Phylogenetic analysis of kobuviruses and astroviruses from Korean wild boars: 2016–2018

Between 2016 and 2018, the prevalence of porcine kobuvirus (PKoV) and porcine astrovirus (PAstV) in Korean wild boars (n = 845) was 28.0% and 10.7%, respectively. Coinfection by both viruses was detected in 5.1% of boars. Phylogenetic analysis revealed that 134 PKoV isolates belonged to diverse lineages within the species Aichivirus C; however, one strain (WKoV16CN-8627) clustered with bovine kobuvirus (Aichivirus B). Forty-seven PAstVs belonged to lineage PAstV4, and only one strain (WAst17JN-10931) was a novel addition to lineage PAstV2. The two viruses were more prevalent in boars weighing ≤ 60 kg than in boars weighing > 61 kg.

As the population density of wild boars (Sus scrofa) increases worldwide, there are concerns that they may transmit infectious pathogens to domestic pigs and other animal species [21]. Indeed, wild boars in Asia and Europe may act as a reservoir for viruses that can cause disease in commercial livestock. Such viruses include classical swine fever virus, African swine fever virus, porcine parvovirus, porcine reproductive and respiratory syndrome virus, and hepatitis E virus [22]. In this study, we examined the circulation of PKoV and PAstV in wild boars in South Korea and investigated their prevalence and genetic diversity.
Of the 845 wild boars captured by hunters during 2016-2018 (a 3-year period), 729 weighed between 6 kg and 300 kg. Based on weight, we classified the wild boars as ≤ 10 kg, 11-30 kg, 31-60 kg, and ≥ 61 kg ( Table 1). The remaining 116 boars lacked information and were classified as "unknown". None of the fecal samples obtained from the captured wild boars were diarrheic. Boars were hunted and captured randomly in the mountainous areas of nine provinces: Gyeonggi (n = 75), Gangwon (n = 112), Chungbuk Handling Editor: Akbar Dastjerdi. For analysis, fecal samples (1 g) were resuspended in 10 volumes of phosphate-buffered saline containing 1% gentamicin (Gibco Life Technologies, Paisley, UK) and then centrifuged (5,000 × g for 10 min) to obtain a clarified supernatant. Viral RNA was extracted from 0.2 ml of fecal supernatant using an RNeasy Mini Kit (QIAGEN, Valencia, CA, USA), followed by reverse transcription into viral cDNA using random hexanucleotide primers (Nano-Helix, Daejeon, Korea). To detect enteric viruses (PKoV and PAstV), PCR was carried out using primers targeting conserved regions within the 3D gene of PKoV and the RNA-dependent RNA polymerase (RdRp)-ORF2 region of PAstV as described previously [9,20]. The amplified genes were sequenced at CosmoGenetech (CosmoGenetech Co., Daejeon, Korea) using specific primers and an ABI Prism 3730xi DNA Sequencer (Applied Biosystems, Foster City, CA, USA). Clustal X 1.83 software was used to align these sequences with reference sequences obtained from the Gen-Bank database [23]. Phylogenetic trees for PKoV and PAstV were constructed [24] by the neighbor-joining method [25] using MEGA 6.0 software, with the Kimura 2-parameter model as the measure of genetic distance.
Overall, 28.0% (237/845, including unknown samples) were PKoV positive, and the prevalence rates were 33 In terms of geographical prevalence, PKoV and PAstV were detected in most provinces; however, PAstV was not detected on Jeju Island (Fig. 1A). The prevalence of PKoV in Gyeongnam (42.2%, 35/83) was significantly higher than that on Jeju Island, which had the lowest PKoV infection rate (13.3%, 2/15) (p < 0.05) (Fig. 1A). In 2018, the prevalence of PKoV infection in wild boars was 35.1%, compared with 18.7% in 2016 and 24% in 2017 (Fig. 1B). Gyeongbuk showed the highest rates of PAstV infection (19.2%, 32/167) (Fig. 1A). The emergence of PAstV in Korean wild boars was first reported in 2011; however, the prevalence was only 0.7% (1/146) [11]. Recently (between 2016 and 2018), PAstV infections have increased: from 3.2% in 2016, to 8.0% in 2017, to 15.9% in 2018 (Fig. 1B). These findings indicate that the enteric viruses PKoV and PAstV are being transmitted at an increasing rate within the wild boar population of South Korea. Therefore, additional investigations of farm pigs will be required to clarify whether the increase in the number of virus-infected wild boars threaten domestic pigs.
Of the 237 PKoV isolates detected in Korean wild boars, 135 were used for phylogenetic analysis (again, redundant sequences were excluded). To investigate the genetic relationships among kobuvirus sequences, we constructed a phylogenetic tree based on 195 partial 3D gene sequences of PKoV, including 60 global PKoV strains (Fig. 2B). The neighbor-joining tree showed that the 134 Korean wild boar PKoV strains clustered within diverse lineages belonging to the species Aichivirus C. Most Korean wild boar PKoVs clustered with domestic pig strains identified in the USA, Japan, Thailand, China, Korea, Spain, and Hungary. Two Korean wild boar strains (WKoV17GN-11321 and WKo-V18GN-11819) were closely related to the WB-1-HUN strain isolated from Hungarian wild boars [28] (Fig. 2B).
In conclusion, we show here that PKoV and PAstV are circulating nationwide in Korean wild boars, and that there is no correlation between prevalence of the virus and the province or the weight of the animals. PKoV strain WKo-V16CN-8627 from Korean wild boars showed the most similarity to bovine kobuvirus strains, suggesting natural transmission of kobuviruses between pigs and cattle. The data also suggest that PAstV-2 infecting Korean wild boars could be involved in interspecies transmission to domestic pigs.