Korean Journal of
Veterinary Service

Influenza is an infectious respiratory disease caused by influenza virus infection. Symptoms in humans range from mild issues such as fever, headache, sore throat, runny nose, cough, muscle pain, and fatigue, to severe complications like pneumonia. Influenza viruses are enveloped negative-sense single-strand RNA viruses with a segmented genome, belonging to the Orthomyxoviridae family. They are classified into four types: Influenza A, B, C, and D viruses (IAV, IBV, ICV, and IDV), each type characterized by a different number of segments. Influenza A and B viruses have eight segments which include RNA polymerase subunits and viral glycoproteins, hemagglutinin (HA) and neuraminidase (NA). In contrast, influenza C and D viruses have only seven RNA segments. Among the four types of influenza viruses, IAV can infect a variety of domestic animals, poultry, and wild birds, including humans. It also constantly mutates through antigenic drift and shift to evade the host’s immune response (Krammer et al., 2018). Of the influenza virus types, only IAV, ICV, and IDV, can infect swine with zoonotic potential (Yu et al., 2021). The first reports of influenza-like disease in pigs emerged in both the United States and Europe during the 1918 human influenza pandemic (Brockwell-Staats et al., 2009).

Influenza in pigs, similar to that in humans, is a highly contagious disease characterized by symptoms such as fever, coughing, sneezing, nasal discharge, lethargy, and low appetite. It is caused by the type A influenza virus. The respiratory tract cells of pigs possess receptor sialyloligosaccharides that include both N-acetylneuraminic acid-α2,3-galactose, which binds to avian influenza viruses, and N-acetylneuraminic acid-α2,6-galactose, which binds to mammalian influenza viruses (Ito et al., 1998). Consequently, pigs are known to act as an intermediary that allows influenza viruses to rearrange and adapt within the host which necessitates it to detect the prevalence of influenza in pigs to prevent influenza from threatening public health (Brockwell-Staats et al., 2009; Tizard, 2021).

However, in Korea, the prevalence of ICV and IDV as compared to swine IAV has not been extensively studied. Therefore, this study attempted to determine the prevalence of ICV and IDV along with IAV in Korean swine farms.

Sample collection

A total of 1,802 respiratory samples, including 1,602 nasal swabs and 200 oral fluids were collected from swine at forty pig farms across nine provinces of Korea: Gyeonggi, Gangwon, Chungbuk, Chungnam, Jeonbuk, Jeonnam, Gyeongbuk, Gyeongnam, and Jeju. Veterinarians collected these samples between October 31 to December 14, 2023. The samples were transported to the laboratory on ice in 24 hours and categorized into seven groups based on the age and health staus of the swine: farrowing pigs (1 to 4 weeks), weaning pigs (5 to 9 weeks), growing pigs (10 to 13 weeks), finishing pigs (over 13 weeks), gilts, sows, and sick pigs.

RNA extraction

Each nasal swab was immersed in 1 mL of phosphate-buffered saline and vortexed for 30 seconds. Each oral fluid sample was vortexed for 3 to 4 seconds and then incubated at room temperature for 20 minutes to facilitate the sedimentation of particulates. The clear supernatant was carefully transferred to a 2.0 mL microtube before RNA extraction. Total RNA was extracted from 200 µL of both nasal swabs and oral fluids, resulting in an elution volume of 50 to 70 µL using a HiQ Viral DNA/RNA Kit v1.0 (BioD, Korea) with a NanoPrep32 Nucleic Acid Extractor, following the manufacturer’s instructions. The extracted RNAs were stored at −80℃ until further analysis.

Reverse transcription PCR

A one-step gel-based RT-PCR method was used for the detection of IAV in RNA samples. The primers specific to the nucleoprotein (NP) of influenza A viruses (Richt et al., 2004) were refined for enhanced amplification. The forward primer used was 5’-AAG CAG GGT AGA TAA TCA CTC A-3’, and the reverse primer used was 5’-TAT GGG TCC TCC AGT TTT C-3’. A volume of 1 µL RNA was reverse transcribed and amplified using 10 µL SuPrimeScript RT-PCR Premix (2X) (GenetBio, Korea, with 1 µL of each primer and 7 µL of water, making a total volume 20 µL. The one-step RT-PCR was conducted at 50℃ for 30 minutes, followed by an incubation at 95℃ for 10 seconds. The amplification process included 35 cycles of denaturation at 95℃ for 30 seconds, followed by annealing at 55℃ for 30 seconds, extension at 72℃ for 1 minute, and a final extension at 72℃ for 5 minutes, using T100 Thermal Cycler. The amplification products were electrophoresed and visualized on 2% agarose gels.

A two-step real-time RT-PCR was used to detect ICV and IDV. Reverse transcription for cDNA synthesis was performed using the WizScript^TM cDNA Synthesis Kit (Wizbiosolutions Inc, Korea), incorporating 3.5 µL of RNA in place of water, as specified in the kit’s protocol, to enhance the efficiency of cDNA synthesis. Further, cDNA was amplified using real-time PCR on the ABI 7500 Fast Real-Time PCR System (Applied Biosystems, USA). The amplification was performed with Premix Ex Taq^TM (Probe qPCR) (Takara Korea Biomedical Inc, Korea) with gene specific primers and probes for the M gene of ICV (Pabbaraju et al., 2013) and the PB1 gene of IDV (Faccini et al., 2017) in a total reaction volume of 25 µL, following the established protocol.

Prevalence of IAV, ICV, and IDV in farm samples

From the 40 farms tested in Korea, IAV was detected in 23 farms (57.05%). The rate of positive samples in IAV-infected farms was 4.0%. Specifically, 43 nasal swabs (2.7%) out of 1,602 and 29 oral fluids (14.5%) out of 200 were positive for IAV. However, ICV and IDV were not detected in any farm (Table 1).

Table 1 . Detection of IAV, ICV and IDV in farm samples including nasal swabs and oral fluids.

Samples	Positive farms and samples (positive rate %)
	IAV			ICV			IDV
	Farms	Samples		Farms	Samples		Farms	Samples
Nasal swabs	16/40 (40.0%)	43/1,602 (2.7%)		0/40 (0%)	0/1,602 (0%)		0/40 (0%)	0/1,602 (0%)
Oral fluids	18/40 (45.0%)	29/200 (14.5%)		0/40 (0%)	0/200 (0%)		0/40 (0%)	0/200 (0%)
Total	23/40 (57.5%)	72/1,802 (4.0%)		0/40 (0%)	0/1,802 (0%)		0/40 (0%)	0/1,802 (0%)

Prevalence of IAV by province

As shown in Fig. 1 and Table 2, the farms and swine samples were classified and analyzed by province. Then, differences in prevalence rates were observed. Every farm in Chungbuk Province was positive for IAV, and 88.9% of farms in Chungnam Province and 80% of farms in Jeonbuk were also IAV positive. Farms in Jeju Island had a positive rate of 66.7%, while those in Gyeonggi Province had a positive rate of 62.5%. The positive rates in these provinces were higher than the overall positive rate of total farms in Korea. Jeonnam and Gyeonbuk Provinces showed lower positive rates than the positive rate of total farms, at 40% and 16.7%, respectively, and all farms in Gangwon and Gyeongnam Provinces were negative. In swine samples, the highest positive rate for IAV was found in Jeonbuk Province at 8.4%, followed by Jeju Island, Chungnam, Chungbuk, and Gyeonggi Provinces at 6.7%, 5.5%, 4.4%, and 4.2%, respectively.

Table 2 . Positive rates of IAV by provinces in Korea.

Province	Swine samples		Positive samples (positive rate %)	Positive farms (positive rate %)
	Nasal swabs	Oral fluids
Gyeonggi (GG)	319	40	15/359 (4.2%)	5/8 (62.5%)
Gangwon (GW)	43	5	0/48 (0%)	0/1 (0%)
Chungbuk (CB)	40	5	2/45 (4.4%)	1/1 (100%)
Chungnam (CN)	360	44	22/404 (5.5%)	8/9 (88.9%)
Jeonbuk (JB)	200	25	19/225 (8.4%)	4/5 (80%)
Jeonnam (JN)	200	25	4/225 (1.8%)	2/5 (40%)
Gyeongbuk (GB)	240	30	1/270 (0.4%)	1/6 (16.7%)
Gyeongnam (GN)	80	11	0/91 (0%)	0/2 (0%)
Jeju Island (JJ)	120	15	9/135 (6.7%)	2/3 (66.7%)
Total	1,602	200	72/1,802 (4.0%)	23/40 (57.5%)

Figure 1. The geographic distribution of IAV positive samples in Korea by provinces in Winter, 2023. There are nine provinces in Korea. The color indicates the proportion of positive farms for IAV in each province. The areas without any color are the cities specially separated from the province. GW, Gangwon; GG, Gyeonggi; CB, Chungbuk; CN, Chungnam; GB, Gyeongbuk; GN, Gyeongnam; JB, Jeonbuk; JN, Jeonnam; JJ, Jeju.

Prevalence of IAV by age of swine

The prevalence of IAV in pigs was also analyzed by age. Among the 23 IAV-positive pig farms, the highest rate of positive samples was found in weaning pigs at 8.9%, followed by farrowing pigs at 7.0%. Growing pigs had a rate of 2.9%. The positive rate of gilts was lowest at 1.0%, with sows slightly higher at 1.1%. In addition, IAV incidences were classified and represented by the age within each farm. It was most frequently observed in weaning pigs, with 16 out of 23 farms affected and second most prevalent age group was farrowing pigs, impacting 9 out of 23 farms. In gilt and sow, it was observed at the lowest frequency, occurring in only 1 out of 23 farms (Fig. 2).

Figure 2. Analysis of IAV-positive pigs by age in Korea. (A) Positive rates by age in pigs from Korean Farms. (B) Comparison of IAV prevalence rates in positive farms for IAV by the age. Sick pigs are categorized as a separate group. The blank indicates that there was no swine in the age.

Influenza is a viral zoonotic disease that occurs worldwide in various species, including humans and pigs (Bailey et al., 2018). Due to its broad host range and zoonotic potential, the influenza virus poses a significant threat to public health. Swine serve as a “mixing vessel” for influenza virus reassortment and evolution, which may facilitate the emergence of new strains or subtypes with zoonotic potential. Four types of influenza viruses have been reported in swine, influenza A virus (IAV), influenza B virus (IBV), influenza C virus (ICV), and influenza D virus (IDV). IAV annually affects an estimated 5∼10% of adults and 20∼30% of children in humans (Fischer et al., 2014). IAV is also widespread globally as one of the most common diseases affecting pigs (Li and Robertson, 2021). In the Midwestern United States, the center of US pig farming, the prevalence ranges from 7∼57%, depending on the season (Chamba Pardo et al., 2017). In Europe, the influence of the IAV is greater than that in the US, with the majority of farms positive for the virus, resulting in approximately one out of every two pigs being infected (Kyriakis et al., 2013). Like IAV, ICV can infect humans and pigs both. Although there are not many studies of ICV in swine, a high seropositive rate of 40∼60% and 80∼100% has been observed in human children and adults, respectively (Lee et al., 2019). Moreover, IDV is less known than other influenza viruses because its primary host is cattle, and no case of IDV infection in humans has been found, while studies from US and Luxembourg revealed seroprevalence rates of 9.5% and 5.9%, respectively, in swine (Snoeck et al., 2018; Liu et al., 2020). These figures represent the extent to which influenza viruses affect humans and pigs.

However, the influenza virus has other risks as well. Being a zoonotic disease, it can spread from pigs to humans with high prevalence, particularly in the light of the Influenza A virus, where the risk is more pronounced due to antigenic shift (Yu et al., 2021). The 2009 pandemic influenza A virus, which originated in swine and subsequently spread to humans worldwide, is a clear example (York and Donis, 2013). Significant antigenic changes, such as antigenic shift, are relatively low for ICV, as it primarily infects humans (Zambon, 1999). Nevertheless, there is a report of swine-human transmission, and the 2009 influenza virus pandemic, linked to zoonotic infections in both humans and pigs indicates that continued attention to ICV is essential (Sederdahl and Williams, 2020). These viruses often evolve in ways that enhance their transmissibility and virulence (Sasaki et al., 2022) so even minor antigenic changes in ICV should not be entirely dismissed, as they could potentially lead to adaptations that pose serious health risks both humans and livestock. With the worldwide appearance and high prevalence of influenza viruses, the possibility of interspecies transmission between humans and pigs poses a significant public health threat, emphasizing the necessity for continuous surveillance of influenza viruses in pigs.

The current study was conducted to detect the prevalence of IAV, ICV, and IDV using polymerase chain reaction. The result showed that 57.5% of pig farms and 4% of the total screened samples were positive for IAV, much lower than Europe at both the farm as well as individual animal levels. The prevalence at the farm level and individual level is similar to that of reports of the US in winter season and southeast Asia, respectively (Kyriakis et al., 2013; Lee et al., 2022). Therefore, the current study suggests that there has been no significant change in IAV prevalence compared to the previous years.

The age-wise prevalence of IAV was found to be highest in farrowing pigs, followed by weaning piglets. Young pigs appeared to be more susceptible to IAV compared to older pigs, which is in agreement with the earlier study which shows that IAV most frequently occurs in pigs aged 4 and 8 weeks and the majority of IAV infections take place in piglets aged under 10 weeks (Takemae et al., 2011). However, ICV and IDV were not found in this study suggesting that the infection is not spreading, while IAV was widespread.

In summary, the incidence of IAV was similar to global levels, but the presence of ICV and IDV is not demonstrated in this study. Nonetheless, given the widespread global influenza epidemic, as such a high incidence rate may pose a very high public health risk, continuous monitoring is essential to further reduce the prevalence of IAV in the Korean swine population.

Between October 31, and December 14, 2023, A total of 1,802 nasal swab and oral fluid samples were collected from 40 pig farms in Korea, and screened for IAV, ICV and IDV, using PCR. The study reveals that 57.5% of the farms tested positive for IAV and the prevalence of IAV in pigs varying according to sampling method, the age, and province. Neither ICV nor IDV was detected on any of the farms surveyed. The presence of ICV, IDV is unclear while IAV is widespread. Nonetheless, to mitigate the potential zoonotic risks associated with various influenza subtypes, it is essential to conduct continuous surveillance of influenza viruses in swine populations.

This research was funded by Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MSIT) (RS-2023-00228644).

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