test compound. The fluorescence change resulting from the reaction was detected by use of a 96-well fluorescence plate reader at 535 nm.

Similarly, the inhibition assay of TGEV 3CL^pro was carried out in 200 µl of reaction solution at 25°C for 30 min. The reaction solution contained 2 µg (250 nM) of TGEV 3CL^pro enzyme and 60 µM fluorogenic substrate (Dabcyl-KVSVNSTLQSGLRKMAE-Edans) in a buffer comprising 15 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, and 7.5 mM ß-mercaptoethanol. At least eight different concentrations of test compounds were used to obtain the dose-response curves from which IC₅₀ were determined.

Western blot analysis. The antibodies used in the immunoblot analysis were anti-TGEV N protein (generated by Chi-Min Chen's lab), anti-caspase-3 (Cell Signaling Technology Inc.), and anti-ß-actin (Chemicon International Inc.). The cell lysates, with equal amounts of total protein, underwent sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the separated proteins were electrotransferred to nitrocellulose membranes. The resulting membranes were incubated with blocking solution and primary and secondary antibodies, and the wash procedures followed the manufacturer's recommendations. Antigen-antibody complexes were detected by use of enhanced chemiluminescence detection reagents (Western Lightning Chemiluminescence Reagent Plus; Perkin-Elmer) according to the manufacturer's instructions.

Viral RNA isolation and relative quantification by real-time RT-PCR. Viral RNA for RT-PCR was extracted from cell lysates. Uninfected cell lysates were processed concomitantly. Cultures were processed at 6 hpi with or without drug treatment. Total RNA was extracted by use of TRIzol reagent (Invitrogen). The relative mRNA amounts of the N and 3CL^pro genes were examined by real-time RT-PCR analysis. Reverse transcription was performed with 2 µg of total-RNA samples with oligo(dT) and SuperScript (First-Strand Synthesis system kit; Invitrogen), in a final volume of 20 µl, according to the manufacturer's instructions. The comparative cycle threshold (C_T) method was used for quantification according to the manufacturer's protocol (Applied Biosystems, Foster City, CA). C_T was measured at least in triplicate. Quantitative changes in mRNA expression involved the use of SYBR green Master Mix (Applied Biosystems) in an ABI PRISM 7900 (Applied Biosystems). PCR conditions were as follows: 50°C for 2 min; 95°C for 10 min; 40 cycles of 95°C for 15 s and 60°C for 1 min. The expression level of the samples was normalized to that of ß-actin. The primers designed with Primer Expression, version 3.0 (Applied Biosystems), were as follows: for N protein, 5'-CCAAGGATGGTGCCATGAAC-3' and 5'-GGACTGTTGCCTGCCTCTAGA-3'; for 3CL^pro, 5'-ATGAAGGATGTCCTGGCAGTGT-3' and 5'-ACCACCGTACATTTCTCCTTCAAA-3'; and for ß-actin, 5'-GGCTCAGAGCAAGAGAGGTATCC-3' and 5'-GGTCTCAAACATGATCTGAGTCATCT-3'.

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Screening and identification of small molecules that inhibit the replication of TGEV. A cell-based system involving TGEV-infected ST cells and an IFA was used in a high throughput screen for anti-TGEV agents. The ST cells were pretreated with the test compounds for 2 h prior to infection with TGEV at an MOI of 10. After 6 h of TGEV infection, the ST cells were fixed and subjected to IFA with antibodies specific for the S and N proteins of TGEV. From 20,000 small-molecule compounds screened from the collection of the Division of Biotechnology and Pharmaceutical Research, we obtained 51 hits with 50% or greater inhibition of the expression of the TGEV S and N proteins at 10 µM (data not shown).

Identification of small molecules that inhibit the 3CL^pro activity of TGEV. Since TGEV 3CL^pro is most homologous to SARS CoV 3CL^pro (25), we first used the well-established SARS 3CL^pro inhibitory assay (12) to examine the 51 compounds and found that 5 of them showed potent inhibition (IC₅₀, 2 to 10 µM) of SARS 3CL^pro activity (data not shown). The respective analogues of these inhibitors that share similar core structures were used to validate their inhibition effects. Compound A01652, containing a benzothiazolium core structure, was found to have significant inhibition activity. Therefore, a collection of 150 benzothiazolium compounds were further subjected to the SARS 3CL^pro assay; 8 of these compounds showed IC₅₀ of 2 to 10 µM, and 11 showed IC₅₀ of 10 to 30 µM (Fig. 1; Table 1).

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FIG. 1. Chemical structures of benzothiazolium TGEV inhibitors.

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TABLE 1. Activities of benzothiazolium compoundsa

To further analyze the inhibitory effects of these compounds on TGEV 3CL^pro activity, we purified recombinant TGEV 3CL^pro at 34 kDa (Fig. 2A). The enzyme exerted optimal catalytic activity at pH 7.5 and 25°C. Its kinetic parameters K_m and V_max toward the fluorogenic peptide substrate Dabcyl-KTSAVLQSGFRKME-Edans were determined as 10.9 µM and 2.0 µM/min, respectively (Fig. 2B and C; also data not shown). Most of the SARS 3CL^pro inhibitors also exerted similar inhibitory activity toward TGEV 3CL^pro (Table 1). Among 19 effective inhibitors of SARS 3CL^pro, 10 had IC₅₀ of 2 to 10 µM and 7 had IC₅₀ of 10 to 30 µM for TGEV 3CL^pro activity (Table 1).

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FIG. 2. Expression, purification, and characterization of TGEV 3CLpro. (A) Escherichia coli BL21(DE3) harboring pGEX-6p-TGEV-3CLpro was propagated in Luria-Bertani broth medium in the presence of carbenicillin. Lanes: 1, no isopropyl-ß-D-thiogalactopyranoside (IPTG) induction; 2, induction with 0.5 mM IPTG; 3, elutant from GST affinity chromatography; 4, PreScission protease-cleaved GST-TGEV 3CLpro; 5, purified TGEV 3CLpro after removal of the cleaved GST through a GST affinity column. Positions of molecular weight markers (in thousands) are shown on the left. (B) Kinetics analysis of purified recombinant TGEV 3CLpro. Reaction rates were plotted against varying concentrations of the substrate. (Inset) Double-reciprocal plot for Km determination. (C) Determination of optimal pH for activity of purified recombinant TGEV 3CLpro. The highest activity obtained in this study was set as 100%.

Benzothiazolium 3CL^pro inhibitors exerted anti-TGEV activity in TGEV-infected ST cells. Since the benzothiazolium compounds displayed inhibitory activity against TGEV 3CL^pro, they could inhibit TGEV replicase function and, thus, viral replication and yield. Therefore, we used an IFA to further examine the effects of these benzothiazolium compounds in inhibiting TGEV S and N protein expression. The amounts of TGEV S and N proteins were used as indicators of viral replication in TGEV-infected ST cells. As shown in Fig. 3A, TGEV-infected cells treated with the vehicle dimethyl sulfoxide (DMSO) clearly showed immunostaining for antibodies against TGEV S and N proteins, while uninfected ST cells showed no staining (data not shown). Compounds A38120, A01652, and A05175 significantly inhibited TGEV replication in ST cells (Fig. 3A); EC₅₀ were 6.2, 2.1, and 4.5 µM, respectively (Table 1). Most of the other benzothiazolium compounds tested also inhibited TGEV replication; EC₅₀ ranged from 2 to 7 µM. For one compound, A40437, the EC₅₀ was 17 µM, whereas for five others—A36638, A41369, A09853, A10059, and A33224—the EC₅₀ were greater than 30 µM (Table 1). Additional factors (e.g., cell permeability or susceptibility) for each individual compound might explain differences in inhibitory potency between in vitro recombinant TGEV 3CL^pro and ex vivo TGEV-infected ST cells.

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FIG. 3. Anti-TGEV activities of compounds A38120, A01652, and A05175. (A) IFA of infected cells treated with vehicle (1% DMSO) and 10 µM compound A38120, A01652, or A05175 with antibodies against TGEV S and N protein. (B) Western blot analysis of TGEV N protein in TGEV-infected ST cells after treatment with the compounds at 0.3 to 10 µM. Results are representative of three independent experiments.

The anti-TGEV activities of compounds A38120, A01652, and A05175 were further validated both by Western blot analysis with an antibody specific to N protein (Fig. 3B) and by quantitative RT-PCR (Table 2). The results were consistent with those from the IFA (Fig. 3A). The amplified 3CL^pro region is encoded by the ORF1a/ORF1ab of the TGEV genome (1). TGEV yields and protein levels were reduced dose-dependently upon treatment with A38120, A01652, or A05175. Thus, these benzothiazolium 3CL^pro inhibitors can protect ST cells from TGEV replication.

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TABLE 2. Effects of compounds A38120, A01652, and A05175 on TGEV yielda

Protective effects of benzothiazolium compounds in TGEV-infected ST cells. Western blot analysis revealed caspase-3 activation by the cleavage of procaspase-3 to an active caspase-3 (35-kDa to 17- and 19-kDa polypeptides). TGEV-infected ST cells showed significant levels of caspase-3 activation between 14 and 18 hpi (Fig. 4A). Compounds A38120, A01652, and A05175, as well as five other active benzothiazolium compounds, were examined for their protective effects against caspase-3 activation at 14 and 18 hpi. All of the test benzothiazolium compounds that effectively reduced TGEV replication as determined by IFA (Table 1)—A38120, A05175, A01652, A51894, and A11605—exerted strong protection against caspase-3 activation at 5 µM (Fig. 4B). Compounds A36638, A41369, and A40437, which showed weak anti-TGEV activity, with EC₅₀ greater than 15 µM in the IFA (Table 1), also exhibited insignificant protective effects against caspase-3 activation at 5 µM.

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FIG. 4. Protective effects of benzothiazolium compounds for TGEV-infected ST cells. (A) Activation of caspase-3 by TGEV infection in ST cells at various times postinfection. (B and C) Protective effects of benzothiazolium compounds (5 µM) against TGEV-induced caspase activation at 14 and 18 hpi (B) and against CPE (C) in ST cells. For the CPE assay, the cells were treated with either DMSO (1%) as a vehicle control, benzothiazolium compounds (5 µM), or the caspase inhibitor ZVAD-fmk (100 µM) as a positive control. CPE was observed at 18 to 24 hpi. Results are representative of two independent experiments at 24 hpi.

TGEV-induced CPE is characterized by cell rounding and enlargement, formation of syncytia, and detachment of cells into the medium. As shown in Fig. 4C, consistent with a previous report (8), the caspase inhibitor ZVAD-fmk (100 µM) displayed protection against TGEV-infected cytotoxicity. As expected, compounds A38120, A05175, A01652, A51894, and A11605 at 5 µM showed a trend of protective effects against CPE similar to that for anti-caspase-3 activation (Fig. 4C).

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Significant conservation in substrate specificity exists among the main CoV proteases (11). Consequently, the main proteases for the replicase polyproteins were predicted to have similar conserved processing kinetics in all CoVs. Unlike the 3CL^pro of SARS CoV (9, 12), which has been characterized extensively, the 3CL^pro of TGEV was only partially purified for a study of substrate specificity with a number of CoV main proteases (11). Although the crystal structure of the 3CL^pro of TGEV has been solved, the enzyme has not been characterized in detail (2). We characterized the recombinant 3CL^pro of TGEV with a purity greater than 95%. The K_m (10 µM) of the recombinant 3CL^pro indicates that the 3CL^pro of TGEV is highly efficient and is suitable for in vitro inhibition drug-screening assays. Using these active enzymes, 3CL^pros of TGEV and SARS CoV (12), we found that the effective benzothiazolium compounds had comparable inhibitory activities toward both enzymes. This finding reflects the conserved structures of these two CoV proteases (2, 11, 26).

Apoptosis-associated caspase activation has been documented among CoVs including TGEV, SARS CoV, MHV, and IBV (4, 5, 8, 14-16). The CPE induced by TGEV in ST cells is mainly due to apoptosis induced by TGEV (8). Blocking of viral replication through inhibition of 3CL^pro should result in protection of TGEV-infected ST cells against caspase activation, apoptosis, and subsequent CPE. Since caspase-3 plays a hub role for caspase cascades, conceivably these benzothiazolium 3CL^pro inhibitors exert protective antiapoptotic and anti-CPE effects through anti-caspase-3 activation in TGEV-infected ST cells.

In conclusion, we have identified benzothiazolium 3CL^pro inhibitors as novel small-molecule inhibitors of TGEV, some of which exert potent anti-TGEV activities and thus protect TGEV-infected ST cells against apoptosis and CPE. The in vitro potencies and cytotoxicities of these benzothiazolium 3CL^pro inhibitors will be improved in order to obtain lead compounds for further in vivo efficacy testing and drug development. Molecular docking will be exploited to elucidate the possible interactions between the benzothiazolium inhibitors and TGEV 3CL^pro. Rational drug design coupled with molecular modeling will be used to guide compound optimization. The chosen lead compounds will be subjected to in vivo efficacy studies using TGEV infected, colostrum-deprived piglets (23). Cocrystallization of each effective benzothiazolium compound with TGEV 3CL^pro will demonstrate their interactions and provide information for further modification of the benzothiazolium compounds when TGEV 3CL^pro mutation occurs.

 
This study was funded by the National Science Council of Taiwan (92-2751-B-400-007-Y and 93-2751-B-400-002-Y) and the National Health Research Institutes, Republic of China.

We acknowledge the technology support of Ssu-Hui Wu and Hua-Hao Chiou as well as the valued input of Michel Klein regarding the uses of virus vaccines and antiviral drugs.

 
* Corresponding author. Mailing address: Division of Biotechnology and Pharmaceutical Research, 35, Keyan Road, Zhunan Town, Miaoli County 350, Taiwan, Republic of China. Phone: 886-37-246166, ext. 35715. Fax: 886-37-586456. E-mail: slee{at}nhri.org.tw

Published ahead of print on 20 August 2007.

P.-H.L. and C.-M.C. contributed equally to this work.

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Antimicrobial Agents and Chemotherapy, November 2007, p. 3924-3931, Vol. 51, No. 11
0066-4804/07/$08.00+0     doi:10.1128/AAC.00408-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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