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Chwan-Chuen King

Publications and source records attributed to Chwan-Chuen King.

17 recordsLinked to original sources
PubMed2007

Differences in replication capacity between enterovirus 71 isolates obtained from patients with encephalitis and those obtained from patients with herpangina in Taiwan.

The cellular-tropism and biological characteristics of enterovirus 71 (EV71) isolates in Taiwan (TW) were studied. Growth curve experiments were conducted using cell lines that were possibly exhibited pathogenesis, and RT-PCR and sequencing tests were undertaken to amplify the 5' non-coding region (5'-NCR). The encephalitis isolate EV71 TW98NTU2078 was PBMC-tropic, temperature-resistant (Tr) at 40 degrees C, and easier to replicate in HTB-14 (astrocytoma) than the herpangina isolate EV71 TW98NTU1186 (The viral yields were 100-fold higher than those of the herpangina isolate EV71 TW98NTU1186 at 96 hr post infection.). The herpangina isolate EV71 TW98NTU1186 was non-PBMC-tropic, and temperature-sensitive (Ts) at 40 degrees C. The replication of EV71 TW98NTU1186 in HTB-14 was lower. No EV71 isolate infected HTB-37 (human colon adenocarcinoma cells). The encephalitis EV71 isolate exhibited better replication and transmission in PBMCs and astrocytes than did the EV71 isolate without CNS involvement.

Animals↗
PubMed2006

Slower rates of clearance of viral load and virus-containing immune complexes in patients with dengue hemorrhagic fever.

BACKGROUND: Although previous studies have revealed the contribution of an initial high level of dengue virus replication to the severe and potentially life-threatening diseases dengue hemorrhagic fever (DHF) and dengue shock syndrome, the involvement of dengue virus in the immunopathological processes during the transition from fever to defervescence, which is a critical stage in determining the progression to DHF, has not been appreciated. Previously, we reported that dengue virus can be detected in the immune complexes of patients with DHF during this period. METHODS: We investigated plasma dengue viral load, virus in immune complexes, antibody response, complements, and cytokines for 54 patients with dengue fever (a relatively mild form of disease) and 49 patients with DHF. The patients had confirmed secondary infection with dengue virus type 2 from a large outbreak in southern Taiwan in 2002. RESULTS: Patients with DHF had a significantly higher viral load and a slower rate of clearance than patients with dengue fever. For viral loads >5.7 log RNA copies/mL on the day of defervescence, the positive and negative predictive values for DHF are 0.88 and 0.95, respectively. A higher level and slower decline of dengue virus-containing immune complexes (and a subsequently higher elevation of C5a and soluble interleukin 2 receptor) were found in patients with DHF, compared with patients with dengue fever. CONCLUSIONS: These findings indicate that slower rates of clearance of viral load and virus-containing immune complexes are associated with subsequent immune activation and contribute to the progression of DHF at this critical stage. Moreover, viral load on the day of defervescence can predict cases of DHF.

Adolescent↗
PubMed2006

Nasopharyngeal shedding of severe acute respiratory syndrome-associated coronavirus is associated with genetic polymorphisms.

BACKGROUND: A high initial or peak severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) load in nasopharyngeal specimens was shown to be associated with a high mortality rate. Because all infected individuals were devoid of preeexisting protective immunity against SARS-CoV, the biological basis for the variable virus burdens in different patients remains elusive. METHODS: The nationwide SARS database in Taiwan was analyzed, and genotyping of 281 single-nucleotide polymorphisms (SNPs) of 65 genes was performed for 94 patients with SARS, to identify SNPs for which distribution between patients with or without detectable nasopharyngeal shedding of SARS-CoV was biased. RESULTS: Titers of SARS-CoV shed in nasopharyngeal specimens varied widely, ranging from nondetectable to 10(8) SARS-CoV RNA copies/mL, and they were correlated positively with a high mortality rate (P<.0001, by trend test) and with early death (i.e., death occurring within 2 weeks of the onset of illness) (P=.0015, by trend test). Virus shedding was found to be higher among male patients (P=.0014, by multivariate logistic regression) and among older patients (P=.015, by multivariate logistic regression). Detectable nasopharyngeal shedding of SARS-CoV was associated with polymorphic alleles of interleukins 18 (P=.014) and 1A (P=.031) and a member of NF kappa B complex (reticuloendotheliosis viral oncogene homolog B [RelB]) (P=.034), all of which are proinflammatory in nature, as well as the procoagulation molecule fibrinogen-like protein 2 (P=.008). CONCLUSION: The SARS-CoV load is a determinant of clinical outcomes of SARS, and it is associated with polymorphisms of genes involved in innate immunity, which might be regulated in an age- and sex-dependent manner. The findings of the present study provided leads to genes involved in the host response to SARS-CoV infection; if substantiated with functional studies, these findings may be applicable to other newly emerged respiratory viruses (e.g., the influenza pandemic strain).

Adult↗
PubMed2006

Spatial mapping of temporal risk characteristics to improve environmental health risk identification: a case study of a dengue epidemic in Taiwan.

Mapping uneven events, such as disease cases or pollutants, is a basic but important procedure for analyzing regional relationships and variation in public health and environmental agencies. The purpose of mapping is to find out the spatial clustering of uneven events and identify spatial risk areas, which could lead to potential environmental hazards or epidemics. Meanwhile, more hypotheses could be generated through mapping process for further investigations. This paper proposed a novel spatial-temporal approach to focusing on: (1) how often these uneven cases occur, (2) how long these cases persist and (3) how significant cases occur in consecutive periods across the study area. The proposed model was applied to the dengue fever epidemic in Taiwan in 2002 as a case study, which was the worst epidemic in the last 60 years. This approach provides procedures to identify spatial health risk levels with temporal characteristics and assists in generating hypothesis that will be investigated in further detail.

Data Interpretation, Statistical↗
PubMed2005

Strategically examining the full-genome of dengue virus type 3 in clinical isolates reveals its mutation spectra.

BACKGROUND: Previous studies presented the quasispecies spectrum of the envelope region of dengue virus type 3 (DENV-3) from either clinical specimens or field-caught mosquitoes. However, the extent of sequence variation among full genomic sequences of DENV within infected individuals remains largely unknown. RESULTS: Instead of arbitrarily choosing one genomic region in this study, the full genomic consensus sequences of six DENV-3 isolates were used to locate four genomic regions that had a higher potential of sequence heterogeneity at capsid-premembrane (C-prM), envelope (E), nonstructural protein 3 (NS3), and NS5. The extent of sequence heterogeneity revealed by clonal sequencing was genomic region-dependent, whereas the NS3 and NS5 had lower sequence heterogeneity than C-prM and E. Interestingly, the Phylogenetic Analysis by Maximum Likelihood program (PAML) analysis supported that the domain III of E region, the most heterogeneous region analyzed, was under the influence of positive selection. CONCLUSION: This study confirmed previous reports that the most heterogeneous region of the dengue viral genome resided at the envelope region, of which the domain III was under positive selection pressure. Further studies will need to address the influence of these mutations on the overall fitness in different hosts (i.e., mosquito and human) during dengue viral transmission.

Adult↗
PubMed2005

Neutralizing antibody response and SARS severity.

Using the Taiwan nationwide laboratory-confirmed severe acute respiratory syndrome (SARS) database, we analyzed neutralizing antibody in relation to clinical outcomes. With a linear mixed model, neutralizing antibody titer was shown to peak between week 5 and week 8 after onset and to decline thereafter, with a half-life of 6.4 weeks. Patients with a longer illness showed a lower neutralizing antibody response than patients with a shorter illness duration (p = 0.008). When early responders were compared with most patients, who seroconverted on and after week 3 of illness, the small proportion (17.4%) of early responders (antibody detectable within 2 weeks) had a higher death rate (29.6% vs. 7.8%) (Fisher exact test, p = 0.004), had a shorter survival time of <2 weeks (Fisher exact test, p = 0.013), and were more likely to be > 60 years of age (Fisher exact test, p = 0.01). Our findings have implications for understanding the pathogenesis of SARS and for SARS vaccine research and development.

Adult↗
PubMed2005

Laboratory diagnosis of dengue virus infection: current and future perspectives in clinical diagnosis and public health.

Since no protective vaccine or specific treatments are available for dengue fever/dengue hemorrhagic fever (DHF), accurate diagnosis is critical for the early initiation of specific preventive health measures to curtail epidemic spread and reduce economic losses. Commonly used diagnosis methods for confirming dengue infection involve virus isolation, detection of virus antigen or RNA in plasma or serum or tissues, and the presence of dengue virus-specific antibodies in serum and other body fluids. Recently, several techniques have been developed for rapid laboratory diagnosis of dengue virus, including centrifugation amplification to enhance virus isolation rate, the flow cytometry method for early detection of cultured virus, detecting viral nucleic acid e.g., by nested reverse transcriptase-polymerase chain reaction (RT-PCR), quantitative RT-PCR, nucleic acid sequence-based amplification, and real-time PCR, detecting free viral non-structure antigens, anti-dengue virus immunoglobulin M (IgM) or IgG antibodies by enzyme-linked immunosorbent assay, and differentiation of primary versus secondary dengue virus infection. Newly established methods must be standardized to maintain high quality laboratory performance. Laboratory diagnostics must be tailored to a specific laboratory environment, the objectives of clinical needs and the availability of clinical specimens. Speed and accuracy of diagnosis must be balanced against test cost and availability. Future challenges in the study of dengue and DHF include the application of modern techniques, such as nucleic acid chips, protein chips and new biomarkers to avoid cross-reactivity among different serotypes of dengue viruses and other flaviviruses, plus development of internationally standardized guidelines to improve quality assurance of these advanced laboratory tests.

Antibodies, Viral↗
PubMed2004

Lymphocyte activation and hepatic cellular infiltration in immunocompetent mice infected by dengue virus.

Activation and expansion of dengue virus-specific T cells and abnormal liver functions in dengue patients have been documented. However, it remains to be determined whether T cells are involved in the pathogenic mechanism of dengue virus infection. In this study, immunocompetent C57BL/6 mice were employed to study dengue virus-induced T cell activation. Mice were inoculated with 10(8) PFU dengue virus serotype 2 strain 16681 by the intravenous route. Dengue viral core RNA was detected by RT-PCR in mouse serum, liver, spleen, and brain at different time points after infection. Splenic T cells were activated as evidenced by their expression of CD69 and O-glycosylated CD43 at as early as day 3 after infection. Splenic T cell expression of O-glycosylated CD43 and IFN-gamma production coordinately peaked at day 5. Coincided with the peak of splenic T cell activation was hepatic lymphocyte infiltration and elevation of liver enzymes. Flow cytometric analysis revealed the infiltrating CD8(+) T cell to CD4(+) T cell ratio was 5/3. After a second inoculation of dengue virus, hepatic T cell infiltration and liver enzyme levels increased sharply. The infiltrating hepatic CD8(+) T cell to CD4(+) T cell ratio increased to 5.8/1. A strong correlation was found between T cell activation and hepatic cellular infiltration in immunocompetent mice infected with dengue virus. The kinetics of liver enzyme elevation also correlated with that of T cell activation. These data suggest a relationship between T cell infiltration and elevation of liver enzymes.

Alanine Transaminase↗
PubMed2004

An external loop region of domain III of dengue virus type 2 envelope protein is involved in serotype-specific binding to mosquito but not mammalian cells.

Dengue virus (DV) is a flavivirus and infects mammalian cells through mosquito vectors. This study investigates the roles of domain III of DV type 2 envelope protein (EIII) in DV binding to the host cell. Recombinant EIII interferes with DV infection to BHK21 and C6/36 cells by blocking dengue virion adsorption to these cells. Inhibition of EIII on BHK21 cells was broad with no serotype specificity; however, inhibition of EIII on C6/36 cells was relatively serotype specific. Soluble heparin completely blocks binding of EIII to BHK21 cells, suggesting that domain III binds mainly to cell surface heparan sulfates. This suggestion is supported by the observation that EIII binds very weakly to gro2C and sog9 mutant mammalian cell lines that lack heparan sulfate. In contrast, heparin does not block binding of EIII to mosquito cells. Furthermore, a synthetic peptide that includes amino acids (aa) 380 to 389 of EIII, IGVEPGQLKL, inhibits binding of EIII to C6/36 but not BHK21 cells. This peptide corresponds to a lateral loop region on domain III of E protein, indicating a possible role of this loop in binding to mosquito cells. In summary, these results suggest that EIII plays an important role in binding of DV type 2 to host cells. In addition, EIII interacts with heparan sulfates when binding to BHK21 cells, and a loop region containing aa 380 to 389 of EIII may participate in DV type 2 binding to C6/36 cells.

Amino Acid Sequence↗
PubMed2004

Study of sequence variation of dengue type 3 virus in naturally infected mosquitoes and human hosts: implications for transmission and evolution.

Dengue virus is an arbovirus that replicates alternately in the mosquito vector and human host. We investigated sequences of dengue type 3 virus in naturally infected Aedes aegypti mosquitoes and in eight patients from the same outbreak and reported that the extent of sequence variation seen with the mosquitoes was generally lower than that seen with the patients (mean diversity, 0.21 versus 0.38% and 0.09 versus 0.23% for the envelope [E] and capsid [C] genes, respectively). This was further verified with five experimentally infected mosquitoes (mean diversity, 0.09 and 0.10% for the E and C genes, respectively). Examination of the quasispecies structures of the E sequences of the mosquitoes and of the patients revealed that the sequences of the major variants were the same, suggesting that the major variant was transmitted. These findings support our hypothesis that mosquitoes contribute to the evolutionary conservation of dengue virus by maintaining a more homogenous viral population and a dominant variant during transmission.

Aedes↗
PubMed2003

High levels of plasma dengue viral load during defervescence in patients with dengue hemorrhagic fever: implications for pathogenesis.

Studies of the pathogenesis of dengue hemorrhagic fever (DHF), a potentially life-threatening disease, have revealed the importance of initial high levels of virus replication. However, the possible involvement of virus during the transition from fever to defervescence, a critical stage in determining the severity of disease, has not been appreciated. Using quantitative reverse transcription-polymerase chain reaction, we examined the levels of plasma dengue viral load during both fever and defervescence periods in patients from a DEN-3 outbreak in southern Taiwan in 1998. Higher levels of plasma dengue viral RNA were found in DHF patients than in DF patients. During defervescence, while the level of plasma dengue viral RNA was undetectable in most DF patients, it remains high in all DHF patients. Using a modified immunoprecipitation assay, we demonstrated for the first time that the plasma dengue viruses persisting during defervescence were in the immune complexes for most DHF patients. These findings suggest that continued active viral replication or delay in the clearance of viremia contributes to the pathogenesis of DHF. Moreover, high levels of plasma dengue viral RNA during defervescence may serve as a disease marker for DHF.

Adult↗
PubMed2003

Concurrent infections by two dengue virus serotypes among dengue patients in Taiwan.

The co-circulation of multiple dengue virus serotypes in the same region has been reported in several countries in Southeast Asia as well as in Central and South America for decades. Although outbreaks involving more than one serotype of dengue virus have been reported in Taiwan since 1987, concurrent infection in the same individual by multiple serotypes of dengue virus have never been identified. Using a modified multiplex reverse transcription-polymerase chain reaction assay, we detected and determined the serotypes of 21 dengue patients during an outbreak in southern Taiwan in 2000. While either dengue type 2 or type 3 virus was found in most cases, 2 of the 21 cases were concurrent infections by both dengue type 2 and dengue type 3 viruses. This was further confirmed by sequence analysis of the amplified products. This study reports for the first time that concurrent infections occur in Taiwan, and suggests that future virologic surveillance of dengue outbreak in Taiwan should take this into consideration.

Base Sequence↗
PubMed2002

Intracellular localization and determination of a nuclear localization signal of the core protein of dengue virus.

In dengue virus (DEN) particles, the core protein is a structural protein of the nucleocapsid. The core protein is known to be present in the nucleus of DEN-infected cells but there have been conflicting reports as to whether it is also present in the nucleolus. To clarify this, the intracellular location of the core protein was examined using a monoclonal antibody, 15B11, which was produced in this study. Immunofluorescence staining with this antibody demonstrated that the core protein first appeared in the cytoplasm and then in the nuclei and nucleoli of infected cells. Nuclear localization of the core protein was determined to be independent of other DEN proteins, since recombinant core proteins still entered the nuclei and nucleoli of cells transfected with only the core protein gene. Three putative nuclear localization signal motifs have been predicted to be present on the core protein. Deletion of the first one (KKAR), located at aa 6-9, and mutation of the second one (KKSK), located at aa 73-76, did not eliminate the nuclear localization property of the core protein. The third motif with a bipartite structure, RKeigrmlnilnRRRR, located at aa 85-100, was determined to be responsible for the nuclear localization of the core protein, since the core protein without this motif was located exclusively in the cytoplasm of DEN-infected cells and that this motif mediated nuclear localization of a normally cytoplasmic protein.

Amino Acid Sequence↗
PubMed2002

Detection of dengue virus replication in peripheral blood mononuclear cells from dengue virus type 2-infected patients by a reverse transcription-real-time PCR assay.

While dengue virus is thought to replicate in mononuclear phagocytic cells in vivo, attempts to detect it in peripheral blood mononuclear cells (PBMC) by virus isolation or antigen detection have had variable and generally low rates. In this study, we developed a reverse transcription (RT)-real-time PCR assay to quantify positive- and negative-sense RNA of dengue virus type 2 within the cells. The assay includes an RT step using either sense or antisense primer followed by a real-time PCR step using the designed primers and probe, which target a capsid region highly conserved in dengue virus type 2 strains. It can be used to monitor the dynamic change of intracellular dengue virus RNA species during the course of infection. When this assay is employed in quantification of dengue virus RNA species in PBMC from 10 patients infected with dengue virus type 2, both positive- and negative-sense dengue RNA can be detected, indicating that dengue virus is actively replicating in PBMC in vivo. Moreover, the amounts of negative-sense dengue virus RNA in PBMC correlate very well with the viral load of dengue virus in plasma, suggesting that quantification of negative-sense dengue virus RNA in PBMC may provide another indicator of dengue virus replication in vivo. Use of this convenient, sensitive, and accurate method of quantification in clinical samples from patients with different disease severity would further our understanding of the pathogenesis of dengue.

Animals↗
PubMed2002

Dengue type 3 virus in plasma is a population of closely related genomes: quasispecies.

Using reverse transcription-PCR and clonal sequencing of the dengue virus envelope gene derived from the plasma samples of six patients, we reported for the first time that dengue virus circulates as a population of closely related genomes. The extent of sequence diversity varied among patients, with the mean pairwise proportions of difference ranging from 0.21 to 1.67%. Genome-defective viruses were found in 5.8% of the total number of clones analyzed. Our findings on the quasispecies nature of dengue virus and the defective virus in vivo have implications with regard to the pathogenesis of dengue virus.

Amino Acid Sequence↗
PubMed2002

Risk factors of enterovirus 71 infection and associated hand, foot, and mouth disease/herpangina in children during an epidemic in Taiwan.

OBJECTIVE: In 1998, an enterovirus 71 (EV71) epidemic in Taiwan was associated with hand, foot, and mouth disease (HFMD)/herpangina and involved 78 fatal cases. We measured EV71 seroprevalence rates before and after the epidemic and investigated risk factors associated with EV71 infection and illness. METHODS: Neutralizing antibodies to EV71 were assayed for 539 people before the epidemic and 4619 people of similar ages after the epidemic. Questionnaires, which were completed during household interviews after the epidemic, solicited demographic variables, exposure history, and clinical manifestations. RESULTS: A total of 129 106 cases of HFMD were reported during the epidemic. Age-specific pre-epidemic EV71 seroprevalence rates were inversely related to age-specific periepidemic mortality rates (r = -0.82) or severe case rates (r = -0.93). Higher postepidemic EV71 seropositive rates among children who were younger than 3 years positively correlated with higher mortality rates in different areas (r = 0.88). After the epidemic, 51 (56%) of 91 younger siblings of elder siblings who were EV71-seropositive were EV71-seropositive; otherwise, 2.2% (4 of 186) of younger siblings were EV71-seropositive (matched odds ratio [OR]: 10; 95% confidence interval [CI]: 3.4-29). Stepwise multiple logistic regression revealed other factors associated with EV71 infection to be older age (adjusted OR: 2.5; 95% CI: 1.9-3.4), attendance at kindergartens/child care centers (adjusted OR: 1.8; 95% CI: 1.3-2.5), contact with HFMD/herpangina (adjusted OR: 1.6; 95% CI: 1.2-2.1), greater number of children in a family (adjusted OR: 1.4; 95% CI: 1.1-1.7), and rural residence (adjusted OR: 1.4; 95% CI: 1.2-1.6). Twenty-nine percent of preschool children who were infected with EV71 developed HFMD/herpangina. Younger age and contact with HFMD/herpangina were significant factors for the development of EV71-related HFMD/herpangina in these children. CONCLUSIONS: An increased incidence of EV71 infection in young children occurred more often in geographic areas with increased mortality rates. Intrafamilial and kindergarten transmissions among preschool children were major modes of disease transmission during the widespread EV71 epidemic in Taiwan in 1998.

Child↗