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Introduction
Porcine reproductive and respiratory syndrome (PRRS) is an infectious disease characterized by reproductive disorders in sows along with respiratory signs in piglets and fatteners resulting in significant economic losses in the pig industry worldwide [21,35]. The disease is caused by the PRRS virus (PRRSV), which is classified as a member of the order Nidovirales, family Arteriviridae, and genus Arterivirus [6]. The genome of PRRSV is approximately 15 kb in length and consists of at least nine open reading frames (ORFs) [2,10]. ORF1a and 1b encode the enzymes responsible for replication; ORF2a and ORFs 3, 4, and 5 encode the membrane-associated glycoproteins; ORF2b and 6 encode the non-glycosylated membrane proteins, and ORF7 encodes the nucleoprotein (N) protein [34]. PRRSVs are divided into two genotypes: European (type 1) and North American (type 2) strains. The two genotypes share an approximately 67% similarity at the nucleotide level over the full genome [20,22]. The virus is primarily transmitted by contact with infected pigs but also through feces, urine, semen, and fomites. Additionally, it can be spread indirectly, presumably via aerosol routes and possibly by mechanical vectors [37].
The habitat of wild boar (Sus scrofa) has been destructed with community development and, consequently, at some area, the density and distribution of the wild boar have increased from 2010 to 2011. Without predators or competing animals, the numbers of wild boars have increased regionally [13]. Direct contact between wild boars and domestic pigs may occur rarely because all domestic pigs are reared within farming facilities in Korea. The potential role of wild boars as a reservoir for PRRSV has been reported in France, Germany, and the USA with serological evidence of infection [3,24]. Since the emergence of PRRSV in 1993, PRRS has been widespread in domestic pigs throughout Korea [6,15,17,18,35]. Furthermore, wild boars and domestic pigs have been reported to have the same susceptibility to PRRSV [1]. Monitoring PRRS in wild boars might therefore be an important factor for disease control in domestic pigs. The present study was performed to assess the prevalence of PRRSV in wild boars in Korea and provide information for developing effective PRRS surveillance programs.
Materials and Methods
Serum sample collection and virus propagation
Blood samples collected from 267 wild boars from eight provinces of Korea (Gyeonggi, Gangwon, Chungbuk, Chungnam, Jeonbuk, Jeonnam, Gyeongbuk, and Gyeongnam) were submitted to the Viral Disease Division of Animal, Plant and Fisheries Quarantine and Inspection Agency (Korea) during the hunting season in November 2010~February 2011. Blood samples were taken by hunters from the heart immediately after the wild boars had been shot. The collected samples were stored at 4℃ and transported to the laboratory within 1~2 weeks. The sera were collected by centrifugation for 15 min at 4℃ at 1,500 × g and stored at -20℃ until use. The PRRSV prototype VR-2332 and Lelystad virus (LV) were propagated in MARC-145 cells, and used as positive controls for the reverse transcription-PCR assay.
Serological test
Anti-PRRSV antibody titers were determined using a commercially available HerdChek PRRS 2XR Virus Antibody Test Kit (Idexx Laboratories, USA) according to the manufacturer's instructions. Samples were considered to be positive for PRRSV antibodies if the ratio of sample absorbance to positive control absorbance (S/P) was greater than 0.4.
RT-PCR for the detection and differentiation of PRRSV
Total RNA was extracted from 100 µL of each serum sample using an RNeasy mini kit (Qiagen, Germany) according to the manufacturer's instructions. RT-PCR was carried out using a OneStep RT-PCR kit (Qiagen, Germany) and a PRRSV common primer set (Table 1) derived from the sequences of ORF7 and the 3' non-coding region of the VR-2332 and LV strains. The primer set was designed to detect and differentiate between PRRSV types 1 and 2. The PCR reaction contained 5 µL 5× RT-PCR buffer (2.5 mM MgCl2), 0.4 mM dNTPs, 0.5 µM of each of the four primers shown in Table 1, 1 µL enzyme mix, and 5 µL RNA in a final volume of 25 µL. RT-PCR amplification was carried out as follows: a reverse transcription step at 50℃ for 30 min; RTase inactivation and initial PCR activation at 95℃ for 15 min followed by 35 cycles of denaturation at 94℃ for 20 sec, annealing at 55℃ for 20 sec, and extension at 72℃ for 30 sec, and a final elongation step at 72℃ for 10 min. The amplicons were separated by electrophoresis in a 1.5% agarose gel and stained with ethidium bromide.
Sequencing and phylogenetic analysis of ORF7
For the complete ORF7 sequencing, PCR was performed using SuperScript One-Step RT-PCR with Platinum Taq (Invitrogen, USA) according to the manufacturer's instructions with primers (Table 2) designed to include the full ORF7 area. The PCR amplicons were purified using a MiniElute gel extraction kit (Qiagen, Germany) and cloned using a pGEMT easy vector system (Promega, USA). Sequencing was then performed using a GenomeLab DTCS-Quick Start Kit (Beckman Coulter, USA) and CEQ8000 automated sequencer (Beckman Coulter, USA). Multiple sequence alignment of the individual sequences was performed using CLUSTALX 1.81, and nucleotide sequence identities among the Korean PRRSV isolates were calculated using BioEdit software (Ibis Biosciences, USA).
Phylogenetic reconstructions were generated with PHYLIP (ver. 3.572c) using the neighbor-joining method based on the Kimura two-parameter model [11,16]. Robustness of the phylogenetic analysis was measured by bootstrap analysis with 1,000 replications. Graphic output was produced by TreeView (ver. 1.6.1) [35]. Evolutionary history was inferred using the neighbor-joining method [36]. An optimal tree in which the sum of branch length was 1.90,543,171 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) is shown next to the branches [12]. The tree was drawn to scale with branch lengths in the same units as those of the evolutionary distances used to establish the phylogenetic tree. Evolutionary distances were calculated using the Kimura two-parameter method [11] and are expressed as units of the number of base substitutions per site. The analysis included 40 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 363 positions in the final dataset. Evolutionary analyses were conducted with Molecular Evolutionary Genetics Analysis version 5 (MEGA 5) [29].
Results
Out of the 267 sera tested, four (1.5%) were positive for PRRSV antibodies (Table 3). The ELISA S/P ratios for the positive samples were 0.46, 0.74, 0.77, and 0.91. Eight sera samples (3.0%) were positive for PRRSV antigens (Fig. 1). All PRRSV-positive wild boars were infected with only one genotype. Type 1 virus was detected in three wild boars from two provinces (Gyeonggi and Chungbuk), and type 2 virus was detected in five animals from three provinces (Gyeonggi, Jeonnam, and Gyeongbuk).
Two amplicons (sample No. 49 from Gyeongbuk and sample No. 129 from Chungbuk) from the positive samples were subjected to ORF7 sequencing (Fig. 2). Homology of the deduced amino acid (aa) sequences between the wild boar type 1 virus (sample No. 129) and LV strain was 92.2% (Fig. 2A). Homology between the wild boar type 2 virus (No. 49) and VR-2332 strain was 100% (Fig. 2B). The wild boar type 1 virus had amino acid sequence identities between 96.0% and 98.4% with PRRSVs from domestic pigs in Korea (Table 4). Phylogenetic analysis revealed that the wild boar type 1 virus (No. 129) is closely related to existing PRRSVs recovered from domestic pigs based on ORF7 sequences (Fig. 3). The percentages of identity between ORF7 sequences in wild boar type 1 virus and seven Korean type 1 PRRSVs analyzed in 2007~2009 (D163-1, D82-1, V0773, G210, V1294, KNU-07, and G2448) were found to be 97.2~99.0%.
Discussion
The significance of wild boars as potential vectors or reservoirs for PRRSV and other viral diseases in France, the USA, Italy, and other countries has been evaluated [1,4,8,9,19,24,25,30-32,38]. The wild boar population density in Korea has increased between 1982 and 1997 and it showed an average of 3.7 wild boars per 100 hectares in 2005 [33]. Wild boar blood samples were collected to monitor foot and mouth disease (FMD) and classical swine fever (CSF) in Korea in 2010 and 2011 by Animal, Plant and Fisheries Qurantine and Inspection Agency as a part of National Animal Disease Monitoring Program. In addition to FMD and CSF, the samples were also tested for many other porcine viral diseases. These samples were screened for PRRSVs antibodies and antigens to evaluate PRRS in Korean wild boars. In the present study, prevalence of PRRS in wild boars was not high (less than 3%).
Spread of the virus from domestic pigs to wild boar populations cannot be excluded because of the high prevalence of PRRS in domestic pigs in Korea. Recent data for serological prevalence of PRRS have not been published, but it was estimated to be approximately 70% in the 1990s [7]. Herd prevalence of a newly emerging Korean type 1 PRRSV was 29.4% during 2007~2008 [18].
The N protein encoded by ORF7 is the most abundant, immunogenic, and conserved of all PRRSV proteins. ORF6 and ORF7 are the most conserved nucleotide sequences among the different strains of PRRSV [14,37]. Thus, ORF7 was selected to detect PRRSV in our PCR-based assay. The ORF7 sequence of the wild boar-derived type 2 virus analyzed in the current study was identical to that of the VR-2332 strain, a prototype vaccine virus used worldwide. It is possible that vaccine viruses have spread from domestic pigs to wild boars because vaccination of domestic pigs with an attenuated type 2 VR-2332 is associated with type 2-positive PCR results in Europe [5,23,28].
The wild boar-derived type 1 virus had 92.2% homology with the LV strain, which was prototypic European strain, and very similar identities (96.0% to 98.4%) with PRRSVs from domestic pigs in Korea. ORF7-based phylogenetic analysis showed that the wild boar type 1 virus (No. 129) and other Korean type 1 isolates appeared to be in a cluster of the pan-European subtype 1 proposed by Stadejek et al. [27] and Lee et al. [18]. However, further studies are needed to confirm this finding.
In Korea, some farmers raise their pigs on the edge of mountains with rough fence around the farmland. Sometimes the housed pigs escaped from their pen. Recently, there were increased numbers of reports about damages by wild boars roaming around residential areas including the outskirts of cities. The boars had started to invade residential areas to search for food. Compared to other less densely human and animal populated countries such as the USA and Canada, the environment in Korea provides a better chance for domestic animals to encounter wild animals. It is highly possible for domestic pigs and wild boars to come into contact and exchange pathogens. Recent evidence of this was provided by detection of the classical swine fever virus genome and antibodies in wild boar samples collected in 2011 [26]. Surveys of animal disease in wildlife would be important for national animal disease monitoring programs to determine the status of diseases in specific territories.
In conclusion, the present study showed that PRRSVs are present in Korean wild boar populations. Low prevalence rates of the virus among these animals suggest that PRRS is not endemic in wild boar populations in Korea. Our study explained the need to perform continual investigation of PRRS in wild boar population for efficient national PRRS monitoring.
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