Biomedical subjects

Shin-Ru Shih

Publications and source records attributed to Shin-Ru Shih.

At least 19 recordsLinked to original sources

PubMed2006

Expression, purification and characterization of enterovirus-71 virus-like particles.

AIM: Enterovirus 71 (EV71) has been implicated as the etiological agent responsible for the recent outbreaks of hand, foot and mouth disease associated with severe neurological diseases in the Asia-Pacific region. METHODS: The assembly process was hypothesized to occur via an orchestrated proteolytic processing of the P1 precursor by the viral protease 3CD. To test this hypothesis, we constructed 3 recombinant baculoviruses: Bac-P1 expressing P1; Bac-3CD expressing 3CD; and Bac-P1-3CD co-expressing P1 and 3CD. RESULTS: Both single infection by Bac-P1-3CD and co-infection by Bac-P1 and Bac-3CD resulted in correct cleavage of P1 to yield individual proteins VP0, VP1 and VP3, while the former approach yielded higher VLP production. In the cells, the structural proteins self-assembled into clusters of virus-like particles (VLP) resembling the authentic EV71 particle aggregates. After ultracentrifugation purification, the dispersed VLPs were indistinguishable from the authentic virus in size, appearance, composition and surface epitopes, as determined by SDS-PAGE, Western blot, transmission electron microscopy and immunogold labeling. CONCLUSION: Our data, for the first time, suggest that in insect cells EV71 structural proteins adopt a processing and assembly pathway similar to poliovirus assembly. The preservation of particle morphology and composition suggest that the VLP may be a valuable vaccine candidate to prevent EV71 epidemics.

Animals↗

PubMed2006

Subtyping of influenza A isolates in Taiwan--2003 to 2004.

BACKGROUND/PURPOSE: Some isolated influenza strains are untypable. The possibility that a novel and potentially lethal strain, such as H5 virus, might be one of these untypable strains remains a major concern. This study investigated the nature of untypable influenza strains in Taiwan. METHODS: Subtyping for unsubtyped isolates was conducted by reverse transcription-polymerase chain reaction (RT-PCR) using different primer sets. Sequence determination and comparison, and characterization of viral growth rate were performed. RESULTS: Analysis of viral re-cultures with the different primer sets for RT-PCR revealed that all isolates were of the H3N2 subtype. All isolates resembled A/Fujian/411/02 (H3N2), and were genetically distinct from N2 of A/chicken/Taiwan/1209/03 (H5N2) on phylogenetic analysis. Real-time RT-PCR quantification of viral loads in the 16 clinical isolates revealed that negative results were partially caused by low RNA levels in the culture fluid. Comparison of replication kinetics in Madin-Darby canine kidney cells of recent influenza A H3N2 isolates showed that some Fujian-like isolates from 2003 to 2004 had significantly lower growth rate than Panama-like isolates from 2002. CONCLUSION: The results of this study suggest that some recent isolates may replicate poorly in tissue culture and fail to be typed by current methodology.

Adult↗

PubMed2006

Combining multiplex reverse transcription-PCR and a diagnostic microarray to detect and differentiate enterovirus 71 and coxsackievirus A16.

Cluster A enteroviruses, including enterovirus 71 (EV71) and coxsackievirus A16 (CA16), are known to cause hand-foot-and-mouth disease (HFMD). Despite the close genetic relationship between these two viruses, EV71 is generally known to be a more perpetuating pathogen involved in severe clinical manifestations and deaths. While the serotyping of enteroviruses is mostly done by conventional immunological methods, many clinical isolates remain unclassifiable due to the limited number of antibodies against enterovirus surface proteins. Array-based assays are able to detect several serotypes with high accuracy. We combined an enterovirus microarray with multiplex reverse transcription-PCR to try to develop a method of sensitively and accurately detecting and differentiating EV71 and CA16. In an effort to design serotype-specific probes for detection of the virus, we first did an elaborate bioinformatic analysis of the sequence database derived from different enterovirus serotypes. We then constructed a microarray using 60-mer degenerate oligonucleotide probes covalently bound to array slides. Using this enterovirus microarray to study 144 clinical specimens from patients infected with HFMD or suspected to have HFMD, we found that it had a diagnostic accuracy of 92.0% for EV71 and 95.8% for CA16. Diagnostic accuracy for other enteroviruses (non-EV71 or -CA16) was 92.0%. All specimens were analyzed in parallel by real-time PCR and subsequently confirmed by neutralization tests. This highly sensitive array-based assay may become a useful alternative in clinical diagnostics of EV71 and CA16.

DNA Probes↗

PubMed2006

Antiviral drug discovery targeting to viral proteases.

Proteases fulfill multiple roles in health and disease, and considerable interest has been expressed in the design and development of synthetic inhibitors of disease-related proteases. Virus-encoded proteases have been shown to be involved in the replication of many viruses. The success of anti-HIV-1 therapy using specific and potent protease inhibitors suggests that viral proteases can be the valid molecular targets for the development of antiviral drugs against other viruses. Intensive genetic and biochemical studies have been conducted on viral proteases and new insights and results are rapidly emerging. This work reviews features of viral proteases with respective to the development of effective antiviral therapy.

Antiviral Agents↗

PubMed2006

Genomic signatures of human versus avian influenza A viruses.

Position-specific entropy profiles created from scanning 306 human and 95 avian influenza A viral genomes showed that 228 of 4591 amino acid residues yielded significant differences between these 2 viruses. We subsequently used 15,785 protein sequences from the National Center for Biotechnology Information (NCBI) to assess the robustness of these signatures and obtained 52 "species-associated" positions. Specific mutations on those points may enable an avian influenza virus to become a human virus. Many of these signatures are found in NP, PA, and PB2 genes (viral ribonucleoproteins [RNPs]) and are mostly located in the functional domains related to RNP-RNP interactions that are important for viral replication. Upon inspecting 21 human-isolated avian influenza viral genomes from NCBI, we found 19 that exhibited > or =1 species-associated residue changes; 7 of them contained > or =2 substitutions. Histograms based on pairwise sequence comparison showed that NP disjointed most between human and avian influenza viruses, followed by PA and PB2.

Amino Acid Sequence↗

PubMed2005

Synthesis and antipicornavirus activity of (R)- and (S)-1-[5-(4'-chlorobiphenyl-4-yloxy)-3-methylpentyl]-3-pyridin-4-yl-imidazolidin-2-one.

The new pyridyl imidazolidinone derivative, 1-[5-(4'-chlorobiphenyl-4-yloxy)-3-methylpentyl]-3-pyridin-4-yl-imidazolidin-2-one (+/-)-1a, was synthesized and found to have an excellent antiviral activity against EV71 (IC50 = 0.009 microM). Therefore, both the enantiomers, (S)-(+)-1a and (R)-(-)-1a, have been prepared starting from readily available monomethyl (R)-3-methylglutarate (7) as a useful chiral building block and their antiviral activity was evaluated in a plaque reduction assay. Interestingly, we observed that the enantiomer (S)-(+)-1a was 10-fold more active against enterovirus71 (EV71) (IC50 = 0.003 microM) than the corresponding enantiomer (R)-(-)-1a (IC50 = 0.033 microM). Similar results were found against all five strains (1743, 2086, 2231, 4643, and BrCr) of EV71 tested. This demonstrated that the absolute configuration of the chiral carbon atom at the 3-position of the alkyl linker considerably influenced the anti-EV71 activity of these pyridyl imidazolidinones.

Antiviral Agents↗

PubMed2005

High-efficiency protein expression mediated by enterovirus 71 internal ribosome entry site.

An internal ribosome entry site (IRES) has been used to facilitate the expression of more than one protein in a single transcript. In this study, we examined the translational activities of several IRES elements derived from different RNA viruses. The protein expression of encephalomyocarditis virus (EMCV) IRES is similar to that of hepatitis C virus (HCV) IRES in mammalian cells. Notably, the protein expression of enterovirus 71 (EV71) IRES was 23-fold higher than the efficiency of EMCV IRES following normalization of mRNA transcriptional level. Thus, expression of the secreted alkaline phosphatase (SEAP) reporter protein in mammalian cells may be controlled at desirable levels by using appropriate IRES in the expression vector.

Animals↗

PubMed2005

Design, synthesis, and antipicornavirus activity of 1-[5-(4-arylphenoxy)alkyl]-3-pyridin-4-ylimidazolidin-2-one derivatives.

A series of pyridylimidazolidinone derivatives was synthesized and tested in vitro against enterovirus 71 (EV71). On the basis of compound 33 (DBPR103), introduction of a methyl group at the 2- or 3-position of the linker between the imidazolidinone and the biphenyl resulted in markedly improved antiviral activity toward EV71 with IC(50) values of 5.0 nM (24b) and 9.3 nM (14a), respectively. Increasing the branched chain to propyl resulted in a progressive decrease in activity, while inserting different heteroatoms entirely rendered the compound only weakly active. The introduction of a bulky group (cyclohexyl, phenyl, or benzyl) led to loss of activity against EV71. The 4-chlorophenyl moiety in 14a was replaced with bioisosteric groups such as oxadiazole (28a-d) or tetrazole (32a,b), dramatically improving anti-EV71 activity and selectivity indices. Compounds 14a, 24b, 28b, 28d, and 32a exhibited a strong activity against lethal EV71, and no apparent cellular toxicity was observed. Three of the more potent imidazolidinone compounds, 14a, 28b, and 32b, were subjected to a large group of picornaviruses to determine their spectrum of antiviral activity.

Antiviral Agents↗

PubMed2005

False positive antibody results against human T-cell lymphotropic virus in patients with severe acute respiratory syndrome.

Taiwan suffered from the outbreak of severe acute respiratory syndrome (SARS) in 2003. Our laboratory performed a series of virology and serology tests for SARS patients admitted to our hospital. Cross-reactivity was found when testing for antibody against human T-cell lymphotropic virus (HTLV) in one patient with SARS. Therefore, antibodies against HTLV were examined in paired-sera from 26 SARS patients. ELISA and a neutralization test were used to measure anti-SARS antibodies. Seroconversion for antibody against SARS-CoV was observed in all patients. Surprisingly, with the use of ELISA for HTLV, sera for 13 patients were positive for HTLV (50%), and seroconversion for HTLV was also observed in 10 patients (38.5%). Western blot for HTLV on those 26 paired-sera from 13 HTLV-positive patients displayed 5 positive results for HTLV-I, 7 positive results for HTLV-II, 1 positive result for both HTLV-I and II, 9 negative results for either HTLV-I or HTLV-II, and 4 "indeterminate" results. The findings that antibody to HTLV can be detected in blood samples collected from SARS patients provide important information for safe handling of blood products. Without such knowledge, blood products can be discarded mistakenly even though they contain anti-SARS-CoV antibodies that may be potentially valuable for SARS therapy.

Adolescent↗

PubMed2005

Revealing molecular targets for enterovirus type 71 detection by profile hidden Markov models.

The enterovirus infection in 1998 claimed 78 deaths in Taiwan, with an average of 40 fatalities each year after. Traditional serum-based diagnostic methods often fail to detect enteroviruses due to antigenic changes. As a result, many isolates remain untyped and are absent from the enterovirus surveillance and epidemiological investigations. We present a profile hidden Markov model (HMM) method for molecular typing of enterovirus 71 (EV71). Based on the enteroviral sequences retrieved from GenBank, we build a nucleotide-based and an amino acid-based profile HMM for each EV71 gene using the package HMMER. HMMER bit score-based Z-scores for EV71 and non-EV71 sequences are calculated for each of these profile HMMs. In a genome-wide analysis, we find that the distribution of the EV71 Z-scores and that of the non-EV71 Z-scores have disjoint support for nucleotide-based VP1 profile HMM if the sequence is longer than 150 bases; a VP1-based molecular typing method for EV71 is thus proposed. We also report VP4 an alternative molecular target for detecting EV71, while the two UTRs and all the genes coding the internal proteins cannot be used for such purpose. To demonstrate the performance of the nucleotide-based EV71 VP1 profile HMM, 330 enterovirus VP1 nucleotide sequences newly reported to GenBank are typed with this method. All the EV71 sequences are detected with no error.

Amino Acid Sequence↗

PubMed2005

Laboratory-based surveillance and molecular epidemiology of influenza virus in Taiwan.

A laboratory-based surveillance network of 11 clinical virological laboratories for influenza viruses was established in Taiwan under the coordination of the Center for Disease Control and Prevention (CDC), Taiwan. From October 2000 to March 2004, 3,244 influenza viruses were isolated, including 1,969 influenza A and 1,275 influenza B viruses. The influenza infections usually occurred frequently in winter in the northern hemisphere. However, the influenza seasonality in Taiwan was not clear during the four seasons under investigation. For example, the influenza A viruses peaked during the winters of 2001, 2002, and 2003. However, some isolated peaks were also found in the summer and fall (June to November) of 2001 and 2002. An unusual peak of influenza B also occurred in the summer of 2002 (June to August). Phylogenetic analysis shows that influenza A isolates from the same year were often grouped together. However, influenza B isolates from the year 2002 clustered into different groups, and the data indicate that both B/Victoria/2/87-like and B/Yamagata/16/88-like lineages of influenza B viruses were cocirculating. Sequence comparison of epidemic strains versus vaccine strains shows that many vaccine-like Taiwanese strains were circulating at least 2 years before the vaccine strains were introduced. No clear seasonality of influenza reports in Taiwan occurred in contrast to other more continental regions.

Animals↗

PubMed2004

Synthesis and antienteroviral activity of a series of novel, oxime ether-containing pyridyl imidazolidinones.

A series of novel, oxime ether-containing pyridyl imidazolidinones were synthesized and their antiviral activity was evaluated in a plaque reduction assay. It is very interesting that this class of compounds is specific for human enteroviruses, in particular, enterovirus 71 (EV71). Some derivatives strongly inhibited enterovirus replication with activities higher or comparable to those of the reference compounds such as A1 and A2. Preliminary SAR studies revealed that the chain length of the alkyl linker and the alkyl substituent at the oxime ether group largely influenced the in vitro anti-EV71 activity of this new class of potent antiviral agents. Among this series of compounds synthesized, the pyridyl imidazolidinone with an ethyl oxime ether group located at the para position of the phenoxyl ring (8b) was identified as the most potent enterovirus 71 inhibitor (IC50=0.001 microM) with no apparent cytotoxic effect toward RD (rhabdomyosarcoma) cell lines (CC50>25 microM). Furthermore, this compound has been shown broad-spectrum activity against most of the serotypes of enteroviruses tested in the nanomolar range.

Antiviral Agents↗

PubMed2004

Design, synthesis, and structure-activity relationships of pyrazolo[3,4-d]pyrimidines: a novel class of potent enterovirus inhibitors.

A series of pyrazolo[3,4-d]pyrimidines were synthesized and their antiviral activity was evaluated in a plaque reduction assay. It is very interesting that this class of compounds provide remarkable evidence that they are very specific for human enteroviruses, in particular, coxsackieviruses. Some derivatives proved to be highly effective in inhibiting enterovirus replication at nanomolar concentrations. SAR studies revealed that the phenyl group at the N-1 position and the hydrophobic diarylmethyl group at the piperazine largely influenced the in vitro antienteroviral activity of this new class of potent antiviral agents. It was found that the pyrazolo[3,4-d]pyrimidines with a thiophene substituent, such as compounds 20-24, in general exhibited high activity against coxsackievirus B3 (IC(50) = 0.063-0.089 microM) and moderate activity against enterovirus 71 (IC(50) = 0.32-0.65 microM) with no apparent cytotoxic effect toward RD (rhabdomyosarcoma) cell lines (CC(50) > 25 microM).

Antiviral Agents↗

PubMed2004

Transmission and clinical features of enterovirus 71 infections in household contacts in Taiwan.

CONTEXT: Although enterovirus 71 has caused epidemics associated with significant morbidity and mortality, its transmission has not been thoroughly investigated. OBJECTIVES: To investigate enterovirus 71 transmission and determine clinical outcomes within households. DESIGN, SETTING, AND PARTICIPANTS: Prospective family cohort study to investigate patients at a children's hospital in Taiwan and family members of these patients who had signs and symptoms suggestive of enterovirus 71 between February 2001 and August 2002. Patients and household members underwent clinical evaluations, virological studies, questionnaire-based interviews, and were followed up for 6 months. MAIN OUTCOME MEASURES: Enterovirus 71 infection, defined as a positive viral culture from a throat or rectal swab, or the presence of IgM or a 4-fold increase in neutralizing antibody in serum; and clinical syndromes, defined as asymptomatic; uncomplicated symptomatic; and complicated; with unfavorable outcomes of sequelae or death. RESULTS: Ninety-four families (433 family members) had at least 1 family member with evidence of enterovirus 71 infection. The overall enterovirus 71 transmission rate to household contacts was 52% (176/339 household contacts). Transmission rates were 84% for siblings (70/83); 83%, cousins (19/23); 41%, parents (72/175); 28%, grandparents (10/36); and 26%, uncles and aunts (5/19). Of 183 infected children, 11 (6%) were asymptomatic and 133 (73%) had uncomplicated illnesses (hand, foot, and mouth disease, herpangina, nonspecific febrile illness, upper respiratory tract infection, enteritis, or viral exanthema). Twenty-one percent (39/183) experienced complicated syndromes including the central nervous system or cardiopulmonary failure. During the 6-month follow-up, 10 died and 13 had long-term sequelae consisting of dysfunction in swallowing, cranial nerve palsies, central hypoventilation, or limb weakness and atrophy. Age younger than 3 years was the most significant factor associated with an unfavorable outcome in children (P =.004). Among 87 infected adults, 46 (53%) were asymptomatic, 34 (39%) had nonspecific illnesses of fever, sore throat, or gastrointestinal discomfort, and 7 (8%) had hand, foot, and mouth disease. There were no complicated cases in adults. CONCLUSIONS: Enterovirus 71 household transmission rates were high for children in Taiwan and severe disease with serious complications, sequelae, and death occurred frequently. In contrast, adults had a much lower rate of acquisition of the infection and much less adverse sequelae.

Adolescent↗

PubMed2004

Selective human enterovirus and rhinovirus inhibitors: An overview of capsid-binding and protease-inhibiting molecules.

The absence of effective vaccines for most viral infections highlights an urgent necessity for the design and development of effective antiviral drugs. Due to the advancement in virology since the late 1980s, several key events in the viral life cycle have been well delineated and a number of molecular targets have been validated, culminating in the emergence of many new antiviral drugs in recent years. Inhibitors against enteroviruses and rhinoviruses, responsible for about half of the human common colds, are currently under active investigation. Agents targeted at either viral protein 1 (VP1), a relatively conserved capsid structure mediating viral adsorption/uncoating process, or 3C protease, which is highly conserved among different serotypes and essential for viral replication, are of great potential to become antipicornavirus drugs.

Antiviral Agents↗

PubMed2004

Identification of genes involved in the host response to enterovirus 71 infection.

Enterovirus 71 (EV71) infection may be asymptomatic or may cause diarrhea, rashes, and hand, foot, and mouth disease (HFMD). However, EV71 also has the potential to cause severe neurological disease. To date, little is known about the molecular mechanisms of host response to EV71 infection. In this report, we utilized cDNA microarray to profile the kinetics and patterns of host gene expression in EV71-infected human neural SF268 cells. We have identified 157 genes with significant changes in mRNA expression and performed hierarchical clustering to classify these genes into five different groups based on their kinetics of expression. EV71 infection led to increases in the level of mRNAs encoding chemokines, proteins involved in protein degradation, complement proteins, and proapoptotis proteins. cDNA microarray expression comparisons of EV71- and mock-infected cells also revealed the down-regulation of several genes encoding proteins involved in host RNA synthesis. Expression of interferon-regulated proteins was increased early in the infection and then decreased. Expression of proteins involved in cellular development and differentiation, some oncogenes, and transcription and translation regulators were suppressed and then stimulated late in the infection. Our findings illustrate the overall host response to EV71 infection, and will aid in understanding the host response to this virus.

Enterovirus↗

PubMed2004

Diverse apoptotic pathways in enterovirus 71-infected cells.

Mechanisms related to the neuropathogenesis of enterovirus 71 infection remain unclear. This investigation conducts a comprehensive study of the apoptotic pathways in neural and non-neural cells following enterovirus 71 infection. Infections with enterovirus 71 not only induce classical cytopathic effects in SF268 (human glioblastoma), SK-N-MC (human neuroblastoma), RD, and Vero cells, but also induce classic signs of apoptosis in all cells, including DNA fragmentation and phosphatidylserine translocation. Apoptosis has also been caused by the efflux of cytochrome c from mitochondria, and subsequently by cleavage of caspase 9 in all cells. Activation of caspase 8 followed by cleavage of the proapoptotic protein Bid only occurs in non-neural cells. Results of this study demonstrate that a mitochondrial pathway of apoptosis mediated by activation and cleavage of caspase 9 is a main pathway in enterovirus 71-induced apoptosis, especially for enterovirus 71-infected neural cells.

Animals↗

PubMed2004

Mutation in enterovirus 71 capsid protein VP1 confers resistance to the inhibitory effects of pyridyl imidazolidinone.

Enterovirus 71 is one of the most important pathogens in the family of Picornaviridae that can cause severe complications in the postpoliovirus era, such as encephalitis, pulmonary edema, and even death. Pyridyl imidazolidinone is a novel class of potent and selective human enterovirus 71 inhibitor. Pyridyl imidazolidinone was identified by using computer-assisted drug design. This virologic investigation demonstrates that BPR0Z-194, one of the pyridyl imidazolidinones, targets enterovirus 71 capsid protein VP1. Time course experiments revealed that BPR0Z-194 effectively inhibited virus replication in the early stages, implying that the compound can inhibit viral adsorption and/or viral RNA uncoating. BPR0Z-194 was used to select and characterize the drug-resistant viruses. Sequence analysis of the VP1 region showed that the resistant variants differed consistently by seven amino acids in VP1 region from their parental drug-sensitive strains. Site-directed mutagenesis of enterovirus 71 infectious cDNA revealed that a single amino acid alteration at the position 192 of VP1 can confer resistance to the inhibitory effects of BPR0Z-194.

Amino Acid Sequence↗