Geographic distribution and molecular analysis of porcine reproductive and respiratory syndrome viruses circulating in swine farms in the Republic of Korea between 2013 and 2016

Background Porcine reproductive and respiratory syndrome virus (PRRSV) causes devastating disease characterized by reproductive failure and respiratory problems in the swine industry. To understand the recent prevalence and genetic diversity of field PRRSVs in the Republic of Korea, open reading frames (ORFs) 5 and 7 of PRRSV field isolates from 631 PRRS-affected swine farms nationwide in 2013–2016 were analyzed along with 200 Korean field viruses isolated in 2003–2010, and 113 foreign field and vaccine strains. Results Korean swine farms were widely infected with PRRSVs of a single type (38.4 and 37.4% for Type 1 and Type 2 PRRSV, respectively) or both types (24.2%) with up to approximately 83% nucleotide sequence similarity to prototype PRRSVs (Lelystad or VR2332). Phylogenetic analysis based on the ORF5 nucleotide sequence revealed that Korean Type 1 field isolates were classified as subgroups A, B, and C under subtype 1, while Korean Type 2 field isolates were classified as lineages 1 and 5 as well as three Korean lineages (kor A, B, and C) with the highest infection prevalence in subgroup A (50.5%) and lineage 5 (15.3%) for Type 1 and Type 2 PRRSV, respectively, among ORF5-positive farms. In particular, the lineages kor B and C were identified as novel lineages in this study, and lineage kor B comprised only the field viruses isolated from Gyeongnam Province in 2014–2015, establishing regionally unique genetic characteristics. It has also recently been confirmed that commercialized vaccine-like viruses (subgroup C) of Type 1 PRRSV and NADC30-like viruses of Type 2 PRRSV (lineage 1) are spreading rapidly in Korean swine farms. The Korean field viruses were also expected to be antigenically variable as shown in the high diversity of neutralizing epitopes and N-glycosylation sites. Conclusions This up-to-date information regarding recent field PRRSVs should be taken into consideration when creating strategies for the application of PRRS control measures, including vaccination in the field.

PRRSV is divided into two major genotypes: Type 1 (European type) and Type 2 (North American type) PRRSV. Lelystad and VR2332 are considered reference strains of Type 1 and Type 2 PRRSV, respectively [27]. Type 1 and Type 2 PRRSV exhibit approximately 60% genomic sequence identity, and 20% nucleotide sequence variability within each genotype [28,29]. Owing to the emergence of highly diverse field viruses, global phylogeny using the ORF5 sequence is characterized by many groups (subtypes 1-4 for Type 1 PRRSV and lineages 1-9 for Type 2 PRRSV) within each of the genotypes, composed of genetically and geographically distinct PRRSVs [30,31].
Previous studies have also reported the genetic diversity and phylogeny of PRRSVs circulating in the Republic of Korea [32][33][34]. Type 2 PRRSV has infected swine farms since the mid-1980s [35], while Type 1 PRRSV has spread rapidly since its first detection in 2005 [33]. However, there has been no study regarding the current prevalence and genetic characteristics of PRRSV in PRRS-affected swine farms nationwide. Recent surveys among swine farmers in the Republic of Korea revealed that over 50% of swine farms have suffered from PRRS, which has been recognized as the most economically important disease in this industry. Combined with the introduction of new modified live vaccines into swine farms over the past three years, the active circulation of field viruses has increased concern regarding the establishment of novel genetic components, and the emergence of antigenic or virulent variants. The following study was conducted to investigate the prevalence, genetic characteristics, and phylogeny of Korean field PRRSVs in recent years (2013-2016).

Study design
Clinical samples (lung tissues and sera) submitted to diagnostic labs (Animal and Plant Quarantine Agency and Jeonbuk National University) were collected from pigs (mostly older than 4 weeks old) with poor growth and respiratory illness between 2013 and 2016. All samples were tested by multiplex reverse transcriptionpolymerase chain reaction (RT-PCR) for amplification of ORF7 with primers designed to detect Type 1 and Type 2 PRRSV simultaneously. Virus isolation using ORF7positive samples was attempted in the MARC-145 cell line and porcine alveolar macrophages (PAMs). Selected viral isolates were sequenced for ORF5, and further subjected to genetic analysis and compared with Korean field viruses isolated in 2003-2010, commercial vaccine strains (DV of Porcilis® PRRS, VP-046 of UNISTRAIN® PRRS, Ingelvac PRRS MLV of Ingelvac PRRS® MLV, and P129 of Fostera® PRRS) commonly used in the Republic of Korea, PRRSV prototype strains (VR2332 and Lelystad), and field isolates previously reported from other countries.

RT-PCR
Total RNA was extracted from the lung homogenates and sera using the RNeasy mini kit (Qiagen, Hilden, Germany), in accordance with the manufacturer's instructions. To detect and differentiate between Type 1 and Type 2 PRRSV, samples were first subjected to multiplex RT-PCR using genotype-specific ORF7 primers (Table 1) and a OneStep RT-PCR kit (Qiagen), which can simultaneously amplify PCR products of 398 and 433 base pairs (bp) for Type 1 and Type 2 PRRSV, respectively. The ORF7-positive sera were further subjected to RT-PCR for ORF5 amplification. The ORF5 genes of PRRSV were amplified with two pairs of primers [33] for Type 1 and Type 2 PRRSV (Table 1), generating PCR products of 754 and 716 bp, respectively. The amplification reaction consisted of 2.5 μl of 5× RT-PCR buffer (including 2.5 mM MgCl 2 ), 0.2 mM dNTPs, 0.2 μM of each primer, 0.5 μl of the enzyme mix, and 5 μl of RNA extract in a final volume of 25 μl. RT-PCR was performed by reverse transcription for 30 min at 50°C, termination of reverse transcription for 15 min at 95°C, followed by 35 cycles of 20 s at 94°C, 20 s at 55°C, 30 s at 72°C, and a final extension of 10 min at 72°C in a C1000 Thermal Cycler (Bio-Rad, Hercules, CA, USA). The RT-PCR amplicons were analyzed using 1% agarose gel electrophoresis.
Nucleotide (NT) and amino acid (AA) sequences and phylogenetic tree analysis Amplified ORF5 products (n = 542) were purified using the QIAquick Gel Extraction Kit (Qiagen) and sequenced (Macrogen, Seoul, Korea). Multiple sequence alignment was initially carried out and homology of the NT and AA sequences among the PRRSV isolates was determined with CLC Main Workbench ver. 7.0.3. ORF5 sequences were subjected to analysis of neutralizing epitopes (NEs), putative N-glycosylation sites (Asn-Xaa-Ser/ Thr), and phylogenetic trees compared with those of Korean field viruses (n = 200) isolated between 2003 and 2010, field isolates of foreign countries (n = 109), including PRRSV prototype strains (VR2332 and Lelystad), and vaccine strains (n = 4) used in the Republic of Korea, available in the GenBank database. The phylogenetic tree was constructed using MEGA6 software with the neighbor-joining method and bootstrap values were calculated on 1000 replicates.

Prevalence of PRRSVs in the Republic of Korea from 2013 to 2016
Among the 631 clinical samples subjected to ORF7 RT-PCR, 38.4% (242/631) were positive for Type 1 PRRSV, 37.4% (236/631) were positive for Type 2 PRRSV, and 24.2% (153/631) were positive for both PRRSV genotypes ( Fig. 1). In terms of geographical prevalence by genotype, Type 1 was more prevalent in Gyeonggi and Jeonbuk Provinces, while Type 2 mainly infected in Gyeongbuk Province. Both genotypes were almost equivalently present in the remainder of the provinces.

Discussion
In this study, the recent geographic prevalence and genetic characteristics of Korean PRRSVs were investigated using field isolates circulating in swine farms nationwide. RT-PCR of 631 clinical samples (farms) revealed that 38. 4% (242/631) were infected by Type 1 PRRSV, 37.4% (236/631) were infected by Type 2 PRRSV, and 24.2% (153/631) were simultaneously infected by both Type 1 and Type 2 PRRSV. When compared with a previous report [38], in which the single infection rates of Type 1 or Type 2 PRRSV and co-infection of both types in swine farms were 29.4, 54.4, and 16.2%, respectively, it was noted that Type 1 PRRSV has become highly prevalent in the 10 years following its first isolation in 2005. In addition, the increased number of farms infected with both Type 1 and Type 2 PRRSV suggested that effective control against PRRS in the Republic of Korea might have to be implemented with the use of control measures against both genotypes.
All of the Korean Type 1 PRRS viruses were classified into the subgroups A, B, and C of subtype 1, consistent with a previous report [34]. The majority of the Korean  [39] first reported that a Korean isolate was grouped with DV. However, it is not possible to define genetic relatedness between the field isolate and the vaccine strain due to the lack of sequence information. Subgroup C viruses (5 DV-and 15 VP-046-like strains) isolated in this study showed a phylogenetic closeness to the vaccine strains with greater than 99% NT sequence similarity without geographical predominance. Therefore, it has been suggested that the vaccine-   (1) Korean Type 2 PRRS viruses have been divided into lineages 1, 4, and 5, as well as the Korean lineages kor A, B, and C based on global PRRSV phylogeny [30]. The majority of the Korean Type 2 PRRS field isolates belonged to lineage 5 and the Korean lineages. Lineage 5 viruses were grouped with VR2332-like viruses genetically close to the Ingelvac PRRS MLV vaccine virus, which has been used commercially since 1996. The lineage also included Korean reference field strains, PL97-1 [40], LMY [32,33], and CA-1 [41]. Regarding the emergence of novel Korean lineages, lineages kor B and C were first classified in this study, while lineage kor A was first reported as a Korean lineage in a previous study of field isolates of 2010 [36]. Lineages kor A and C were observed to include field viruses isolated in 2005-2009 and 2013-2016, which indicated that these groups have developed genetic components geographically distinct from those in foreign countries for longer than 10 years. Meanwhile, lineage kor B comprised only recent isolates that originated from Gyeongnam Province between 2014 and 2016, suggesting that this group may have recently established regionally unique genetic properties within the Republic of Korea.
Nine viruses of lineage 1 isolated between 2014 and 2015 were closely related to NADC30, with 92.3-94.9% NT sequence similarity to NADC30, which was one of the representative virulent strains along with MN184 and SDSU73 in the United States [42]. Chinese NADC30-like viruses (HNjz15, JL580, HENAN-HEB, HENAN-XINX, and CHsx140) were also reported to cause obvious clinical symptoms in pigs with virulence comparable to that of NADC30 [43][44][45]. It should be noted that the circulation of NADC30-like viruses in the Republic of Korea has not been previously reported. Although the virulence of NADC30-like viruses is known to be less than that of highly pathogenic PRRS viruses [43], there is a strong need to define the virulence of recent NADC30-like isolates in pigs in Korean farms. There are two possible explanations for the recent emergence of NADC30-like viruses. First, the NADC30like viruses may have recently been introduced by the importation of breeding pigs, likely as Chinese NADC30-like viruses [45]. Second, it has been assumed that NADC30 was introduced 7-10 years ago, circulated in a particular environment (e.g., with wild boars as hosts) based on an estimated mutational change rate (0.78% per year) of ORF5 NTs identified in previous studies [24,46], and was recently transmitted to domestic swine farms in various ways (e.g., wild boar farming).
The NEs and N-glycosylation patterns of GP5 were highly variable, and novel epitopes and N-glycosylation patterns continuously emerged and disappeared in the field viruses, although this was more apparent in field viruses isolated in 2013-2016 than in 2003-2010 (Table 3). As expected, NEs of vaccine strains or PRRSV prototype strains existed in the majority of field isolates (82.2 and 61.0% for Type 1 and Type 2 PRRSV, respectively) without a preference for a certain subgroup or lineage. In particular, NEs of DV (or Lelystad) (WSFADGN) and Ingelvac PRRS MLV (or VR2332) (SHLQLIYNL) were maintained in approximately 80.4 and 54.6% of field isolates, respectively. It has been speculated that the dominance of the two NEs might be due to extensive vaccination using vaccine strains (Ingelvac PRRS MLV and DV) that originated from PRRSV prototype strains. Nevertheless, it has been noted that the emergence of novel NEs has been significantly expedited over time in PRRSV field isolates (8. Regarding the N-glycosylation pattern, it was interesting to note that N-glycosylation sites were highly diverse and changeable in Korean Type 2 PRRS field isolates, while being conserved in Korean Type 1 PRRS field isolates. NEs and the Nglycosylation pattern are closely associated with virus propagation, neutralization, and immunological protection [47][48][49][50][51]. Therefore, it has been suggested that the strong preference for mutation of NEs and the N-glycosylation pattern, particularly in Type 2 PRRSV, might be associated with limited protective efficacy of PRRS vaccination against heterologous viruses in the field, as shown in previous studies [52][53][54]. Type 2 vaccine-like viruses have become highly prevalent since the first use of Ingelvac PRRS MLV in 1996 [32]. In this study, lineage 5, including the Type 2 vaccine strain and vaccine-like viruses, showed the highest prevalence (15.3%) in the field. Likewise, the recent prevalence of subgroup C, including Type 1 vaccine-like viruses, is low (3.69%). However, extensive use of Type 1 live vaccines may result in a dramatically increased prevalence of Type 1 vaccine-like viruses and/or isolation of field viruses sharing a genomic part of Type 1 live (See figure on previous page.) Fig. 3 Multiple alignment of ORF5 amino acid sequences of representative isolates in each subgroup (Type 1 PRRSV) (a) and lineage (Type 2 PRRSV) (b). The representative isolates were selected based on main amino acid sequence of neutralizing epitope of Type 1 PRRSV (residues 29-35) [23] and Type 2 PRRSV (residues 37-45) [37]. The solid line box and gray color box represent neutralizing epitope and decoy epiotpe (residues 27-31) [57], respectively. The dot-line box indicates hypervariable regions [58] vaccine strains in the near future, caused by genetic evolution mechanisms of PRRSV, random point mutations, and homologous recombination [55]. To minimize the circulation of live vaccine strains in the field, we may have to strengthen swine farm bio-security to prevent contamination of live vaccine strains among swine farms vaccinated with live vaccines. At the same time, limited use of live vaccines may be recommended in young pigs (3-8 weeks old) or pregnant gilts/sows of PRRS-affected farms.

Conclusions
This study suggests that PRRS viruses in the Republic of Korea have recently developed genetic characteristics geographically distinct from currently prevalent global PRRS viruses or commercial vaccine strains. Therefore, further study will be required to evaluate how the established genetic characteristics have affected PRRS outbreaks in domestic swine farms, which will provide important information for the implementation of control and preventive measures in swine farms.