Porcine Parvovirus

Overview and Taxonomy of Porcine Parvovirus

Porcine parvovirus (PPV) represents one of the most significant etiological agents of reproductive failure in swine globally, a pathogen that has commanded the attention of veterinary virologists and swine producers alike for over half a century [21, 31]. The disease manifestations associated with PPV infection, stillbirth, mummification, embryonic death, and infertility, are collectively designated under the acronym SMEDI, a syndrome that exacts a profound economic toll on intensive pig production systems worldwide [1, 21]. The World Organisation for Animal Health (WOAH) recognizes PPV as a critical pathogen of reproductive disease, underscoring its importance in international swine health management and trade. While the archetypal virus, PPV type 1 (PPV1), has been recognized since the 1960s, the past two decades have witnessed a remarkable expansion of the known parvovirus landscape in swine, with the discovery of seven additional genotypes (PPV2 through PPV8) that have fundamentally reshaped our understanding of the genus and its evolutionary dynamics [1, 18, 21, 31].

Taxonomic Classification and Phylogenetic Diversity

The taxonomic framework of PPV has undergone considerable revision as molecular detection techniques, particularly high-throughput sequencing and advanced phylogenetic analyses, have revealed an unexpected diversity of parvoviruses circulating in swine populations. Members of the family Parvoviridae, subfamily Parvovirinae, these viruses are small, non-enveloped particles with a linear, single-stranded DNA genome of approximately 4–6 kilobases [1, 21]. The historical classification placed PPV1 within the genus Protoparvovirus as Ungulate parvovirus 1, but the identification of novel genotypes has necessitated a broader taxonomic reassessment [15, 25, 31]. The eight recognized genotypes (PPV1–PPV8) are now distributed across at least three distinct genera within the Parvovirinae subfamily, reflecting deep evolutionary divergence rather than mere strain variation [8, 28].

PPV1 remains the archetypal and most intensively studied member, classified within the genus Protoparvovirus [15, 21]. It is unequivocally established as the primary causative agent of SMEDI syndrome, and its pathogenic mechanisms, including transplacental infection, fetal death, and persistent viremia in naïve gilts, have been thoroughly characterized [6, 21, 31]. Phylogenetic analyses of PPV1 have revealed at least four to five distinct clusters or subgroups, with the 27a-like strains (cluster D) emerging as the predominant field strains in Europe and Asia since the early 2000s [6, 10, 14, 16, 23]. These 27a-like strains exhibit increased viral fitness and altered antigenic profiles compared to classical vaccine strains such as NADL-2, raising important questions regarding vaccine efficacy and the potential for immune escape [14, 23, 31]. Indeed, recent studies have demonstrated that certain amino acid substitutions in the VP2 capsid protein (particularly at position 228) confer a selective advantage, enabling these strains to outcompete older lineages even in vaccinated herds [23].

The classification of the novel genotypes presents a more complex picture. PPV2, PPV3, and PPV4 are assigned to the genus Tetraparvovirus, based on genomic organization and phylogenetic clustering distinct from PPV1 [21, 28]. These viruses were first identified in the early 2000s and have since been detected in swine populations across North America, Europe, Asia, and Africa [5, 13, 18, 27]. PPV5 and PPV6 belong to the genus Protoparvovirus, sharing a closer evolutionary relationship with PPV1 than with the tetraparvoviruses, yet exhibiting unique pathogenic and epidemiological features [12, 13, 21, 34]. PPV7 is classified within the genus Chapparvovirus, a distinct lineage that was first identified in the United States in 2016 and subsequently reported in China, Brazil, Colombia, and several European countries [22, 25, 28, 30]. The most recently described member, PPV8, was initially identified in China in 2022 via high-throughput sequencing of porcine reproductive and respiratory syndrome virus (PRRSV)-positive samples; phylogenetic analysis places it within the genus Protoparvovirus, with the highest sequence homology to PPV1 (31.86–32.68% amino acid identity in the NS1 protein) [15]. Remarkably, retrospective analyses have demonstrated that PPV8 has been circulating in Chinese swine herds since at least 1998, indicating a long-standing but previously unrecognized presence [15]. The global dissemination of PPV8 has been confirmed by subsequent reports in Europe (Hungary and Slovakia) and the Americas (Colombia), suggesting a cosmopolitan distribution that mirrors that of the earlier novel genotypes [3, 9].

Genomic Architecture and Physicochemical Properties

The genomic organization of PPVs is characterized by a compact, efficient arrangement typical of the Parvoviridae. The genome contains two major open reading frames (ORFs): the non-structural ORF encoding the NS1 and NS2 proteins, which are essential for viral DNA replication and transcriptional regulation, and the structural ORF encoding the capsid proteins VP1 and VP2 [21, 31]. The VP2 protein, which constitutes the primary immunogenic component of the viral capsid, is capable of self-assembling into virus-like particles (VLPs) that are morphologically and antigenically similar to native virions [7, 19, 26, 32, 33]. This property has been exploited extensively in the development of subunit vaccines and diagnostic reagents [11, 26, 33].

A notable genomic feature distinguishing the PPV genotypes is their nucleotide composition and CpG island distribution. A comprehensive analysis of PPV1 through PPV8 genomes revealed that PPV1–PPV6 possess AT-rich genomes (GC content ≤50%), whereas PPV7 exhibits a GC content exceeding 50% [8]. The number of CpG islands, which are implicated in epigenetic regulation of viral gene expression through cytosine methylation, varies markedly across genotypes. PPV1, PPV4, PPV5, and PPV6 contain relatively few CpG islands (1–5), while PPV7 contains a moderate number (6–11), and PPV2 and PPV3 harbor a striking abundance (12–16) [8]. This variability in CpG content may reflect differential evolutionary pressures and host–virus interactions, as CpG methylation can influence viral replication, latency, and immune evasion.

The physicochemical stability of PPV is exceptional among mammalian viruses, a feature that has practical implications for both transmission and biosecurity. PPV is remarkably resistant to heat, desiccation, and a wide range of disinfectants, enabling prolonged environmental persistence [4, 24]. Studies evaluating the survival of PPV within insect larvae used for feed production, including mealworms (Tenebrio molitor) and black soldier fly larvae (Hermetia illucens), demonstrated that infectious PPV could be detected for up to 9 days post-ingestion, with viral DNA persisting throughout the study period [4]. This extraordinary stability underscores the importance of rigorous biosecurity protocols and the potential for non-conventional transmission routes (e.g., contaminated feed ingredients) to sustain viral circulation within and between swine herds.

Host Range and Global Distribution

The host range of PPV has traditionally been considered restricted to swine, encompassing both domestic pigs (Sus scrofa domesticus) and wild boar (Sus scrofa), as well as African warthogs (Phacochoerus africanus) [2, 10, 17, 31]. However, the detection of PPV nucleic acids in a wide variety of clinical samples, including serum, oral fluids, processing fluids, feces, lung tissue, and fetal tissues, highlights the ability of these viruses to infect multiple organ systems and to be shed via numerous routes [3, 13, 18, 20, 27]. The epidemiological significance of wild boar populations as reservoirs for PPV transmission to domestic swine has been increasingly recognized. Studies from Serbia, Namibia, and various European and African countries have documented high seroprevalence rates (up to 56% in some wild boar populations) and the circulation of genetically diverse PPV strains, including those closely related to virulent 27a-like lineages [2, 10, 17].

Globally, PPV1 is enzootic in virtually all swine-producing regions, with the exception of a few countries where stringent eradication programs have been implemented [21]. The novel genotypes (PPV2–PPV8) exhibit similarly widespread distributions, having been detected in North America, South America, Europe, Asia, and Africa [1, 3, 5, 12, 13, 18, 25, 27, 29]. The prevalence of these viruses varies considerably depending on the genotype, geographical region, age group, and sample type. For instance, studies in South Korea reported detection rates ranging from 7.9% (PPV1) to 32.6% (PPV2) in lung samples [13], while investigations in China and Europe have documented coinfection rates exceeding 50% for combinations of PPV genotypes [18, 27]. The expanding list of recognized PPV genotypes, the high prevalence of coinfections, and the potential for synergistic interactions with other swine pathogens (particularly porcine circovirus type 2 [PCV2] and PRRSV) collectively underscore the need for continuous molecular surveillance and a refined understanding of the taxonomy, evolution, and pathogenic potential of the genus Parvoviridae as it pertains to swine health.

Molecular Pathogenesis and Genomic Evolution of Porcine Parvovirus

Porcine parvovirus (PPV) represents a paradigm of viral adaptation, wherein a structurally simple single-stranded DNA (ssDNA) virus has evolved a sophisticated molecular arsenal to subvert host cellular machinery, establish persistent infections, and undergo remarkable genomic diversification. The molecular pathogenesis of PPV is orchestrated primarily by the non-structural protein NS1 and the structural capsid protein VP2, which collectively drive viral replication, cellular cytotoxicity, and immune evasion while simultaneously driving the virus’s evolutionary trajectory from the classical PPV1 to eight distinct genotypes (PPV1–PPV8) [1, 21]. Understanding the interplay between these molecular mechanisms and the selective pressures exerted by host immunity and vaccination is critical for interpreting the emergence of novel strains and designing next-generation countermeasures.

Molecular Determinants of Viral Replication and Cytopathicity

The Multifunctional NS1 Protein: A Master Regulator of Pathogenesis

The NS1 protein of PPV is a multifunctional phosphoprotein essential for viral DNA replication, transcriptional regulation, and cytotoxicity. Its pathogenic potential is intimately linked to its capacity to manipulate host cell signaling pathways and induce programmed cell death. NS1 is a nucleus-targeting protein that must traverse the nuclear envelope to initiate viral DNA replication. Seminal work by Cao et al. demonstrated that NS1 exhibits dynamic nucleocytoplasmic shuttling, mediated by two functional nuclear export signals (NES1 at aa 602–608 and NES2 at aa 283–291) that operate through the chromosome region maintenance 1 (CRM1)-dependent pathway, and a bipartite nuclear localization signal (NLS at aa 256–274) that engages importins α5 and α7 [45]. The absolute necessity of these trafficking signals for viral replication was underscored by the failure to rescue mutant viruses lacking NES or NLS motifs, highlighting the NS1 nuclear cycle as a critical vulnerability for antiviral intervention [45].

Once in the nucleus, NS1 drives viral DNA replication but simultaneously triggers profound cellular stress responses. PPV infection activates endoplasmic reticulum (ER) stress selectively through the protein kinase R-like ER kinase (PERK) signaling pathway, without engaging inositol-requiring enzyme 1 (IRE1) or activating transcription factor 6 (ATF6) [52]. This PERK-mediated ER stress, while initially a host defense, paradoxically inhibits PPV replication through the induction of C/EBP homologous protein (CHOP)-dependent apoptosis, suggesting a delicate balance between viral replication and host cell survival [52]. However, NS1 has evolved countermeasures to this antiviral state. The protein activates the nuclear factor-κB (NF-κB) signaling pathway through a Toll-like receptor 2 (TLR2)-dependent mechanism, leading to upregulation of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) [47]. This NF-κB activation appears to be a double-edged sword: while it may promote inflammation and viral replication, it also renders the virus susceptible to anti-inflammatory interventions such as ferulic acid, which downregulates NS1 expression and suppresses the NF-κB inflammasome axis [49].

Induction of Apoptosis and Necroptosis: Dual Pathways to Cell Death

PPV-induced cell death is a central feature of its pathogenesis, particularly in placental tissues where it mediates reproductive failure. The virus employs both apoptotic and necroptotic pathways, with NS1 playing a pivotal role in both. NS1 induces mitochondria-mediated intrinsic apoptosis through the generation of reactive oxygen species (ROS), disruption of mitochondrial membrane potential, and activation of caspase-9, but not caspase-8 [48]. Mechanistically, NS1 downregulates anti-apoptotic molecules Bcl-2 and Mcl-1 while upregulating pro-apoptotic factors Bax, P21, and P53, culminating in cytochrome c release and caspase cascade activation [48, 50]. This apoptotic pathway is further potentiated by the upregulation of Bid and Bad, and can be effectively blocked by ferulic acid through inhibition of Bid-related signaling [50].

Remarkably, PPV has also evolved the capacity to induce necroptosis, a regulated form of necrosis, as an alternative cell death pathway. Xu et al. demonstrated that PPV infection of porcine placental trophoblast cells (PTCs) triggers Z-nucleic acid-binding protein 1 (ZBP1)-mediated necroptosis through the RIPK3/MLKL axis, independent of caspase-8 [37]. This necroptotic pathway is dependent on viral DNA sensing, as UV-inactivated virus failed to induce necroptosis, while a mutant virus lacking the translation initiation codon retained this capacity [37]. The existence of dual death pathways suggests that PPV has evolved to exploit host cell death machinery for viral dissemination, with necroptosis potentially serving as a backup mechanism when apoptosis is inhibited, ensuring viral release and propagation.

Autophagy: A Double-Edged Sword in Viral Replication

PPV has evolved sophisticated strategies to hijack host autophagy machinery to promote its replication. Infection of PTCs triggers autophagy through the AMPK/Raptor/mTOR pathway, leading to inhibition of mTORC1 activation and upregulation of Beclin 1 and LC3II [41]. This autophagic response is proviral, as treatment with the mTOR activator MHY1485 suppressed autophagy and reduced viral replication, while AMPK knockout inhibited Raptor activation and decreased autophagosome formation [41]. The functional significance of autophagy extends beyond mere replication enhancement. Zhang et al. demonstrated that in the presence of caspase inhibitors, PPV induces non-apoptotic cell death characterized by lysosomal damage, which is associated with autophagy [53]. Induction of complete autophagic flux with rapamycin promoted viral replication and lysosomal damage, whereas inhibition of autophagy increased apoptosis and reduced viral replication [53]. These findings reveal a complex interplay where autophagy serves as a proviral mechanism that also modulates the mode of cell death, favoring non-apoptotic pathways that may reduce inflammatory responses and facilitate persistent infection.

Host-Virus Interactions: Immune Evasion and Signaling Subversion

Manipulation of Innate Immune Signaling

PPV has evolved multiple strategies to subvert the host innate immune response, particularly through manipulation of Toll-like receptor (TLR) signaling and interferon pathways. The NS1 protein directly interacts with host proteins to modulate antiviral responses. Du et al. identified chaperonin-containing T-complex polypeptide complex subunit 5 (CCT5) as a critical NS1-interacting partner that mediates the interaction between NS1 and coatomer protein complex subunit ε (COPƐ) [43]. This interaction suppresses interferon-β expression, thereby promoting viral replication. The dependency on NS1’s N-terminal 36–42 amino acid motif for CCT5 binding provides a potential target for therapeutic intervention [43].

CD38, a transmembrane protein with diverse functions in immune regulation, has emerged as a key mediator of PPV pathogenesis. Zheng et al. demonstrated that CD38 enhances TLR9 expression and activates the NLRP3 inflammasome following PPV infection [44]. CD38 deficiency inhibited TLR9 upregulation, reduced interferon-α and Mx1 expression, and suppressed ROS levels and NLRP3 transcription [44]. Furthermore, CD38 deficiency activated SIRT1 expression and impaired PPV proliferation, suggesting that CD38 represents a host factor exploited by the virus to enhance replication through multiple signaling axes [44].

Alternative Splicing and the NS1/NS2 Ratio

A unique aspect of PPV molecular biology is the production of NS2 mRNA through alternative splicing of NS1 mRNA, yet the mechanism regulating this process remained elusive until recently. Chen et al. identified SYNCRIP (synaptotagmin-binding cytoplasmic RNA-interacting protein), a member of the hnRNP family, as a critical regulator of PPV NS1 mRNA splicing [46]. SYNCRIP was significantly upregulated by PPV infection both in vitro and in vivo, and was found to directly bind the 3′-terminal site of NS1 mRNA to promote cleavage into NS2 mRNA [46]. Remarkably, overexpression of SYNCRIP reduced NS1 mRNA and protein levels, while deletion impaired NS2 mRNA formation and NS2/NS1 ratio, ultimately reducing viral replication [46]. This finding reveals a novel mechanism of viral gene expression regulation and identifies SYNCRIP as a potential antiviral target.

Proteomic and Transcriptomic Landscapes of PPV Infection

High-throughput approaches have provided unprecedented insights into the cellular reprogramming induced by PPV infection. Wang et al. conducted a comprehensive proteomic analysis of PPV-infected PK-15 cells, identifying 32 differentially expressed proteins at 6 hours post-infection (hpi) and 345 at 36 hpi [38]. Gene ontology and KEGG enrichment analysis revealed significant involvement of toll-like receptor signaling, tumor necrosis factor signaling, and viral carcinogenesis pathways [38]. Notably, poly(rC) binding protein 1 (PCBP1) was found to be downregulated following PPV infection, and functional validation confirmed that PCBP1 inhibits PPV replication, identifying it as a potential host restriction factor [38].

Complementary transcriptomic analysis by Lu et al. revealed a temporal evolution of the host response, with 547 differentially expressed genes (DEGs) at 24 hpi and 1,765 at 48 hpi [39]. Importantly, 149 genes associated with various forms of cell death were upregulated at both time points, underscoring the multifaceted nature of PPV-induced cytotoxicity [39]. These findings establish a foundation for understanding the complex molecular interplay between PPV and its host, revealing multiple vulnerabilities that could be exploited for therapeutic intervention.

Genomic Evolution and Emergence of Novel Genotypes

Phylogenetic Diversity and Classification

The genomic landscape of PPV has undergone a dramatic transformation over the past two decades, expanding from a single recognized genotype (PPV1) to eight distinct species (PPV1–PPV8) [1, 21]. This diversification reflects both improved detection capabilities through high-throughput sequencing and genuine viral evolution driven by selective pressures from host immunity and vaccination. Guo et al. identified PPV8 in 2021 through metagenomic sequencing of PRRSV-positive samples from Guangdong province, China, with a nearly full-length genome of 4,380 nucleotides sharing only 16.23–44.18% sequence identity with PPV1–7 [15]. The relatively highest homology to PPV1 and its phylogenetic placement within the genus Protoparvovirus suggest a common ancestry, yet PPV8’s distinct clade indicates substantial divergence [15]. Subsequent detection of PPV8 in Hungary and Slovakia, where 68% of farms tested positive, demonstrated its rapid global dissemination, with oral fluids showing the highest detection rates [3]. The identification of PPV8 in Colombia further confirmed its intercontinental spread and association with porcine respiratory disease [9].

The evolutionary dynamics of PPV7 have been particularly well-characterized. Wang et al. estimated that PPV7 likely originated around 2004, with nucleotide substitution rates of 8.01 × 10⁻⁴ and 2.19 × 10⁻³ per site per year for NS1 and Cap genes, respectively, rates approaching those of RNA viruses [28]. Phylogenetic analysis of global PPV7 strains revealed two major clades with Chinese strains as the most likely ancestral lineage, suggesting that China may serve as an epicenter for PPV7 evolution [28]. The identification of eight subtypes (PPV7a–PPV7h) in Fujian Province alone, with PPV7h as the predominant subtype, underscores the remarkable genetic plasticity of this virus [40].

Recombination and Genomic Plasticity

Recombination serves as a major driver of PPV evolution, facilitating the emergence of novel strains with altered pathogenic potential. Lyu et al. identified recombination events in four of 29 sequenced PPV7 genomes from Fujian Province, with evidence of partial nucleotide deletions or insertions contributing to genetic diversity [40]. Similarly, Li et al. reported a PPV7 strain from domestic pigs that originated from recombination between two wild boar isolates (JX15-like and JX38-like), demonstrating that the wild boar reservoir serves as a genetic nursery for novel variants [18]. Studies from Inner Mongolia further confirmed recombination as a driving force, with strains NM-19 and NM-4 arising from recombination between two Chinese PPV7 strains, and amino acid deletions in the capsid protein potentially altering receptor binding and immunogenicity [22].

The structural basis for capsid plasticity has been illuminated by studies of PPV7 VP2. Vargas-Bermúdez et al. identified a 15-nucleotide (five amino acid) insertion in the VP2 capsid protein of Colombian PPV7 strains, located in a coil region that did not significantly alter the predicted 3D structure but could affect B cell epitope presentation [25]. Wang et al. similarly reported insertions of 3 to 15 nucleotides in the cap gene of Guangxi PPV7 strains, resulting in capsid proteins ranging from 470 to 474 amino acids compared to the reference strain’s 469 [30]. These findings suggest that the PPV7 capsid has evolved an unusual degree of structural flexibility that may facilitate immune evasion.

Antigenic Evolution and Vaccine Escape

The emergence of PPV1 variants with reduced neutralization by vaccine-induced antibodies has been a major concern for the swine industry. Streck et al. demonstrated that amino acid substitutions characteristic of the 27a-like strains, which have become predominant in Europe since the early 2000s, confer increased viral fitness and decreased neutralization activity by sera raised against commercial vaccines [23]. Using a series of mutants in a NADL-2 background, they identified a 27a-specific neutralizing epitope around amino acid 228 of VP2, and determined that substitutions at positions 215 and 378 significantly enhanced replication efficiency [23]. Importantly, while partial immune escape was observed, absolute immune escape was not evident, suggesting that increased viral fitness, rather than complete antigenic drift, may be the primary driver of 27a strain success [23].

The global phylogeography of PPV1, as elucidated by Franzo et al., reveals a complex history of international dissemination. Analysis of VP2 sequences from eight African countries demonstrated that 27a-like strains were more frequently detected than NADL-8-like strains, a pattern mirroring that observed in high-income regions [10]. Phylogeographic reconstruction suggested that the African PPV1 landscape has been shaped by multiple importation events from Europe and Asia, highlighting the role of international animal movement in viral spread [10]. Vereecke et al. provided a temporal perspective, estimating that PPV1 has evolved since approximately 1855 at a rate of 4.71 × 10⁻⁵ nucleotide substitutions per site per year, with the expansion of swine populations and vaccination intensity influencing viral dissemination [14]. Their reclassification of PPV1 into four phylogenetic groups (PPV1a–PPV1d) provides a standardized framework for monitoring strain emergence and evaluating vaccine cross-protection [14].

Selective Pressures and Evolutionary Constraints

Despite the remarkable diversity observed across PPV genotypes, selective pressure analyses reveal that the virus is predominantly under purifying (negative) selection. Li et al. demonstrated that VP2 sequences of PPV1–7 are largely under negative selection, with only a few positive selection sites detected in PPV7 VP2 [18]. Similarly, Deng et al. calculated dN/dS ratios for NS1 and VP2 genes of Chinese PPV1 isolates, confirming that both genes are under negative selection [16, 51]. This purifying selection suggests that the majority of mutations are deleterious and eliminated, yet the virus maintains sufficient genetic plasticity to generate antigenic variants. The emergence of PPV1c (NADL-8-like) and PPV1d (NADL-2-like) groups, along with the highly virulent 27a-like strains, indicates that even under negative selection, specific amino acid substitutions can confer selective advantages [14, 16].

The role of CpG islands in PPV evolution represents an intriguing epigenetic dimension. Xu et al. demonstrated that PPV1–6 have AT-rich genomes (GC content ≤50%), whereas PPV7 exhibits GC content >50% [8]. CpG island distribution varies dramatically across genotypes: PPV1, PPV4, PPV5, and PPV6 contain only 1–5 CpG islands, while PPV7 contains 6–11, and PPV2 and PPV3 contain 12–16 [8]. This differential CpG island distribution may influence host immune recognition through TLR9-mediated sensing of unmethylated CpG motifs, potentially contributing to the differential pathogenicity and tissue tropism observed among genotypes [8].

Molecular Epidemiology and Global Dissemination

The application of advanced molecular diagnostic tools has revealed the true extent of PPV diversity and co-infection dynamics. Cao et al. developed a SYBR Green-based multiplex real-time PCR capable of simultaneous detection of PPV, PRV, and PCV3, with detection limits as low as 3.67 copies/μL for PPV, enabling accurate diagnosis of co-infections that are increasingly recognized as the norm rather than the exception in commercial swine herds [35]. Studies from Korea demonstrated that PPV2 was the most prevalent genotype in lung samples (32.6%), and that co-infection with PCV2 and PRRSV significantly modulated viral detection rates: PPV1 and PPV6 were more common in PCV2-positive samples, while PPV2 and PPV7 were associated with PRRSV infection [13].

The detection of PPV2 in Colombia with an overall prevalence of 37.6% and its presence in both respiratory cases and stillborn fetuses suggests a broader pathogenic role beyond the classical SMEDI syndrome [5]. Nelsen et al. provided mechanistic evidence for this association, demonstrating by in situ hybridization that PPV2 localizes predominantly to alveolar macrophages in porcine respiratory disease complex (PRDC) lungs, with viral load correlating with macrophage numbers [42]. The detection of PPV5 in Russia, with overall prevalence of 8.9% and successful propagation in primary porcine testicular cells, further expands the geographic range and tissue tropism of these emerging viruses [36].

The persistence of PPV in the environment and its potential for vector-borne transmission represent additional dimensions of its molecular epidemiology. Lecocq et al. demonstrated that PPV genomic DNA could be detected in mealworm and black soldier fly larvae up to 9 days post-ingestion, and infectious virus could be recovered from mealworm larvae for at least 3 days, with real-time PCR detecting viral RNA up to 9 days [4]. The exceptional stability of PPV, including resistance to heat and low pH, facilitates its persistence in insect vectors and contaminated environments, contributing to its ubiquitous presence in swine herds worldwide and complicating eradication efforts [4, 24].

Epidemiology and Genetic Diversity of Emerging Strains (PPV1–PPV8)

The epidemiological landscape of porcine parvoviruses has undergone a profound transformation over the past two decades, expanding from a single recognized pathogen, PPV1, to a complex constellation of eight distinct genotypes (PPV1–PPV8) with global distribution, intricate co-infection dynamics, and rapidly evolving genetic architectures. This paradigm shift, driven largely by advances in high-throughput sequencing and molecular surveillance, has revealed that the porcine parvoviral ecosystem is far more heterogeneous and ecologically intricate than previously appreciated [1, 21]. The epidemiological and genetic diversity of these emerging strains presents formidable challenges for disease control, vaccine design, and the fundamental understanding of parvoviral pathogenesis in swine populations worldwide.

Global Distribution and Prevalence Patterns of Novel PPV Genotypes

The classical view of porcine parvovirus as a monotypic pathogen responsible for SMEDI syndrome (stillbirth, mummification, embryonic death, and infertility) has been irrevocably altered by the successive discovery of PPV2 through PPV8. Epidemiological investigations across multiple continents have documented that these novel genotypes are not merely sporadic findings but are, in fact, widely endemic in commercial swine herds. In China, a systematic investigation of 435 clinical samples collected from eight provinces between 2016 and 2020 revealed that PPV2 and PPV3 were the most prevalent genotypes, each detected in 22.53% of samples, with an overall PPV positivity rate of 55.40% [18]. This study further demonstrated that PPV1–PPV7 co-infections were detected in 27.36% of samples, involving two to five genotypes simultaneously, underscoring the complexity of parvoviral ecology in endemic regions [18].

The global reach of these emerging genotypes is striking. In Korea, comprehensive surveillance of serum, lung, and fecal samples from 2018 to 2020 documented detection rates ranging from 7.9% for PPV1 to 32.6% for PPV2 in lung tissues, with fattening pigs exhibiting the highest prevalence across all genotypes, from 6.4% for PPV1 to 36.5% for PPV6 [13]. Similarly, in Poland, testing of 271 oral fluids, 1244 serum samples, and 1238 fecal samples from 19 farms revealed that PPVs are widely disseminated, with oral fluids yielding the highest detection rates (10.7% for PPV1 to 48.7% for PPV2), and growing-finishing pigs consistently demonstrating the most frequent viral detection [27]. Fattening pigs have emerged as a consistent epidemiological reservoir across studies, with infections persisting into the late fattening period, suggesting chronic or persistent infection dynamics for several genotypes, particularly PPV2 [13, 27].

The African continent, historically underrepresented in PPV surveillance efforts, has recently yielded critical phylogeographic data. Analysis of 71 partial VP2 sequences from eight African countries (Burkina Faso, Côte d’Ivoire, Kenya, Mozambique, Namibia, Nigeria, Senegal, and Tanzania) collected between 2011 and 2021 revealed that the African PPV1 landscape has been largely shaped by multiple importation events from Europe and Asia, with 27a-like strains predominating over less virulent NADL-8-like strains [10]. This pattern mirrors that observed in high-income regions and highlights the role of international swine movement in shaping global PPV epidemiology [10]. In Namibia specifically, warthogs (Phacochoerus africanus) were found to harbor PPV1 at a prevalence of 31% (13/42), with co-infections with African swine fever virus and PCV2 documented, confirming that wild African suids serve as reservoirs for these viruses [17].

The most recently discovered genotype, PPV8, was first identified in China in 2022 through high-throughput sequencing of PRRSV-positive samples from Guangdong province, with retrospective analysis demonstrating that the virus had been circulating in Chinese swine herds since at least 1998 [15]. Subsequent surveillance of 211 clinical samples collected across 12 Chinese regions from 1990 to 2021 identified 37 PPV8-positive samples (17.5%), spanning 24 years and demonstrating long-term endemic circulation [15]. The detection of PPV8 has now extended beyond Asia. In Europe, systematic sampling of 2230 serum, 233 oral fluid, and 115 processing fluid samples from 23 Hungarian and 2 Slovakian pig farms between 2020 and 2023 revealed PPV8 on 65% of Hungarian farms and both Slovakian farms, with oral fluids showing the highest positivity rates, reaching 100% in some herds [3]. On the American continent, PPV8 was first detected in Colombia, where 6 of 146 (4.1%) pigs exhibiting porcine respiratory disease tested positive, marking the first report of this genotype outside China [9].

Co-infection Dynamics and Ecological Interactions

The epidemiological significance of emerging PPV genotypes cannot be appreciated in isolation, as co-infection with other swine pathogens, particularly PCV2, PCV3, and PRRSV, appears to be the rule rather than the exception. In Colombia, investigation of PPV2 in lung samples from pigs with respiratory disease and stillbirths revealed an overall prevalence of 37.6% (62/165), with the most common dual co-infections involving PPV2/PCV2 (7.5%) and PPV2/PRRSV (4%) in the respiratory group, while triple co-infections of PPV2/PCV2/PRRSV were detected in 15% of respiratory samples and 21% of stillbirth samples [5]. Quadruple co-infections (PPV2/PCV2/PCV3/PRRSV) were observed exclusively in the respiratory group (5.5%), suggesting that cumulative viral burden may exacerbate clinical outcomes [5].

In Korea, statistical analyses of co-factor associations revealed distinct patterns: PCV2-positive lung samples with or without PRRSV co-infection exhibited significantly higher detection rates of PPV1 and PPV6, whereas PPV2 and PPV7 prevalence was significantly elevated in PRRSV-infected lungs regardless of PCV2 status [13]. PPV5 was detected significantly more frequently in samples harboring both PCV2 and PRRSV, suggesting genotype-specific ecological niches and potential synergistic interactions [13]. These findings align with studies from China, where co-infection of PPV7 with PCV2 and PCV3 was common, and PPV7-positive samples demonstrated significantly higher PCV3 copy numbers compared to PPV7-negative samples, supporting the hypothesis that PPV7 may stimulate PCV3 replication [55].

The clinical significance of these co-infections is increasingly recognized. PPV2 has been demonstrated to be predominantly associated with macrophages in porcine respiratory disease complex (PRDC), with in situ hybridization localizing PPV2 infection to alveolar macrophages and other cells in lungs exhibiting interstitial pneumonia [42]. Importantly, in one-third of PPV2-positive lung samples identified by qPCR, no other known respiratory viruses were detected by metagenomic sequencing, suggesting that PPV2 may act as a primary respiratory pathogen in some contexts [42]. Similarly, PPV2 alone was detected in stillbirths, providing evidence for its involvement in porcine reproductive failure (PRF) beyond its established association with PRDC [5].

Genetic Diversity, Recombination, and Evolutionary Dynamics

The genetic architecture of emerging PPV genotypes is characterized by remarkable plasticity, driven by both point mutations and recombination events. Whole-genome sequencing of PPV7 strains from Fujian Province, China, revealed that 29 newly obtained genomes shared 90.0–97.2% nucleotide identity with each other and 88.9–98.1% identity with 128 reference strains from China and other countries [40]. Phylogenetic analysis classified these strains into eight subtypes (PPV7a–PPV7h), with PPV7h emerging as the predominant subtype in Fujian [40]. Notably, recombination analysis identified inferred recombination events in four strains, and six strains exhibited partial nucleotide deletions or insertions, highlighting the dynamic nature of PPV7 evolution [40]. In South China, PPV7 strains from Fujian and Guangdong provinces demonstrated remarkable subtype diversity, with PPV7e and PPV7f being most prevalent in Fujian and Guangdong, respectively, and a putative recombinant strain (21FJ09) identified using SimPlot and Recombination Detection Program 4 software [20].

The capsid protein-encoding region of PPV7 exhibits extraordinary variability. In Guangxi, China, analysis of 105 PPV7-positive serum samples from 385 pigs (27.3% positivity) revealed that 16 strains contained insertions of 3 to 15 nucleotides in the middle of the cap gene, resulting in Cap proteins encoding either 470 or 474 amino acids compared to the 469-amino-acid reference strain [30]. This represents the first report of sequence variation within the cap gene leading to increased amino acid length in PPV7 Cap protein [30]. In Colombia, four PPV7 strains were found to harbor a 15-nucleotide insertion (five amino acids) in the VP2 capsid protein (positions 540–554 nt; 180–184 aa), which phylogenetic analysis showed to differentiate two well-defined evolutionary groups [25]. Despite this insertion, three-dimensional structural modeling indicated that the additional residues are located in a coil region and do not significantly alter overall protein conformation or suppress potential B cell epitopes, though epitope lengthening could affect immune recognition [25].

PPV1 itself has undergone significant evolutionary change since its initial characterization. Sequencing of 10 PPV1 strains isolated from northern China revealed eight amino acid substitutions in the NS1 protein and fourteen substitutions in the VP2 protein compared to the reference strain NADL-2 [6]. The JX strain, which exhibited reduced neutralizing activity against commercially available vaccine-induced antibodies in vitro, was selected for challenge experiments in pregnant sows, where it caused viremia, mild edema, inflammation of respiratory and reproductive tissues, and fetal infection after penetrating the placental barrier [6]. This strain demonstrates the emergence of vaccine-escape variants with retained pathogenic potential, a phenomenon of increasing concern.

Phylogeographic analysis of PPV1 using extensive VP1/VP2 sequencing has revealed that the virus has evolved since approximately 1855 (95% HPD: 1737–1933) at a rate of 4.71 × 10⁻⁵ nucleotide substitutions per site per year [14]. This analysis identified four relevant phylogenetic groups: PPV1a (G1), PPV1b (G2 or 27a-like), PPV1c (e.g., NADL-8), and PPV1d (e.g., NADL-2). While most European strains belong to PPV1a or PPV1b, Asian and American G2 strains, along with some European strains, were divided into virulent PPV1c and attenuated PPV1d groups [14]. The increase in swine population size, vaccination coverage, and biosecurity management practices were identified as factors influencing PPV1 spread [14]. Critically, amino acid substitutions observed in the 27a-like strains have been demonstrated to significantly increase viral fitness and decrease neutralization activity of serum samples raised against commercial vaccines and old virus strains, defining a 27a-specific neutralizing epitope around amino acid 228 of the VP2 capsid protein [23]. This suggests a scenario reminiscent of canine parvovirus evolution, where immune selection and enhanced fitness combined to drive global replacement of original virus strains by new antigenic types [23, 31].

Genomic and Structural Variation Across Genotypes

The genomic diversity across PPV1–PPV8 extends to fundamental features such as nucleotide composition and CpG island distribution. Analysis of CpG islands in the genomes of PPV1–PPV8 revealed that PPV1–6 possess AT-rich genomes (GC content ≤50%), whereas PPV7 exhibits a GC content exceeding 50% [8]. The number of CpG islands varies dramatically: PPV1, PPV4, PPV5, and PPV6 contain 1–5 CpG islands; PPV7 contains moderate numbers (6–11); while PPV2 and PPV3 contain the most (12–16) [8]. These differences may have profound implications for epigenetic regulation of viral gene expression and host-virus interactions, as CpG methylation is a well-established mechanism influencing viral life cycles [8].

The newly discovered PPV8 provides an illustrative example of the genetic distance separating these emerging genotypes. The nearly full-length genome of PPV8 strain GDJM2021 is 4380 nucleotides in length, encoding two overlapping open reading frames for NS1 and VP1 [15]. Genomic sequence analysis demonstrated that PPV8 shares only 16.23–44.18% nucleotide identity with PPV1–7, with the highest homology to PPV1. The NS1 protein of PPV8 shares 31.86–32.68% amino acid sequence identity with those of PPV1 and porcine bufavirus (PBuV), forming an independent branch adjacent to members of the genus Protoparvovirus [15]. This degree of divergence underscores the extraordinary genetic plasticity within the porcine parvoviral evolutionary lineage.

Genetic Characterization in Understudied Regions

Emerging data from regions previously lacking PPV surveillance are filling critical gaps in the global epidemiological picture. In Russia, the first identification of PPV6 was reported in four of seven sampled regions, with an overall prevalence of 9.4% (49/521 serum samples), predominantly in fattening pigs [12]. Phylogenetic analysis of the Russian isolate revealed high homology with strains from Spain, suggesting potential transboundary transmission pathways within Europe and Asia [12]. Similarly, PPV5 was detected in 8.9% of samples across 11 of 20 investigated pig farms in Russia, with isolates demonstrating high nucleotide identity with globally detected strains [36]. The successful propagation of both PPV5 and PPV6 in primary porcine testicular cells and established cell lines (SPEV and SK, respectively) provides essential tools for future functional studies [12, 36].

In East Africa, the Democratic Republic of Congo reported the first detection of PPV3 in domestic pig farms without reproductive failure, with high nucleotide sequence similarity suggesting a common viral source [29]. In India, molecular detection of PPV1 in 14.3% of aborted and stillborn fetuses from Tamil Nadu and Kerala revealed two sequences belonging to cluster E, representing the first report of this cluster in southern India and demonstrating amino acid variations at positions 59, 215, 228, and 314 of the VP2 protein [54]. In Argentina, PPV1 was detected in 17 of 131 mummies and stillbirths from normal deliveries in vaccinated sows, with all isolates related to the NADL-2 strain but maintaining amino acid differences at positions 436 (S→P) and 565 (R→K), suggesting that current vaccines reduce but do not eliminate transplacental infection [58].

Epidemiological Implications for Disease Control

The cumulative evidence from global epidemiological surveillance reveals several critical findings with direct implications for disease management. First, the high prevalence of novel PPV genotypes in fattening pigs coupled with persistent infection through the late finishing period suggests that these viruses may establish chronic infections with prolonged shedding, facilitating continuous circulation within herds [13, 27]. Second, the widespread co-infection with PCV2, PCV3, and PRRSV, themselves pathogens of major economic importance, raises the possibility that PPVs may act as co-factors exacerbating disease severity, either through direct synergistic effects or through immunomodulation [13, 18, 55]. Third, the documented emergence of vaccine-escape variants (particularly the 27a-like PPV1 strains) and the limited cross-protective efficacy of existing vaccines against heterologous strains [14, 23, 56, 57] underscore the urgent need for updated vaccine antigens that reflect current circulating genetic diversity. Fourth, the detection of PPV8 in both Europe and the Americas within two years of its initial discovery in China [3, 9, 15] illustrates the rapid global dissemination of these emerging viruses, likely facilitated by international trade in swine and swine products. Finally, the detection of PPVs in wild boar populations, warthogs

Clinical Manifestations and Reproductive Disorders in Swine

Porcine parvovirus (PPV) stands as a singularly formidable etiological agent within the global swine industry, responsible for a spectrum of reproductive pathologies that are collectively devastating to swine productivity and economic sustainability. The clinical hallmark of classic PPV1 infection is the SMEDI syndrome, an acronym denoting stillbirth, mummification, embryonic death, and infertility, a constellation of reproductive failures that has defined the clinical perception of this pathogen for decades [1, 21]. However, the contemporary understanding of PPV pathogenesis now extends far beyond this classic paradigm, encompassing an increasingly complex landscape of novel genotypes (PPV2 through PPV8), intricate co-infection dynamics, and previously unrecognized tissue tropisms that manifest in respiratory, enteric, and systemic disease [3, 5, 13, 15, 27]. This section provides an exhaustive, mechanistic dissection of the clinical manifestations of PPV infection, integrating molecular pathogenesis with field-level epidemiological observations to furnish a comprehensive clinical portrait of this ubiquitous swine pathogen.

The Classic Reproductive Syndrome: SMEDI and Its Molecular Underpinnings

The preeminent clinical consequence of PPV1 infection is reproductive failure in naïve or inadequately immunized breeding females, particularly primiparous gilts, which are most vulnerable due to their lack of prior exposure and the waning of maternally derived antibodies [60, 67]. The clinical expression of SMEDI is exquisitely dependent on the gestational stage at which infection occurs, a temporal determinant that dictates whether the outcome is embryonic resorption, fetal mummification, stillbirth, or the birth of live, but persistently infected, piglets. Infection prior to day 35 of gestation, when the fetus is immunologically immature and unable to mount a protective inflammatory response, typically results in embryonic death and resorption, often manifesting as irregular returns to estrus or a reduced litter size that may go clinically undetected [6, 21, 64]. As gestation progresses, between approximately days 35 and 70, the fetus acquires the capacity to mount an immune response but remains highly susceptible to the cytopathic effects of viral replication. Infection during this critical window leads to the hallmark lesions of fetal mummification, wherein the fetus dies and is subsequently dehydrated and desiccated within the sterile uterine environment, a process vividly demonstrable upon necropsy of affected litters [58, 59, 63]. After day 70, the fetus is immunocompetent and can survive infection, yet may be stillborn or born weak, often with detectable viremia and persistent viral shedding, thereby serving as a reservoir for horizontal transmission within the farrowing house [6, 54, 58, 65].

The molecular mechanism driving this gestational-stage-dependent pathology is rooted in the exquisite tropism of PPV for rapidly dividing cells, particularly those of the fetal trophoblast, endothelium, and myocardium. The nonstructural protein NS1, a multifunctional helicase and endonuclease, is the primary cytotoxic effector, orchestrating a cascade of cellular damage that culminates in placental insufficiency and fetal death. Specifically, NS1 triggers intrinsic, mitochondria-mediated apoptosis in porcine placental trophoblast cells (PTCs) via the induction of reactive oxygen species (ROS) accumulation, loss of mitochondrial membrane potential, and the subsequent activation of caspase-9 and caspase-3 [48, 49]. This NS1-driven apoptosis is profoundly potentiated by its ability to translocate to the nucleus via the importin α/β pathway and to shuttle back to the cytoplasm via CRM1-dependent nuclear export, activity essential for viral replication and the induction of cellular damage [45]. Furthermore, beyond canonical apoptosis, PPV infection of PTCs can induce necroptosis, a programmed, lytic form of cell death, through a Z-nucleic acid-binding protein 1 (ZBP1)-dependent pathway that activates RIPK3 and MLKL, bypassing caspase-8 [37]. This necroptotic pathway, triggered by the detection of viral DNA, causes plasma membrane rupture and the release of damage-associated molecular patterns (DAMPs), exacerbating local inflammation and placental damage. The virus further hijacks cellular machinery by inducing autophagy via the AMPK/Raptor/mTOR signaling cascade, a process that paradoxically promotes viral replication while simultaneously suppressing apoptotic cell death in the early stages of infection [41, 53]. This complex interplay of cell death pathways, apoptosis, necroptosis, and autophagy, creates a dynamic pathological environment within the placenta, ultimately disrupting the maternal-fetal interface and leading to the tissue damage characteristic of SMEDI [39, 52].

Expanding the Clinical Spectrum: The Role of Novel PPV Genotypes (PPV2–PPV8)

The discovery of seven additional porcine parvovirus genotypes (PPV2 through PPV8) since the early 2000s has fundamentally reshaped the understanding of PPV-associated disease, revealing a far more diverse and clinically nuanced pathogen complex than previously appreciated. While PPV1 remains the primary agent of SMEDI, mounting evidence implicates several of these novel viruses in a broader spectrum of clinical disorders, including porcine respiratory disease complex (PRDC), enteric disease, and systemic illness [5, 13, 15, 27, 42].

Among the novel genotypes, PPV2 has emerged as the most consistently associated with respiratory disease. Large-scale epidemiological investigations have demonstrated a high prevalence of PPV2 in lung tissues from pigs with PRDC, with detection rates ranging from 37.6% to 39% across diverse geographic regions, including Colombia, Korea, and the United States [5, 13, 42]. Importantly, in situ hybridization has localized PPV2 nucleic acids primarily within alveolar macrophages and, to a lesser extent, within other pulmonary immune cells, strongly suggesting a tropism for the respiratory immune system [42]. The presence of PPV2 within macrophages in areas of lymphohistiocytic interstitial pneumonia provides a plausible mechanistic link to PRDC: viral infection of these sentinel cells may dysregulate their phagocytic and cytokine-secreting functions, impairing pulmonary immunity and rendering the lung more susceptible to secondary bacterial or viral pathogens. Histopathological evaluation of PPV2-positive lungs from pigs with respiratory disease has consistently revealed variable degrees of histiocytic or lymphohistiocytic interstitial pneumonia, often accompanied by neutrophilic bronchopneumonia [5]. Notably, PPV2 has been detected as a sole infectious agent in a significant proportion of PRDC cases (approximately 16% in one study), where no other known respiratory viruses (PRRSV, PCV2, IAV) were identified by metagenomic sequencing, suggesting that PPV2 itself may be capable of inducing pulmonary pathology under certain conditions [42].

PPV3 also appears to play a role in the respiratory disease complex, frequently co-detected with other pathogens. In a systematic investigation of PPV1–7 in China, PPV2 and PPV3 were the most prevalent genotypes overall, each detected in 22.53% of samples, and their presence was significantly associated with nursery and finishing pigs, the age groups most clinically affected by PRDC [18]. The detection of PPV3 in clinically healthy pigs without reproductive failure in the Democratic Republic of Congo underscores its potential for subclinical circulation, but this does not preclude its role as a co-factor in disease exacerbation [29]. Similarly, PPV5 and PPV6 have been identified in association with respiratory disease and, intriguingly, PPV6 has been shown to induce typical apoptotic features in infected cells in vitro, including DNA fragmentation, chromatin margination, and nuclear condensation, paralleling the cytopathology of PPV1 in placental tissues [12, 13].

PPV7, first identified in 2016, has rapidly been detected across the globe, including in the USA, China, South Korea, Poland, Brazil, and Colombia, and is consistently found at high prevalence rates, often exceeding 25% in clinically diseased pig populations [20, 25, 27, 30, 40]. The clinical significance of PPV7 remains an area of active investigation, but compelling evidence links it to both reproductive and respiratory manifestations. In sows that experienced reproductive failure, PPV7 was detected at a significantly higher rate in PCV3-positive samples (51.2%) compared to PCV3-negative samples (14.4%), and the copy number of PCV3 was significantly elevated in PPV7-positive sera, suggesting that PPV7 may act as a co-factor that potentiates PCV3 replication and, consequently, reproductive pathology [55]. This synergistic interaction is further supported by studies showing that PPV7 co-infection with PCV2 or PCV3 is highly prevalent in lung tissue from pigs with respiratory disease [20, 55]. The PPV7 capsid protein (Cap) exhibits a high nucleotide substitution rate (2.19 × 10⁻³ substitutions/site/year), and its antigenic drift, characterized by insertions and deletions in the VP2-capsid protein, may facilitate immune evasion and the emergence of strains with altered tropism [25, 28].

The most recently described genotype, PPV8, was first identified in China in 2022 and has since been detected in Europe (Hungary and Slovakia) and the Americas (Colombia) [3, 9, 15]. Its clinical role is still being elucidated, but its detection in oral fluids (up to 100% positivity in some herds) and its co-occurrence with PCV2 and PRRSV in pigs with porcine respiratory disease strongly implicate it in the respiratory disease complex [3, 9]. The widespread presence of PPV8 across continents, with high genetic similarity to the original Chinese strain but evidence of local evolution, highlights the dynamic nature of the PPV landscape and the need for continuous surveillance [3, 15].

Co-infection Dynamics and the Synergistic Exacerbation of Disease

One of the most critical clinical realities of PPV infection is its near-universal occurrence within a complex milieu of co-infecting pathogens. The concept of syndemic disease, whereby two or more pathogens interact synergistically to produce a clinical outcome more severe than the sum of their individual effects, is exquisitely illustrated by PPV and its interactions with porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), and pseudorabies virus (PRV) [5, 32, 35, 61, 62, 66]. The clinical differentiation of these co-infections is notoriously challenging, as they share overlapping clinical features such as respiratory distress, reproductive failure, and wasting, necessitating advanced molecular diagnostics for accurate etiological attribution [35, 61, 62].

The interaction between PPV and PCV2 is perhaps the most extensively documented and clinically impactful synergy. PPV infection is recognized as a potent trigger for the development of postweaning multisystemic wasting syndrome (PMWS) and PCV2-associated reproductive disease. Mechanistically, PPV is thought to provide a “helper” function for PCV2 replication, possibly through the induction of cellular proliferation and the provision of replication factors that PCV2, a small, replication-deficient circovirus, requires [32, 33]. Epidemiological studies consistently demonstrate that PCV2-positive pigs have significantly higher detection rates of PPV1 and PPV6 compared to PCV2-negative animals, and that the viral load of PCV2 is often elevated in the presence of PPV co-infection [13, 55]. The clinical consequence is a more severe and prolonged course of disease, characterized by more pronounced wasting, respiratory distress, and a higher incidence of reproductive failure in breeding herds. In experimentally challenged pigs, dual infection with PCV2d and PPV2 resulted in a synergistic increase in pulmonary pathology and lymphoid depletion compared to single-agent infections [66]. The development of combined VLP vaccines targeting both PCV2 Cap and PPV VP2 has proven highly effective in mitigating this synergism, significantly reducing clinical signs and improving average daily weight gain in PMWS-affected herds [32].

The interaction between PPV and PRRSV is equally complex and clinically significant. PRRSV, an arterivirus that causes severe reproductive and respiratory disease, is frequently co-detected with PPV in cases of reproductive failure and PRDC. In a comprehensive study of PPV1–7 prevalence in Korea, the detection of PPV2 and PPV7 was significantly higher in PRRSV-infected lung samples, regardless of PCV2 status, suggesting a specific interaction between these parvoviruses and PRRSV [13]. This association is particularly pronounced in stillborn piglets and aborted fetuses, where triple infections (PPV2/PCV2/PRRSV) are common, accounting for up to 21% of cases [5]. The mechanism of synergy likely involves PRRSV-mediated immunosuppression, particularly its ability to dysregulate alveolar macrophage function and impair type I interferon responses, creating a permissive environment for enhanced parvovirus replication. Conversely, PPV-induced apoptosis and necroptosis within the placenta and lung may exacerbate the tissue damage initiated by PRRSV, leading to more severe reproductive and respiratory outcomes [37, 48]. The field-level impact is substantial: herds with high PRRSV prevalence often experience more severe and prolonged SMEDI outbreaks when PPV is also circulating, underscoring the need for comprehensive vaccination strategies that address both pathogens [67, 68].

The interaction with PRV (Aujeszky’s disease virus) further complicates the clinical picture. PRV, a herpesvirus, causes a range of neurological, respiratory, and reproductive signs that can closely mimic those of PPV infection. Co-infection with these two agents has been documented in wild boar and domestic swine populations, and the differentiation of the etiology of reproductive failure in such cases requires molecular confirmation [2, 35, 61]. The development of multiplex real-time PCR assays capable of simultaneously detecting PRV, PPV, and PCV3 has been a critical advancement, enabling the rapid identification of co-infections and facilitating targeted intervention strategies [35].

Subclinical Infection and the Carrier State: Implications for Herd Health

A critical aspect of PPV epidemiology that profoundly influences clinical management is the existence of a subclinical carrier state. The virus can persist in apparently healthy pigs, particularly in growing-finishing animals, without causing overt clinical signs, yet these animals serve as a continuous source of viral shedding within the herd [13, 27, 58]. Studies utilizing highly sensitive molecular detection methods have consistently identified PPV DNA in serum, feces, and oral fluids from clinically normal pigs, with detection rates in fattening pigs often exceeding those in younger or older age groups [13, 27]. For example, in Korea, the highest detection rates of PPV1–7 were found in the 10–20-week age group, with prevalence rates for PPV2 reaching 36.5% in this cohort [13]. Similarly, in Poland, PPVs were detected in oral fluids from apparently healthy finisher pigs at rates ranging from 10.7% for PPV1 to 48.7% for PPV2 [27]. This chronic, low-level circulation is particularly insidious because it can lead to the gradual seroconversion of the breeding herd without triggering clinical suspicion, only to result in a sudden outbreak of SMEDI when a naïve cohort of gilts is introduced or when herd immunity wanes [58, 60].

The subclinical carrier state is also evident in the detection of PPV in fetal tissues from sows without any history of reproductive failure. In a seminal study from Argentina, PPV DNA was detected in mummies and stillborn piglets from a commercial farm that maintained a rigorous vaccination program and reported no overt reproductive problems [58]. Furthermore, in another Argentine study, PPV1 was detected in 17 of 131 mummies and stillbirths from normal deliveries, with the NADL-2-like strain identified, suggesting that even vaccines based on this strain do not provide sterilizing immunity [58, 65]. These findings have profound clinical implications: they indicate that current vaccination protocols, while highly effective at reducing the incidence and severity of SMEDI, do not fully prevent transplacental infection. The virus can cross the placenta and infect a subset of fetuses, causing death, while the remainder of the litter appears normal. This “silent” infection can result in a subtle reduction in the number of live-born piglets and an increase in the percentage of mummified fetuses that may go unnoticed unless a thorough post-mortem examination of all stillbirths and placentas is conducted [58, 63].

The detection of PPV7 in serum, feces, and saliva of apparently healthy pigs further illustrates the subclinical potential of the novel genotypes [20]. In a study of South Chinese pig farms, PPV7 was detected in blood, stool, and saliva at high rates, but was absent from breast milk, suggesting that lactogenic transmission

Advances in Diagnostic Methods for Porcine Parvovirus Detection

The detection of porcine parvovirus (PPV) has undergone a profound transformation over the past five years, driven by the recognition of eight distinct genotypes (PPV1–PPV8), the high prevalence of co-infections with other swine pathogens, and the urgent need for rapid, field-deployable diagnostic tools that can operate in resource-limited settings. The traditional diagnostic paradigm, reliant on virus isolation, hemagglutination inhibition (HI) assays, and conventional endpoint polymerase chain reaction (PCR), has given way to a sophisticated arsenal of molecular, serological, and spectroscopic platforms that offer unprecedented sensitivity, specificity, and multiplexing capacity. This section provides an exhaustive analysis of these diagnostic advances, dissecting the underlying mechanisms, comparative performance characteristics, and epidemiological applications of each methodological category.

Multiplex Real-Time PCR Platforms: The New Gold Standard

The simultaneous detection of multiple pathogens from a single clinical specimen has become a cornerstone of modern swine diagnostics, particularly given the complex co-infection dynamics that characterize porcine respiratory disease complex (PRDC) and reproductive failure syndromes. The development of SYBR Green I-based multiplex real-time PCR assays represents a significant breakthrough, leveraging differential melting temperature (Tm) analysis to discriminate amplicons without the need for multiple fluorescent probes. Cao et al. [35] established a triplex assay capable of simultaneously detecting pseudorabies virus (PRV), PPV, and porcine circovirus type 3 (PCV3) by exploiting distinct melting peaks at 90°C, 84°C, and 80°C, respectively. This assay demonstrated exceptional analytical sensitivity, with detection limits of 3.67 copies/μL for PPV and high linearity (R² ≥ 0.995), and outperformed conventional PCR in clinical sample testing across nine regions of Guangdong Province, China [35]. The elegance of this approach lies in its simplicity, SYBR Green chemistry is cost-effective and requires no probe synthesis, while maintaining the quantitative capabilities essential for viral load monitoring.

A more advanced iteration of this technology is the TaqMan probe-based quadruplex real-time PCR developed by Quan et al. [61], targeting Torque teno sus virus 1 (TTSuV1), PCV2, PRV, and PPV simultaneously. By designing four pairs of primers and specific probes against conserved regions (the Rep gene of PCV2, the gE gene of PRV, and the VP2 gene of PPV), the assay achieved a detection limit of 10 copies/μL for each target with no cross-reactivity against a panel of eight other swine viruses, including African swine fever virus (ASFV), classical swine fever virus (CSFV), and porcine reproductive and respiratory syndrome virus (PRRSV) [61]. The inter-assay and intra-assay coefficients of variation ranged from 0.33% to 1.43%, demonstrating exceptional reproducibility. When applied to 150 clinical samples, this method revealed positive rates of 11.33% for PPV, highlighting the value of multiplex approaches for comprehensive surveillance in herds where co-infections are the norm rather than the exception [61].

The need to discriminate among all known PPV genotypes has driven the development of more expansive multiplex PCR panels. Kim et al. [72] designed a seven-plex conventional PCR assay targeting PPV1 through PPV7, utilizing seven sets of genotype-specific primers. This assay exhibited a detection limit of 3 × 10³ viral copies and demonstrated high specificity when testing various combinations of viruses in field samples, including serum, lung, lymph node, and fecal specimens [72]. The diagnostic utility of this approach was further validated by Li et al. [18], who developed a panel of individual PCR assays for PPV1–7 and applied it to 435 clinical samples collected from eight Chinese provinces between 2016 and 2020. Their systematic investigation revealed that 55.40% of samples were positive for at least one PPV genotype, with PPV2 and PPV3 being the most prevalent (both 22.53%), and co-infections involving two to five genotypes detected in 27.36% of samples [18]. Importantly, co-infection with PCV2 was observed in 22.30% of samples, underscoring the synergistic interactions between parvoviruses and circoviruses that complicate clinical diagnosis and disease management.

For smaller genotyping panels, Rajkhowa et al. [62] developed a one-step triplex PCR assay for the simultaneous detection of PCV2, PPV, and CSFV, three pathogens responsible for reproductive failure with overlapping clinical presentations. This assay amplified targeted fragments of 478 bp (PCV2), 275 bp (PPV), and 172 bp (CSFV) with a sensitivity of 300 pg of mixed viral genomic nucleic acid and no cross-amplification with PRRSV, Japanese encephalitis virus (JEV), or porcine group A rotavirus [62]. The inclusion of CSFV in this panel is particularly relevant for regions where classical swine fever remains endemic, allowing for rapid discrimination between viral causes of reproductive disorders that require fundamentally different control strategies.

Isothermal Amplification and CRISPR-Based Detection: Point-of-Care Revolution

The most transformative advances in PPV diagnostics have emerged from the convergence of isothermal nucleic acid amplification technologies with CRISPR-Cas effector systems. These platforms eliminate the need for thermal cycling equipment, dramatically reduce time to result, and can be coupled with visual readout modalities such as lateral flow dipsticks (LFDs), enabling deployment in field settings, abattoirs, and small-scale farms with minimal laboratory infrastructure.

The recombinase polymerase amplification (RPA)-CRISPR/Cas12a assay developed by Wen et al. [69] for PPV7 exemplifies this paradigm. By designing five CRISPR RNAs (crRNAs) targeting a highly conserved region within the NS1 gene, the authors optimized crRNA-05 at 200 nM and achieved a detection limit of 100 copies/μL, with confirmation via both fluorescence and lateral flow detection [69]. The specificity was absolute, only PPV7 DNA samples returned positive results, and the clinical validation on 50 lung tissue samples from diseased pigs demonstrated a positivity rate of 58% (29/50) compared to 44% (22/50) by conventional PCR, representing a 14% increase in diagnostic sensitivity [69]. This enhanced detection capability is particularly valuable for PPV7, which often circulates at low viral loads and may be missed by less sensitive methods.

A related approach using enzymatic recombinase amplification (ERA) instead of RPA was reported by Wei et al. [70], who developed an ERA-CRISPR/Cas12a system targeting the VP2 gene of PPV. The isothermal reaction operates at 37°C, and the detection limit was determined to be 3.75 × 10² copies/μL with no cross-reactivity against other porcine viruses [70]. The integration of lateral flow dipstick readout makes this system particularly attractive for resource-limited settings, as the results are visible to the naked eye without specialized equipment. Similarly, He et al. [71] coupled recombinase-aided amplification (RAA) with lateral flow dipstick for PPV detection, achieving a detection limit of 10² copies/μL of recombinant plasmid, 6.38 × 10⁻⁷ ng/μL PPV DNA, and 10⁻¹ TCID₅₀/mL of virus, with complete specificity against other swine pathogens [71]. The RAA reaction proceeds at 37°C within 15 minutes, making it one of the fastest nucleic acid amplification methods available for PPV.

The biological basis for the enhanced sensitivity of CRISPR-based diagnostics compared to conventional PCR lies in the collateral cleavage activity of Cas12a. Upon specific recognition of the target DNA by the crRNA-guide complex, Cas12a undergoes a conformational change that activates its non-specific single-stranded DNase activity, cleaving reporter molecules (typically fluorophore-quencher pairs) and generating an amplified signal that is proportional to the initial target concentration [69, 70]. This signal amplification cascade, combined with the pre-amplification step (RPA or ERA), enables the detection of targets present at ultra-low concentrations while maintaining exquisite sequence specificity.

Serological and Nanobody-Based Immunoassays

While molecular detection methods provide definitive evidence of active infection, serological assays remain indispensable for monitoring vaccine-induced immunity, determining herd-level exposure status, and conducting large-scale epidemiological surveys. The development of PPV virus-like particle (VLP)-based enzyme-linked immunosorbent assays (ELISAs) represents a significant improvement over traditional whole-virus antigen preparations, offering enhanced specificity, reproducibility, and biosafety.

Gao et al. [19] demonstrated the utility of recombinant PPV VP2 proteins expressed in E. coli that self-assemble into VLPs, which were purified to 95% purity using multi-step chromatography. The VLP-based indirect ELISA (I-ELISA) was validated against 487 clinical pig serum samples and showed excellent concordance with a commercial PPV kit, while offering the advantages of tag-free VLPs and cost-effective production in a prokaryotic system [19]. The self-assembly of VP2 into VLPs preserves conformational epitopes that are critical for antibody recognition, thereby improving diagnostic accuracy compared to assays using denatured or partially purified antigens.

A revolutionary advance in serological diagnostics has been the development of nanobody-based reagents for PPV detection. Lu et al. [74] generated five VP2-specific nanobodies (single-domain antibodies derived from camelid heavy-chain antibodies) by immunizing Bactrian camels and subsequently engineered nanobody-horseradish peroxidase (HRP) and nanobody-enhanced green fluorescent protein (EGFP) fusions. Using PPV-VP2-Nb19 as the capture antibody and PPV-VP2-Nb56-HRP fusion as the detection antibody in a sandwich ELISA format, the assay achieved 92.1% agreement with real-time PCR for detecting PPV in clinical samples [74]. Additionally, a direct fluorescent assay using PPV-VP2-Nb12-EGFP fusion as a probe was developed for detecting PPV in infected ST cells, showing 81.5% agreement with real-time PCR [74]. The advantages of nanobodies over conventional monoclonal antibodies include their small size (approximately 15 kDa), high stability, ease of recombinant production, and the ability to engineer fusion proteins that eliminate the need for secondary antibodies, substantially reducing assay time and cost.

The limitations of current serological tools for assessing vaccine efficacy were highlighted by Renzhammer et al. [60], who examined 450 serum samples from twelve farms using both ELISA and HI assays. Alarmingly, 65% of gilts vaccinated twice with commercial vaccines were seronegative by HI assay, and while 98% of twelve-week-old pigs had detectable HI titers (1:10–1:640), only 30% were positive by ELISA [60]. This discordance between serological methods underscores the need for standardized, validated assays that can reliably distinguish between vaccine-induced immunity and natural infection, particularly given the documented emergence of vaccine-escape variants such as the 27a-like strains [6, 14, 23].

RAMAN Spectroscopy: A Label-Free Diagnostic Frontier

An innovative and fundamentally different approach to PPV detection has emerged from the application of RAMAN spectroscopy, a vibrational spectroscopic technique that provides a molecular fingerprint of biological samples without the need for labels, probes, or prior target amplification. Gogone et al. [75] investigated the use of RAMAN spectroscopy with a 633 nm laser to discriminate between swine testis (ST) cells infected with PPV, PCV2, and uninfected controls. Principal component analysis coupled with linear discriminant analysis (PCA-LDA) achieved sensitivity rates of 95.55% for PCV2-infected cells and 97.77% for PPV-infected cells, with a Leave-One-Out validation algorithm demonstrating 99.97% accuracy [75].

The biological basis for this discrimination lies in the distinct biochemical changes induced by viral infection, including alterations in protein secondary structure, lipid composition, and nucleic acid content. Extensive band assignment revealed that specific spectral regions corresponding to proteins (amide I at 1650–1680 cm⁻¹, amide III at 1230–1300 cm⁻¹), lipids (CH₂ bending at 1440–1460 cm⁻¹), and DNA/RNA (phosphate backbone vibrations at 1080–1100 cm⁻¹) were most important for distinguishing infected from uninfected cells [75]. While RAMAN spectroscopy currently requires fixed cells and sophisticated instrumentation, its potential for rapid, non-destructive, high-throughput screening, particularly when integrated with microfluidic platforms and machine learning algorithms, positions it as a promising future diagnostic modality for swine viral diseases.

Diagnostic Considerations for Emerging Genotypes and Epidemiological Applications

The discovery of novel PPV genotypes (PPV2–PPV8) has necessitated the development and validation of detection methods capable of identifying these emerging viruses, many of which are associated with subclinical infections or complex disease presentations. The first detection of PPV8 in Europe, reported by Igriczi et al. [3], was achieved through the development of a real-time quantitative PCR method targeting the viral genome. Analysis of 2,230 serum samples, 233 oral fluid samples, and 115 processing fluid samples from Hungarian and Slovakian pig farms revealed PPV8 in 65% of Hungarian farms and both Slovakian farms, with oral fluids showing the highest positivity rates (up to 100% in some herds) [3]. This study demonstrated that oral fluids are a superior diagnostic matrix for detecting PPV8, likely due to high viral shedding in oropharyngeal secretions.

Similarly, the first detection of PPV8 on the American continent was reported by Vargas-Bermúdez and Jaime [9] in Colombian pigs with porcine respiratory disease, using conventional PCR targeting the NS1 gene. Phylogenetic analysis confirmed that Colombian isolates belonged to the genus Protoparvovirus and were highly similar to the Chinese prototype strain [9]. The detection of PPV8 in co-infections with PCV2 and PRRSV highlights the importance of comprehensive diagnostic panels that include this newly identified genotype.

For PPV7, which has been detected in numerous countries since its identification in 2016, SYBR Green I real-time PCR assays have been developed that demonstrate 1,000-fold higher sensitivity than conventional PCR, with detection limits as low as 35.6 copies [73]. These assays have been instrumental in revealing PPV7 prevalence rates of 25.73% in Fujian Province, China [40], 28.72% in eastern Inner Mongolia [22], and 27.3% in Guangxi, China [30]. The high genetic diversity of PPV7, including the identification of eight subtypes (PPV7a–PPV7h) [40] and evidence of recombination events [20, 40], underscores the need for diagnostic methods that can adapt to ongoing viral evolution.

The detection of PPV in non-traditional sample matrices has expanded the epidemiological utility of diagnostic testing. Lecocq et al. [4] demonstrated that PPV genomic DNA could be detected by PCR in black soldier fly larvae and mealworm larvae up to 9 days post-ingestion, with infectious virus detectable in mealworm homogenates for at least 3 days by immunoperoxidase staining and up to 9 days by real-time PCR. These findings have implications for feed safety and biosecurity, as insect larvae are increasingly used as protein sources in animal feed and could potentially serve as mechanical vectors for PPV

Current Vaccine Strategies and Their Limitations in Cross-Protection

The control of porcine parvovirus (PPV)-induced reproductive failure has historically relied upon prophylactic vaccination, a strategy that has been widely implemented in breeding herds globally for decades. However, the evolving landscape of PPV genomics, the emergence of novel genotypes (PPV2–PPV8), and the documented antigenic drift within PPV1 strains have collectively exposed significant vulnerabilities in the cross-protective efficacy of existing vaccine platforms. A critical, evidence-based appraisal of current vaccine strategies, encompassing conventional inactivated whole-virus vaccines, novel subunit and virus-like particle (VLP) vaccines, and vectored approaches, is essential to understand the mechanisms underpinning these immunological gaps and to chart a path toward next-generation interventions [1, 21].

Conventional Inactivated and Modified-Live Vaccine Platforms

The cornerstone of PPV prophylaxis for decades has been the use of inactivated whole-virus vaccines, typically based on classical PPV1 strains such as NADL-2 or related isolates. These vaccines are formulated with oil or aqueous adjuvants and are administered to gilts and sows prior to breeding to induce neutralizing antibodies against the viral capsid, primarily the VP2 protein [11, 21]. While these vaccines have been instrumental in reducing the incidence of SMEDI syndrome (stillbirth, mummification, embryonic death, infertility), their limitations in cross-protection have become increasingly apparent.

Antigenic Drift and Strain Specificity: The emergence of the 27a-like strains in Europe around the turn of the millennium, followed by their global dissemination, has been a watershed event in PPV vaccinology. Phylogenetic analyses have demonstrated that these strains belong to distinct clusters (e.g., PPV1b or G2) that are genetically and antigenically divergent from the vaccine prototype NADL-2 [14, 16]. Critically, studies using antisera raised against commercial vaccines (Porcilis® Parvo, Eryseng® Parvo, ReproCyc® ParvoFLEX) have revealed significantly reduced neutralizing activity against 27a-like field strains compared to homologous vaccine strains, with the magnitude of this reduction varying by vaccine product [14, 23]. This phenomenon is driven by specific amino acid substitutions in the VP2 capsid protein. For instance, residues around position 228 of VP2 have been identified as part of a 27a-specific neutralizing epitope; mutations at this site (e.g., Q228E) can decrease antibody binding and neutralization [23]. Furthermore, systematic reverse genetics studies have demonstrated that certain substitutions found in field isolates (e.g., at positions 215, 228, 314) not only alter antigenicity but also increase viral fitness in cell culture, suggesting that these strains are under dual selective pressure from both immune evasion and replicative advantage [23, 31]. The recent characterization of Chinese isolates, such as the JX strain, which shows reduced neutralizing activity against commercial vaccine sera in vitro and retains pathogenicity in pregnant sows, underscores the ongoing nature of this antigenic drift [6].

Incomplete Protection Against Fetal Infection: A critical limitation of many inactivated vaccines is their inability to consistently prevent transplacental infection, even when they reduce clinical disease. Several field and experimental studies have documented the detection of PPV1 DNA in mummified or stillborn fetuses from sows that were rigorously vaccinated [58, 65]. For example, a study in Argentina detected PPV1 (closely related to NADL-2) in 17 out of 131 fetuses from normal deliveries on a farm using a commercial vaccine, demonstrating that the vaccine reduced but did not eliminate fetal infection [58]. Similarly, an experimental evaluation of three commercial vaccines revealed that while all prevented fetal mummification after a heterologous challenge, only the ReproCyc® ParvoFLEX subunit vaccine successfully prevented viremia in the gilts themselves, indicating that inactivated whole-virus vaccines may be less effective at blocking systemic viral spread [56]. This incomplete protection highlights a fundamental gap: current vaccines may primarily limit the pathological consequences of infection (e.g., fetal death) rather than sterilizing immunity, thereby allowing low-level viral replication and shedding that perpetuates transmission cycles within herds [56, 57].

Duration of Immunity and Maternally Derived Antibody Interference: The immunological window of protection afforded by conventional vaccines is finite. Studies on the duration of immunity have shown that while some subunit vaccines can protect for at least six months post-vaccination [82], the decay of maternally derived antibodies (MDA) in piglets is exponential and highly variable. A recent investigation into MDA dynamics on twelve farms found that 98% of 12-week-old pigs had detectable hemagglutination inhibition (HI) antibodies, but only 30% were seropositive by ELISA, indicating a decline to sub-protective levels by weaning age [60]. This MDA interference can also compromise the priming of active immunity in young animals if vaccination is administered too early, creating a window of susceptibility. Furthermore, a striking finding was that 65% of gilts vaccinated twice with certain commercial vaccines were seronegative by HI assay, suggesting that routine serological monitoring may fail to confirm successful immunization [60]. This poor concordance between serological tools and true protective status poses a practical challenge for herd-level management.

Novel Subunit and Virus-Like Particle (VLP) Vaccines

In response to the limitations of traditional inactivated vaccines, significant efforts have been directed toward the development of next-generation vaccines based on the VP2 protein, which is the primary immunogen capable of inducing neutralizing antibodies and self-assembling into VLPs [11, 19, 33]. These platforms offer several advantages, including the absence of infectious virus, defined antigenic content, and the potential for improved safety profiles.

VP2 Subunit Vaccines: The baculovirus-based VP2 subunit vaccine ReproCyc® ParvoFLEX has been extensively evaluated and demonstrates superior immunogenicity compared to some conventional products. It induces earlier seroconversion, effectively prevents viremia after heterologous challenge, and provides robust fetal protection [56, 82]. Its safety profile across all reproductive stages has been experimentally validated [83]. However, even this advanced platform has limitations. While it protects against PPV1 strains, it is not designed to cover the novel parvoviruses (PPV2–PPV8) that are highly prevalent in swine populations and are increasingly implicated in reproductive and respiratory disease complexes [1, 13, 18]. Furthermore, the immunogenicity of the VP2 antigen is heavily dependent on the choice of adjuvant. Studies comparing adjuvants for a VP2 subunit vaccine found that ISA 201VG (a water-in-oil emulsion) induced significantly higher HI and neutralizing antibody titers than IMS 1313VG, and provided complete protection against the HN2019 strain, whereas the commercial inactivated vaccine provided only incomplete protection [11]. This highlights that even a superior antigen requires optimal delivery systems to maximize cross-protective potential.

VLP-Based Vaccines: VLP vaccines, produced by expressing the VP2 protein in E. coli, Kluyveromyces marxianus, or baculovirus systems, mimic the native viral capsid structure and are highly immunostimulatory [7, 19, 26, 33]. E. coli-derived PPV VLPs, when formulated with a water-in-oil-in-water adjuvant, elicited high HI and neutralizing antibody titers in guinea pigs and pigs, and conferred complete fetal protection in primiparous gilts [33]. The K. marxianus platform achieved an unprecedented yield of 2.5 g/L of VLPs, with high purity and immunogenicity in mice, suggesting a scalable and cost-effective alternative for large-scale production [26]. Chimeric VLP strategies have also been explored, where the PPV VP2 scaffold is used to present heterologous epitopes from other pathogens, such as foot-and-mouth disease virus (FMDV) SAT2. These chimeric VLPs successfully induced specific antibodies against both PPV and FMDV, demonstrating the versatility of the VP2 platform as a bivalent vaccine vector [7]. Despite these advances, VLPs based on a single PPV1 VP2 sequence face the same fundamental constraint: they cannot be expected to provide robust cross-protection against the antigenically diverse Tetraparvovirus and Chapparvovirus species (PPV2–PPV8) that are now circulating widely [1, 18]. Moreover, the immunodominance of VP2 epitopes may actually divert the immune response away from conserved epitopes across different PPV species, potentially limiting heterologous protection.

Vectored and Combination Vaccine Approaches

To address the issue of co-infections and to broaden protection, several vectored and multi-pathogen combination vaccines have been developed.

Recombinant Pseudorabies Virus (PRV) Vectors: A promising approach involves engineering the PRV genome to express the PPV VP2 protein alongside immunomodulatory cytokines such as porcine interleukin-6 (IL-6). The recombinant virus rPRV-VP2-IL6 induced PPV-specific antibodies and lymphocyte proliferation in mice and provided partial protection against virulent PPV challenge, while simultaneously protecting against PRV [79]. This bivalent or trivalent strategy addresses the common clinical scenario of co-infection, but the vector itself (PRV) carries inherent risks, including residual virulence and the potential for reversion to pathogenicity, which limits its use in naïve herds.

Combination VLP Vaccines for PCV2 and PPV: Given the frequent co-detection of PPV with porcine circovirus type 2 (PCV2) and their synergistic role in postweaning multisystemic wasting syndrome (PMWS), combined PCV2 Cap and PPV VP2 VLP vaccines have been developed using E. coli expression systems. These combined VLPs induced stronger humoral and cellular immune responses than either monovalent vaccine alone and significantly improved growth performance in PMWS-affected piglets [32]. This demonstrates that combining VLPs from different pathogens can be safe and effective, but again, the cross-protective coverage remains limited to PPV1.

Adjuvant-Driven Enhancements: The immune response to PPV vaccines can be profoundly influenced by the adjuvant. Chitosan derivatives like N-2-HACC have been shown to enhance the HI antibody response and provide complete protection against homologous challenge, but they predominantly stimulate humoral immunity with minimal cellular immune activation [77, 80]. In contrast, Acanthopanax senticosus polysaccharide-loaded calcium carbonate nanoparticles (CaCO₃–ASPS–PEI) not only enhanced PPV-specific IgG and cytokine production but also promoted a balanced Th1/Th2 response, potentially offering broader immunological coverage [78]. Propolis flavonoid adjuvants have also demonstrated the ability to enhance both humoral and cellular immunity, including increased interferon-γ and interleukin-2/4 levels, which could be critical for controlling intracellular viral replication [76]. These studies underscore that the choice of adjuvant is not merely a technical detail but a central determinant of the qualitative and quantitative nature of the immune response, and thus a key variable in achieving cross-protection.

Fundamental Limitations: The Challenge of Novel Parvoviruses

The most profound limitation of current vaccine strategies is their exclusive focus on PPV1. Since 2000, at least seven novel parvovirus species (PPV2–PPV8) have been identified globally, and epidemiological surveys reveal their widespread prevalence [1, 12, 13, 15, 18, 20, 27, 28, 81]. In China, for example, co-infections with two to five different PPV genotypes were detected in 27.36% of clinical samples [18]. In Korea, PPV2 was the most prevalent (up to 32.6% in lung samples), and its detection was significantly associated with PRRSV-positive samples, suggesting a role in the porcine respiratory disease complex (PRDC) [13]. The recent detection of PPV8 in Europe and the Americas indicates that these viruses are not geographically restricted and are actively spreading [3, 9, 15]. The cap proteins of these novel PPVs are highly divergent from PPV1 VP2, with sequence identities often falling below 50% [15, 28]. Consequently, vaccine-induced antibodies against PPV1 VP2 are unlikely to neutralize these heterologous viruses. Furthermore, evidence suggests that PPV7 may stimulate PCV2 and PCV3 replication, and PPV2 has been localized within alveolar macrophages in PRDC-affected lungs, indicating that these viruses are not mere bystanders but active contributors to disease pathogenesis [42, 55]. The lack of cross-protection against this expanding family of parvoviruses represents a critical gap that cannot be addressed by simply refining existing PPV1-centric vaccine platforms.

Selective Pressure and Evolution: There is mounting evidence that widespread vaccination may itself be driving the antigenic evolution of PPV1. The emergence of 27a-like strains with increased replicative fitness and reduced susceptibility to vaccine-induced antibodies mirrors the evolutionary trajectory of canine parvovirus, where the original virus was completely replaced by new antigenic types [23, 31]. The observation that the PPV1 genome substitution rate (approximately 4.71 × 10⁻⁵ substitutions/site/year) approaches that of RNA viruses, and that VP2 genes are under positive selection at specific codons, suggests that vaccine escape variants will continue to emerge [14]. A phylogeographic analysis of PPV1 in Africa highlighted multiple importation events from Europe and Asia, with 27a-like strains predominating, emphasizing the international dissemination of these fitter variants and the inadequacy of local vaccine strains to control them [10]. This dynamic necessitates a proactive, rather than reactive, approach to vaccine strain selection, possibly incorporating antigens from circulating field strains as has been done for influenza and SARS-CoV-2.

Insufficient Control of Viral Shedding: Even the most efficacious vaccines currently available have not consistently been shown to eliminate viral shedding. While ReproCyc® ParvoFLEX prevented viremia and transplacental infection, other commercial vaccines significantly reduced but did not eliminate the presence of PPV in fetuses [56, 57]. The detection of PPV1 in oral fluids, feces, and serum of growing pigs on farms with high vaccination coverage indicates that vaccine-induced immunity is leaky, allowing viral circulation within the herd [27, 60]. This persistent circulation is particularly problematic in settings of high population density and continuous farrowing, where it provides a reservoir for the emergence of new antigenic variants. The failure to block mucosal transmission may also explain the continued detection of PPV in wild boar populations, which can act as a source of re-introduction to domestic herds [2, 17].

In summary, while current vaccine strategies, from conventional inactivated vaccines to sophisticated subunit and VLP platforms, have been instrumental in managing PPV1-associated disease, they are fundamentally constrained by antigenic drift within PPV1, a lack of coverage against novel and emerging PPV genotypes (PPV2–PPV8), and an inability to consistently induce sterilizing immunity or block viral shedding. Addressing these limitations will require a paradigm shift toward multi-genotype vaccines that incorporate conserved antigens or immunogens derived from the full spectrum of pathogenic parvoviruses, combined with advanced adjuvants capable of driving both broad humoral and T-cell-mediated immunity.

Future Directions in Vaccine Design and Disease Control

The landscape of porcine parvovirus (PPV) vaccinology and disease management is undergoing a fundamental transformation, driven by the convergence of several critical discoveries: the identification of eight distinct PPV genotypes (PPV1–PPV8) with global distribution [1, 3, 9, 13, 15, 18], the emergence of vaccine-escape variants such as the 27a-like strains [14, 23, 31], and an increasingly sophisticated understanding of viral pathogenesis at the molecular level [37-39, 41, 43, 44, 46-50, 52, 53]. The path forward demands a departure from conventional empirical vaccine development toward a rationally designed, multi-pronged strategy that integrates structural biology, systems immunology, advanced adjuvant technology, and real-time molecular epidemiology. The World Organisation for Animal Health (WOAH) and the Food and Agriculture Organization (FAO) recognize PPV as a significant constraint to global swine productivity, and the future control paradigm must address the virus's extraordinary genetic plasticity and its capacity for synergistic co-infection with pathogens such as porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), and the emerging PCV3 [5, 13, 35, 55, 61, 62].

Next-Generation Antigen Design: Beyond Classical Whole-Virus Vaccines

The limitations of current inactivated and modified-live vaccines are now well-documented. Vaccination with commercial PPV1-based products has demonstrably failed to prevent transplacental infection in some settings, as evidenced by the detection of PPV1 in mummified fetuses and stillborn piglets from vaccinated sows in Argentina and other regions [58, 65]. Furthermore, antisera raised against classical vaccine strains such as NADL-2 exhibit reduced neutralizing activity against the currently predominant 27a-like strains, which have acquired amino acid substitutions in the VP2 capsid protein that enhance viral fitness and partially abrogate antibody binding [14, 23]. This antigenic drift, reminiscent of the evolution of canine parvovirus, underscores the urgent need for vaccines that reflect contemporary circulating strains.

The future of PPV vaccinology lies squarely in the realm of virus-like particle (VLP) technology and rationally engineered subunit vaccines. The VP2 protein of PPV1 possesses the intrinsic capacity to self-assemble into immunogenic VLPs that mimic the native virion structure without containing infectious genetic material [11, 19, 26, 32, 33]. Critically, the baculovirus expression system has been leveraged to produce VP2-based subunit vaccines, such as ReproCyc® ParvoFLEX, which have demonstrated superior efficacy compared to traditional inactivated products, conferring complete fetal protection against heterologous PPV1 challenge and preventing viremia in gilts [56, 82, 83]. These findings provide a clear proof-of-concept that molecularly defined antigens can outperform complex whole-virus preparations. The next frontier involves expanding this VLP platform to encompass the antigenic diversity of the novel PPVs. For PPV7, immunoinformatic analyses have identified conserved linear B-cell epitopes and CD8 T-cell epitopes within the Cap protein that offer promising targets for a broadly protective multi-epitope vaccine [28]. Similarly, the VP2 capsid protein of PPV7 exhibits sequence insertions and deletions that may alter antigenic surface topology, necessitating careful epitope selection to ensure coverage across the eight PPV7 subtypes (PPV7a–PPV7h) now recognized in China [25, 30, 40].

Perhaps the most innovative direction is the development of chimeric VLPs that serve as bivalent or multivalent vaccine platforms. The PPV VP2 protein tolerates the insertion of exogenous epitopes within its surface-exposed loops, as demonstrated by the successful generation of chimeric PPV-SAT2-VLPs expressing T- and B-cell epitopes from foot-and-mouth disease virus (FMDV) serotype SAT2 [7]. This approach opens the door to creating combination vaccines that simultaneously target PPV, PCV2, and other endemic swine pathogens. The construction of recombinant pseudorabies virus (PRV) expressing PPV VP2 and porcine IL-6 (rPRV-VP2-IL6) represents another vector-based strategy that has induced robust PPV-specific humoral and cellular immunity while conferring protection against PRV challenge in mice [79]. The challenge moving forward will be to optimize the antigenic payload, ensure proper VLP assembly with foreign epitopes, and rigorously evaluate these chimeric constructs in the target species, pregnant gilts, against heterologous challenge with contemporary field strains.

Adjuvant Innovation: Immunomodulation and Targeted Delivery

The efficacy of next-generation PPV vaccines will be inextricably linked to advances in adjuvant technology. Current commercial vaccines rely on oil emulsions or aluminum-based adjuvants that primarily drive humoral immunity but often fail to elicit robust cellular responses, particularly cytotoxic T lymphocyte (CTL) activity necessary for clearing intracellular virus from placental tissues. The emerging paradigm emphasizes the use of immunostimulatory molecules that engage specific innate immune pathways to shape the adaptive response. For instance, the polysaccharide adjuvant N-2-hydroxypropyl trimethyl ammonium chloride chitosan (N-2-HACC) has been shown to significantly enhance hemagglutination inhibition (HI) antibody titers and provide 100% protective efficacy against PPV challenge in sows, although it did not stimulate T-cell proliferation or type-1 cytokine secretion [77, 80]. This suggests that for PPV, robust humoral immunity alone may be sufficient for fetal protection, but the exploration of adjuvants that also trigger Th1-dominated responses may be critical for controlling co-infections with PCV2 and PRRSV.

Nature-derived compounds offer a rich source of novel adjuvants. Propolis flavonoid extracts, and particularly the isolated compound ferulic acid, have demonstrated both direct antiviral activity against PPV, inhibiting viral replication by suppressing NS1-induced apoptosis and the TLR4/NF-κB inflammasome axis, and potent immunoenhancing effects when used as vaccine adjuvants [49, 50, 76]. The ability of propolis flavonoids to upregulate IgM, IgG subclasses, IL-2, IL-4, and IFN-γ suggests they can bridge humoral and cellular immunity [76]. Future vaccine formulations may incorporate such multifunctional adjuvants to simultaneously provide antiviral protection and immunological enhancement.

Nanoparticle-based delivery systems represent another quantum leap in adjuvant design. Calcium carbonate nanoparticles loaded with Acanthopanax senticosus polysaccharide (ASPS) and coated with polyethylenimine (CaCO₃-ASPS-PEI) have been shown to activate macrophages, upregulate co-stimulatory molecules (CD86, MHCII), and induce a potent Th1/Th2 hybrid immune response when complexed with PPV antigen [78]. The electrostatic attraction between positively charged nanoparticles and negatively charged cell membranes facilitates antigen uptake by dendritic cells, enhancing cross-presentation and T-cell priming. Future research should focus on optimizing nanoparticle size, surface charge, and antigen-loading capacity for PPV vaccines, as well as evaluating their safety and efficacy in pregnant sows, a population with unique immunological considerations.

Harnessing Molecular Pathogenesis for Therapeutic and Vaccination Strategies

A deep understanding of PPV-host interactions at the molecular level is revealing novel targets for antiviral intervention and vaccine enhancement. PPV NS1 protein is a multifunctional hub that orchestrates viral DNA replication, induces nucleocytoplasmic shuttling via CRM1- and importin α/β-dependent pathways, and triggers host cell apoptosis through mitochondria-mediated, ROS-dependent mechanisms [45, 48]. The discovery that chaperonin CCT5 binds NS1 and facilitates its interaction with COPε to suppress type I interferon signaling [43] provides a druggable target: small molecules that disrupt the NS1-CCT5 interaction could attenuate viral replication. Similarly, the identification of SYNCRIP as a critical factor for alternative splicing of NS1 mRNA to generate NS2 mRNA, a process essential for PPV DNA replication, opens another avenue for antiviral targeting [46].

The role of programmed cell death pathways in PPV pathogenesis is particularly relevant to vaccine design and disease control. PPV infection of porcine placental trophoblast cells (PTCs) induces necroptosis via a ZBP1/RIPK3/MLKL signaling axis that is triggered by viral DNA [37]. This inflammatory form of cell death likely contributes to placental damage and reproductive failure. Concurrently, PPV activates autophagy through the AMPK/Raptor/mTOR pathway to promote viral replication, while also inducing ER stress and PERK-mediated apoptosis as a host restriction mechanism [41, 52, 53]. Understanding how to modulate these pathways, for example, using pharmacological inhibitors of autophagy (3-MA, ATG5 knockdown) that reduce PPV replication [53], could yield adjunct therapies to complement vaccination in outbreak settings. Furthermore, the discovery that the host protein PCBP1 inhibits PPV replication [38] and that CD38 activates the TLR9/IFN-α and NLRP3/CASP1 pathways upon infection [44] provides endogenous immunological levers that could be potentiated by strategic vaccination or immunomodulators.

Advanced Diagnostic Platforms for Targeted Disease Control

The ability to rapidly and accurately detect PPV infections, including discrimination among eight genotypes and co-infections with other swine pathogens, is a prerequisite for rational disease control. The future of PPV diagnostics is shifting toward multiplex, point-of-care, and isothermal amplification technologies that bypass the need for expensive thermocyclers and skilled personnel. The development of SYBR Green-based multiplex real-time PCR assays capable of simultaneously detecting PPV, PCV3, and PRV with melting curve discrimination [35], as well as quadruplex TaqMan-based methods for TTSuV1, PCV2, PRV, and PPV [61], represents the current gold standard for high-throughput laboratory surveillance. However, these methods require sophisticated instrumentation and are ill-suited for on-farm use.

The CRISPR/Cas revolution has already yielded field-deployable diagnostic tools for PPV. The combination of recombinase polymerase amplification (RPA) with CRISPR/Cas12a has achieved a detection limit of 100 copies/μL for PPV7, with results readable by fluorescence or lateral flow dipstick within 30 minutes at 37°C [69]. A similar ERA-CRISPR/Cas12a system targeting the VP2 gene of PPV1 demonstrated a detection limit of 3.75 × 10² copies/μL with no cross-reactivity against other porcine viruses [70]. The recombinase-aided amplification (RAA) coupled with lateral flow dipstick (LFD) has pushed sensitivity even further, detecting 10² copies/μL of recombinant plasmid and 10⁻¹ TCID₅₀/mL of virus within 15 minutes at a constant 37°C [71]. These platforms are inherently portable, cost-effective, and compatible with crude sample lysates, making them ideal for surveillance in resource-limited settings and for rapid outbreak response. Future iterations should incorporate multiplexing capabilities to simultaneously differentiate PPV1–PPV8 and key co-pathogens such as PCV2, PCV3, and PRRSV.

Nanobody-based immunoassays offer another transformative diagnostic modality. Recombinant nanobodies derived from camelid heavy-chain antibodies can be engineered as fusion proteins with horseradish peroxidase (Nb-HRP) or enhanced green fluorescent protein (Nb-EGFP) to create direct detection probes that eliminate the need for secondary antibodies [74]. A sandwich ELISA using PPV VP2-specific nanobodies achieved 92.1% agreement with real-time PCR for PPV detection in clinical samples, while a direct fluorescent assay using Nb12-EGFP enabled visualization of PPV in infected cells [74]. The small size, high stability, and modular nature of nanobodies make them ideal building blocks for multiplexed biosensors and lateral flow devices. Even Raman spectroscopy has shown promise as a label-free diagnostic tool, achieving 99.97% accuracy in discriminating PPV-infected from uninfected cells based on molecular fingerprints of proteins, lipids, and nucleic acids [75]; while currently a research tool, miniaturization of Raman systems could eventually enable real-time, non-invasive detection.

Interrupting Transmission: Biosecurity and Integrated Control

Future disease control must extend beyond vaccination to encompass comprehensive biosecurity and transmission interruption strategies. The extraordinary environmental stability of PPV, its resistance to heat, low pH, and chemical disinfectants, poses unique challenges for farm hygiene. The finding that PPV can remain infectious within mealworm (Tenebrio molitor) larvae for up to nine days post-ingestion [4] highlights the potential for insect vectors to perpetuate viral circulation in feed systems. As the swine industry increasingly explores insect-based protein sources for feed, strategies to inactivate PPV in these substrates will be essential. Non-thermal plasma (NTP) treatment has demonstrated a minimum four-log reduction of PPV infectivity after 120 minutes of oxygen plasma exposure [24], offering a promising sterilization method for feed ingredients and medical devices.

The role of wild boar populations as reservoirs for PPV transmission is increasingly recognized. Serological surveys in Serbia have detected PPV in 56% of wild boars, while studies in Namibia documented 31% positivity in warthogs, often in co-infection with African swine fever virus [2, 17]. These findings underscore the need for coordinated surveillance at the wildlife-domestic animal interface, particularly in regions with extensive free-range pig production. Vaccination of wild boar populations using oral baits containing thermostable PPV VLPs or vectored vaccines (e.g., recombinant PRV) could be explored as a strategy to reduce environmental viral load, though logistical and regulatory hurdles remain substantial.

The Road Ahead: Systems Immunology, Multi-Omics, and Precision Vaccinology

The ultimate goal of PPV research is to transition from reactive disease management to predictive control. This will require the integration of multi-omics data, genomics, transcriptomics, proteomics, and metabolomics, to define the molecular signatures of protective immunity against PPV. Proteomic profiling of PK-15 cells infected with PPV has already revealed differential expression of proteins in the toll-like receptor, TNF, and viral carcinogenesis pathways [38], while transcriptomic analyses at 24 and 48 hours post-infection have identified over 1,700 differentially expressed genes involved in multiple cell death modalities [39]. These datasets will inform the rational design of vaccines that elicit the precise combination of neutralizing antibodies, CTL responses, and trained immunity required to prevent transplacental infection.

Furthermore, the application of reverse genetics systems, such as the DNA-launched infectious clone carrying double genetic markers (His-tag and Flag-tag) developed for PPV [84], will enable high-throughput screening of antiviral compounds and the systematic dissection of virulence determinants. This platform can be used to engineer rationally attenuated vaccine strains with precisely defined deletions in the NS1 NES or NLS motifs, which have been shown to be essential for viral replication [45]. Such designer vaccines could combine the safety of inactivated products with the immunogenicity of live vaccines.

In conclusion, the future of PPV vaccine design and disease control lies in the convergence of structural vaccinology, advanced adjuvants, molecular pathogenesis, and field-deployable diagnostics. The era of one-size-fits-all vaccination is over; the path forward demands genotype-specific or multi-genotype VLP-based vaccines, rationally adjuvanted to drive protective immunity, deployed within a framework of real-time molecular surveillance enabled by CRISPR-based point-of-care testing. Global organizations including WOAH and FAO should prioritize the harmonization of PPV surveillance protocols and the establishment of a centralized sequence database to track the emergence of antigenic variants, much as the WHO does for influenza. Only through such an integrated, science-driven approach can the swine industry hope to stay ahead of this remarkably adaptable pathogen.

References

[1] Liu Y, Chen Y, Shang Y, Deng X, Hao H. Porcine Parvovirus in China: Recent Advances, Epidemiology, and Vaccine Strategies. Viruses. 2025. DOI: https://doi.org/10.3390/v17091262

[2] Jezdimirovic N, Savic B, Milovanović B, Glišić D, Ninković M, Kureljušić J, et al.. Molecular Detection of Porcine Cytomegalovirus, Porcine Parvovirus, Aujeszky Disease Virus and Porcine Reproductive and Respiratory Syndrome Virus in Wild Boars Hunted in Serbia during 2023. Veterinary Sciences. 2024. DOI: https://doi.org/10.3390/vetsci11060249

[3] Igriczi B, Dénes L, Schönhardt K, Balka G. First Report of Porcine Parvovirus 8 in Europe: Widespread Detection and Genetic Characterization on Commercial Pig Farms in Hungary and Slovakia. Animals. 2024. DOI: https://doi.org/10.3390/ani14131974

[4] Lecocq A, Alencar ALF, Lazov CM, Rajiuddin S, Bøtner A, Belsham G. Use of a Novel Feeding System to Assess the Survival of a Very Stable Mammalian Virus, Porcine Parvovirus, Within Black Soldier Fly (Hermetia illucens) Larvae: A Comparison with Mealworm (Tenebrio molitor) Larvae. Pathogens. 2024. DOI: https://doi.org/10.3390/pathogens13121038

[5] Vargas-Bermúdez DS, Mainenti M, Naranjo-Ortíz MF, Mogollón J, Piñeyro P, Jaime J. First Report of Porcine Parvovirus 2 (PPV2) in Pigs from Colombia Associated with Porcine Reproductive Failure (PRF) and Porcine Respiratory Disease Complex (PRDC). Transboundary and Emerging Diseases. 2024. DOI: https://doi.org/10.1155/2024/1471536

[6] Deng H, Wang M, Cong G, Fu F, Feng L. Genetic characteristics and pathogenicity of variant porcine parvovirus 1 in northern China.. Veterinary Microbiology. 2024. DOI: https://doi.org/10.1016/j.vetmic.2024.110274

[7] Li Q, Ma X, Shen Y, Dai J, Nian X, Shang X, et al.. Chimeric Porcine Parvovirus VP2 Virus-like Particles with Epitopes of South African Serotype 2 Foot-and-Mouth Disease Virus Elicits Specific Humoral and Cellular Responses in Mice. Viruses. 2024. DOI: https://doi.org/10.3390/v16040621

[8] Xu M, Zhou Z, Xiong R, Zhang L, Yu C, Liu Q. CpG islands: Features and distribution in the genomes of porcine parvovirus.. Polish journal of veterinary sciences. 2024. DOI: https://doi.org/10.24425/pjvs.2024.151734

[9] Vargas-Bermúdez D, Jaime J. The first report of porcine parvovirus 8 (PPV8) on the American continent is associated with pigs in Colombia with porcine respiratory disease. Archives of Virology. 2024. DOI: https://doi.org/10.1007/s00705-024-06099-z

[10] Franzo G, Zerbo H, Ouoba B, Dji-Tombo AD, Kindo MG, Sawadogo R, et al.. A Phylogeographic Analysis of Porcine Parvovirus 1 in Africa. Viruses. 2023. DOI: https://doi.org/10.3390/v15010207

[11] Ling Z, Zhang H, Chen Y, Sun L, Zhao J. A Subunit Vaccine Based on the VP2 Protein of Porcine Parvovirus 1 Induces a Strong Protective Effect in Pregnant Gilts. Vaccines. 2023. DOI: https://doi.org/10.3390/vaccines11111692

[12] Komina A, Anoyatbekova A, Krasnikov N, Yuzhakov A. Identification and in vitro characterization of a novel porcine parvovirus 6 in Russia. Veterinary research communications. 2023. DOI: https://doi.org/10.1007/s11259-023-10226-7

[13] Kim S, Kim J, Kim J, Park G, Jeong C, Kim W. Prevalence of porcine parvovirus 1 through 7 (PPV1-PPV7) and co-factor association with PCV2 and PRRSV in Korea. BMC Veterinary Research. 2022. DOI: https://doi.org/10.1186/s12917-022-03236-1

[14] Vereecke N, Kvisgaard LK, Baele G, Boone C, Kunze M, Larsen L, et al.. Molecular epidemiology of Porcine Parvovirus Type 1 (PPV1) and the reactivity of vaccine-induced antisera against historical and current PPV1 strains. Virus Evolution. 2022. DOI: https://doi.org/10.1093/ve/veac053

[15] Guo Y, Yan G, Chen S, Han H, Li J, Zhang H, et al.. Identification and genomic characterization of a novel porcine parvovirus in China. Frontiers in Veterinary Science. 2022. DOI: https://doi.org/10.3389/fvets.2022.1009103

[16] Deng H, Cong G, Wang H, Hu Z, Shi D, Shi H, et al.. Isolation, characterization, and phylogenetic analysis of two new porcine parvovirus 1 isolates from Northern China. Virus Research. 2023. DOI: https://doi.org/10.1016/j.virusres.2023.199247

[17] Molini U, Franzo G, Settypalli TB, Hemberger M, Khaiseb S, Cattoli G, et al.. Viral Co-Infections of Warthogs in Namibia with African Swine Fever Virus and Porcine Parvovirus 1. Animals. 2022. DOI: https://doi.org/10.3390/ani12131697

[18] Li J, Xiao Y, Qiu M, Li X, Li S, Lin H, et al.. A Systematic Investigation Unveils High Coinfection Status of Porcine Parvovirus Types 1 through 7 in China from 2016 to 2020. Microbiology spectrum. 2021. DOI: https://doi.org/10.1128/Spectrum.01294-21

[19] Gao Y, Wang H, Wang S, Sun M, Fang Z, Liu X, et al.. Self-Assembly of Porcine Parvovirus Virus-like Particles and Their Application in Serological Assay. Viruses. 2022. DOI: https://doi.org/10.3390/v14081828

[20] Zhang X, Zheng C, Lv Z, Xue S, Chen Y, Liu Y, et al.. Genetic and epidemic characteristics of porcine parvovirus 7 in the Fujian and Guangdong regions of southern China. Frontiers in Veterinary Science. 2022. DOI: https://doi.org/10.3389/fvets.2022.949764

[21] Streck AF, Truyen U. Porcine Parvovirus.. Current Issues in Molecular Biology. 2019. DOI: https://doi.org/10.21775/9781910190913.06

[22] Wen S, Song Y, Lv X, Meng X, Liu K, Yang J, et al.. Detection and Molecular Characterization of Porcine Parvovirus 7 in Eastern Inner Mongolia Autonomous Region, China. Frontiers in Veterinary Science. 2022. DOI: https://doi.org/10.3389/fvets.2022.930123

[23] Streck AF, Canal C, Truyen U. Viral Fitness and Antigenic Determinants of Porcine Parvovirus at the Amino Acid Level of the Capsid Protein. Journal of Virology. 2021. DOI: https://doi.org/10.1128/JVI.01198-21

[24] Bras FL, Carré G, Aguemon Y, Colin M, Gellé M. Inactivation of Enveloped Bovine Viral Diarrhea Virus and Non-Enveloped Porcine Parvovirus Using Low-Pressure Non-Thermal Plasma. Life. 2021. DOI: https://doi.org/10.3390/life11121292

[25] Vargas-Bermúdez D, Rendon-Marin S, Ruíz-Sáenz J, Mogollón D, Jaime J. The first report of porcine parvovirus 7 (PPV7) in Colombia demonstrates the presence of variants associated with modifications at the level of the VP2-capsid protein. PLoS ONE. 2021. DOI: https://doi.org/10.1371/journal.pone.0258311

[26] Yang D, Chen L, Duan J, Yu Y, Zhou J, Lu H. Investigation of Kluyveromyces marxianus as a novel host for large‐scale production of porcine parvovirus virus‐like particles. Microbial Cell Factories. 2020. DOI: https://doi.org/10.1186/s12934-021-01514-5

[27] Miłek D, Woźniak A, Guzowska M, Stadejek T. Detection Patterns of Porcine Parvovirus (PPV) and Novel Porcine Parvoviruses 2 through 6 (PPV2–PPV6) in Polish Swine Farms. Viruses. 2019. DOI: https://doi.org/10.3390/v11050474

[28] Wang D, Mai J, Yang Y, Wang N. Porcine Parvovirus 7: Evolutionary Dynamics and Identification of Epitopes toward Vaccine Design. Vaccines. 2020. DOI: https://doi.org/10.3390/vaccines8030359

[29] Bisimwa PN, Wasso DS, Bantuzeko F, Aksanti CB, Tonui R, Birindwa AB, et al.. First investigation on the presence of porcine parvovirus type 3 in domestic pig farms without reproductive failure in the Democratic Republic of Congo. Veterinary and Animal Science. 2021. DOI: https://doi.org/10.1016/j.vas.2021.100187

[30] Wang W, Cao L, Sun W, Xin J, Zheng M, Tian M, et al.. Sequence and phylogenetic analysis of novel porcine parvovirus 7 isolates from pigs in Guangxi, China. PLoS ONE. 2019. DOI: https://doi.org/10.1371/journal.pone.0219560

[31] Mészáros I, Olasz F, Cságola A, Tijssen P, Zádori Z. Biology of Porcine Parvovirus (Ungulate parvovirus 1). Viruses. 2017. DOI: https://doi.org/10.3390/v9120393

[32] Liu G, Qiao X, Chang C, Hua T, Wang J, Tang B, et al.. Reduction of Postweaning Multisystemic Wasting Syndrome-Associated Clinical Symptoms by Virus-Like Particle Vaccine Against Porcine Parvovirus and Porcine Circovirus Type 2. Viral immunology. 2020. DOI: https://doi.org/10.1089/vim.2019.0201

[33] Hua T, Zhang D, Tang B, Chang C, Liu G, Zhang X. The immunogenicity of the virus-like particles derived from the VP2 protein of porcine parvovirus.. Veterinary Microbiology. 2020. DOI: https://doi.org/10.1016/j.vetmic.2020.108795

[34] Sun P, Bai C, Zhang D, Wang J, Yang K, Cheng B, et al.. SYBR Green-based real-time polymerase chain reaction assay for detection of porcine parvovirus 6 in pigs.. Polish journal of veterinary sciences. 2020. DOI: https://doi.org/10.24425/pjvs.2020.132766

[35] Cao L, Lv W, Li A, Yang L, Zhou F, Wen F, et al.. A SYBR green I-based multiplex real-time PCR for simultaneous detection of pseudorabies virus, porcine circovirus 3 and porcine parvovirus. BMC Veterinary Research. 2025. DOI: https://doi.org/10.1186/s12917-024-04440-x

[36] Anoyatbekova A, Komina A, Vlasova NN, Kononova E, Gulyukin A, Krasnikov N, et al.. Molecular Detection of Porcine Parvovirus 5 in Domestic Pigs in Russia and Propagation of Field Isolates in Primary Porcine Testicular Cells. Veterinary Sciences. 2025. DOI: https://doi.org/10.3390/vetsci12060535

[37] Xu N, Du Q, Cheng Y, Nie L, Ma P, Feng D, et al.. Porcine parvovirus infection induces necroptosis of porcine placental trophoblast cells via a ZBP1-mediated pathway. Veterinary Research. 2024. DOI: https://doi.org/10.1186/s13567-024-01410-x

[38] Wang L, Song Y, Xu M, Zhang C, Zhang L, Xia L, et al.. Proteomics analysis of PK-15 cells infected with porcine parvovirus and the effect of PCBP1 on PPV replication. Microbiology spectrum. 2024. DOI: https://doi.org/10.1128/spectrum.03914-23

[39] Lu T, Song X, Zhao L, Ma X. Analysis of differentially expressed genes related to cell death in porcine kidney-15 cells at 24 and 48 hours post porcine parvovirus infection.. American Journal of Veterinary Research. 2024. DOI: https://doi.org/10.2460/ajvr.24.06.0164

[40] Lyu Z, Zhang X, Xue S, Yang X, Liu J, Fan K, et al.. Detection and genetic evolution analysis of porcine parvovirus type 7 (PPV7) in Fujian Province.. Infection, Genetics and Evolution. 2023. DOI: https://doi.org/10.1016/j.meegid.2023.105515

[41] Zhang X, Ma P, Shao T, Xiong Y, Du Q, Chen S, et al.. Porcine parvovirus triggers autophagy through the AMPK/Raptor/mTOR pathway to promote viral replication in porcine placental trophoblasts. Veterinary Research. 2022. DOI: https://doi.org/10.1186/s13567-022-01048-7

[42] Nelsen A, Lin C, Hause B. Porcine Parvovirus 2 Is Predominantly Associated With Macrophages in Porcine Respiratory Disease Complex. Frontiers in Veterinary Science. 2021. DOI: https://doi.org/10.3389/fvets.2021.726884

[43] Du Q, Zhang X, Xu N, Ma M, Miao B, Huang Y, et al.. Chaperonin CCT5 binding with porcine parvovirus NS1 promotes the interaction of NS1 and COPƐ to facilitate viral replication.. Veterinary Microbiology. 2022. DOI: https://doi.org/10.1016/j.vetmic.2022.109574

[44] Zheng Y, Xu Y, Xu W, Cao S, Yan Q, Huang X, et al.. CD38 Enhances TLR9 Expression and Activates NLRP3 Inflammasome after Porcine Parvovirus Infection. Viruses. 2022. DOI: https://doi.org/10.3390/v14061136

[45] Cao L, Fu F, Chen J, Shi H, Zhang X, Liu J, et al.. Nucleocytoplasmic Shuttling of Porcine Parvovirus NS1 Protein Mediated by the CRM1 Nuclear Export Pathway and the Importin α/β Nuclear Import Pathway. Journal of Virology. 2021. DOI: https://doi.org/10.1128/JVI.01481-21

[46] Chen S, Miao B, Chen N, Chen C, Shao T, Zhang X, et al.. SYNCRIP facilitates porcine parvovirus viral DNA replication through the alternative splicing of NS1 mRNA to promote NS2 mRNA formation. Veterinary Research. 2021. DOI: https://doi.org/10.1186/s13567-021-00938-6

[47] Jin X, Yuan Y, Zhang C, Zhou Y, Song Y, Wei Z, et al.. Porcine parvovirus nonstructural protein NS1 activates NF-κB and it involves TLR2 signaling pathway. Journal of Veterinary Sciences. 2020. DOI: https://doi.org/10.4142/jvs.2020.21.e50

[48] Zhang J, Fan J, Li Y, Liang S, Huo S, Wang X, et al.. Porcine Parvovirus Infection Causes Pig Placenta Tissue Damage Involving Nonstructural Protein 1 (NS1)-Induced Intrinsic ROS/Mitochondria-Mediated Apoptosis. Viruses. 2019. DOI: https://doi.org/10.3390/v11040389

[49] Ma X, Guo Z, Zhang Z, Li X, Liu Y, Zhao L, et al.. Ferulic Acid Protects against Porcine Parvovirus Infection-Induced Apoptosis by Suppressing the Nuclear Factor-κB Inflammasome Axis and Toll-Like Receptor 4 via Nonstructural Protein 1. Evidence-Based Complementary and Alternative Medicine. 2020. DOI: https://doi.org/10.1155/2020/3943672

[50] Ma X, Guo Z, Zhang Z, Li X, Wang X, Liu Y, et al.. Ferulic acid isolated from propolis inhibits porcine parvovirus replication potentially through Bid-mediate apoptosis.. International Immunopharmacology. 2020. DOI: https://doi.org/10.1016/j.intimp.2020.106379

[51] Deng S, Han Z, Zhu M, Fan S, Zhang J, Huang Y, et al.. Isolation and phylogenetic analysis of a new Porcine parvovirus strain GD2013 in China.. Journal of Virological Methods. 2020. DOI: https://doi.org/10.1016/j.jviromet.2019.113748

[52] Cao L, Xue M, Chen J, Shi H, Zhang X, Shi D, et al.. Porcine parvovirus replication is suppressed by activation of the PERK signaling pathway and endoplasmic reticulum stress-mediated apoptosis. Virology. 2019. DOI: https://doi.org/10.1016/j.virol.2019.09.012

[53] Zhang X, Xiong Y, Zhang J, Shao T, Chen S, Miao B, et al.. Autophagy Promotes Porcine Parvovirus Replication and Induces Non-Apoptotic Cell Death in Porcine Placental Trophoblasts. Viruses. 2019. DOI: https://doi.org/10.3390/v12010015

[54] Parthiban S, Sowndhraya RKV, Raja P, Parthiban M, Ramesh A, Raj GD, et al.. Molecular detection of porcine parvovirus 1–associated reproductive failure in southern India. Tropical Animal Health and Production. 2022. DOI: https://doi.org/10.1007/s11250-022-03194-8

[55] Mai J, Wang D, Zou Y, Zhang S, Meng C, Wang A, et al.. High Co-infection Status of Novel Porcine Parvovirus 7 With Porcine Circovirus 3 in Sows That Experienced Reproductive Failure. Frontiers in Veterinary Science. 2021. DOI: https://doi.org/10.3389/fvets.2021.695553

[56] Noguera M, Vela A, Kraft C, Chevalier M, Goutebroze S, Paz Xd, et al.. Effects of three commercial vaccines against porcine parvovirus 1 in pregnant gilts.. Vaccine. 2021. DOI: https://doi.org/10.1016/j.vaccine.2021.05.042

[57] Born Evd, Elzen Pvd, Kilsdonk Ev, Hoeijmakers M, Segers R. An octavalent vaccine provides pregnant gilts protection against a highly virulent porcine parvovirus strain. BMC Veterinary Research. 2020. DOI: https://doi.org/10.1186/s12917-020-2272-3

[58] Serena M, Serena M, Cappuccio J, Cappuccio J, Metz GE, Metz GE, et al.. Detection and molecular characterization of porcine parvovirus in fetal tissues from sows without reproductive failure in Argentina. Heliyon. 2019. DOI: https://doi.org/10.1016/j.heliyon.2019.e02874

[59] Trinh TTH, Do VT, Do V, Vu-Khac H. Isolation and characterization of porcine parvovirus in Vietnam. Veterinary World. 2024. DOI: https://doi.org/10.14202/vetworld.2024.1530-1537

[60] Renzhammer R, Truyen U, Buchebner B, Baumgartner G, Kobialka R, Wahed AEE, et al.. Duration of maternally derived antibodies of porcine parvovirus in growing pigs and presence of antibodies in gilts and sows vaccinated with three different parvovirus vaccines. Porcine Health Management. 2024. DOI: https://doi.org/10.1186/s40813-024-00361-1

[61] Quan F, Geng Y, Wu Y, Jiang F, Li X, Yu C. Development and application of a quadruplex real-time PCR method for Torque teno sus virus 1, Porcine circovirus type 2, pseudorabies virus, and porcine parvovirus. Frontiers in Cellular and Infection Microbiology. 2024. DOI: https://doi.org/10.3389/fcimb.2024.1461448

[62] Rajkhowa S, Choudhury M, Pegu S, Sarma D, GUPTA VM. Development of a novel one‐step triplex PCR assay for the simultaneous detection of porcine circovirus type 2, porcine parvovirus and classical swine fever virus in a single tube. Letters in Applied Microbiology. 2022. DOI: https://doi.org/10.1111/lam.13732

[63] – L, Hemalatha S, Nagarajan K, Raj GD, Thangavelu A, Balasubramanyam D. Molecular Detection of Porcine Parvovirus in Swine with Reproductive Failure. Indian Journal of Animal Research. 2022. DOI: https://doi.org/10.18805/ijar.b-4861

[64] . porcine parvovirus. CABI Compendium. 2022. DOI: https://doi.org/10.1079/cabicompendium.68597

[65] Serena M, Dibárbora M, Olivera V, Metz GE, Aspitia CG, Pereda A, et al.. Evidence of porcine circovirus type 2 and co-infection with ungulate protoparvovirus 1 (porcine parvovirus) in mummies and stillborn piglets in subclinically infected farm.. Infection, Genetics and Evolution. 2021. DOI: https://doi.org/10.1016/j.meegid.2021.104735

[66] Opriessnig T, Karuppannan AK, Halbur P, Calvert J, Nitzel G, Matzinger SR, et al.. Porcine circovirus type 2a or 2b based experimental vaccines provide protection against PCV2d/porcine parvovirus 2 co-challenge.. Vaccine. 2020. DOI: https://doi.org/10.1016/j.vaccine.2020.01.013

[67] Sánchez-Matamoros A, Camprodón A, Maldonado J, Pedrazuela R, Miranda J. Safety and long-lasting immunity of the combined administration of a modified-live virus vaccine against porcine reproductive and respiratory syndrome virus 1 and an inactivated vaccine against porcine parvovirus and Erysipelothrix rhusiopathiae in breeding pigs. Porcine Health Management. 2019. DOI: https://doi.org/10.1186/s40813-019-0118-9

[68] Garcia-Morante B, Noguera M, Kraft C, Bridger P. Field evaluation of the safety and compatibility of a combined vaccine against porcine parvovirus 1 and porcine reproductive and respiratory syndrome virus in breeding animals. Porcine Health Management. 2019. DOI: https://doi.org/10.1186/s40813-019-0138-5

[69] Wen S, She L, Dang S, Liao A, Li X, Zhang S, et al.. Development of a RPA-CRISPR/Cas12a based rapid visual detection assay for Porcine Parvovirus 7. Frontiers in Veterinary Science. 2024. DOI: https://doi.org/10.3389/fvets.2024.1440769

[70] Wei J, Li Y, Cao Y, Liu Q, Yang K, Song X, et al.. Rapid and Visual Detection of Porcine Parvovirus Using an ERA-CRISPR/Cas12a System Combined With Lateral Flow Dipstick Assay. Frontiers in Cellular and Infection Microbiology. 2022. DOI: https://doi.org/10.3389/fcimb.2022.879887

[71] He Y, Chen W, Fan J, Fan S, Ding H, Chen J, et al.. Recombinase-Aided Amplification Coupled with Lateral Flow Dipstick for Efficient and Accurate Detection of Porcine Parvovirus. Life. 2021. DOI: https://doi.org/10.3390/life11080762

[72] Kim S, Jeong C, Nazki S, Lee S, Baek Y, Jung Y, et al.. Evaluation of a multiplex PCR method for the detection of porcine parvovirus types 1 through 7 using various field samples. PLoS ONE. 2021. DOI: https://doi.org/10.1371/journal.pone.0245699

[73] Li Y, Yu Z, Bai C, Zhang D, Sun P, Peng M, et al.. Development of a SYBR Green I real-time PCR assay for detection of novel porcine parvovirus 7.. Polish journal of veterinary sciences. 2021. DOI: https://doi.org/10.24425/pjvs.2021.136791

[74] Lu Q, Li X, Zhao J, Zhu J, Luo Y, Duan H, et al.. Nanobody‑horseradish peroxidase and -EGFP fusions as reagents to detect porcine parvovirus in the immunoassays. Journal of Nanobiotechnology. 2020. DOI: https://doi.org/10.1186/s12951-019-0568-x

[75] Gogone ICVP, Ferreira GH, Gava D, Schaefer R, Paula-Lopes FFd, Rocha RdA, et al.. Applicability of Raman spectroscopy on porcine parvovirus and porcine circovirus type 2 detection.. Spectrochimica Acta Part A - Molecular and Biomolecular Spectroscopy. 2020. DOI: https://doi.org/10.1016/j.saa.2020.119336

[76] Ma X, Guo Z, Li Y, Yang K, Li X, Liu Y, et al.. Phytochemical Constituents of Propolis Flavonoid, Immunological Enhancement, and Anti-porcine Parvovirus Activities Isolated From Propolis. Frontiers in Veterinary Science. 2022. DOI: https://doi.org/10.3389/fvets.2022.857183

[77] Zhao K, Gao Y, Hu G, Wang L, Cui S, Jin Z. N-2-Hydroxypropyl Trimethyl Ammonium Chloride Chitosan as Adjuvant Enhances the Immunogenicity of a VP2 Subunit Vaccine against Porcine Parvovirus Infection in Sows. Vaccines. 2021. DOI: https://doi.org/10.3390/vaccines9091027

[78] Li X, Zhang Z, Guo Z, Ma X, Ban X, Xinghui S, et al.. Acanthopanax senticosus polysaccharide-loaded calcium carbonate nanoparticle as an adjuvant to enhance porcine parvovirus vaccine immune responses. . 2021. DOI: https://doi.org/10.1016/J.MEDIDD.2021.100094

[79] Zheng H, Wang L, Fu P, Zheng L, Chen H, Liu F. Characterization of a recombinant pseudorabies virus expressing porcine parvovirus VP2 protein and porcine IL-6. Virology Journal. 2020. DOI: https://doi.org/10.1186/s12985-020-1292-8

[80] Mo Z, Wanying Q, Sun Y, Liang L, Jin Z, Cui S, et al.. Water-soluble N-2-Hydroxypropyl trimethyl ammonium chloride chitosan enhanced the immunogenicity of inactivated porcine parvovirus vaccine vaccination on sows against porcine parvovirus infection.. Immunology Letters. 2020. DOI: https://doi.org/10.1016/j.imlet.2020.04.014

[81] Wang Y, Yang K, Wang J, Wang X, Zhao L, Sun P, et al.. Detection and molecular characterization of novel porcine parvovirus 7 in Anhui province from Central-Eastern China.. Infection, Genetics and Evolution. 2019. DOI: https://doi.org/10.1016/j.meegid.2019.03.004

[82] Garcia-Morante B, Noguera M, Klocke SE, Sommer K, Bridger P. Duration of immunity against heterologous porcine parvovirus 1 challenge in gilts immunized with a novel subunit vaccine based on the viral protein 2. BMC Veterinary Research. 2020. DOI: https://doi.org/10.1186/s12917-020-02394-4

[83] Garcia-Morante B, Noguera M, Klocke SE, Sommer K, Kaiser T, Haist V, et al.. A novel subunit vaccine based on the viral protein 2 of porcine parvovirus: safety profile in bred pigs at different stages of the reproduction cycle and in offspring. Heliyon. 2019. DOI: https://doi.org/10.1016/j.heliyon.2019.e02593

[84] Chen S, Miao B, Chen N, Zhang X, Zhang X, Du Q, et al.. A novel porcine parvovirus DNA-launched infectious clone carrying stable double labels as an effective genetic platform.. Veterinary Microbiology. 2020. DOI: https://doi.org/10.1016/j.vetmic.2019.108502