Molecular Detection of Severe Fever with Thrombocytopenia Syndrome Virus in Korean Domesticated Pigs

Introduction

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne disease caused by a novel SFTS virus (SFTSV) in the family of Bunyaviridae that was first reported in China in 2011: it is characterized by high fever, thrombocytopenia, leukopenia, gastrointestinal symptoms, and multiple-organ dysfunction in severe cases in humans (Yu et al. 2011). Although human-to-human transmission has been reported, SFTSV is transmitted primarily by the cattle tick Haemaphysalis longicornis, which is known as a major vector for SFTSV (Kim et al. 2013, Luo et al. 2015). In Korea, the incidence of SFTS has been increased annually since the first SFTS case was reported in 2013 (Choi et al. 2016). Moreover, SFTSV antigens have been detected in cats, dogs, and wild animals in Korea (Lee et al. 2017, Oh et al. 2016). However, surveillance of the prevalence of SFTSV in animals is still insufficient in Korea. Therefore, the aim of this study was to investigate the prevalence of SFTSV in domesticated pigs (Sus scrofa domesticus) in Korea.

Materials and Methods

Domesticated pigs raised by confined ranging in small farms (<50 pigs) adjacent to mountainous areas were selected for this study from all eight provinces of Korea from July to October 2013. Blood samples were collected and centrifuged to harvest the sera. RNA was extracted from 200 μL serum using the Gene-spin™ Viral DNA/RNA Extraction Kit (iNtRON Biotechnology, Seongnam, Korea) according to the manufacturer's instructions. To amplify the 346 bp S segment of SFTSV, one-step RT nested PCR was conducted using SFTSV genome-specific primer sets NP-2F (5′-CATCATTGTCTTTGCCCTGA-3′) and NP-2R (5′-AGAAGACAGAGTTCACAGCA-3′) for the first round of PCR, and N2F (5′-AAYAAGATCGTCAAGGCATCA-3′) and N2R (5′-TAGTCTTGGTGAAGGCATCTT-3′) for the nested PCR, as described previously (Oh et al. 2016).

The amplified PCR products were purified using a Gel Extraction Kit (Qiagen, Hilden, Germany) and were cloned into the pGEM-T Easy Vector (Promega, Madison, WI, USA) followed by transformation into Escherichia coli JM109. The plasmid DNA was purified using MG Plasmid SV miniprep (Macrogen, Seoul, Korea) and was sent to Cosmogenetech (Seoul, Korea) for sequencing. To compare the relationship between the sequences from this study and other deposited sequences in GenBank, sequences were aligned using Clustal X V. 2.1 and analyzed with MEGA 7. Phylogenetic trees were constructed using the Maximum Likelihood method based on the Kimura two-parameter model and the data set was resampled 1000 times to generate bootstrap values.

Results

A total of 240 sera were collected from 30 domesticated pigs (5 pigs per farm, 48 farms) in eight provinces (Gwangwon-do, Gyeonggi-do, Gyengsangbuk-do, Gyengsangnam-do, Chungchengbuk-do, Chungcheongnam-do, Jeollabuk-do, and Jeollanam-do). Four sera in Gyeonggi-do (n = 1), Gyeongsangbuk-do (n = 2), and Gyeongsangnam-do (n = 1) tested positive for SFTSV by RT-PCR. The obtained 346 bp S segment sequences of SFTSV were deposited in GenBank (Accession Nos. MG922499–MG922502). Pig 26 (MG922499) and Pig 200 (MG922501) were identical and corresponded to previous reported sequences (KP747610 and KP967531) belonging to a Japanese clade (Fig. 1). Pig 185 (MG922500) and Pig 211 (MG922502) were identical and corresponded to a previous reported sequence (KP967530) belonging to a Chinese clade (Fig. 1).

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FIG. 1.

Phylogenetic analysis of SFTSV based on the partial S segment (346 bp). The sequences identified in this study are indicated by bold letters. Evolutionary history was inferred by using the maximum likelihood method based on the Kimura 2-parameter model (1,000 bootstrap replicates). The percentage of trees in which associated taxa clustered together is shown next to the branches. Scale bar indicates number of nucleotide substitutions per position. SFTSV, severe fever with thrombocytopenia syndrome virus.

Discussion

Previous studies in China have shown that the seroprevalence of SFTSV in livestock varies (3.1–82.8%) according to the animal species (Jiao et al. 2015, Niu et al. 2013, Li et al. 2014). Especially in domesticated pigs, the seroprevalence of SFTSV (3.1–4.7%) was lower than in any other livestock in China (Niu et al. 2013, Li et al. 2014). In addition, to the best of our knowledge, only one study has reported the molecular detection of SFTSV RNA in 2.6% (22/839) of domesticated pigs using quantitative real-time PCR in China (Niu et al. 2013). These findings suggest that domesticated pigs may have lower exposure opportunities to SFTSV due to their raising methods, thicker skin, less hair, and other unknown factors. However, a domesticated pig-derived SFTSV sequence has never been reported until now.

In this study, we identified SFTSV sequences from domesticated pigs; a total of 1.7% (4/240) of domesticated pigs tested positive for SFTSV. Unfortunately, comparing the relationship between SFTSV sequences obtained from Chinese and Korean domesticated pigs was impossible. However, Korean wild boar (S. scrofa)–derived SFTSV sequences were available and were identical to the obtained SFTSV sequences from domesticated pigs in this study. Moreover, the acquired SFTSV sequences from several animals and ticks in Korea were distinct from human SFTSV strains based on phylogenetic analysis (Fig. 1). Although this result does not explain how SFTSV is transmitted or circulates in domesticated animals, we confirmed that a strain differing from the human-derived SFTSV strain was present in animals in Korea.

Based on phylogenetic studies of SFTSV, this virus has been classified into Chinese and Japanese clades (Yoshikawa et al. 2015). To date, many SFTSV strains in Korea have been more closely related to Japanese clades than to Chinese clades (Yoshikawa et al. 2015, Lee et al. 2017). However, this was not observed from our results. In this study, two of the four (50%) obtained sequences were included in the Japanese clade, whereas the other two sequences (50%) were related to the Chinese clade. Although this study had some limitations, including the small sample size and only four positive samples, these results in combination with those of previous studies indicate that the prevalence of SFTSV strains related to the Chinese cluster is higher in animals than in humans.

In conclusion, this study represents the first identification of SFTSV sequences from domesticated pigs and the first molecular detection of SFTSV in domesticated pigs in Korea. Moreover, our results, in combination with those of previous studies, suggest that a new subclade of SFTSV, different from that in humans, might be present in livestock. Therefore, further surveillance of various animal species and large-scale sampling, including genome analysis of SFTSV, is necessary to monitor the incidence of SFTSV in animals and to better understand the role of animals in the life cycle of SFTSV.