Porcine Epidemic Diarrhea Virus Variants Research Articles

Epidemiological monitoring and genetic variation analysis of pathogens associated with porcine viral diarrhea in southern China from 2021 to 2023.

Large-scale outbreaks of virus-associated severe diarrhea have occurred in pig populations since 2010. To investigate the prevalence and genetic evolution of the diarrhea-associated viruses responsible for the outbreaks, we tested 1,791 diarrhea samples collected from 213 pig farms in five provinces in southern China between 2021 and 2023. The test results showed that porcine epidemic diarrhea virus (PEDV) was the most frequently detected virus. The prevalence rates ranged from 47.40 to 52.22% in samples and 76.06% (162/213) in pig farms. Porcine rotavirus (PoRV) was the second common virus, with prevalence rates ranging from 25.81 to 50.81% in samples and 72.77%(155/213) in pig farms. Porcine delta coronavirus (PDCoV) was the third common virus, with prevalence rates ranging from 16.33 to 17.48% in samples and 38.50% (82/213) in pig farms. The detection rates of both transmissible gastroenteritis virus (TGEV) and porcine acute diarrheal syndrome coronavirus (SADS-CoV) were very low, less than 1.01% in samples and less than 3.76% in pig farms. In this study, we found SADS-CoV only in piglet diarrhea samples from Jiangxi, Guangdong, and Guangxi provinces in China, with a prevalence rate of 5.16% (11/213) in pig farms. Co-infection with these diarrhea-associated viruses is a common occurrence. The most common co-infections were PEDV and PoRV, with a prevalence rate of 6.64% (119/1,791), followed by PDCoV and PoRV, with a prevalence rate of 4.19% (75/1,791). Phylogenetic analyses showed that PEDV and PEDV variants prevalent in southern China during the past three years clustered into genotype GIIb and recombinant PEDV subtypes. Among the currently endemic PEDV, the most common mutations occurred in the collagenase equivalent (COE) and epitope regions of the spike gene. PoRV strains were mainly dominated by the G9 subtype, followed by the G5, G3 and G4 subtypes. Our results suggest that variant PEDV, PDCoV and PoRV are the main pathogens of swine diarrhea, and singular- or co-infection with pathogenic enteric CoV is common in pig herds in southern China. Therefore, prevention and control of porcine viral diarrhea should be given high attention.

Porcine Epidemic Diarrhea Virus: Etiology, Epidemiology, Antigenicity, and Control Strategies in China.

Porcine epidemic diarrhea virus (PEDV) is a porcine enteric coronavirus, which is one of the main causative agents of porcine epidemic diarrhea (PED), with 100% morbidity and 80-100% mortality in neonatal piglets. Since 2010, large-scale PED caused by highly pathogenic variants of PEDV has occurred successively in China and other countries in the world, posing a great threat to the global pig industry. It has been demonstrated in many investigations that the classic attenuated vaccine strain, PEDV CV777, is insufficient to fully protect against the PEDV variants. Moreover, the maternally derived antibodies elicited by inactivated vaccines also cannot completely protect piglets from infection. In addition, feedback feeding poses a risk of periodic PEDV recurrence in pig farms, making it challenging to successfully limit the spread of PEDV in China. This review focuses on the etiology, epidemiology, antigenicity, and control strategies of PEDV in China and provides information for the formulation of effective control measures.

Drug repurposing screens identify Tubercidin as a potent antiviral agent against porcine nidovirus infections

The emergence of new coronaviruses poses a significant threat to animal husbandry and human health. Porcine epidemic diarrhea virus (PEDV) is considered a re-emerging porcine enteric coronavirus, which causes fatal watery diarrhea in piglets. Currently, there are no effective drugs to combat PEDV. Drug repurposing screens have emerged as an attractive strategy to accelerate antiviral drug discovery and development. Here, we screened 206 natural products for antiviral activity using live PEDV infection in Vero cells and identified ten candidate antiviral agents. Among them, Tubercidin, a nucleoside analog derived from Streptomyces tubercidicus, showed promising antiviral activity against PEDV infection. Furthermore, we demonstrated that Tubercidin exhibited significant antiviral activity against both classical and variant PEDV. Time of addition assay showed that Tubercidin displayed a significant inhibitory effect on viral post-entry events but not during other periods. Molecular docking analysis indicated that Tubercidin had better docking efficiency and formed hydrophobic interactions with the active pocket of RNA-dependent RNA polymerase (RdRp) of PEDV and other nidoviruses. Additionally, Tubercidin can effectively suppress other porcine nidoviruses, such as SADS-CoV and PRRSV, demonstrating its broad-spectrum antiviral properties. In summary, our findings provide valuable evidence for the antiviral activity of Tubercidin and offer insights into the development of new strategies for the prevention and treatment of coronavirus infections.

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.

Mini-review: microbiota have potential to prevent PEDV infection by improved intestinal barrier.

Porcine epidemic diarrhea virus (PEDV) infection poses a significant threat to the global pig industry. Current prevention and control strategies are inadequate in protecting pigs from new PEDV variants. This review aims to examine the relationship between PEDV and intestinal microbes, and investigate whether modulating intestinal microbes could affect PEDV infection. The mechanisms by which various intestinal microbes affect viral infection were initially introduced. Intestinal microbes can influence enteric viral infection through direct contact, such as binding, or by affecting interferons (IFNs) production and the intestinal barrier. Influencing the intestinal barrier by microbes can impact PEDV infection in young piglets. To narrow down the range of microbes that may influence PEDV infection, this review summarized microbes that change after infection. Short chain fatty acids (SCFAs), bacterial cell components, and toxins from microbes were identified as important mediators affecting PEDV infection. SCFAs primarily strengthen the intestinal barrier and inhibit intestinal inflammation, while bacterial cell components and toxins are more likely to damage the intestinal barrier. Therefore, this review hypothesizes that fecal transplantation, which allows the host to colonize more SCFAs-producing microbes, may prevent PEDV infection. However, these hypotheses require further proof, and the transplantation of intestinal microbes in pigs requires more exploration.

Fangchinoline inhibits the PEDV replication in intestinal epithelial cells via autophagic flux suppression.

Animal and human health are severely threatened by coronaviruses. The enteropathogenic coronavirus, porcine epidemic diarrhea virus (PEDV), is highly contagious, leading to porcine epidemic diarrhea (PED), which causes large economic losses in the world's swine industry. Piglets are not protected from emerging PEDV variants; therefore, new antiviral measures for PED control are urgently required. Herein, the anti-PEDV effects and potential mechanisms of fangchinoline (Fan) were investigated. Fan dose-dependently inhibited a PEDV infection at 24 h post-infection (EC50 value = 0.67 μM). We found that Fan mainly affected the PEDV replication phase but also inhibited PEDV at the attachment and internalization stages of the viral life cycle. Mechanistically, Fan blocked the autophagic flux in PEDV-infected cells by regulating the expression of autophagy-related proteins and changing PEDV virus particles. In summary, Fan inhibits PEDV infection by blocking the autophagic flux in cells. Our findings will help develop new strategies to prevent and treat PEDV infection.

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.

Phylogenetic analysis and molecular characterization of the co-infection of the new variant of the porcine epidemic diarrhea virus and the novel porcine kobuvirus isolated from piglets with diarrhea.

Porcine epidemic diarrhea virus (PEDV) is a virus that can cause diarrhea in pigs, resulting in significant economic losses to the pig industry. The mutation of the virus and its co-infection with other enteroviruses leads to poor control of PEDV infection. In this study, we found that the diarrhea outbreak in a pig farm in Shandong Province was mainly caused by PEDV infection. Through high-throughput sequencing, we also detected one other diarrhea-related virus (porcine kobuvirus). In the phylogenetic analysis and molecular characterization of the detected PEDV S gene and PKV, it was found that the S gene of the PEDV strain detected in this study (named SD22-2) had more mutations than the CV777 strain. The highest homology between PKV (named SD/2022/China) detected in this study and other strains was only 89.66%. Based on polyprotein, we divided SD/2022/China strains into a new grouping (designated group 4) and detected recombination signals. In summary, SD22-2 detected in this study is a new PEDV variant strain, and SD/2022/China strain might be a novel PKV strain. We also found the co-infection of the new PEDV variant and the novel PKV isolated from piglets with diarrhea. Our data suggested the importance of continuous surveillance of PEDV and PKV.

Hypericum japonicum extract inhibited porcine epidemic diarrhea virus in vitro and in vivo.

Porcine epidemic diarrhea virus (PEDV) infection causes lethal watery diarrhea and high mortality in neonatal piglets, leading to huge economic losses in the global swine industry. Currently, the existing commercial vaccines cannot fully control PEDV, so it is urgent to develop effective antiviral agents to complement vaccine therapy. In the present study, we investigated the antiviral effect of Hypericum japonicum extract (HJ) against PEDV in vivo and in vitro. In in vitro assays, HJ could directly inactivate PEDV strains; moreover, it inhibited the proliferation of PEDV strains in Vero or IPI-FX cells at its non-cytotoxic concentrations. Time of addition assays revealed that HJ mainly inhibited PEDV at the later stages of the viral life cycle. In in vivo, compared with the model group, HJ could reduce the viral titers in the intestines of infected piglets, and improve their intestinal pathological, indicating that HJ could protect the newborn piglets from highly pathogenic PEDV variant infection. Furthermore, this effect may be related to the fact that HJ can not only directly inhibit viruses, but also regulate the structure of intestinal microbiota. In conclusion, our results indicate that Hypericum japonicum could inhibit PEDV replication in vitro and in vivo and might possess the potential to develop as the anti-PEDV drug.

Correlation between the IgG/IgA Antibody Response against PEDV Structural Protein and Virus Neutralization.

Porcine epidemic diarrhea (PED) is a highly contagious intestinal infectious disease caused by porcine epidemic diarrhea virus (PEDV). Large-scale outbreaks of PEDV have caused huge economic losses to the pig industry since 2010. Neutralizing antibodies play a pivotal role in protecting piglets from enteric infections. However, there has been no systematic report on the correlations between neutralizing antibody titers (NTs) and absorbance values of IgG or IgA to all PEDV individual structural proteins in clinical serum, fecal, and colostrum samples. In this study, the spike protein S1 domain (S1), membrane protein (M), envelope protein (E), and nucleocapsid protein (N) of the variant PEDV strain AH2012/12 were expressed and purified by using the human embryonic kidney (HEK) 293F expression system. A total of 92 clinical serum samples, 46 fecal samples, and 33 colostrum samples were collected, and the correlations between IgG or IgA absorbance values and NTs were analyzed. R2 values revealed that anti-S1 IgA absorbance values show the highest agreement with NTs in all serum, fecal, and colostrum samples, followed by the N protein. The correlations between anti-E or M IgA and NTs were very low. However, in the colostrum samples, both IgG and IgA to S1 showed high correlations with NTs. In addition, compared with E and M, the highest correlations of IgA absorbance values were with N and S1 in serum and fecal samples. Overall, this study revealed the highest correlation between NTs and IgA to PEDV S1 protein. Therefore, the diagnostic method with anti-S1 IgA can be used as a powerful tool for assessing the immune status of pigs. IMPORTANCE The humoral immune response plays an important role in virus neutralization. Against PEDV, both IgG and the mucosal immune component IgA play roles in virus neutralization. However, which plays a more prominent role and whether there are differences in different tissue samples are not clearly reported. Additionally, the relationship between IgG and IgA against individual structural proteins and viral neutralization remains unclear. In this study, we systematically determined the relationship between IgG and IgA against all PEDV structural proteins and viral neutralization in different clinical samples and found the highest correlation between neutralization activity and IgA to PEDV S1 protein. Our data have important guiding implications in the evaluation of immune protection.

Phylogenetic and Spatiotemporal Analyses of Porcine Epidemic Diarrhea Virus in Guangxi, China during 2017–2022

Since 2010, porcine epidemic diarrhea virus (PEDV) has swept across China and spread throughout the country, causing huge economic losses. In this study, 673 diarrhea samples from 143 pig farms in Guangxi during 2017-2022 were collected and detected for PEDV. Ninety-eight strains were selected for S1 gene analyses and these strains were classified into four subgroups (G1b, G2a, G2b and G2c), accounting for 1.02 (1/98), 75.51 (74/98), 16.33 (16/98) and 7.14% (7/98) of the total, respectively. Importantly, an increased number of strains in the G2c subgroup was found from 2019 onwards. Bayesian analysis revealed that Guigang may have been the epicenter of PEDVs in Guangxi. In addition, Guigang was identified as the primary hub from which PEDVs spread via two routes, namely Guigang-Wuzhou and Guigang-Laibin. Moreover, several coinfections of novel PEDV variants bearing large deletions in the partial S1 protein and PEDVs possessing an intact partial S1 protein were found in pigs. Further recombination analyses indicated that two of the strains, 18-GXNN-6 and 19-GXBH-2, originated from intra-genogroup recombination. Together, our data revealed a new profile of PEDV in Guangxi, China, which enhances our understanding of the distribution, genetic characteristics and evolutionary profile of the circulating PEDV strains in China.

Porcine Epidemic Diarrhea Virus nsp7 Inhibits MDA5 Dephosphorylation to Antagonize Type I Interferon Production.

Porcine epidemic diarrhea virus (PEDV) is a reemerging enteropathogenic coronavirus that causes high mortality in piglets and has catastrophic effects on the global pig industry. PEDV-encoded nonstructural protein 7 (nsp7) is an important component of the viral replication and transcription complex, and a previous study reported that it inhibits poly(I:C)-induced type I interferon (IFN) production, but the mechanism by which this occurs remains unclear. Here, we demonstrated that ectopic expression of PEDV nsp7 antagonized Sendai virus (SeV)-induced interferon beta (IFN-β) production, as well as the activation of transcription factors interferon regulatory factor 3 (IRF3) and nuclear factor-kappa B (NF-κB) in both HEK-293T and LLC-PK1 cells. Mechanistically, PEDV nsp7 targets melanoma differentiation-associated gene 5 (MDA5) and interacts with its caspase activation and recruitment domains (CARDs), which sequester the interactions between MDA5 and the protein phosphatase 1 (PP1) catalytic subunits (PP1α and PP1γ), thereby suppressing MDA5 S828 dephosphorylation and keeping MDA5 inactive. Furthermore, PEDV infection attenuated MDA5 multimerization and MDA5-PP1α/-γ interactions. We also tested the nsp7 orthologs of five other mammalian coronaviruses and found that all of them except severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nsp7 inhibited MDA5 multimerization and SeV- or MDA5-induced IFN-β production. Collectively, these results suggest that the inhibition of MDA5 dephosphorylation and multimerization may be a common strategy employed by PEDV and some other coronaviruses to antagonize MDA5-mediated IFN production. IMPORTANCE Since late 2010, a reemerging porcine epidemic diarrhea virus variant with high pathogenesis has swept through most pig farms in many countries, resulting in significant economic losses. Coronavirus nonstructural protein 7 (nsp7), conserved within the family Coronaviridae, combines with nsp8 and nsp12 to form the viral replication and transcription complex that is indispensable for viral replication. However, the function of nsp7 in the infection and pathogenesis of coronaviruses remains largely unknown. Our present study demonstrates that PEDV nsp7 specifically competes with PP1 for binding MDA5 and impedes the PP1-mediated dephosphorylation of MDA5 at S828, thereby blocking MDA5-mediated IFN production, revealing the complex mechanism utilized by PEDV nsp7 to efficiently escape host innate immunity.

Construction and immunogenicity of a trypsin-independent porcine epidemic diarrhea virus variant.

Porcine epidemic diarrhea virus (PEDV) is a re-emerging enteropathogenic coronavirus that causes high mortality in neonatal piglets. The addition of trypsin plays a crucial role in the propagation of PEDV, but also increases the complexity of vaccine production and increases its cost. Previous studies have suggested that the S2' site and Y976/977 of the PEDV spike (S) protein might be the determinants of PEDV trypsin independence. In this study, to achieve a recombinant trypsin-independent PEDV strain, we used trypsin-dependent genotype 2 (G2) PEDV variant AJ1102 to generate three recombinant PEDVs with mutations in S (S2' site R894G and/or Y976H). The three recombinant PEDVs were still trypsin dependent, suggesting that the S2' site R894 and Y976 of AJ1102 S are not key sites for PEDV trypsin dependence. Therefore, we used AJ1102 and the classical trypsin-independent genotype 1 (G1) PEDV strain JS2008 to generate a recombinant PEDV carrying a chimeric S protein, and successfully obtained trypsin-independent PEDV strain rAJ1102-S2'JS2008, in which the S2 (amino acids 894-1386) domain was replaced with the corresponding JS2008 sequence. Importantly, immunization with rAJ1102-S2'JS2008 induced neutralizing antibodies against both AJ1102 and JS2008. Collectively, these results suggest that rAJ1102-S2'JS2008 is a novel vaccine candidate with significant advantages, including no trypsin requirement for viral propagation to high titers and the potential provision of protection for pigs against G1 and G2 PEDV infections.

In silico and in vitro evaluation of antiviral activity of wogonin against main protease of porcine epidemic diarrhea virus.

The high mortality rate of weaned piglets infected with porcine epidemic diarrhea virus (PEDV) poses a serious threat to the pig industry worldwide, demanding urgent research efforts related to developing effective antiviral drugs to prevent and treat PEDV infection. Small molecules can possibly prevent the spread of infection by targeting specific vital components of the pathogen's genome. Main protease (Mpro, also named 3CL protease) plays essential roles in PEDV replication and has emerged as a promising target for the inhibition of PEDV. In this study, wogonin exhibited antiviral activity against a PEDV variant isolate, interacting with the PEDV particles and inhibiting the internalization, replication and release of PEDV. The molecular docking model indicated that wogonin was firmly embedded in the groove of the active pocket of Mpro. Furthermore, the interaction between wogonin and Mpro was validated in silico via microscale thermophoresis and surface plasmon resonance analyses. In addition, the results of a fluorescence resonance energy transfer (FRET) assay indicated that wogonin exerted an inhibitory effect on Mpro. These findings provide useful insights into the antiviral activities of wogonin, which could support future research into anti-PEDV drugs.`.

Identification of niclosamide as a novel antiviral agent against porcine epidemic diarrhea virus infection by targeting viral internalization

Porcine epidemic diarrhea virus (PEDV), an enteropathogenic coronavirus, has catastrophic impacts on the global pig industry. However, there remain no effective drugs against PEDV infection. In this study, we utilized a recombinant PEDV expressing renilla luciferase (PEDV-Rluc) to screen potential anti-PEDV agents from an FDA-approved drug library in Vero cells. Four compounds were identified that significantly decreased luciferase activity of PEDV-Rluc. Among them, niclosamide was further characterized because it exhibited the most potent antiviral activity with the highest selectivity index. It can efficiently inhibit viral RNA synthesis, protein expression and viral progeny production of classical and variant PEDV strains in a dose-dependent manner. Time of addition assay showed that niclosamide exhibited potent anti-PEDV activity when added simultaneously with or after virus infection. Furthermore, niclosamide significantly inhibited the entry stage of PEDV infection by affecting viral internalization rather than viral attachment to cells. In addition, a combination with other small molecule inhibitors of endosomal acidification enhanced the anti-PEDV effect of niclosamide in vitro. Taken together, these findings suggested that niclosamide is a novel antiviral agent that might provide a basis for the development of novel drug therapies against PEDV and other related pathogenic coronavirus infections.

Resistance of Field-Isolated Porcine Epidemic Diarrhea Virus to Interferon and Neutralizing Antibody.

Variant porcine epidemic diarrhea virus (PEDV), belonging to the genogroup G2b, has higher pathogenicity and mortality than classical PEDV, belonging to the genogroup G1a. To understand the pathogenesis of the G2b PEDV, we examined the resistance of the G2b PEDV to interferon (IFN) and neutralizing antibodies, which are important for controlling PEDV infection. We found that the G2b PEDV showed higher resistance to IFN than G1a PEDV. The G1a PEDV could replicate in IFN-deficient Vero cells, but not in IFN-releasing porcine alveolar macrophages, whereas the G2b PEDV showed similar infectivity in both types of cells. We also found that G2b PEDV was not effectively blocked by neutralizing antibodies, unlike G1a PEDV, suggesting differences in the antigenicity of the two strains. These results provide an understanding of the occurrence of variant PEDV and its pathogenesis.

Molecular Characterization and Phylogenetic Analysis of a Variant Recombinant Porcine Epidemic Diarrhea Virus Strain in China

Simple SummaryWe successfully isolated and identified PEDV strain SC-YB73. The sequence analysis of the SC-YB73 genome identified a six-nucleotide insertion in the E gene, which has not previously been detected in PEDV strains. The phylogenetic analysis based on the complete genome showed that SC-YB73 was clustered in variant subgroup GII-a, which is widely prevalent in the Chinese pig population. The recombination analysis suggested that SC-YB73 originated from the recombination of GDS47, US PEDV prototype-like strains TW/Yunlin550/2018, and COL/Cundinamarca/2014. In future research, we aim to evaluate the function of E-gene insertions using in vitro cellular culture and in vivo animal experiments.Since 2010, a variant of porcine epidemic diarrhea virus (PEDV) has re-emerged in several provinces of China, resulting in severe economic losses for the pork industry. Here, we isolated and identified a variant PEDV strain, SC-YB73, in Guangdong Province, China. The pathological observations of jejunum showed atrophy of villi and edema in the lamina propria. The sequence analysis of the viral genome identified a six-nucleotide insertion in the E gene, which has not previously been detected in PEDV strains. Furthermore, 50 nucleotide sites were unique in SC-YB73 compared with 27 other PEDV strains. The phylogenetic analysis based on the complete genome showed that SC-YB73 was clustered in variant subgroup GII-a, which is widely prevalent in the Chinese pig population. The recombination analysis suggested that SC-YB73 originated from the recombination of GDS47, US PEDV prototype-like strains TW/Yunlin550/2018, and COL/Cundinamarca/2014. In the present study, we isolated and genetically characterized a variant PEDV strain, thus providing essential information for the control of PED outbreaks in China.

Comparative transcriptomic analysis of porcine epidemic diarrhea virus epidemic and classical strains in IPEC-J2 cells

In recent years, porcine epidemic diarrhea (PED) has become widespread and caused huge economic losses for the global pig industry. There is growing evidence that frequent outbreaks of diarrhea are caused by the variants of porcine epidemic diarrhea virus (PEDV) with high pathogenicity. Herein, an epidemic strain of PEDV HLJ strain was isolated and characterized from Heilongjiang Province of China, and the whole genomic expression profile of intestinal porcine epithelial cells (IPEC-J2) infected with HLJ strain was investigated in comparison with classical CV777 strain. A total of 26,851 genes were identified, of these, 25,880 were known genes and 971 were novel genes. There were 258 differentially expressed genes (DEGs) identified between PEDV HLJ-infected and uninfected cells at 24 h post infection (hpi), and 201 DEGs between PEDV HLJ and CV777 infection. A comparative analysis revealed that 258 DEGs were enriched in 468 gene ontology (GO) terms and mapped to 179 KEGG pathways, and 201 DEGs in 1120 GO terms and mapped to 115 KEGG pathways for HLJ-infected cells in contrast to the uninfected and CV777-infected cells, respectively. Specifically, PEDV HLJ strain could activate anti-viral innate immune response and inflammation more intensively than CV777, in which mRNA levels of interferon (IFN-β), chemokines (CCL5 and CXCL10) and pro-inflammatory cytokines (IL-8 and TNF-α) were induced earlier and more strongly. Subsequently, 20 DEGs and 5 proteins were selected and validated by real-time fluorescence quantitative PCR (RT-qPCR) and western blot, and the results were consistent with the transcriptomic analysis. Overall, this study may be helpful for understanding the pathogenesis mechanism of PEDV variants, and contribute to the effective prevention and control of PEDV infection.

Antigenicity Alternations of Variant PEDV S Protein Disclosed by Linear B Cell Epitope Mapping.

The spike protein (S) plays a crucial role in porcine epidemic diarrhea virus (PEDV) infection and induces neutralizing antibodies. Mutations of the S protein are supposed to provide the main antigenic shift leading to the antigenic escape of PEDVs. It is therefore a significant question how much accumulation of antigenic shift could lead to the antigenic escape of the variant PEDV. To provide an answer in the study, B cell epitopes (BCEs) on the S protein of the PEDV vaccine strain CV777 (SCV777) and variant strain SD2014 (SSD2014) were mapped using biosynthetic peptides and rabbit anti-PEDV S serum. Seventy-nine and 68 linear BCEs were identified from SCV777 and SSD2014, respectively. While 66.2% of the BCEs of SSD2014 could be recognized by anti-SCV777 serum and 67.1% of SCV777 BCEs could be recognized by anti-SSD2014 serum, more than 40% of the BCEs identified using anti-SCV777 serum on SCV777 could not be recognized by anti-SSD2014 serum and vice versa. The completely shared BCEs took low percentages of 29.4% and 25.3% for SSD2014 and SCV777, respectively. These results indicate a low conservation of antigenicity of the S protein compared to a relatively high amino acid sequence similarity of 92.2% between the two strains. The study provided a BCE shift reference of PEDV antigenic escape and surveillance control.

Establishment of enzyme-linked immunosorbent assays based on recombinant S1 and its truncated proteins for detection of PEDV IgA antibody

Porcine epidemic diarrhea virus (PEDV) can infect pigs of all ages, especially piglets. PEDV has spread across Asia since the 1980s. The highly virulent variant PEDV broke out on a large scale and caused huge economic losses to the pig industry in late 2010 in China. Rapid detection methods with high specificity and sensitivity are urgently needed for the diagnosis and control of the disease. In this study, we divided the PEDV S1 gene into three segments and constructed the recombinant plasmids pFastBac1-S1T1 (aa 21–279), pFastBac1-S1T2 (aa 280–539) and pFastBac1-S1T3 (aa 540–788), which carry the different antigenic regions of the S1 gene. Truncated S1 proteins PEDV-S1T1/S1T2/S1T3 were obtained by a Bac-to-Bac expression system, with protein sizes of 36 kDa, 38 kDa and 38 kDa, respectively. Recombinant proteins presented high reactivity with the monoclonal antibody against PEDV and positive pig serum. Based on full-length S1 protein and these truncated proteins, we established indirect ELISA methods for the detection of PEDV IgA antibody. A total of 213 clinical serum samples were tested by the above indirect ELISA methods, and IFA was used as the gold standard. ROC curves revealed a significant correlation between S1-ELISA and S1T2-ELISA with a 0.9134 correlation coefficient and favourable sensitivity and specificity of S1-ELISA (93.24%, 95.68%) and S1T2-ELISA (89.33%, 94.16%). Our results also indicated that serum with higher neutralizing activity (SNT ≥ 40) had a higher IgA antibody level based on S1-ELISA, S1T1-ELISA and S1T2-ELISA. In conclusion, both S1-ELISA and S1T2-ELISA can be used as candidate systems for detecting anti-PEDV IgA antibody titers in serum, which can reflect the level of neutralizing activity in pigs after natural infection or vaccination. The above research results provide a basis for the prevention and control of PEDV and can be used in the detection of host anti-infective immunity and evaluation of vaccine immune effects.