Whole-genome sequencing and genetic characteristics of representative porcine reproductive and respiratory syndrome virus (PRRSV) isolates in Korea

Porcine reproductive and respiratory syndrome (PRRS) is one of the most important epidemic diseases affecting the global pig industry and was first identified in the United States in 1987 [1]. The economic impact of the disease in the United States, the pork industry of which accounts for approximately 60% of global pig production, has been estimated at $664 million annually [2, 3]. The causative agent, porcine reproductive and respiratory virus (PRRSV), is an enveloped, single-stranded positive-sense RNA virus with a genome that is approximately 15 kb in length and encodes a 5′ untranslated region (UTR), at least 11 open reading frames (ORFs), a 3′ UTR and a 3′-poly(A) tail [4, 5]. PRRSV is classified into two genotypes, namely, PRRSV1 (EU-type, prototype strain Lelystad virus) and PRRSV2 (NA-type, prototype strain VR-2332 virus), which share only approximately 60% similarity at the nucleotide level [6, 7].

The mutation rate of RNA viruses is high due to the lack of 3′–5′ exonuclease proofreading ability of RNA-dependent RNA polymerase (RdRp), and the calculated rate of nucleotide substitutions in PRRSV is the highest reported for an RNA virus [8, 9]. Among the structural genes of PRRSV, the ORF5 gene encodes the major viral envelope protein GP5, which plays an important role in viral assembly, infectivity and neutralizing antibody induction [10‐12]. As ORF5 exhibits high genetic diversity, it is widely used for phylogenetic analysis [13]. Based on the genetic relationships among ORF5 sequences, PRRSV2 is divided into nine lineages, lineage 1 to lineage 9 (L1 to L9), and PRRSV1 is divided into four subtypes, subtype 1 to subtype 4 (sub1 to sub4) [14‐16].

In South Korea, PRRSV2 was first detected in the mid-1980s [17], while PRRSV1 was first detected in 2005 and has since spread rapidly [18, 19]. PRRSV1 has been recognized as highly prevalent in Korea and is classified into subgroups A (sub1A), B (sub1B) and C (sub1C) of subtype 1 (Pan-European PRRSV1), with the majority of isolates belonging to sub1A [14, 20]. PRRSV2 has been spreading across the nation for decades, and its genetic diversity has continued to increase through independent evolution, forming unique nation-specific clades designated lineages K and A (LKA), B (LKB) or C (LKC), which are distinct from prevalent global PRRSV strains and commercially modified live vaccine (MLV) strains [14, 21].

Although PRRSV ORF5 has been a very useful tool that has provided insight into the epidemiology of PRRSV, it accounts for only 5% of the whole genome. Thus, whole-genome sequencing (WGS) studies are urgently needed to provide a more complete picture of the virus and obtain identify the remaining 95% of genomic information for prediction of genetic variations [22, 23]. Indeed, based on the whole-genome information of PRRSV strains, studies have identified recombination between two wild-type PRRSV strains [21, 24, 25], between wild-type PRRSV and a MLV strain [26‐28] and between MLV strains [29]. RNA recombination has been experimentally suggested to occur in vitro and in vivo in two different strains of PRRSV [30, 31]. It not only results in the generation of novel PRRSV genotypes but is also associated with increases in virulence [32]. Additionally, WGS of PRRSV enabled the analysis of insertion and deletion (INDEL) patterns within the genome. Deletion is often observed in PRRSVs. For example, nonstructural protein 2 (NSP2), which is the largest PRRSV protein, can tolerate amino acid (aa) deletions and foreign gene insertions [33], and it has been reported to be linked to certain patterns, such as a 131-aa discontinuous deletion in NSP2 of MN184- or NADC30-like PRRSV [34], a 100-aa continuous deletion in NSP2 of NADC34-like PRRSV [35], and a 30-aa discontinuous deletion in NSP2 of a highly pathogenic PRRSV strain in China [36].

Since high-throughput next-generation sequencing (NGS) became available in 2005 (Roche 454 FLX platform), NGS technology has become an essential tool for nearly all fields of biological research [37]. However, NGS is not yet in routine use for veterinary testing, and recent studies have attempted to utilize NGS technology for PRRSV WGS based on several different methods and sequencing platforms [23, 38, 39]. Sequence-independent, single-primer amplification (SISPA) is a random priming method that allows the enrichment of the viral genome in only a few steps [40, 41]. The value of the high sensitivity of NGS combined with target-independent genome amplification has been proven via the identification of novel viruses from various samples in previous studies [42‐45]. However, to the authors’ knowledge, NGS utilizing the SISPA method (SISPA-NGS) has not been tested or applied in PRRSV.

As the majority of PRRSV genomic sequences submitted to GenBank were contributed by the United States and China [46], the two leading pig-raising countries, the need to identify the prevalent features and genetic variation of unique Korean strains at the whole-genome level has increased [47]. Thus, the purpose of this study was to establish and assess the SISPA-NGS method with the Illumina iSeq 100 platform for PRRSV WGS and to characterize genomic features at the whole-genome level by sequencing archived Korean PRRSV strains.

The epidemiology of PRRSV has been investigated largely by sequencing the ORF5 gene and classifying virus lineages based on ORF5 phylogenetic analysis since the ORF5 lineage classification system was established [15, 16]. However, due to the error-prone nature of PRRSV RdRp and frequent recombination among field strains or vaccine strains, an urgent need for WGS and genome-based analysis has been demonstrated recently. In Korea, ORF5 sequencing has been used in most diagnostic laboratories and has identified PRRSV outbreaks and the emergence of Korean nation-specific lineages [14, 18‐20, 62, 65]. However, the origin and genetic characteristics of these novel lineages remain unclear due to the limited available whole-genome information, as only 12 PRRSV whole-genome sequences are available in the GenBank database. Furthermore, recent reports of recombinant strains in Korea indicate that ORF5 sequencing is no longer sufficient for the complete identification or classification of PRRSV isolates [21, 47, 61, 66]. Thus, the purposes of this study were to establish a WGS method for retrieving PRRSV whole-genome sequences and to analyze the PRRSV genetic diversity in Korea at the whole-genome level.

WGS provides an unsurpassed level of genetic information. In this study, we compared cDNA preparation methods using random hexamers with or without RNA fragmentation and SISPA with or without RNA fragmentation [see Additional file 1]. SISPA is a random priming method that allows the enrichment of a viral genome in only a few steps, which is very useful for obtaining genome sequences from RNA viruses or complex clinical samples, as no prior sequence information is needed [43]. As expected, more virus reads could be generated from samples using SISPA than by using non-SISPA methods [see Additional file 3], resulting in the retrieval of complete genome sequences with a higher depth of coverage [see Additional files 4 and 5]. Moreover, we applied a 300,000 Da MWCO membrane to concentrate PRRSV particles from the cell-cultured supernatant as previously described [49], resulting in a higher initial virus titer, shown as a lower Cq value [see Additional file 3]. Such virus filtration or concentration protocols are common methods for capturing and sequencing unknown virus pools from samples with large volumes, such as ocean water samples [67, 68]. With the advantages mentioned above, the established SISPA-NGS method coupled with virus concentration in this study enabled the successful WGS of cell-cultured PRRSV isolates without prior sequence information (Table 1 and Additional file 6). However, the detection limit based on the Cq value (18.96) was lower than that indicated in a previous report that used the MiSeq platform (20.6 ~ 23.6) [38] (Fig. 1). This might have been due to the reduced data output of the iSeq100 platform, which allows smaller batch sizes than higher-throughput sequencers, such as the MiSeq system [69], resulting in a less sufficient depth of coverage for de novo assembly. It will be necessary to test the established NGS method with clinical samples and other sequencers in the future.

The Korean PRRSV strains newly sequenced in this study were classified into distinct lineages by the ORF5 lineage classification system as previously reported in Korea [14, 48]: subtype 1A, subtype 1C, lineage 1, lineage 5, lineage KOR A (LKA), KOR B (LKB), and KOR C (LKC) (Fig. 2a). With regard to PRRSV1 (EU type), phylogenetic analysis of the whole genome showed that Korean PRRSV subtype 1A viruses are highly divergent from viruses identified from other countries and share a low level of identity with each other (Fig. 2b), which is consistent with previous studies [18, 19]. As PRRSV1 continued to become increasingly prevalent and to impose an enormous economic burden in Korea, two PRRSV1 MLVs [Unistrain PRRS (Amervac) and Porcilis PRRS)] were introduced in Korea in 2014 [66]. A vaccine-like strain (subtype 1C) isolated in this study (strain JB15-E-M17-JB) shares > 99% nucleotide homology with the Unistrain PRRS or SHE strain at the whole-genome level (Fig. 2b). PRRSV1 subtype 1A strains isolated in Korea after 2015 (JB15-E-P47-GB, CBNU0495, and JBNU-19-E01 strains) are highly divergent from those isolated before 2015 (KNU-07, E38, and D40 strains) at the whole-genome level (Fig. 2b). As NSP2 INDEL pattern analysis indicated a consistent 19-aa deletion among PRRSV1 subtype 1A strains (Fig. 3a and Additional file 7) and no recombination signal was detected among PRRSV1 strains, it is suspected that increased independent evolution of Korean PRRSV1 subtype 1A is ongoing in the field. Indeed, a recent study in Korea revealed that PRRSV1 MLV vaccination increased viral genetic heterogeneity and the emergence of new glycosylation sites among Korean subtype 1A viruses when comparisons were conducted before and after vaccine adoption in the field [66]. Continuous surveillance of the dynamics of PRRSV1 evolution in Korea should be further performed at the whole-genome level.

With regard to PRRSV2, vaccine variants of Ingelvac MLV (RespPRRS_MLV) with high nucleotide homology (> 95%) and a consistent NSP2 INDEL pattern were confirmed, except that the JB15-N-M8 strain had a unique 150-aa deletion in the HV region (Figs. 2b, 3b and Additional file 8). In lineage 1, NADC30-like strains (sublineage 1.8) that have emerged since 2015 in Korea [14] and non-NADC30-like strains that were closely related to the ISU30 strain at the whole-genome level were detected based on whole-genome phylogenetic analysis (Fig. 2b). These non-NADC30-like viruses (strains JBNU-19-N01 and JBNU-20-N01) were first isolated in Korea in 2017 [48] and were classified into sublineage 1.6 (close to strain NADC31) based on ORF5 (Fig. 2a). However, these viruses were confirmed to be recombinants of NADC30 and NADC34 through RDP and SimPlot analyses (Table 2 and Additional file 9). The NADC34-like (or RFLP 1–7-4) lineage, which emerged in the United States in 2014, is a highly pathogenic cluster of lineage 1 that causes dramatic abortion ‘storms’ in sow herds and high mortality among piglets [35]. It was subsequently detected in China beginning in 2017 [70], and some Chinese NADC34-like strains were confirmed to be recombinants of NADC34 as the major parent and NADC30 or ISU30 as the minor parent, with recombination in the NSP2 or ORF4-5 region [71]. Likewise, the lineage 1 recombinants identified in this study showed recombination involving NADC34 as the major parent and NADC30 as the minor parent, with recombination signals in the NSP2 and NSP10 regions (Table 2 and Additional file 9). Although it is not clear whether the observed recombination occurred among Korean field isolates or the recombinant strains were initially imported from outside of the country, lineage 1 viruses have been confirmed to be spreading extensively in Korea since 2017 [48]. Thus, the clinical manifestations and continual surveillance of these lineage 1 recombinants should be further assessed and monitored.

Recombination is a pervasive phenomenon among PRRSV isolates and is an important strategy for the generation of viral genetic diversity as a result of increased virulence and/or the generation of novel genotypes [30, 32, 72‐74]. As variants of PRRSV2, Korean lineages (LKA, B and C) have been found to be prevalent in Korean swine herds, but the origin of these nation-specific lineages is currently unknown [14, 48]. Considering the epidemiology of PRRSV2 in Korea, it is suspected that genomic recombination between the LKC and Ingelvac MLV (RespPRRS_MLV) strains might have been involved in the generation of LKB (Fig. 5). Following the first identification of PRRSV2 in Korea in the 1980s [17], the Ingelvac MLV was commercialized in Korea in 1996 [75]. LKA and LKC have been isolated from Korean swine herds since 2003 and 2005, respectively [62, 75]. A study that investigated ORF5 sequences collected from 2003 to 2016 indicated that LKB was first detected in samples collected since 2014 [14]. In this study, multiple recombination signals were detected among Korean lineages, with K07-2273 (LKC) as the major parent and Ingelvac MLV as the minor parent of the strains isolated in the early 2010s (Table 2 and Fig. 4). The analysis of NSP2 INDEL patterns suggested that the Korean lineages originated from the same parents, as identical discontinuous 131-aa deletion patterns were identified in the HV region of NSP2 (Fig. 3b and Additional file 8), which is a well-established pattern in NADC30-like or MN184-like viruses [34, 76]. Among the recombinants identified among the isolates from the early 2010s, the GGYC45 strain (isolated in 2010) is suspected to be the ancestor of current LKB strains, as it is the earliest strain that possesses the ORF5 gene of LKB, since LKB strains emerged in 2014. Given that Korean PRRSV2 evolved independently with genetic heterogeneity in antigenic regions of structural proteins [61], recombination between the Korean-specific lineage (LKC) and the MLV strain in NSP regions, together with evolution in the structural protein regions that resulted in the LKB-like ORF5 gene, might have induced the emergence of a novel sublineage. Although more evidence should be collected and analyzed, it is well known that the extensive use of live-attenuated vaccines contributes to the increased PRRSV genetic diversity in the field [77, 78], and several studies have reported PRRSV2 recombinants involving MLVs [79, 80]. Since multiple lineages of PRRSV2, including MLV variants, Korean lineages and imported lineage 1 viruses are circulating in Korea [48], there is a potential risk of recombination between different lineages. Further research on PRRSV evolution followed by continuous surveillance at the whole-genome level in Korea is needed.

In summary, we established NGS using the SISPA protocol for successful WGS of PRRSV field isolates. Genomic sequence analysis revealed continuous independent evolution of PRRSV1 and evidence of multiple recombination events between PRRSV2 MLV variants and Korean lineages that are prevalent in Korea. Additionally, potential recombinant NADC30-like and NADC34-like strains could be detected in this study. As Korea is an isolated country with very tight borders, the independent evolutionary dynamics observed in Korea could contribute to the general knowledge of PRRSV evolution. Our study highlights the importance of continued monitoring of PRRSV and new vaccine strategies for more efficient control of the virus.