Cheo Lin

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.

A Dengue Vector Surveillance by Human Population-Stratified Ovitrap Survey for Aedes (Diptera: Culicidae) Adult and Egg Collections in High Dengue-Risk Areas of Taiwan

ABSTRACT Aedes aegypti L. is the primary dengue vector in southern Taiwan. This article is the first report on a large-scale surveillance program to study the spatial-temporal distribution of the local Ae. aegytpi population using ovitraps stratified according to the human population in high dengue-risk areas. The sampling program was conducted for 1 yr and was based on weekly collections of eggs and adults in Kaohsiung City. In total, 10,380 ovitraps were placed in 5,190 households. Paired ovitraps, one indoors and one outdoors were used per 400 people. Three treatments in these ovitraps (paddleshaped wooden sticks, sticky plastic, or both) were assigned by stratified random sampling to two areas (i.e., metropolitan or rural, respectively). We found that the sticky plastic alone had a higher sensitivity for detecting the occurrence of indigenous dengue cases than other treatments with time lags of up to 14 wk. The wooden paddle alone detected the oviposition of Ae. aegypti throughout the year in this study area. Furthermore, significantly more Ae. aegypti females were collected indoors than outdoors. Therefore, our survey identified the whole year oviposition activity, spatial-temporal distribution of the local Ae. aegypti population and a 14 wk lag correlation with dengue incidence to plan an effectively proactive control.

Comparison of the Efficacy of CO2-Baited and Unbaited Light Traps, Gravid Traps, Backpack Aspirators, and Sweep Net Collections for Sampling Mosquitoes Infected with Japanese Encephalitis Virus

ABSTRACT: Two field studies were conducted to determine the efficacy of mosquito collection methods for species composition, species abundance, and Japanese encephalitis virus infection rates in Taiwan. Traps evaluated included John W. Hock (JH) model UD black light traps, JH model 1012 new standard miniature CDC light traps, JH model 1712 CDC gravid traps, and Taiwan-made Pest-O-Lite light traps. Backpack aspirators and sweep nets were also used to collect the resting population. Culex tritaeniorhynchus in all studies and Mansonia uniformis in the Taipei areas were the two most abundance species collected. Dry ice-baited UD black light traps were effective in regard to species diversity, species abundance, and Japanese encephalitis virus infection rates. The unbaited Pest-O-Lite light traps collected significantly more female mosquitoes than the UD black light traps but performed similarly with regard to species diversity and male mosquito collection. Most mosquitoes collected by Pest-O-Lite light traps were dried and not suitable for virus detection. Dry ice-baited CDC light traps collected significantly fewer mosquitoes than other light traps. Although CO2-baited UD black light traps with octenol attracted more mosquitoes, no statistical significance was found compared to CO2-baited UD black light traps without octenol. Japanese encephalitis viruses were isolated from half of the positive pools in UD black light traps and CDC light traps.

Susceptibility of Aedes albopictus and Aedes aegypti to three imported Chikungunya virus strains, including the E1/226V variant in Taiwan

BACKGROUND/PURPOSE An E1/226V variant Chikungunya virus (CHIKV) efficiently transmitted by Aedes albopictus to humans poses a significant threat to public health for those areas with the presence of Aedes albopictus, including Taiwan. METHODS We infected three imported CHIKV isolates including the E1/226V variant with Ae. albopictus and Aedes aegypti in the laboratory to understand the disease risk. Viral RNA was measured by real time reverse transcription polymerase chain reaction. RESULTS The viral susceptibility varied by virus strain and mosquito species and strain. The Asian virus strain started to replicate at 5-6 days post infection (dpi) with the maximum virus yield, ranging from 10(3.63) to 10(3.87) at 5-10 dpi in both species. The variant CHIKV Central/East/South African (CESA) virus genotype replicated earlier at 1 dpi with the maximum virus yield ranging from 10(5.63) to 10(6.52) at 3-6 dpi in Ae. albopictus females while the nonvariant virus strain replicated at 1-2 dpi with the maximum virus yield ranging from 10(5.51) to 10(6.27) at 6-12 dpi. In Ae. aegypti, these viruses replicated at 1-2 dpi, with maximum yields at 4-5 dpi (range from 10(5.38) to 10(5.62)). CONCLUSION We concluded that the risk of CHIKV in Taiwan is high in all distribution areas of Ae. aegypti and Ae. albopictus for the CESA genotype and that the E1/226V variant virus strain presents an even higher risk.