Identification of the Effects of 5-Azacytidine on Porcine Circovirus Type 2 Replication in Porcine Kidney Cells.

Porcine circovirus type 2 (PCV2) is the main pathogen causing post-weaning multisystemic wasting syndrome (PMWS), which mainly targets the body's immune system and poses a serious threat to the global pig industry. 5-Azacytidine is a potent inhibitor of DNA methylation, which can participate in many important physiological and pathological processes, including virus-related processes, by inhibiting gene expression. However, the impact of 5-Aza on PCV2 replication in cells is not yet clear. We explored the impact of 5-Aza on PCV2 infection utilizing PK15 cells as a cellular model. Our objective was to gain insights that could potentially offer novel therapeutic strategies for PCV2. Our results showed that 5-Aza significantly enhanced the infectivity of PCV2 in PK15 cells. Transcriptome analysis revealed that PCV2 infection activated various immune-related signaling pathways. 5-Aza may activate the MAPK signaling pathway to exacerbate PCV2 infection and upregulate the expression of inflammatory and apoptotic factors.

Vertical transmission of porcine circovirus-like virus P1 in BALB/c mice.

BACKGROUND: Porcine circovirus-like virus P1 is the animal virus with the smallest genome discovered so far, and it has become widely distributed in the Chinese mainland in recent years. RESULTS: In this study, a BALB/c mouse model was used to reveal P1 infection in female reproductive systems and the vertical transmission of the virus. The female reproductive system, including the ovary and uterus, was harvested on day 14 postinfection and examined for pathological lesions. One-day-old mice without colostrum born from infected or uninfected mothers were collected, and P1 virus distribution in the different organs was investigated. During the trials, all the mice showed no clinical symptoms or gross lesions. However, stillbirth did occur in groups infected with the P1 virus. P1 nucleic acid was detected in the heart, liver, spleen, lung, kidney, and brain tissues of 1-day-old mice born from infected mice. Microscopic lesions in P1-infected female mice were characterized by necrosis of the ovarian follicular granulosa cells and abscission, follicular atresia, necrosis of the endometrial epithelial and uterine glandular epithelial cells, and hyperplasia of the squamous endometrial epithelium. The spermatocytes in the seminiferous tubules of the infected male mice were disorderly arranged, and the germ and Sertoli cells were shed, necrotic, and decreased in number. Immunohistochemical results identified P1-positive particles in the nucleus and cytoplasm of cells from the ovary and uterus of female mice. CONCLUSIONS: This study shows that the P1 virus could cause pathological damage to the reproductive system of female mice and could be transmitted vertically.

Identification of Cell Types and Transcriptome Landscapes of Porcine Epidemic Diarrhea Virus-Infected Porcine Small Intestine Using Single-Cell RNA Sequencing.

Swine coronavirus-porcine epidemic diarrhea virus (PEDV) with specific susceptibility to pigs has existed for decades, and recurrent epidemics caused by mutant strains have swept the world again since 2010. In this study, single-cell RNA sequencing was used to perform for the first time, to our knowledge, a systematic analysis of pig jejunum infected with PEDV. Pig intestinal cell types were identified by representative markers and identified a new tuft cell marker, DNAH11. Excepting enterocyte cells, the goblet and tuft cells confirmed susceptibility to PEDV. Enrichment analyses showed that PEDV infection resulted in upregulation of cell apoptosis, junctions, and the MAPK signaling pathway and downregulation of oxidative phosphorylation in intestinal epithelial cell types. The T cell differentiation and IgA production were decreased in T and B cells, respectively. Cytokine gene analyses revealed that PEDV infection downregulated CXCL8, CXCL16, and IL34 in tuft cells and upregulated IL22 in Th17 cells. Further studies found that infection of goblet cells with PEDV decreased the expression of MUC2, as well as other mucin components. Moreover, the antimicrobial peptide REG3G was obviously upregulated through the IL33-STAT3 signaling pathway in enterocyte cells in the PEDV-infected group, and REG3G inhibited the PEDV replication. Finally, enterocyte cells expressed almost all coronavirus entry factors, and PEDV infection caused significant upregulation of the coronavirus receptor ACE2 in enterocyte cells. In summary, this study systematically investigated the responses of different cell types in the jejunum of piglets after PEDV infection, which deepened the understanding of viral pathogenesis.

First detection and complete genome analysis of porcine circovirus-like virus P1 and porcine circovirus-2 in yak in China.

Porcine circovirus-like virus P1, like porcine circovirus type 2 (PCV2), is a potential pathogen of post-weaning multisystemic wasting syndrome in swine. Yaks are a valuable species and an iconic symbol of the Tibet Plateau which is the highest and largest plateau in the world. In this study, a total of 105 yak diarrheal samples, collected from 13 farms in Linzhi in the Tibet Plateau from January 2019 to December 2021, that were screened for P1 and PCV2 by polymerase chain reaction, 10.48% (n = 11) were positive for P1, 4.76% (n = 5) for PCV2, and 5.71% (n = 6) were positive for coinfection of P1 and PCV2. In addition, the whole genomes of eight P1 strains and eight PCV2 strains were sequenced. Alignment of deduced amino acid sequences of P1 ORF1 and PCV2 ORF2 gene revealed that ON012566 had one unique amino acid mutation at residues 137 (T to P). This mutation has important implication for the study of virus virulence, tissue tropism, and immune response. Phylogenetic analysis shows that the yak-origin P1 strains in this study with cattle-origin P1 reference strains were grouped into one cluster. The yak-origin PCV2 (ON012566) and a buffalo-origin PCV2 (KM116514) reference strain clustered in the same branch in the PCV2b regions. Meanwhile, the remaining PCV2 strains and buffalo-origin PCV2 reference strain (ON012565) clustered in the PCV2d regions. To summarize, to our knowledge, this is the first report on the molecular prevalence and genome characteristics of P1 and PCV2 in yaks in the world and will contribute to further study of the molecular epidemiology, source, and evolution of P1 and PCV2 strains.

Nonstructural Protein 1 of Variant PEDV Plays a Key Role in Escaping Replication Restriction by Complement C3.

Zoonotic coronaviruses represent an ongoing threat to public health. The classical porcine epidemic diarrhea virus (PEDV) first appeared in the early 1970s. Since 2010, outbreaks of highly virulent PEDV variants have caused great economic losses to the swine industry worldwide. However, the strategies by which PEDV variants escape host immune responses are not fully understood. Complement component 3 (C3) is considered a central component of the three complement activation pathways and plays a crucial role in preventing viral infection. In this study, we found that C3 significantly inhibited PEDV replication in vitro, and both variant and classical PEDV strains induced high levels of interleukin-1ß (IL-1ß) in Huh7 cells. However, the PEDV variant strain reduces C3 transcript and protein levels induced by IL-1ß compared with the PEDV classical strain. Examination of key molecules of the C3 transcriptional signaling pathway revealed that variant PEDV reduced C3 by inhibiting CCAAT/enhancer-binding protein ß (C/EBP-ß) phosphorylation. Mechanistically, PEDV nonstructural protein 1 (NSP1) inhibited C/EBP-ß phosphorylation via amino acid residue 50. Finally, we constructed recombinant PEDVs to verify the critical role of amino acid 50 of NSP1 in the regulation of C3 expression. In summary, we identified a novel antiviral role of C3 in inhibiting PEDV replication and the viral immune evasion strategies of PEDV variants. Our study reveals new information on PEDV-host interactions and furthers our understanding of the pathogenic mechanism of this virus. IMPORTANCE The complement system acts as a vital link between the innate and the adaptive immunity and has the ability to recognize and neutralize various pathogens. Activation of the complement system acts as a double-edged sword, as appropriate levels of activation protect against pathogenic infections, but excessive responses can provoke a dramatic inflammatory response and cause tissue damage, leading to pathological processes, which often appear in COVID-19 patients. However, how PEDV, as the most severe coronavirus causing diarrhea in piglets, regulates the complement system has not been previously reported. In this study, for the first time, we identified a novel mechanism of a PEDV variant in the suppression of C3 expression, showing that different coronaviruses and even different subtype strains differ in regulation of C3 expression. In addition, this study provides a deeper understanding of the mechanism of the PEDV variant in immune escape and enhanced virulence.

Porcine circovirus-like virus P1 activates pancreatic secretion pathway by interacting with CHRM3 protein.

The porcine circovirus-like virus P1, a member of the circovirus family, causes post-weaning multisystemic wasting syndrome (PMWS) in weaned piglets with progressive wasting as the main clinical symptom. The pancreatic secretion pathway induces pancreatic acinar cells to secrete various digestive enzymes and as such is an important signaling pathway for the digestive system and somatic growth. This study examined the effects and mechanism of P1 virus infection on the pancreatic secretion pathway. The experiment was conducted by transfecting double-copy plasmid P1 into PK-15 and 3D4 cells and by infecting cells with the P1 virus. Samples were collected at various times after transfection or infection. The pathway's transcription and translation levels of CHRM3, Gq, PLC-ß2, PRKCA, Rab3D, RhoA, Rac1, and amyA proteins were detected by real-time PCR and Western blots; these analyses confirmed that the P1 virus infection could upregulate the expression level of key pancreatic secretion signaling molecules. Then, we confirmed that the VP1 protein of the P1 virus could interact with the pathway initiation protein CHRM3 using Co-IP, pull-downs, and confocal fluorescence microscopy. Finally, we demonstrated that the VP1 protein activates the pancreatic secretory pathway through the CHRM3 protein. In conclusion, this study demonstrated that the P1 virus can interact with the CHRM3 receptor protein to activate the pancreatic secretion pathway and promote the secretion of various digestive enzymes downstream of the pathway, thereby providing a basis for P1 virus pathogenesis.

Nearly 20 Years of Genetic Diversity and Evolution of Porcine Circovirus-like Virus P1 from China.

Porcine circovirus-like virus P1 can infect many kinds of animals and mainly causes postweaning multisystemic wasting syndrome. In China, the genetic diversity, variation, and evolutionary processes of this virus have not been described yet. To improve our knowledge of its genetic diversity, evolution, and gene flow, we performed a bioinformatics analysis using the available nucleotide sequences of the P1 virus; among them, 12 nucleotide sequences were from ten pig farms in Jiangsu Province in this epidemiological survey, and 84 sequences were downloaded from GenBank. The P1 sequences showed a rich composition of AT nucleotides. Analyses of the complete genomic sequences were polymorphic and revealed high haplotype (gene) diversity and nucleotide diversity. A phylogenetic analysis based on the NJ method showed that all P1 virus sequences formed two distinct groups: A and B. High genetic differentiation was observed between strains from groups A and B. The codon usage pattern of P1 was affected by dinucleotide compositions. Dinucleotide UU/CC was overrepresented, and dinucleotide CG was underrepresented. The mean evolutionary rate of the P1 virus was estimated to be 3.64 × 10-4 nucleotide substitutions per site per year (subs/site/year). The neutrality tests showed negative values. The purifying selection and recombination events may play a major driving role in generating the genetic diversity of the P1 population. The information from this research may be helpful to obtain new insights into the evolution of P1.

Interaction of porcine circovirus-like virus P1 capsid protein with host proteins.

BACKGROUND: Porcine circovirus-like virus P1 is a relatively new kind of virus that is closely related to the post-weaning multisystemic wasting syndrome, congenital tremors, and abortions in swine. The molecular mechanisms of P1 virus infection and pathogenesis are fully unknown. To analyze P1 and its host interactions, we used a yeast two-hybrid (Y2H) assay to identify cellular proteins interacting with the Cap of the P1 virus. In this study, the Cap of the P1 virus exhibited no self-activation and toxicity to yeast cells and was used as bait to screen the Y2H library prepared from the pancreas tissue. RESULTS: Five cellular proteins (EEP, Ral GDS, Bcl-2-L-12, CPS1, and one not identified) were found to interact with P1 Cap. The interaction between Cap and Ral GDS was confirmed by co-immunoprecipitation. CONCLUSIONS: Our data are likely to support the future investigation of the underlying mechanism of P1 infection and pathogenesis.

Quantitative proteomic analysis reveals that serine/threonine kinase is involved in Streptococcus suis virulence and adaption to stress conditions.

The eukaryotic-type serine/threonine kinase of Streptococcus suis serotype 2 (SS2) performs critical roles in bacterial pathogenesis. In this study, isobaric tags for relative and absolute quantification (iTRAQ) MS/MS were used to analyze the protein profiles of wild type strain SS2-1 and its isogenic STK deletion mutant (Δstk). A total of 281 significant differential proteins, including 147 up-regulated and 134 down-regulated proteins, were found in Δstk. Moreover, 69 virulence factors (VFs) among these 281 proteins were predicted by the Virulence Factor Database (VFDB), including 38 downregulated and 31 up-regulated proteins in Δstk, among which 15 down regulated VFs were known VFs of SS2. Among the down-regulated proteins, high temperature requirement A (HtrA), glutamine synthase (GlnA), ferrichrome ABC transporter substrate-binding protein FepB, and Zinc-binding protein AdcA are known to be involved in bacterial survival and/or nutrient and energy acquisition under adverse host conditions. Overall, our results indicate that STK regulates the expression of proteins involved in virulence of SS2 and its adaption to stress environments.

Co-infection analysis of bacterial and viral respiratory pathogens from clinically healthy swine in Eastern China.

Porcine respiratory disease complex (PRDC) is one of the most challenging health concerns for pig production worldwide. The aim of the present study was to determine the prevalence of pathogens associated with PRDC, including porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) and bacterial agents, such as Streptococcus suis, Haemophilus parasuis and Actinobacillus pleuropneumoniae, in clinically healthy pigs in Eastern China. Molecular detection revealed positive single-pathogen detection rates of 59.9%, 27.2%, 52.3%, 33.2% and 0.4% for PCV2, PRRSV, S. suis, H. parasuis and A. pleuropneumoniae, respectively. Co-infection with more than one pathogen was frequently detected in these samples, with PCV2/S. suis, H. parasuis and PCV2/H. parasuis mixed infection rates of 35.4%, 33.2% and 21.6%, respectively, and PCV2/S. suis/H. parasuis and PRRSV/PCV2/S. suis co-infection rates of 21.6% and 6.2%, respectively. These results suggest that mixed infections are prevalent among PRDC cases in swine, which may pose a greater threat to the health of herds compared with single-pathogen infections.

Characterization of porcine circovirus-like virus P1 replication and lesions in BALB/c mice.

Five-week-old male BALB/c mice were inoculated intraperitoneally with a single (sP1) or multiple doses (mP1) of porcine circovirus-like virus P1 or cell culture medium. None of the mice exhibited clinical signs or gross lesions throughout the study. However, the body weights of the mP1 mice were significantly decreased, and the mice inoculated with P1 exhibited viral replication, seroconversion, and microscopic lesions. P1 nucleic acid was detected in the heart, liver, spleen, lung, bladder, testis, brain, thymus, and pancreatic tissues. Special P1 antibody was found in the P1-inoculated mice. Microscopic lesions in the sP1 and mP1 mice were characterized by interstitial pneumonia, including edema in the connective tissue around the pulmonary vessels, mild inflammatory cell infiltrate, thickened alveolar walls, myocardial necrosis, and dissolution of Purkinje cell nuclei. The results showed that the P1 virus could infect BALB/c mice. Thus, BALB/c mice may serve as models for P1 research.

In vitro and In vivo Antibacterial Effects of Nisin Against Streptococcus suis.

Nisin is a promising therapeutic candidate because of its potent activity against Gram-positive bacteria. The present study aimed to describe the in vitro and in vivo antibacterial effects of nisin against Streptococcus suis, an important zoonotic pathogen. The minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) of nisin against different S. suis strains ranged from 0.12 to 4.0 µg/mL and from 0.25 to 8.0 µg/mL, respectively. Time-killing curve assays illustrated that nisin killed 100% of tested virulent S. suis strains within 4 h when used at 2× MIC, which indicates the rapid bactericidal activity of nisin against the bacteria. Transmission and scanning electron microscopy revealed that nisin destroyed S. suis cell membrane integrity and affected its cellular ultrastructure, including a significantly wrinkled surface, intracellular content leakage, and cell lysis. In addition, nisin inhibited biofilm formation by S. suis in a concentration-dependent manner and exhibited strong degrading activities against preformed biofilms. More importantly, nisin displayed antimicrobial activity against S. suis infection in vivo. Upon treatment with 5.0-10 mg/kg nisin solution, the survival rates of mice challenged with a lethal dose of virulent S. suis virulent ranged 87.5-100%. Nisin significantly decreased bacterial proliferation and translocation in the mouse spleen, brain, and blood. These results indicate that nisin has potential as a novel antimicrobial agent for the clinical treatment and prevention of infection caused by S. suis in animals.

Genomic Rearrangement and Recombination of Porcine Circovirus Type 2 and Porcine Circovirus-Like Virus P1 in China.

Porcine circovirus type 2 (PCV2) belongs to the genus Circovirus of the family Circoviridae, and it has been associated with porcine circovirus (associated) disease (PCVD or PCVAD) in pigs. PCVAD is the generic term for a series of disease syndromes that have caused economic losses to the pig industry worldwide. Since the discovery of PCV2 in the late 1990s, the virus has continued to evolve, and novel genotypes have continued to appear. Moreover, there has been recombination between different genotypes of PCV2. This review attempts to illustrate some progress concerning PCV2 in genome rearrangement and genomic recombination with non-PCV2-related nucleic acids, particularly focusing on the porcine circovirus-like virus P1 formed by the recombination of PCV2. The presence of rearranged PCV2 genomes can be demonstrated both in vivo and in vitro, and these subviral molecules ranged from 358 to 1,136 bp. Depending on whether it has the ability to encode a protein, the agents formed by PCV2 recombination can be divided into two categories: porcine circovirus-like viruses and porcine circovirus-like mini agents. We mainly discuss the porcine circovirus-like virus P1 regarding genomic characterization, etiology, epidemiology, and pathogenesis. Further research needs to be conducted on the pathogenicity of other porcine circovirus-like viruses and porcine circovirus-like mini agents and the effects of their interactions with PCV2, especially for the porcine circovirus-like mini agents that do not have protein-coding functions in the genome.

Development of a Novel Double Antibody Sandwich Quantitative Enzyme-Linked Immunosorbent Assay for Detection of Porcine Epidemic Diarrhea Virus Antigen.

Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea and dehydration in sucking piglets with a high mortality rate. Here, we developed a double antibody sandwich quantitative enzyme-linked immunosorbent assay (DAS-qELISA) for detection of PEDV using a specific monoclonal antibody against PEDV N protein and anti-PEDV rabbit serum. Using DAS-qELISA, the detection limit of recombinant PEDV N protein and virus titer were approximately 1 µg/L and 102.0 TCID50/ml, respectively. A total of 90 intestinal and 237 fecal samples were then screened for the presence of PEDV using DAS-qELISA and reverse transcriptase PCR (RT-PCR). DAS-qELISA had a high specificity of 98.1% and sensitivity of 93.5%. The accuracy rate between DAS-qELISA and RT-PCR was 95.7%. More importantly, the viral antigen concentrations remained unchanged before and after one inactivated vaccine preparation by using the DAS-qELISA. These results suggest DAS-qELISA could be used for antigen detection of inactivated vaccine samples and clinical samples. It is a novel method for diagnosing diseases and evaluation of the PEDV vaccine.

Complete genome sequences of porcine circovirus-like virus P1 mutants with 163 amino acids in the capsid protein.

Porcine circovirus-like virus P1 is a novel circovirus that was originally detected in China in 2005. Here, we report the genome sequences of P1 isolates JS02, JS03, and HuN06, each with 163 amino acids in its capsid protein. The complete genome of each of these isolates contains 649 nucleotides and has a T insertion at position 207. Phylogenetic analysis based on complete genome sequences of 18 P1 reference strains grouped 16 P1 sequences from this study into one cluster, with the JS02, JS03, and HuN06 isolates forming an independent clade. However, phylogenetic analysis based on amino acid sequences of the capsid protein showed that the JS02, JS03, and HuN06 strains were on the same large branch with PCV2, distinct from other P1 isolates. These results help us to understand the origin and evolution of P1.

First molecular identification of porcine circovirus-like agents in dogs and cats in China.

Porcine circovirus-like agents comprise two types of viruses: porcine circovirus-like viruses (P1, P2, P3, and P4) and porcine circovirus-like mini agents (PCVL258, PCVL264, PCVL201, and PCVL347). Of these, P1 has been identified in pigs, cattle, goats, and rabbits in China; P2, P3, P4, PCVL258, and PCVL264 have been identified in pigs; and PCVL201 and PCVL347 have been identified in cattle. The purpose of this study was to determine whether dogs and cats have been exposed to porcine circovirus-like agents. We screened 158 serum samples from diseased dogs and 41 from cats in China by PCR and nucleotide sequencing. In dogs, approximately 18% (n = 28) were positive for P1, 17% (n = 26) for PCVL258, and 9% (n = 14) for PCVL264; in cats, 17.1% (n = 7) were positive for P1, 9.8% (n = 4) for P4, and 14.6% (n = 6) for PCVL258. The P1 genomes in this study consisted of 648 nucleotides (nt) and shared 96.8 to 100% nt identity with other P1 genomes in GenBank. The P4 genome shared 98.3 to 100% nt identity with other reported P4 genomes, and PCVL258 and PCVL264 showed 100% nt identity with previously reported genomes. To our knowledge, this is the first report on molecular characterization of porcine circovirus-like agents in dogs and cats. Further studies are needed to clarify the epidemiology, evolution, and pathogenesis of porcine circovirus-like agents in dogs and cats.

Basigin-CyP elevated porcine circovirus type2 replication.

Porcine circovirus type2 (PCV2) is a member of the circoviridae family. PCV2 was identified as the main pathogen of postweaning multisystemic wasting syndrome (PMWS) in weaned piglets and causes massive economic loss. Basigin, is a transmembrane glycoprotein belonging to the immunoglobulin superfamily; which is also a receptor for cyclophilins. CyP belongs to the immunophilin family that has peptidyl-prolyl cis-trans isomerase activity. Basigin-CyP interaction affects the replication stages of several viruses. In this study, we found that Basigin could elevate the replication of PCV2, and the Basigin only affected the replication stage rather than adsorption or endocytosis stages. In addition, the ligands of Basigin, CyPA and CyPB also elevated the replication of PCV2. Basigin-CyP interation was necessary for elevating PCV2 replication; At last, CyPs were proved to promote the replication of PCV2 by activating ERK signaling.

PCV2 inhibits the Wnt signalling pathway in vivo and in vitro.

Porcine circovirus type 2 (PCV2) is an important pathogen of the current pig industry. The Wnt signalling pathway plays an important role in the growth of young animals. In this study, we mainly elucidated the relationship between PCV2 and the Wnt signalling pathway. In an in vivo experiment in mice, we demonstrated the downregulatory effects of PCV2 infection on expression levels of downstream components of the Wnt signalling pathway. Weight loss in mice was reversed by activating the Wnt signalling pathway, and the body weight was still significantly higher than that in mice infected with PCV2. We detected levels of growth hormone (GH) in the liver and sera, which showed that GH was also downregulated in mice challenged with PCV2. Lithium chloride, the activator of Wnt signalling, upregulated GH, albeit to a significantly lesser degree than that in corresponding non-stimulated mock mice. In vitro studies showed that PCV2 infection downregulated protein expression of ß-catenin and mRNA expression of matrix metallopeptidase-2 (Mmp2), downregulated protein expression of ß-catenin in the cytoplasm and nucleus, and reduced the activity of the TCF/LEF promoter, demonstrating that PCV2 inhibited activation of the Wnt signalling pathway in vitro. Finally, we found that Rep protein of PCV2 might be responsible for the inhibitory effect.

Nonstructural protein 6 of porcine epidemic diarrhea virus induces autophagy to promote viral replication via the PI3K/Akt/mTOR axis.

Porcine epidemic diarrhea virus (PEDV) has caused, and continues to cause, severe economic losses to the swine industry worldwide. The pathogenic mechanism and immune regulatory interactions between PEDV and the host remain largely unknown. In this study, the interaction between autophagy and PEDV replication in intestinal porcine epithelial (IPEC-J2) cells was investigated. The effects of the structural and nonstructural proteins of PEDV on the autophagy process and the autophagy-related signaling pathways were also examined. The results shown that PEDV replication increased the autophagy flux in IPEC-J2 cells, and that autophagy was beneficial to PEDV replication, which may be one of the reasons for the rapid damage to intestinal epithelial cells and the enhanced virulence of PEDV in both newborn piglets and finishing pigs. When autophagy was pharmacologically induced by rapamycin, PEDV replication increased from 8.5 × 105 TCID50/mL to 8.8 × 106 TCID50/mL in IPEC-J2 cells. When autophagy was pharmacologically suppressed by hydroxychloroquine, PEDV replication decreased from 8.5 × 105 TCID50/mL to 7.9 × 104 TCID50/mL. To identify which PEDV proteins were the key inducers of autophagy, all 4 structural proteins and 17 nonstructural proteins of PEDV were eukaryotic expressed. It was found that the nonstructural protein 6 (nsp6) and ORF3 of PEDV were able to induce significant autophagy in IPEC-J2 cells, but the other proteins were unable to induce autophagy. It was indicated that nsp6-induced autophagy mainly occurred via the PI3K/Akt/mTOR signaling pathway. The results accelerate the understanding of the biology and pathogenesis of PEDV infection and provide new insights into the development of effective therapeutic strategies.

A flagellin-adjuvanted inactivated porcine epidemic diarrhea virus (PEDV) vaccine provides enhanced immune protection against PEDV challenge in piglets.

Since late 2010, outbreaks of porcine epidemic diarrhea (PED) have been reported in the swine industry in China. A variant PEDV strain that differs from strain CV777 causes prevalent PEDV infections which commercial vaccines based on CV777 cannot provide complete protection. In this study, we designed a new vaccine based on the epidemic PEDV strain AH2012/12, adjuvanted with flagellin, a mucosal adjuvant that induces mucosal and systemic production of IgA. Three groups of pregnant sows were immunized twice, with a 14-day interval, with PEDV adjuvanted with flagellin, PEDV alone, or PBS before farrowing, and newborn piglets from each group were selected and challenged with PEDV. Immunization with this vaccine elicited high levels of IgG, IgA, and neutralizing antibodies in the serum and colostrum of sows, and newborn piglets were protected against PEDV while suckling. This study should guide the prevention and control strategies for PEDV infection, thereby reducing the losses associated with this virus.