Advances in porcine epidemic diarrhea virus research: genome, epidemiology, vaccines, and detection methods

Abstract

Porcine epidemic diarrhea (PED) is a highly contagious intestinal disease caused by the porcine epidemic diarrhea virus (PEDV). The economic impact of PEDV on the global pig industry has been significant, resulting in considerable losses. This paper presents a review of the latest research progress on PEDV genome, molecular epidemiology, vaccine development, and molecular detection methods. It was determined that the genetic diversity of the PEDV spike (S) gene was closely associated with the epidemiological trend of PEDV. The prevalence of S gene variants of different genotypes exhibited variability across regions and pig populations. Epidemiological analyses have demonstrated that PEDV can be transmitted via multiple routes, including direct contact, airborne aerosol, and water source contamination. With regard to vaccine research, the available vaccines can be classified into several categories, including live-attenuated vaccines, inactivated vaccines, subunit vaccines, bacterial vector vaccines, viral vector vaccines, mRNA vaccines, etc. Each of these has distinctive characteristics in terms of immunogenicity, protection efficiency, and safety. Molecular detection methods, including PCR-based methods, isothermal amplification techniques, immunological assays, and biosensors, play an important role in the diagnosis and monitoring of PEDV. Furthermore, this paper examines the current developments in PEDV research and identifies the key areas of future investigation. The objective of this paper is to establish a theoretical foundation for the prevention and control strategies of PED, and to provide a point of reference for further research on the genomics, epidemiology, vaccine development and detection methods of PEDV.

Introduction

Porcine epidemic diarrhea virus (PEDV) is a pathogen belonging to the genus Alphacoronavirus in the family Coronaviridae of the order Nidovirales. It represents a significant economic threat to the global pig industry [1, 2]. Since its initial identification in the United Kingdom and Belgium in 1971, PEDV has garnered considerable attention, particularly in the 2013 outbreak in the United States, which resulted in substantial economic losses [3–5]. PEDV is capable of infecting pigs of all ages, but is particularly lethal to neonatal piglets, with a mortality rate of up to 95%. The disease is primarily characterized by acute watery diarrhea, vomiting, and dehydration [6, 7].

PEDV infection primarily affects the villous intestinal epithelium of the small intestine, resulting in atrophy of the intestinal villi, disruption of the tight junctions of intestinal cells, reduction in the amount of mucin, and a decrease in the activity of digestive enzymes. These effects contribute to malabsorption and dyspepsia [6, 8]. Furthermore, PEDV has the potential to infect alveolar macrophages, resulting in lung lesions [9]. The clinical symptoms are analogous to those observed in cases of porcine transmissible gastroenteritis (TGE), although the transmission of PEDV is relatively slower and the mortality rate is slightly lower [2]. The primary modes of transmission for PEDV include direct or indirect contact with infected pigs, ingestion of contaminated feces or vomitus, and contact with contaminated equipment, vehicles, or other surfaces [10, 11].

In recent years, there has been a notable focus on the mutation and recombination of PEDV. The emergence of the G1 and G2 genegroups, especially the high pathogenicity in lactating piglets, indicates the virus’s adaptive and evolutionary capacity [12]. Group G1 (classical strains) are typically regarded as low-pathogenicity or mild strains. In contrast, group G2 strains, particularly subtypes G2a and G2b, exhibit higher pathogenicity and have been associated with mortality rates reaching 100%, particularly in suckling piglets [13]. Currently, a number of different genotypes of PEDV strains are present in swine herds, which increases the complexity of the measures required for defense and control [14, 15].

The impact of mutations in the PEDV genome, particularly in the S gene, has been twofold. Firstly, they have affected the transmission and pathogenicity of the virus, leading to global epidemics. Secondly, they have challenged the protective efficacy of existing vaccines. Concurrently, these mutations have prompted the refinement and enhancement of molecular detection techniques, thereby ensuring the accurate identification and expeditious control of emerging virus strains, thus safeguarding the global pig industry from the threat of PED [16]. In light of the aforementioned considerations, this paper will concentrate on providing an up-to-date account of the research progress made on PEDV genomic variants, epidemiology, vaccine studies and detection methods. The aim is to offer the pig industry a scientific foundation upon which to base future decisions and actions.

PEDV genome

Genome and structures of PEDV

PEDV is an enveloped, single- and positive-stranded RNA virus with a diameter of approximately 95–190 nm and a surface with protrusions of approximately 18 nm in length [4]. The genome of PEDV is approximately 28 kb in length and contains a 5′-cap structure, a 3′-poly(A) tail, and seven open reading frames (ORFs). Of these, ORF1a and ORF1b encode two large polyprotein precursors (pp1a and pp1ab), which are cleaved into 16 nonstructural proteins (nsp1-nsp16) by virus-encoded proteases [17]. The nonstructural proteins are instrumental in the transcription and replication of viral RNA [18, 19].

ORFs 2−6 encode four structural proteins, including spike (S), membrane (M), envelope (E), and nucleocapsid (N) proteins, as well as an accessory protein, ORF3 [18]. Of these, the S protein is the principal surface glycoproteins of PEDV, playing a pivotal role in receptor binding, membrane fusion, and viral entry into host cells [20]. The S protein is situated on the envelope of the virus and contains four neutralizing epitopes, namely COE, SS2, SS6, and 2C10 [21, 22]. Additionally, they represent the primary targets for the induction of neutralizing antibodies [23, 24]. PEDVs from genegroups G1 and G2 exhibit differences in the amino acid sequence of the S protein. Various amino acid insertion and deletion mutations have been observed in the S protein of group G2 PEDV. These mutations may result in the inactivation of the glycosylation site of the S protein or the introduction of new glycosylation sites, which may lead to alterations in the viral antigenicity, receptor-binding capacity, and immune escape ability [25]. The M and N proteins are involved in the assembly of viral particles and nucleocapsid formation, respectively. The auxiliary protein ORF3 is believed to be an ion channel protein, although its precise function remains unclear [26].

Genomic variation and typing of PEDV

In recent years, the application of high-throughput sequencing technology has facilitated the analysis of the whole genome sequence of PEDV, thereby revealing the evolutionary dynamics of the virus [27]. A comprehensive analysis of the PEDV S gene and other structural genes revealed the existence of multiple genotypes, which were primarily classified into two major genegroups: G1 and G2. The S gene of group G2 PEDV exhibits a greater number of amino acid mutations, particularly in the N-terminal domain of S1, in comparison to group G1 [28]. And the ORF3 gene of group G2 may also undergo natural truncations, which may accelerate the weakening of viral virulence [29, 30]. The G1 and G2 clusters can be further subdivided into subgroups, including G1a, G1b, G1c, G2a, G2b, G2c, and G2d [1, 31–34]. The genetic diversity observed in PEDV strains isolated from different regions and at different times reflects the evolutionary history and geographical distribution characteristics of the virus.

G2 strains are predominantly endemic in China and the United States. An outbreak of a highly virulent PEDV variant in southern China in 2010 resulted in a severe PED epidemic with an extremely high mortality rate [35]. In China, the endemic strains of PEDV have undergone a gradual evolutionary process, evolving from early classical strains to highly virulent mutant strains [36, 37]. These mutant strains exhibit a considerable number of mutations and deletions in pivotal regions, such as the S gene, which contribute to augmented virulence and diminished protective efficacy of existing vaccines [38–40]. Furthermore, inter-viral recombination events have accelerated the evolutionary process of PEDV, resulting in the emergence of recombinant strains with novel properties [41–44]. The emergence and rapid dissemination of highly virulent mutant strains have resulted in significant losses for the swine industry [45–48]. Similarly, Asian countries such as Korea [49–53], Japan [54, 55], Vietnam [56–58], and the Philippines [59, 60] are experiencing comparable challenges, and the rising prevalence and mutation of PEDV are a significant concern. In the United States, the most prevalent strains of PEDV are NON-INDEL (USA/Kansas29/2013) and S-INDEL (USA/OH851/2014), which are both categorized as type G2 [61, 62]. Experimental infections have demonstrated that non-INDEL strains exhibit heightened pathogenicity and mortality compared to S-INDEL strains, despite the latter being diagnosed at a lesser frequency [63, 64]. Subsequently, the detection of PEDV strains has been reported in other countries, including Canada, Mexico, Austria, Belgium, France, Germany, and Italy [5, 65–67].

Epidemiology of PEDV

Since its initial identification in Europe, PEDV has proliferated at an accelerated rate in Asia, where it has become pervasive. The epidemiological characteristics of PEDV exhibit considerable variation across regions and over time (Table 1) [68, 69]. The initial outbreak of PEDV was documented in Japan in 1982, and the virus subsequently disseminated to countries and regions including China, Korea, Thailand, and Vietnam, where it persists as a significant threat to the pig industry in these regions [61, 70–72]. Despite the long history of endemicity of PEDV in Europe and the United States, new outbreaks and virus mutations have occurred in recent years [73–77]. The first detection of the virus in the United States was in 2013, after which it spread rapidly to Canada and Mexico, causing severe losses to the local pig industry [5, 67]. In Europe, a 2016 outbreak on a small pig farm in Hungary demonstrated that PEDV affected all animals, with mortality predominantly observed in piglets, reaching up to 30% [78].

Table 1.

Global epidemiology of PEDV over the past decade

Years	Countries	Targets	Positivity rate (no.)	Refs.
2011–2012	China	Viral RNA	92.7% (n = 288)	[44]
2010–2012	China	Viral RNA	54.9–69.2% (n = 378)	[90]
2011–2012	China	Viral RNA	72.3% (n = 577, samples), 79.7% (n = 177, farms)	[46]
2013–2014	Japan	Viral RNA	19.5% (n = 1269, farms)	[91]
2007–2014	Italy	Viral RNA	74% (n = 51)	[73]
2012–2014	China	Viral RNA	24.2% (n = 314)	[81]
2013–2014	Japan	Viral RNA	72.5% (n = 204, samples), 88.5% (n = 87, farms)	[55]
2014–2015	China	Viral RNA	92.3% (n = 129, samples), 94.0% (n = 67, farms)	[69]
2014–2015	Philippines	Viral RNA	29.4% (n = 34)	[59]
2015–2016	China	Viral RNA	84.2% (n = 70)	[41]
2015–2016	China	Viral RNA	92.6% (n = 27)	[39]
2015–2016	China	Viral RNA	65.5% (n = 252)	[92]
2016	China	Viral RNA	28.9% (n = 1272)	[31]
2016–2017	Italy	Viral RNA	3.8% (n = 444)	[93]
2015–2017	China	Viral RNA	19.6% (n = 398)	[83]
2016–2017	China	Viral RNA	52.6% (n = 116)	[94]
2012–2018	China	Viral RNA	50.2–62.1% (n = 2987, samples), 96.4% (n = 168, farms)	[95]
2014–2018	China	Viral RNA	35.8% (n = 645, samples), 47.7% (n = 156, farms)	[32]
2016–2018	Mexico	Viral RNA	77.9% (n = 68)	[96]
2017–2018	China	Viral RNA	19.1% (n = 672)	[97]
2015–2019	China	Viral RNA	51.7% (n = 575)	[98]
2017–2019	Spain	Viral RNA	38.7% (n = 106, farms)	[99]
2018–2019	China	Viral RNA	47.0% (n = 34)	[100]
2019	Vietnam	IgG	88.8% (n = 600, samples), 96.7% (n = 30, farms)	[101]
2019–2020	China	Viral RNA	62.2% (n = 413)	[102]
2011–2021	China	Viral RNA	49.8% (n = 149,869)	[103]
2011–2021	China	Viral RNA	40% (n = 65)	[42]
2017–2021	China	IgA	64.0% (n = 882, samples), 52.2% (n = 303, farms)	[33]
2019–2021	Mexico	IgG	61.7% (n = 1054)	[104]
2019–2021	China	Viral RNA	37.5% (n = 176)	[105]
2017–2022	China	Viral RNA	53.9% (n = 673, samples), 79.9% (n = 143, farms)	[43]
2021	Mexico	Viral RNA	67.6% (n = 74)	[106]
2022–2023	China	Viral RNA	19.7% (n = 462)	[107]
NA	China	Viral RNA	38.1% (n = 160)	[108]
2021–2022	China	Viral RNA	51.8%(n = 112)	[109]
2021–2023	China	Viral RNA	49.7% (n = 1791, samples), 76.1% (n = 213, farms)	[110]
2022–2023	China	Viral RNA	8.8% (n = 5483)	[111]

It is noteworthy that PEDV is frequently co-infected with other viruses, including porcine deltacoronavirus (PDCoV) [79], porcine circovirus type 2 (PCV2) [80], PCV4 [42], porcine kobuvirus (PKV) [81], transmissible gastroenteritis virus (TGEV) [82], Porcine rotavirus A (PoRV-A) [82], porcine sapovirus (PSaV) [83], porcine bocavirus (PBoV) [84], swine enteric coronavirus (SeCoV) [85], swine acute diarrhea syndrome coronavirus (SADS-CoV) [86], and porcine torovirus (PToV) [87]. Such mixed infections may serve to increase the complexity of the virus, thereby complicating the prevention and control measures that must be employed.

Furthermore, recent years have seen a significant focus on the persistence of PEDV in the environment, with particular attention paid to its ability to survive in marine environments. In the United States, although the risk of transmission of PEDV and PDCoV via migratory birds is low, the persistence of PEDV in the environment has prompted concerns [88]. An experimental study of PEDV persistence in the French coastal environment demonstrated a negative correlation between seawater temperature and the persistence of infectious viruses in seawater. This suggests that infectious viruses may survive in seawater for a shorter period of time during the warm season [89]. These findings have significant implications for the assessment of the persistence of coronaviruses in coastal environments and the associated risk of transmission.

PEDV vaccines

The mutations and recombination of PEDV have not only altered the genetic characteristics of the virus, but have also had a profound impact on its pathogenicity and vaccine efficacy. The emergence of highly virulent mutant strains has significantly diminished the protective efficacy of existing vaccines, even leading to instances of immunization failure [98]. Meanwhile, recombination events among viruses may also result in the generation of recombinant strains with novel characteristics, thereby introducing additional challenges to the prevention and control of the disease [112]. It is therefore crucial to enhance the monitoring and investigation of PEDV mutation and recombination in order to develop efficacious prevention and control strategies [100]. A quick search in the Web of Science database using the term “porcine epidemic diarrhea virus vaccine” returned > 1000 publications (Fig. 1). As illustrated in Fig. 1, there has been a general increase in the number of publications related to PEDV vaccines globally over the past two decades. This trend not only reflects the continued research efforts of the scientific community in this area, but also serves to underscore the remarkable advancements that have been made in the field of vaccine research.

Fig. 1.

Publications in the Web of Science database from 2004 to 2024 (as of December 20) on the topic of PEDV vaccines

Traditional vaccine strategies

Live attenuated vaccine (LAV) utilizes a treated, attenuated strain of PEDV that is still capable of replicating within the host. These vaccines stimulate a protective response from the immune system of the pig as it is able to multiply in the host, but not enough to cause disease [113]. A fully attenuated PEDV LAV candidate can be constructed by introducing multiple virulence-related mutations that help maintain the genetic stability of the virus and reduce the risk of the virus regaining virulence.

Inactivated vaccines are vaccines in which the live virus or live virus components have been rendered incapable of replication and thus unable to cause disease. Inactivated vaccines contain killed PEDV or some of its components that are unable to reproduce or cause disease [114]. However, they are sufficient to activate the immune system to recognize and remember the foreign material. The inactivation process typically entails the treatment of the virus with a chemical agent, such as formaldehyde, with the objective of preserving the protective epitope structure of the virus while simultaneously eliminating its capacity to replicate or regain virulence.

Novel vaccine strategies

RNA interference (RNAi) technology: RNAi technology in vaccine development involves the use of small interfering RNAs (siRNAs) to specifically target and silence viral genes, providing a potent and specific silencing effect that can be harnessed for antiviral therapies [115]. Recent studies have demonstrated that the construction of shRNA expression plasmids targeting the PEDV N, M, and S genes and their successful transfection into Vero cells have led to the identification of two highly efficient shRNAs (pSilencer4.1-M1 and pSilencer4.1-N). These shRNAs have been shown to inhibit the invasion and replication of PEDV with high specificity and efficiency [116]. This finding not only corroborates the potential of RNAi technology in PEDV control but also provides a theoretical foundation for the prospective development of RNAi-based antiviral drugs.

Messenger RNA (mRNA) vaccines: The utilization of mRNA for the encoding of specific antigenic proteins represents a crucial aspect of the immune system’s response to viral infection [117]. These mRNAs are internalized by cells, where they direct the production of antigenic proteins derived from the virus. This process ultimately stimulates the immune system to mount an effective immune response. The vaccine is non-infectious and non-integrating, encodes multiple antigens to enhance the immune response, and can be structurally modified to improve stability and reduce immunogenicity [118]. The rapid advancement of mRNA technologies has led to a significant focus on their potential use in the development of a vaccine for PEDV [119]. The ability of mRNA vaccines to respond rapidly and efficiently offers a promising avenue for controlling PEDV.

Nanovector vaccines: Nanovectors are targeted delivery vehicles that facilitate the transport of nanoscale materials [120]. The use of nanovectors has been demonstrated to markedly enhance the density of antigenic epitopes in comparison to traditional protein subunit vaccines, thereby facilitating the generation of more potent neutralizing antibodies and cellular immune responses. The use of glycyrrhizinic acid-based lipid nanoparticle complexes has been shown to result in favorable immunogenicity and anti-inflammatory effects in the development of vaccines against PEDV [121]. Additionally, by using coated PEDV-loaded microspheres (700–900 μm in diameter), researchers were able to effectively induce high levels of specific mucosal immunity in pigs [122]. The nanovector vaccines not only enhance the efficiency of antigen delivery but also augment the protective efficacy of the vaccine by modulating the immune response.

Genetically engineered vaccines: Genetically engineered vaccines work by using recombinant DNA technology to introduce precise genetic modifications that result in cell-mediated responses and memory lymphocyte responses [123]. The advantages of genetically engineered vaccines include the ability to elicit targeted immune responses, enhanced safety through the use of programmed properties, and the versatility of genes encoding multiple antigens or immune system proteins. By employing reverse genetics technology, researchers have successfully constructed vaccine candidates based on full-length complementary DNA (cDNA) clones of PEDV [124]. These vaccines offer a versatile platform for viral genome manipulation and provide a robust foundation for the development of new vaccines and coronavirus vectors [125].

The effect of lactic acid bacteria in the control of PEDV

The potential of lactic acid bacteria (LAB) as probiotics in the control of PEDV has also been the subject of considerable research [126]. It has been demonstrated that LAB can mitigate the mortality and diarrhea symptoms associated with PEDV infection, as well as inhibit viral replication by regulating the expression of host intestinal barrier genes [127]. Furthermore, the engineering of Lactobacillus plantarum to contain PEDV S1 has resulted in the induction of specific immunity to PEDV in guinea pigs, thereby establishing a foundation for the subsequent development of an oral vaccine [128]. In addition, the administration of ZnO resulted in a reduction in diarrhea in PEDV-infected piglets, along with the production of antiviral and anti-inflammatory effects in the same subjects [129].

Vaccines reported for the prevention of PEDV

A review of the literature revealed that various PEDV vaccines have been reported in recent years (Table 2). These were compiled and analyzed with a focus on vaccine types, immunization targets, construction strategies, and the evaluation of clinical outcomes. These vaccines are designed to be flexible, capable of providing protection against multiple subtypes of viruses, and are produced in high quality with favorable safety profiles.

Table 2.

Vaccines reported in recent years for the prevention and control of PEDV

Type of vaccines	Immunization targets	Preparation strategies	Experimental animals	Vaccine efficacy	Refs.
Live-attenuated vaccine	PEDV	A cold-adapted live-attenuated vaccine was prepared by short-term passage of a toxic PEDV isolate at successively lower temperatures in Vero cells	Piglets	The vaccine markedly improved piglet survival and significantly reduced the severity of post-challenge diarrhea and PEDV fecal shedding. In addition, a strong antibody response to PEDV was detected in the serum of the immunized sows and in the colostrum as well as in the serum of their offspring	[130]
Live-attenuated vaccine	PEDV	Generation of live-attenuated virus by serial passaging of highly virulent G2b PEDV in Vero cells	Piglets	This sow vaccination regimen completely protected of lactating piglets from toxic G2b challenge, as evidenced by an increase in survival from 0 to 100% and a significant reduction in the intensity of diarrhea, including the amount and duration of PEDV fecal shedding	[131]
Inactivated vaccine	PEDV	Production of an inactivated vaccine using the PEDV isolate	Piglets	Vaccination increased the survival rate of piglets challenged with PEDV and reduced the severity of diarrhea, including fecal shedding of virus. In addition, a strong neutralizing antibody response to PEDV was verified in immunized sows and their offspring	[114]
Subunit vaccine	PEDV	Recombinant S1 protein was expressed and generated	Sow, piglets	Vaccination of sows induced S1-specific IgG and IgA antibodies, which were passively transferred to piglets. In addition, high virus-neutralizing titers were observed in the sera of vaccinated sows and their piglets	[132]
Subunit vaccine	PEDV	A flagellin-adjuvanted PEDV vaccine	Piglets	Vaccination not only enhanced antibody responses to serum IgG/IgA, mucosal IgA, and serum neutralizing antibodies, but also improved IFN-γ and IL-4 production	[133]
Subunit vaccine	PEDV	A plant-based COE variant generated from G2a PEDV	Piglets	Inoculated piglets produced high levels of PEDV-specific IgG antibodies, COE-IgA-specific antibodies, neutralizing antibodies and IFN-γ responses	[134]
Subunit vaccine	PEDV	Two distinct S protein fragments cloned and expressed as glutathione S-transferase -tagged fusion proteins in Escherichia coli	Mice	The inoculation of mice resulted in the induction of high levels of serum IgG and neutralizing antibodies	[135]
Subunit vaccine	PEDV, CSFV	A subunit vaccine based on the CSFV E2 protein and the PEDV S1 protein	Mice	The vaccine elicited high neutralizing antibody titers and IgG1/IgG2a, as well as induced high IL-4 expression	[136]
Bacterial vector vaccine	PEDV	Expression of PEDV S1 glycoprotein by Lactobacillus casei	Mice	Antibody levels increased significantly after oral administration of recombinant bacteria. Extracellular cytokines were detected on day 42 after immunization, indicating high levels of humoral and cellular immune responses in the mice	[137]
Bacterial vector vaccine	PEDV	Use of L. johnsonii as an antigen delivery vehicle to generate a recombinant strain expressing COE protein	Sows	Anti-PEDV-specific serum IgG, IgA, IgM and mucosal secretory immunoglobulin A (SIgA) antibodies were induced in sows after vaccination	[138]
Bacterial vector vaccine	PEDV	An oral recombinant L. casei vaccine for the delivery of COE protein	Mice	Anti-PEDV-specific IgG antibodies with PEDV-neutralizing activity were detected in serum in immunized mice, accompanied by elevated levels of mucosal SIgA antibodies in the reproductive tract, intestinal mucus, and feces	[139]
Bacterial vector vaccine	PEDV	The S1 region of the PEDV S gene was inserted into Lactococcus lactis to produce a recombinant plasmid	Mice	Elevated levels of anti-PEDV IgG and SIgA were detected in orally inoculated mice. And the vaccine stimulated the proliferation of spleen lymphocytes and induced high levels of IL-4 and IFN-γ in the mice	[140]
Bacterial vector vaccine	PEDV	L. casei expresses a dendritic cell (DC)-targeting peptide fused with the COE antigen	Piglets	The vaccine promoted lymphocyte proliferation and effectively protected piglets from PEDV infection. In addition, the vaccine worked in the anti-inflammatory response of mesenteric lymph nodes during PEDV infection	[141]
Bacterial vector vaccine	PEDV	L. casei strains expressing the PEDV COE antigen or a fusion of COE and the M-cell targeting peptide Co1 were constructed	Mice	The vaccine could effectively induce mucosa, humoral and Th2-type cellular immune responses against PEDV infection by oral administration	[142]
Bacterial vector vaccine	PEDV	A DC-targeting oral vaccine using L. casei to deliver DC-targeted peptide fused with COE antigen	Mice	The vaccine effectively elicited the SIgA- and IgG-based immune response, and the vaccine expressing the DC-targeting peptide fused with the COE antigen was more immune-efficient	[143]
Bacterial vector vaccine	PEDV	PEDV spike protein B-cell linear epitope on L. mucosae G01 S-layer surface	Mice	The vaccine induced the production of IgA secreted in the gut and feces of mice. Serum IgG levels are also elevated. In addition, the levels of cytokines IL-2, IL-4, IFN-γ, and IL-17 were significantly increased	[144]
Bacterial vector vaccine	PEDV	Recombinant L. casei expressed PEDV N protein	Mice	The vaccine could induce specific PEDV IgA and IgG responses and activated mucosal and systemic immune responses	[145]
Bacterial vector vaccine	PEDV	Recombinant L. casei expressed PEDV S1 and N proteins	Mice	Both L. casei-expressed S1 and N proteins could elicit mucosal and systemic immune responses, but the latter elicited a more pronounced response	[146]
Bacterial vector vaccine	PEDV	Integration of the PEDV S1 gene into the L. paracasei genome	Mice, piglets	The vaccine could cause mucous membrane, humoral and cellular immune responses. Moreover, the vaccine demonstrated efficacy in protecting piglets from PEDV infection	[147]
Bacterial vector vaccine	PEDV	Eukaryotic expression recombinant plasmids carrying the S1 and S2 epitopes of PEDV were transferred into L. acidophilus	Mice, sows	The inoculated mice exhibited elevated levels of anti-PEDV-specific IgG and SIgA antibodies, as well as robust cellular immune responses. Furthermore, the vaccine has been demonstrated to elicit specific systemic and mucosal immune responses in pregnant sows	[148]
Bacterial vector vaccine	PEDV	Recombinant L. casei strains expressing PEDV S protein and OMP16 protein from Brucella abortus	Mice	The analysis of the fecal samples from the immunized mice revealed the presence of elevated levels of IgG, neutralizing antibodies, IL-4, IL-10, and INF-γ	[149]
Bacterial vector vaccine	PEDV	Recombinant yeast expressing the PEDV S1 gene	Mice, piglets	Recombinant yeast could be used as a PEDV vaccine vector to induce high levels of IgG and IgA against PEDV in pigs	[150]
Viral vector vaccine	PEDV, PoRV	The ORF3 gene of the attenuated PEDV was replaced with the PoRV VP7 gene by a reverse genetic system	Piglets	Vaccine-immunized piglets produced specific IgG and IgA in serum and saliva. In addition, both PoRV and PEDV neutralizing antibodies were produced in these piglets	[151]
Viral vector vaccine	PEDV, CSFVa	A recombinant CSFV expressed the antigenic domain of PEDV	Rabbits, pigs	The inoculation of animals resulted in a notable elevation in PEDV-specific antibodies and IFN-γ within the serum	[152]
Viral vector vaccine	PEDV, ORFVb	An ORFV vector expressing the PEDV full-length spike protein	Piglets	PEDV-specific IgG, IgA, and neutralizing antibodies were detected in inoculated piglets. In addition, vaccinated piglets exposed to live PEDV exhibited augmented neutralizing antibody responses	[153]
Viral vector vaccine	PEDV, ORFV	An ORFV vector expressing PEDV full-length spike protein	Piglets	The vaccination of piglets induced strong serum IgG, IgA and neutralizing antibody responses to PEDV. It also protected pigs from clinical signs of PED and reduced virus shedding in feces during the infection challenge	[154]
Viral vector vaccine	PEDV, VSVc	An attenuated recombinant VSV vector expressing PEDV spike protein	Piglets	The vaccine effectively induced PEDV-specific immunity in pigs by intramuscular rather than intranasal immunization. In addition, immunization of sows with the vaccine provided protective lactic acid-producing immunity against toxic G2b PEDV challenge in piglets	[155]
Viral vector vaccine	PEDV, BVDVd	Construction of a BVDV chimeric virus expressing an antigenic fragment of PEDV spike	BALB/c mice	IgG antibodies against BVDV and PEDV was observed in inoculated mice	[156]
Viral vector vaccine	PEDV, baculovirus	Baculovirus as a vaccine vector to express PEDV full-length S protein and G2b PEDV S1 protein	Mice, piglets	The levels of anti-PEDV S-specific IgG were significantly increased in vaccinated mice and piglets. And immunized piglets exhibited milder clinical signs and less fecal virus shedding	[157]
Viral vector vaccine	PEDV, PRRSVe	A non-toxic PRRSV as a vector to express G2 PEDV S protein	Piglets	Vaccination induced systemic homologous neutralizing antibodies against PEDV and PRRSV to reduce viral anemia	[158]
Nanovaccine	PEDV	Three species of trimeric nanovaccines constructed based on Trimer-Tag technology, namely S1 trimer, COE trimer, and receptor-binding domain trimer	Mice, sows and piglet	Immunization with S1-trimer induced high levels of humoral immunity containing PEDV-specific IgG and IgA. Simultaneously, S1-trimer-induced mucosal IgA responses and systemic IgG responses exhibited high titers of virus-neutralizing antibodies in vitro	[159]
Minimally replicative vaccine	PEDV	PEDV was heat-treated and exposed to RNAse to prepare vaccines with intact viral structure/antigenicity but highly attenuated replication capacity	Piglets	Strong PEDV spike protein-specific and virus-neutralizing antibody responses were induced in vaccinated piglets. And all vaccinated pigs exhibited protection against fecal virus shedding and intestinal pathology	[160]
Virus-like particle (VLP)	PEDV, TGEV	A recombinant vaccine targeting PEDV S and TGEV S proteins	Piglets	The vaccine effectively stimulated the production of neutralizing antibodies against PEDV and TGEV. And the vaccine induced elevated levels of IFN-γ, IL-2, and IL-4 in the peripheral blood of piglets and enhanced cytotoxic T cell activity	[161]
RNA vaccine	PEDV	Five shRNAs expression plasmids targeting the N, M and S genes of PEDV were constructed and transfected into Vero cells	Cell cultures	Two shRNAs demonstrated the capacity to safeguard cells from PEDV invasion with remarkable specificity and efficacy. Additionally, the expression of two shRNAs was observed to significantly inhibit the replication of PEDV	[116]
mRNA vaccine	PEDV	Two lipid nanoparticle-encapsulated mRNA vaccines encoded either the full-length PEDV spike proteins or a multiepitope chimeric spike protein	Piglets	The mRNA vaccine provided effective antibody responses and antigen-specific T-cell responses to immunized piglets. Following a viral challenge, active and passive immunization protected piglets against PEDV	[119]

^aCSFV indicates classical swine fever virus

^bORFV indicates orf virus

^cVSV indicates vesicular stomatitis virus

^dBVDV indicates bovine viral diarrhea virus

^ePRRSV indicates porcine reproductive and respiratory syndrome virus

PEDV detection methods

As illustrated in Fig. 2, the number of publications pertaining to PEDV detection has exhibited an upward trajectory over the past two decades. This trend reflects not only the unwavering commitment of the scientific community to the advancement of detection methods but also the substantial progress made in detection technologies through interdisciplinary collaboration. Detection methods, including in situ hybridization, polymerase chain reaction (PCR)-based assays, isothermal amplification techniques, immunological assays, biosensors, and so forth, play a pivotal role in the diagnosis and monitoring of PEDV.

Fig. 2.

Publications in the Web of Science database from 2004 to 2024 (as of December 20) on the topic of PEDV detection

Nucleic acid tests

In situ hybridization

In situ hybridization (ISH) visualizes and localizes the location of target nucleic acids by using labeled nucleic acid probes that specifically bind to specific nucleic acid sequences on tissue sections, cells or chromosomes.

In the context of PEDV detection, ISH is capable of accurately localizing viral RNA in tissue sections, which is of critical importance for understanding the distribution and replication sites of the virus within the host. For instance, it has been demonstrated that PEDV replicates primarily in the villous epithelial cells of the jejunum and ileum, whereas no signal is observed in the cecum and colon [162]. This precise localization information is crucial for elucidating the pathomechanism of the virus and developing efficacious therapeutic strategies. Additionally, ISH is applicable to formaldehyde-fixed, paraffin-embedded tissues, which is advantageous in circumstances where solely fixed tissue samples are accessible [163]. Moreover, ISH offers the additional benefit of providing detail and tissue structure at the cellular level, which is advantageous for studying small numbers of PEDV-infected cells and lesions in the same section [163]. The technique is advantageous for retrospective studies, as it enables the detection of viral nucleic acids in fixed tissue, even when fresh tissue is no longer available.

In summary, ISH techniques provide more comprehensive and in-depth results in specific situations, making them valuable for understanding viral pathomechanisms, guiding therapy, and conducting retrospective studies.

Reverse transcription-polymerase chain reaction

Reverse transcription-polymerase chain reaction (RT-PCR) is a technique that reverses the transcription of an RNA template into cDNA, which is then used as a template for PCR amplification.

The results of the RT-PCR demonstrated excellent sensitivity and specificity in the detection of PEDV. The method demonstrated the capacity to detect PEDV viral loads as low as 10⁴ TCID₅₀ mL^–1, exhibiting no cross-reactivity with other viruses, including TGEV and PoRV, thus ensuring the accuracy of the results [164].

It was gratifying to observe that RT-PCR was capable of detecting PEDV not only in the initial stages of viral infection but also in experimentally vaccinated piglet samples within a day of vaccination [165]. This characteristic renders RT-PCR an efficacious instrument for expeditious diagnosis of PEDV infection.

Remarkably, the surface of articles treated with disinfectants (e.g., phenol, sodium hypochlorite, oxidizers, etc.) can still be positive for PEDV RNA using RT-PCR. However, these PEDVs are no longer infectious [166]. Consequently, the differentiation of infectious PEDV represents a further area of investigation.

Multiplex RT-PCR

Multiplex RT-PCR is a technique that simultaneously detects multiple target RNA molecules in the same PCR system, thereby enhancing the efficiency and throughput of the detection process.

For PEDV detection, researchers have developed multiplex RT-PCR techniques that are able to differentiate between various strains of the virus, including wild-type and vaccine strains, based on the analysis of the S and ORF3 genes [167, 168]. These methods demonstrate high sensitivity, with a detection limit as low as 1 × 10^1.7 TCID₅₀ 100 μL^–1, and good specificity without cross-reactivity. The pervasive occurrence of variant PEDV was additionally substantiated through the analysis of samples procured from disparate geographical locations within China.

Additionally, given that PEDV and other porcine diarrhea viruses frequently co-infect and are challenging to distinguish clinically, the researchers devised several multiplex RT-PCR systems to achieve the concurrent detection of multiple pathogens. For instance, dual RT-PCR can detect both PEDV and PDCoV or TGEV [169, 170], while triplex or quadruplex RT-PCR can detect several pathogens other than PEDV, including TGEV, PDCoV, SADS-CoV, PoRV-A, PSaV, PCV2, CSFV, PRRSV, porcine sapelovirus, etc., which significantly enhanced the diagnostic efficiency and accuracy [80, 82, 171–173]. Notably, the storage temperature had a significant effect on multiplex RT-PCR for the detection of viral nucleic acids in fecal samples [174].

Nested RT-PCR

Nested RT-PCR (nRT-PCR) is a technique that incorporates a nested primer amplification step into the process of RT-PCR. The process commences with the transcription of RNA into cDNA through reverse transcription, which is then subjected to two rounds of amplification using two distinct pairs of PCR primers. The initial pair of primers amplifies a longer fragment, while the subsequent pair of primers (termed “nested primers”) bind within the first-round amplification product to amplify a shorter, more specific fragment, thereby increasing the sensitivity and specificity of the assay [175].

Further, Li et al. developed a dual nested RT-PCR technique capable of detecting both PEDV and PDCoV in a single reaction and clinical sample validation demonstrated its accuracy [79]. In addition, a triplex nRT-PCR method was developed for PEDV, TGEV and PRV-A [176]. Jung et al. validated the reliability of multiplex RT-nPCR for the detection of PEDV and TGEV in formalin-fixed, paraffin-embedded tissue samples with 100% compliance compared with in situ hybridization, which shows its promising application [177].

Fluorescence quantitative RT-PCR

Fluorescence quantitative RT-PCR (RT-qPCR) is a technique that combines reverse transcription and fluorescence quantitative PCR for the precise detection and analysis of RNA targets. The technique initially involves the reversal transcription of RNA into cDNA, followed by the real-time monitoring of alterations in the quantity of the resulting product during PCR amplification through the use of fluorescent probes or dyes [178].

The researchers successfully established a detection system for PEDV N and S genes by optimizing the RT-qPCR method and evaluated its transmission potential in experimental pigs [179]. The results demonstrated that the N gene-based assay exhibited superior performance compared to the assay targeting S gene. And PEDV was detected in the rectal mucus of all infected pigs approximately two weeks post-infection. Additionally, a TaqMan RT-qPCR was developed to differentiate between PEDV and attenuated vaccine strains, exhibiting high sensitivity and good reproducibility [180]. Zhou et al. demonstrated that TaqMan RT-qPCR outperformed RT-PCR and SYBR Green I-based RT-qPCR in terms of analytical sensitivity [181]. Furthermore, Wang et al. developed a one-step RT-qPCR method that can effectively differentiate between PEDV vaccine strains and wild-type strains, thereby providing an efficient and cost-effective technological platform for accurate investigation of clinical data [182]. It is also evident that nucleic acid extraction methods exert a non-negligible influence on the test results [183].

Multiplex RT-qPCR

The necessity for the establishment of multiple enterovirus assays is driven by two key factors: firstly, the extreme similarity of the clinical signs and pathologic-anatomical changes observed in piglets infected with PEDV and other epidemic diarrhea viruses; and secondly, the potential for co-infection.

The dual qPCR method based on SYBR Green I or EvaGreen has been successfully employed for the simultaneous detection of PEDV, PCV3, PCV4, PBoV3/4/5, and rotaviruses, etc. [42, 84, 184, 185].

To further address the need for the simultaneous detection of multiple pathogens in complex samples, researchers have developed multiplex RT-qPCR techniques. These techniques employed the use of distinct fluorescently labeled probes or primers in conjunction with specific melting curve analysis, thereby enabling the differential detection of a vast array of porcine enteroviruses, including TGEV, PEDV, PCV2, PDCoV, SADS-CoV, PoRV-A, PoRV-C, PKV, PSaV, and PToV, etc. [15, 83, 85–87, 107–109, 111, 186–193]. The detection limit was as low as several copies per microliter with good reproducibility and repeatability, providing a reliable basis for the diagnosis of mixed infections.

The objective of related researches has been the detection and differentiation of PEDV mutant strains [194]. By designing specific primers and TaqMan probes, the researchers have successfully established dual and multiplex real-time PCR methods that can distinguish between PEDV mutant strains and classical strains, providing crucial technical support for epidemic prevention and control [195, 196].

Droplet digital PCR

Droplet digital PCR (ddPCR) is an absolute quantitative analytical technique based on single-molecule PCR [197]. It distributes the samples into a large number of independent tiny reaction units, and each unit carries out the PCR reaction independently. An endpoint assay determines whether amplification has occurred in each unit, and the initial sample copy number concentration is calculated based on a Poisson distribution. The ddPCR exhibits ultra-high sensitivity and accuracy, enabling absolute quantitative detection of trace nucleic acids.Zhou et al. developed a ddPCR method targeting the PEDV M gene, which demonstrated a detection limit of 0.15 copies/μL, a notable improvement over that of real-time PCR method (10 copies/μL) [198]. And the ddPCR method showed excellent linearity (R² > 0.99) within the template concentration range of 1 to 1 × 10⁴ copies μL⁻¹. The results of the ddPCR and real-time PCR methods were validated by 150 clinical samples, and were found to be in complete agreement. Similarly, a ddPCR method for PEDV was established by Cao et al., which was also able to detect PEDV RNA at an ultra-low concentration (0.26 copies μL⁻¹) [199]. The ddPCR-based PEDV detection methods exhibited notable advantages in terms of analytical sensitivity, offering crucial technical support for the precise prevention and control of PED.

NanoPCR

In recent years, the application of nanoparticles in molecular diagnostics have markedly enhanced the sensitivity and specificity of pathogen detection. A variety of PCR methods based on functionalized nanoparticles (nanoPCRs) have demonstrated notable advantages in the detection of viruses such as PEDV and TGEV.

By conducting a systematic optimization of functionalized magnetic beads and gold nanoparticles, Xing and colleagues developed a ultrasensitive nanoparticle DNA probe-based PCR (UNDP-PCR) method for the detection of PEDV [200]. This method can effectively enrich viral RNA in fecal samples, forming MMPs-RNA-AuNPs complexes that can then be specifically detected by PCR amplification. In comparison to conventional RT-PCR, UNDP-PCR exhibits a sensitivity that is 400-fold higher, accompanied by excellent reproducibility and high specificity. In the detection of fecal samples, the positive detection rate of UNDP-PCR was significantly higher than that of RT-PCR and SYBR Green RT-qPCR, indicating considerable potential for application. Moreover, a study investigated the potential of nanoPCR assay for the PEDV N gene [201]. The method exhibits a detection limit of 2.7 × 10⁻⁶ ng μL⁻¹ of PEDV RNA and demonstrates high specificity.

Of interest, to detect both PEDV and TGEV, researchers developed a dual nanoPCR method [202]. The method was based on the design of primers based on the N genes of both viruses and performed well in screening clinical samples from four provinces in China. Further, a dual UNDP-PCR based on functionalized magnetic bead enrichment and gold nanoparticles can also be used for the simultaneous detection of PEDV and TGEV, providing an efficient, cost-effective, and reliable technology for virus detection at the preclinical level [203].

Other novel PCR-based assays

To enhance the sensitivity of detecting PEDV and TGEV in fecal samples, the investigators devised a multiplex RT-PCR-based dot blot hybridization, which markedly increased the detection sensitivity of both PEDV and TGEV (by a factor of 1000 and 100, respectively) [204].

It is important to acknowledge that while the RT-qPCR technique is highly sensitive in detecting viruses, it is challenging to differentiate between infectious and non-infectious viruses. It has been demonstrated that a method integrating propidium monoazide (PMA) with qPCR can effectively discern infectious viruses [205]. The combination of PMA treatments with heat or UV inactivation offers a novel approach to the assessment of viral infectivity. The viability RT-qPCR method, optimized by Puente et al., enabled selective detection of infectious and heat-inactivated PEDV by screening different photoactivatable dyes and platinum compounds [206]. Furthermore, a method enhanced the estimation of PEDV infectivity by combining Triton X-100 with viability markers. And it was demonstrated that viral RNAs could be detected following treatment at various temperatures. However, the viable virus exhibited loss of activity after 10 s of treatment at temperatures exceeding 76 °C [207].

To block non-specific amplification, the researchers developed a double priming oligonucleotide (DPO) system (Fig. 3). As shown in Fig. 3, the DPO system comprises two distinct primer fragments of different lengths, connected by a polydeoxyinosine (poly(I)) linker [208]. The low melting temperature of deoxyinosine (I) allows the poly(I) linker to form a bubble structure in the primer, which facilitates the separation of the primer into a longer 5′ segment and a shorter 3′ segment at the annealing temperature. This inhomogeneity in nucleotide distribution results in the 5′ segment exhibiting a preferential binding to the template DNA, while the 3′ segment displays a specific binding to the target site. This prevents non-specific annealing and ensures that the DPO system only performs target-specific extensions. A DPO-based multiplex RT-qPCR assay provided a novel strategy for the accurate differentiation of TGEV, PEDV, PDCoV, and PoRV [97]. Moreover, the multiplex RT-PCR method based on the DPO system streamlined the primer design and enhanced the detection efficiency, offering a cost-effective and user-friendly detection tool for the expedient diagnosis of major intestinal RNA viruses in pigs [209]. Additionally, the universal primer-pentaplex PCR method enabled the simultaneous detection and differentiation of five porcine diarrhea-associated viruses through short-cycle multiplex amplification [210].

Fig. 3.

Schematic representation of DPO-based PCR strategies [97, 208]

Interestingly, the development of the panCoV RT-PCR assay offered a novel approach for the detection of human and animal coronaviruses, including δ-CoVs [211]. The method demonstrated 100% sensitivity and specificity in swine biological samples, providing substantial technical support for the surveillance of outbreaks.

Isothermal amplification methods

Loop-mediated isothermal amplification

Loop-mediated isothermal amplification (LAMP) was developed by Notomi et al. in 2000 as a rapid and sensitive nucleic acid amplification method. It is capable of completing nucleic acid amplification under isothermal conditions (60–65 °C) in less than 1 h [212]. The technique is distinguished by the configuration of four to six specific primers for six to eight regions of the target gene and the incorporation of Bst DNA polymerase to facilitate efficient amplification, rendering it well-suited for on-site rapid detection.

For PEDV detection, researchers have developed a number of assays based on LAMP, including reverse transcription LAMP (RT-LAMP), visual RT-LAMP, and real-time RT-LAMP methods [213–217]. These methods have markedly enhanced the sensitivity, specificity, and rapidity of the assay by optimizing primer design, amplification conditions, or integrating other techniques.

Furthermore, it has been demonstrated that the LAMP technology is not only effective for the identification of individual viruses, but also for the concurrent detection of multiple viruses through multiplex amplification strategies [218]. For instance, the triplex LAMP method combined with a lateral flow device (LFD) could simultaneously detect multiple viruses, including PEDV, PoRV, PBoV, and PCV2 [219, 220].

To address the complexity and risk of cross-species transmission of porcine enteric CoVs, the researchers also developed a multiplex nucleic acid detection platform based on CRISPR/Cas12a and multiplex RT-LAMP, as well as a real-time RT-LAMP system [221, 222]. By integrating advanced molecular techniques and automated equipment, these platforms facilitate the rapid and accurate detection of multiple diarrhea-type CoVs, providing substantial support for disease prevention and control.

Polymerase spiral reaction

Polymerase spiral reaction (PSR) is a novel isothermal nucleic acid amplification technique that employs a specific hybrid primer design and Bst DNA polymerase with strand displacement activity to achieve rapid nucleic acid amplification under a constant temperature condition. Only two primers are required for nucleic acid amplification, which makes PSR a potentially valuable tool for the point-of-care testing (POCT) of pathogenic microorganisms.

Wang et al. designed specific primers based on the conserved region of ORF3 and established a rapid reverse transcription PSR (RT-PSR) method for the detection of PEDV [223]. The method required only 15 min for reverse transcription, followed by 50 min of incubation at a constant 62 °C to detect the presence of PEDV, which resulted in a notable reduction in the overall detection period. Most notably, the RT-PSR method exhibited high specificity and a low detection limit (1 order of magnitude lower than that of RT-PCR), thereby providing robust technical support for the early and accurate detection of PEDV.

Recombinase polymerase amplification

Recombinase polymerase amplification (RPA) is an isothermal nucleic acid amplification technique developed by Piepenburg et al. in 2006 [224]. The technique combines the synergistic action of recombinase, single-stranded DNA binding protein (SSB), and strand-replacing DNA polymerase to achieve rapid DNA amplification at a constant temperature (37–42 °C).

In consideration of the diversity of PEDV strains, the researchers devised a real-time reverse transcription RPA (RT-RPA) method for PEDV based on ORF1 deletion sequences [225]. This approach proved effective in differentiating attenuated vaccine strains from wild-type strains. Another study developed a real-time RT-RPA assay based on the PEDV N gene, which demonstrated the capacity to detect both classical and mutated PEDV strains [226]. The detection performance of clinical samples was excellent, exhibiting high diagnostic concordance with RT-PCR.

In order to achieve multiple detection, a research team designed multiple pairs of specific primers for the conserved sequences of PEDV and PDCoV, and successfully established a dual RT-RPA assay by optimizing the reaction conditions [227]. The method demonstrated high sensitivity (1 × 10² copies μL^–1) and specific amplification for both viruses, validating its potential as an alternative to RT-qPCR.

Moreover, Ma et al. developed an RT-RPA approach targeting the PEDV N gene, which, when coupled with LFD, enabled visual detection with a detection limit as low as 10² copies μL^–1 and a high compliance rate (96.5%) with RT-PCR detection of clinical samples [228]. The combination with immunochromatography permitted the rapid and highly sensitive detection of PEDV, thereby validating its effectiveness in practical applications [229].

Recombinase-aided amplification

Recombinase-aided amplification (RAA) is a technique for nucleic acid amplification that employs enzymes such as recombinase, SSB, and DNA polymerase under isothermal conditions (37–42 °C) [230]. This technology enables the rapid amplification of nucleic acids by locating and unraveling specific regions in double-stranded DNA through the combination of recombinase and primers. This is followed by the prevention of recombination of single-stranded DNA with the help of SSB proteins. Finally, the synthesis of new strands is completed under the action of DNA polymerase.

In view of the challenge posed by PEDV mutant strains on the efficacy of vaccine protection, Yin et al. devised an innovative approach combining the CRISPR/Cas13a system with RAA to rapidly differentiate between PEDV wild-type strains and attenuated vaccine strains [231]. In addition, the combination of reverse transcription RAA with portable instrumentation has been shown to offer notable advantages in the detection of PEDV clinical samples [232]. The optimized primer and probe targeting the N gene enabled the detection of PEDV in less than 30 min, with good specificity and a low detection limit (10 copies per reaction).

Of interest, Zhao et al. developed an alternative approach by combining RAA with Pyrococcus furiosus Argonaute (PfAgo) to detect PEDV, obviating the need for complex instrumentation [233]. And the entire process did not necessitate product purification, offering a novel solution for rapid on-site diagnosis of PEDV.

Enzymatic recombinase amplification

Enzymatic recombinase amplification (ERA) employs a combination of specific recombinase, exonuclease, polymerase, and other multi-enzyme systems to achieve the targeted amplification of trace nucleic acids at a constant temperature range of 37–42 °C [234]. ERA is distinguished by its capacity to function at low temperatures, high sensitivity, and rapid amplification.

Wu et al. established a dual ERA system that was capable of effectively differentiating between the two viruses, PEDV and PoRV-A, and demonstrated excellent sensitivity [235]. The method was validated with 64 clinical samples, and the results were found to be in complete agreement with those obtained using a commercially available kit.

Furthermore, the novel integration of reverse transcription ERA and CRISPR-Cas12a system offers a promising approach for the expeditious identification of PEDV wild-type and attenuated vaccine strains [234]. This protocol facilitates direct observation of the assay results under LED blue light through amplification of ORF3-specific fragments and subsequent detection of Cas12a/crRNA complexes. The method demonstrated a high degree of sensitivity and specificity, thereby ensuring the accuracy of the detection results.

Insulated isothermal PCR

Insulated isothermal PCR (iiPCR) is a nucleic acid amplification technique that follows the fundamental principles of PCR. The reaction vessel is heated by a single heat source at the bottom of the instrument, based on the Rayleigh-Bénard principle of natural convection [236]. This results in the spontaneous formation of a continuous temperature gradient in the internal fluid, which in turn leads to the rapid PCR amplification processes of denaturation, annealing, and extension at the corresponding temperature levels.

In the context of global outbreaks of PEDV and PDCoV, the capacity to diagnose rapidly has become a critical factor in the prevention and control of these diseases. Zhang et al. investigated the potential use of iiPCR on a portable POCKIT™ device for field detection [237]. The results demonstrated that both PEDV/PDCoV reverse transcription-iiPCRs exhibited high specificity, and the analytical sensitivity was validated through in vitro transcribed RNA and cell culture analyses.

Cross-priming amplification

Cross-priming amplification (CPA) represents an efficient and rapid method for amplifying target sequences, whereby multiple pairs of specific cross-primers are employed [238]. In the reaction system, the cross primers recognize and bind to different regions of the target sequence, thereby initiating the strand displacement reaction and successive amplification cycles. This allows for the amplification of a large number of nucleic acid sequences at a single constant temperature.

Wang et al. developed a rapid method for the detection of PEDV by combining CPA and a nucleic acid test strip (CPA-NATS) [239]. Amplification was conducted using specific primers, and product detection was accomplished through the use of a labeled probe and NATS. The optimized CPA-NATS method exhibits comparable sensitivity and specificity to PCR, with a detection limit as low as 10⁻⁶ dilution, and is suitable for application to clinical diagnosis.

Nanopore sequencing

Nanopore sequencing is a single-molecule sequencing technology that employs nanopores to detect alterations in electrical signals generated by the movement of individual DNA or RNA molecules, thereby determining base sequences [240]. The principal advantage of this technology is its capacity to sequence raw DNA and RNA directly, without the necessity for PCR amplification, due to its ability to achieve a long-read length. Nanopore sequencing has considerable potential for application in a number of areas, including genome splicing, full-length transcriptome analysis, and the detection of large fragment structural variations.

A nanopore sequencing-based metagenomic next-generation sequencing protocol for unbiased identification of the major porcine viruses PRRSV and PEDV was successfully established by Chen et al. [241]. The results demonstrated that the detection limits of the method were 2.3 × 10² and 9 × 10⁴ copies per reaction for PRRSV and PEDV, respectively, and exhibited a high degree of concordance with the PCR results. Moreover, the protocol demonstrated the capacity to accurately identify single or multiple viral infections in a single sample, thereby illustrating its potential utility in the diagnosis of complex diseases. Another study demonstrated that nanopore sequencing is an effective method for detecting PEDV and PoRV-A, producing high-quality reads within a short period after the initiation of sequencing. This further validates the utility of nanopore sequencing in the field of viral diagnostics [242].

Immunological methods

Enzyme-linked immunosorbent assays

Enzyme-linked immunosorbent assay (ELISA) is an immunoassay that employs enzyme-labeled antibodies or antigens to interact with specific antigens or antibodies present in the samples to be tested. This interaction results in a quantitative determination of the antigens or antibodies through an enzyme-catalyzed color development reaction. Depending on the detection strategies, ELISAs currently available for PEDV detection mainly include indirect, sandwich, and competitive methods. (Fig. 4). Figure 4 provides a schematic illustration of the three ELISAs mainly utilized for the detection of PEDV. This figure demonstrates the distinct mechanisms and applications of each ELISA in antigen or antibody detection [243]. In the context of PEDV prevention and control, the development of a novel ELISA assay based on antigenic proteins of mutant strains is of paramount importance for the monitoring of antibody levels in pigs following immunization. Given that PEDV primarily infects piglets through the transfer of maternal antibodies, specifically SIgA and IgG, present in colostrum and milk, and that PEDV IgA can also serve as an effective diagnostic antibody [244].

Fig. 4.

Schematic presentation of three types of ELISA used for the detection of PEDV [243]

In indirect ELISA, a signal is generated to quantify the antigen through the binding of a non-specific antibody to the antigen to be tested. This is then detected by using a specific antibody and an enzyme-labeled secondary antibody [245]. To address the varying antigenic components of PEDV (including S, M, COE, and N proteins), the research team developed multiple indirect ELISA methods, markedly enhancing detection performance by optimizing key component concentrations and reaction conditions [246–249]. In particular, the ELISA based on the spike protein (S1) demonstrated efficacy in the detection of antibodies produced in the early stages of infection and antibodies directed against PEDV in clinical samples [250]. The recombinant M protein-based ELISA was found to be an effective method for distinguishing PEDV from other porcine coronaviruses [251]. Furthermore, the ELISA based on recombinant COE protein exhibited high sensitivity and specificity, rendering it an appropriate choice for mass screening and clinical diagnosis [252]. In addition, ELISAs based on recombinant spike (including S1 and S12), whole virus, and recombinant N proteins demonstrated excellent detection performance [253–257]. Additionally, the researchers developed an indirect ELISA method based on the prevalent strains of PEDV for the detection of IgA antibodies [258]. This method was successfully applied to the detection of IgA antibodies in serum and oral fluid samples, thereby providing a powerful tool for the assessment of mucosal immune responses.

Sandwich ELISA is a frequently utilized ELISA methodology in which two antibodies with disparate epitopes (typically a capture antibody and a detection antibody) are concurrently bound to the target to form a “sandwich” structure, thereby facilitating highly sensitive and specific detection of the target antigen. Fan et al. developed a double antibody sandwich quantitative ELISA (DAS-qELISA) for the detection of PEDV, which achieved high sensitivity and specificity through the use of PEDV N protein-specific monoclonal antibodies and rabbit polyclonal antibodies [259]. In comparison to RT-PCR, the method demonstrated a 95.7% detection compliance and stability for antigen detection in both inactivated vaccine and clinical samples. The validity of monoclonal antibody-based DAS-ELISA for the detection of PEDV also confirmed in the study conducted by Sozzi et al. [260]. Furthermore, the researchers developed ELISAs based on the PEDV S protein using genetic engineering techniques and monoclonal antibody technology, which demonstrated comparable sensitivity and specificity to RT-PCR [261, 262].

The competitive ELISA technique is achieved through competition between the substance to be measured and the enzyme-labeled antigen. Both competitive blocking ELISA developed by Rodák et al. and monoclonal antibody-based competitive ELISA demonstrated high sensitivity and are suitable for mass screening and surveillance of PEDV [263, 264]. In addition, multiple serologic assays such as indirect ELISA, blocking ELISA, fluorescent microsphere immunoassay, and fluorescent focus neutralization assay have shown high sensitivity and specificity, providing a powerful tool for PEDV antibody detection [265]. Notably, nanobodies show potential in PEDV detection due to their unique advantages, and the anti-PEDV N protein nanobodies screened by Ma et al. through phage display technology were successfully applied in the development of blocking ELISA [266]. And the novel blocking ELISA based on biotinylated nanobodies developed by the team is rapid, low-cost and highly specific and reliable for PEDV antibody detection and vaccine efficacy assessment [267].

Immunochromatographic tests

The colloidal gold-based immunochromatographic tests (ICTs) is an immunoassay based on colloidal gold labeling. It is a rapid and straightforward qualitative or semi-quantitative detection technique that relies on the specific binding of a particular antibody or antigen to the substance under examination. Researchers have developed a variety of ICTs for PEDV antibodies and antigens in porcine serum and intestinal mucosal immune systems, as well as PEDV in fecal samples. these methods utilized recombinant N proteins, SIgA, viscous solvent, and mAbs to demonstrate high sensitivity and specificity and some of the methods possess good storage stability [268–273]. In particular, the AuNP dimer synthesized by Ag ion-welding strategy was used as a labeling probe, which further enhanced the sensitivity of PEDV detection and provided an innovative technological tool for POCT [274].

In addition, the europium nanoparticle (EuNP)-based ICT demonstrated a highly effective tool for the rapid diagnosis and epidemiological surveillance of PEDV. An optimized ICT was developed by Xu et al. [275]. The utilization of an EuNPs-labeled mAb as a fluorescent probe markedly enhanced the sensitivity of the assay and mitigated the background fluorescence interference. Another study developed a straightforward and expeditious ICT system integrating Eu(III) microparticles, which enables the quantitative detection of PEDV [276]. The methodology employed a fluorescent strip reader to quantify the fluorescence peak height ratio between the test and control lines, thereby facilitating the highly sensitive and specific identification of PEDV.

Other novel immunoassays

In recent years, researchers have achieved a number of innovative results in the field of PEDV monoclonal antibody development and its rapid detection technology. A focus on key PEDV proteins, including the S protein and its S1/S2 junction region, the S1 subunit, and the N protein, has led to the successful preparation of a variety of high-specificity and high-affinity mAbs through gene coding, recombinant expression, and hybridoma techniques [277–282]. These mAbs have demonstrated excellent performance in a variety of detection platforms, including ELISA, indirect immunofluorescence assay, and flow cytometry. They are not only useful for diagnosing PED but also serve as a powerful tool for further research on virus-host interactions and disease mechanisms.

Furthermore, researchers have devised a multitude of expeditious detection methods founded upon disparate principles. Among these techniques, Liao et al. successfully prepared an anti-PEDV RdRp polyclonal antibody and demonstrated the ability to detect PEDV through immunofluorescence assay and western blotting [283]. High-throughput neutralization test enabled the objectification and automation of the testing process through imaging cytometry, thereby significantly reducing the reading time and facilitating the long-term storage and evaluation of the data [284, 285]. The one-step immunoassay facilitated rapid and efficient PEDV detection by streamlining the diagnostic process [286]. A multiplex immunoassay enabled the simultaneous detection of multiple pathogens, thereby enhancing the efficiency of the test [287]. Quantum dots-labeled ICT demonstrated superior performance in the detection of clinical samples, exhibiting high sensitivity and specificity [288]. Furthermore, an amplified luminescent proximity homogeneous assay linked immunosorbent assay (AlphaLISA) exhibited the benefits of a no-wash process, high sensitivity, rapidity, and a minimal sample requirement (Fig. 5) [289]. AlphaLISA is a homogeneous, no-wash assay based on the proximity-dependent luminescence of microbeads. As illustrated in Fig. 5, the coupling of donor and acceptor microbeads to antibodies specific to PEDV facilitates the formation of a close proximity between the microbeads, thereby enabling energy transfer and the emission of chemiluminescent signals. The paper-based ICT, with its low cost and ease of use, demonstrated considerable promise as a tool for use in pig production systems [290]. These technologies have markedly enhanced the sensitivity, specificity, and convenience of PEDV detection, rendering them particularly well-suited for large-scale screening and surveillance in the field.

Fig. 5.

Schematic diagram of the principle of AlphaLISA [289, 291]

Biosensors

Biosensors utilize biological recognition elements to react specifically with target substances, converting the generated biological signals into measurable signals (e.g., electrochemical, optical, thermal, or acoustic) through transducers. As illustrated in Fig. 6, electrochemical biosensors are capable of detecting target molecules through the observation of current or voltage fluctuations. Optical biosensors utilize light signals to detect target molecules. Thermal biosensors quantify alterations in heat generation resulting from interactions. And acoustic biosensors are capable of detecting alterations in the propagation characteristics of sound waves [292]. Biosensors are widely used in the fields of environmental monitoring and food safety.

Fig. 6.

Schematic diagram of the principle of biosensors [292]

El-Tholoth et al. developed a low-cost, field-deployable 3D-printed microfluidic device for the combined detection of PEDV, TGEV, and PDCoV, with a performance comparable to the RT-qPCR method [293]. Jiang et al. realized a rapid detection of porcine pathogens through a high-throughput microfluidic chip, which significantly shortened the detection cycle and reduced the risk of infection transmission [294].

In the field of electrochemical immunosensors, researchers have developed innovative ultra-sensitive sandwich-type immunosensors [295]. The co-deposition of graphene and Prussian blue was employed to enhance the sensitivity, specificity, and stability of the sensor, which was successfully applied to PEDV detection. Another study demonstrated the potential of a portable immunosensor based on floating gate carbon nanotubes field-effect transistors for rapid identification of PEDV with high sensitivity and specificity [296].

In addition, Victorious et al. developed a rapid on-farm detection method for PEDV using a dual-electrode electrochemical chip and a barcode-released electroactive aptamer, which significantly reduced the time between sample collection and result generation [297]. To address the issue of co-infection with PEDV and rotavirus, the researchers developed a multi-stable AuNPs-based lateral flow immunosensor, which enabled the simultaneous detection of both viruses with high sensitivity and accuracy [298].

Of interest, the ultra-sensitive photoelectrochemical sensing platform based on the LAMP method exhibited exceptional performance in the quantitative detection of PEDV [299]. The extremely low detection limit and wide linear range offer promising avenues for the detection of other pathogens.

Other novel assays

Cruz et al. developed a focus formation assay (FFA) based on Vero cells and PAP staining techniques for the detection and titration of PEDV in microculture systems [300]. The FFA method demonstrated comparable titer estimation capabilities to the standard plaque assay, but with a shorter incubation time and higher sensitivity to field isolates, thus representing an alternative method. Moreover, negative staining electron microscopy techniques and dark-field microscopy-based gold nanorod probe-assisted counting methods have been shown to possess distinctive capabilities in the identification of viral particles, thus offering a highly effective tool for the detection of viruses [301, 302].

In regard to virus concentration technology, research has demonstrated that glass wool is an effective medium for concentrating a range of viruses, including PEDV, in water samples with varying pH levels [303]. The concentration effect can be further optimized through the incorporation of specific additives. Moreover, single-domain antibodies (sdAbs) have been employed in the detection and isolation of PEDV due to their distinctive properties [304]. The construction of nanoprobes and immunomagnetic beads enabled the efficient imaging of PEDV and the separation of clinical samples. Furthermore, Avicel, a mixture of microcrystalline cellulose and carboxymethyl cellulose, has been employed for the detection of viral plaques [305].

The development of multi-detection methods and microfluidic microarray systems has further facilitated the rapid diagnosis of PEDV. The Luminex xTAG platform and microfluidic RT-LAMP chip have demonstrated high sensitivity and the capacity for simultaneous detection of multiple diarrhea-causing pathogens, respectively, which align with the requirements for on-site diagnosis [306, 307]. The electrochemiluminescence platform has markedly enhanced the sensitivity and specificity of PEDV detection through an innovative signal amplification strategy, thereby paving the way for novel coronavirus assays [308, 309].

Smartphone-based readers and ATP bioluminescence technology also demonstrated the potential for portability and real-time feedback in the detection of PEDV. The former enabled on-site quantitative analysis of PEDV by incorporating ambient light sensors, thereby enhancing the potential applications of ICT [310]. And the latter provided real-time feedback on the cleanliness of livestock trailers through the detection of surface ATPs, providing a reliable means of monitoring cleaning and disinfection procedures [311].

Discussion and future prospects

Genomic variation and epidemiology of PEDV

The current epidemiological status of PEDV worldwide evinces its rapid geographic spread, high variability, and substantial economic and health impact on the swine industry.

The genomic variation of PEDV represents a significant factor in its epidemiological characterization and vaccine development. In particular, the high variability of the S gene has resulted in the emergence of multiple genotypes. The G2 genotype has become the predominant prevalent strain globally, which appears to be associated with the use of G2-targeted vaccines. The evolutionary pattern of G2 viruses exhibits a geographic bias, with rapid evolution in Korea and the highest recombination in China [103]. Furthermore, the identification of additional genotypes, including G2c and G2d, has introduced an additional layer of complexity to the epidemiology of PEDV, thereby exacerbating the challenges associated with prevention and control [1, 31–34].

Mutations in the S gene not only affect the biological properties of PEDV, such as pathogenicity and histophilicity, but may also alter its resistance to vaccines [312]. The commercially available vaccines and drugs that have been developed are primarily effective against the classical G1 strains that were prevalent before 2010. However, they are not as effective against the G2 variants [313]. In light of these findings, it is evident that a deeper understanding of the relationship between genotypes and PEDV prevalence is crucial for the development of isotype-specific vaccines against specific genotypes. Furthermore, given the geographic distribution and cross-species transmissibility of PEDV genotypes, global surveillance and study of PEDV is imperative for comprehending its molecular evolution, epidemiology, development of prevention and control strategies, and development of effective vaccines. The monitoring of PEDV epidemiologic dynamics can facilitate the acquisition of new insights that may inform the prevention and control of PEDV and other coronaviruses.

PEDV vaccine research

The initial research conducted into the development of a vaccine to combat PEDV focused on the utilization of traditional methodologies, namely live-attenuated and inactivated vaccines [314]. It has been demonstrated that inactivated vaccine candidates based on G2a strains exhibit greater promise than G2b-based candidates for the development of an efficacious vaccine against the current highly virulent pandemic PEDV strain [315].

The development of novel vaccines, including subunit vaccines, viral vector vaccines, bacterial vector vaccines, and mRNA vaccines, has garnered attention due to their potential for enhanced safety and production efficiencies. These vaccines are regarded as promising solutions to address the limitations of existing vaccines. Remarkably, mRNA vaccines are currently at the vanguard of PEDV vaccine research due to their potential for expeditious design and production. And the development of polyvaccines and polyvalent vaccines also represents a significant avenue for future investigation, particularly in the context of co-infection with porcine enteroviruses.

Areas of interest for future researches include the pathogenic mechanism of PEDV, the quantitative relationship between neutralizing antibodies and protective power, the optimal immunization strategy for sows, the genetic stability and safety of live attenuated vaccines, and the phenomenon of antibody-dependent enhancement. The findings of these studies will provide a theoretical basis and practical guidance for the development of more effective and safer vaccines for PEDV.

PEDV detection methods

Significant advances have been made in the field of PCR-based methods for PEDV. Multiplexed RT-PCR represents an effective improvement in detection efficiency; however, primer design remains a challenging and validated process. RT-qPCR offers the ability to monitor and quantify the virus in real time, though it is a costly method [316]. nRT-PCR demonstrates high sensitivity and specificity, though it is susceptible to aerosol contamination. ddPCR does not require standards, but it is costly and requires high analytical requirements for the results. NanoPCR is highly sensitive, but further research and validation are necessary. Each of these methods has its own advantages and disadvantages, and the optimal choice should be made according to the specific requirements. Further research is needed to enhance the performance and reduce the cost, and to investigate the potential for joint application in order to meet the needs of high-throughput and high-sensitivity testing.

The optimization of nucleic acid detection methods, particularly isothermal amplification technology, offers novel avenues for expedient and cost-effective detection of PEDV. However, primer design, RNA quality, and operation progress affect the accuracy. LAMP is a simple and rapid method, but it is susceptible to aerosol contamination. RPA, RAA, and ERA are performed at room temperature, but the primer design and reaction system are complicated. iiPCR is simple to perform, but the equipment is specific. Of interest, the integration of these technologies with test strips and microfluidic chips serves to enhance the convenience of testing and the applicability of POCT. CRISPR-based isothermal assays show great potential in developing next-generation PEDV molecular diagnostics due to their high sensitivity, specificity and reliability.

Immunoassays for PEDV are indispensable for evaluating the efficacy of immunization, monitoring the progress of vaccination, and forecasting the duration of immunization. Although indirect ELISAs, sandwich ELISAs and competitive ELISAs are widely utilized, their applications are constrained by the financial and storage limitations associated with the specific antibodies used in these assays [317, 318]. The N protein of PEDV is an optimal antigen for early diagnosis; however, ELISA based on the spike protein performs better in vaccinated animals [319]. Moreover, IgG ELISA kits, despite their efficacy, need to take into account age-related discrepancies in detection rates and antibody persistence [320]. Notably, ICTs offer significant advantages in POCT due to their brief testing duration, user-friendliness and cost-effectiveness [321]. Of interest, oral fluid-based testing may offer a straightforward and “animal-friendly” approach to sample collection for nucleic acid- and antibody-based PEDV monitoring in pigs [322].

In the future, the development of immunological assays and nucleic acid detection methods for PEDV will continue to prioritize the enhancement of sensitivity, specificity, rapidity, and cost-effectiveness. Monoclonal antibody technology and recombinant proteins will be employed with greater frequency in immunological assays, with the objective of enhancing the accuracy of ELISAs and ICTs [243]. In the field of nucleic acid detection, isothermal amplification technologies are anticipated to assume a more prominent role in future PEDV detection, due to their rapidity and efficiency. Moreover, next-generation sequencing will furnish invaluable insights into the genomic heterogeneity and evolution of PEDV, thereby facilitating the identification of emerging or re-emerging viruses. Of particular interest is the fact that nanomaterials and nanotechnology, due to their distinctive physicochemical characteristics, demonstrate considerable promise for PEDV detection, especially in enhancing the sensitivity, speed, and simplicity of detection [323]. The advancement of these technologies will further improve the accuracy and efficiency of PEDV detection, thereby providing robust technical support for the prevention and control of PED.

Conclusion

In recent years, researchers have made notable advancements in the field of PEDV research, including significant progress in understanding the virus’s genome, epidemiology, vaccine development, and detection methods. Researchers have gained a deeper understanding of PEDV genomic variation, which provides the basis for analysis of the genetic evolution and typing of the virus. The global epidemic of PEDV remains a significant concern and requires enhanced surveillance, prevention, and control measures. Although traditional vaccines remain in use, the development of novel vaccines, such as mRNA vaccines, is advancing to provide more efficient and safer means of defense and control. Immunological and nucleic acid detection methods have become the main means of PEDV detection. Moreover, novel detection technologies, including next-generation sequencing and methods based on nanomaterials and biosensors, are being developed to provide more options for rapid and accurate detection.

These advances provide significant support for the control of PEDV transmission and the promotion of the healthy development of pig farming. Furthermore, it is our hope that the review of these research results will provide new ideas and methods for research in related fields. Given the considerable variability and complexity of PEDV, further in-depth research is required in the future. Through interdisciplinary collaboration and sustained innovation, we anticipate the realization of more efficacious PEDV prevention and control strategies on a global scale, thereby facilitating the healthy development of pig farming.

Author contributions

LLZ: Conceptualization, Formal analysis, Investigation, Writing—original draft, Writing—review & editing. YZ: Methodology, Formal analysis, Investigation, Writing—review & editing. JYS: Conceptualization, Formal analysis, Investigation, Writing—review & editing. LS: Conceptualization, Methodology, Investigation. PH: Conceptualization, Formal analysis, Investigation. JBY: Conceptualization, Methodology, Formal analysis. YZ: Conceptualization, Methodology, Formal analysis, Writing—review & editing. QPS: Conceptualization, Methodology, Formal analysis, Resources, Writing—review & editing.

Funding

This work was supported by the Natural Science Fund Project of Jiangsu Vocational College of Agriculture and Forestry (Nos. 2022kj32, 2023kj08) and the Natural Science Fund Project of Colleges in Jiangsu Province (No. 22KJB180001), and the College Students’ Innovation and Entrepreneurship Training Program (No. 202313103017Y).

Data availability

Data availability is not applicable to this article as no new data were created or analyzed in this study. No datasets were generated or analysed during the current study.

Declarations

Conflict of interest

The authors declare no competing interests.