The porcine reproductive and respiratory syndrome virus (PRRSV), the causal pathogen of porcine reproductive and respiratory syndrome (PRRS), primarily infects swine herds, causing reproductive disorders in pregnant sows, including miscarriage, the occurrence of stillbirths and mummified fetuses, as well as respiratory diseases in pigs of all ages (Sha et al., 2023). Since the initial isolation of PRRSV in Europe and the Americas in the early 1990s (Collins et al., 1992; Wensvoort et al., 1991), PRRS has become globally prevalent in many countries. In 2006, an outbreak of highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS), characterized by high fever and mortality in pigs of all ages, occurred in China and spread rapidly to many Asian countries (Guo et al., 2013). The incidence of HP-PRRS ranged from 50 to 100%, with mortality rates varying between 20 and 100%, resulting in the culling of approximately 40 million pigs in China in 2006–2007 (An et al., 2011), causing huge economic losses to the swine industry. However, given a range of complicating factors, including genetic diversity, immunosuppression, and secondary infections, the complete eradication of PRRS in the global swine industry presents significant challenge (Cai et al., 2023). Consequently, PRRS continues to pose a serious economic threat to the global swine industry.

PRRSV, a member of the viral genus Arterivirus, within the family Arteriviridae and order Nidovirales, is an enveloped, single-stranded positive-sense RNA virus (Adams et al., 2017). The PRRSV genome is approximately 15 kb in length and encodes 11 open reading frames (ORFs) (Zhang et al., 2023). ORF1a and ORF1b encode two polyproteins, namely, pp1a and pp1ab, respectively, which are subsequently cleaved by protein hydrolases to generate 16 non-structural proteins, whereas ORF2a, ORF2b, ORFs3–7, and ORF5a encode eight corresponding structural proteins (Chen et al., 2023). At present, PRRSV is divided into two distinct species, Betaarterivirus suid 1 (PRRSV-1) and Betaarterivirus suid 2 (PRRSV-2), which have approximately 60% nucleotide homology (Brinton et al., 2021; Lu et al., 2014). Among the viral proteins, the GP5 protein encoded by ORF5 is characterized by the highest structural diversity between PRRSV-1 and PRRSV-2 strains. At the amino acid level, the homology of PRRSV-1 and PRRSV-2 GP5 is between 50 and 55%, and this protein is thus commonly used in genetic sequence analysis (Lunney et al., 2010; Zhao et al., 2022). On the basis of ORF5 sequences, PRRSV-1 can be classified into four subtypes, namely, subtype 1 (Global), subtype 1 (Russia), subtype 2, and subtype 3, whereas PRRSV-2 can be classified into nine lineages and 37 sublineages (Li et al., 2022; Shi et al., 2010). However, Yim-Im et al. (2023) refined a phylogenetic classification system based on the PRRSV-2 ORF5, reclassifying PRRSV-2 into 11 lineages (L1-L11), 21 sublineages (L1A-L1F, L1H-L1J, L5A-L5B, L8A-L8E, and L9A-L9E), as well as the 5 taxa within L1C (L1C.1-L1C.5). Although partial sequences of PRRSV-1 strains isolated in Vietnam have been uploaded to the GenBank database, to the best of our knowledge, there have been no systematic studies on PRRSV-1 conducted to date, unlike PRRSV-2, for which there have been a number of studies. In 2007, an outbreak of highly pathogenic PRRS (HP-PRRS) was detected in Vietnam, which spread rapidly nationwide (Lee et al., 2019a). In the same year, Feng et al. (2008) isolated suspected PRRSV strains for the first time, and on the basis of bioinformatics analysis, revealed that these were strains of the PRRSV-2 species, with a 99% nucleotide homology to PRRSV-2 strains that were prevalent in China in 2006. However, as early as 2002, Kamakawa et al. (2006) had already detected PRRSV on free-range pig farms in Vietnam, thereby indicating that PRRS was prevalent in Vietnam prior to 2007. Do et al. (2016) found that among PRRSV field strains isolated from 2007 to 2015, all were of PRRSV-2 sub-lineages 8.7 and 5.1. Whereas for strains isolated in 2021, Nguyen et al. (2022) detected those of lineage 8, sublineage 8.7, lineage 1, and sublineage 1.4.

Since the 2007 PRRS outbreak, although numerous PRRS vaccines had been introduced in Vietnam (Lee et al., 2019b), until 2021, many swine farms in Vietnam on which pigs had for long been vaccinated with PRRS attenuated vaccines continued to experience PRRS outbreaks, and it was accordingly speculated that mutations in the GP5 were reducing the protective efficacy of these vaccines (Nguyen et al., 2022). To confirm this supposition and gain further insights into the genetic variations of the PRRSV GP5 gene in Vietnam from 2007 to 2023, we obtained 344 PRRSV GP5 sequences from the GenBank database and used these to construct phylogenetic trees. Among these sequences, we selected 52 PRRSV-1 and 80 PRRSV-2 GP5 sequences for amino acid and nucleotide homology analyses and amino acid sequence alignment, along with analyses of potential recombination events. Our findings in this study will provide a theoretical foundation for understanding future epidemiological trends in PRRS and for the development of vaccine-based prevention and control measures in Vietnam.

Since the first reported outbreak of HP-PRRS in China in 2006, this disease has spread rapidly throughout other Asian countries, including Vietnam, the Philippines, and Thailand (Do D. T. et al., 2015), and the continuous mutation and recombination of PRRSV present a considerable challenge to the global swine industry (Li et al., 2016). The GP5 protein of this virus, which is essential for virus assembly and infection, and also important for the induction of neutralizing antibodies, is characterized by a notable variability, thereby identifying it as a useful focus for studying the genetic diversity of PRRSV (Jantafong et al., 2015). On the basis of the GP5 gene of phylogenetic analyses revealed that in recent years, PRRSV-2 lineage 8 strains have been the prevalent strains in Vietnam. The strains of this lineage have been established to be more closely related to lineage 1 strains and most distantly related to those of lineage 5 strains, whereas PRRSV-1 subtype 1 (global) strains were closer to subtype 3 strains and distant from subtype 1 (Russia). A comparison of the evolutionary trees constructed using the ML and NJ methods revealed no differences in the genetic distances between PRRSV-1 and PRRSV-2, or among different PRRSV-2 lineages. This observation may be related to the selected PRRSV strain gene from Vietnam. The emergence of HP-PRRS was first reported in China in 2006, and in the following year, an outbreak of HP-PRRS was reported in northern Vietnam, which subsequently spread rapidly to central and southern regions (Do T. D. et al., 2015; Tian et al., 2007). Between 2008 and 2012, Thuy et al. (2013) isolated 32 PRRSV strains of the PRRSV-2 sublineage 8.7 in Vietnam, a majority of which were found to be closely related to the JXA1-2006 strain isolated from China. It has been proposed that PRRS outbreaks are also associated with climatic factors (Alkhamis et al., 2017). Given the close geographical proximity of northern Vietnam to China, it is speculated that these factors may have contributed to a close correlation in the PRRSV strains transmitted between Vietnam and China. In 2021, PRRSV-2 lineage 1 strains were reported in Vietnam for the first time, along with the isolation of a vaccine-like strain, VN-HVLC1-2021 (belonging to L8E) (Nguyen et al., 2022). Accordingly, it is speculated that there may have been variation or recombination in the PRRSV vaccine strains used on Vietnamese farms. In this regard, the phylogenetic trees constructed in this study reveal that the PRRSV-2 L8E strains are the most abundant among those isolated in Vietnam, whereas L1A strains are the least abundant. Although to date there have been no relevant systematic reports, we found that whereas PRRSV-1 strains were isolated in Vietnam in 2016, PRRSV-2 L1A strains appeared in 2021, the number of L5A strains exhibited a gradual increase in 2022, thereby highlighting the emerging complexity of the epidemiological and genetic evolution of PRRSV on Vietnamese swine farms. Although our analyses in this study were constrained to a certain extent by the limited number and variety of PRRSV sequences obtained from strains isolated in Vietnam, on the basis of the types of PRRSV strains isolated in recent years, it would be predicted that the prevalence of PRRSV-1 strains, along with PRRSV-2 lineage 1 and 5 strains may gradually increase in the future.

In this study, we accordingly assessed 52 PRRSV-1 and 80 PRRSV-2 strains to investigate the nucleotide and amino acid homologies and amino acid sequence alignments of the GP5 protein, which thereby enabled us to establish the genetic diversity of this protein in Vietnam and predict potential evolutionary trends. Our findings revealed nucleotide and amino acid homologies of 76.1–100.0% and 75.2–100.0%, respectively, for PRRSV-1 strain GP5 proteins and corresponding values of 81.8–100.0% and 81.1–100.0% for PRRSV-2 strain GP5 proteins. Among these, PRRSV-1 subtype 1 (Global) exhibited the lowest homology and the most distant phylogenetic relationship with subtype 1 (Russia). Furthermore, we established that the GP5 gene sequences of the Vietnamese PRRSV-2 strains CT.C1-2012 and HG.RV2-2012 are identical to that of the Chinese PRRSV-2 strain 09HUB1-2009 (100%), whereas we detected no close similarities regarding PRRSV-1 strains, thereby tending to indicate that the evolutionary trends of PRRSV strains in Vietnam and China are both similar and distinct.

N-glycosylation sites on the GP5 protein are essential for viral replication and play important roles in receptor binding, viral infectivity, and the induction of immune responses (Wei et al., 2012). It has been shown that different N-glycosylation site mutations have differing effects on PRRSV, including loss of polysaccharide residues in N³⁴, N⁴⁴, and N⁵¹ enhancing immunogenicity, whereas those at N³³, N³⁴, and N³⁵ may enhance viral virulence; moreover, mutations in neutralizing epitopes can also lead to changes in PRRSV virulence and immune escape (Luo et al., 2023). Furthermore, specific mutations in N-glycosylation sites, such as D³⁷N, D⁵⁶N, S⁶⁰N, and G⁶³N, have been established to be closely associated with immune evasion, inhibition of host viral recognition, and loss of host neutralization capacity (Hsueh et al., 2023). In the present study, we identified ten N-glycosylation site mutations in the GP5 protein of Vietnamese PRRSV strains (4 in PRRSV-1, 6 in PRRSV-2), particularly at residues 33, 34, 35 and 51 of the PRRSV-2 GP5 protein, which may potentially play an important role to immune evasion or altered PRRSV virulence. All four glycosylation sites of PRRSV-1 were conserved, with mutations observed in only a few individual strains; whereas among the Vietnamese PRRSV-2 strains, the N⁵¹ site was relatively conserved, with mutations observed only in the strain VNUA-PRRSV-HN1-22-2022. Guo et al. (2019) have previously found that mutations to the 39^th and 57^th amino acids of GP5 contribute to virus evasion of host immune responses, and the 187–200 amino acid positions of the GP5 positions in both PRRSV-1 and PRRSV-2 were instrumental in the cross-neutralization reaction of antibody formation (de Lima et al., 2006). Compared with the findings reported by Nguyen et al. (2022), we identified certain identical amino acid mutation sites (aa 39, 52–61, 187–200) in the PRRSV-2 GP5 protein, which may confer the virus with the ability to evade host immunity; and certain different amino acid mutation sites were identified in sublineage L1A, including a novel deletion mutation (aa 37), a lineage-specific mutation (L⁴⁷I) and four mutations specific (F²⁶I, D⁶¹E, L¹⁴⁵V, and E¹⁷⁰I). In contrast, among the Vietnamese PRRSV-1 strains, with the exception of EuroViet-01-2016 and EuroViet-02-2016, which exhibited mutations at amino acid positions 187–200, no other strains demonstrated mutations at these sites. Furthermore, two specific mutations were identified in PRRSV-1 subtype 1 (Global): H⁵C and A²⁰¹V. However, the association of these mutations with the epidemiological and genetic variation trends of PRRSV strain in Vietnam will necessitate further investigation.

It has been established that PRRSV spreads between farms via the transfer of infected animals and infected semen, aerosols, and items contaminated with the virus (Lee et al., 2019a), and Zimmerman et al. (1997) found that birds may be involved in the transmission of PRRSV. Accordingly, given that many small-scale farms in Vietnam operate mixed livestock farming systems (Lee et al., 2019b), the potential for virus transmission from avian species to pigs cannot be ruled out. Notably, such small-scale swine farms account for 70% of the total pig production in Vietnam, and farmers rearing pigs on these farms rarely use PRRSV vaccine (Lee et al., 2019a). Consequently, given the inadequate vaccine coverage and lack of effective biosecurity measures, these small-scale farms are at a heightened risk of PRRS outbreaks. Outbreaks are, however, by no means confined to these smallholdings, as PRRS outbreaks still occur on swine farms that have been conducting vaccination over prolonged periods, which would indicate that new mutations and antigen changes in PRRSV strains may have led to immune evasion. In addition, Vietnamese farmers continue to sell sick pigs in order to reduce the economic losses caused by PRRS, thereby making the prevention and control of PRRS even more challenging (Huong et al., 2016).

To summarize, in this study, we sought to examine nucleotide and amino acid homologies among different strains of PRRSV and characterized amino acid mutations in these strains based on analyses of the genetic dynamics of 271 PRRSV GP5 sequences obtained from Vietnamese strains isolated between 2007 to 2023. Although the GP5 protein of this virus has been established to show significant variability and is prone to recombination events, we detected no relevant recombination in this study, and we suspect that the recombination events may exist in other ORFs. During the course of PRRS epidemics, PRRSV strains undergo continuous mutation and evolution, thereby giving rise to numerous novel strains that can compromise the efficacy of vaccines. Moreover, as a consequence of economic imperatives and a lack of biosecurity awareness, small-scale farms in Vietnam tend to be characterized by low PRRSV vaccination rates and unrestricted pig trade, thereby presenting considerable challenges for ensuring effective PRRS prevention and control. On the basis of our analysis of PRRSV GP5 sequences obtained from strains isolated in different years, we provide a theoretical foundation for further research and the development of novel vaccines based on the GP5 gene.

In this study, we established that from 2007 to 2023, the PRRSV-1 strains prevalent in Vietnam were of subtype 1 (Global), along with sublineages L1A, L5A, and L8E PRRSV-2 strains, among which, L8E strains have tended to be the predominant type, followed by L5A, whereas L1A became prevalent only from 2021. Phylogenetic analysis based on sequences of the GP5 gene enabled us to assess the genetic diversity of PRRSV strains in Vietnam, and using such information, continuous monitoring of PRRSV GP5 genetic variation should be conducted to strengthen PRRSV prevention and control measures in this country.

On the basis of a survey of reports related to PRRSV in Vietnam, it has been established that the outbreaks of PRRS epidemics in the country can largely be traced to small-scale swine farms that account for the highest proportion of Vietnamese pig production. This can be ascribed to the fact that these farms tend to be based on mixed livestock and poultry breeding systems, combined with low vaccination rates against PRRSV and a lack of biosecurity awareness among pig farmers. Nevertheless, even on farms in which livestock have been routinely vaccinated against PRRS, herds still exhibit clinical symptoms related to PRRS, which provides evidence to indicate that PRRSV strains mutate and recombine to evade vaccine-promoted immunity, and thus highlights the inability of vaccines currently administered in Vietnam to effectively prevent and control PRRS. Given that the PRRSV GP5 protein shows high variability, and that mutations in the amino acid sequence can variously influence the virulence and immune evasion of PRRSV, as well as the neutralization capacity of the host, in-depth analyses of trends in the genetic variation of this protein could provide a theoretical basis for the development of novel vaccines and the establishment of effective prevention and control measures. Moreover, it is imperative that Vietnam further strengthens the cultivation of biosafety awareness among small-scale farmers and implement relevant biosafety measures, which, we anticipate, would contribute to the future control or even eradication of PRRS.