Three-year surveillance of culicine mosquitoes (Diptera: Culicidae) for flavivirus infections in Incheon Metropolitan City and Hwaseong-si of Gyeonggi-do Province, Republic of Korea

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Three-Year Surveillance of Culicine Mosquitoes (Diptera: Culicidae) for Flavivirus Infections in Incheon Metropolitan City and Hwaseong-Si of Gyeonggi-do Province, Republic of Korea Seung JEGAL , Hojong JUN , Myung-Deok KIM-JEON , Seo Hye PARK , Seong Kyu AHN , Jinyoung LEE , Young Woo GONG , Kwangsig JOO , Mun Ju KWON , Jong Yul ROH , Wook-Gyo LEE , Woojoo LEE , Young Yil BAHK , Tong-Soo KIM PhD PII: DOI: Reference:

S0001-706X(19)30425-5 https://doi.org/10.1016/j.actatropica.2019.105258 ACTROP 105258

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Acta Tropica

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Please cite this article as: Seung JEGAL , Hojong JUN , Myung-Deok KIM-JEON , Seo Hye PARK , Seong Kyu AHN , Jinyoung LEE , Young Woo GONG , Kwangsig JOO , Mun Ju KWON , Jong Yul ROH , Wook-Gyo LEE , Woojoo LEE , Young Yil BAHK , Tong-Soo KIM PhD , ThreeYear Surveillance of Culicine Mosquitoes (Diptera: Culicidae) for Flavivirus Infections in Incheon Metropolitan City and Hwaseong-Si of Gyeonggi-do Province, Republic of Korea, Acta Tropica (2019), doi: https://doi.org/10.1016/j.actatropica.2019.105258

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Three-Year Surveillance of Culicine Mosquitoes (Diptera: Culicidae) for Flavivirus Infections in Incheon Metropolitan City and Hwaseong-Si of Gyeonggi-do Province, the Republic of Korea

Seung JEGAL1#, Hojong JUN2#, Myung-Deok KIM-JEON1, Seo Hye PARK2, Seong Kyu AHN2, Jinyoung LEE2, Young Woo GONG1, Kwangsig JOO1, Mun Ju KWON1, Jong Yul ROH3, Wook-Gyo LEE3, Woojoo LEE4, Young Yil BAHK5*,, Tong-Soo KIM2* 1

Department of Infectious Diseases Diagnosis, Incheon Metropolitan City Institute of Public

Health and Environment, Incheon 22320, Korea; 2Department of Tropical Medicine, Inha University School

of Medicine, Incheon 22212, Korea; 3Division of Vectors and Parasitic

Diseases, Korea Centers for Disease Control and Prevention, Osong 28159, Korea; 4

Department of Statistics, Inha University,

Incheon 22212, Korea; 5Department of

Biotechnology, College of Biomedical and Health Science, Konkuk University, Chungju 27478, Korea

Running title: Three-year surveillance of Culicine Mosquitoes (Diptera: Culicidae) for Flavivirus Infections

*

Corresponding authors:

Tong-Soo KIM, PhD Young Yil BAHK, PhD

#

1

These authors contributed equally to this work.

Abstract Japanese encephalitis virus (JEV) is a single stranded positive sense RNA virus of the genus Flavivirus that belongs to family Flaviviridae and emerged as one of the most pivotal form of viral encephalitis. The virus is transmitted to humans by mosquito vector and is an etiological agent of acute zoonotic infection. In this study, we investigated distribution and density over 3-year period in central regions of Korean peninsula. We selected two cities as mosquito-collecting locations and subdivided them into five collection sites; downtown Incheon Metropolitan City as a typical urban area, and the Hwaseong-si area as a rural area. A total of 35,445 female culicine mosquitoes were collected using black light traps or BG SentinelTM traps from March to November 2016-2018.

Aedes (Ae.) vexans nipponii was the

most frequently collected specimens (48.91%), followed by Culex (Cx.) pipiens (32.05%), Ochlerotatus (Och.) dorsalis (13.58%), Och. koreicus (1.68%), and Cx. tritaeniorhynchus (1.49%). In the urban area, Cx. pipiens was the predominant species (92.21%) and the other species accounted for <5% of the total mosquitoes collected. However, in the rural area, Ae. vexans nipponii had the highest population (61.90%), followed by Och. dorsalis (17.10%), Cx. tritaeniorhynchus (1.84%) and Och, koreicus (1.78%). Culicine mosquitoes were identified at the species level, placed in pools of up to 30 mosquitoes each, and screened for flavivirus RNA using the SYBR Green-based RT-PCR. Three of the assayed 1,092 pools were positive for Chaoyang virus from Ae. vexans nipponii and Japanese encephalitis virus from Cx. pipiens. The maximum likelihood estimations (the estimated number of virus-positive mosquitoes/1,000 mosquitoes) for Ae. vexans nipponii positive for Chaoyang virus and Cx. pipiens for Japanese encephalitis virus were 3.095 and 0.20, respectively. The results of our study demonstrate that although mosquito-borne diseases were not detected in the potential vectors, enhanced monitoring and long-term surveillance of these vector viruses are of great public health importance.

2

Keywords: mosquito, flavivirus, Tsutsugamushi, vector-borne diseases, long-term surveillance, climate change, urban infection

1. Introduction With the impact of climate change due to global warming on public health becoming widely recognized, vector-borne diseases have garnered increased focus (Kovats and Haines, 1995; Githeko et al., 2000; Ogden, 2017; McMichael et al., 2006) and feasible changes in the prevalence of vector-borne diseases have attracted particular attention. Climate change is an unequivocal phenomenon and is expected to accelerate in the future, especially in the Korean Peninsula where the extent of climate change is largely being felt (Min et al., 2013). The Korean tropical rain belt called ‘the Jangma front’ forms in the western North Pacific takes approximately 4-5 weeks to traverse the Korean Peninsula. In recent years, the Jangma front has been moving more quickly, taking less than 3 weeks resulting in more extreme weather (warm and severe cold spells) and locally heavy showers. In addition, the annual mean temperature has been increasing at a rate of 0.52°C per decade, this increase in rate is significantly larger over urbanized areas (Chung et al., 2004). The incidence and geographic distribution of vector-borne diseases are anticipated to change as a result (Rogers et al., 2013). In addition, the possibility of noticeable foreign infectious diseases, such as yellow fever, malaria, West Nile fever, and dengue fever, occurring in Korea is growing every year rather than decreasing despite rapid economic development, and cases of inflow of dengue fever have been confirmed in Korea (KCDC, 2016). Meanwhile, Singapore focused on vector mosquito control from 1960 to 1970 to effectively prevent the spread of dengue fever (Ool et al., 2006). Emerging communicable infectious diseases continue to threaten public health, in particular, and, since Ae. albopictus, a major vector for dengue virus infection, has been discovered and collected in Korea and Cx. pipiens, a vector of West Nile fever, is widely distributed throughout Korea (KCDC, 2016, Chang et al., 2018, Park and Cho, 2014), it has 3

become necessary to monitor these vectors as a measure against the inflow of these organisms, which are elegant transmission vectors for a number of infectious agents covering both parasites and pathogens being a major threat to public health. In many region of the world, mosquitoes are responsible for the transmission of numerous severe disease-causing pathogens, thus being a major threat to human health (Reegan et al., 2016). Mosquito-borne infections contribute to the public burden causing millions of deaths and hundreds of millions of cases annually (World Health Organization, 2013). In fact, they are considered to rank first place as vectors of human infectious diseases. In Mosquito Week of

2014, Bill Gates, a co-founder of Microsoft Corporation and the Bill

and Melinda Gates Foundation, announced that the deadliest animal in the world is the mosquito due to it carrying devastating diseases (Gates, 2014). The three major genera of mosquitoes are Anopheles, which is responsible for the transmission of malaria; Culex, which is a vector of filariasis and several arboviruses; and Aedes, which transmits dengue fever, Zika fever, chikungunya fever, and yellow fever viruses. The major mosquito-borne diseases that pose a great threat to public health are dengue fever, chikungunya fever, malaria, filariasis, Zika fever, yellow fever, and West Nile fever, especially in tropical and subtropical regions (Mackenzie et al., 2004; WHO, 2016). High-profile elimination is underway for many infectious diseases. It is generally accepted that there is a strong association between vector mosquito density and disease, and a strong association between climate variables and mosquito abundance and the consequential increase in disease transmission (Li et al., 2014; Wilke et al., 2017; Chaves et al., 2012). The genus Flavivirus (family Flaviviridae) comprises a diverse group of viruses. The mosquito-borne flaviviruses, which are the most recognized since they are causative pathogens in humans and worldwide, are potential arthropod-borne pathogens that cause lifethreatening diseases such as Japanese encephalitis, yellow fever, Rift Valley fever, dengue fever, West Nile fever, and Zika fever (Braks et al., 2014; Blitvich and Firth, 2015; 4

Holbrook, 2017; Weaver et al., 2016); these diseases cause significant morbidity and mortality worldwide.

Japanese encephalitis virus (JEV) is distributed in eastern and

southern Asia. Cx tritaeniorhynchus is the major vector mosquito of JEV in most part of Asia and other Culex mosquitoes play roles as transmission vectors (van den Hurk et al., 2009). Furthermore, it is evident that Japanese encephalitis is endemic in Korea and the appearance of JEV genotype V with Cx. bitaeniorhynchus may mark the beginning of a genotype shift (Takhampunya et al., 2011; Kim et al., 2015). The detection of JEV genotype V from Cx. bitaeniorhynchus mosquitoes was in Gyeonggi-do in 2010 and this case was the third case following the case reports Malaysia and China (Mohammed et al., 2011; Li et al., 2011). In Korea, Japanese encephalitis is once a major public health problem and has declined since 1980s due to improved living conditions, a mosquito eradication program and a national vaccination program (Sohn, 2000). In this study, to monitor and reduce the potential for the autochthonous transmission of imported mosquito-borne viruses as a result of climate change in Korea, we evaluated the species composition, diversity, abundance, and distribution of mosquitoes and their pathogens in three locations in the downtown area of Incheon Metropolitan City (Incheon) and in a cowshed in the Hwaseong-Si area. This study was designed and practiced as a project of Metropolitan Center for Vector Surveillance in Climate Change in Inha University, College of Medicine, funded from KCDC. Since the spread of mosquito-borne infectious diseases from foreign countries cannot be prevented through quarantine only, continuous and systematic attention and powerful surveillance are required. The results of this study could be the basis for future epidemiological studies and risk assessment of mosquito-borne pathogens affected by climate change in Korea. 2. Materials and methods 2.1. Collection sites From March to November, 2016~2018, observational, descriptive and prospective 5

monitoring was

performed to identify any circulating flaviviruses in different mosquito

species in some areas in the

middle of the Korean Peninsula. Vector mosquitoes were

collected using black light traps or BG

SentinelTM traps (Biogents, Regensburg,

Germany) for a 24 hr period every two weeks in four regions: downtown Incheon (Bupyeong dong, Yeonhui dong, and Shinheung dong) as a typical urban area, and the Hwaseong-si area (a cowshed in Hogok-ri, Gyeonggi-do (Province)) as a rural area. Table 1 presents the geographic coordinates recorded using a handheld GPS tracker where traps were set for the collection of mosquitoes. Incheon is a city located in northwestern Korea, bordering on the Seoul Metropolitan City and Gyeonggi-do. It has a humid subtropical/continental climate (Kottek et al., 2006) and is of average size compared with the rest of Korea’s cities. Despite the predominant continental climate, traces of an oceanic climate can be detected in Incheon due to its location on the coast. The Köppen-Geiger climate classification for Incheon is Dwa (Continental, Dry summer and

Hot summer).

The mean annual temperature for the year

in Incheon is 12.1°C while the highest temperature recorded 38.9°C (August 16, 1949) and the lowest -21.0°C (January 11, 1931). The mean annual precipitation is 1234.4 mm, which is low compared to other regions on similar latitudes. Hwaseong-si is a city in Gyeonggi-do Province, Korea located in the western area of the Korean Peninsula and has the largest area of farmland of any region in Gyeonggi-do Province. The temperature in winter is low along the coast as it is located in the lower plains and is close to the Yellow Sea, where the water is shallow. The mean annual temperature for the year in Hwaseong is 11.5°C and the mean annual precipitation is at 1,259 mm. The climate classification of the Hwaseong-si area based on the Köppen-Geiger method is also Dwa.

2.2. Collection of mosquitoes A total of four traps were set up at each of the study sites. All materials collected were transported to the Laboratory of the Department of Infectious Diseases Diagnosis, Incheon 6

Metropolitan City Institute of Public Health and Environment. Adult mosquitoes were continuously collected using black light traps and BG Sentinel traps for 24 hr at 2-week intervals from March to November, 2016-2018. Mosquitoes were transferred from the traps into Styrofoam cooler on wet ice, and transported to the laboratory. Female mosquitoes were identified and morphologically confirmed using taxonomic keys (Tanka et al., 1979; Lee, 1999; Ree, 2003) and were compared with standard specimens in the collection of the School of Medicine, Inha University and the Korea Centers for Disease Control and Prevention (KCDC). The abbreviations for the mosquito genera and subgenera follow the standardization proposed by Reinert (2001). The collected mosquitoes were pooled (1 - 50/pool) by species, collection site, and time of survey and quickly transferred to microcentrifuge tubes at 4 °C, where their species were identified and they were assayed for flaviviruses including West Nile virus, JEV, yellow fever virus, and dengue fever virus. A total of 1,092 mosquito pools were analyzed for flavivirus identification. 2.3. RNA extraction and quantitative real-time (qRT)-PCR analysis of mosquito pools for flavivirus detection To improve the extraction and to achieve high efficiency and reduced variability in the total RNA yields, bead beaters with a QIAampTM viral RNA mini kit were used in accordance with the manufacturer’s instructions (Qiagen 52904, Qiagen GmbH, Hilden, Germany). RNA was extracted from the homogenized mosquito samples after which the extracted total RNA was resuspended in RNase-free water containing RNAsinTM Plus RNase inhibitor (Promega, Madison, WI, USA) and then stored at -70°C until use. qRT-PCR assays were conducted using a Verso SYBR Green 1-step qRT-PCR kit (Thermo AB4104, Thermo-Fisher Scientific, Waltham, MA, USA) in accordance with the manufacturer’s protocol for the detection of pathogens in mosquitoes (Yang et al., 2010). The species-specific primers for nonstructural protein

5

(NS

5)

gene

used

were

GCCATATGGTACATCATGTGGCTGGGA-GC-3’, 7

as

follows:

FL-R3

FL-F1

(antisense)

(sense)

5’-

5’-GTKATTCT-

TGTGTCCCAWCCGGCTGTGTCATC-3’

and

FL-R4

(antisense)

5’-

GTGATGCGRGTGTCCCAGC-CRGCKGTGTCATC-3’ (amplified DNA band size 202 bp). qRT-PCR was performed using 1.0 step SYBR RT-PCR buffer, each with 1

PrimeScripTM 1-step enzyme mix, 12.5

2X1

primer, and 1 pg to 100 ng total RNA in a 25

total reaction volume. qRT-PCR conditions for each reaction were 30 min at 50°C, followed by 10 min at 95°C, then 15 s at 95°C, 20 s at 58°C and 30 s 72°C for 45 cycles, and finally annealed for 1 min at 60°C. Samples that generated a detectable fluorescence signal when assayed were determined as flavivirus positive by monitoring the increase in fluorescence of a dye labeled oligonucleotide probe (Chao et al., 2007; Yang et al., 2010). The amplification products were sequenced in both directions in an automated sequencer for flavivirus sequence confirmation. The PCR products were then sent to Macrogen company (Macrogen Korea, Seoul, Korea), where the obtained DNA sequences were edited and assembled for flavivirus classification. 2.4. Statistics We test whether a certain trend of the proportion of flavivirus RNA positive mosquitoes exists over time using a simple logistic regression. R (version 3.5.0, The R foundation for statistical computing, Vienna, Austria) was used for the analysis. 3. Results 3.1. Collection of mosquitoes A total of 35,445 (1,092 pools) female mosquitoes comprising 14 species (Ae. albopictus, Ae.

vexans,

Armigeres

subalbatus,

Coquillettidia

ochracea,

Cx.

inatomii,

Cx.

bitaeniorhynchus, Cx. orientalis, Cx. pipiens, Cx. tritaeniorhynchus, Cx. vagans, Mansonia uniformis, Och. dorsalis, Och. koreicus, Och. togoi) were collected and assayed for the presence of flaviviruses from March to November 2016-2018 (Table 2) at three locations in the downtown of Incheon and one location in a cowshed in the Hwaseong-si area. Ae. vexans nipponii was the most frequently collected mosquito (n=17,333, 48.91%), followed by 8

Cx. pipiens (n=11,360, 32.05%), Och. dorsalis (n=4,812, 13.58%), Och. koreicus (n=594, 1.68%), and Cx. tritaeniorhynchus (n=529, 1.49%). The remaining nine species comprised <1.00% of the total number of mosquitoes collected. The mosquitoes had periods of high prevalence (Fig. 1): Ae. vexans nipponii populations increased from early May until early September peaking in early July; Cx. pipiens populations increased from late March to early November, peaking in late June and Och. dorsalis increased from early April to early October peaking in late May (Fig. 1 and Table 2); and Ae. vexans nipponii females showed bimodal collection rates, with the highest numbers collected during the first week of July (4,191/6,218 (67.40%)) and then high again during the third week of August (2,215/2,971 (74.55%)) (Fig. 1 and Table 2). However, there were prevalence patterns for the urban and rural areas and as expected, fewer mosquitoes were collected in the urban area compared with the rural area of Hwaseong, a finding similar to many studies in other nations (Honnen and Monaghan, 2017). Tables 3 and 4 describe the population densities of the collected culicine female mosquitoes by species between the downtown Incheon areas and the cowshed in Hwaseong (Fig. 2). Cx. pipiens was the predominant species in the urban areas (n=6,956 from a total of 7,544 (92.21%)) and the other species made up <5% of the total mosquitoes collected during the collection period. However, in the rural area (the cowshed in Hwaseong), Ae. vexans nipponii had the highest population (n=17,272 from a total 27,901 (61.90%)), followed by Och. dorsalis (n=4,770, 17.10%), Cx. tritaeniorhynchus (n=513, 1.84%) and Och. koreicus (n=498, 1.78%) (Table 5). The other species made up <1% of the total mosquitoes collected during the study period. The population densities of these species were too low to be considered important vectors in our study sites. 3.2. Pathogens in mosquito vectors The qRT-PCR amplicons from the pooled mosquitoes were sequenced, and the sequences were edited and assembled to obtain small fragments. From the analysis of the derived nucleotide sequences for matching genotypes using the NCBI-BLAST service, three pools of 9

Cx. pipiens and Ae. vexans nipponii of the 1,092 tested pools were positive for flavivirus: one comprised JEV and two Chaoyang virus (Table 6). The JEV-positive pool was phylogenetically identified as genotype V and was from Cx. pipiens, whereas the two Chaoyang virus pools were from Ae. vexans nipponii (Cook et al., 2006; Lee et al., 2013). The maximum likelihood estimations (MLE) (the estimated number of virus-positive mosquitoes/1,000 mosquitoes) for Ae. vexans nipponii positive for the Chaoyang virus and Cx. pipiens for JEV were 3.095 and 0.20, respectively (Table 6). These mosquitoes were collected from downtown Incheon (Bupyeong-dong) during the first week in September 2018 and at the cowshed in

Hwaseong during the second week in September and the second week

in October 2017. Ae. vexans nipponii, the most frequently captured mosquito (48.91%), is an anthropophilic species common to Korea and is known to be a mosquito host for the Chaoyang virus (an insect-specific virus), a newly reported flavivirus in Korea (Kim et al., 2004; Lee et al., 2013; Takhampunya et al., 2014). However, the role of the Chaoyang virus in animal or human diseases is currently unknown. Cx. pipiens is a nuisance mosquito, which widely exists throughout Korea, was the second most frequently collected mosquito (32.05%) (Table 3) and the most frequently captured mosquito in the urban areas (92.21%) (Table 3). However, the other types of flavivirus were unidentified in the study sites with Verso SYBR Green-based 1-step qRT-PCR assay. We test whether a certain trend of the proportion of flavivirus RNA positive mosquitoes exists over time using a simple logistic regression. The outcome variable is the number of flavivirus RNA positive mosquitoes from 2016 to 2018, and the covariate is the year. The regression coefficient associated with the year is 0.3432 (se=0.1159), and it implies that there is a statistically significant increasing trend. However, this trend is based on only three observations so a further follow-up is needed to confirm this finding.

10

4. Discussion and conclusion To date, the climate of Korea has gradually changed into a subtropical one, and this might make the Korean Peninsula environmentally suitable for the proliferation of vector mosquitoes in the near future (Yeom, 2017). In addition, owing to the effect of urbanization and the continuous increase in international travelers, the incidence of mosquito-borne diseases, such as Japanese encephalitis, chikungunya fever, and Zika virus fever is also increasing. Currently, the indigenous mosquito-borne infectious diseases in Korea are vivax malaria and Japanese encephalitis, which occur in limited areas (Lee et al., 2015;Bahk et al., 2018). Although no cases of other flavivirus diseases, such as dengue fever, chikungunya fever, Zika virus fever, yellow fever, and West Nile fever, have been reported to be indigenous to Korea yet, it is highly likely that based on the existence of the vector mosquitoes in Korea, the situation might change if they are introduced from outside the country. Flavivirus is a genus of viruses in the family Flaviviridae and had attracted little attention until 2000, when infections were reported in urban areas throughout the tropical and subtropical regions of the world (Blitvich and Firth, 2015). Most of these viruses are transmitted by the bite of infected arthropods with mosquitoes and ticks being the major vectors. In fact, global climate warming has had an indirect effect on the increase in the number of patients with vector-borne diseases but a direct effect on the expansion of the infected areas of the vectors, such as mosquitoes and ticks. In this

respect,

controlling continuous and systematic vector monitoring is required. Human infections with most of these viruses are incidental, as humans are unable to replicate the virus to sufficiently high titers to reinfect the arthropods needed to continue the viral lifecycle, with the exceptions of the yellow fever, dengue and Zika fever viruses (Chambers et al., 1990). In Korea, these viruses acquired considerable attention in 2016 when more than 100 imported cases of dengue fever were reported in addition to 16 imported cases of Zika fever from January to October 2016 (KCDC, 2016). In addition, Lee et al. (2018) demonstrated that higher temperatures 11

have momentously augmented the potential threat of domestic outbreaks of the diseases in Korea. In a nationwide survey in Korea, Kim et al. (2015) studied the population densities of culicine mosquitoes and reported that Ae. vexans nipponii was the highest collected mosquito (50.7%), followed by C. tritaeniorhynchus (36.6%), Cx. pipiens (7.5%), Och. dorsalis (2.1%) and Cx. bitaeniorhynchus (1.3%). In contrast, our monitoring identified that the population density for Ae. vexans nipponii was 49.13% (17,333/35280) and those of Cx. pipiens, Och. dorsalis, Och. koreicus, and

Cx. tritaeniorhynchus were 31.78%, 13.62%, 1.68%, and

1.50%, respectively. Cx. tritaeniorhynchus is the primary vector throughout Asia, including Korea, and Cx. vishnui and Cx bitaeniorhynchus have been recently implicated as vectors of Japanese encephalitis in Korea, India and other parts of the world (Rosen, 1986; Takhampunya et al., 2011; Kim et al., 2011). From 2011 to 2016, a total of 131 clinical cases of Japanese encephalitis including 17 deaths were reported in Korea (KCDC, 2017). Although approximately 1,600 cases of Japanese encephalitis were reported annually until the 1970s, the cases decreased to 131 including 17 deaths in Korea from 2011 to 2016 due to the introduction of a vaccine and a mandatory vaccination program (KCDC, 2017; Bae et al., 2018). The number of Japanese encephalitis cases show a seasonal pattern and most cases are reported between August and November (Jung et al., 2016). JEVs are generally classified into five genotypes based on similarities in the E gene. Genotypes II and III were isolated before 1951, and genotype III was isolated in the 1980s, , genotype I after the 1990s, , genotype V from Cx. bitaeniorhynchus, Cx. orientalis, and Cx. pipiens since 2010 (Yun et al., 2010; Bae et al., 2018). The appearance of JEV genotype V in Korea in Cx. bitaeniorhynchus from 2010 may mark the beginning of a significant genotype shift (Mohammed et al., 2011; Li et al., 2011). Chaoyang virus was primarily isolated and identified from Ae. vexans in rural corrals in Chaoyang city in Liaoning province in China (Wang et al., 2009; Liu et al., 2011). This virus 12

induces cytopathic effects in C6/36 cells resulting to cell deformation, disordered arrangement, aggregation and death. However, the potential public and veterinary health concerns of this virus remain to be determined (Liu et al., 2011). In conclusion, we tried to determine the status of flaviviral diseases transmitted by mosquitoes, and investigate their target vector diversity, abundance and distribution in Incheon and Hwaseong. There are significant public health implications requiring effective surveillance and clear and routine communications of findings to regional and global partners. Although mosquito-borne diseases were not detected in the potential vectors in this study, monitoring patients with suspected mosquito-borne diseases should be given precedence to reduce potential viral transmission to resident populations. Currently, in addition to the existing driving forces of vector-borne diseases, such as seasonal variation, socio-economic status, vector control programs, environmental changes and drug resistance, evidences suggest that climate variability has a direct influence on the epidemiology of vector-borne disease (Githeko et al., 2000). This evidence has been assessed at the global basis to elucidate the feasible consequences of the future climate change in view of the increased likelihood of climate change. In field, the fraction of changes in mosquito-borne diseases attributable to global climate change is still uncertain and this indistinctness is a big burden to evidence-based health strategies. However, it is evident that the global-warming scenarios of climate change have fueled prospective view that mosquito vectors could increase in distribution, reproduction and activity and finally give rise to augmentation in the prevalence of mosquito-borne pathogens causing public health burdens. Given increasing global travel and global warming, the risk of invasion of mosquito-borne diseases via human activity and transportation has become incessant, thus, enhanced monitoring and long-term surveillance of these vector viruses are of great public health significance.

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Acknowledgements This work was supported by the Korea Centers for Disease Control and Prevention (4851304-320) in the Republic of Korea.

Conflict of interest The authors declare that they have no conflicts of interests.

References Bae, W., Kim J.H., Kim, J., Lee, J., Hwang, E.S., 2018. Changes of epidemiological characteristics of Japanese encephalitis viral infection and birds as a potential viral transmitter in Korea. J. Korean Med. Sci. 33(9), e70. https://doi.org/10.3346/jkms.2018.33.e70. Bahk, Y.Y., Lee, H.-W., Na, B.-K., Kim, J., Jin, K., Hong, Y.-S., Kim, T.-S., 2018. Epidemiological characteristics of re-emerging vivax malaria in the Republic of Korea. Korean J. Parasitol. 56(6), 531-543. https://doi.org/10.3347/kjp.2018.56.6531. Blitvich, B.J., Firth, A.E., 2015. Insect-specific flaviviruses: A systematic review of their discovery, host range, mode of transmission, superinfection exclusion potential and genomic organization. Viruses 7, 1927-1959. https://doi:10.3390/v7041927. Braks, M., van Ginkel, R, Wint, W., Sedda, L., Sprong, H., 2014. Climate change and public health policy: translating the science. Int. J. Environ. Res. Public Health 11, 13-29. https://doi:10.3390/ijerph110100013. Chambers, T.J., Hahn, C., Galler, R., Rice, C.M., 1990. Flavivirus genome organization, expression, and replication. Ann. Rev. Miciobiol. 44, 649-688. https://doi:10.1146/annrev.mi.44.100190.003245. Chang, K.-S., Kim, G.-H., Ha, Y.-R., Jeong, E.K., Kim, H.-C., Klein, T.A., Shin, S.H., Kim, E.J., Jegal, S., Chung, S.J., Ju, Y.-R., Jee, Y.M., 2018. Monitoring and control of Aedes 14

albopitus, a vector of Zika virus, near residences of imported Zika virus patients during 2016 in South Korea. Am. J. Trop. Med. Hyg. 98, 166-172. https://doi:10.4269/ajtmh.170587. Chao, D.-Y, Davis, B.S., Chang, G.-J., 2007. Development of multiplex real-time reverse transcriptase PCR assays for detecting eight medically important Flaviviruses in mosquitoes. J. Clin. Microbiol. 45(2), 584-589. https://doi:10.1128/JCM.00842-06. Chaves, L.F., Morrison, A.C., Kitron, U.D., Scott, T.W., 2012. Nonlinear impacts of climate variability on the density-dependent regulation of an insect vector of disease. Glob. Change Biol. 18, 457-468. https://dx.doi.org/10.1111/j.1365-2486.2011.02522.x. Chung, Y.-S., Yoon, M.-B., Kim, H.-S., 2004. On climate variations and changes observed in South Korea. Climate Change 66, 151-161. Cook, S., Holmes, E.C., 2006. A multigene analysis of the phylogenetic relationships among the flaviviruses (Family: Flaviviridae) and the evolution of vector transmission. Arch. Virol. 151, 309-325. https://doi:10.1007/s00705-005-0626-6. Gates, B., 2015. The Gates Notes. Available from https://www.gatesnotes.com/Health/MostLethal-Animal-Mosquito-Week. Githeko, A.K., Lindsay, S.W., Confalonieri, U.E., Patz, J.A., 2000. Climate change and vector-borne diseases: a regional analysis. Bull. World Health Organ. 78, 1136-1147. Holbrook, M.R., 2017. Historical perspectives on Flavivirus research. Viruses 9, 97-115. https://doi:10.3390/v9050097. Honnen, A.-C., Monaghan, M.T., 2017. City-dwellers and country folks: Lack of population differentiation along an urban-rural gradient in the mosquito Culex pipiens (Diptera: Culidae). J. Insect Sci. 17(5), 1-9. https://doi:10.1093/jisesa/iex086. Jung, C.W., Yang, T.U., Hong, J.I., 2016. Epidemiology of Japanese encephalitis in Korea, 2011-2015. Public Health Wkly. Rep. 9(12), 211-216. KCDC, 2016. Disease Web Statistics System. Available at: 15

http://is.cdc.go.kr/dstat/jsp/stat/stat0001.jsp. Accessed July 20, 2017. KCDC, 2017. Infectious disease agent surveillance. https://www.cdc.go.kr/CDC/info/CdcKrInfo0403. Jsp?menuIds=HOME001-MNU1134MNU1153-MNU0046. Updated 2017. Accessed Aug. 28, 2017. Kim, H., Cha, G.W., Jeong, Y.E., Lee, W.G., Chang, K.S., Roh, J.Y., Yang, S.C., Park, C., Shin, E.H., 2015. Detection of Japanese encephalitis virus genotype V in Culex orientalis and Culex pipiens (Diptera Culicidae) in Korea. PLoS ONE 10(2), e0116547. https://doi:10.1371/journal.pone.0116547. Kim, H.C., Chong, S.T., Pike, J.G., O’Guinn, M.L., Pacha, L.A., Lee, H.C., Klein, T.A., 2004. Sesonal prevalence of mosquitoes collected from light traps in the Republic of Korea, 2002. Entomol. Res. 34(3), 177-186. https://doi.org/10.1111/j.1748-5967.2004.tb00111.x Kim, H.C., Klein, T.A., Takhampunya, R., Evans, B.P., Mingmongkolchai, S., Kengluecha, A., Grieco, J., masuoka, P., Kim, M.S., Chong, S.T., Lee, J.K., Lee, W.J., 2011. Japanese encephalitis virus in culicine mosquitoes (Diptera: Culicidae) collected at Daeseongdong, a village in the demilitarized zone of the Republic of Korea. J. Med. Entomol. 48, 1250-1256. https://doi:10.1603/me11091. Kim, H.C., Takhampunya, R., Tippayachai, B., Chong, S.T., Park, J.Y., Kim M.S., Seo, H.J., Yeh, J.Y., Lee, W.J,. Lee, D.K., Klein, T.A., 2015. Japanese encephalitis virus in culicine mosquitoes (Diptera: Culidae) of the Republic of Korea, 2008-2010. Mil. Med. 180 (2), 158167. https://doi:10.7205/MILMED-D-14-00206. Kottek, M., Grieser, J., Beck, C., Rudolf, B., Rubel, F., 2006. World map of the KoppenGeiger climate classification updated. Meteorol. Z. 15 (3), 259–263. https://doi:10.1127/0941-2948/2006/0130. Kovats, S., Haines, A., 1995. The potential health impacts of climate change: an overview. Med. War. 11, 168-178. Lee, J.S., Grubaugh, N.D., Kondig, J.P., Turell, M.J., Kim, H.C., Klein, T.A., O’Guinn, M.L., 16

2013. Isolation and genomic characterization of Chaoyang virus strain ROK144 from Aedes vexans nipponii from the Republic of Korea. Virology 435, 220-224. http://doi:10.1016/j.virol.2012.10.020. Lee, H., Kim, J.E., Lee, S., Lee, C.H., 2018. Potential effects of climate change on dengue transmission dynamics in Korea. PLoS ONE 13(6), e0199205. https://doi.org/10.1371/kournal.pone.0199205. Lee, H.Y., Park, Y.J., Park, O., 2015. Incidence and epidemiology of Japanese Encephalitis in Korea. Public Health Wkly. Rep. 8, 433-436. Lee, K.W., 2014. A revision of the illustrated taxonomical keys to genera and species of female mosquitoes of Korea, p40. Seoul, Korea, Department of the Army, 5th medical detachment, 168th

medical battalion, 18th medical command, 1999. Available at

http://www.afpmb.org/content/literature-retrieval-system. Li, M.H., Fu, S.H., Chen, W.X., Wang, H.Y., Guo, Y.H., Liu, Q.Y., Li, Y.X., Luo, H.M., Da, W., Duo, Ji D.Z., Ye, X.M., Liang, G.D., 2011. Genotype V Japanese encephalitis virus is emerging. PLoS Negl. Trop. Dis. 5(7), e1231. https://doi:10.1371/journal.pntd.0001231. Li, Y., Kamara, F., Zhou, G., Puthiyakunnon, S., Li, C., Liu, Y., Zhou, Y., Yao, L., Yan, G., Chen, X.-G., 2014. Urbanization increases Ardes albopictus larval habitats and accelerates mosquito development and survivorship. PLoS Negl. Trop. Dis. 8, e3301. https://dx.doi.org/10.1371/journal.pntd.0003301. Liu, H., Gao, X., Liang, G., 2011. Newly recognized mosquito-associated viruses in mainland China, in the last two decades. Virol. J. 8, 68. http://www.virologyj.com/contents/8/1/68. Mackenzie, J.S., Gubler, D.J., Peterson, L.R., 2004. Emerging flaviviruses: the spread and resurgence of Japanese encephalotis, West Nile and dengue viruses. Nat. Med. 10, S98-S109. https://doi:10.1038/nm1144. McMichael, A.J., Woodruff, R.E., Hales, S., 2006. Climate change and human health: Present and future risks. Lancet 367, 859-869. https://doi:10.1016/S0140-6736(06)68079-3. 17

Min, S.K., Zhang, X., Zwiers, F., Shiogama, H., Tung, Y.S., Wehner, M., 2013. Multimodel detection and attribution of extreme temperature changes. J. Climate 26, 7430. Mohammed, M.A., Galbraith, S.E., Radford, A.D., Dove, W., Takasaki, T., Kurane, I., Solomon, T., 2011. Molecular phylogenetic and evolutionary analyses of Muar strain of Japanese encephalitis virus reveal it is the missing fifth genotype. Infect. Genet. Evol. 11(5), 855-862. https://doi:10.1016/j.meegid.2011.01020. Ogden, N.H., 2017. Climate change and vector-borne diseases of public health significance. FEMS Microbiol. Lett. 364, fnx186. https://doi:10.1093/femse/fnx186. Ool, E.E., Goh, K.T., Gubler, D.J., 2006. Dengue prevention and 35 years of vector control in Singapore. Emerg. Infect. Dis. 12, 887-893. https://doi:10.3201/10.3201/eid1206.051210. Ree, H.I., 2003. Taxonomic review and revised keys of the Korean mosquitoes (Diptera: Culicidae). Korean J. Entomol. 33, 39-52. Reegan, A.D., Ceasar, S.A., Paulraj, M.G., Ignacimuthu, S., Al-Dhabi, N.A., 2016. Current status of

genome editing in vector mosquitoes: A review. Biosci. Trends 10(6), 424-432.

https://doi:10.5582/bst.2016.01180. Reinert, J.E., 2001. Revised list of abbreviations for genera and subgenera of Culicidae (Diptera) and notes on genetic and subgenetic changes. J. Am. Mosq. Control Assoc. 17, 5155. Rogers, D.J., Suk, J.E., Semenza, J.C., 2013. Using global maps to predict the risk of dengue in Europe. Acta Trop. 129, 1-14.

https://doi:10.1016/j.actatropica.2013.08.008.

Rosen, L., 1986. The natural history of Japanese encephalitis virus. Ann. Rev. Microbiol. 40, 395-414. Sohn, Y.M., 2000. Japanese encephalitis immunization in South Korea: Past, present, and future.

Emerg. Infect. Dis. 6, 17-24. https://doi:10.3201/eid0601.000103.

Takhampunya, R., Kim, H.-C., Tippayachai, B., Kengluecha, A., Klein, T.A., Lee, W.-J., Grieco, J., Evans, B.P., 2011. Emergence of Japanese encephalitis virus genotype V in the 18

Republic of Korea. Virol. J. 8, 449. https://www.virollogyj.com/content/8/1/449. Takhampunya, R., Kim, H.-C., Tippayachai, B., Lee, D.K., Lee, W.J., Chong, S.T., Kim, M.S., Lee, J.S., Klein, T.A., 2014. Distribution and mosquito hosts of Chaoyang virus, a newly reported flavivirus from the Republic of Korea. J. Med. Entomol. 51(2), 464-474. https://doi:10.1603/me13033. Tanaka, K., Mizusawa, K., Saugsad, E.S., 1979. A revision of the adult and larval mosquitoes of Japan (including the Ryuku Archipelago and the Ogasawara Islands) and Korea (Diptera: Culicidae). Contrib. Am. Entomol. Instit. 16, 1-987. van den Hurk, A.F., Ritchie, S.A., Mackenzie, J.S., 2009. Ecology and geographical expansion of Japannese encephalitis virus. Annu. Rev. Entomol. 54, 17-35. https://doi:10.1146/annurev.ento.54.110807.090510. Wang, Z.S., An, S.Y., Wang, Y., Han, Y., Guo, J.Q., 2009. A new virus of Flavivirus: Chaoyang virus isolated in Liaoning province. Chin. Pub. Health 25(7), 769-772 (in Chinese). Weaver, S., Charlier, C., Vasilakis, N., Lecuit, M., 2016. Zika, chikungunya, and other emerging vector-borne viral diseases. Annu. Rev. Med. 69, 1-14. https://doi:10.1146/annurev-med-05715-105122. WHO, 2016. WHO Director-General summerizes the outcome of the Emergency Committee regarding clusters of microcephaly and Guillain-Barre syndrome. https://www.who.int/mediacenter/news/statements/2016/emergency-committee-zikamicrocephaly/en/2016 (accessed September 2, 2016). World Health Organization, 2013. World health report: research for universal health coverage. Technical Report. https://www.who.int/whr/en (Accessed August 15, 2013). Wilke, A.B.B., Medeiros-Sousa, A.R., Ceretti-Junior, W., Marrelli, M.T., 2017. Mosquito populations dynamics associated with climate variations. Acta Trop. 166, 343-350. https://dx.doi.org/10.1016/j.actatropica.2016.10.025. 19

Yang, C.F, Chen, C.F., Su, CL., Teng, H.J., Lu, L.C., Lin C, Wang, C.Y., Shu, P.Y., Huang, J.H., Wu, H.S., 2010. Screening of mosquitoes using SYBR Green I-based real-time RTPCR with group-specific primers for detection of Flaviviruses and Alphaviruses in Taiwan. J. Virol. Methods 168, 147-151. https://doi:10.1016/jviromet.2010.05.006. Yeom, J.S., 2017. Current status and outlook of mosquito-borne diseases in Korea. J. Korean Med. Assoc. 60(6), 468-474. https://doi.org/10.5124/jkma.2017.60.6.468 (In Korean). Yun, S.M., Cho, J.E., Ju, Y.R., Kim, S.Y., Ryou, J., Han, M.G., Choi, W.Y., Jeong, Y.E., 2010. Molecular epidemiology of Japanese Encephalitis virus circulating in South Korea. Virol. J. 7, 127. https://doi:10.1186/1743-422X-7-127.

20

Figure legends

Fig. 1. Distribution of the total population densities of each mosquito species collected from March to November 2016-2018 depending on the collection time.

Fig. 2. Comparison of the distribution of total population densities between urbanized downtown Incheon and rural Hwaseong from March to November 2016-2018 depending on the collection time.

21

Tables

Table 1. Locations and geographic coordinates of mosquito collection. Collection Sites

Bupyeong-dong,

Study

Collection Method

Environment

(Latitude/Longitude

s

)

Downtown

Black light traps

Downtown

Black light traps

Downtown

BG SentinelTM traps

Hwanseong-si

22

37.468351N/126. 63484E

Incheon Hogok-ri,

37.528529N/126.65 8841E

Incheon Shinheung-dong,

37.498796N/126.72 4164E

Incheon Yeonhui-dong,

GPS Coordinates

Cowshed

Black light traps

37.065760N/126.46 3750E

Table 2. Total number of female mosquitoes collected in this study with black light trap and BG at every other week from Mar. to Nov. 2016~2018.

Mar. Collectio n time

Apr.

May

3

5

1s

3r

2n

rd

th

t

d

d

4th

Jun. 2n

4th

Jul. 1st

Aug.

3rd

1st

3rd

d

Sep. 2n

4th

Oct. 1st

d

Nov.

3r

2n

d

d

Total

4th

Species Ae.

352

albopictus

10 0

0

0

0

1

0

8

0

10

33

76

46

9

2 31

18

0

(0.99 0

0

%)

Ae.

17,33

vexans

3

nipponii 5

0

0

4

68

12

18

20

41

20

12

22

13

31

16

9

34

74

11

91

10

02

15

99

1

7

1 8

3

(48.9 0

Ar.

177

Subalbatu s

(0.50 0

0

0

0

1

5

22

15

27

16

24

16

25

18

6

2

0

0

Coq.

(0.03 0

0

0

0

0

0

0

0

7

0

1

1

0

0

0

0

0

0

Cx.

%) 63

bitaeniorh

(0.18 0

0

0

0

0

2

0

0

2

2

2

39

6

6

2

2

0

0

Cx. orientalis

%) 9

Ochracea

ynchus

1%)

%) 7

0

0

0

0

0

0

0

0

0

0

3

1

2

0

0

1

0

0

(0.02 %)

Cx. pipiens

6

6

4

4

11

26

11

20

16

15

10

42

55

55

68

7

2

5

3

0

71

57

50

51

98

7

9

8

5

6

4

3

2

3

5

11,36 13

(32.0 5%)

Cx.

172

inatomii

(0.49 1

23

0

0

0

0

0

11

49

0

47

33

0

25

0

6

0

0

0

0

%)

Cx.

529

tritaeniorh ynchus

0

0

0

0

0

0

0

0

0

0

0

10

23

16

4

1

0

(1.49 33

1

0

0

%)

Cx.

6

vagans

(0.02 0

0

0

0

0

4

2

0

0

0

0

0

0

0

0

0

0

0

Man.

29

uniformis

(0.08 0

0

0

Och.

1

5

dorsalis

3

1

50

11

83

33

10

18

21

8

4

2

59

9

8

0

3

9

0

0

0

0

0

0

0

12

7

2

5

2

0

1

0

0

0

%) 4,812

88

12

21

38

4

8

3

(13.5 7

0

0

Och.

8%) 594

Koreicus 0

0

0

1

4

7

79

15

17

10

3

2

6

(1.68 28

5

6

20

10

3

0

0

Och.

%) 3

togoi

(<0.0 0

0

0

0

0

0

0

0

0

0

3

0

0

0

0

0

0

0

Total

24

%)

1%) 35,44

1

5

6

5

1

6

8

6

13

26

40

45

62

39

26

29

24

13

13

7

43

1

(100

2

7

0

4

10

82

44

74

18

41

58

71

63

28

05

7

8

3

%)

Table 3. Total number of female mosquitoes collected at downtown of Incheon Metropolitan City with black light trap and BG at every other week from Mar. to Nov. 2016~2018.

Collection time

Mar.

Apr.

May

Jun.

3r

5t

1

3r

2n

4t

2n

4t

d

h

st

d

d

h

d

h

Jul. 1st

Aug.

3r

1st

d

Sep.

Oct.

3r

2n

4t

d

d

h

1st

Nov.

3r

2n

4t

d

d

h

Total

Species Ae.

349

albopictus

10 0

0

0

0

1

0

8

0

10

33

75

45

8

(4.63 31

18

20

0

0

Ae. vexans

%) 61

nipponii

(0.81 0

0

0

0

1

11

4

5

2

23

8

0

3

4

0

0

0

0

Ar.

%) 2

Subalbatus

(0.03 0

0

0

0

0

0

0

0

1

0

0

0

1

0

0

0

0

0

%)

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Coq. Ochracea Cx.

7

bitaeniorhyn chus

(0.09 0

0

0

0

0

2

0

0

2

1

1

0

0

0

1

0

0

0

Cx.

%) 6

orientalis

(0.08 0

0

0

0

0

0

0

0

0

0

2

1

2

0

0

1

0

0

Cx. pipiens

%) 6,956

6

3

3

2

5

7

4

0

0

0

0

19

54

84

59

88

61

32

52

48

66

62

42

1

(92.21

97

7

3

7

0

4

0

1

7

5

3

1

0

3

%)

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Cx. inatomii Cx.

16

tritaeniorhy nchus

25

(0.21 0

0

0

0

0

0

0

0

0

0

0

1

12

2

1

0

0

0

%)

Cx. vagans

6 (0.08 0

0

0

0

0

4

2

0

0

0

0

0

0

0

0

0

0

0

Man.

%) 1

uniformis

(0.01 0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

0

0

0

%)

Och.

42

dorsalis

(0.56 0

0

0

0

3

0

15

0

0

8

0

0

5

10

1

0

0

0

Och.

%) 96

Koreicus

(1.27 0

0

0

1

2

6

7

9

5

10

12

5

6

20

10

3

0

0

Och. togoi

%) 3 (0.04

0

0

0

0

0

0

0

0

0

0

3

0

0

0

0

0

0

0

Total

7,544

2

26

%)

6

3

3

10

22

57

86

61

95

71

37

66

55

69

64

42

1

(100

5

7

5

4

0

9

1

0

9

1

2

4

2

5

5

0

3

%)

Table 4. Total number of female mosquitoes collected at Hwaseong-Gun Area with black light trap and BG at every other week from Mar. to Nov. 2016~2018.

Mar. Collectio n time

Apr.

3

5

1s

3r

rd

th

t

d

May 2nd

4th

Jun. 2nd

4th

Jul. 1st

Aug.

3rd

1st

3rd

Sep. 2nd

Oct.

Nov.

4t

1s

3

2

4

h

t

rd

nd

th

Total

Species Ae.

3

albopictus

(0.01 0

0

0

0

0

0

0

0

0

0

1

1

1

0

0

0

0

0

Ae.

17,27

vexans nipponii 5

0

0

4

3

1

68

12

18

20

41

19

11

22

13

0

6

8

23

70

06

89

87

94

15

96

7

7

2 1 8

3

(61.9 0

Ar. 1 0

0

0

0

1

5

22

15

26

16

24

16

24

8

(0.63 6

2

0

0

Coq.

%) 9

Ochracea

(0.03 0

0

0

0

0

0

0

0

7

0

1

1

0

0

0

0

0

0

Cx.

%) 56

bitaeniorhy

(0.20 0

0

0

0

0

0

0

0

0

1

1

39

6

6

1

2

0

0

Cx.

%) 1

orientalis

(<0.0 0

0

0

0

0

0

0

0

0

0

1

0

0

0

0

0

0

0

Cx.

1%) 4,404

pipiens

1 4

2

5

1

16

63

62

12

10

66

48

10

8

10

60

7

8

6

32

7

2

1

3

2

2

(15.7 5

0

Cx.

8%) 172

inatomii

(0.62 1

27

0%) 175

Subalbatus

nchus

%)

0

0

0

0

11

49

0

47

33

0

25

0

6

0

0

0

0

%)

Cx.

1

tritaeniorh ynchus

513

10

21

5

3

(1.84

0

0

0

0

0

0

0

0

0

0

0

3

9

8

2

1

0

0

%)

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Cx. vagans Man.

28

uniformis

(0.10 0

0

0

Och.

1

5

dorsalis

3

1

49

11

82

33

10

17

21

8

4

9

59

4

8

0

5

9

0

0

0

0

0

0

0

12

7

2

5

88

2

0

0

0

2

3

4,770

11

0

8

(17.1

9

8

2

7

0

0

0

0

Och.

%)

0%) 498

Koreicus

14

16

(1.78

0

0

0

0

2

1

72

4

7

96

16

0

0

0

0

0

0

0

%)

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Och. togoi Total

27,90

1 0

28

2

1

5

4

2

12

24

34

37

56

29

19

25

3

9

06

62

65

13

08

82

47

99

7

6

1

17

7

1

3

1

99

6

0

2

8

(100 0

%)

Table 5. Trap index I(TI) of female mosquitoes collected at downtown of Incheon Metropolitan City and Hwaseong-Gun Area, and number of pools tested for the flavivirus detection from Mar. to Nov. 2016-2018. (mosquitoes/night/trap) Ae .

Species

Ae. vex

al b

Locations

Ar .

Co q.

C x.

C x.

su b

oc h

in a

bit

Cx. ori

Cx pip

C x.

C x.

tri

va g

Ma Oc. n. dor uni

O c.

Oc. tog

Tot al

ko r

Bupye

No

ong

.

0

6

0

0

0

0

2

655

0

0

0

1

9

0

673

%

0

0.8

0

0

0

0

0.3

97.

0

0

0

0

1.

0

100

0

33

2

44

0

59

9

34

Po Downt

ol

0

4

0

0

0

0

0

0

0

1

8

own Yeonh

No

111

1,24

ee

.

3

48

0

0

0

2

4

9

16

5

1

36

10

1

4

%

0.

3.8

0

0

0

0.

0.3

89.

1.

0.

0.

2.8

0.

0.0

100

24

6

16

2

95

29

40

08

9

80

8

ol

3

20

1

3

53

6

3

1

11

8

1

Shinhe

No

34

ung

.

6

Po 0

0

518

109 5,62

7

2

0

0

5

0

2

0

1

0

5

77

2

7

0.1

0.

0

0

0.

0

92.

0

0

0

0.0

1.

0.0

100

5

2

04

9

37

4

Po ol

27

4

2

5

22

1

No

34

.

9

61

2

0

0

7

6

6

16

6

1

42

96

3

4

%

4.

0.8

0.

0

0

0.

0.0

92.

0.

0.

0.

0.5

1.

0.0

100

63

1

03

09

8

21

21

08

01

6

27

4

30

28

2

5

5

236

6

4

1

17

38

2

% 6.1

Total

0

09

0

0

4

09

0

139

0

1

0

695

205 7,54

po ol

29

0

0

373

No Hwase

Cowsh

ong

ed

172

17

17

440

51

47

49

.

3

72

5

9

2

56

1

4

3

0

28

70

8

0

27,9 01

%

0.

61.

0.

0.

0.

0.

<0.

15.

1.

0

0.

17.

1.

0

100

01

90

63

03

62

20

01

78

84

10

10

78

2

366

26

3

19

9

1

137

15

7

116

18

0

719

35

173

17

113

52

48

59

No.

2

33

7

9

2

63

7

60

9

6

29

12

4

3

45

%

0.

48.

0.

0.

0.

0.

0.0

32.

1. 0.0

0.

13.

1.

<0.

100

99

91

50

03

49

18

2

05

49

08

58

68

01

Po ol

Total

17

0

2

Poo l

13 32

394

28

3

19

14

6

373

21

4

7

3

Ae. alb; Aedes albopictus, Ae. vex; Aedes vexans, An. sin; Anopheles sinensis, Ar. sub; Armigeres subalbatus, Coq. och; Coquillettidia ochracea, Cx. ina: Culex inatomii, Cx. bit; Culex bitaeniorhynchus, Cx. ori; Culex orientalis, Cx. pip; Culex pipiens, Cx. tri; Culex tritaeniorhynchus, Cx. vag; Cullex vagans, Man. uni; Mansonia uniformis, Oc. dor; Ochlerotatus dorsalis, Oc. kor; Ochlerotatus koreicus, Oc. tog; Ochlerotatus togoi

30

35,4

1,09 56

2

2

Table 6. Number (%) of culicine mosquotoes species collected and pools assayed and positive for flaviviral RNAby qPCR and determination of the maximum likelihood estimation (MLE) (Estimated number of flaviviral RNA positibe mosquitoes per 1,000) in this study, 2016-2018.

Species

Total No. of tested (%)

Ae. albopictus

352

0.99%

32

17,333

61.90%

394

Ae. vexans nipponii

No. of tested pools

Ar. subalbatus

177

0.50%

28

Coq. Ochracea

9

0.03%

3

63

0.18%

14

7

0.02%

6

11,360

32.05%

373

172

0.49%

19

529

1.49%

21

6

0.02%

4

29

0.08%

7

4,812

13.58%

133

594

1.68%

56

3

<0.01%

Cx. bitaeniorhynchus Cx. orientalis Cx. pipiens Cx. inatomii Cx. tritaeniorhynchus Cx. vegans Man. Uniformis Och. dorsalis Och. koreicus Och. togoi

31

Flavivirus (MLE)

Location

Collection time

Chaoyang (1.64)

Hwaseong

Sep. 2nd wk (2017)

Hwaseong

Oct. 1st wk (2017)

Chaoyang (4.55)

Bupyeong JEV-5 (0.20)

2

Sep. 1st wk (2018)