Pei-Yun Shu

Development of Group- and Serotype-Specific One-Step SYBR Green I-Based Real-Time Reverse Transcription-PCR Assay for Dengue Virus

ABSTRACT A quantitative one-step SYBR Green I-based reverse transcription (RT)-PCR system was developed for the detection and differentiation of four different dengue virus serotypes in acute-phase serum samples. A set of group- and serotype-specific primer pairs was designed against conserved sequences in the core region and evaluated for clinical diagnosis. A linear relationship was obtained between the amount of input RNA and cycle threshold (Ct) value over a range of 10 to 107 PFU per ml of cell culture-derived dengue viruses. The detection limit of the group-specific primer pair was between 4.1 and 43.5 PFU/ml for four dengue serotypes. The detection limit of each of the serotype-specific primer pairs was calculated to be 10 PFU/ml for dengue virus serotype 1 (DEN-1), 4.6 PFU/ml for DEN-2, 4.1 PFU/ml for DEN-3, and 5 PFU/ml for DEN-4. Comparisons between the one-step SYBR Green-based RT-PCR assay and the conventional cell culture method in the clinical diagnosis of dengue virus infection from acute-phase serum samples of confirmed dengue patients were performed. The results showed that 83 and 67% of 193 acute-phase serum samples tested were positive by the one-step SYBR Green-based RT-PCR method and cell culture method, respectively. Further analysis showed that the one-step SYBR Green-based RT-PCR method could detect twice as many acute-phase serum samples with positive dengue-specific immunoglobulin M (IgM) and/or IgG antibodies than cell culture method. Our results demonstrate the potential clinical application of the one-step SYBR Green I-based RT-PCR assay for the detection and differentiation of dengue virus RNA.

Fever Screening at Airports and Imported Dengue

Airport fever screening in Taiwan, July 2003–June 2004, identified 40 confirmed dengue cases. Results obtained by capture immunoglobulin (Ig) M and IgG enzyme-linked immunoassay, real time 1-step polymerase chain reaction, and virus isolation showed that 33 (82.5%) of 40 patients were viremic. Airport fever screening can thus quickly identify imported dengue cases.

Potential application of nonstructural protein NS1 serotype-specific immunoglobulin G enzyme-linked immunosorbent assay in the seroepidemiologic study of dengue virus infection: correlation of results with those of the plaque reduction neutralization test.

ABSTRACT An NS1 serotype-specific indirect enzyme-linked immunosorbent assay (ELISA) was developed to differentiate primary and secondary dengue virus infections and serotypes of primary dengue virus infection. For this report, we carried out retrospective seroepidemiologic studies on serum samples collected from residents of Liuchiu Hsiang, Pingtung County, an isolated island in southern Taiwan during 1997-1998. The results demonstrated that good correlation existed between dengue virus NS1 serotype-specific immunoglobulin G (IgG) ELISA and dengue virus plaque reduction neutralization test (PRNT). Our data suggested that NS1 serotype-specific IgG ELISA could replace PRNT for seroepidemiologic studies to differentiate Japanese encephalitis and dengue virus infections and for dengue virus serotyping.

Concurrent Isolation of Chikungunya Virus and Dengue Virus from a Patient with Coinfection Resulting from a Trip to Singapore

ABSTRACT We report two cases of imported infection in patients who had returned to Taiwan from Singapore: one was coinfected with chikungunya virus and dengue virus type 2, and the other was infected with the same dengue virus. Both viruses were successfully isolated from the coinfected case by using antibody neutralization and a plaque purification technique.

Molecular Characterization and Phylogenetic Analysis of Dengue Viruses Imported into Taiwan during 2008–2010

We present our surveillance results on imported dengue cases in Taiwan during 2008-2010. A total of 734 imported dengue patients were identified. The travelers were arriving from 18 countries, including Southeast Asia, the Indian subcontinent, South Pacific islands, and Latin America. Gene sequences from 358 dengue virus (DENV) isolates were used to perform phylogenetic analyses, thus, providing an updated view of the geographic distribution and dynamic transmission of DENV strains circulating in these countries. Our results suggest that the DENV-1 genotype I and DENV-2 Cosmopolitan genotype comprise the predominant DENV strains circulating in Southeast Asian countries. The DENV-3 Genotype III strain was found to be newly emerging in several Southeast Asian countries, however, the Asian genotype 2 and the Asian/American genotype of DENV-2 strains appeared to be less prevalent in Southeast Asia. Furthermore, imported dengue viruses are representative of the overall patterns of serotype/genotype frequencies of dengue outbreaks that occurred in Taiwan.

Dengue Virus Serotyping Based on Envelope and Membrane and Nonstructural Protein NS1 Serotype-Specific Capture Immunoglobulin M Enzyme-Linked Immunosorbent Assays

ABSTRACT Envelope and membrane (E/M) and nonstructural protein NS1 serotype-specific capture Immunoglobulin M (IgM) enzyme-linked immunosorbent assays (ELISAs) were developed to differentiate four dengue virus serotypes. A total of 93 anti-dengue virus IgM-positive serum samples collected between days 5 and 45 of illness from 59 confirmed dengue patients were analyzed. The results showed that positive serotype specificity could be identified for 86.1 and 47.6% of serum samples tested for E/M-specific IgM antibodies versus 83.3 and 42.9% of serum samples tested for NS1-specific IgM antibodies from patients with primary and secondary dengue virus infections, respectively. Dual analyses with both E/M and NS1 serotype-specific capture IgM ELISAs showed that positive serotype specificity could be correctly identified for 98.6 and 61.9% of all of the primary and secondary serum samples tested, respectively. These findings suggested that E/M and NS1 serotype-specific capture IgM ELISAs have the potential to be of use in dengue virus serotyping.

Characteristics of dengue epidemics in Taiwan

Dengue fever is caused by dengue viruses (DENVs) and is the most common arboviral disease in tropical and subtropical regions of the world, with more than 50 million cases recorded every year. Dengue viruses (serotypes 1e4) are mosquito-borne members of the genus Flavivirus in the family Flaviviridae. In recent decades, the number of dengue cases reported worldwide and the number of countries with endemic dengue has increased dramatically because of the enlarging habitat of the mosquito vectors Aedes sp, growing numbers of susceptible human hosts, and increasing spread of DENVs through rapid and frequent global travel. Dengue disease can manifest in the form of the mild dengue fever or the more severe and potentially fatal dengue hemorrhagic fever/dengue shock syndrome, which has a fatality rate as high as 10e15% depending on the availability of health care. Currently there is no vaccine or therapeutic agent available against dengue fever. Dengue is not considered endemic in Taiwan and the constant importation of DENVs from the neighboring Southeast Asian countries through close commercial links and air travel is responsible for the local outbreaks each year. The dengue hemorrhagic fever cases in Taiwan are highly correlated with advanced age and secondary DENV infection. Studies on molecular epidemiology and phylogenetic analyses of dengue genome sequences reveal that each local outbreak was caused by a single imported DENV strain that disappears with the ending of each outbreak. Table 1 shows the serotype and genotype of DENVs isolated from major dengue epidemics (with more than 10

Screening of dengue virus in field-caught Aedes aegypti and Aedes albopictus (Diptera: Culicidae) by one-step SYBR green-based reverse transcriptase-polymerase chain reaction assay during 2004-2007 in Southern Taiwan.

We carried out virological surveillance of dengue virus (DENV) in field-caught Aedes mosquitoes during 2004-2007 to estimate the monthly prevalence of infected females in dengue high-risk areas of Taiwan. A total of 92,892 Aedes aegypti (43,133 females and 49,759 males) and 79,315 Aedes albopictus (57,319 females and 21,996 males) adults were collected, grouped into 25,654 pools, and processed for virus detection using a one-step SYBR Green-based real-time reverse transcriptase-polymerase chain reaction assay. DENVs were periodically and sympatrically detected in Ae. aegypti females in accordance with major dengue outbreaks and the corresponding dengue serotypes. Only 0.2% of 7628 pools of Ae. aegypti females were positive for DENVs. This resulted in an overall estimated infection rate (maximum likelihood estimation) of 0.970 per 1000 mosquitoes (95% confidence interval [CI] = 0.53-1.65). The total monthly infection rates ranged from 0.50 to 2.23 per 1000 mosquitoes (95% CI = 0.03-10.71). When sampling areas were scaled down to the city level, monthly infection rates increased to 0.73-12.59 (95% CI = 0.06-59.19). Monthly infection rates over all sampling areas and at the city level increased significantly by month. All positive pools were collected in July (one pool), August (two pools), September (one pool), October (three pools), November (four pools), and December (one pool). All four virus serotypes were detected in mosquitoes, which were consistent with dengue serotypes infecting humans in 2004 (DENV-4), 2005 and 2006 (DENV-2 and DENV-3), and 2007 (DENV-1). Our results provide supporting evidence that, in general, DENV infection rates were low in local Aedes mosquito population during 2004-2007 and that transovarial transmission may not be occurring or is occurring at much lower rates than evidenced in some endemic countries.

Screening of mosquitoes using SYBR Green I-based real-time RT-PCR with group-specific primers for detection of Flaviviruses and Alphaviruses in Taiwan

Surveillance for infectious agents carried by mosquitoes is important for predicting the risk of vector-borne infectious diseases. In this study, a method was established to mass-screen mosquitoes for viral infections. The assay detected the viral load of 4 dengue virus (DENV) serotypes (DENV-1, DENV-2, DENV-3, and DENV-4), the Japanese encephalitis virus (JEV), the Sindbis virus and the Chikungunya virus at 1PFU/mL (determined by real-time RT-PCR) in 36.64-43.45 cycles. This method was applied to 75,364 field-captured mosquitoes that were grouped into 10,343 pools. Japanese encephalitis viruses were detected in 25 pools of 906 Culex tritaeniorhynchus females and a single pool of 44 Cx. fuscocephala females. These viruses were isolated from half of the positive pools. Dengue viruses were detected in 2 pools of 43 Aedes aegypti females. Additionally, mosquitoes that were infected orally with dengue viruses in the laboratory were also used to verify the test. The best detection times for individual mosquitoes after being fed virally-contaminated blood were at day 0 and day 10. The number of mosquitoes detected per pool was up to one infected mosquito plus 59 non-infected mosquitoes; the appropriate storage substances for holding samples within 24h included ice cubes and dry ice. This method, combined with a robust and automated RNA-extraction method and a 96 well real-time RT-PCR machine, allows the processing of a large number of samples at once, making it a powerful tool for monitoring simultaneously local and emerging vector-borne infectious diseases of Flaviviruses and Alphaviruses. This study is the first to quantify the viral load in individual mosquitoes over the course of a 16-day extrinsic incubation period.

Phylogenetic analysis of 56-kDa type-specific antigen gene of Orientia tsutsugamushi isolates in Taiwan.

Scrub typhus is a rickettsial disease transmitted to humans through the bite of chigger mites infected with Orientia tsutsugamushi, and is an endemic disease in Taiwan. To elucidate the molecular epidemiology of O. tsutsugamushi, the complete open reading frame of the 56-kDa type-specific antigen gene sequence of strains isolated from scrub typhus patients were determined and analyzed. A total of 116 isolates of O. tsutsugamushi were successfully isolated from patients infected in diverse geographic origins including Taiwan and three offshore islets, Kinmen, Matsu, and Penghu between May 2006 and December 2007. Sequence analysis revealed that 22 distinct sequence types could be identified that were broadly distributed in different clusters of the phylogenetic tree. Most of the isolates belong to Karp, Kawasaki, and Kuroki genotypes and are closely related to strains from Thailand, Japan, and Korea, whereas unique isolates different from other countries were also found in Taiwan. Distinct seasonal distributions were found in different sequence types. Some sequence types caused disease in the cold season, whereas others caused disease in the warm season.