Korean Journal of
Veterinary Service

Avian influenza viruses (AIVs) belong to the Orthomyxoviridae family and have negative-sense RNA genomes with eight segments (Webster et al, 1992). They are divided into 16 hemagglutinin (HA) and 9 neuraminidase (NA) subtypes based on their surface glycoproteins (Kuchipudi et al, 2018). The H5 and H7 subtypes of low-pathogenic AIVs (LPAIVs) can mutate into highly pathogenic avian influenza viruses (HPAIVs) that cause substantial economic losses in the poultry industry (Garcia et al, 1996; Gonzales et al, 2012). Some H5 and H7 subtypes can also infect and elicit severe clinical symptoms in humans (Sutton, 2018).

Wild aquatic birds, especially those in the Anseriformes and Charadriiformes orders, are the primary reservoir for AIVs (Stallknecht et al, 1988). The viruses normally replicate in the gastrointestinal or respiratory tracts of wild birds and can be transmitted to poultry through the fecal-oral route (Wille et al, 2018). Moreover, wild birds often shed high concentrations of AIVs in their feces even when they display no clinical signs of infection (Brown et al, 2014).

The migration of wild birds along nine major global migratory flyways has been suggested to facilitate the circulation of AIVs (Hill et al, 2012; Hansen et al, 2022). South Korea is located on the largest of these avian routes, the East Asian-Australasian Flyway (EAAF) (Hansen et al, 2022), which brings many migratory birds to South Korea during the cold season to overwinter and breed (Choi et al, 2022). Such migratory birds were found to have played a critical role in the global spread of the H5N8 subtype of HPAIVs in 2014, which had first been detected in South Korea and later caused worldwide economic damage (Samantha et al, 2016). Routine monitoring of wild birds in South Korea is therefore necessary to prevent such outbreaks in the future. In this study, we collected active AIV surveillance data from wild bird habitats and identified AIVs prevalence in South Korea from 2019 to 2022.

Sample collection

Fresh fecal samples of wild birds were collected every Korean winter season from 2019 to 2022 from a total of 100 wild bird habitats located near Ganwolho, Gamcheon, Gokgyocheon, Geumgang, Geumgangho, Geumhogang, Dongrimj, Dongjingang, Mangyeonggang, Bonggangcheon, Bongseonji, Bunamho, Sapgyocheon, Seongamji, Ansim Wetland, Yedangji, Joenjucheon, Cheongmicheon, Pungseocheon and Pungjeonji (Fig. 1). The study sites where large numbers of wild birds have used and antigen of AIVs were frequently detected were selected. We collected only fresh and well-distinguished single samples. Specimens were transported to our laboratory facilities immediately and stored at 4℃ until analyses.

Figure 1. Geographical locations of the sampling sites in South Korea (red dots). The map was created using QGIS software.

Virus isolation

Each fecal sample was suspended in phosphate-buffered saline (PBS, pH 7.4) containing antibiotics (100 U/μL of penicillin and 100 U/μL of streptomycin) and centrifuged at 2,800 g for 10 min. Supernatants of five samples were pooled and filtered through a sterile 0.45-μm filter (GVS, USA). The resulting solutions were inoculated into 9∼11-day-old specific pathogen-free (SPF) embryonated chicken eggs, incubated at 37℃ for 3∼4 days, and chilled at 4℃ to harvest allantoic fluids. An HA test was conducted according to the World Organization for Animal Health (OIE) recommendations (Stear, 2005). The HA-positive pooling fecal samples were inoculated individually into SPF embryonated chicken egg using the method previously described and further processed for viral extraction.

Viral RNA detection, subtyping, and species identification

Viral RNA was extracted from HA-positive allantoic fluids using a Miracle-AutoXT Automated Nucleic Acid Extraction System (iNtRON Biotechnology, South Korea). A LiliF^Ⓡ AIV M Real-time RT-PCR Kit (iNtRON Biotechnology, Korea) protocol was applied to detect AIVs. Subtypes were determined by RT-qPCR using the TOP-real™ One-step RT qPCR Kit (Enzynomics, South Korea) with previously described primers (Hoffmann et al, 2016). Host DNA was extracted from the fecal samples and species were identified using mitochondrial cytochrome C oxidase I gene, as previously described (Gao et al, 2009).

Prevalence of AIVs

We isolated 97 AIVs from a total of 7,590 fecal samples (Table 1). The yearly prevalence of AIVs was 0.83% (27/3,248), 1.48% (37/2,499), and 1.27% (33/1,843) from 2019 to 2022, respectively. The prevalence was higher in winter 2020/2021 than during the same months 1 year before and after. We detected higher prevalence in the samples from September to November than in those collected during the remaining months of every year during the study period. The AIVs were isolated from Pungseocheon, Sapgyocheon, Bonggangcheon, Gokgyocheon, Bunamho, Joenjucheon, Mangyeonggang, Dongjingang and Geumgang and the prevalence was 2.22%, 4.40%, 1.28%, 2.37%, 1.66%, 1.37%, 1.81%, 1.38% and 0.64%, respectively (Table 2). The highest rate of AIVs isolation showed in Sapgyocheon (4.40%).

Table 1 . Prevalence of AIVs from 2019 to 2022 in South Korea.

Year	Number of collected samples	Number of AIV isolates	Prevalence of AIV (%)
2019.10	944	10	1.05
2019.11	932	11	1.18
2019.12	740	2	0.27
2020.01	130	0	0.00
2020.02	330	3	0.90
2020.03	172	1	0.58
2019.10∼2020.03	3,248	27	0.83
2020.09	100	18	18
2020.10	229	9	3.93
2020.11	200	1	0.5
2020.12	650	4	0.61
2021.01	640	3	0.46
2021.02	630	1	0.16
2021.03	50	1	2
2020.09∼2021.02	2,499	37	1.48
2021.11	708	28	3.95
2021.12	635	5	0.78
2021.11∼2022.01	1,843	33	1.79
Total	7,590	97	1.27

Table 2 . Numbers of AIVs detected in wild bird species.

Location		Chungcheong-do						Jeolla-do				Total
		Pungseo-cheon	Sapgyo-cheon	Bonggang-cheon	Gokgyo-cheon	Bunam-ho		Jeonju-cheon	Mangyeong-gang	Dongjin-gang	Geum-gang
Number of collected samples		90	454	233	253	120		290	2,422	576	1,243
Number of AIVs isolates (Prevalence of AIV, %)		2 (2.22)	20 (4.40)	3 (1.28) 33 (2.86)	6 (2.37)	2 (1.66)		4 (1.37)	44 (1.81)	8 (1.38) 64 (1.41)	8 (0.64)	97
Numbers of host	A. platyrhynchos		14	2	5	1		3	39	5	1	70
	A. poecilorhyncha	2	5	1	1			1	3	2		15
	A. albifrons		1			1					4	6
	A. anser										2	2
	A. falcata/A. penelope								2			2
	A. crecca									1		1
	A. fabalis										1	1

Prevalence of host species

The hosts of all identified AIVs belonged to the order Anseriformes. Most were detected in the species Anas platyrhynchos (72%; n=70), followed by Anas poecilorhyncha (15.4%; n=15), Anser albifrons (6.1%; n=6), Anser anser (2.0%; n=2), Anser falcata/Anas penelope (2.0%; n=2), Anas crecca (1.0%; n=1), and Answer fabalis (1.0%; n=1) (Table 2). In particular, two or more host species of AIVs were detected in most of studied site and the most diverse host species were identified in Geumgang.

Prevalence of HA and NA subtypes

We isolated 25 HA-NA subtypes of AIVs, including nine HA (H3, H4, H5, H6, H7, H8, H9, H10 and H11) and eight NA (N1, N2, N3, N4, N6, N7, N8 and N9) (Table 3). The most frequently detected HA and NA subtypes was H5 (16.49%; n=16) and N6 (20.62%; n=20), respectively, except mixed subtypes. Both H4N6 (12.37%; n=12) and H5N3 (12.37%; n=12) were the most prevalent subtypes. Fig. 2 also shows yearly distributions of subtypes from 2019 to 2022. H4, H5 and H6 subtype identified in all seasons. The most diverse subtypes of AIVs were detected in Mangyeonggang.

Table 3 . Numbers of haemagglutinin (HA) and neuraminidase (NA) subtypes during AIVs surveillance in wild birds in South Korea, 2019∼2022.

Location		Chungcheong-do						Jeolla-do				Total (prevalence, %)
		Pungseo-cheon	Sapgyo-cheon	Bonggang-cheon	Gokgyo-cheon	Bunam-ho		Jeonju-cheon	Mangyeong-gang	Dongjin-gang	Geum-gang
Numbers of subtypes
H3	N6		3									3 (3.09)
	N8		3									3 (3.09)
	N9		1									1 (1.03)
H4	N2									1		1 (1.03)
	N3				1							1 (1.03)
	N6	2	1	1	1				7			12 (12.37)
H5	N2										3	3 (3.09)
	N3			1					11			12 (12.37)
	N8							1				1 (1.03)
H6	N1		2	1		1						4 (4.12)
	N2								7		1	8 (8.24)
	N6		3									3 (3.09)
	N8							1			1	2 (2.06)
H7	N1								1			1 (1.03)
	N6								1			1 (1.03)
	N7								1	1		2 (2.06)
	N9							1				1 (1.03)
H8	N4								1			1 (1.03)
H9	N2					1			2			3 (3.09)
H10	N4								1		1	2 (2.06)
	N7									1		1 (1.03)
H11	N2									2		2 (2.06)
	N3								4			4 (4.12)
	N8										1	1 (1.03)
	N9								5			5 (5.15)
Mixed subtypes			5		4				2	3	1	15 (15.4)
HxNx			2					1	1			4 (4.12)

Figure 2. Hemagglutinin (HA) and neuraminidase (NA) subtypes proportions of the AIVs from 2019 to 2020 in South Korea. The number of AIVs were described in table.

Global prevalence of AIV was 0.06% (0.30∼1.21%) (Barbara et al, 2017). However, the prevalence of AIVs in wild birds depends on host species, seasonality and locations (Munster et al, 2007). The higher prevalence of AIVs compared to that of global showed in this study (1.27%) and this result was thought to be due to active surveillance conducted in winter season and wild bird habitats.

In 2019/2020, HPAIVs were neither detected in wild bird feces nor at any poultry farms in South Korea (Jeong et al, 2020). However, in 2020/2021, the H5N8 subtype was identified at 109 poultry farms, and the novel H5N1 virus entered the country in late 2021 (Sagong et al, 2022). The higher prevalence of AIVs recorded in 2020/2021 might be explained by the fact that more active surveillance systems were implemented in wild bird habitats to prevent HPAIV outbreaks.

Most migratory birds returned to mid-central and western provinces of South Korea (Jeolla-do and Chungcheong-do) (Lee et al, 2020). We isolated AIVs in Chungcheong-do and Jeolla-do province and this result shows high population of wild birds is related to high isolation rates of AIVs. According to the National Institute of Biological Resources in South Korea, the migration season of wild birds can be divided into four stages: October to November, December to January, February to March, and April to August (Lee et al, 2020). Stage 1 represents autumn migration, and during this season, most migratory wild birds arrive in South Korea from China or Russia (Lee et al, 2020). In an earlier study conducted in South Korea, isolation rates of AIVs in wild bird feces increased during stage 1 but decreased during stage 2, which is consistent with the results of the current study (Shin et al, 2015). These findings suggest that many wild birds that are infected with AIVs migrate to South Korea during the autumn season. AIV surveillance is therefore particularly needed from October to November to prevent the spread of AIVs from wild birds to poultry farms.

The isolation rate of AIVs was especially high in the mallard (Anas platyrhynchos) and the spot-billed duck (Anas poecilorhyncha) and this result is consistent with the results of previous study (Munster et al, 2007). In addition, not only LPAIVs but also HPAIVs have been detected in these avian species earlier (Hill et al, 2012). Moreover, Anas platyrhynchos infected with HPAIVs have been found to exhibit no clinical signs of infection but have high titers of AIVs (Keawcharoen et al, 2008). Previous studies have also shown that HPAIV-positive poultry farms are usually geographically close to wild bird habitats, suggesting that these birds might play a key role in spreading AIVs (Jeong et al, 2014; Kim et al, 2021). Therefore, contact between wild birds and poultry farms should be prevented.

The H4 subtype was distributed worldwide and showed high prevalence in wild birds (Tang et al, 2020). The H4N6 subtype was detected yearly during the study period, which is consistent with previous research. The H5 and H7 subtypes were also isolated, but except for the H5N8 virus, all other H5 and H7 subtypes were LPAIVs. In 2019/2020 and 2020/2021 mixed subtypes of AIVs were isolated in a single wild bird fecal sample, suggesting that coinfection with multiple subtypes of AIVs occurred in wild birds. Coinfection of wild birds by AIVs is important source of reassortment events that generate novel strains of AIVs (Fuller et al, 2010). Thus, even if LPAIVs, continuous genetic analysis of AIVs is required.

AIVs have zoonotic potential, with some subtypes able to infect mammals including humans (Mostafa et al, 2018). The US Centers for Disease Control and Prevention (CDC) have reported that most AIVs that cause severe symptoms and lead to death in humans belong to the H7N9 and H5N1 subtypes of HPAIVs. Human infections with AIVs have occurred, albeit not in South Korea. The World Health Organization International Health Regulations (WHO IHR) recorded 132 such cases from January 1, 2018 to April 4, 2022, and H9N2 LPAIV was identified as the most common virus subtype. Thus, since human infections caused by not only HPAIVs but also LPAIVs have been confirmed continuously and considering that H9N2 LPAIVs are frequently detected in wild birds in South Korea, continuous and active surveillance of AIVs in wild birds is essential to prevent their spread to mammals.

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (2019R1A6A1A03033084), as well as by a Government-sponsored R&D Fund for infectious disease research (GFID; HG18C0084). We would also like to express our gratitude to the editors of the Writing Center at Jeonbuk National University for their skilled English-language assistance.

No potential conflict of interest relevant to this article was reported.