First detection and molecular characterization of porcine epidemic diarrhea virus (PEDV) in India: evidence of a new variant in Karnataka

Abstract

Background

Porcine epidemic diarrhea (PED) is a significant pig disease causing high mortality in suckling pigs and high morbidity across all age groups. It is highly prevalent in Southeast Asia, posing a threat of transboundary transmission to India. Although antibodies were detected as early as 2003 in Assam, there was no evidence of viral detection or molecular characterization until this study. This study reports the first clinical outbreak of PED in India, followed by the detection and genetic characterization of PED virus (PEDV) during 2022-23.

Methods

The outbreak was characterized, and fecal samples (n = 21) were collected from affected pigs. These samples were screened for PEDV using RT-PCR, targeting the N, S, and M genes. Serosurveillance was conducted in eight districts, and serum samples (n = 339) were tested for PEDV antibodies using ELISA. Partial N, S, and M gene sequencing, followed by phylogenetic analysis using MEGA v11.0.13, was performed to identify the prevailing genotype and variations in the coding region.

Results

This study identified the first clinical outbreak of PEDV in India, with morbidity rates of 55-57.1% and symptoms including yellow watery diarrhea, vomiting, and abdominal pain. PEDV was confirmed in 17 of 21 fecal samples by amplifying the N, S, and M genes. Serosurveys showed seropositivity in Mandya (2.8%), Bengaluru Rural (6.6%), and Kolar (21.6%), districts indicating PEDV circulation in the state of Karnataka, India. The phylogenetic analysis of the S and M genes placed our study sequences within the Genotype 2a (G2a) clade, aligning with other known G2a strains. In contrast, the phylogenetic tree of the N gene clustered our sequences within the Genotype 1a (G1a) clade suggesting potential recombination. The Indian PEDV strains clustered with strains of China, with unique amino acid substitutions in the S gene, particularly in the receptor binding region.

Conclusion

This study reports the first clinical outbreak of PED in India and identifies the circulating genotype of PEDV. The study emphasizes the need for large-scale surveillance studies to understand the disease’s status. Understanding PEDV’s genetic diversity and evolution is essential to develop area-specific vaccines to mitigate the disease impact on India’s pig population.

Background

Porcine epidemic diarrhea (PED) is an economically significant enteric disease in pigs, causing high mortality in suckling piglets, though pigs of all ages are susceptible with morbidity rates reaching up to 100% [1]. PED is caused by the porcine epidemic diarrhea virus (PEDV), of genus Alphacoronavirus and family Coronaviridae is an enveloped, positive-sense, single-stranded RNA virus with a 28 kb genome. Clinically, the disease is characterized by yellow watery diarrhea, vomiting, anorexia, weight loss, and signs of abdominal pain. PEDV targets enterocytes, leading to significant intestinal damage [2].

The virus was first identified in the UK and Belgium in the 1970s and was isolated two years later [3]. Although the disease was controlled in Europe, it has become one of the leading causes of piglet diarrhea in Asia [4], with high prevalence reported in China, South Korea, Japan, and Vietnam [5,6,7,8]. In India, the pig population is largely concentrated in the northeastern states and piglet diarrhea is a frequently observed condition. Laboratory investigations of clinical samples have detected pathogens such as E. coli, Salmonella, Rotavirus, and Picobirnavirus, which occur either individually or as mixed infections [9,10,11]. Although PEDV had not been detected in the country until the current study, serological evidence suggests probable circulation of PEDV in the pig population [12].

As a transboundary animal disease, PEDV can easily spread across borders through various means, including the trade of live animals and animal products, thus posing a persistent threat to regions with interconnected pig value chains (13–14). India, with its extensive and informal pig trade networks, particularly between Northeast India and Southeast Asia, is at considerable risk of PEDV introduction, reintroduction and dissemination. The Indian states of Mizoram and Nagaland share an international border with Myanmar, while Arunachal Pradesh shares a border with China. Although reports of PEDV from Myanmar are not available, the disease is endemic in most Southeast Asian countries, including China, Vietnam, Thailand, and the Philippines. Hence, the risk of disease spread to India is high. Therefore, understanding the PEDV circulating in India and its genetic relatedness with strains of other countries is crucial for identifying the potential origins of PEDV in India and strengthening biosecurity measures to prevent future infections. Previous studies have shown the existence of an informal pig trade network connecting Northeast India to South India, which can potentially spread diseases to the rest of the country [15].

Surveillance is an important means of early detection of cases in new geographical region. Early detection of PEDV is crucial for control and prevention of the disease spread [16]. The PEDV genome encodes several structural proteins, with the Nucleocapsid (N), Membrane (M), and Spike (S) proteins being particularly crucial for virus detection and molecular characterization. The N gene encodes the nucleocapsid protein, which is highly conserved and often used as a target for diagnostic assays due to its abundance and stability [6]. The M gene encodes the membrane protein, integral to viral assembly and morphogenesis, and plays a role in inducing host immune responses, making it another key target for molecular diagnostics. The S gene encodes the spike protein, which is responsible for virus attachment and entry into host cells [17]. This gene exhibits significant genetic variability, making it essential for studying viral evolution, pathogenesis, and for distinguishing between different PEDV strains [17]. The variability in the S gene, in particular, allows for the identification of new variants and contributes to understanding the virus’s spread and epidemiology.

Genotyping of porcine epidemic diarrhea virus (PEDV) is crucial for understanding its genetic diversity, evolution, and epidemiology. Different genotypes of PEDV have been identified globally, each potentially varying in virulence, transmissibility, and host range. Genotyping revealed distinct genetic lineages and the emergence of new variants, which may have implications for disease management and vaccine development [18–19]. In particular, genotyping has been instrumental in tracking the spread of PEDV strains across regions and identifying evolutionary changes that may impact disease dynamics [20]. Understanding the genetic characteristics of circulating PEDV strains through genotyping is essential for implementing targeted control measures, such as vaccine development tailored to specific genetic variants, and for enhancing surveillance strategies to monitor virus evolution and emergence of new strains [21].

Serosurveillance of PEDV is essential to know the disease status in the country. The positive rate in sero-surveillance can vary significantly depending on several factors, including the endemicity of the disease, population being tested, the timing of the sampling relative to an outbreak, and the sensitivity and specificity of the assays used. In general, sero-surveillance studies in swine populations have reported positive rates ranging from 5.5% [22] to over 61.66% [23]. The surveillance study in India reported PEDV seropositivity of 21.2% from North East region of the country [12]. India being a vast country with diverse agroclimatic zones and varied husbandry practices highlights the need for broader surveillance to assess the disease’s spread and impact on swine health and productivity. PEDV is a highly contagious pathogen associated with severe morbidity and mortality in piglets, representing a potential threat to swine populations where data on prevalence are sparse [2]. Conducting seroprevalence studies provides critical insights into PEDV exposure and immunity levels, informing biosecurity, herd management, and vaccination strategies. The Enzyme-Linked Immunosorbent Assay (ELISA) is a valuable tool for such studies, due to its sensitivity, specificity, and adaptability for large-scale screening [17]. Using PEDV viral proteins, such as the spike (S) and nucleocapsid (N) proteins, as coating antigens, ELISA enables reliable antibody detection, supporting disease surveillance efforts where routine screening is limited [6]. Establishing baseline seroprevalence, particularly in regions like India’s Northeast, can help guide early interventions and reduce the risk of outbreaks.

In light of above observations, outbreak of piglet diarrhea was attended in the pig population of Karnataka (South India) with the objectives of detecting and genetically characterizing PEDV and performing serosurveillance.

Materials and methods

Outbreak description and sample collection

In 2022–2023, two outbreaks of atypical piglet diarrhea, unresponsive to antibiotic treatment, were investigated in Bengaluru rural district of Karnataka, India. Clinical samples (fecal swabs and serum) were collected from affected pigs following standard protocols. Data on the number of cases, deaths, and the total pig population were gathered for both outbreaks.

To assess the antibody prevalence of PEDV in the pig population, a pilot sero-survey was conducted across eight districts of Karnataka, India, involving semi-organized pig farms. The selected districts were characterized by active pig farming with semi-intensive structure comprising breeders, growers and finishers. Assuming high disease risk and potential for spread, a total of 339 pig serum samples were randomly collected from the selected districts. The serum samples were screened for PEDV antibodies using a commercial indirect ELISA kit (ID Screen^® PEDV Indirect). The kit detects the antibodies against nucleoprotein of PEDV.

PCR based screening of clinical samples

A total of 21 fecal samples were processed to detect PEDV by PCR. RNA was extracted from a 10% fecal suspension of each sample using QIAamp Viral RNA Mini Kit (Qiagen), a viral RNA extraction kit. The presence of PEDV was screened using a nucleocapsid (N) gene-specific RT-PCR method [24]. PCR products were separated on a 2.0% agarose gel, and the results were documented using a gel documentation system (Vilber-Eppendorf, India Pvt. Ltd.). Additional screening for PEDV genes, specifically the spike (S) and membrane protein (M) genes, was performed using established RT-PCR protocols [25, 26]. Concurrently, samples were screened for TGEV using recognized RT-PCR protocol [27].

Nucleotide sequencing and genotyping of PEDV

PCR-amplified products of the N (394 bp), M (675 bp), and S (651 bp) genes were gel-purified and sequenced commercially using Sanger sequencing. The positive control (extracted PEDV viral RNA from infected cells) used in RT-PCR was donated by Dr. Linda J. Saif, Distinguished University Professor, Center for Food Animal Health, OARDC, College of Veterinary Medicine, The Ohio State University, USA. The nine S sequences from this study were deposited in GenBank with accession number. The N-terminal subunit of the spike protein (S), N and M gene sequences obtained in this study were aligned with reference sequences (n-26) from global PEDV strains using the Muscle module of MEGA version 11.0.13. To evaluate the genetic relatedness of the PEDV strains circulating in Karnataka, India, with those from various global regions, phylogenetic trees were constructed for each gene segment (N, M, and S) using reference sequences from GenBank (19 N gene references, 21 M gene references, and 26 S gene references). Phylogenetic analysis included 9, 6, and 19 sequences of the S, M, and N genes, respectively, obtained from this study. The phylogenies were built using the Maximum likelihood method and the General Time Reversible (GTR) model in MEGA version 11.0.13 [28]. Bootstrapping (1000 replicates) was performed to assess the reliability of the phylogenetic relationships.

Genome recombination analysis

The S, M, and N gene sequences were analysed individually for potential recombination events, using reference PEDV sequences from China, Vietnam, Japan, South Korea, Germany, and the USA. The sequences retrieved from GenBank, along with those generated in this study, were aligned using MEGA version 11.0.13 [28]. The aligned sequences were then examined for recombination signals using the RDP4 software, which integrates seven recombination detection methods: SiScan, BOOTSCAN, Chimaera, GENECONV, MaxChi, RDP, and 3Seq [29].

Results

Clinical manifestation and descriptive epidemiology

The clinical manifestation of the disease was characterized by yellow, watery diarrhea, vomiting, anorexia, and signs of abdominal pain (Fig. 1). Despite five days of antibiotic treatment, affected pigs showed no improvement. Descriptive epidemiology revealed high morbidity rates (55-57.1%), varying mortality rates (0.2-8%), and case fatality rates (0.5–16%) (Table S1). Deaths were attributed to severe dehydration, with most fatalities occurring in pigs less than two months old.

Fig. 1

Clinical manifestation of Porcine Epidemic Diarrhea. Dehydrated piglets with rough haircoat (A), Yellow watery diarrhea (B, C, D & E)

Table 1 Screening of PED serum and fecal Samples among pig populations in various districts of Karnataka, India (2022-23)

N, S, and M gene specific RT-PCR confirms the diarrhea due to PEDV

A total of 21 fecal samples from both outbreaks were screened for PEDV viral RNA targeting the nucleocapsid gene (N), and 17 samples tested positive by RT-PCR (Fig. S1). N gene-positive samples were further screened for S and M genes (Fig. S1). Representative amplicons of N, S and M genes were sequenced and confirmed to be PEDV. Although virus isolation attempts have been unsuccessful in four blind passages thus far, further passages are currently in progress. The serum samples (n = 21) tested negative for PEDV antibodies, and all samples were also negative for Transmissible Gastroenteritis Virus (TGEV).

Serological surveys revealed a wider circulation of PEDV in Karnataka, India

According to the 20th Livestock Census-2019, the pig population is more concentrated in the districts of northern Karnataka, India, while organized pig farming activity is more prevalent in southern Karnataka. A total of 339 serum samples from eight different districts of southern Karnataka were screened for PEDV antibodies using a commercial ELISA kit (ID Screen^® PEDV Indirect) (Table 1). The positive rate in general was 5.3% (18/339) in sero-surveillance with seropositivity in Mandya (2.8%), Bengaluru Rural (6.6%), and Kolar (21.6%) (Fig. 2). This suggests the wider circulation of PEDV in the pig population of Karnataka, India.

Fig. 2

PED seropositivity among the pig population of sampled eight districts of Karnataka, India during 2022-23. The map depicts the percent seropositivity (The maps were prepared using QGIS version 3.22.1)

PEDV circulating in Karnataka is genotypically unique

Phylogenetic analysis based on partial S gene (636 bp) and deduced amino acid sequences showed that the 9 PEDVs from this study (GeneBank Acc. No. PP066287 - PP066295) formed a distinct cluster, branching with a maximum of 97% relation with the G2a strains (GD1, HNZMD-25, CH-JS-XZ672-2020) circulating in China since 2012 (Fig. 3). S1 similarity with other genotypes ranged from 93.4 to 96.8%, with nucleotide mismatches up to 23 with G2a and up to 42 with G1a type (Table 2).

Fig. 3

Phylogenetic analysis of PEDV based on partial Spike gene sequence: Phylogenetic analysis of partial S gene nucleotide sequences of Indian PEDV. Multiple sequence alignments were performed using the MUSCLE module in MEGA version 11.0.13 and phylogeny was constructed using the Maximum Likelihood method with the General Time Reversible model and Bootstrap value of 1000 replicates. The scale bar indicates the nucleotide substitution per site. Black squares indicate PEDV sequences from the current study

The deduced amino acid sequence of the S gene of the PEDVs from this study, compared with the amino acid sequence of the S protein (NC_003436) of the reference strain (CV777), which is also a vaccine strain, revealed unique amino acid changes in Indian PEDVs at five different locations: A528G, D542E, K563N, D644G, and I659V. Notably, three of these changes occurred in collagenase equivalent regions (COE) (Fig. 4).

Table 2 PEDV S gene homology analysis of Indian strains (GeneBank Acc. No. PP066287 - PP066295) * with reference genotypes using NCBI-BLAST

The phylogenetic analysis of the M gene sequences from this study (PQ594396 to PQ594401) revealed that they cluster within the G2a genotype clade. The G2a clade, which includes reference sequences from countries like China, Japan, South Korea, the United States, and various other regions, represents a highly prevalent and widely distributed genotype. The sequences in this study grouped closely with these international G2a strains, suggesting potential genetic similarity and shared evolutionary history (Fig. 5).

The phylogenetic analysis of partial N gene sequences from our study (PQ594402 to PQ594414) shows that all sequences cluster predominantly within the G1a genotype clade having strains from Germany (1987), Switzerland (2001), China (2006), and South Korea (2010). Bootstrap values, particularly 99% for the G1a clade, indicate strong support for this classification (Fig. 6).

The phylogenetic analysis of PEDV sequences of this study based on partial S, N and M gene revealed that, the analysis of the S and M genes placed our study sequences within the G2a clade, aligning with other known G2a strains. In contrast, the phylogenetic tree of the N gene clustered our sequences within the G1a clade. This discordance in genotypic classification between the S/M and N genes suggests potential recombination. However, the recombination analysis using RDP4 on each of the S, M, and N genes did not detect any recombination events within these individual genes when analysed separately.

Fig. 4

Schematic representation of deduced amino acid sequence analysis of S1 domain of PEDV S protein. The strains (labelled as “Sequences from this study”) from the current study showed mutations at five different locations viz., A528G, D542E, K563N, D644G and I659V of which, three missense changes were in collagenase equivalent regions (COE). The mutations were identified in reference to the CV777 reference strain of PEDV (G1a subgenogroup), however the representative sequences of other subgenogroups (G1b, G2a & G2b) were also included for comparison. The visualization of multiple sequence alignment was generated using Geneious prime [30]

Fig. 5

Phylogenetic analysis of PEDV based on partial membrane protein gene sequences: Phylogenetic analysis of partial M gene nucleotide sequences of Indian PEDV. Multiple sequence alignments were performed using the MUSCLE module in MEGA version 11.0.13 and phylogeny was constructed using the Maximum Likelihood method with the General Time Reversible model and Bootstrap value of 1000 replicates. The scale bar indicates the nucleotide substitution per site. Black squares indicate PEDV sequences from the current study

Fig. 6

Phylogenetic analysis of PEDV based on partial nucleocapsid gene sequences: Phylogenetic analysis of partial N gene nucleotide sequences of Indian PEDV. Multiple sequence alignments were performed using the MUSCLE module in MEGA version 11.0.13 and phylogeny was constructed using the Maximum Likelihood method with the General Time Reversible model and Bootstrap value of 1000 replicates. The scale bar indicates the nucleotide substitution per site. Black squares indicate PEDV sequences from the current study

Discussion

Porcine epidemic diarrhea virus (PEDV) is an emerging pig disease in India, necessitating continuous surveillance, rapid detection, and comprehensive molecular characterization to understand its epidemiology, virulence, and evolution. This research reports for the first time the clinical outbreak of PED among the pig population in India, followed by comprehensive molecular characterization to understand the circulating genotype and seropositivity in pig populations.

The observed morbidity rates of 57.1% and 55% in these outbreaks align with those reported in other Asian countries experiencing PED outbreaks [31]. Notably, the relatively low mortality and morbidity rates identified in our study are characteristic of non-pandemic PEDV strains [31], contrasting with the significantly higher morbidity rate of 100% associated with virulent or pandemic PEDV strains [2]. However, to gain a more comprehensive understanding of PED infection dynamics in the region and to obtain reliable estimates of disease morbidity and mortality, further investigation into a larger number of outbreaks is necessary.

This study conclusively identifies the presence of PEDV in diarrheal outbreaks within the pig population of Karnataka, India, utilizing PCR and gene sequencing techniques. In our PEDV outbreak investigation, we targeted the Nucleocapsid (N), Membrane (M), and Spike (S) genes using RT-PCR because they play critical roles in viral detection, pathogenesis, and evolution. The N gene is highly conserved across PEDV strains, making it a reliable marker for sensitive detection, especially for general diagnostics [32]. The M gene, involved in viral morphogenesis, is similarly conserved, allowing for the detection of structural variations [33]. The S gene, though more variable, is crucial for identifying different genotypes and tracking mutations related to viral infectivity and immune. Including these three genes ensures comprehensive detection of PEDV while capturing both conserved and variable regions, which is critical for understanding viral diversity and epidemiological dynamics. This approach is consistent with common diagnostic methods such as RT-PCR targeting the N gene [34]and S gene sequencing for strain genotyping [35], both of which have been widely adopted. Our multi-gene detection method enhances the robustness of PEDV diagnostics in nonendemic countries and is particularly valuable in characterizing the outbreak strain’s properties and ensuring accurate diagnosis.

Sero-surveillance at the face of outbreak is critical to know the prevalence and spread of the PEDV within defined geographical regions. In the current study, the serum samples collected from clinical cases from the outbreak were negative which might be due to the timing of sample collection relative to the infection phase [16]. However, the serum samples collected from pigs from three of the eight districts surveyed have shown seropositivity suggesting wider circulation of PEDV in pig population of Karnataka state. The varied seropositivity among the three positive districts might be due to differences in farm management practices, biosecurity measures, and pig movement patterns [36]. The sero-surveillance data is crucial for understanding the epidemiology of the outbreak and for identifying high-risk areas that require targeted control efforts [37].

The S1 portion of the S gene in PEDV is often used as a genetic marker to differentiate PEDV genotypes due to its rapid rate of evolution [38]. Phylogenetic analysis based on the partial S gene and deduced amino acid sequences showed that the PEDVs from this study were related to G2a strains circulating in China but they formed a distinct cluster suggesting potentially new variant. Currently, there are no universal consensus guidelines for defining new genogroups or subgenogroups of PEDV. Based on existing literature [19, 39, 40], a 2% divergence at the nucleotide level within the S gene is typically used to delineate subgenogroups. While four subgenogroups are identified as classical types (G1a, G1b, G2a, and G2b), the two new subgenotypes (G2c and G2d) are described with a few inconsistencies [40]. Due to the redundancy of the genetic code, not all nucleotide changes result in significant alterations of the amino acid sequence. The deduced amino acid sequence of the S protein, especially the rapidly evolving S1 domain with the neutralization epitope region, is frequently used to differentiate PEDV genotypes. A seven amino acid substitution in the neutralizing epitopes (499–638 amino acid region of the S protein) differentiates the G1 and G2 genogroups. Further, the classification of the G2c subgenogroup is based on three unique amino acid changes in addition to shared substitutions with the G1a and G2a subgenogroups [19]. The branching of the Indian cluster, which showed 97.0% relatedness with the G2a subgenogroup and exhibits five unique amino acid substitutions along with one shared substitution with G2a, suggests the potential emergence of a new subgenogroup. The detection of this novel variant in the Indian subcontinent likely indicates its early presence [12] and independent evolution, influenced by geographical isolation, host adaptation, and other factors driving the macro and microevolution of RNA viruses [41]. However, it is important to note that our findings are based on partial gene sequences. Further studies involving virus isolation and whole-genome sequencing are essential to confirm the circulation of potentially new genotypes in the region.

Subgenogroups of G2 have displayed characteristic geographical distributions. Most G2a subgenogroup strains are found in the Americas, with a few exceptions in China and Japan, while G2b subgenogroup strains are predominantly endemic to Asia, particularly China, South Korea, and Japan. G2c subgenogroup strains are mainly found in Europe, with some occurrences in the USA and China [19, 42]. The detection of a potentially new variant in our study reinforces the notion of spatial clustering and independent evolution of PEDV in Karnataka, South India. The unorganized pig sector and unstructured trading systems between regions and along the international borders pose a high risk of disease spread. Studies have previously demonstrated a strong association between swine trade and the spread of PEDV in China and across the world [43].

Further, the amino acid changes in the neutralizing epitope region might affect the effectiveness of available vaccines, indicating the need for new vaccine candidates. However, it would be intriguing to determine the genotypes of PEDV in Northeast India, which accounts for nearly 50% of India’s pig population and has traditional trade links with Myanmar, a gateway to Southeast Asia.

The genotypic characterization by S gene phylogeny is strengthened by similar nature of clustering in M gene phylogeny. In contrast the N gene grouped them within G1a. This discordance could be due to historical recombination events or independent selective pressures on different genomic regions of PEDV [44–47]. Studies previously did identify the recombinants of G2a and G1a and grouped such recombinants in to new subgroup [19]. Although no recombination events were detected within the S, M, or N genes individually using RDP4, this does not rule out the possibility of whole-genome recombination events that may involve larger genomic regions beyond these single genes. Our findings are consistent with previous reports indicating recombination as a driver of genetic diversity in coronaviruses, including PEDV [42]. Further whole-genome sequencing and recombination analysis would be necessary to fully elucidate the recombination landscape in PEDV and determine whether genome-wide recombination contributes to the observed genetic divergence in our study sequences. Further, we admit that in our study, the phylogenetic analysis was conducted using partial sequences of the S, M, and N genes, which presents certain limitations. While these gene fragments provide valuable insights into the genetic relatedness of our study sequences, they may not capture the full extent of genomic variation seen in whole-genome analyses. Whole-genome sequencing would provide a more comprehensive view, allowing us to detect potential recombination events and better resolve the genetic lineages.

The difficulty in distinguishing piglet diarrhea caused by PED from other porcine gastroenteric diseases, such as transmissible gastroenteritis (TGE), rotavirus, or bacterial infections, may have contributed to the delayed identification of this disease [48]. Systematic investigation, including timely and appropriate sampling, facilitated the detection of this pathogen in our study. However, attempts to isolate the virus have been unsuccessful after four blind passages. This could be due to amino acid changes in the receptor-binding site of the viral protein or a low viral load in the clinical samples. Previous research has indicated that isolating PEDV from clinical samples is challenging because of low viral loads and the mutation in structural proteins of the virus [49–50]. Additionally, some studies have observed that the virus titre may decrease during serial passages in cell culture [16]. PEDV with mutations in S gene sometimes required up to 20 passages before the virus adapts to the cell culture and result in success isolation [51].

Conclusion

The current study reports the first detection of PEDV in India, identifying a possible new variant circulating in the pig population of Karnataka having unique amino acid substitution within the receptor binding region. Our study highlights the importance of analysing multiple genes for accurate genotyping and suggests that while S and M genes provide a stable classification within G2a, variations in the N gene’s evolutionary history may reflect complex genomic dynamics. Future research incorporating whole-genome data will be essential for a more comprehensive understanding of PEDV evolution and recombination in India. Serosurvey revealed that broader circulation of the virus within the state. Further, comprehensive studies are needed to understand the molecular epidemiology of PEDV in India, and systematic sero-surveillance is essential to ascertain the disease’s burden.