Molecular Characterization of the Nsp2 and ORF5 (ORF5a) Genes of PRRSV Strains in Nine Provinces of China During 2016–2018

Porcine reproductive and respiratory syndrome virus (PRRSV) causes a highly contagious disease and brings huge economic losses to commercial pork production worldwide. PRRSV causes severe reproductive failure in sows and respiratory distress in piglets. To trace the evolution of PRRSV in pigs with respiratory diseases in some regions of China, 112 samples were collected from nine provinces in China during 2016–2018. All samples were detected by RT-PCR and analyzed by the Nsp2/ORF5 (ORF5a)-genes-phylogeny. Sequence analysis and recombination analysis were conducted on the Nsp2/ORF5 (ORF5a) genes of the identified strain in the study. The RT-PCR result shown that the positive rate of PRRSV was 50.89% (57/112). Phylogenetic analysis showed that the identified PRRSV strains were all NA genotype and belonged to lineage 1, 3, and 8. The Nsp2 gene of identified PRRSV strains exhibited nucleotide homologies of 53.0 ~ 99.8%, and amino acid homologies of 46.8 ~ 99.7%. The ORF5 gene of identified PRRSV strains exhibited nucleotide homologies of 82.4 ~ 100%, and amino acid homologies of 79.6 ~ 100%. Sequence analysis revealed that a discontinuous 30-amino-acid deletion (positions 481 and 533–561) and a 131-amino-acid discontinuity deletion (positions 323–433, 481, and 533–551) in Nsp2 of PPRSV isolates; all identified strains in this study may be wild strains, and most identified strains may be highly virulent strains. Sequence analysis of ORF5 and ORF5a revealed that the mutation sites of GP5 were mainly concentrated in the signal peptide and epitopes region, while the mutation sites of ORF5a were mainly concentrated in the transmembrane and the intramembrane region. The recombination analysis indicated that there may be multiple recombination regions in identified strains, and the recombination pattern was more complex. This study showed that the prevalent PRRSV strain in some regions of China was still HP-PRRSV, while NADC30 strain also occupied a certain proportion; different types of PRRSV strains showed different patterns and variation in China. This study suggested that the monitoring of PRRSV prevalence and genetic variation should be further strengthened.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes a highly contagious disease and brings huge economic losses to commercial pork production worldwide. PRRSV causes severe reproductive failure in sows and respiratory distress in piglets. To trace the evolution of PRRSV in pigs with respiratory diseases in some regions of China, 112 samples were collected from nine provinces in China during 2016-2018. All samples were detected by RT-PCR and analyzed by the Nsp2/ORF5 (ORF5a)-genes-phylogeny. Sequence analysis and recombination analysis were conducted on the Nsp2/ORF5 (ORF5a) genes of the identified strain in the study. The RT-PCR result shown that the positive rate of PRRSV was 50.89% (57/112). Phylogenetic analysis showed that the identified PRRSV strains were all NA genotype and belonged to lineage 1, 3, and 8. The Nsp2 gene of identified PRRSV strains exhibited nucleotide homologies of 53.0 ∼ 99.8%, and amino acid homologies of 46.8 ∼ 99.7%. The ORF5 gene of identified PRRSV strains exhibited nucleotide homologies of 82.4 ∼ 100%, and amino acid homologies of 79.6 ∼ 100%. Sequence analysis revealed that a discontinuous 30-amino-acid deletion (positions 481 and 533-561) and a 131-amino-acid discontinuity deletion (positions 323-433, 481, and 533-551) in Nsp2 of PPRSV isolates; all identified strains in this study may be wild strains, and most identified strains may be highly virulent strains. Sequence analysis of ORF5 and ORF5a revealed that the mutation sites of GP5 were mainly concentrated in the signal peptide and epitopes region, while the mutation sites of ORF5a were mainly concentrated in the transmembrane and the intramembrane region. The recombination analysis indicated that there may be multiple recombination regions in identified strains, and the recombination pattern was more complex. This study showed that the prevalent PRRSV strain in some regions of China was still HP-PRRSV, while NADC30 strain also occupied a certain proportion; different types of PRRSV strains showed different patterns and variation in China. This study suggested that the monitoring of PRRSV prevalence and genetic variation should be further strengthened.
Keywords: porcine reproductive and respiratory syndrome virus, Nsp2 gene, ORF5 gene, ORF5a gene, genetic evolution INTRODUCTION Porcine reproductive and respiratory syndrome (PRRS) is a major threat to the global swine industry, causing significant economic losses each year. The causative agent is PRRS virus (PRRSV), a member of the Arteriviridae family, order Nidoviridales. PRRSV is a single positive-strand RNA virus with a genome length of ∼15.4 kb (1,2). The PRRSV contains at least 10 open reading frames (ORFs), which are ORF1a, ORF1b, ORF2a, ORF2b, ORF5a, and ORF3 ∼ 7 from the 5' to the 3' untranslated regions (UTR) (3,4). ORF1a and ORF1b are cleaved into at least 13-16 non-structural proteins (Nsps) by a complex proteolytic cascade (3).
PRRSV was first reported in commercial pigs by the United States in 1987 (5), and the disease quickly spread worldwide with frequent break outs. PRRSV is still considered a highly contagious disease in the pig industry and creates huge economic losses (1,6,7). PRRSV was divided into two genotypes: the European genotype (type I) and North American genotype (type II) (3). There are three main subtypes of PRRSV (type II) isolates in Chinese pig populations: classical PRRSV (type II) including CH-1a, S1, and BJ-4; highly pathogenic PRRSV (HP-PRRSV) including JXA1, HuN4, and TJ; and NADC30like PRRSV including JL580, CHsx1401, and HNjz15 (8). The genetic characteristic of HP-PRRSV isolates have a discontinuous 30-amino-acid deletion in Nsp2, and NADC30-like PRRSV isolates have a discontinuous 131-amino-acid deletion in Nsp2 (9,10). PRRSV has mutated in the epidemic process to produce new strains due to the high frequency of gene mutation and recombination, in which new strains often have stronger environmental adaptations. The above factors have made the PRRSV epidemic more complicated, and it also brings great difficulties to disease prevention (11,12). Nsp2 and ORF5 (ORF5a) are highly variable and ORF5 is associated with the neutralizing epitope (13,14). They are usually used as target genes for PRRSV molecular epidemiological surveillance.
This study intends to reveal the prevalence and genetic evolution of PRRSV during 2016-2018 in different regions of China. The current study used the Nsp2 and ORF5 (ORF5a) genes to analyze the genetic evolution of the identified PRRSV strains. Our aim is to provide a theoretical basis for further monitoring of genetic variations of PRRSV in China.

Sampling
In total, 112 samples of the lung or lymph node tissues from pigs with respiratory diseases were collected between 2016 and 2018 in nine provinces or municipalities of China, including Heilongjiang, Jilin, Liaoning, Hubei, Jiangsu, Jiangxi, Zhejiang, Hebei, and the Inner Mongolia Autonomous Region. The lung or lymph node tissues were ground to powder with liquid nitrogen and diluted with three volumes of phosphate-buffered saline (PBS). The samples were centrifuged at 5,000 × g for 15 min at 4 • C and the supernatants were transferred to a 1.5 mL tube. The genomic RNA was extracted from the supernatant using a commercial TIANamp Stool RNA Kit (Tiangen Biotech Co., Ltd, Beijing, China). The viral cDNA was synthesized using Moloney murine leukemia virus (RNaseH-) reverse transcriptase (Novoprotein Scientific Inc., Shanghai, China) in conjunction with six-random-nucleotide primers. The extracted genomic RNA and cDNA was stored at −80 • C.

PCR Detection and Sequencing of PRRSV Strains
The primer of ORF5 full-length gene (including complete ORF5a gene) can be found in the report by Cao et al. (15). A pair of primers of Nsp2 gene were designed based on the alignment of published PRRSV genome sequences obtained from the NCBI GenBank database. Primer information is shown in Table 1. The amplification reactions were carried out in a 25 µL reaction volume containing 12.5 µl of EmeraldAmp R PCR Master Mix (2×Premix) (TaKaRa Biotechnology Co., Ltd., Dalian, China), 0.5 µM of the forward primer, 0.5 µM of the reverse primer, 1 µL of cDNA, and an appropriate volume of double-distilled (dd) H 2 O. The cycling parameters of ORF5 gene were: 36 cycles of 94 • C for 30 s, 55 • C for 30 s, and 72 • C for 1 min, followed by a final extension at 72 • C for 10 min. The cycling parameters of Nsp2 gene were: 35 cycles of 95 • C for 30 s, 57.6 • C for 30 s, and 72 • C for 3 min, followed by a final extension at 72 • C for 10 min. The PCR products were analyzed by electrophoresis in a 1% agarose gel under UV light, and the samples with positive results were recorded. After the amplification, products were purified using the AxyPrep DNA Gel Extraction kit (A Corning Brand, Suzhou, China), and cloned into pGM-T Vector (TaKaRa Biotechnology Co., Ltd., Dalian, China). Each fragment was sequenced at least three times. All nucleotide

Phylogenetic and Sequence Analysis of PRRSV Strains
For the phylogenetic analysis, the Nsp2 and ORF5 (ORF5a) genes of PRRSV reference strains were retrieved from the NCBI nucleotide database as reference sequences. Detailed information and the GenBank number of PRRSV reference strains is shown in Supplementary Table 1. To construct phylogenetic trees, nucleotide sequences of the target gene using the ClustalX alignment tool in the MEGA 6.06 software (16). Neighbor-joining phylogenetic trees were constructed with 1,000 bootstrap replicates and the remaining default parameters in the MEGA 6.06 software. The generated phylogenetic tree was annotated using the online software ITOL (https://itol.embl.de/) (17). The PRRSV-identified strains and reference strains were analyzed by MegAlign program in DNASTAR TM 5.06 software. Nucleotide/amino acid homology of Nsp2 and ORF5 (ORF5a) genes of PRRSV-identified strains, and reference strains were gained using the Pairwise/Multiple Align function in Geneious Prime software. recombinant events, five or more methods were identified as gene recombination and P < 0.05 in RDP4.0 software. The strains in this event were determined to be recombinant strains.
In addition, the detected recombination events were further confirmed by SimPlot 3.5.1.     Frontiers in Veterinary Science | www.frontiersin.org the phylogenetic tree constructed with the ORF5 gene. With the rapid growth of sequence deposition into the databases, it would be complicated for the diversity of PRRSV sequences. sublineage. This further suggests HLJ/2017/1127a is likely to be generated by recombinant strains in lineage 3 and sublineage 8.7. The classical PRRSV (type II) ATCC VR-2332 was used as the reference standard; the identified strain HLJ/2017/1127c had the same mutation pattern with vaccine strain CH-1R, which lacked a V at position 630 aa in the Nsp2. The identified strains of sublineage 8.7 and lineage 3 all showed a discontinuous 30amino-acid deletion (positions 481 and 533-561) that conforms to the classical deletion mutation pattern of the HP-PRRSV-like strain. Excluding HLJ/2018/410, all strains identified in lineage 1 showed 131-amino-acid discontinuity deletion (positions 323-433, 481, and 533-551), that conforms to the classical deletion mutation pattern of the NADC30-like strain (Figures 3-6). The prevalent PRRSV strain in some regions of China was still HP-PRRSV, while NADC30 strain also occupied a certain proportion.  Table 4). The mutation sites of ORF5 were mainly concentrated in the signal peptide and epitopes region. GP5 virulence-related sites showed that nine of the 39 identified strains had mutated at the position 13th aa Frontiers in Veterinary Science | www.frontiersin.org (R→ Q). A total of 12 identified strains of sublineage 8.7 and lineage 1 had mutated at position 151 aa (R→ K), The 137th aa of all identified strains was conservative and was S (Figure 7). It is observed that all identified strains may be wild strains, and most identified strains may be highly virulent strains in the study.  (Figure 8). This shows that the mutation sites of ORF5a were mainly concentrated in the transmembrane region and the intramembrane region.

Recombination Analysis
The recombination analysis of Nsp2 gene showed that there were five potential recombination events ( Table 6).
The recombination analysis of Nsp2 gene showed that the recombinant strains in event 1 and 2 were produced by recombination of lineage 1 and sublineage 8.7 (Supplementary Figure 1). The high frequency mutation and recombination make the virus gain more genetically diverse (19). This recombination pattern is the most common in PRRSV recombinant strains in China, and animal tests have confirmed that the virulence of some recombinant strains is higher than the prototype strain NADC30 (9). The identified strain HLJ/2017/1127a in recombinant event 3 was produced by recombination of lineage 3 and sublineage 8.7 wild strain. The main parental strain was FZ06A, while the minor parental strain was QYYZ. Previous studies have shown that the low virulence prototype strain QYYZ even became highly virulent after recombination with the vaccine strain derived from HP-PRRSV (20). The identified strain JS/NT/2017/14b in recombinant event four showed that the main parental strain HLJ/2017/1127b belongs to subline 8.7, while the minor parental strain JS/NT/2017/14a belongs to subline 8.7. Three recombinant strains in recombinant event five were from the same origin as JS/NT/2017/14b, but the recombinant sites are different. The recombination analysis of ORF5 genes showed that the recombinant event included four recombinant strains of lineage 1 ( Table 7). The main parental strain CY1-1604 belongs to lineage 1, while the minor parental strain GS2008 belongs to sublineage 8.7 (Supplementary Figure 2). Combined with the recombination analysis of Nsp2 gene, the identified strain HLJ/HEB/2016/1031 (a, b) was also recombined in ORF5 gene, which indicated that there may be multiple recombination regions in identified strains, and the recombination pattern was more complex.

DISCUSSION
Since the outbreak of HP-PRRSV in 2006, PRRSV has been widely spread across the world. In previous studies, the positive rate of PRRSV was shown to be 55.21% (7,490/11,3567) in 29 provinces of China in 2012-2015 (21). In Central and Southern China, there was a positive rate of 50.62% (530/1,047) of PRRSV among 257 pig farms (22). In our study, the total positive rate of PRRSV was 50.89% (57/112) in nine provinces of China from 2016 to 2018, which was in accordance with the above scholars. PRRS is one of the most prevalent and threatening infectious diseases in Chinese pig farms. Nsp2 and ORF5 (ORF5a) genes have the highest variability in PRRSV genome and are used as main target genes for PRRSV genetic variation. Phylogenetic tree analysis showed that all the 56 PRRSV strains identified in this study belong to the North American genotype and were distributed in lineage 1, 3, and 8 according to Shi et al. (18). In this study, the HP-PRRSV strain accounted for the highest proportion of epidemic strains in China; the NADC30-like strain had increased gradually, which was in accordance with the results of Gao et al. (23). However, some studies have shown that the NADC30-like strain in some regions of China have replaced the HP-PRRSV strain, and has become a new dominant strain (22). The rising infection rate of HP-PRRSV and NADC30-like strains may lead to a significant decrease in the effective protection rate of vaccines on pig farms.
The sequence alignments of Nsp2 gene revealed that the identified strain HLJ/2017/1127c in subline 8.1 had a high similarity with vaccine strain CH-1R, and existed a V deletion in the 630aa, suggesting that the identified strain may be a vaccine strain or a recombinant strain of a vaccine strain. Sequence alignments identified a discontinuous 30-amino-acid deletion (positions 481 and 533-561) and a 131-amino-acid discontinuity deletion (positions 323-433, 481, and 533-551) in Nsp2 of PPRSV isolates. JX/FC/2017/914(c, a1, b1) had the same deletion pattern as PRRSV strains HeN1401 and HeN1601, isolated by Zhang et al. (24). The recombinant analysis of the two epidemic strains revealed that HeN1401 and HeN1601 strains were generated by the recombinant weak vaccine strains TJbd14-1 and NADC30 (24). This further suggests that the identified strain JX/FC/2017/914(c, a1, b1) may also be a recombinant strain.
The GP5 protein sequences of different subline strains showed high similarity with the representative strains of the subline. The mutation sites of GP5 were mainly concentrated in the signal peptide and epitopes region. But some identified strains also have some amino acid consistent mutations in immune-related regions. Allende et al. found that nine amino acid site mutations may be closely related to the virulence of the PRRSV and that two sites (13 and 151 aa) were located in GP5 protein. The   GP5 protein of high virulence strains generally were shown as R 13 and R 151 (25). Wesley et al. showed that the 137aa of GP5 protein can distinguish the attenuated vaccine strain (A 137 ) and the wild strain (S 137 ) (26). Therefore, the above three amino acid sites are often used to predict the virulence of PRRSV strains. The sequence analysis of GP5 protein showed that only one mutation pattern (R 13 → Q 13 and R 151 → K 151 ) existed in this study. Nine identified strains had mutated at position 13 aa, and 12 identified strains mutated at position 151 aa. In addition, the 137 aa of all identified strains is S. The results suggest that all identified strains may be wild strains, and most identified strains may be highly virulent strains in nine provinces of China during 2016-2018. Studies have shown that ORF5a protein is essential for viral viability and infectivity (27,28). There is fairly limited information available on current genetic variations of PRRSV ORF5a gene (29). Therefore, this study explored the genetic variation of ORF5a gene of PRRSV epidemic strains in China by molecular biological methods. The ORF5a protein generally encoded 46-51 amino acids of which ORF5a protein of PRRSV strains encoded 46 amino acids in lineage 1 and 8, and 51 amino acids in lineage 3 and 5 (30). All the ORF5a proteins identified in this study encoded 46 amino acids. Compared with the reference strain, the identified strains also showed high sequence similarity, and the mutation sites of ORF5a were mainly concentrated in the transmembrane region and the intramembrane region, while the other region was highly conserved. Our study demonstrated the existence of multiple different strains in the same region and extensive genetic mutation of PRRSV in China from 2016 to 2018.
The recombination analysis indicated that there may be multiple recombination regions in identified strains, and the recombination pattern was more complex. At present, many studies have shown that PRRSV strain in lineage 1 is prone to recombinant mutation, and some of the recombinant strains are more virulent than others (10,19). Although the recombination pattern of the virus identified in this study is in accordance with that reported by some previous scholars, the change of pathogenicity of PRRSV by gene recombination is not absolute. This study only carried out partial gene (Nsp2 and ORF5) recombination analysis without the virus isolation and whole genome sequences of PRRSV, so the recombination of whole genome sequences is more complicated and different.

CONCLUSION
This study showed that PRRSV infection was prevalent in nine provinces of China from 2016 to 2018, and the prevalent PRRSV strain in most regions was still HP-PRRSV, while the NADC30 strain also occupied a certain proportion. There was a discontinuous 30-amino-acid deletion (positions 481 and 533-561) and a 131-amino-acid discontinuity deletion (positions 323-433, 481, and 533-551) in Nsp2 of PPRSV isolates. All identified strains in this study may be wild strains, and most identified strains may be highly virulent strains. This study identified highly homologous HP-PRRSV variants with distinct genetic mutation, which contributes to further analyzing the epidemics and evolution of PRRSV in the field.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.