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Article

Genetic Variability and Recombination of the NSP2 Gene of PRRSV-2 Strains in China from 1996 to 2021

1
School of Life Science and Engineering, Foshan University, Foshan 528000, China
2
Gladstone Institutes of Virology and Immunology, University of California, San Francisco, CA 94158, USA
*
Authors to whom correspondence should be addressed.
These authors contributed equally to this work.
Vet. Sci. 2023, 10(5), 325; https://doi.org/10.3390/vetsci10050325
Submission received: 14 March 2023 / Revised: 18 April 2023 / Accepted: 28 April 2023 / Published: 29 April 2023

Abstract

:

Simple Summary

Genetic variability and recombination of the NSP2 gene are of great significance in gaining an in-depth understanding of the prevalence of PRRSV in China over the past 25 years. We compared the nucleotide and amino acid homologies of the NSP2 sequences of different PRRSV-2 lineages, and examined phylogenetic relationships based on an analysis of the NSP2 sequences of 122 strains. What is more, recombination analysis revealed the occurrence of five recombinant events among the 135 selected PRRSV-2 strains. These results provide a theoretical foundation for evolution and epidemiology of the spread of PRRSV.

Abstract

Porcine reproductive and respiratory syndrome (PRRS) is one of the most serious infectious diseases that detrimentally affects the pig industry worldwide. The disease, which is typically difficult to control, is an immunosuppressive disease caused by the porcine reproductive and respiratory syndrome virus (PRRSV), the genome of which (notably the NSP2 gene) undergoes rapid mutation. In this study, we sought to determine the genetic variation in the PRRSV-2 NSP2 gene in China from 1996 to 2021. Strain information was obtained from the GenBank database and analyzed from a molecular epidemiological perspective. We compared the nucleotide and amino acid homologies of the NSP2 sequences of different PRRSV-2 lineages, and examined phylogenetic relationships based on an analysis of the NSP2 sequences of 122 strains. The results revealed that NADC-30-like strains, which are represented by lineage 1, and HP-PRRSV strains, which are represented by lineage 8, were the most prevalent in China from 1996 to 2021. Close similarities were detected in the genetic evolution of lineages 3, 5, and 8. For nucleotide and amino acid sequence comparisons, we selected representative strains from each lineage, and for the NSP2 among different PRRSV-2 strains, we accordingly detected homologies of 72.5–99.8% and 63.9–99.4% at the nucleotide and amino acid levels, respectively, thereby indicating certain differences in the degrees of NSP2 amino acid and nucleotide variation. Based on amino acid sequence comparisons, we identified deletions, insertions, and substitutions at multiple sites among the NSP2 sequences of PRRSV-2 strains. Recombination analysis revealed the occurrence of five recombinant events among the 135 selected PRRSV-2 strains, and that there is a high probability of recombination of lineage 1 strains. The findings of this study enabled us to gain an in-depth understanding of the prevalence of PRRSV in China over the past 25 years and will contribute to providing a theoretical basis for evolution and epidemiology of the spread of PRRSV.

1. Introduction

Porcine reproductive and respiratory syndrome (PRRS) is a highly prevalent infectious disease caused by the porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV infection causes immunosuppression; reproductive disorders in pregnant sows, including miscarriage, premature birth, and mummified fetuses; and respiratory diseases in piglets, thereby having particularly detrimental impacts on the pig industry worldwide. PRRS was first discovered in North Carolina in 1987, and the LV and VR2332 strains were subsequently isolated from infected pigs in Europe and America in 1991 and 1992, respectively [1,2]. At present, PRRSV-1 and PRRSV-2 were classified as the species Betaarterivirus suid 1 and Betaarterivirus suid 2, respectively. PRRSV-2 strains are mainly prevalent in China, wherein PRRSV was isolated for the first time in 1996 [3]. In 2006, a highly pathogenic PRRSV (HP-PRRSV) was detected, which was associated with a high fatality rate among piglets [4,5]. More recently, in 2012, a new strain type, NADC30, appeared in China [6]. PRRSV continues to recombine and mutate, which accordingly presents considerable challenges with respect to the prevention and control of PRRS [7].
PRRSV, a single-stranded positive-sense RNA virus, is a member of the genus Arterivirus in the family Arterivirus of the order Nidoviridae. It comprises a 15 kb genome containing 11 open reading frames (ORFs), namely, ORF1a, ORF1b, ORF2a, ORF2b, ORF3-7, ORF5a, and ORF1aTF, that overlaps the non-structural protein (NSP) 2-encoding region of ORF1a [8]. Among these, ORF1a and ORF1aTF translationally encode at least 12 NSPs, including NSP1α, NSP1β, NSP2-related proteins (NSP2N, NSP2TF, and NSP2), NSP3, NSP4, NSP5, NSP6, NSP7α, NSP7β, and NSP8 [9,10]; NSP2 at 2.9 kb in length is the longest. NSP2 and ORF5 are highly variable and ORF5 is associated with the neutralizing epitope. They are usually used as target genes for PRRSV molecular epidemiological surveillance [11]. Since the time of its isolation in the United States in 2001, the MN184 strain has been found to have discontinuously lost 131 amino acids in NSP2 sequence [12], whereas isolates obtained during the HP-PRRSV outbreak in China in 2006 were found to have discontinuously lost 30 amino acids from NSP2 sequence [5]. Similarly, the NSP2 sequence of the NADC34-like PRRSV strains recently isolated in China was found to have undergone a continuous loss of 100 amino acids. Studies have demonstrated that the recombinant strain HLJ/2017/1127a is a result of the recombination of the FZ06A and QYYZ strains. The recombination breakpoint is located at 1892–2730 (1742–2442) [13].
Shi et al. proposed a systematic classification of PRRSV-2 based on ORF5 gene in 2011, dividing PRRSV-2 into 9 lineages and 37 subfamilies [14]. Given the increasing number of PRRSV recombinant strains and increases in recombinant frequency in recent years, the epidemic situation of PRRSV in our country has become progressively more complex, indicating that recombination of PRRSV has played an important role in virus evolution [15,16]. Moreover, this continual variation and recombination of the PRRSV genome also hampers measures currently taken to control the incidence and spread of PRRS. Consequently, timely monitoring of the patterns of PRRSV mutation is essential for ensuring effective epidemic evolution and epidemiology. To this end, in this study, we compared the nucleotide and amino acids homologies of PRRSV-2 NSP2 among the strains of different lineages, and analyzed phylogenetic relationships, thereby enabling us to gain more understanding of genetic variation in the NSP2 protein. These findings will contribute to establishing a theoretical basis for assessing future epidemic trends in PRRS and for identifying changes in the NSP2 sequence that these viruses use to evade host immunity, thereby perpetuating the genetic evolution of PRRSV.

2. Materials and Methods

2.1. The Dataset

One hundred and twenty-two Chinese PRRSV-2 strains and 13 US PRRSV-2 strains in the NCBI website GenBank database were chosen, including those of lineage 1, 3, 5, and 8 (Table 1 and Table 2). There are a few reasons why these strains were selected. First, the year needs to be included in different years from 1996 to the present year. Second, the strains are representative of PRRSV strains frequently reported in other pieces of literature. Third, the commonly used vaccine strains needed to be selected.

2.2. Phylogenetic Analysis

Phylogenetic analysis of the NSP2 gene was based on the reference strains sequence information shown in Table 1. The comparison was first performed using the Clustal W method in the MegAlign function of DNAStar software (version 7.0) and then using the neighbor-joining (NJ) method of MEGA software (version 7.0) with 1000 bootstrap replicates. It was then analyzed using the Maximum Likelihood (ML) method of PhyloSuite software (version 1.2.2) with 1000 bootstrap replicates.

2.3. Alignment of NSP2 Nucleotide Sequences

For the purpose of determining similarities among the NSP2 nucleotide sequences of different PRRSV lineages, we analyzed the reference strains information shown in Table 1 based on the Clustal W method in the MegAlign function of DNAStar software.

2.4. Alignment of NSP2 Amino Acid Sequences

Similarities among NSP2 amino acid sequences were analyzed using the Clustal W method, based on reference strains information, and multi-sequence alignment analysis was performed using the BioEdit software (version 7.2).

2.5. Recombination Analysis

When potential recombinant events were detected based on RDP software (version 4.0), GENECONV, BootScan, MaxChi, Chimera, SiScan, and 3eq analyses, five or more methods were identified as genetic recombination and P < 0.05 in RDP software. The strains thus identified were considered recombinant strains. In addition, we used SimPlot (version 3.5.1) to confirm the detected recombination events.

3. Results

3.1. Phylogenetic Analysis

Based on the global PRRSV classification system and NSP2 sequence information in the GenBank database, we selected the NSP2 sequences of 122 PRRSV-2 strains for phylogenetic analysis (Table 1). The phylogenetic tree constructed using these sequences revealed that the PRRSV-2 strains prevalent in China could be classified into four lineages, namely, lineages 1, 3, 5, and 8 (Figure 1 and Figure 2). Among these, lineage 3 is represented by GM2-2011, QYYZ-2011, and FJFS-2012, which appear to be closely related, whereas lineage 1 and lineage 8 strains appear to be separated by comparatively large genetic distances. Since the advent of PRRSV in China, lineage 8, which includes the classical PRRSV strains (CH-1a-like) prevalent before 2006 and the HP-PRRSV-like strains circulating after 2006, has predominated. However, since 2013, lineage 1 strains, also referred to as NADC30-like strains, have spread rapidly across the country, with clinical detection rates comparable to those of HP-PRRSV-like lineage 8 strains.

3.2. Nucleotide Similarity

In order to further examine the genetic variation in NSP2 that has occurred during the course of PRRSV evolution, we selected 15 strains from each of the aforementioned four lineages for nucleotide homology analyses, and thereby determined evolutionary relationships among the different lineages at the nucleotide level (Figure 3). We accordingly detected a nucleotide homology of between 72.5% and 99.8% among the NSP2 protein of different PRRSV-2 strains, of which the CHsx1401-2014 strain showed the lowest homology of 72.5% with the QYYZ-2011 and GM2-2011 strains. Contrastingly, the nucleotide sequence of the BJ-4-1996 strain was found to be highly similar to that of the RespPRRS MLV-1994 strain, with a homology of 99.8%. With respect to each of the four assessed lineages, we obtained homology values of 86.0% to 91.2% among lineage 1 strains, 92.4% to 98.8% among lineage 3 strains, 99.4% to 99.8% among lineage 5 strains, and 92.3% to 99.6% among lineage 8 strains, of which the homologies of classic-type CH-1a-like strains were between 92.3% and 99.3% and those of HP-PRRSV-like strains were between 99.4% and 99.6%. The largest differences in NSP2 sequences were detected in lineage 1 (NADC-30-like) strains, and we speculate that this reflects extensive mutation and recombination among these strains. Lineages 3 and 5 were found to be highly similar at the nucleotide level, and the clinical detection of these two lineages in China is generally very low.

3.3. Amino Acid Sequence Similarity

Similar to our analysis of nucleotide similarities, we also examined the genetic variation of the PRRSV NSP2 protein at the amino acid level among 15 representative strains of the four lineages, in order to gain an understanding of the evolutionary relationship among amino acids sequences in each lineage (Figure 4). The results revealed amino acid homologies of between 63.9% and 99.4% among the different PRRSV-2 strains, of which that between CHsx1401-2014 and GM2-2011 at 63.9% was the lowest. The highest similarities were detected between the TJ-2006 strain and strains HUN4-2006 and JXA1-2006, with homologies reaching 99.4%, which was found to be inconsistent with the differences among these strains detected at the nucleotide level. In this regard, it is conceivable that amino acid substitution between lineage 1 and 5 strains is more common. In terms of each lineage, we detected amino acid homologies of 80.8% to 87.8%, 88.8% to 98.3%, 98.7% to 99.3%, and 87.9% to 99.4% among lineage 1, 3, 5, and 8 strains, respectively. Of these, homologies of between 89.6% and 98.7%, and 99.2% and 99.4% were obtained for the classical type CH-1a-like and HP-PRRSV-like strains, respectively. The largest differences in NSP2 sequences were identified in lineage 1 (NADC-30-like) strains, which is consistent with the nucleotide alignment results.

3.4. Amino Acid Sequence Alignment

The NSP2 nucleotide sequences of the 15 selected strains of each lineage were initially translated into amino acid sequences, for which we subsequently performed a multi-sequence comparative analysis (Figure 5). We accordingly detected differences in the lengths of PRRSV-2 NSP2 amino acid sequences, characterized by significant variability in the sequences encoding NSP2, including deletions, insertions, and substitutions at different amino acid sites. As previously stated, lineage 1 and 8 NADC-30-like and HP-PRRSV-like strains are currently the most prevalent in China, and have long been detected in the country, during which time they have caused substantial economic losses in the pig industry. Based on our brief analysis of amino acid mutations in the highly pathogenic lineage 8 strains, we detected 30 discontinuous deletions at amino acid residues 481, and 533 to 561 in the HP-PRRSV representative strains HUN4-2006, JXA1-2006, and TJ-2006, which is consistent with the findings of a previous study [4]. Moreover, we identified high amino acid homologies of up to 99.4% among these strains, with up to only 10 amino acid differences. In the TJ-2006 strain, these changes are located at positions 8, 194, 213, 253, 296, 488, 504, 544, 745, and 796, at which we detected proline, asparagine, histidine, glutamic acid, leucine, valine, serine, alanine, and asparagine residues, respectively. In JXA1-2006, changes were identified at positions 8, 253, 296, 488, 745, and 796, at which we detected the substitution of threonine, valine, phenylalanine, valine, and glycine, respectively, whereas in the HUN4-2006 strain, substitutions of threonine, tyrosine, phenylalanine, methionine, and proline were detected at positions 8, 194, 213, 488, 504, and 544, respectively. Furthermore, in representative strains of lineage 1, we detected different degrees of amino acid deletion at positions 481, 320–323, 325–345, 347–381, 383–429, 431–434, and 502–520, whereas in representative lineage 3 strains, in addition to the deletion mutations, two amino acid deletions were found at positions 300 and 301, and there has been a continuous insertion of 36 amino acids at positions 817–852.

3.5. Recombinant Analysis

To gain a more complete picture of the recombination of PRRSV-2, we added some classic strains from the United States (Table 2). Recombinant analysis of the NSP2 gene revealed seven potential recombinant events by RDP4 (Table 3), among which, five potential recombinant events was verified by SimPlot. According to the RDP4 results, the credibility of all seven recombination events is high with a statistical significance of P < 0.05. Events 2 and 3 were detected in lineage 1 strains, involving a recombination of lineage 1 and 8 sequences (Figure 6). The primary and secondary parental strains of recombinant strain SD-YL1712-2017 are BJ2021-2021 and JSTZ1712-12-2017, respectively; the primary and secondary parental strains of recombinant strain MN184A-2001 are NADC30-2008 and QHD3-2017, respectively. Similarly, recombinant events 4, 5, and 7 were detected in lineage 1 strains, resulting from recombination among these strains. The primary and secondary parental strains of recombinant strain NADC30-2017 are SD-A19-2015 and FJ1402-2014, respectively; the primary and secondary parental strains of recombinant strain SCya18-2018 are SD17-36-2017 and ZJXS1412-2014, respectively; and the primary and secondary parental strains of recombinant strain SCcd2020-2020 are JS2020-2020 and SCcd17-2017, respectively. Among lineage 8 strains, we detected recombinant events 1 and 6, resulting from recombination between lineage 5 and 8 strains. In the case of recombinant strain CH-1R-2008, the primary and secondary parental strains are GD3-2005 and CH-1a-1996, respectively, whereas the primary and secondary parental strains of recombinant strain Ingelvac-ATP-1999 are CC-1-2006 and HB-2(sh)-2001, respectively. However, SimPlot verified that no recombination occurred in events 3 and 5 (Figure 7).

4. Discussion

Our analysis of PRRSV evolution covering a period of approximately 25 years revealed apparent changes in the highly variable region of the NSP2 protein. In order to gain a comprehensive insight into evolutionary changes in the NSP2 genetic evolution, we performed phylogenetic analyses based on the NSP2 sequences of 122 selected PRRSV-2 strains, the results of which revealed the genetically close evolutionary distances of lineage 3 and 5 strains. Lineage 3 comprises variants that have emerged since 2010, the transmission of which has been recorded primarily in southern China (Jiangxi, Fujian, Guangdong, and Guangxi provinces) with a clinical detection rate of less than 10% [17,18]. In contrast, although lineage 5 (BJ-4-like/VR2332-like) strains appeared as early as 1996, these do not appear to be prevalent in China and have a low clinical detection rate [19]. In China, lineage 8 (HP-PRRSV-like) and lineage 1 (NADC30-like) strains have become the predominant strains, which is consistent with previous reports, which may be attributable to the fact that these strains are characterized by high genetic variation and recombinant properties, which are features facilitating the evasion of immune surveillance promoted by existing vaccines. Zhao et al. [20] reported that NADC30-like PRRSV has gradually become the most prevalent genotype in Sichuan Province. Yu et al. [21] proofed NADC30-like PRRSVs are undergoing a decrease in population genetic diversity. Zhou et al. [22] found that the NADC30 and the HP-PRRSV strains mainly circulated in southwest China.
For each of the four assessed lineages, we selected different representative strains for comparisons of nucleotide and amino acid sequences, which revealed nucleotide and amino acid homologies of 72.5% to 99.8% and 63.9% to 99.4%, respectively, among the NSP2 proteins of different PRRSV-2 strains. We speculate that these strains have undergone relatively limited mutation during the course of genetic evolution, and accordingly have been unable to effectively evade vaccine-mediated immunity and host immune surveillance, although further studies are necessary to ascertain specific details in this regard. Currently, lineage 1 and 8 strains are the prevalent epidemic strains in China, among which, the NADC30-like strains appear to be characterized by a high frequency to recombinant events, thereby enabling these viruses to adapt to changing environmental pressures during long-term evolution [6]. Further, it is speculated that the NSP2 sequence has also undergone a corresponding series of changes as a consequence of mutation and recombination of the different lineage 1 strains. Moreover, we detected high amino acid homologies between lineages 3 and 5 strains, which is consistent with the patterns observed in the nucleotide comparison.
Compared with the classical PRRSV strains, HP-PRRSV strains are characterized by a discontinuous deletion of (29 + 1) amino acids, whereas for NADC30-like strains there is evidence of a discontinuous deletion of 131 amino acids. Lineage 3 strains of PRRSV-2, specifically GM2-2011, QYYZ-2011, and FJFS-2012, exhibit 37 amino acid insertions at positions 817–853 of NSP2. Comparison of amino acid sequences among different PRRSV-2 strains reveals variations in deletions, insertions, and substitutions at multiple sites, which suggest differences in antigenicity.
There are some reports showing the recombination of NSP2 [13,23]. Taking NADC30-like strains as an example, new deletion of NADC30-like strains occurs, which makes it easier to recombine. Recombinant analysis performed for PRRSV-2 NSP2 revealed a total of five recombinant events among the 135 selected strains, a majority of which appear to have occurred between lineage 1 and 8 strains, possibly because there are many types of PRRSV vaccines in the Chinese market (inactivated vaccines: KV; activated vaccines: Ingelvac PRRS MLV, MLV CH-1R, MLV R98, MLV JXA1-R, MLV TJM-F92, MLV HuN4-F112, MLV GDr180), and the abuse of vaccines is also the main reason why PRRSV is prone to recombination. There is no solid evidence that the changes in NSP2 gene affects the recombination of PRRSV-2. Maybe through reverse genetics, mutation, or deletion of NSP2, the results of recombination can be further observed. So far, no such result has been reported. We will conduct such research in the future. Fang et al. found a high frequency of interlineage recombination hot spots in NSP9, so we hypothesize that changes in the NSP2 gene can affect PRRSV recombination. For the clinical severity, variation of NSP2 sequence was not related to viral virulence. Studies have shown that virulence is related to PRRSV NSP9 and NSP10 (NSP9 and NSP10 contribute to the fatal virulence of HP-PRRSV emerging in China), and two residues in NSP9 contribute to the enhanced replication and pathogenicity of HP-PRRSV [24]. Although HP-PRRSV derived vaccines have contributed to reducing the severity of clinical signs and restricting the spread of PRRS to a certain extent, their efficacy still falls far short of that initially anticipated. Moreover, the frequent use of vaccines can have a number of undesirable side effects, including the anti-virulence of live attenuated vaccines [25,26], viral recombination, and a significant increase in the rate of mutation [27,28,29].
Compared with the results of Jiang [13], our study focuses on the PRRSV-2 NSP2 gene, which we have divided into four lineages. We conducted nucleotide and amino acid similarity analyses, as well as sequence alignment and recombination analysis on NSP2. Although today we have a better understanding of the clinical pathogenesis of PRRS, molecular epidemiology, viral proliferation, pathogenesis, immune response, and immune evasive mechanisms, PRRSV is still not effectively controlled. In the past, investigators tended to focus on amino acid mutations in the hypervariable region (NSP2, ORF5) of the virus genome; however, in recent years, several strain recombination events have been discovered, and the frequency of recombination appears to be rising. In this regard, Zhao et al. [30] have confirmed that the highly pathogenic JL580 strain has a mixed genetic background of NADC30-like, HP-PRRSV, and local strains, and Chen et al. [31] concluded that HBap4-2018 is a new PRRSV strain with HP-PRRSV and NADC30-like PRRSV as primary and secondary parental strains, respectively, which retains most of the virulence-related regions of the HP-PRRSV genome and has been shown to be highly pathogenic to piglets. Moreover, Li et al. [32] have established that the HNyc15 strain is derived from the recombination of VR2332 and CH-1a gene fragments. The increases in recombinant strains and high rates of recombination in recent years indicate that recombination has played a very important role in the evolution of the virus. PRRSV NSP2 gene is prone to deletion, insertion, and recombination, and recombination plays an important role in the evolution of viruses. Therefore, we speculate that the change in NSP2 gene may affect the evolution of PRRSV. From the perspective of the prevention and control of PRRSV, it is accordingly imperative to ascertain the epidemiological characteristics of PRRSV, establish the molecular mechanisms underlying the high variability and recombinant frequency, and elucidate determinants of the variable virulence observed among different PRRSV strains.

5. Conclusions

From 1996 to 2021, PRRSV-2 isolates in China have been categorized into lineages 1, 3, 5, and 8, based on variations in their NSP2 genes. Lineages 1 and 8 have been found to be predominant in the prevalence of PRRS in China. Additionally, the removal of the NSP2 sequence prior to 2006 has been identified as a key factor driving the evolution of PRRSV and has also contributed to the evolution of virus. In recent years, recombination has played a significant role in the evolution of PRRSV-2. Therefore, monitoring the NSP2 gene of PRRSV-2 can serve as a reference for effectively controlling PRRS in China in the future.

Author Contributions

Writing—original draft preparation, H.Z. and Q.L.; writing—review and editing, Y.Z. and H.S.; software, G.L.; methodology, W.K.; funding acquisition, L.H.; visualization, M.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by National Natural Science Foundation of China (31902279).

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 conflict of interest.

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Figure 1. A NJ phylogenetic tree was constructed based on the NSP2 sequences of 122 PRRSV strains, using MEGA software (version 7.0) with 1000 bootstrap replicates. The circles (●) refer to the representative virus strains of each lineage in China.
Figure 1. A NJ phylogenetic tree was constructed based on the NSP2 sequences of 122 PRRSV strains, using MEGA software (version 7.0) with 1000 bootstrap replicates. The circles (●) refer to the representative virus strains of each lineage in China.
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Figure 2. A ML phylogenetic tree was constructed based on the NSP2 sequences of 122 PRRSV strains, using PhyloSuite software (version 1.2.2) with 1000 bootstrap replicates.
Figure 2. A ML phylogenetic tree was constructed based on the NSP2 sequences of 122 PRRSV strains, using PhyloSuite software (version 1.2.2) with 1000 bootstrap replicates.
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Figure 3. Results of similarity analysis performed for NSP2 nucleotide sequences from representative strains of PRRSV lineages 1, 3, 5, and 8. Clustal W method in MegAlign function of DNAStar software was used to analyze the similarity of NSP2 nucleotides. The 15 representative strains selected were CHsx1401-2014, JL580-2013, NADC30-2008 of lineage 1, GM2-2011, QYYZ-2011, FJFS-2012 of lineage 3, VR2332-1992, BJ-4-1996, RespPRRS MLV-1998 of lineage 5. CH-1a-1996, CH-1R-2008, CH2002-2009 of classical CH-1a-like in lineage 8, JXA1-2006, TJ-2006, HUN4-2006 of highly pathogenic HP-PRRSV-like in lineage 8.
Figure 3. Results of similarity analysis performed for NSP2 nucleotide sequences from representative strains of PRRSV lineages 1, 3, 5, and 8. Clustal W method in MegAlign function of DNAStar software was used to analyze the similarity of NSP2 nucleotides. The 15 representative strains selected were CHsx1401-2014, JL580-2013, NADC30-2008 of lineage 1, GM2-2011, QYYZ-2011, FJFS-2012 of lineage 3, VR2332-1992, BJ-4-1996, RespPRRS MLV-1998 of lineage 5. CH-1a-1996, CH-1R-2008, CH2002-2009 of classical CH-1a-like in lineage 8, JXA1-2006, TJ-2006, HUN4-2006 of highly pathogenic HP-PRRSV-like in lineage 8.
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Figure 4. Results of similarity analysis performed for NSP2 amino acid sequences from representative strain of PRRSV lineages 1, 3, 5, and 8. Clustal W method in MegAlign function of DNAStar software was used to analyze the similarity of NSP2 amino acids. The 15 representative strains selected were CHsx1401-2014, JL580-2013, NADC30-2008 of lineage 1, GM2-2011, QYYZ-2011, FJFS-2012 of lineage 3, VR2332-1992, BJ-4-1996, RespPRRS MLV-1998 of lineage 5. CH-1a-1996, CH-1R-2008, CH2002-2009 of classical CH-1a-like in lineage 8, JXA1-2006, TJ-2006, HUN4-2006 of highly pathogenic HP-PRRSV-like in lineage 8.
Figure 4. Results of similarity analysis performed for NSP2 amino acid sequences from representative strain of PRRSV lineages 1, 3, 5, and 8. Clustal W method in MegAlign function of DNAStar software was used to analyze the similarity of NSP2 amino acids. The 15 representative strains selected were CHsx1401-2014, JL580-2013, NADC30-2008 of lineage 1, GM2-2011, QYYZ-2011, FJFS-2012 of lineage 3, VR2332-1992, BJ-4-1996, RespPRRS MLV-1998 of lineage 5. CH-1a-1996, CH-1R-2008, CH2002-2009 of classical CH-1a-like in lineage 8, JXA1-2006, TJ-2006, HUN4-2006 of highly pathogenic HP-PRRSV-like in lineage 8.
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Figure 5. NSP2 amino acid sequence alignment for representative strains of PRRSV lineages 1, 3, 5, and 8. BioEdit software (version 7.2) was used to analyze amino acids of NSP2. The 15 representative strains selected were CHsx1401-2014, JL580-2013, NADC30-2008 of lineage 1, GM2-2011, QYYZ-2011, FJFS-2012 of lineage 3, VR2332-1992, BJ-4-1996, RespPRRS MLV-1998 of lineage 5. CH-1a-1996, CH-1R-2008, CH2002-2009 of classical CH-1a-like in lineage 8, JXA1-2006, TJ-2006, HUN4-2006 of highly pathogenic HP-PRRSV-like in lineage 8. The contents in the black box represent the deletion and insertion of amino acids.
Figure 5. NSP2 amino acid sequence alignment for representative strains of PRRSV lineages 1, 3, 5, and 8. BioEdit software (version 7.2) was used to analyze amino acids of NSP2. The 15 representative strains selected were CHsx1401-2014, JL580-2013, NADC30-2008 of lineage 1, GM2-2011, QYYZ-2011, FJFS-2012 of lineage 3, VR2332-1992, BJ-4-1996, RespPRRS MLV-1998 of lineage 5. CH-1a-1996, CH-1R-2008, CH2002-2009 of classical CH-1a-like in lineage 8, JXA1-2006, TJ-2006, HUN4-2006 of highly pathogenic HP-PRRSV-like in lineage 8. The contents in the black box represent the deletion and insertion of amino acids.
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Figure 6. RDP (version 4.0) verification of NSP2 gene recombination events. The horizontal axis represents position in alignment, the vertical axis represents pairwise identity. (a) is the RDP analysis result of the recombinant strain CH-1R; (b) is the RDP analysis result of the recombinant strain SD-YL1712-2017; (c) is the RDP analysis result of the recombinant strain MN-184A-2001; (d) is the RDP analysis result of the recombinant strain NADC30-2017; (e) is the RDP analysis result of the recombinant strain SCya18-2018; (f) is the RDP analysis result of the recombinant strain Ingelvac-ATP-1999; (g) is the RDP analysis results of the recombinant strain SCcd2020-2020.
Figure 6. RDP (version 4.0) verification of NSP2 gene recombination events. The horizontal axis represents position in alignment, the vertical axis represents pairwise identity. (a) is the RDP analysis result of the recombinant strain CH-1R; (b) is the RDP analysis result of the recombinant strain SD-YL1712-2017; (c) is the RDP analysis result of the recombinant strain MN-184A-2001; (d) is the RDP analysis result of the recombinant strain NADC30-2017; (e) is the RDP analysis result of the recombinant strain SCya18-2018; (f) is the RDP analysis result of the recombinant strain Ingelvac-ATP-1999; (g) is the RDP analysis results of the recombinant strain SCcd2020-2020.
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Figure 7. Simplot verification of NSP2 gene recombination events. Simplot (version 3.5.1) was used to confirm the recombination events which are detected in RDP (version 4.0) software. The horizontal axis represents position, the vertical axis represents percentage of permuted trees. The different colors depict different PRRSV strains, and blue lines represent the major parent; red lines represent minor parent. (a) is the Simplot validation result of the recombinant strain CH-1R; (b) is the Simplot validation result of the recombinant strain SD-YL1712-2017; (c) is the Simplot validation result of the recombinant strain MN-184A-2001; (d) is the Simplot validation result of the recombinant strain NADC30-2017; (e) is the Simplot validation result of the recombinant strain SCya18-2018; (f) is the Simplot validation result of the recombinant strain Ingelvac-ATP-1999; (g) is the Simplot validation results of the recombinant strain SCcd2020-2020.
Figure 7. Simplot verification of NSP2 gene recombination events. Simplot (version 3.5.1) was used to confirm the recombination events which are detected in RDP (version 4.0) software. The horizontal axis represents position, the vertical axis represents percentage of permuted trees. The different colors depict different PRRSV strains, and blue lines represent the major parent; red lines represent minor parent. (a) is the Simplot validation result of the recombinant strain CH-1R; (b) is the Simplot validation result of the recombinant strain SD-YL1712-2017; (c) is the Simplot validation result of the recombinant strain MN-184A-2001; (d) is the Simplot validation result of the recombinant strain NADC30-2017; (e) is the Simplot validation result of the recombinant strain SCya18-2018; (f) is the Simplot validation result of the recombinant strain Ingelvac-ATP-1999; (g) is the Simplot validation results of the recombinant strain SCcd2020-2020.
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Table 1. The reference sequence of each PRRSV NSP2 strains in China.
Table 1. The reference sequence of each PRRSV NSP2 strains in China.
YearAeraSequenceStrainGenbank Accession NumberBased on the ORF5 Lineage
1996ChinaNSP2CH-1aAY0326268
1996ChinaNSP2BJ-4AF3318315
2001ChinaNSP2HB-1(sh)AY1503128
2001ChinaNSP2HB-2(sh)AY2623528
2002ChinaNSP2CH2002EU880438.28
2002ChinaNSP2HB-1HQ2336058
2003ChinaNSP2HN1AY457635.15
2003ChinaNSP2CH2003EU880440.28
2003ChinaNSP2GS2003EU8804425
2004ChinaNSP2NVSL 97-7895AY5459858
2004ChinaNSP2CH2004EU880439.28
2005ChinaNSP2GD3GU2695418
2005ChinaNSP2SHBEU864232.18
2006ChinaNSP2Ingelvac ATPDQ9880808
2006ChinaNSP2Prime PacDQ7797918
2006ChinaNSP2HEB1EF1124478
2006ChinaNSP2CC-1EF1534868
2006ChinaNSP2FZ06AMF3705578
2006ChinaNSP2JX143 EU7087268
2006ChinaNSP2TJEU8602488
2006ChinaNSP2HUB1EF0759458
2006ChinaNSP2Hunan-1EU200965.18
2006ChinaNSP2JXA1EF1124458
2006ChinaNSP2HuN1EF198112.18
2006ChinaNSP2HUN4EF635006.18
2007ChinaNSP2Em2007EU2626038
2007ChinaNSP2SHHEU1068888
2007ChinaNSP2LNEU1095028
2007ChinaNSP2HN07-1KX7663788
2007ChinaNSP2SY0608EU1440798
2007ChinaNSP2GDEU825724.18
2007ChinaNSP2HQ-5EU255920.18
2007ChinaNSP2GDY2/2007EU200964.18
2007ChinaNSP2WUH1EU1874848
2008ChinaNSP2XH-GDEU6241178
2008ChinaNSP2YN2008EU8804358
2008ChinaNSP2JN-HSHM0161588
2008ChinaNSP2YN9GU2327388
2008ChinaNSP2JXA1-p80FJ5488538
2008ChinaNSP2GDBY1GQ3744428
2008ChinaNSP2CH-1REU8078408
2008ChinaNSP2PRRSV01FJ1756875
2009ChinaNSP2SY0909HQ3158378
2009ChinaNSP2ZP-1HM0161598
2009ChinaNSP2SX-1GQ8576568
2009ChinaNSP2WUH2EU6783528
2009ChinaNSP2CH2003EU8804408
2009ChinaNSP2JXA1-P120KC4227278
2009ChinaNSP2SD1-100GQ9149975
2009ChinaNSP2GS2002EU8804415
2009ChinaNSP2CH2002EU8804388
2010ChinaNSP2JN-HSHM0161588
2010ChinaNSP210FS-GDJX1926348
2010ChinaNSP2JXJX3176498
2010ChinaNSP2QY2010JQ7436663
2010ChinaNSP2GX1003JX9122498
2011ChinaNSP2YNJX8576985
2011ChinaNSP2GM2JN6624243
2011ChinaNSP2QYYZJQ3087983
2011ChinaNSP2HH08JX6791798
2012ChinaNSP2FJFSKP9984763
2012ChinaNSP2HZ-31KC4451385
2012ChinaNSP2SH1211KF6784348
2012ChinaNSP2NT1KP1794028
2013ChinaNSP2BJ-F150KP8903428
2013ChinaNSP2HeNan-A9KJ5464128
2013ChinaNSP2HENAN-XINXKF6119051
2013ChinaNSP2HLJB1KT3517408
2013ChinaNSP2JL580KR706343.11
2013ChinaNSP2FJW05KP8609111
2014ChinaNSP2NMG2014KM0000668
2014ChinaNSP2GD1404MF6697208
2014ChinaNSP2ZJXS1412MF6697228
2014ChinaNSP2CHsx1401KP8616251
2014ChinaNSP2TJbd14-1KP7429868
2014ChinaNSP2FJ1402KX1691911
2015ChinaNSP2XJzx1KX6892338
2015ChinaNSP2HNyc15KT9450183
2015ChinaNSP2SC-dMF3752618
2015ChinaNSP2HeN1501MF7664728
2015ChinaNSP2HeN1502MF7664738
2015ChinaNSP2SD-A19MF3752608
2015ChinaNSP2HZL1501MF6697218
2015ChinaNSP2TJnh1501KX5102698
2015ChinaNSP2GDsgKX6210033
2015ChinaNSP215LY02-FJKU2154178
2015ChinaNSP2HNjZ15KT9450171
2015ChinaNSP2FJZ03KP8609091
2016ChinaNSP2YN2008EU8804358
2016ChinaNSP2HeNhxKX7663791
2016ChinaNSP2HeN1601MF7664748
2016ChinaNSP2CQwz16KY620008.18
2016ChinaNSP2SCcd16KY620011.18
2016ChinaNSP2HB-1AH015834.28
2016ChinaNSP2JXwn06EF6410088
2016ChinaNSP215LN1KX8154238
2017ChinaNSP2SD-YL1712MT7085008
2017ChinaNSP2SD17-36MH1210618
2017ChinaNSP2SD17-38MH0688788
2017ChinaNSP2QHD2MH1673878
2017ChinaNSP2JSTZ1712-12MK9060268
2017ChinaNSP2QHD3MH1673888
2017ChinaNSP2XJ17-5MK7598538
2017ChinaNSP2SCN17MH0784908
2017ChinaNSP2SCcd17MG9140678
2017ChinaNSP2GZgy17MK1445428
2017ChinaNSP2NADC30MH500776.11
2017ChinaNSP2FJWQ16KX7582498
2017ChinaNSP2FJM4KY4128888
2017ChinaNSP2JX-CHKY4957808
2017ChinaNSP2SH-CHKY4957818
2018ChinaNSP2SCya18MK1445438
2018ChinaNSP2Henan-BY3MN550954.18
2019ChinaNSP2Henan-JZ29MN550957.18
2019ChinaNSP2SDRZ01MZ3229568
2020ChinaNSP2SD-QD-2101MZ1729718
2020ChinaNSP2SXSZ-2020MW8807728
2020ChinaNSP2JS2020MZ3429008
2020ChinaNSP2rJXA1-RMT1633148
2020ChinaNSP2SCcd2020MW8031348
2021ChinaNSP2BJ2021OK0952998
2021ChinaNSP2HY21OL687155.18
Table 2. The reference sequence of each PRRSV NSP2 strains in US.
Table 2. The reference sequence of each PRRSV NSP2 strains in US.
YearAeraSequenceStrainGenbank Accession NumberBased on the ORF5 Lineage
1992USANSP2VR2332EF536003.15
1993USANSP2ATCC VR2332U87392.35
1996USANSP2SDSU73JN654458.18
2003USANSP2JA142AY424271.18
1998USANSP2RespPRRS MLVAF066183.45
1999USANSP2Ingelvac ATPDQ988080.18
1999USANSP2MLV RespPRRS/ReproAF1591498
2001USANSP2MN184ADQ1760191
2002USANSP2P129AF494042.18
2004USANSP2NVSL 97-7895AY545985.18
2006USANSP2Prime PacDQ779791.18
2008USANSP2NADC30JN6544591
2008USANSP2NADC31JN6601501
Table 3. Recombination analysis of Nsp2 gene.
Table 3. Recombination analysis of Nsp2 gene.
Recombination EventRecombinant Strains (Lineages)Main Parental Strain (Lineages)Minor Parental Strain (Lineages)Recombinant BreakpointRecombination Analysis Method
1CH-1R-2008 (8)GD3-2005 (8)CH-1a-1996 (8)63~2935 (1884~1892)RDP (P = 2.349 × 10−149) GENECONV (P = 8.169 × 10−145) BootScan (P = 1.298 × 10−113) MaxChi (P = 1.507 × 10−46) Chimaera (P = 3.911 × 10−48) SiScan (P = 3.619 × 10−63)
3seq (P = 3.334 × 10−8)
2SD-YL1712-2017 (8)BJ2021-2021 (8)JSTZ1712-12-2017 (8)7~2485 (784~823)RDP(P = 1.915 × 10−64) GENECONV (P = 6.699 × 10−96) BootScan (P = 4.816 × 10−98) MaxChi (P = 5.325 × 10−27) Chimaera (P = 1.423 × 10−25) SiScan (P = 3.001 × 10−31)
3seq (P = 3.726 × 10−9)
3MN184A-2001 (1)NADC30-2008 (8)QHD3-2017 (8)63~2519 (1027~1061)RDP (P = 5.700 × 10−72) GENECONV (P = 1.524 × 10−45) BootScan (P = 8.197 × 10−71) MaxChi (P = 3.496 × 10−21) Chimaera (P = 6.284×10−22) SiScan (P = 1.075 × 10−29)
3seq (P = 5.226 × 10−55)
4NADC30-2017 (8)SD-A19-2015 (8)FJ1402-2014 (1)772~889 (1414~1520)RDP (1.308 × 10−31) GENECONV (P = 2.888 × 10−27) BootScan (P = 9.744 × 10−32) MaxChi (P = 4.598 × 10−16) Chimaera (P = 4.563 × 10−16) SiScan (P = 5.757 × 10−19)
3seq (P = 2.526 × 10−43)
5SCya18-2018 (8)SD17-36-2017 (8)ZJXS1412-2014 (8)17~2539 (352~482)RDP (P = 1.874 × 10−30) GENECONV (P = 6.122 × 10−8) BootScan (P = 1.347 × 10−29) MaxChi (P = 7.742 × 10−10) Chimaera (P = 7.922 × 10−12) SiScan (P = 1.804 × 10−10)
3seq (P = 1.276 × 10−10)
6Ingelvac-ATP-1999 (8)CC-1-2006 (8)HB-2(sh)-2001 (8)33~2894 (907~1536)RDP (P = 5.359 × 10−10) GENECONV(NS) BootScan (P = 7.970 × 10−12) MaxChi (P = 2.038 × 10−14) Chimaera (P = 2.298 × 10−10) SiScan (P = 5.881 × 10−6)
3seq (P = 1.257 × 10−8)
7SCcd2020-2020 (8)JS2020-2020 (8)SCcd17-2017 (8)762~903 (2112~2255)RDP (P = 2.347 × 10−4) GENECONV(NS) BootScan (P = 3.109 × 10−3) MaxChi (P = 1.818 × 10−7) Chimaera(NS) SiScan (P = 4.693 × 10−16)
3seq (P = 2.094 × 10−13)
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Zhang, H.; Luo, Q.; Zheng, Y.; Sha, H.; Li, G.; Kong, W.; Huang, L.; Zhao, M. Genetic Variability and Recombination of the NSP2 Gene of PRRSV-2 Strains in China from 1996 to 2021. Vet. Sci. 2023, 10, 325. https://doi.org/10.3390/vetsci10050325

AMA Style

Zhang H, Luo Q, Zheng Y, Sha H, Li G, Kong W, Huang L, Zhao M. Genetic Variability and Recombination of the NSP2 Gene of PRRSV-2 Strains in China from 1996 to 2021. Veterinary Sciences. 2023; 10(5):325. https://doi.org/10.3390/vetsci10050325

Chicago/Turabian Style

Zhang, Hang, Qin Luo, Yajie Zheng, Huiyang Sha, Gan Li, Weili Kong, Liangzong Huang, and Mengmeng Zhao. 2023. "Genetic Variability and Recombination of the NSP2 Gene of PRRSV-2 Strains in China from 1996 to 2021" Veterinary Sciences 10, no. 5: 325. https://doi.org/10.3390/vetsci10050325

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