Porcine Epidemic Diarrhea Virus Strains Research Articles

Molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus in Xinjiang, China, from 2020 to 2022.

In recent years, the pig industry in Xinjiang, China, has been severely impacted by outbreaks of porcine epidemic diarrhea (PED), despite vaccination efforts. In this study, we investigated the genetic characteristics of currently prevalent porcine epidemic diarrhea virus (PEDV) strains in the region. We collected 548 samples from animals with suspected PED on large-scale pig farms in Xinjiang. Of these, 258 tested positive for PEDV by RT-PCR, yielding an overall positivity rate of 47.08%. S1 gene sequencing and phylogenetic analysis were conducted on 23 randomly selected RT-PCR-positive samples. Three endemic strains of PEDV (PEDV/CH/XU/2020, PEDV/CH/XK/2020, and PEDV/CH/XA/2020) were isolated, and their complete genome sequences were analyzed for evidence of genetic recombination. Sequence comparison of the S gene indicated significant variations in the S1 gene of the Xinjiang strains compared to the vaccine strains CV777, AJ1102, and LWL, with 90.2%-98.5% nucleotide sequence identity. Notably, both the N-terminal and C-terminal domains of the S protein showed significant variation. Genetic evolutionary analysis identified the GIIa subtype as the dominant genotype among the epidemic strains in Xinjiang. Recombination analysis revealed inter-subtype recombination events in the PEDV/CH/XK/2020 and XJ1904-34 strains. These findings highlight the extensive genetic variation in the predominant GIIa genotype of PEDV in Xinjiang, which does not match the genotype of the currently used vaccine strains. These data may guide further efforts toward the development of effective vaccines for the control of PED.

Effects of porcine epidemic diarrhea virus infection on CD21+ B cells activation

Porcine epidemic diarrhea virus (PEDV) is a devastating pathogen of acute- gastrointestinal infectious diseases, which can cause vomiting, diarrhea, dehydration and high morbidity and mortality among neonatal piglets. Humoral immunity plays a vital role in the host anti-PEDV infection process, but the mechanism of PEDV-induced B-cell immune response remains unknown. In this study, the effects of PEDV infection on CD21+ B cell activation were systematically analyzed through animal experiments. Enzyme-linked immunosorbent assays (ELISA) revealed that low levels of serum-specific IgA, IgM, or IgG were detected in piglets after PEDV infection, respectively. Serum interleukin (IL)-6 levels increased significantly at 4 d after infection, and the levels of IL-4, B-cell activating factor (BAFF), interferon (IFN)-γ, transforming growth factor (TGF)-β and IL-10 decreased at 7 d after infection. Fluorescence-activated cell sorting (FACS) showed that expression levels of CD21, MHC Ⅱ, CD40, and CD38 on B cell surfaces were significantly higher. In contrast, the proportions of CD21+IgM+ B cells were decreased in peripheral blood mononuclear cells (PBMCs) from the infected piglets. No differences were found in the percentage of CD21+CD80+ and CD21+CD27+ B cells in PBMCs from the infected piglets. In addition, the number of CD21+B cells in PBMCs stimulated with PEDV in vitro was significantly lower. No significant change in the mRNA expression of BCR molecules was found while the expression levels of paired immunoglobulin-like receptor B (PIR-B), B cell adaptor molecule of 32 kDa (Bam32) and BAFF were decreased. In conclusion, our research demonstrates that virulent strains of PEDV profoundly impact B cell activation, leading to alterations in phenotypic expression and BCR signaling molecules. Furthermore, this dysregulation results in compromised specific antibody secretion and perturbed cytokine production, highlighting the intricate immunological dysfunctions induced by PEDV infection.

Genetic evolution and phylogenetic analysis of porcine epidemic diarrhea virus strains circulating in and outside China with reference to a wild type virulent genotype CHYJ130330 reported from Guangdong Province, China.

During the last decade, porcine epidemic diarrhea virus has detrimental consequences on swine industry, due to severe outbreaks especially in the suckling piglets. In March 2013, an outbreak was reported on a commercial swine farm in Guangdong Province, Southern China. A wild-type PEDV strain named as CHYJ130330 was identified, complete genome was sequenced and deposited in GenBank (accession no. KJ020932). The molecular epidemiological including evolutionary characteristics and pathogenicity assessment were explored during this study with particular interest and focus to develop this candidate strain for new vaccine. The isolates from China pre- and post-2013 shared 96.5-97.2% and 97-99% nt identity respectively with wild-type CHYJ130330 strain which during experimental studies has demonstrated high virulence and 100% mortality in 104 TCID50 group piglets within 5days. The 22 reference strains selected from other parts of the world shared 98-99% identity with our sequence except Chinese (CV777) and S. Korean (vir.DR13, SM98 and atten.DR13) strains sharing 96.8, 97.6, 96.6 and 97.1% identity respectively. The phylogenetic tree revealed most strains reported after 2013 in GII genogroup while the prototype (CV777), S.korean and earlier Chinese (JS2008, 85-7mutant, Atten.vaccine, SD-M, LZC and CH/S) were GI Group. The amino acid sequence of CHYJ130330 E and M protein is highly conserved while ORF3 and N protein having 9 and 17 amino acid substitutions respectively in comparison to CV777 strain. The comparison of full length genome and the structural proteins revealed variations signifying that PEDV variant strains are still the main source of outbreaks in spite of continuous vaccination and also explain the variable trend of large scale outbreaks during this decade as compared to sporadic tendency of disease found before 2010. It is evident from this study that Chinese strains display significant level of mixing with the strains reported from other countries. The strain CHYJ130330 was also adapted successfully to Vero cell line and has shown high virulence in piglets. The information/findings will be helpful to develop a strategy for control of PEDV and have also shown that CHYJ130330 strain has strong virulence and is a more popular clinical strain in recent years, which has the potential to be developed into PEDV vaccine.

Comparison of B cells' immune response induced by PEDV virulent and attenuated strains.

Porcine epidemic diarrhea virus (PEDV) is an acute, highly contagious enterovirus that infects pigs of all ages. The B cells are important for antigen presentation, antibody production, and cytokine secretion to resist infection. However, the role of B cells in PEDV infection remains unclear. In this study, the effects of PEDV virulent (QY2016) and attenuated strains (CV777) on B cells sorted from neonatal piglets, nursery piglets, and gilts were investigated. The results showed that PEDV-QY2016 and PEDV-CV777 could significantly increase the expression of CD54 and CD27 in B cells from neonatal piglets. The percentages of CD80, MHC II, and IgM expressed on neonatal piglet B cells infected with PEDV-QY2016 were significantly lower than those expressed on the B cells infected with PEDV-CV777. Both PEDV-QY2016 and PEDV-CV777 could stimulate IFN-α and GM-CSF secretions in neonatal piglet B cells; IL-1, IFN-α, and IL-4 secretion in nursery piglet B cells; and IL-1, TGF-β secretion, and GM-CSF in gilt B cells. Furthermore, both PEDV-QY2016 and PEDV-CV777 could induce the secretion of IgA, IgM, and IgG in nursery piglet B cells but could not induce the secretion of IgA, IgM, and IgG in neonatal piglet B cells. The secretion of IgA, IgM, and IgG was significantly higher by the PEDV-CV777 strains infected B cells than those by the PEDV-QY2016 strains infected gilt B cells. In conclusion, the surface molecule expression, cytokine secretion, and antibody production of B cells induced by PEDV are closely related to the ages of pigs and the virulence of the PEDV strain.

Inhibition of PEDV viral entry upon blocking N-glycan elaboration

Porcine Epidemic Diarrhea Virus (PEDV) poses a significant threat to the global swine industry, demanding a thorough understanding of its cellular invasion mechanism for effective interventions. This study meticulously investigates the impact of O- and N-linked glycans on PEDV proteins and host cell interaction, shedding light on their influence on the virus's invasion process.Utilizing CRISPR-Cas9 technology to inhibit cell surface O- and N-linked glycan synthesis demonstrated no discernible impact on virus infection. However, progeny PEDV strains lacking these glycans exhibited a minor effect of O-linked glycans on virus infection. Conversely, a notable 40% reduction in infectivity was observed when the virus surface lacked N-linked glycans, emphasizing their pivotal role in facilitating virus recognition and binding to host cells.Additionally, inhibition studies utilizing kifunensine, a natural glycosidase I inhibitor, reaffirmed the significant role of N-linked glycans in virus infection. Inhibiting N-linked glycan synthesis with kifunensine substantially decreased virus entry into cells and potentially influenced spike protein expression.Assessment of the stability and recovery potential of N-linked glycan-deficient strains underscored the critical importance of N-glycans at various stages of the virus lifecycle. In vivo experiments infecting piglets with N-glycan-deficient strains exhibited milder clinical symptoms, reduced virus excretion, and less severe pathological lesions compared to conventional strains.These findings offer promising translational applications, proposing N-glycosylation inhibitors as potential therapeutic interventions against PEDV. The utilization of these inhibitors might mitigate virus invasion and disease transmission, providing avenues for effective antiviral strategies and vaccine development. Nonetheless, further research is warranted to elucidate the precise mechanisms of N-linked glycans in PEDV infection for comprehensive clinical applications.

Isolation and Identification of a Tibetan Pig Porcine Epidemic Diarrhoea Virus Strain and Its Biological Effects on IPEC-J2 Cells.

Porcine epidemic diarrhoea virus (PEDV) is a coronavirus that can cause severe watery diarrhoea in piglets, with high morbidity and mortality rates, seriously hindering the healthy development of the global swine industry. In this study, we isolated a strain of PEDV from Tibetan pigs and named it CH/GS/2022. Subsequently, we screened the apoptosis signals of PEDV-infected IPEC-J2 cells and studied the correlation between apoptosis signals and cell apoptosis. The results showed that different infections of PEDV induced different degrees of apoptosis in cells, and PEDV-induced cell apoptosis was dose-dependent. We then detected the expression of the p53, p38, JNK, Bax, and Bcl-2 genes in the apoptosis signal pathway. The results showed that 24 h after PEDV infection, the expression of the p53, p38, JNK, and Bax genes in IPEC-J2 cells increased significantly, while the expression of the Bcl-2 gene decreased significantly (p < 0.05). Subsequently, we used Western blot to detect the protein levels of these five genes, and the results showed that PEDV infection upregulated the expression of p53, p38, JNK, and Bax proteins (p < 0.05) while downregulating the expression of Bcl-2 protein (p < 0.05). Thus, it was initially inferred that PEDV infection could regulate cell apoptosis by activating the p53, p38, and JNK signalling pathways. Finally, we further investigated the apoptosis of the cells through the use of inhibitors. The results indicated that the p53 inhibitor Pifithrin-α has a significant inhibitory effect on the expression of the p53 protein after PEDV infection and can reverse the expression levels of Bax and Bcl-2 proteins. This suggested that p53 is involved in PEDV-induced cell apoptosis. Similarly, the p38 MAPK inhibitor SB203580 has an inhibitory effect on the expression of the p38 protein and can reverse the expression levels of Bax and Bcl-2 proteins. This suggested that p38 is also involved in PEDV-induced cell apoptosis. On the other hand, the JNK inhibitor SP600125 has no inhibitory effect on the expression of the JNK protein after PEDV infection, but the expression levels of Bax and Bcl-2 proteins have changed. Furthermore, it is noteworthy that SP600125 can inhibit the activity of apoptotic proteins but not their levels, resulting in reduced cell apoptosis. These preliminary results indicated that JNK may be involved in PEDV-induced IPEC-J2 cell apoptosis.

Porcine epidemic diarrhea virus strain CH/HLJ/18 isolated in China: characterization and phylogenetic analysis

BackgroundPorcine epidemic diarrhea (PED) is an infectious disease of the digestive tract caused by the porcine epidemic diarrhea virus (PEDV), characterized by vomiting, severe diarrhea, and high mortality rates in piglets. In recent years, the distribution of this disease in China has remarkably increased, and its pathogenicity has also increased. PEDV has been identified as the main cause of viral diarrhea in piglets. This study aimed to understand the genetic evolution and diversity of PEDV to provide a theoretical basis for the development of new vaccines and the prevention and treatment of PED.MethodsA PEDV strain was isolated from the small intestine of a diarrheal piglet using Vero cells. The virus was identified using reverse transcription-polymerase chain reaction (RT-PCR), indirect immunofluorescence assay (IFA), and transmission electron microscopy. The whole genome sequence was sequenced, phylogenetic analysis was conducted using MEGA (version 7.0), and recombination analysis was performed using RDP4 and SimPlot. The S protein amino acid sequence was aligned using Cluster X (version 2.0), and the S protein was modeled using SWISS-MODEL to compare differences in structure and antigenicity. Finally, the piglets were inoculated with PEDV to evaluate its pathogenicity in newborn piglets.ResultPEDV strain CH/HLJ/18 was isolated. CH/HLJ/18 shared 89.4–99.2% homology with 52 reference strains of PEDV belonging to the GII-a subgroup. It was a recombinant strain of PEDV BJ-2011-1 and PEDV CH_hubei_2016 with a breakpoint located in ORF1b. Unique amino acid deletions and mutations were observed in the CH/HLJ/18 S protein. The piglets then developed severe watery diarrhea and died within 7 d of inoculation with CH/HLJ/18, suggesting that CH/HLJ/18 was highly pathogenic to newborn piglets.ConclusionA highly pathogenic recombinant PEDV GII-a strain, CH/HLJ/18, was identified in China, with unique deletion and mutation of amino acids in the S protein that may lead to changes in protein structure and antigenicity. These results will be crucial for understanding the prevalence and variation of PEDV and for preventing and controlling PED.

AEN Suppresses the Replication of Porcine Epidemic Diarrhea Virus by Inducing the Expression of Type I IFN and ISGs in MARC-145 Cells.

Apoptosis-enhancing nuclease (AEN), which shares close evolutionary relationships with the interferon-stimulated gene 20 protein (ISG20) homologs in humans, is a member of the DEDDh exonuclease family. Numerous studies on various pathogens have identified the essential roles of ISG20 in inhibiting virus replication. However, the fundamental functions of AEN during viral infection remain largely unknown. This study discovered that AEN expression was significantly upregulated in MARC-145 cells infected with Porcine epidemic diarrhea virus (PEDV) strain 85-7. In contrast, the amount of AEN protein decreased as viral replication increased. It was found that PEDV nsp1 and nsp5 mediated the decrease in AEN production, suggesting that an increase in AEN was not conducive to virus replication. By comparing AEN and its exonuclease-inactive mutant AEN-4A, we determined that the antiviral activity of AEN was independent of its exonuclease function. qPCR analyses revealed that AEN and AEN-4A could induce a significant increase in the transcription levels of IFN-α, IFN-β, and ISGs (OASL, IFI44, IFIT2, ISG15, Mx1, Mx2), and that AEN-4A has a higher induction ability. Overexpression of AEN and AEN-4A in MARC-145 cells targeting IFN-β knockdown or IFN-deficient Vero cells showed reduced or a complete loss of antiviral activity of both, suggesting that AEN may activate the type I IFN immune response and promote the expression of ISGs, thereby inhibiting PEDV replication. Taken together, our data prove the novel mechanism of AEN-mediated virus restriction.

Clinical, Pathological and Virological Outcomes of Tissue-Homogenate-Derived and Cell-Adapted Strains of Porcine Epidemic Diarrhea Virus (PEDV) in a Neonatal Pig Model.

Porcine epidemic diarrhea virus (PEDV) is characterized by diarrhea, vomiting, dehydration, and high mortality rates in neonatal piglets. Two distinct genogroups, S-INDEL (G1a, G1b) and non-S INDEL (G2a, G2b, and G2c), circulate worldwide and are characterized by varying degrees of virulence. Here, we compared the early pathogenesis of a PEDV S-INDEL strain obtained from intestine homogenate (CALAF-HOMOG) or adapted to cell culture by 22 passages (CALAF-ADAP) and a virulent non-S INDEL strain (PEDV-USA) in newborn piglets. After orogastric inoculation of PEDV strains, body weight, temperature and clinical signs were monitored for 48 hpi. Pathological studies were performed at 48 hpi and RNA extracts from jejunal content (at 48 hpi) and rectal swabs (at 0 and 48 hpi) were tested for the presence of PEDV RNA as well as sequenced and compared to the inoculum. Piglets inoculated with PEDV-USA and CALAF-HOMOG isolates showed more severe weight loss, diarrhea, villi fusion and atrophy compared to CALAF-ADAP inoculated piglets. The viral load of rectal swabs was higher in the PEDV-USA inoculated group, followed by CALAF-HOMOG and CALAF-ADAP isolates. Similarly, viral RNA load in jejunal content was comparable among PEDV-USA and CALAF-HOMOG inoculated piglets and higher than that of CALAF-ADAP ones. The comparison of three full PEDV sequences of the inocula with the corresponding ones of pigs after 48 hpi yielded a nucleotide identity >99.9%. This study highlights variations in virulence among S-INDEL and non-S INDEL strains and between S-INDEL isolates obtained from homogenate and cell culture.

Pathological and immunohistochemical assessment of the impact of three different strains of swine enteric coronaviruses in the intestinal barrier

Swine enteric coronaviruses, such as porcine epidemic diarrhea virus (PEDV) or transmissible gastroenteritis virus (TGEV), have risen concern for the porcine industry and research community due to the increase in their virulence, their potential recombination capacity and the emergence of new variants. This in vivo study aims to compare the impact of three different strains of swine enteric coronaviruses [(two G1b (S-INDEL) PEDV strains and a recombinant TGEV-PEDV or Swine enteric coronavirus (SeCoV)] in the intestine of 3-weeks-old infected piglets, focusing on the pathology and main components of the intestinal barrier, including the number of goblet cells, and the expression of IgA as well as FoxP3, a regulatory T cell marker. Severity of lesions was evidenced in the three infected groups and was highly correlated with the viral load in feces and the frequency of viral antigen-positive cells. Furthermore, higher cellular death together with an increase in the expression of the FoxP3 marker was detected in the duodenum and jejunum of infected animals at 3 days post-infection. Our results highlight a recruitment of FoxP3+ cells in the small intestine of infected animals which may represent a response to the tissue damage caused by viral replication and cell death. Further studies should be addressed to determine the potential role of these cells during swine enteric coronavirus infections.

Molecular Characterization of Porcine Epidemic Diarrhea Virus from Field Samples in South Korea.

Porcine epidemic diarrhea virus (PEDV) is a highly contagious enteric pathogen of swine. PEDV has been a major problem in the pig industry since its first identification in 1992. The aim of this study was to investigate the diversity, molecular characteristics, and phylogenetic relationships of PEDVs in field samples from Korea. Six PEDVs were identified from the field samples, and the full spike (S) glycoprotein gene sequences were analyzed. A phylogenetic analysis of the S gene sequences from the six isolates revealed that they were clustered into the G2b subgroup with genetic distance. The genetic identity of the nucleotide sequences and deduced amino acid sequences of the S genes of those isolates was 97.9-100% and 97.4-100%, respectively. A BLAST search for new PEDVs revealed an identity greater than 99.5% compared to the highest similarity of two different Korean strains. The CO-26K equivalent (COE) epitope had a 521H→Y/Q amino acid substitution compared to the subgroup G2b reference strain (KNU-1305). The CNU-22S11 had 28 amino acid substitutions compared to the KNU-1305 strain, which included two newly identified amino acid substitutions: 562S→F and 763P→L in the COE and SS6 epitopes, respectively. Furthermore, the addition and loss of N-linked glycosylation were observed in the CNU-22S11. The results suggest that various strains of PEDV are prevalent and undergoing evolution at swine farms in South Korea and can affect receptor specificity, virus pathogenicity, and host immune system evasion. Overall, this study provides an increased understanding of the prevalence and control of PEDV in South Korea.

Failure to experimentally infect 10 days-old piglets with a cell culture-propagated infectious stock of a classical genotype 1a porcine epidemic diarrhea virus.

Porcine epidemic diarrhea virus (PEDV) causes enteric disease in pigs of all ages. PEDV can be grouped into G1 (classical strains) and G2 (variant strains) based on sequence differences in the spike gene. Although several pathogenesis studies using contemporary strains of PEDV have been conducted to date, there is limited information on the pathogenesis of historical PEDV strains in contemporary pigs. This study aimed to investigate the clinical disease course of 10 days-old pigs infected with a classical European G1a PEDV strain from the 1980s which was last passaged in pigs in 1994. Sequencing results confirmed that the virus inoculum was a PEDV strain closely related to the prototype CV777 strain. The PEDV stock was serially passaged three times in Vero cells, and the P3 infectious virus stock was used to inoculate the pigs. A total of 40 pigs were inoculated using the oral route. Pigs showed no enteric disease signs, and PEDV shedding was not detected for 44 days post-inoculation (dpi). At necropsy at 3 (5 pigs) or 7 dpi (5 pigs), no lesions were observed in intestinal sections, which were negative for PEDV antigen by immunohistochemistry. In addition, no IgG or IgA PEDV-specific antibodies in serum or fecal samples for 35 dpi further indicates a lack of infection. Titration of the leftover thawed and refrozen PEDV virus stock inoculum showed that the virus stock retained its infectivity in Vero cell culture and the porcine small intestine enterocytes cell line IPEC-J2. The reasons for the loss of infectivity in pigs are unknown. In conclusion, we showed that a classical G1a PEDV strain successfully propagated in cell cultures could not orally infect 40 piglets.

Whole-Genome, Recombinant, and Phylogenetic Analysis of Porcine Epidemic Diarrhea Virus Strain CH/JSXZ/2015

Background. Porcine epidemic diarrhea virus (PEDV) is an important pathogen causing highly contact infectious intestinal infections in pigs belonging to the family Coronaviridae, genus Coronavirus, which can cause porcine epidemic diarrhea (PED). Since 2010, outbreaks of PEDV variants have caused great economic losses to the swine industry worldwide. Our study will provide the basis for discovering the key points of PEDV variation and help in understanding the trend of popularity and evolution of PEDV in China. Methods. We amplify the complete PEDV CH/JSXZ/2015 genome sequence from naturally infected piglets in Xuzhou, Jiangsu Province, China, by RT-PCR. The comparative genome circle graph, characterization and phylogenetic analysis, and recombination in the PEDV CH/JSXZ/2015 and other PEDVs are analyzed by bioinformatics analysis software. Results. At the whole-genome level, CH/JSXZ/2015 showed the highest nucleotide identity (99.5%) with CH/GDZHDM/1401 strain (KR153326.1 and KX016034.1) and the lowest similarity (93.5%) to 85-7-mutant1 strain. CH/JSXZ/2015 S amino acid identity was 90.8%–99.8% compared with other strains. CH/JSXZ/2015 ORF3 amino acid identity was 91.2%–100.0% compared with other PEDV strains. Phylogenetic analysis based on the whole genome, S, and ORF3 revealed that CH/JSXZ /2015 is most genetically close to the CH/GDZHDM/1401 strain (KR153326.1 and KX016034.1) isolated in China in 2014. In addition, we revealed that the CH/GDZHDM/1401 (KX016034.1) strain, PEDV 1842/2016 ITA, and CH/JSXZ /2015 had recombination, and the position of recombination was 1–5530 and 28185–28544 bp. 5531–28184 bp of JSXZ are highly homologous to CH/GDZHDM/1401 (KX016034.1), while 1–5530 and 28185–28544 bp of JSXZ are highly homologous to 1842/2016 ITA. These results helped discover PEDV variation rules and provided a basis for preventing and controlling PEDV. Conclusions. Our study revealed that PEDV CH/JSXZ/2015 was a variant strain, the current epidemic strain in China. The CH/GDZHDM/1401 (KX016034.1) strain, PEDV 1842/2016 ITA (KY111278) and CH/JSXZ /2015 had recombination.

Inhibitory effects of quercetin on porcine epidemic diarrhea virus in vitro and in vivo

Porcine epidemic diarrhea (PED) is an acute, severe, highly contagious disease. Porcine epidemic diarrhea virus (PEDV) strains are prone to mutation, and the immune response induced by traditional vaccines may not be strong enough to be effective against the virus. Therefore, there is an urgent need to develop novel anti-PEDV drugs. This study aimed to explore the therapeutic effects of quercetin in PEDV infections in vitro (Vero cells) and in vivo (suckling piglets). Using transmission electron microscopy and laser confocal microscopy, we found that PEDV infection promotes the accumulation of lipid droplets (LDs). In vitro, studies showed that quercetin inhibits LD accumulation by down-regulating NF-κB signaling and IL-1β, IL-8, and IL-6 levels, thereby inhibiting viral replication. In vivo, studies in pigs demonstrated that quercetin can effectively relieve the clinical symptoms and intestinal injury caused by PEDV. Collectively, our findings suggest that quercetin inhibits PEDV replication both in vivo and in vitro, which provides a new direction for the development of PED antiviral drugs.

Isolation, identification, and pathogenicity of porcine epidemic diarrhea virus.

Porcine epidemic diarrhea (PED) is an enterophilic infectious disease caused by the porcine epidemic diarrhea virus (PEDV), which can lead to dehydration-like diarrhea in piglets with a mortality rate of up to 100%, causing huge economic losses to the global pig industry. In this study, we isolated two PEDV strains, FS202201 and JY202201, from diarrheal samples collected from two new PED outbreak farms in 2022. We performed phylogenetic analysis of the S gene and whole gene sequence. The effects of the different mutations on viral pathogenicity were investigated using piglet challenge experiments. The results showed that both strains belong to the G2c subtype, a widely prevalent virulent strain. Compared with FS202201, JY202201 harbored substitution and deletion mutations in nsp1. Both FS202201 and JY202201 infected piglets showed severe diarrhea and significant intestinal tissue lesions at an infection dose of 104 TCID50/mL, with a mortality rate of 50%; however, JY202201 required an additional day to reach mortality stabilization. An infection dose of 103 TCID50/mL reduced diarrhea and intestinal tissue lesions in piglets, with mortality rates of the two strains at 16.7% and 0%, respectively. In addition, PEDV was detected in the heart, liver, spleen, lungs, kidneys, mesenteric lymph nodes, stomach, large intestine, duodenum, jejunum, and ileum, with the highest levels in the intestinal tissues. In conclusion, this study enriches the epidemiology of PEDV and provides a theoretical basis for the study of its pathogenic mechanism and prevention through virus isolation, identification, and pathogenicity research on newly identified PED in the main transmission hub area of PEDV in China (Guangdong).

Genetic signatures associated with the virulence of porcine epidemic diarrhea virus AH2012/12.

Porcine epidemic diarrhea (PED) caused by PED virus (PEDV) remains a big threat to the swine industry worldwide. Vaccination with live attenuated vaccine is a promising method to prevent and control PED, because it can elicit a more protective immunity than the killed vaccine, subunit vaccine, and so on. In this study, we found two obvious deletions in the genome of a high passage of AH2012/12. We further confirmed the second deletion which contains seven amino acids at the carboxy-terminus of the S2 gene and the start codon of ORF3 can reduce its pathogenicity in vivo. Animal experiments indicated that the recombinant PEDV with deleted carboxy-terminus of S gene showed higher IgG, IgA, neutralization antibodies, and protection effects against virus challenge than the killed vaccine. These data reveal that the engineering of the carboxy-terminus of the S2 gene may be a promising method to develop live attenuated vaccine candidates of PEDV.

Deletion of a 7-amino-acid region in the porcine epidemic diarrhea virus envelope protein induces higher type I and III interferon responses and results in attenuation in vivo.

Porcine epidemic diarrhea virus (PEDV) leads to enormous economic losses for the pork industry. However, the commercial vaccines failed to fully protect against the epidemic strains. Previously, the rCH/SX/2016-SHNXP strain with the entire E protein and the rCH/SX/2015 strain with the deletion of 7-amino-acid (7-aa) at positions 23-29 in E protein were constructed and rescued. The pathogenicity assay indicated that rCH/SX/2015 is an attenuated strain, but rCH/SX/2016-SHNXP belongs to the virulent strains. Then, the recombination PEDV (rPEDV-EΔaa23-aa29)strain with a 7-aa deletion in the E protein was generated, using the highly virulent rCH/SX/2016-SHNXP strain (rPEDV-Ewt) as the backbone. Compared with the rPEDV-Ewt strain, the release and infectivity of the rPEDV-EΔaa23-aa29 strain were significantly reduced in vitro, but stronger interferon (IFN) responses were triggered both in vitro and in vivo. The pathogenicity assay showed that the parental strain resulted in severe diarrhea (100%) and death (100%) in all piglets. Compared with the parental strain group, rPEDV-EΔaa23-aa29 caused lower mortality (33%) and diminished fecal PEDV RNA shedding. At 21 days, all surviving pigs were challenged orally with rPEDV-Ewt. No pigs died in the two groups. Compared with the mock group, significantly delayed and milder diarrhea and reduced fecal PEDV RNA shedding were detected in the rPEDV-EΔaa23-aa29 group. In conclusion, the deletion of a 7-aa fragment in the E protein (EΔaa23-aa29) attenuated PEDV but retained its immunogenicity, which can offer new ideas for the design of live attenuated vaccines and provide new insights into the attenuated mechanism of PEDV. IMPORTANCE Porcine epidemic diarrhea virus (PEDV) causes high mortality in neonatal piglets and remains a large challenge to the pork industry. Unfortunately, no safe and effective vaccines are available yet. The pathogenesis and molecular basis of the attenuation of PEDV remain unclear, which seriously hinders the development of PEDV vaccines. This study found that the rPEDV carrying EΔaa23-aa29 mutation in the E protein induced significantly higher IFN responses than the parental virus, partially attenuated, and remained immunogenic in piglets. For the first time, PEDV E was verified as an IFN antagonist in the infection context and identified as a virulence factor of PEDV. Our data also suggested that EΔaa23-aa29 mutation can be a good target for the development of live attenuated vaccines for PEDV and also provide new perspectives for the attenuated mechanism of PEDV.

Full-length genome sequence of porcine epidemic diarrhea virus strain SDTA13-2020.

We report here the complete genome sequence of porcine epidemic diarrhea virus (PEDV) strain SDTA13-2020, isolated from a suckling piglet with watery diarrhea in Shandong, China. The isolate is genetically close to other recent Chinese G2 genotype PEDVs and distinct from the classical PEDVs.

The Characterization and Pathogenicity of a Recombinant Porcine Epidemic Diarrhea Virus Variant ECQ1.

Porcine epidemic diarrhea virus (PEDV), a re-emerging enteropathogenic coronavirus, has become the predominant causative agent of lethal diarrhea in piglets, resulting in huge economic losses in many countries. Furthermore, the rapid variability of this virus has increased the emergence of novel variants with different pathogenicities. In this study, 633 fecal samples collected from diarrheic piglets in China during 2017-2019 were analyzed, and 50.08% (317/633) of these samples were PEDV-positive. The full-length spike (S) genes of 36 samples were sequenced, and a genetic evolution analysis was performed. The results showed that thirty S genes belonged to the GII-a genotype and six S genes belonged to the GII-b genotype. From the PEDV-positive samples, one strain, designated ECQ1, was successfully isolated, and its full-length genome sequence was determined. Interestingly, ECQ1 is a recombinant PEDV between the GII-a (major parent) and GII-b (minor parent) strains, with recombination occurring in the S2 domain of the S gene. The pathogenicity of ECQ1 was assessed in 5-day-old piglets and compared with that of the strain EHuB2, a representative of GII-a PEDV. Although both PEDV strains induced similar fecal viral shedding in the infected piglets, ECQ1 exhibited lower pathogenicity than did EHuB2, as evidenced by reduced mortality and less severe pathological changes in the intestines. These data suggest that PEDV strain ECQ1 is a potential live virus vaccine candidate against porcine epidemic diarrhea.

Transcriptome Profiling of Vero E6 Cells during Original Parental or Cell-Attenuated Porcine Epidemic Diarrhea Virus Infection.

Porcine epidemic diarrhea virus (PEDV) has led to significant economic losses in the global porcine industry since the emergence of variant strains in 2010. The high mutability of coronaviruses endows PEDV with the ability to evade the host immune response, which impairs the effectiveness of vaccines. In our previous study, we generated a highly cell-passaged PEDV strain, CT-P120, which showed promise as a live attenuated vaccine candidate by providing satisfactory protection against variant PEDV infection in piglets. However, the mechanism by which the attenuated CT-P120 adapts to cells during passage, resulting in increased replication efficiency, remains unclear. To address this question, we conducted a comparative transcriptomic analysis of Vero E6 cells infected with either the original parental strain (CT-P10) or the cell-attenuated strain (CT-P120) of PEDV at 6, 12, and 24 h post-infection. Compared to CT-P10, CT-P120 infection resulted in a significant decrease in the number of differentially expressed genes (DEGs) at each time point. Functional enrichment analysis of genes revealed the activation of various innate immune-related pathways by CT-P10, notably attenuated during CT-P120 infection. To validate these results, we selected eight genes (TRAF3, IRF3, IFNL1, ISG15, NFKB1, MAP2K3, IL1A, and CCL2) involved in antiviral processes and confirmed their mRNA expression patterns using RT-qPCR, in line with the transcriptomic data. Subsequent protein-level analysis of selected genes via Western blotting and enzyme-linked immunosorbent assay corroborated these results, reinforcing the robustness of our findings. Collectively, our research elucidates the strategies underpinning PEDV attenuation and immune evasion, providing invaluable insights for the development of effective PEDV vaccines.