Day-Yu Chao

The incidence of varicella and herpes zoster in Taiwan during a period of increasing varicella vaccine coverage, 2000-2008.

The introduction and the widespread use of the varicella vaccine in Taiwan has led to a 75-80% decrease in the incidence of varicella in children. However the vaccines long-term impact on the incidence of herpes zoster (HZ) has attracted attention. By controlling gender, underlying diseases, and age effects, a Poisson regression was applied on the 2000-2008 chart records of 240 000 randomly selected residents who enrolled in the Universal National Health Insurance. The results show that, as the vaccine coverage in children increases, the incidence of varicella decreases. However, the incidence of HZ increased even before the implementation of the free varicella vaccination programme in 2004, particularly in females. The increase in the incidence of HZ cannot be entirely and directly attributed to the widespread vaccination of children. Continuous monitoring is needed to understand the secular trends in HZ before and after varicella vaccination in Taiwan and in other countries.

Re-assess Vector Indices Threshold as an Early Warning Tool for Predicting Dengue Epidemic in a Dengue Non-endemic Country

Background Despite dengue dynamics being driven by complex interactions between human hosts, mosquito vectors and viruses that are influenced by climate factors, an operational model that will enable health authorities to anticipate the outbreak risk in a dengue non-endemic area has not been developed. The objectives of this study were to evaluate the temporal relationship between meteorological variables, entomological surveillance indices and confirmed dengue cases; and to establish the threshold for entomological surveillance indices including three mosquito larval indices [Breteau (BI), Container (CI) and House indices (HI)] and one adult index (AI) as an early warning tool for dengue epidemic. Methodology/Principal Findings Epidemiological, entomological and meteorological data were analyzed from 2005 to 2012 in Kaohsiung City, Taiwan. The successive waves of dengue outbreaks with different magnitudes were recorded in Kaohsiung City, and involved a dominant serotype during each epidemic. The annual indigenous dengue cases usually started from May to June and reached a peak in October to November. Vector data from 2005–2012 showed that the peak of the adult mosquito population was followed by a peak in the corresponding dengue activity with a lag period of 1–2 months. Therefore, we focused the analysis on the data from May to December and the high risk district, where the inspection of the immature and mature mosquitoes was carried out on a weekly basis and about 97.9% dengue cases occurred. The two-stage model was utilized here to estimate the risk and time-lag effect of annual dengue outbreaks in Taiwan. First, Poisson regression was used to select the optimal subset of variables and time-lags for predicting the number of dengue cases, and the final results of the multivariate analysis were selected based on the smallest AIC value. Next, each vector index models with selected variables were subjected to multiple logistic regression models to examine the accuracy of predicting the occurrence of dengue cases. The results suggested that Model-AI, BI, CI and HI predicted the occurrence of dengue cases with 83.8, 87.8, 88.3 and 88.4% accuracy, respectively. The predicting threshold based on individual Model-AI, BI, CI and HI was 0.97, 1.16, 1.79 and 0.997, respectively. Conclusion/Significance There was little evidence of quantifiable association among vector indices, meteorological factors and dengue transmission that could reliably be used for outbreak prediction. Our study here provided the proof-of-concept of how to search for the optimal model and determine the threshold for dengue epidemics. Since those factors used for prediction varied, depending on the ecology and herd immunity level under different geological areas, different thresholds may be developed for different countries using a similar structure of the two-stage model.

The changing epidemiology of varicella incidence after implementation of the one-dose varicella vaccination policy

The varicella vaccine has been available in the Taiwan market since July 1997. Beginning 1998-1999, Taipei City and Taichung City/County as the early launch areas included the varicella vaccine in their free pediatric vaccination programs. By contrast, the national free vaccination program was not implemented until 2004. We aim to investigate the changing epidemiology of varicella incidence through an analysis of age-period-cohort effects. With the greatest decrease in varicella incidence occurring in children aged below 6, the incidence of varicella shifted to older age groups as reflected in different birth cohorts. The current study provides important implications for the current vaccination policy.

Nonstructural Protein 1-Specific Immunoglobulin M and G Antibody Capture Enzyme-Linked Immunosorbent Assays in Diagnosis of Flaviviral Infections in Humans

ABSTRACT IgM antibody- and IgG antibody-capture enzyme-linked immunosorbent assays (MAC/GAC-ELISAs) targeted at envelope protein (E) of dengue viruses (DENV), West Nile virus, and Japanese encephalitis virus (JEV) are widely used as serodiagnostic tests for presumptive confirmation of viral infection. Antibodies directed against the flavivirus nonstructural protein 1 (NS1) have been proposed as serological markers of natural infections among vaccinated populations. The aim of the current study is to optimize an IgM and IgG antibody-capture ELISA (MAC/GAC-ELISA) to detect anti-NS1 antibodies and compare it with anti-E MAC/GAC-ELISA. Plasmids to express premembrane/envelope (prM/E) or NS1 proteins of six medically important flaviviruses, including dengue viruses (DENV-1 to DENV-4), West Nile virus (WNV), and Japanese encephalitis virus (JEV), were constructed. These plasmids were used for the production of prM/E-containing virus-like particles (VLPs) and secreted NS1 (sNS1) from COS-1 cells. Archived clinical specimens from patients with confirmed DENV, JEV, and WNV infections, along with naive sera, were subjected to NS1-MAC/GAC-ELISAs before or after depletion of anti-prM/E antibodies by preabsorption with or without VLPs. Human serum specimens from previously confirmed DENV infections showed significantly enhanced positive-to-negative (P/N) ratios for NS1-MAC/GAC-ELISAs after the depletion of anti-prM/E antibodies. No statistical differences in sensitivities and specificities were found between the newly developed NS1- and VLP-MAC/GAC-ELISAs. Further application of the assays to WNV- and JEV-infected serum panels showed similar results. A novel approach to perform MAC/GAC-ELISAs for NS1 antibody detection was successfully developed with great potential to differentiate antibodies elicited by the tetravalent chimeric yellow fever-17D/dengue vaccine or DENV infection.

Scanning mutagenesis studies reveal a potential intramolecular interaction within the C-terminal half of dengue virus NS2A involved in viral RNA replication and virus assembly and secretion

ABSTRACT The NS2A protein of dengue virus (DENV) has eight predicted transmembrane segments (pTMSs; pTMS1 to pTMS8). NS2A has been shown to participate in RNA replication, virion assembly, and the host antiviral response. However, the role of the amino acid residues within the pTMS regions of NS2A during the virus life cycle is poorly understood. In the study described here, we explored the function of DENV NS2A by introducing a series of double or triple alanine substitutions into the C-terminal half (pTMS4 to pTMS8) of NS2A in the context of a DENV infectious clone or subgenomic replicon. Fourteen (8 within pTMS8) of 35 NS2A mutants displayed a lethal phenotype due to impairment of RNA replication by a replicon assay. Three NS2A mutants with mutations within pTMS7, the CM20, CM25, and CM27 mutants, displayed similar phenotypes, low virus yields (>100-fold reduction), wild-type-like replicon activity, and low infectious virus-like particle yields by transient trans-packaging experiments, suggesting a defect in virus assembly and secretion. The sequencing of revertant viruses derived from CM20, CM25, and CM27 mutant viruses revealed a consensus reversion mutation, leucine (L) to phenylalanine (F), at codon 181 within pTMS7. The introduction of an L181F mutation into a full-length NS2A mutant, i.e., the CM20, CM25, and CM27 constructs, completely restored wild-type infectivity. Notably, L181F also substantially rescued the other severely RNA replication-defective mutants with mutations within pTMS4, pTMS6, and pTMS8, i.e., the CM2, CM3, CM13, CM31, and CM32 mutants. In conclusion, the results revealed the essential roles of pTMS4 to pTMS8 of NS2A in RNA replication and/or virus assembly and secretion. The intramolecular interaction between pTMS7 and pTMS4, pTMS6, or pTMS8 of the NS2A protein was also implicated. IMPORTANCE The reported characterization of the C-terminal half of dengue virus NS2A is the first comprehensive mutagenesis study to investigate the function of flavivirus NS2A involved in the steps of the virus life cycle. In particular, detailed mapping of the amino acid residues within the predicted transmembrane segments (pTMSs) of NS2A involved in RNA replication and/or virus assembly and secretion was performed. A revertant genetics study also revealed that L181F within pTMS7 is a consensus reversion mutation that rescues both RNA replication-defective and virus assembly- and secretion-defective mutants with mutations within the other three pTMSs of NS2A. Collectively, these findings elucidate the role played by NS2A during the virus life cycle, possibly through the intricate intramolecular interaction between pTMS7 and other pTMSs within the NS2A protein.

Virus-Like Particle Secretion and Genotype-Dependent Immunogenicity of Dengue Virus Serotype 2 DNA Vaccine

ABSTRACT Dengue virus (DENV), composed of four distinct serotypes, is the most important and rapidly emerging arthropod-borne pathogen and imposes substantial economic and public health burdens. We constructed candidate vaccines containing the DNA of five of the genotypes of dengue virus serotype 2 (DENV-2) and evaluated the immunogenicity, the neutralizing (Nt) activity of the elicited antibodies, and the protective efficacy elicited in mice immunized with the vaccine candidates. We observed a significant correlation between the level of in vitro virus-like particle secretion, the elicited antibody response, and the protective efficacy of the vaccines containing the DNA of the different DENV genotypes in immunized mice. However, higher total IgG antibody levels did not always translate into higher Nt antibodies against homologous and heterologous viruses. We also found that, in contrast to previous reports, more than 50% of total IgG targeted ectodomain III (EDIII) of the E protein, and a substantial fraction of this population was interdomain highly neutralizing flavivirus subgroup-cross-reactive antibodies, such as monoclonal antibody 1B7-5. In addition, the lack of a critical epitope(s) in the Sylvatic genotype virus recognized by interdomain antibodies could be the major cause of the poor protection of mice vaccinated with the Asian 1 genotype vaccine (pVD2-Asian 1) from lethal challenge with virus of the Sylvatic genotype. In conclusion, although the pVD2-Asian 1 vaccine was immunogenic, elicited sufficient titers of Nt antibodies against all DENV-2 genotypes, and provided 100% protection against challenge with virus of the homologous Asian 1 genotype and virus of the heterologous Cosmopolitan genotype, it is critical to monitor the potential emergence of Sylvatic genotype viruses, since vaccine candidates under development may not protect vaccinated humans from these viruses. IMPORTANCE Five genotype-specific dengue virus serotype 2 (DENV-2) DNA vaccine candidates were evaluated for their immunogenicity, homologous and heterologous neutralizing (Nt) antibody titers, and cross-genotype protection in a murine model. The immunity elicited by our prototype vaccine candidate (Asian 1 genotype strain 16681) in mice was protective against viruses of other genotypes but not against virus of the Sylvatic genotype, whose emergence and potential risk after introduction into the human population have previously been demonstrated. The underlying mechanism of a lack of protection elicited by the prototype vaccine may at least be contributed by the absence of a flavivirus subgroup-cross-reactive, highly neutralizing monoclonal antibody 1B7-5-like epitope in DENV-2 of the Sylvatic genotype. The DENV DNA vaccine directs the synthesis and assembly of virus-like particles (VLPs) and induces immune responses similar to those elicited by live-attenuated vaccines, and its flexibility permits the fast deployment of vaccine to combat emerging viruses, such as Sylvatic genotype viruses. The enhanced VLP secretion obtained by replacement of ectodomain I-II (EDI-II) of the Cosmopolitan genotype vaccine construct (VD2-Cosmopolitan) with the Asian 1 EDI-II elicited significantly higher total IgG and Nt antibody titers and suggests a novel approach to enhance the immunogenicity of the DNA vaccine. A DENV vaccine capable of eliciting protective immunity against viruses of existing and emerging genotypes should be the focus of future DENV vaccine development.

Re-Model the Relation of Vector Indices, Meteorological Factors and Dengue Fever

Background: Dengue is the most rapidly expanding and spreading mosquito-borne viral disease in tropical and subtropical countries. In Taiwan, dengue incidence clustered in Southern part, especially Kaohsiung in the past decade. Aim: The spatial and temporal patterns of dengue transmission in Taiwan from 2005 to 2012 were examined to investigate the occurrence of dengue fever (DF) patients and its association with immature and adult mosquito indices, and its interaction with meteorological factors and household density. Methods: Three databases were spatially and temporally linked, including the comprehensive chart records of DF cases and vector surveillance data in Kaohsiung, as well as the meteorological and environmental information from 2005 to 2012. A case-crossover study design was used to explore the effects of mosquito indices and weather on the risks of DF, and conditional logistic regression was applied to estimate the odds ratios (OR). Results: Results showed immature mosquito indices had significant positive association with DF in the medium and high household density areas (e.g., adjusted ORs of Breteau index were 1.04, 95% CI=[1.02, 1.06] and 1.06, CI=[1.04, 1.08] respectively), while adult mosquito index was significant to all low/med/high household densities (adjusted ORs of Aedes aegypti index were 1.29, CI=[1.23,1.36]; 1.49, CI=[1.37,1.61] and 1.3, CI=[1.21,1.39] respectively). Meanwhile, combination with 2-week lag rainfall, 2-month lag rainfall, 2-week lag temperature and relative humidity, resulted better prediction of DF incidence. Conclusion: Meteorological conditions affect DF occurrence in a nonlinear way, and a single time-point rainfall variable is insufficient to fit it. Our study suggested that short-lag (last 2 weeks) conditions of moderate rainfall, moderate temperature and high humidity, in combination with a long-lag heavy rainfall were related to higher probability of DF incidence. BI and CI are useful predictors for DF occurrence in medium and high household density areas, but not in the low density areas.

Influenza vaccination and the endurance against air pollution among elderly with acute coronary syndrome

OBJECTIVE Air pollution, weather condition and influenza are known risk factors of acute coronary syndrome (ACS) among elderly people. The influenza vaccine (IV) has been shown to reduce major cardiovascular events. The purpose of this study was to compare resistance to air pollution and weather factors causing ACS between vaccinated and less-vaccinated elderly people. METHODS A case-crossover design was applied to 1835 elderly ACS patients who were obtained from the 1-million sample of Taiwan National Health Insurance Research Data with inclusion criteria: (1) the first diagnosis of ACS was in cold season and at age 68 or more, (2) had received the free IV program at least once during the period 3years before the ACS. They were stratified into two groups: 707 had received flu vaccinations for all the 3years and the remaining 1128 had not. The measurements of air pollutants, temperature, and humidity corresponding to each of the 3days prior to the ACS diagnosis date were retrieved from the data banks of the Taiwan Environmental Protection Administration and Central Weather Bureau. FINDINGS Increases in air pollution concentrations of CO, NO2, PM10 or PM2.5 and decreases in temperature significantly influenced the risk of ACS for the non-continuously vaccinated elderly population; however, less significant effects were observed for the continuously vaccinated population. CONCLUSION Consecutive influenza vaccination may potentially offer resistance against the detrimental effects of air pollution and changes in temperature in frail elderly adults with ACS. Future studies are needed to directly assess the interaction effect between the vaccination and environmental factors on ACS.

The Temporal Trend of Influenza-Associated Morbidity and the Impact of Early Appearance of Antigenic Drifted Strains in a Southeast Asian Country

Globally, influenza infection is a major cause of morbidity and mortality in the elderly, who are suggested to be the major target group for trivalent influenza vaccine (TIV) vaccination by World Health Organization. In spite of an increasing trend in vaccine coverage rates in many countries, the effect of vaccination among the elderly in reducing hospitalization and mortality remains controversial. In this study, we conducted a historical cohort study to evaluate the temporal pattern of influenza-associated morbidity among persons older than 64 years over a decade. The temporal patterns of influenza-associated morbidity rates among the elderly older than 64 years indicated that Taiwans elderly P&I outpatient visits have been decreasing since the beginning of the 1999–2000 influenza season; however, hospitalization has been increasing despite significant increases in vaccine coverage. The propensity score logistic regression model was implemented to evaluate the source of bias and it was found that the TIV-receiving group had a higher propensity score than the non-receiving group (P<0.0001). In order to investigate the major factors affecting the temporal pattern of influenza-associated morbidity, we then used the propensity score as a summary confounder in a multivariate Poisson regression model based on the trimmed data. Our final models suggested that the factors affected the temporal pattern of morbidity differently. The variables including co-morbidity, vaccination rate, influenza virus type A and B isolation rate were associated with increased outpatient visits and hospitalization (p<0.05). In contrast, variables including high propensity score, increased 1°C in temperature, matching vaccine strains of type A/H1N1 and type B were associated with decreased outpatient visits and hospitalization (p<0.05). Finally, we assessed the impact of early appearance of antigenic-drifted strains and concluded that an excess influenza-associated morbidity substantial trends toward higher P&I hospitalization, but not outpatient visits, during the influenza season with early appearance of antigenic-drifted strains.

Comprehensive evaluation of differential serodiagnosis between Zika and dengue viral infection

Diagnostic testing for Zika virus (ZIKV) or dengue virus (DENV) infection can be accomplished by a nucleic acid detection method; however, a negative result does not exclude infection due to the low virus titer during infection depending on the timing of sample collection. Therefore, a ZIKV- or DENV-specific serological assay is essential for the accurate diagnosis of patients and to prevent potential severe health outcomes. A retrospective study design with dual approaches of collecting human serum samples for testing was developed. All serum samples were extensively evaluated by using both non-infectious virus-like particles (VLPs) and soluble non-structural protein 1 (NS1) in the standard immunoglobulin M (IgM) antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA). Both VLP- and NS1-MAC-ELISAs were found to have similar sensitivity for detecting anti-premembrane/envelope and NS1 antibodies from ZIKV-infected patient sera. Group cross reactive (GR)-antibody-ablated homologous fusion peptide-mutated (FP)-VLPs consistently showed higher P/N values than homologous wild-type VLPs. Therefore, FP-VLPs were used to develop the algorithm for differentiating ZIKV from DENV infection. Overall, the sensitivity and specificity of the FP-VLP-MAC-ELISA and the NS1-MAC-ELISA were each higher than 80% with no statistical significance. A novel approach to differentiate ZIKV from DENV infection serologically has been developed. The accuracy can reach up to 95% when combining both VLP and NS1 assays. In comparison to current guidelines using neutralization tests to measure ZIKV antibody, this approach can facilitate laboratory screening for ZIKV infection, especially in regions where DENV infection is endemic and capacity for neutralization testing does not exist.