Variations in NSP1 of Porcine Reproductive and Respiratory Syndrome Virus Isolated in China from 1996 to 2022

Since its successful isolation in China in 1995, the porcine reproductive and respiratory syndrome virus (PRRSV) has been mutating into highly pathogenic strains by constantly changing pathogenicity and genetic makeup. In this study, we investigated the prevalence and genetic variation of nonstructural protein 1 (NSP1) in PRRSV-2, the main strain prevalent in China. After formulating hypotheses regarding the biology of the NSP1 protein, the nucleotide and amino acid similarity of NSP1 were analyzed and compared in 193 PRRSV-2 strains. The results showed that NSP1 has a stable hydrophobic protein with a molecular weight of 43,060.76 Da. Although NSP1 lacked signal peptides, it could regulate host cell signaling. Furthermore, NSP1 of different strains had high nucleotide (79.6–100%) and amino acid similarity (78.6–100%). In the amino acid sequence comparison of 15 representative strains of PRRSV-2, multiple amino acid substitution sites were found in NSP1. Phylogenetic tree analysis showed that lineages 1 and 8 had different evolutionary branches with long genetic distances. This study lays the foundation for an in-depth understanding of the nature and genetic variation of NSP1 and the development of a safe and effective vaccine in the future.


Introduction
Porcine reproductive and respiratory syndrome (PRRS), an acute and highly contagious infectious disease caused by the PRRS virus (PRRSV), is characterized by adverse reproductive outcomes and respiratory symptoms in pigs [1]. Regardless of age, most PRRSV-infected pigs experience breathing difficulties [2]. When infected, pregnant sows suffer from miscarriages, premature births, stillbirths, and mummified fetuses. Boars experience shortness of breath and reduced semen quality [3]. Infected piglets become anorexic, breathless, and prone to coughing [4]. Sick pigs infected with PRRS often die from secondary infections, such as pleurisy, streptococcosis, and gasping disease [5]. PRRS has inflicted heavy economic losses on pig farms [6] and gravely threatened the development of Chinese pig industry.
PRRSV is a single-stranded positive-sense enveloped RNA virus. The diameter of the viral particles is between 50 and 65 nm, and the surface has apparent spikes [5]. The nucleocapsid is a three-dimensional symmetrical icosahedral with a diameter between 25 and 35 nm [7,8]. The PRRSV genome is approximately 15 kb long and contains eight open reading frames (ORFs) [9,10]. The replicase gene ORF1 is the largest ORF at approximately 12 kb, which accounts for 80% of the viral genome. The position of ORF1 is after Because NSP1 is involved in inhibiting host cell immune regulation, promoting PRRSV replication and helping PRRSV evade the intrinsic antiviral immunity of the host [45], we used NSP1 as the target gene for the genetic evolution analysis of PRRSV. Additionally, NSP1α has been found to suppress the production of IFN-β, while NSP1β hinders both the synthesis and signaling of IFN, effectively blocking the production of IFN-I in the body. Zhao's experiments have demonstrated that the NSP1β mutant vaccine strain can effectively combat the reversion to virulence that often occurs after vaccination, while also stimulating a specific natural immune response [46]. This suggests that further investigation into genetic mutations in NSP1 could potentially lead to the development of vaccines that offer improved cross protection and higher immune efficacy.
In this study, the genetic variation of PRRSV NSP1 was investigated by analyzing the nucleotide and amino acid sequences of NSP1 of 193 PRRSV-2 strains to construct a phylogenetic tree, and the amino acid sequences of NSP1 were compared to reflect the amino acid substitution of NSP1 in different lineages. The findings of this study provide a basis for further screening of amino acid loci affecting PRRSV virulence and for further studies on the regulatory role of NSP1 amino acid locus changes on the host cellular immune response and the impact on PRRS pathogenesis. We also discussed the role of NSP1 in regulating the immune response of host cells. Hence, our study on NSP1 mutations provides a theoretical basis for preparing a PRRSV vaccine to facilitate the prevention and control of PRRS. The approval number of the Foshan University Ethical committee is SYXK-2020-0235.

Source of Sequences
The nucleotide information of NSP1 of the 193 PRRSV strains used in the experiments was obtained from GenBank, and the amino acid sequences of the virulent strains were translated on EditSeq function of DNASTAR software (version 7.0, Madison, WI, USA).

Nucleotide Sequence Comparison in NSP1
The MegAlign function of DNASTAR software (version 7.0, Madison, WI, USA) was used to analyze the nucleotide sequence similarity in NSP1. The information about reference strains is shown in Table 1.

Amino Acid Sequence Comparison in NSP1
The similarity of NSP1 amino acid sequences was analyzed using the Clustal W method in the MegAlign function of DNASTAR software (version 7.0, Madison, WI, USA). The amino acid sequences of representative strains of each lineage were compared using MEGA software (version 7.0; Center for Evolutionary Medicine and Informatics, Tempe, AZ, USA). GeneDoc [49] annotated the amino acid sequence comparison chart. The information about reference strains is shown in Table 1.

Phylogenetic Tree Analysis
The neighbor-joining tree (NJ) and the maximum likelihood tree (ML) of 193 strains of PRRSV based on NSP1 were generated using MEGA software (version 7.0; Center for Evolutionary Medicine and Informatics, Tempe, AZ, USA). In testing the neighborjoining tree, the phylogeny test w MEGA software (version 7.0; Center for Evolutionary Medicine and Informatics, Tempe, AZ, USA) [50] as the bootstrap method, the number of bootstrap replications was 1000, and the p-distance was utilized. In testing the maximum likelihood tree, the model was the Tamura-Nei model. iTOL (iTOL: Interactive Tree Of Life (embl.de)) [51] annotated the phylogenetic tree. The information about 192 strains is shown in Table 1.

Stability Coefficient and Signal Peptide Prediction of NSP1
ExPASy analysis showed that the predicted molecular weight of NSP1 was 43,060.76 Da, and the instability coefficient was 52.76, implying that it is an unstable protein. The fat solubility coefficient was 83.99, and the theoretical isoelectric point was 8.71. The total mean of hydrophilicity was −0.145, indicating that NSP1 is a hydrophobic protein. SignalP-6.0 estimated that the protein encoded by NSP1 had no signaling peptides.
Because the nucleotide similarity analysis plot of 193 strains of NSP1 was too large, we selected representative strains of each lineage for nucleotide analysis to obtain a comparative plot and elucidate the genetic variation of NSP1 in the evolution of PRRSV. As illustrated in Figure 1
Because the amino acid sequence similarity plot of 193 strains of NSP1 was too large, we selected representative strains of each spectrum for nucleotide sequence analysis to obtain a comparative plot and elucidate the genetic variation of NSP1 in the evolution of PRRSV. As shown in Figure 2, the amino acid similarity was 89.3-91.6% in the lineage 1 group, 87.5% in the lineage 3 group, 99.5% in the lineage 5 group, 90.1-100% in the lineage 8 group, 97.7-100% in the C-PRRSV-like group, and 99.7-100% in the HP-PRRSV-like group. Among these, the amino acid sequence in the lineage 3 group showed the most significant difference (Figure 2).
Because the amino acid sequence similarity plot of 193 strains of NSP1 was too large, we selected representative strains of each spectrum for nucleotide sequence analysis to obtain a comparative plot and elucidate the genetic variation of NSP1 in the evolution of PRRSV. As shown in Figure 2, the amino acid similarity was 89.3-91.6% in the lineage 1 group, 87.5% in the lineage 3 group, 99.5% in the lineage 5 group, 90.1-100% in the lineage 8 group, 97.7-100% in the C-PRRSV-like group, and 99.7-100% in the HP-PRRSV-like group. Among these, the amino acid sequence in the lineage 3 group showed the most significant difference (Figure 2).

Amino Acid Sequence Alignment of NSP1 of Representative Strains
The amino acid sequences of the representative strains of each lineage were compared using MEGA software (Figure 3). The amino acid sequence of NSP1 PRRSV-2 consisted of 383 amino acid residues, with some substitutions and no additions or deletions. The NSP1 amino acid sequence was generally relatively conservative, particularly the 1-97 sites with low substitution frequency. However, among the 182-291 and 302-378 sites, the sequences of various pedigrees were different, and some substitutions were observed. Finally, the amino acid substitution site of the new wild strain GDZQ-2021 was consistent with the traditional representative strains of HP-PRRSV-like and C-PRRSV-like groups.

Phylogenetic Tree Analysis
Phylogenetic tree analysis showed that the QYYZ and QY2010 strains of lineage 3 had close genetic distance with SDRZ01, HH08, CH2002, and CH-1a strains of lineage 8.1, whereas the VR2332 strain of lineage 5 had a long genetic distance with HNyc15, CHsx1401, and BJ-F1501 strains of lineage 8.7 (Figures 4 and 5).

Amino Acid Sequence Alignment of NSP1 of Representative Strains
The amino acid sequences of the representative strains of each lineage were compared using MEGA software (Figure 3). The amino acid sequence of NSP1 PRRSV-2 consisted of 383 amino acid residues, with some substitutions and no additions or deletions. The NSP1 amino acid sequence was generally relatively conservative, particularly the 1-97 sites with low substitution frequency. However, among the 182-291 and 302-378 sites, the sequences of various pedigrees were different, and some substitutions were observed. At

Phylogenetic Tree Analysis
Phylogenetic tree analysis showed that the QYYZ and QY2010 strains of lineage 3 had close genetic distance with SDRZ01, HH08, CH2002, and CH-1a strains of lineage 8.1, whereas the VR2332 strain of lineage 5 had a long genetic distance with HNyc15, CHsx1401, and BJ-F1501 strains of lineage 8.7 (Figures 4 and 5).

Discussion
In recent years, PPRSV has been consistently evolving and enhancing virulence, owing to genetic recombination and variation [52]. PRRS was first identified in the United States and Europe in 1986 and 1990 [53]. PRRSVs with antigenicity close to the United States-based strain were isolated in Japan in 1990 [54]. PRRSV appeared in China in 1995. In 1996, the virus was isolated by Guo et al. and named CH-1a [55]. In 1997, Yang et al. isolated BJ-4 [56]; CH-1a and BJ-4 are North-America-based strains. After spreading in China, the genetic diversity of PRRSV gradually increased, and the amino acids of NSP2 and ORF5 are highly pathogenic [57,58]. After the PRRSV outbreak in China, Tian et al. compared genomics through a trial. The analysis determined that the strain was of a North American genotype with high nucleotide sequence similarities with the Chinese HB-1 strain. Moreover, the NSP2 of the strain was analyzed using bioinformatics analysis. The results confirmed a discontinuous absence of 30 amino acids, which may have contributed to its high pathogenicity [59]. In 2014, Liu et al. compared 36 isolated PRRSV

Discussion
In recent years, PPRSV has been consistently evolving and enhancing virulence, owing to genetic recombination and variation [52]. PRRS was first identified in the United States and Europe in 1986 and 1990 [53]. PRRSVs with antigenicity close to the United States-based strain were isolated in Japan in 1990 [54]. PRRSV appeared in China in 1995. In 1996, the virus was isolated by Guo et al. and named CH-1a [55]. In 1997, Yang et al. isolated BJ-4 [56]; CH-1a and BJ-4 are North-America-based strains. After spreading in China, the genetic diversity of PRRSV gradually increased, and the amino acids of NSP2 and ORF5 are highly pathogenic [57,58]. After the PRRSV outbreak in China, Tian et al. compared genomics through a trial. The analysis determined that the strain was of a North American genotype with high nucleotide sequence similarities with the Chinese HB-1 strain. Moreover, the NSP2 of the strain was analyzed using bioinformatics analysis. The results confirmed a discontinuous absence of 30 amino acids, which may have contributed to its high pathogenicity [59]. In 2014, Liu et al. compared 36 isolated PRRSV strains and glycoprotein 5 of eight vaccine strains. Similarity comparison and amino acid analysis revealed that the 2014 isolates had variations in the neutralizing expression of independent and N-glycosylation sites compared with the first subset of strains [60]. In 2017, Long confirmed through amino acid sequence analysis of NSP2 that the NADC34-like strain was introduced to China from North America. This strain and China's native strain, HP-PRRSV, eventually recombined to form a new strain, SCcd2020 [61]. Therefore, PRRSV diversity is ever increasing.
Although the changes in NSP1 were not as evident as those in the highly variable NSP2 and ORF5, NSP1 still underwent considerable variation that impacted PRRSV evolution. Our analyses showed that NSP1 is a hydrophobic protein without any signaling peptides, indicating that NSP1 is not transportable and may be synthesized in the cytoplasm or organelle matrix from proteins composed of free ribosomes that can enter the cytosol and not from secreted or membrane proteins. NSP1 has high hydrophobicity, which is conducive to forming an α-helix and the inward folding of protein to form a secondary structure; therefore, it is considered stable. Nucleotide and amino acid similarity analyses of 193 PRRSV-2 NSP1 proteins showed that the nucleotide similarity was 79.6-100%, and the amino acid similarity was 78.6-100%. As shown in Figure 1, the lineage 3 strain FJFS had high similarity with the lineage 8 strain TJ, which was as high as 94.2%. This finding indicates that NSP1 undergoes genetic variation during evolution; moreover, there may be gene recombination between the two lineages, which needs to be further verified by recombinant analysis of the strains of the two lineages. Furthermore, lineage 3 is a late-onset strain, and lineage 8 is common in China, meaning that their survival rates differ despite their high nucleotide similarity in NSP1. This result implies that the effect of NSP1 may not be the main factor affecting the survival of PRRSV in the host, which warrants further study. As illustrated in Figure 2, the amino acid sequence of NSP1 had a high similarity. Regarding the differences within the lineage amino acid sequences, lineages 3 and 8 had the largest and smallest differences, respectively. The similarity within a pedigree was higher than that between pedigrees.
Based on the above results, we conclude that during the evolution of PRRSV, NSP1 is relatively conserved. However, genetic variation still occurs, and there may be recombination between strains of different pedigrees, which is worthy of further experimental confirmation. In the comparative analysis of amino acid sequences, NSP1 amino acids underwent substitutions at individual sites, and no additions or deletions were observed ( Figure 3). The variation in NSP1 was more conserved than that of NSP2 and ORF5. In previous studies, Shi found that the smallest region of the carboxyl-terminal extension required for NSP1α to inhibit IFN-β transcription was the 167-176 amino acid site, in which the 176th amino acid and zinc finger structure played the most important roles [34,62]. Replacing the 176th amino acid significantly impacted the inhibition of IFN-β transcription. Shi et al. also found that Cys-270 and His-339 were the catalytic residues of NSP1β; therefore, if Cys were mutated to Ser or His to Ala, NSP1β would lose its enzymatic activity [38]. Wang et al. revealed that KPNA1 degradation and IFN-mediated signal inhibition decreased when residue 19 of NSP1β changed from Val to Ile [44]. These studies indicate that amino acid substitution is related to the viral inhibition of IFN transcription, which impacts virus virulence, phagocytosis, and cleavage.
The phylogenetic tree analyses in Figures 4 and 5 show a relatively large genetic distance between lineages 1 and 8. Zhou et al. proved a genetic recombination between these two strains [62], which increased the survival rate of PRRSV in the host. Therefore, these two lineages are common in China. In genetics, species are determined by conserved, irreplaceable sequences that ensure species stability. In contrast, the differences within species are caused by non-conservative sequences. The recombination and variations in genetic information leads to new strains with different viability and virulence properties [63].
As NSP1 is a conserved sequence, its characteristics enable PRRSV to influence host cells by effectively regulating their immune response, inhibiting signal transduction in specific cases, and blocking their tolerance to inflammation. As the main protease of PRRSV, NSP1 can be used to study corresponding drugs according to their characteristics. In previous studies, Xue et al. studied the three-dimensional structure of PRRSV NSP1β and revealed that the CTD possessed a cleavage site of Trp-Tyr-Gly203 ↓ Ala-Gly-Lys. In contrast, the NTD had strict metal-dependent nuclease activity. In addition, their mutagenesis studies confirmed that Lys18 and Glu52 in the NTD surface-charged region contributed to NSP1β nuclease activity [64]. These results can aid in developing more multi-target drugs in the future. Shi et al. found that HnRNP A2/B1 overexpression can enhance PRRSV virulence without affecting the IFN-β transcription signaling pathway, implying a different mechanism is involved [34]. These studies provide a theoretical basis for future vaccine development. The findings of the present study on the genetic variation of NSP1 pave the way for further research on its role in vaccine development and the pathogenesis, prevention, and control of PRRSV.

Conclusions
The data showed that the strains of the same lineage and different lineages had high nucleotide and amino acid similarity regarding NSP1. The amino acid sequence is relatively conserved, and there are amino acid substitutions at some sites. The study discusses the role of NSP1 in the PRRS and contributes to the monitoring of the evolution of PRRSV in China and the development of methods to prevent and control PRRS. The genetic variation analysis of NSP1 in this study may provide a theoretical basis for the potential impact of NSP1 genetic variation on PRRSV virulence and merits further study in the future. It also provides a basis for future research into more cross-protective vaccines.