Porcine reproductive and respiratory syndrome (PRRS) is an acute infectious disease caused by the PRSS virus (PRRSV). PRRS first appeared in Europe and North America in the 1980s (Wang et al., 2019). Subsequently, the disease quickly spread worldwide and caused huge economic losses in the swine industry. The clinical symptoms of PRRS are mainly caused by reproductive disorders in sows and respiratory diseases in piglets (Fang et al., 2022).

Porcine reproductive and respiratory syndrome virus (PRRSV) is a member of the Arteriviridae family in the order Nidovirales (Adams et al., 2017). PRRSV is an enveloped, positive single-stranded RNA virus (Chen et al., 2020). The PRRSV genome is approximately 15 kilobases (kb) in length and contains at least 10 open reading frames (ORFs) and two untranslated regions at the 5′ and 3′ ends of the genome (Zhang et al., 2022). ORF1a and ORF1b encode at least 16 nonstructural proteins (Nsps) which are related to replication (Fang and Snijder, 2010; Fang et al., 2012). ORF2a, ORF2b, ORF3–5, ORF5a, ORF6, and ORF7 encode eight viral structural proteins (Wu et al., 2001). Among the NSPs, Nsp2 is the largest PRRSV nonstructural protein which contains a highly variable region. Because this region usually observed mutations, insertions, or deletions, Nsp2 could be a unique pointer for monitoring the genetics and evolution of PRRSVs (Saenglub et al., 2020). ORF5 encodes the major structural protein glycoprotein 5 (GP5). Because of the high variability of ORF5, GP5 is usually used for phylogenetic analyses and classification of PRRSV isolates (Murtaugh et al., 2010). GP5 is an important immunogenic protein, which contains several primary neutralizing epitopes (PNEs) and N-linked glycosylation sites (NGSs; Ostrowski et al., 2002). PNEs can produce non-neutralizing antibodies against GP5 and tend to delay the production of neutralizing antibodies against PRRSV (Stoian and Rowland, 2019). The deletion of NGSs in GP5 alters the susceptibility of the virus to the host’s neutralizing antibodies through a phenomenon known as “glycan shielding,” thereby evading vaccine-induced immune responses (Rupasinghe et al., 2022).

Based on the distinct genetic and antigenic diversity, PRRSV isolates were divided into two major genotypes: the European genotype (PRRSV-I) and the North American genotype (PRRSV-II; Kuhn et al., 2016). The prototype representative strains were Lelystad and VR-2332, respectively (Fang et al., 2022; Zhang et al., 2022). These two genotypes share only 60% nucleotide identity (Shi et al., 2010a). PRRSV-I has been divided into four genetically distinct subtypes (Fitzgerald et al., 2020). Based on a comprehensive of ORF5 sequences, PRRSV-II has been classified into nine lineages. Most of the PRRSV-II were identified in the United States and distributed in North America and Asia (Paploski et al., 2019, 2021; Kikuti et al., 2021). Although PRRSV-I and PRRSV-II co-exist in the Chinese swine herds, PRRSV-II is predominant in China (Gao et al., 2017; Guo et al., 2018). The prevalent PRRSV-II strains in China were clustered into four lineages: lineage 1, lineage 3, lineage 5 (sublineage 5.1), and lineage 8 (sublineage 8.7). In 1996, the first isolated PRRSV strain CH-1a was reported in China, which belongs to lineage 8 (Valicek et al., 1997). CH-1a was recognized as an ancestor strain of classical PRRSV in China, and its presence suggested that PRRSV was widespread in China. Then in 2006, an undefined swine endemic with high incidence and mortality broke out in Jiangxi Province, China. It was later determined that the pathogen of this endemic was a PRRSV variant with a characteristic unique discontinuous deletion of 30 amino acids in Nsp2, which was named highly pathogenic PRRSV (HP-PRRSV) belonging to sublineage 8.7 (An et al., 2007; Tong et al., 2007). Since then, HP-PRRSV gradually became the main PRRSV type causing epidemics in mainland China (Peng et al., 2017; Liang et al., 2019). HP-PRRSV presently also circulating in most Southeast Asian countries including Laos, Vietnam, Cambodia, Bhutan, and India (Rajkhowa et al., 2022). The representative sublineage 5.1 strain BJ-4 was isolated in 1997, showing 99.6% genome homology with VR2332 (Peng et al., 2017; Liang et al., 2019). In 2010, the widely occurring lineage 3 strains QYYZ and GM2 were reported in Southern China (Shi et al., 2010b). Lineage1 is known to have a long epidemic history since it initially appeared in Canada in the 1990s. Since the initial outbreak in North America in the early 2000s, its representative strain is MN184 (Ramos et al., 2022). In 2013, a new cluster of lineage1.8 named NADC30 spread from Canada to the United States (Yu et al., 2021). At the same time, NADC30-like PRRSV isolates were reported in Henan Province, China. The detection rates of NADC30-like strains in China have gradually increased. PRRSV strain NADC30, which has a discontinuous 131-amino-acid deletion in Nsp2 and is moderately virulent, was first isolated in the United States (Brockmeier et al., 2012). In 2014, the sublineage 1.5 representative strain NADC34 started to emerge in the United States and caused large abortion outbreaks and high mortality rates in piglets (van Geelen et al., 2018). Isolate NADC34 has a continuous deletion of 100 amino acids in Nsp2. From 2015 to 2017, various NADC34-like (PRRSV 1-7-4) strains were identified in Peru, and one whole genome of NADC34-like strain was reported from South Korea (Ramirez et al., 2019; Kim et al., 2022). At the same time, two novel PRRSV NADC34-like isolates (LNWK96 and LNWK130) were reported in Liaoning Province, China (Zhang et al., 2018). Since then, different regions of China (Heilongjiang, Henan, Fujian, and Jiangsu) have reported NADC34-like PRRSV strains (Zhang et al., 2018; Liu et al., 2019; Xie et al., 2020). Epidemiological investigations suggest that NADC34-like PRRSV has become endemic in China. PRRSV has high gene mutation and recombination frequencies, and in the epidemic process, new strains have developed. These factors have led to increased diversification and prevalence of clinical strains, impeding disease prevention (Wenhui et al., 2012; Lu et al., 2015).

In this study, we aimed to reveal the prevalence and genetic evolution of PRRSV during 2017–2022 in different regions of Eastern China. We have also identified various PRRSV strains and spotted critical amino acid variation among them based on ORF5 and Nsp2 proteins. Our study provides a theoretical basis for further monitoring of PRRSV variants in China.

PRRS is one of the most devastating swine diseases caused by PRRSV and is broadly distributed across the globe. In this study, we have collected 231 samples from different pig farms across four provinces in Eastern China between the year 2017 and 2022 and screened them for PRRSV. Thirteen PRRSV isolates were obtained from samples and their sequences were deposited to GeneBank for further analysis and characterization. The evolutionary history of PRRSV outbreaks in China is roughly divided into three stages (era). The first stage occurred from 1995 to 2006, when typical strains of PRRSV were dominant (Zhou and Yang, 2010). The second stage existed from the year 2006 to 2012, and strain HP-PRRSV became most prevalent (Li et al., 2011). The third stage started in 2012 and is currently going on, in this stage HP-PRRSV is spotted as the main circulating strain, however, co-circulation of new mutants strains, i.e., NADC30-like, GM2, and NADC34-like have been reported in pig farms from some areas across China (Peng et al., 2017; Liang et al., 2019). According to a survey (Chen et al., 2020) HP-PRRSV strain was found to be the most prevalent (51.57%) PRRSV strain circulating in China between the years 2017 to 2018, and NADC30-like strains also accounted for a large part in PPRSV Soutbreaks (43.30%). Meanwhile, the first NADC34-like PRRSV strain was reported in Liaoning Province, and then some NADC34-like PRRSV strains were reported in different regions of China (Zhang et al., 2018; Liu et al., 2019; Xie et al., 2020). As the ORF5 and Nsp2 coding regions are highly variable, so their genomes are used for PRRSV mutational analysis, virus species determination, and molecular epidemiological characteristics (Cha et al., 2004; Shi et al., 2010a). In this study, two neighbor-joining phylogenetic trees were also constructed and their percentage identities were calculated with other isolated and reference strains based on ORF5 and Nsp2 genomes. All 13 strains were found to have lower nucleotide and amino acid identity with the European strains and higher identity with the North American and Chinese PRRSV strains. Most of the isolated strains were HP-PRRSV, which contain discontinuous deletions of 1 and 29 amino acids in the Nsp2 region. The lineage 1 strains also accounted for a large part. Among all 13 strains, three different strains appeared in the Lianyungang pig farm in Jiangsu previously during the years 2017 and 2018, containing PRRSV classic strains, HP-PRRSV, and NADC30-like strains. The epidemic characteristics from the isolated strains were found consistent with the previous and current PRRSV epidemic situation. We also isolated two NADC34-like strains (i.e., JS-KS-2021-12 and JS-NJ-2022-01) from the Jiangsu province, which contain continuous deletions of 100 amino acids. In late 2021 and early 2022, two NADC34-like strains were isolated from the Jiangsu province by Yuan et al. (2022). Yuan et al. (2022) and Zhu et al. (2022) emphasizing that NADC34-like strains have gradually spread and that they have become a potential endemic strain in China. Therefore, further research into NADC34-like strains is necessary.

GP5 has relevant sites for determining viral virulence and plays a key role in the induction of the immune response (Zhang et al., 2017). The amino acids at positions 13 and 151 can be used to distinguish between virulent and attenuated strains. In this study, five isolated strains containing both amino acids at positions R¹³ and R¹⁵¹ were found to be highly virulent. These isolates caused abortion in sows with depression, anorexia, hyperthermia, and dyspnea in piglets. Three isolates having R13 or R151 amino acids were moderately virulent and induced moderate clinical signs (i.e. hyperthermia, anorexia, and moderate respiratory signs). The rest of the strains caused milder clinical symptoms, with elevated body temperature in piglets accompanied by loss of appetite and depression. GP5 has several epitopes, including a decoy epitope, a PNE, T cell epitopes, and B cell epitopes. In this study, we found that most strains had amino acid mutations in T cell and B cell epitopes. The PNE (37–45 aa) and the decoy epitope (27–31 aa) of GP5 play important roles in inhibiting the immune response. Some critical amino acid substitutions in these positions may lead to the ability of the virus to evade recognition by neutralizing antibodies, resulting in the failure of live attenuated cellular vaccine strains (Ostrowski et al., 2002). In this study, eight strains belonging to sublineage 5.1 and lineage8 had the same amino acid in antigenic epitopes compared to their representative strains. Strain ZJ-JX-2017-04, which belongs to lineage 3, had an amino acid mutation (²⁷ALVSA³¹ → ²⁷ALVNA³¹). Compared to lineage 1 representative strains NADC30 and NADC34, four strains belonging to lineage 1 had some amino acid mutations and deletions. Three strains had a mutation from ²⁷ALVNA³¹ to ²⁷ALVSA³¹, and strain JS-LYG-2018-06 had a special mutation to ²⁷VLINA³¹. There were few amino acid variations in the PNE compared to reference strains. Only strain AH-BB-2018 had a mutation at position 41 from L⁴¹ to V⁴¹. These mutations may result in a lack of immune cross-protection between different PRRSV strains.

N-glycosylation of viral proteins is a complex post-translational modification that affects protein folding, viral entry, receptor interactions, immune escape and pathogenesis (Das et al., 2011; Rupasinghe et al., 2022). GP5 contains five NGSs, including N³⁰, N^32/33/34, N³⁵,N⁴⁴, and N⁵¹, which determine the antigenic properties and viral susceptibility to neutralizing antibodies (Akter et al., 2021). The potential NGSs located upstream of N⁴⁴ were relatively variable while N⁴⁴ and N⁵¹ are highly conserved in United States and European strains. Mutations in the N⁴⁴ residue resulted in non-infectious offspring (Ansari et al., 2006). Relevant studies have confirmed that the expression of N-glycosylated GP5 mutants in PRRSV significantly increased the level of neutralizing antibodies in infected piglets. For the missing glycosylation site, it exposes neutralizing epitopes that are masked by glycosylation, thereby enhancing immunogenicity and susceptibility of GP5 protein to neutralizing antibodies (Vu et al., 2011; Peng et al., 2017). The lack of an N-glycosylation site at N⁵¹ has been shown to increase the sensitivity of the virus to antibody neutralization and enhance the ability of the virus to establish a cognate NAb response (Vu et al., 2011). The 13 strains isolated in this study were subjected to NGS prediction, and two to five NGSs were predicted. Among them, the AH-BB-2018-01 strain is deleted at N51, and the immune failure caused by this strain may be caused by the reduction of NGS.

Currently, commercial RespPRRS MLV vaccines are widely used for the prevention and control of PRRS in China. However, previous studies and this study indicated that commercial vaccines could not provide complete protection against HP-PRRSV, NADC30-like, and NADC34-like strains. Thus, additional PRRS prevention strategies must be developed.

In conclusion, we have investigated the epidemiological status of PRRSV in Eastern China between the years 2017 and 2022. We have found that highly pathogenic (HP-PRRSV) remained the predominant circulating PPRSV type in the field. The detection rates of NADC30-like and NADC34-like strains have increased in recent years, accounting for a large proportion of PRRSV. Furthermore, several critical mutations have been spotted within Nsp2 and GP5 proteins of isolated strains which may involve in immune escape. This study facilitates further analysis of PRRSV prevalence.

The data presented in the study are deposited in the “National Center for Biotechnilogy Information” repository, accession number “ON357667-ON35767”.

JW, XW, and ZM conceived the study. LZ, YY, and QX analyzed the data. LZ wrote the manuscript for submission. ZG, JZ, BL, YQ, and KL participated in the design of the study, performed the data collection and analysis, and commented on the manuscript. All authors contributed to the article and approved the submitted version.

The study was supported by the Shanghai Agriculture Applied Technology Development Program, China (nos. X2022-02-08-00-12-F01195 and X2021-02-08-00-12-F00770 awarded to JW).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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