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Article

Recombination and Genetic Diversity Analysis of Porcine Reproductive and Respiratory Syndrome 1 Nonstructural Protein 2 Genes in China

by
Chen Lv
1,
Baoyi Guan
1,
Jiankun Pang
1,
Weili Kong
2,
Ruining Wang
3,
Lin Wang
4,
Mengmeng Zhao
1,* and
Hang Zhang
1,*
1
Guangdong Provincial Key Laboratory of Animal Molecular Design and Precise Breeding, School of Animal Science and Technology, Foshan University, Foshan 528225, China
2
Gladstone Institutes of Virology and Immunology, University of California, San Francisco, CA 94158, USA
3
College of Veterinary Medicine, Henan University of Animal Husbandry and Economy, Zhengzhou 450046, China
4
Institute of Cancer Sciences, University of Glasgow, Glasgow G12 8QQ, UK
*
Authors to whom correspondence should be addressed.
Genes 2025, 16(5), 507; https://doi.org/10.3390/genes16050507
Submission received: 19 March 2025 / Revised: 23 April 2025 / Accepted: 25 April 2025 / Published: 28 April 2025
(This article belongs to the Section Animal Genetics and Genomics)
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Abstract

:
Background: Porcine reproductive and respiratory syndrome (PRRS) has been present in China for about 30 years, and because of the high mutability of PRRSV, it causes huge economic losses to pig enterprises every year. PRRSV-2 is widely prevalent in China, and the detection rate of PRRSV-1 is also on the rise. Nonstructural protein 2 (NSP2) is a highly variable protein with multiple biological functions, such as PRRSV replication, which plays an important role in understanding PRRSV variation and epidemic alerts. Objectives: The epidemic characteristics and recombination of PRRSV-1 NSP2 are still unknown. The purpose of this study is to study the epidemic characteristics of PRRSV-1 NSP2 and lay a foundation for the prevention and control of PRRSV-1. Methods: In this study, we collected several PRRSV-1 and PRRSV-2 NSP2 gene sequences for gene sequence and recombination analyses, aiming to analyze the recombination pattern and genetic variation in the PRRSV-1 NSP2 genes in China. Results: The genetic similarity results showed that the 69 PRRSV-1 NSP2 gene sequences collected in this study showed nucleotide similarity ranging from 67.3% to 100.0% and amino acid similarity ranging from 64.3% to 100.0%. Amino acid sequence comparison showed that PRRSV-1 had more amino acid deletion or substitution sites than PRRSV-2. NSP2 also contains special amino acid regions such as the highly immunogenic region. PRRSV-1 can be categorized into four strains, NMEU09-1-like, BJEU06-1-like, HKEU-16-like and Amervac-like isolates, and are at different positions in the ML and NJ phylogenetic trees. In the ninety selected PRRSVs, six recombination events were detected using recombination analysis, two of which occurred in Chinese PRRSV-1 strains. Therefore, sequence analysis of NSP2 helps us to understand the prevalence and variation in PRRSV-1 in China over the past two decades and provides a theoretical basis for studying the epidemiology and evolution of NSP2.

1. Introduction

Porcine reproductive and respiratory syndrome (PRRS), also known as “pig blue ear disease”, is caused by the PRRS virus (PRRSV) and is a highly contagious disease. China experiences a high rate of losses in pig production, often attributed to reinfections from other diseases like pseudorabies, circovirus disease, and bacterial infections, including Escherichia coli in piglets [1]. In 1991 and 1992, the Lelystad virus (LV) strain was obtained in Europe, while the VR2332 strain was isolated from swine in the United States [2,3]. Currently, it is believed that two distinct species belonging to the Betaarterivirus suid 1 and Betaarterivirus suid 2 exist, also known as PRRSV-1 and PRRSV-2, respectively [4]. In 1996, PRRSV-2, also known as CH-1a, was first discovered in Harbin, China, with its complete genome sequence deposited in GenBank in the same year [5]. In 1997, an outbreak of PRRSV-1, also known as B13, was first reported in Beijing, China [6]. PRRSV-2 is mostly found in Asia and North America, in addition to PRRSV-1. PRRSV-2 is exclusively found in the southern part of the American continent, whereas PRRSV-1 is more common in Europe. Nonetheless, PRRSV-2 has also been observed in Western Europe. With the widespread spread of PRRSV in China, mutations and recombinations have occurred. These recombinations occur not only in wild strains but also between wild strains and modified live vaccines (MLV) and even between two different MLV vaccine strains [7,8]. The emergence of recombination phenomena has led to the emergence of new recombinant strains of PRRSV and the phenomenon of virulence reintroduction, which has made the clinical situation of preventing and controlling the PRRS outbreak even more severe and complicated.
PRRSV is an RNA virus with a positive-sense genome enclosed within an envelope. The genome is approximately 15 kb and consists of eleven open reading frames (ORFs) [9]. Specifically, the genome of Lelystad measures 15,111 bp, while the genome of VR2332 is slightly larger at 15,182 bp. ORF1a is responsible for translating at least ten nonstructural proteins (NSPs), including NSP1α/β, NSP1-6, NSP7α/β, and NSP8. Nonstructural protein 2 (NSP2) is the largest replicase cleavage product of PRRSV, which is about 2.9 kb and highly variable, and is the most variable protein in the PRRSV genome [10,11,12,13,14]. NSP2 contains six structural domains, including the N-terminal hypervariable region-I (HV-I), the cysteine protease PLP2 region, the structure of the peptidase C33, the 500–700 aa hypervariable region-II (HV-II) and the C-terminal hydrophobic transmembrane region (TM), in which the PLP2 functional domain has both cis- and trans-cleavage activity, cleaving its own peptide to produce multiple NSP2 subunits (NSP2a-f) or cleaving the region between NSP2 and NSP3 [13,15,16]. Among these, the NSP 2TF and NSP 2N proteins can increase the functional complexity of the NSP2 region of the viral replicase, and both share the N-terminal PLP2 structural domain with NSP2 [17,18]. In addition, HV-II is responsible for the lower amino acid similarity of NSP2 in both the same and different subtypes of strains compared to other more conserved proteins of PRRSV [19]. NSP2 is often used together with the GP5 gene as a subject of molecular epidemiological investigations as well as mutational analyses of PRRSV and plays an important role in understanding viral variation and outbreak alerts.
PRRSV-1 ORF5 sequences from GenBank were analyzed by Shi et al., who identified three subtypes: Subtype I (Russian, Global), Subtype II (Belarus, Lithuania, Russian), and Subtype III [20]. Initially discovered in Western Europe, Subtype I of PRRSV-1 has subsequently spread to the Americas and Asia, and it can be further categorized into 12 branches. Meanwhile, Subtype II and III are primarily found in Eastern Europe and Russia. The majority of PRRSV-1 isolates in China belong to Global Subtype I. There are four subgroups of PRRSV-1. Chen et al. [21] categorized PRRSV-1 into NMEU09-1-like, BJEU06-1-like, Amervac-like, and HKEU-16-like subtypes. China has a high incidence of PRRSV-2, which has led to most studies being focused on investigating PRRSV-2. In recent years, there has been an increase in the incidence of PRRSV-1, thus emphasizing the need for further research on the function of NSP2 in PRRSV-1 strain [8,22,23]. This study aimed to understand recombination events and genetic diversity of NSP2 in PRRSV-1 strains. To compare nucleotide and amino acid similarities, multiple amino acid sequence alignments were performed, phylogenetic relationships were examined, and recombination events were analyzed. These results will contribute to monitoring of the epidemic patterns of PRRSV-1.

2. Materials and Methods

2.1. Strain Collection

Sixty-nine PRRSV-1 NSP2 strains (twenty-five from China and forty-four from overseas) and twenty-one PRRSV-2 strains were carefully selected from the NCBI website (Table 1). In this study, PRRSV-1 strains that have appeared in China and abroad, including classical PRRSV-1 strains and strains that have appeared in recent years, were included in order to comprehensively analyze the genetic variation in the PRRSV-1 NSP2 gene over time and across multiple strains and to help differentiate between domestic and foreign PRRSV-1 strains. In addition, some representative PRRSV-2 strains were selected for comparative analysis to determine the genetic evolutionary differences between the two genotypes.

2.2. Sequence Analysis of the PRRSV-1 NSP2

We examined sixty-nine PRRSV-1 NSP2 sequences for nucleotide and amino acid similarities. The CDs of 69 PRRSV-1 NSP2 strains downloaded from the NCBI website (https://www.ncbi.nlm.nih.gov/ (accessed on 4 April 2025)) were processed into nucleotide sequences using the EditSeq function in the DNAStar software (Version 7.0, Madison, WI, USA), where the nucleotide sequences were further processed into amino acid sequences, and subsequently all sequences were aligned using the Clustal W method in the MegAlign function, and the final output of the nucleotide and amino acid similarity comparison results. In addition, the NSP2 nucleotide sequences of 34 PRRSV-1 and 6 PRRSV-2 strains were selected, and these 34 PPRSV-1 strains are strains from different countries and times, including classical and vaccine strains. Then, they were translated into amino acid sequences in MegAlign, and the amino acid sequences were also processed using the Clustal W method to output the amino acid sequence comparison results. Finally, some regions were labeled and annotated using Adobe Photoshop (version 2022).

2.3. Phylogenetic Analysis

Phylogenetic evaluation of ninety PRRSV NSP2 sequences (Table 1) was performed using the maximum likelihood (ML) method and the neighbor-joining (NJ) method with 1000 bootstrap replicates of MEGA software (Version 11, Mega Limited, Auckland, New Zealand). Subsequently, the sequences were annotated using a web-based tool called The Interactive Tree of Life (https://itol.embl.de (accessed on 15 April 2025)).

2.4. Recombination Analysis

Seven methods (Chimaera, BootScan, 3Seq, GeneConv, SiScan, MaxChi, and RDP) of RDP software (version 4.101) were used to identify potential recombination events. Recombination events that were detected by at least six methods with a p-value of less than 0.05 were significant. In addition, recombination events identified through SimPlot (version 3.5.1) were subjected to validation.

3. Results

3.1. Analysis of Nucleotide and Amino Acid Similarity of the PRRSV-1 NSP2

In this research, twenty-five Chinese and forty-four overseas PRRSV-1 NSP2 sequences were analyzed to assess their nucleotide similarity (Table 2 and Table S1). The findings revealed that the nucleotide similarity of the PRRSV-1 NSP2 was 67.3–100.0%. The similarity between Lena and lena (100.0%) was the highest, whereas that between Tyu16 and BE_08V156 was 67.3%. The amino acid similarity of PRRSV-1 NSP2 was between 64.3% and 100.0%. Similarities between lena and Lena (100.0%) were the highest, whereas similarity between Tyu16 and 15HEN1_EU (64.3%) was the lowest.

3.2. Amino Acid Sequence Alignment

To evaluate the diversity of PRRSV-1 and PRRSV-2 NSP2 amino acid sequences, thirty-four PRRSV-1 NSP2 and six PRRSV-2 NSP2 sequences were selected for multiple sequence alignments (Figure 1). The results confirmed a higher number of amino acid deletion sites in PRRSV-1 NSP2 than in PRRSV-2, indicating lower similarity in PRRSV-1 NSP2. These deletion sites were primarily located within amino acid positions 32–42, 151–153, 193–197, 216–219, 242–247, 387–408, 513–524, 538–570, 585–596, 761–768 and 799–819, and there are a number of scattered positive sites. Furthermore, areas with higher mutation rates were largely centered at positions 32–42, 242–247, 513–524, 538–570, and 799–819 of the amino acid sequences. The variation in NSP2 among PRRSV-1 did not display a specific pattern, and deletion of NSP2 was the highest at the 538–570 site. In addition, mutations and deletions of amino acids similarly occur in the highly immunogenic region of NSP2 and in the region associated with PRRSV replication.

3.3. Analysis of Phylogenetic Analysis

Phylogenetic tree analysis showed that the 90 strains used in the treatment could be divided into two branches, PRRSV-1 and PRRSV-2 (Figure 2 and Figure 3). PRRSV-1 could be further divided into two branches, and the larger branch could be divided into Amervac-like isolates, BJEU06-1-like isolates, HKEU16-like isolates and NMEU09-1-like isolates. In the ML phylogenetic tree, among the NMEU09-1-like isolates, the FJQEU14 and NMEU09-1 strains were genetically closer to PRRSV-2, and the BJEU06-1-like isolates were the most genetically related to PRRSV-1. In the NJ phylogenetic tree, Amervac-like isolates were most closely genetically related to PRRSV-1, and BJEU06-1-like isolates and NMEU09-1-like isolates were more closely genetically related. Notably, the Amervac PRRS vaccine strain showed a close genetic relationship with the SHE strains in both results, and both the European representative LV strain and PRRSV-LV4.2.1 were within the same evolutionary branch.

3.4. Recombinant Analysis

This study aimed to improve our comprehension of the genetic development of PRRSV-1 NSP2 through the analysis of recombination events in NSP2 sequences obtained from a sample of ninety PRRSV strains. We conducted the analysis of the collected sequences using RDP software (version 4.101), revealing six possible occurrences of recombination. These six instances of recombination were substantiated by more than five distinct algorithms, suggesting their strong credibility and statistical significance (p < 0.05) (Table 3 and Figure 4). A total of four sets of recombination events were predicted for the PRRSV-1 strain. In the first recombination event, NVDC-NM1, which was the recombinant strain, contained LNEU12 as the main parental strain and BJEU06-1-2006 as the minor parental strain. Similarly, in the second recombination event, HU18861-2016, which was the recombinant strain, contained JB15-E-M17-JB as the main parental strain and HU18755-2016 as the minor parental strain. In the third recombination event, Porcilis_DV-MLV, which was the recombinant strain, contained PRRSV-LV4.2.1 as the main parental strain and D40 as the minor parental strain. Finally, in the fourth recombination event, HLJB1, which was the recombinant strain, contained BJEU06-1 as the main parental strain and JB15-E-M17-JB as the minor parental strain. In addition, recombination events 1 and 5 were recombinations that occurred in PRRSV-2. JL580 and FJFS strains were the two strains that underwent recombination, FJZ03 and HUN4 strains were their main parental strains, and HUB1 and QYYZ strains were their minor parental strains, respectively. We utilized SimPlot (Version 3.5.1) to conduct our assessment of the six recombination events, confirming their validity (Figure 5). The results showed that recombination events 1, 2, 3, and 6 were verified as recombination events by SimPlot, which contained one recombination event within PRRSV-2 and three within PRRSV-1.

4. Discussion

Since its emergence, PRRS has caused great economic losses to the global pig industry due to its highly infectious nature, and the emergence of new mutant strains with the continuous spread and mutation of PRRSV has also brought great pressure to the clinical prevention and control of PPRS [3,24]. PRRSV-1 and PRRSV-2 outbreaks in China occurred in 1997 and 1996, respectively [5,6]. Now PRRSV-1 has been sporadically endemic and is relatively mildly pathogenic, causing mainly reproductive problems in pigs and occasional cases of respiratory distress, including fever [25]. Consequently, the majority of studies have focused on PRRSV-2. Nevertheless, in recent years, the occurrence of PRRSV-1 has risen in several areas of China, which have reported the identification of PRRSV-1 [26]. The emergence of PRRSV-1 has been reported in major importing countries of Chinese breeding pigs, and the emergence of PRRSV-1 has been reported in all of these countries [27,28]; however, China prohibits the use of PRRSV-1 modified live vaccine, so it is highly likely that PRRSV-1 in China originates from imported breeding pigs. Yu et al. [29] showed that PRRSV-1 strains first entered China as the LV-like PRRSV strain by 2006 and then formed an independent branch in the spread of the virus, and the most recent introduction occurred before 2009, which may be related to the trade of pigs between China and Denmark. In recent years, PRRSV-1 has been reported in several provinces and regions of China. Therefore, conducting epidemiologic studies of PRRS and monitoring genetic variation in PRRSV-1 are essential for the prevention and control of PRRS epidemics [26,29,30].
The NSP2 protein, the most mutated protein of PRRSV, has six structural domains with different functions and plays important functions in PRRSV assembly, transcription, replication, and regulation of immune responses through interactions with PRRSV self and host proteins [15,17,31]. Replication of the NSP2 gene occurs in a bilayer membrane vesicle located in the cytoplasm close to the nucleus. This region is crucial for the replication and transcription of PRRSV. In addition, specific regions of the NSP2 protein (amino acid regions 323–433, 628–747, and 727–747) annotated in Figure 1 have a critical role in PRRSV replication. Previous research has demonstrated that transfecting PRRSV NSP2 plasmid into cells and establishing a stable NSP2 cell line can be used to measure the virus concentration. These experiments showed that NSP2 can promote PRRSV proliferation [32], suggesting that the 727–747 amino acid region of NSP2 may be related to the replication capacity of PRRSV [33]. Wang et al. [32] reported that NSP2 enhances the replication of PRRSV in Marc-145 cells. Conversely, when NSP2 is interfered with in Marc-145 cells, it leads to the inhibition of PRRSV replication. Further investigation of the mechanism by which NSP2 promotes PRRSV replication revealed that the NSP2 C-terminal region is independent of PRRSV replication [34]. However, the amino acid regions 323–433 and 628–747 within NSP2 play a vital role in the PRRSV replication process [35]. Liu et al. [36] further proved that deletion of the 628–727 amino acid region of NSP2 in the TJM strain could inhibit PRRSV proliferation by truncating the transient expression of the protein.
A distinctive feature of PRRSV is the excessive number of mutations, especially in the NSP2 gene, which displays various types of mutations such as deletion, recombination, and insertion, resulting in noticeable variations in the NSP2 proteins [37]. The similarity between the LV and VR2332 strains was only 40.0%. The CH-1a strain of PRRSV-2, first discovered in China, shares 80.0% similarity with NSP2 of the VR2332 strain from the United States. According to Zhang et al. [38], the KZ2018 strain, which is also known as PRRSV-1, shares approximately 88.6% similarity with the LV strain and has 81.9–90.8% similarity with other PRRSV-1 strains found in China. Shen et al. [39] studied the entire nucleotide sequence of the genomic RNA of vaccine strain SP. These findings revealed that the vaccine strain SP exhibited only 78.0% nucleotide sequence similarity with the European LV strain. The most significant variation was observed in the C-terminus of NSP2, wherein 36 and 155 amino acids were inserted. This insertion sequence did not show any similarity with equivalent arterial virus proteins. The notable differences in NSP2 among various PRRSV isolates indicate its potential utility as a marker to distinguish between PRRSV genotypes. Furthermore, NSP2 of the PRRSV-1 KNU-07 strain, which was initially isolated in South Korea in 2009, exhibited a deletion of 60 amino acids [40]. Consequently, NSP2 is commonly employed as a target gene to analyze the evolution of PRRSV. In this study, 25 Chinese PRRSV-1 NSP2 sequences and 44 foreign PRRSV-1 NSP2 sequences were obtained from the NCBI database and analyzed for genetic similarity. The findings indicated that the amino acid and nucleotide resemblance varied from 64.3 to 100.0% and 67.3 to 100.0%, respectively. Zhang et al. [41] reported that the amino acid and nucleotide similarities in PRRSV-2 NSP2 ranged from 72.5 to 99.8% and 63.9 to 99.4%, respectively. The degree of variation in the amino acid sequence of PRRSV-1 NSP2 was higher than that of PRRSV-2 NSP2, but the degree of variation in nucleotide sequence was comparable to that of PRRSV-2 NSP2, which may be attributed to the accumulation of nonsynonymous mutations in some key loci of PRRSV-1 strains under the positive selection pressure and the occurrence of adaptive changes in genes in order to adapt to the epidemiology in China. In addition, the diversity of sample sources is also one of the important reasons affecting the results of gene sequence analysis, and samples from different countries and regions may have different genotypes and genealogical relationships under them. In our investigation, the analysis of amino acid sites between PRRSV-1 and PRRSV-2 NSP2 by aligning thirty-four PRRSV-1 and six PRRSV-2 NSP2 sequences indicated that PRRSV-1 NSP2 showed a higher number of deletion and substitution sites than PRRSV-2. In addition, PRRSV NSP2 has a highly immunogenic region, 21-840aa [42,43], which has multiple B-cell linear epitopes, and different parts of the region showed mutations and deletions of the locus. The results of the amino acid variation (Figure 1) showed that different strains had different degrees of variability in the hypervariable and hyperimmunogenic regions, which could be an adaptive evolution of PRRSV to facilitate immune escape and adaptation of the virus itself for better transmission. It can be seen that the deletion and mutation of amino acids in the NSP2 region of PRRSV-1 pose an obstacle to the effective use of existing vaccines but also provide new directions for the design of new vaccines.
Recombination and mutation are important factors in the evolution of RNA viruses, and recombinant viruses can inherit adaptive features from parental strains to enhance host adaptation and transmission. PRRSV exhibits significant genetic diversity due to its high-frequency mutation and recombination properties, leading to new strains that break through the efficacy of existing vaccine protection [44,45]. The prevalence of PRRSV-1 strains in China may be associated with vaccinated breeding pigs, while factors such as quarantine loopholes and latent infections contribute to the genetic recombination of PRRSV-1, and some of the recombinant strains have demonstrated enhanced pathogenicity and transmission capacity [46], which has resulted in a double blow to the Chinese swine industry from both PRRSV-1 and PRRSV-2. Analyzing the recombination events of PRRSV-1 strains in China and abroad is helpful in revealing its evolutionary pattern in China. When new strains are isolated in the clinic, the combination of phylogenetic tree analysis can rapidly trace back the parental strains and provide a theoretical basis for vaccine selection. The results of our study show that the 90 strains used in the phylogenetic trees constructed based on the ML and NJ methods could be categorized into two genotypes, PRRSV-1 and PRRSV-2, but the division of the similar strains within PRRSV-1 was different in different evolutionary trees. The NMEU09-1-like and BJEU06-1-like strains were genetically closer in the phylogenetic trees constructed by both the ML and NJ methods, and the NMEU09-1-like strain could be similarly divided into two parts. It is worth noting that the HLJB1 strain belonged to the BJEU06-1-like lineage in Chen et al.’s [21] study, whereas it did not belong to any of the sub-lineages in the present study, but both were genetically close to the Amervac-like lineage, which may be due to factors such as algorithmic updating resulting from the iteration of the analysis software. In addition, HKEU-16-like strains were more independent in both phylogenetic trees. However, the results of the analysis of the complete genome sequences varied. Specifically, BJEU06-1-like was more closely genetically related to NMEU09-1-like and Amervac-like strains. This finding further confirms the close genetic linkage between BJEU06-1-like and NMEU09-1-like strains [8]. The reason for this difference may also lie in the significant variation observed in the PRRSV-1 NSP2 gene. Therefore, based on the results of this study, it is possible to classify PRRSV-1 solely on the basis of the NSP2 gene. In our study, six potential cases of recombination were identified. Notably, two of the recombination cases were observed in China, and both involved Chinese parental strains, and all six recombinant strains belonged to or were very close to four subspectrums, including Amervac-like strains. However, Chen et al. [47] indicated that the formation of the HLJB1 isolate resulted from a recombination occurrence between the Amervac vaccine variant and the BJEU06-1 strain. The discrepancy in these discoveries might have arisen because Chen et al. conducted an exhaustive analysis of the complete sequence for identifying recombination cases, while our research solely concentrated on the NSP2 sequence.
In China, vaccination plays a key role in the prevention and control of PRRS, with a major focus on domestically produced vaccines against PRRSV-2 [48]. For PRRSV-1, there are many international vaccines, such as Suvaxyn PRRS MLV (Zoetis, Belgium, USA) and ReproCyc PRRS EU (Boehringer Ingelheim, Ingelheim am Rhein, Germany, etc.) The PRRSV NSP2 region is particularly important in vaccine research because it tolerates deletion of the deletion fragment and maintains stable expression of the inserted genes. Currently, despite the extensive understanding of the pathogenesis, molecular epidemiology, and immune response of PRRSV, timely monitoring of genetic changes in PRRSV could provide a valuable reference for the effective management of PRRS in China in the coming years.

5. Conclusions

From the first appearance of PRRSV-1 in China in 1997 to 2022, the prevalence of PRRSV-1 in China has been more sporadic than that of PRRSV-2, but in recent years, its prevalence has been on the rise. NSP2 is a protein with a high degree of PRRSV variability, which can be involved in various aspects of PRRSV replication and immunomodulation. The PRRSV-1 strains that emerged in China were categorized into four different sub-lineages: NMEU09-1-like, HKEU-16-like, BJEU06-1-like, and Amervac-like, and the PRRSV-1 NSP2 sequences showed a greater diversity of substitutions, deletions, and mutations than the PRRSV-2 NSP2 sequences. In addition, recombination analysis showed that recombination occurred between both PRRSV-1 and PRRSV-2 strains. By analyzing the gene sequence of PRRSV-1 NSP2, we can visualize its genetic evolution and mutation trends during the more than two decades of its prevalence in China and provide a theoretical basis to guide the further study of the biological function of NSP2 and the development of a novel vaccine.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/genes16050507/s1, Figures S1 and S2: Amino acid sequence alignment of the PRRSV-1 NSP2; Table S1: Analysis of nucleotide and amino acid similarity of the PRRSV-1 NSP2.

Author Contributions

Conceptualization, C.L. and J.P.; methodology, C.L. and J.P.; software, C.L. and H.Z.; validation, C.L. and B.G.; formal analysis, C.L. and J.P.; investigation, C.L. and B.G.; resources, C.L. and M.Z.; data curation, C.L. and H.Z.; writing—original draft preparation, C.L. and H.Z.; writing—review and editing, C.L., M.Z. and H.Z.; visualization, M.Z. and H.Z.; supervision, W.K., R.W., L.W. and M.Z.; project administration, M.Z. and H.Z.; funding acquisition, M.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the characteristic innovation project of the Guangdong Provincial Department of Education (2023KTSCX128) and the National Natural Science Foundation of China (31902279) from M.Z.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All datasets are available in the main manuscript. The dataset supporting the conclusions of this article is included within the article.

Conflicts of Interest

The authors declare no conflicts of interest.

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Genes 16 00507 g001
Figure 1. NSP2 amino acid sequence alignment effects of representative PRRSV strains. The colored bars in the plot range from blue to red, with high to low levels of variation at that amino acid site. Amino acid similarity sites are denoted by “.”. Red areas represent PRRSV-1 NSP2 amino acid deletion sites, the blue background represents a highly immunogenic region that NSP2 has, the green background represents a region of NSP2 related to PRRSV replication, and the recombinant strains are shown in purple boxes.
Figure 1. NSP2 amino acid sequence alignment effects of representative PRRSV strains. The colored bars in the plot range from blue to red, with high to low levels of variation at that amino acid site. Amino acid similarity sites are denoted by “.”. Red areas represent PRRSV-1 NSP2 amino acid deletion sites, the blue background represents a highly immunogenic region that NSP2 has, the green background represents a region of NSP2 related to PRRSV replication, and the recombinant strains are shown in purple boxes.
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Genes 16 00507 g002
Figure 2. Phylogenetic evaluation of the PRRSV NSP2 gene was performed using the ML method. Sixty-nine PRRSV-1 and twenty-one PRRSV-2 strains were chosen for the analysis. PRRSV-2 strains are highlighted using a red background color, while PRRSV-1 strains are highlighted using a blue background color. Chinese PRRSV-1 strains are represented by triangles (▲), PRRSV-1 vaccine strains are indicated with squares (■), recombinant strains are represented by triangles (★), and the numbers represent the branch lengths.
Figure 2. Phylogenetic evaluation of the PRRSV NSP2 gene was performed using the ML method. Sixty-nine PRRSV-1 and twenty-one PRRSV-2 strains were chosen for the analysis. PRRSV-2 strains are highlighted using a red background color, while PRRSV-1 strains are highlighted using a blue background color. Chinese PRRSV-1 strains are represented by triangles (▲), PRRSV-1 vaccine strains are indicated with squares (■), recombinant strains are represented by triangles (★), and the numbers represent the branch lengths.
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Genes 16 00507 g003
Figure 3. Phylogenetic evaluation of the PRRSV NSP2 gene was performed using the NJ method. A total of sixty-nine PRRSV-1 strains and twenty-one PRRSV-2 strains were chosen for the analysis. PRRSV-2 strains are highlighted using a red background color, while PRRSV-1 strains are highlighted using a blue background color. Chinese PRRSV-1 strains are represented by triangles (▲), PRRSV-1 vaccine strains are indicated with squares (■), recombinant strains are represented by triangles (★), and the numbers represent the branch lengths.
Figure 3. Phylogenetic evaluation of the PRRSV NSP2 gene was performed using the NJ method. A total of sixty-nine PRRSV-1 strains and twenty-one PRRSV-2 strains were chosen for the analysis. PRRSV-2 strains are highlighted using a red background color, while PRRSV-1 strains are highlighted using a blue background color. Chinese PRRSV-1 strains are represented by triangles (▲), PRRSV-1 vaccine strains are indicated with squares (■), recombinant strains are represented by triangles (★), and the numbers represent the branch lengths.
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Figure 4. Potential recombination events in the NSP2 gene as detected using RDP (Version 4.101). The horizontal axis indicates the position in alignment, while the vertical axis illustrates pairwise identity. Panels (a−f) correspond to the RDP software predictions for each recombinant strain in Table 3, respectively.
Figure 4. Potential recombination events in the NSP2 gene as detected using RDP (Version 4.101). The horizontal axis indicates the position in alignment, while the vertical axis illustrates pairwise identity. Panels (a−f) correspond to the RDP software predictions for each recombinant strain in Table 3, respectively.
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Genes 16 00507 g005
Figure 5. Verification of recombination events in the NSP2 gene through SimPlot (Version 3.5.1). The horizontal axis denotes the position, and the vertical axis signifies the percentage of permuted trees. Panels (a–f) correspond to the validation results of SimPlot software for recombination events for each recombinant strain in Figure 4, respectively. The main parental strain is depicted by the blue line, the minor parental strain by the green line, and the control strain by the red line.
Figure 5. Verification of recombination events in the NSP2 gene through SimPlot (Version 3.5.1). The horizontal axis denotes the position, and the vertical axis signifies the percentage of permuted trees. Panels (a–f) correspond to the validation results of SimPlot software for recombination events for each recombinant strain in Figure 4, respectively. The main parental strain is depicted by the blue line, the minor parental strain by the green line, and the control strain by the red line.
Genes 16 00507 g005
Table 1. Information on the reference sequence of PRRSV NSP2.
Table 1. Information on the reference sequence of PRRSV NSP2.
YearAeraStrainGenBank
Accession Number		Type
1991NetherlandsLelystad virusM96262PRRSV-1
1992BelgiumBE_92V058MW448197PRRSV-1
1996Belgium96V198MK876228PRRSV-1
1999NetherlandsMLV-DVKJ127878PRRSV-1
2001USASD-01-08DQ489311PRRSV-1
2003ChinaHK3KF287129PRRSV-1
2003USAEuroPRRSVAY366525PRRSV-1
2003BelgiumBE_03V140MW053394PRRSV-1
2004ChinaHK5KF287130PRRSV-1
2004ChinaHK8KF287128PRRSV-1
2004ChinaHK10KF287131PRRSV-1
2004NetherlandsPRRSV LV4.2.1AY588319PRRSV-1
2006ChinaBJEU06-1GU047344PRRSV-1
2006United Kingdom215-06OP047897PRRSV-1
2007ChinaHKEU16EU076704PRRSV-1
2007BelarusLenaJF802085PRRSV-1
2008BelgiumBE_08V156MW053397PRRSV-1
2008BelaruslenaJF802085PRRSV-1
2009ChinaSHEGQ461593PRRSV-1
2009ChinaNMEU09-1GU047345PRRSV-1
2009SpainAmervac PRRSGU067771PRRSV-1
2010BelarusSU1-BelKP889243PRRSV-1
2011ChinaGZ11-G1KF001144PRRSV-1
2011ChinaNVDC-FJKC492506PRRSV-1
2011ChinaNVDC-NM1JX187609PRRSV-1
2011ChinaNVDC-NM2KC492504PRRSV-1
2011ChinaNVDC-NM3KC492505PRRSV-1
2011South KoreaD40MZ287330PRRSV-1
2012ChinaLNEU12KM196101PRRSV-1
2012GermanyGER12-720789OP529852PRRSV-1
2013ChinaFJEU13KP860912PRRSV-1
2013RussiaWestSib13KX668221PRRSV-1
2013Belgium13V117KT159249PRRSV-1
2013BelgiumBE_13V246MW053396PRRSV-1
2013BelgiumBE_13V173MW053395PRRSV-1
2013Belgium13V091KT159248PRRSV-1
2013BelgiumBE_13V200MW053399PRRSV-1
2013BelgiumBE_13V153MW053398PRRSV-1
2013SpainES13-49_P85MK024325PRRSV-1
2013BelgiumBE_13V264MW053400PRRSV-1
2014ChinaFJQEU14KP860913PRRSV-1
2014ChinaHLJB1KT224385PRRSV-1
2014GermanyDE14-3073_P85MK024324PRRSV-1
2014DenmarkPorcilis_DV-MLVMT311646 PRRSV-1
2014BelgiumBE_14V023MW053401PRRSV-1
2014PolandPL14-02_P85MK024327PRRSV-1
2014ItalyPR40/2014MF346695PRRSV-1
2014ItalyIT14-32_P85MK024326PRRSV-1
2015China15HEN1_EUKX967492PRRSV-1
2015South KoreaJB15-E-M17-JBMZ287329PRRSV-1
2015South KoreaJB15-E-P47-GBMZ287328 PRRSV-1
2016South KoreaCBNU0495MZ287327PRRSV-1
2016FrancePRRS-FR-2016-56-11-1MH018883PRRSV-1
2016HungaryHU19401/2016MH463457PRRSV-1
2016HungaryHU24924/2016MH463459PRRSV-1
2016HungaryHU19483/2016MH463458PRRSV-1
2016HungaryHU18861/2016MH463456PRRSV-1
2016HungaryHU18755/2016MH463455PRRSV-1
2016RussiaTyu16MT008024PRRSV-1
2017ChinaHENZMD-10KY363382PRRSV-1
2018ChinaKZ2018MN550991PRRSV-1
2018ChinaEUGDHD2018MK639926PRRSV-1
2018ChinaNPUST-2789-3W-2MN242825PRRSV-1
2019South KoreaJBNU-19-E01MW847781PRRSV-1
2019DenmarkDK-2019-10166-107MN603982PRRSV-1
2020ChinaSC-2020-1MW115431PRRSV-1
2020ChinaTZJ226OP566682PRRSV-1
2020ChinaTZJ637OP566683PRRSV-1
2022AustriaAUT22-97OP627116PRRSV-1
1992USAVR2332EF536003.1PRRSV-2
1996ChinaBJ-4AF331831PRRSV-2
1996ChinaCH-1aAY032626PRRSV-2
2003ChinaHN1AY457635.1PRRSV-2
2006ChinaTJEU860248PRRSV-2
2006ChinaHUB1EF075945PRRSV-2
2006ChinaJXA1EF112445PRRSV-2
2006ChinaHUN4EF635006.1PRRSV-2
2008ChinaPRRSV01FJ175687PRRSV-2
2009ChinaSD1-100GQ914997PRRSV-2
2010ChinaQY2010JQ743666PRRSV-2
2011ChinaGM2JN662424PRRSV-2
2011ChinaQYYZJQ308798PRRSV-2
2012ChinaHZ-31KC445138PRRSV-2
2012ChinaFJFSKP998476PRRSV-2
2013ChinaJL580KR706343.1PRRSV-2
2013ChinaFJW05KP860911PRRSV-2
2015ChinaGDsgKX621003PRRSV-2
2015ChinaHNjZ15KT945017PRRSV-2
2015ChinaFJZ03KP860909PRRSV-2
2017ChinaNADC30MH500776.1PRRSV-2
Table 2. Evaluation of nucleotide and amino acid similarities among sixty-nine PRRSV-1 NSP2.
Table 2. Evaluation of nucleotide and amino acid similarities among sixty-nine PRRSV-1 NSP2.
Tyu16Lena
15HEN1_EUa 64.3 (aa)
lena b 100 (nt, aa)
BE_08V156a 67.3 (nt)
a indicates the lowest similarity. b indicates the highest similarity. nt indicates the nucleotide, aa indicates the amino acid.
Table 3. Recombination analysis of the PRRSV NSP2 gene using RDP.
Table 3. Recombination analysis of the PRRSV NSP2 gene using RDP.
Recombination
Event		Recombinant StrainRecombinant
Breakpoint		Recombination Analysis Method (p-Value)
Main
Parental Strain		Minor
Parental Strain		RDPGENECONVBootScanMaxChiChimaeraSiScan3seq
1JL5802134–2205
(3328–3386)		1.509 × 10−641.123 × 10−54NS4.298 × 10−272.053 × 10−291.630 × 10−381.020 × 10−11
FJZ03HUB1
2NVDC-NM13718–66
(1204–1301)		4.846 × 10−305.298 × 10−268.873 × 10−261.700 × 10−204.974 × 10−166.034 × 10−341.020 × 10−11
LNEU12BJEU06-1-2006
3HU18861-20163641–35
(713–884)		6.887 × 10−309.225 × 10−295.293 × 10−321.287 × 10−61.224 × 10121.179 × 10−221.020 × 10−11
JB15-E-M17-JBHU18755-2016
4Porcilis_DV-MLV881–940
(1090–1198)		2.414 × 10−271.319 × 10−273.401 × 10−121.545 × 10−107.047 × 10−107.332 × 10−81.020 × 10−11
PRRSV-LV4.2.1D40
5FJFS746–945
(3590–14)		5.124 × 10−56.492 × 10−202.196 × 10−231.345 × 10−131.414 × 10−81.543 × 10−453.633 × 10−25
HUN4QYYZ
6HLJB12783–3002
(3678–33)		2.268 × 10−105.446 × 10−61.729 × 10−85.043 × 10−73.227 × 10−44.590 × 10−105.884 × 10−3
BJEU06-1JB15-E-M17-JB
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Lv, C.; Guan, B.; Pang, J.; Kong, W.; Wang, R.; Wang, L.; Zhao, M.; Zhang, H. Recombination and Genetic Diversity Analysis of Porcine Reproductive and Respiratory Syndrome 1 Nonstructural Protein 2 Genes in China. Genes 2025, 16, 507. https://doi.org/10.3390/genes16050507

AMA Style

Lv C, Guan B, Pang J, Kong W, Wang R, Wang L, Zhao M, Zhang H. Recombination and Genetic Diversity Analysis of Porcine Reproductive and Respiratory Syndrome 1 Nonstructural Protein 2 Genes in China. Genes. 2025; 16(5):507. https://doi.org/10.3390/genes16050507

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Lv, Chen, Baoyi Guan, Jiankun Pang, Weili Kong, Ruining Wang, Lin Wang, Mengmeng Zhao, and Hang Zhang. 2025. "Recombination and Genetic Diversity Analysis of Porcine Reproductive and Respiratory Syndrome 1 Nonstructural Protein 2 Genes in China" Genes 16, no. 5: 507. https://doi.org/10.3390/genes16050507

APA Style

Lv, C., Guan, B., Pang, J., Kong, W., Wang, R., Wang, L., Zhao, M., & Zhang, H. (2025). Recombination and Genetic Diversity Analysis of Porcine Reproductive and Respiratory Syndrome 1 Nonstructural Protein 2 Genes in China. Genes, 16(5), 507. https://doi.org/10.3390/genes16050507

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