PHWR Public Health Weekly Report

Flavivirus, a member of the family Flaviviridae and the genus Orthoflavivirus, is a positive-sense single-stranded RNA virus that is transmitted and disseminated through arthropod (primarily mosquitoes or ticks) vectors. Notably, mosquito-borne flaviviruses constitute a group of pathogens that are responsible for the development of significant public health concerns, including but not limited to Dengue fever, Yellow fever, Zika virus infection, West Nile fever, and Japanese encephalitis [1]. The classification of Flavivirus is based on its clinical symptoms, which are indicative of its association with either the gastrointestinal system (e.g., Yellow fever, Dengue fever, and Zika virus infection) or the nervous system (e.g., West Nile fever, Japanese encephalitis, and Zika virus infection). While the majority of infections across all strains are asymptomatic, some can lead to severe, life-threatening complications, including hemorrhage, encephalitis, myelitis, paralysis, or congenital malformations.

Historically, the distribution of mosquito-borne infectious diseases was largely restricted to specific countries or regions, depending on the habitat distribution of particular mosquito species that served as vectors for these pathogens. However, global warming has contributed to the proliferation of mosquito populations, thereby increasing the risk of infectious disease transmission. For example, the geographical distribution of Dengue fever has expanded significantly, with the number of affected countries rising from three in the 1940s to 129 in the 2010s [2]. Similarly, the Zika virus infection has become endemic in 73 countries (62 countries since 2015 and 11 countries in the past).

Meanwhile, the spread of mosquitoes capable of transmitting infectious diseases and the transmission of infectious diseases among regions or countries is closely linked to human and cargo movement. Advances in transportation technologies, such as air and maritime travel, have facilitated the long-distance dispersal of mosquitoes. Reduced travel time has been shown to increase the survivability of mosquitoes aboard vehicles, thereby increasing the likelihood of their survival and potential for disease transmission at their destination [3]. In response, the World Health Organization recommends surveillance at airports and seaports [4] to monitor mosquito species distribution and virus carriage, thereby aiding in the prevention of infectious disease spread. Notably, a recent surge in Dengue fever cases has been reported in Southeast Asia. With the increase in international travel following the coronavirus disease 2019 pandemic, there have been ongoing reports of domestic cases involving infections acquired abroad (primarily in the Philippines, Vietnam, Thailand, and Indonesia) [5].

Therefore, the Regional Center for Disease Control and Prevention has been conducting vector surveillance in quarantine regions to prevent the introduction of mosquito-borne infectious diseases from foreign countries into the Republic of Korea (ROK) and to monitor disease-carrying mosquito populations. This article aims to present the findings of the surveillance conducted in 2023.

1. Mosquito Collection

Five Regional Centers for Disease Control and Prevention and 13 National Quarantine Stations collaborated to monitor mosquitoes carrying foreign infectious diseases and pathogens in quarantine areas, including airports and seaports in ROK. Mosquitoes were collected twice a month from June to October at a total of 29 sites (six airports and 23 seaports) (Table 1). Each collection site was characterized by high human or cargo traffic, such as cargo loading docks within quarantine zones or traveler pathways. Mosquitoes were primarily collected from environments with water, trees, and vegetation, such as sewers. Mosquito collection was conducted using BG-Sentinel (BG) traps and black light (BL) traps, with the choice of trap type depending on the environmental conditions of each site. Additionally, dry ice was used as a mosquito attractant at certain locations. Sampling equipment was deployed at 10:00 AM on the day of collection and remained in operation for 24 hours a day, until 10:00 AM the following day. BL traps were installed in low-light areas at night, positioned 1.5 m above the ground. BG traps were placed at ground level in areas with dense vegetation, which provide favorable conditions for the proliferation of Aedes spp. When both trap types were used simultaneously, they were positioned at least 10 m apart to prevent interference between them.

Table 1 Sampling sites and trap models

Regional center	Quarantine station	Site	Trap
Capital	Incheon Airport	Incheon International Airport terminal (EG1)	BL
		Incheon International Airport Observatory Deck	BL
	Incheon	Incheon Inner Port street park	BG
		Incheon Inner Port GATE-1	BG
		Incheon Inner Port GATE-3	BL
	Donghae	Donghae Port International Passenger terminal	BL
		Samcheok Port Sampyocement	BL
Chungcheong	Gunsan	Gunsan Port Pier 1	BGa)
		Gunsan Port Pier 5	BGa)
		Janghang Port	BGa)
	Pyeongtaek	Pyeongtaek Marine Center	BL, BGa)
		Cheongju International Airport	BL, BG
	Daesan office	Daesan office	BGa)
Honam	Mokpo	Gotbawi	BLa)
		Muan International Airport	BG
	Yeosu	Korea National Oil Corporation	BLa)
		Yeosu Port (Expo Port)	BLa)
	Jeju	Jeju Ferry Passenger Terminal	BGa)
		Jeju Port & Transport Trade Union Federation	BGa)
Gyeongbuk	Ulsan	Main Port Pier 2	BG
		Ulsan Quarantine station	BG
	Pohang	Pohang Quarantine station	BL
		Songrim Park	BG
Gyeongnam	Busan	Busan Quarantine station	BL
		Gamcheon Port	BG
	Masan	Masan Quarantine station offical residence	BL
		Gyeongnam Regional Government Complex	BL
	Gimhae	International Flights baggage claim	BL
		International Flights Cargo terminal	BL

BL=black light trap; BG=BG-Sentinel trap. ^a)Collection site with dry ice..

2. Mosquito Species Identification and Pathogen Detection

The collected mosquitoes were pretreated at –70°C before sorting and identification. They were then grouped into sets of up to 30 individuals based on species, collection time, and collection site. Then, RNA extraction was performed following the uniform homogenization of mosquito tissue using the FastPrep-24 apparatus (MP Biomedicals). The extracted RNA was subsequently analyzed for gene detection using the Clear-MD^Ⓡ Flavivirus Real-time reverse transcription polymerase chain reaction detection kit (Invirustech) to confirm the presence of Flavivirus in the mosquitoes. The amplification of flaviviruses was confirmed through melting curve analysis, and suspected positive specimens were subjected to sequencing for final verification.

3. Utilization of Results

Regional statistical data were uploaded to the Integrated Vector Surveillance System (VectorNet) within The Epidemic Infectious Disease System (https://eid.kdca.go.kr). The consolidated results were then sent to the National Quarantine Station and shared with Health Center in the quarantine areas on a monthly basis.

1. Mosquito Collection

In 2023, a total of 4,721 mosquitoes representing 14 species were collected through the infectious disease vector surveillance project in quarantine zones. Notably, no non-native disease-carrying mosquito species, such as Aedes aegypti or Haemogogus spp., were detected. The most prevalent mosquito species identified at airports and seaports in ROK was Culex pipiens, a vector for West Nile fever, accounting for 2,580 individuals (54.6% of the total specimens collected). This was followed by Culex tritaeniorhynchus, a vector for Japanese encephalitis, with 570 individuals (12.1%), and Aedes albopictus, which transmits Dengue fever and Zika virus infection, with 493 individuals (10.4%). Other collected species included Mansonia spp., Armigeres spp., and Anopheles spp., which are known malaria vectors (Table 2).

Table 2 Distribution of the mosquitoes by species in 2023

Genus	Species	No. of mosquitoes (%)
Culex	Culex pipiens	2,580 (54.6)
	Culex tritaeniorhynchus	570 (12.1)
	Culex bitaeniorhynchus	8 (0.2)
	Culex inatomii	122 (2.6)
	Culex orientalis	12 (0.3)
Aedes	Aedes albopictus	493 (10.4)
	Aedes vexans	25 (0.5)
	Aedes lineatopennis	12 (0.3)
	Ochlerotatus koreicus	154 (3.3)
	Ochlerotatus togoi	51 (1.1)
	Ochlerotatus dorsalis	296 (6.3)
Coquillettidia	Coquillettidia ochracea	19 (0.4)
Armigeres	Armigeres subalbatus	142 (3.0)
Anopheles	Anopheles spp.	237 (5.0)
Total		4,721 (100.0)

An analysis of mosquito collection by region revealed that the highest number of mosquitoes were collected in Honam region (n=2,576), followed by Gyeongnam region (n=704), Capital region (n=668), Gyeongbuk region (n=477), and Chungcheong region (n=296). In terms of mosquito species diversity by region, the Capital region (n=14)>Gyeongnam and Honam regions (n=10 each)>Chungcheong region (n=7)>Gyeongbuk region (n=6) were in the following order. Regarding species distribution, Culex pipiens and Aedes albopictus were prevalent in all regions except Gyeongnam region, where Culex tritaeniorhynchus (58.7%) accounted for the highest proportion (Figure 1).

Figure 1. Overview of major mosquito species distributed by Regional Center for Disease Control and Prevention

2. Mosquito Density by Month and Sampling Equipment

To determine the density of mosquito species over time, the distribution of species was analyzed by month. From June through September, the number of mosquitoes collected averaged over 1,000 per month before declining sharply in October. The prevalence of mosquito species varied throughout the collection period. Culex pipiens, the most prevalent mosquito species in ROK, exhibited the highest density in June (76.2%), followed by a gradual decline through October. However, no statistically significant differences in dominance were observed among the primary species, with the most prevalent species accounting for an average of 54.6% of the total mosquito population each month. The monthly density of Culex tritaeniorhynchus, a vector for Japanese encephalitis, a domestically endemic infectious disease, was low in June but increased sharply in August and September (from 1.0% in June to 28.1% in August). Meanwhile, Aedes albopictus showed a steady increase before declining, with the overall mosquito population dropping sharply in October (Figure 2).

Figure 2. Distribution of the mosquito species by month in 2023

Next, the species composition of mosquitoes collected was compared between BG and BL traps to analyze the characteristics of each collection device. In BG traps, which primarily attract diurnal Aedes spp. such as Aedes albopictus, the species composition was dominated by Culex pipiens (65.4%) and Aedes albopictus (28.0%), together accounting for over 90% of the total species collected. In BL traps, which attract nocturnal mosquitoes through light, Culex pipiens (51.7%) and Culex tritaeniorhynchus (15.3%) were the most common species. In addition to Culex spp., a diverse range of mosquitoes was collected, including Ochlerotatus dorsalis (8.2%), Anopheles gambiae (5.8%), and Aedes albopictus (4.5%) (Figure 3).

Figure 3. Mosquito species distribution by trap model
(A) BG-Sentinel trap. (B) Black light trap.

3. Pathogen Surveillance

Mosquitoes collected from quarantine areas were tested for the presence of flaviviruses responsible for Dengue fever, Yellow fever, West Nile fever, Japanese encephalitis, and Zika virus infection. Detection of flaviviruses was conducted on 4,484 mosquitoes, excluding malaria-transmitting Anopheles gambiae (237 mosquitoes). Japanese encephalitis virus (Genotype V) was detected in one Culex pipiens (10 individuals) collected at Incheon Port, out of a total of 567 samples tested. No evidence of the other four flaviviruses (Dengue fever, Yellow fever, West Nile fever, or Zika virus infection) was found (Table 3).

Table 3 Distribution of the number of mosquitoes and pools by Regional Center for Disease Control and Prevention

		Capital	Chungcheong	Honam	Gyeongbuk	Gyeongnam	Total
Populations		591	268	2,452	475	698	4,484
No. of pools		142	74	184	58	109	567
Result	Positive	1	0	0	0	0	1
	Negative	141	74	184	58	109	566

Quarantine areas, including airports and seaports, serve as entry points for international travelers and cargo into the country and have been identified as high-risk locations for the presence of mosquitoes carrying infectious diseases. Therefore, this project focused on monitoring mosquitoes carrying overseas infectious diseases in the quarantine areas of domestic airports and seaports, with the objective of analyzing the distribution and density of mosquito species. As a result, a total of 4,721 mosquitoes from 14 species were collected. While mosquitoes not native to ROK, such as Aedes aegypti and Haemogogus spp., were not identified, Culex pipiens (54.6%), Culex tritaeniorhynchus (12.1%), and Aedes albopictus (10.4%), which are capable of transmitting mosquito-borne infectious diseases, were collected. Subsequent analysis of the sampling results by location revealed regional variation in mosquito abundance and species composition. In four regions (Capital region, Chungcheong, Honam, and Gyeongbuk regions), the predominant species were Culex pipiens and Aedes albopictus, both of which are known vectors for infectious diseases, accounting for the majority of the collected samples. In Gyeongnam region, Culex tritaeniorhynchus exhibited the highest sampling rate, likely due to the presence of rice fields in the quarantine area. Additionally, Anopheles spp., the primary malaria vectors, were predominantly collected in the west coast regions (Capital region>Chungcheong region>Honam region).

The occurrence of an imported mosquito-borne disease depends on three key conditions: first, the introduction of a virus that is not endemic to the country; second, the presence of a vector capable of mediating virus transmission; and third, an ecological and climatic environment conducive to the vector’s ability to transmit the virus [6]. While exotic mosquito species carrying causative agents of Dengue fever can be introduced into a country, potentially causing outbreaks and becoming endemic, endemic mosquito species (e.g., Aedes albopictus, Aedes vexans, Culex pipiens) can also cause outbreaks by feeding on infected individuals. For instance, Japan, which shares a similar latitude and climate with ROK, experienced a Dengue fever outbreak in 2014, infecting 162 individuals in Tokyo’s Yoyogi park. This outbreak was attributed to Aedes albopictus, which fed on the blood of a Dengue virus-infected individual who had contracted the virus abroad [7]. The dominant mosquito species identified in the quarantine areas during this surveillance project were Culex pipiens (54.6%), Culex tritaeniorhynchus (12.1%), and Aedes albopictus (10.4%), which is consistent with the findings of community mosquito surveillance in ROK [8]. In ROK, the proliferation of disease-transmitting species, including Aedes albopictus (a primary vector for Dengue fever, Yellow fever, and Zika virus infection), Culex pipiens and Aedes vexans (which transmit West Nile fever), and Culex tritaeniorhynchus (a vector for Japanese encephalitis), has been observed to increase in both frequency and density [8,9]. This shift in the proportion of domestic mosquito species suggests a higher likelihood of an epidemic should an infectious disease be introduced into the country.

Density of mosquito are known to decrease when daily precipitation exceeds 75 mm or when total precipitation exceeds 150 mm over a 15-day period [9]. According to the annual climate report for 2023 from the Korea Meteorological Administration, precipitation during the summer rainy season totaled 660.2 mm nationwide, up from 356.7 mm in a typical year. Additionally, the average number of precipitation days per month was 22.1 days, representing a 28% increase compared to the normal average of 17.3 days. Therefore, the observed decline in mosquito numbers collected during the summer months (July and August) relative to June can likely be attributed to the elevated precipitation levels and the increased number of precipitation days. However, in 2023, due to the ongoing effects of global warming and the El Niño phenomenon, the average global temperature reached its highest level since the onset of industrialization (14.98°C). In ROK, the average annual temperature exceeded normal levels by 1.2°C [10]. This acceleration in climate change is expected to lead to a higher prevalence of mosquitoes and an extended mosquito season, thereby increasing the likelihood of introducing and establishing non-native species, such as Aedes aegypti, a primary vector for Dengue fever, in ROK [3,6].

Meanwhile, the judicious selection of collection equipment tailored specifically for mosquito surveillance has been demonstrated to enhance the efficacy of such efforts. Equipment used for collecting adult mosquitoes includes mosquito magnet traps, light traps, and BG traps. Among these, BG traps are the most commonly used, as they are designed to attract Aedes aegypti and Aedes albopictus, vectors for Dengue fever, Yellow fever, and Zika virus infection, using the BG-Lure attractant [11]. These traps can be installed in both urban centers and rural or unpopulated areas, as they can remain operational as long as there is a power supply. While no non-native species were observed during the surveillance, Aedes albopictus, Aedes vexans (a vector of West Nile fever), and Ochlerotatus dorsalis (a vector of both West Nile fever and Japanese encephalitis) were collected, confirming the effectiveness of the equipment (Figure 3A). The efficacy of BL traps is optimal in the absence of ambient light, making them particularly suitable for the collection of nocturnal mosquitoes. However, the diversity of species collected may depend on the characteristics of the surveillance site, including its proximity to forests, human settlements, or standing water bodies. A comparison of species distribution revealed that, in addition to Culex spp., Aedes spp., such as Ochlerotatus dorsalis, Aedes albopictus, and Ochlerotatus koreicus, accounted for approximately 20%. Furthermore, malaria vectors, including Anopheles spp. (5.8%), Mansonia spp., and Armigeres spp., were identified (Figure 3B).

The mosquitoes collected during this surveillance project were tested for five flaviviruses. While none of the pathogens responsible for Dengue fever, West Nile fever, Yellow fever, or Zika virus infection were identified, Japanese encephalitis virus was detected in mosquitoes collected from the seaport area. These findings suggest that proactive surveillance of mosquito species in quarantine areas, coupled with pathogen detection, could serve as a promising strategy for the early identification of foreign infectious diseases entering the country. Given the ongoing effects of global warming and the increasing number of countries where Dengue fever is endemic, the importance of mosquito surveillance in quarantine zones—which serve as primary entry points for internationally transmitted infectious diseases—is growing. It is crucial to sustain this surveillance program to promptly detect disease-carrying mosquitoes in quarantine zones and prevent the spread of pathogens into the country.

Ethics Statement: Not applicable.

Funding Source: None.

Acknowledgments: We acknowledge to stuffs in the National Quarantine Station for collecting mosquitoes in airport and seaport areas, and the Division of Laboratory Diagnosis Analysis, Korea Regional Center for Disease Control and Prevention in Korea Disease Control and Prevention Agency for mosquitoes classification and detecting pathogens. Additionally, we would like to thank Division of Vectors and Parasitic Diseases, Department of Disease Diagnosis and Analysis, Korea Disease Control and Prevention Agency for their fundamental supports.

Conflict of Interest: The authors have no conflicts of interest to declare.

Author Contributions: Conceptualization: HSY, DHK. Data curation: SHJ, YIJ. Formal analysis: SHJ, YIJ. Investigation: HSY, YIJ. Methodology: SHJ, EJL. Project administration: SHJ, MGJ. Resources: SHJ, YJG. Software: SHJ, SHL. Supervision: HSY, MGJ. Validation: EJL, SDP. Visualization: SHJ, YIJ. Writing – original draft: SHJ, DHK. Writing – review & editing: SHJ, DHK, HSY.