Porcine Epidemic Diarrhea Virus Antagonizes Host IFN-λ-Mediated Responses by Tilting Transcription Factor STAT1 toward Acetylation over Phosphorylation To Block Its Activation.

Porcine epidemic diarrhea virus (PEDV) is the main etiologic agent causing acute swine epidemic diarrhea, leading to severe economic losses to the pig industry. PEDV has evolved to deploy complicated antagonistic strategies to escape from host antiviral innate immunity. Our previous study demonstrated that PEDV downregulates histone deacetylase 1 (HDAC1) expression by binding viral nucleocapsid (N) protein to the transcription factor Sp1, inducing enhanced protein acetylation. We hypothesized that PEDV inhibition of HDAC1 expression would enhance acetylation of the molecules critical in innate immune signaling. Signal transducer and activator of transcription 1 (STAT1) is a crucial transcription factor regulating expression of interferon (IFN)-stimulated genes (ISGs) and anti-PEDV immune responses, as shown by overexpression, chemical inhibition, and gene knockdown in IPEC-J2 cells. We further show that PEDV infection and its N protein overexpression, although they upregulated STAT1 transcription level, could significantly block poly(I·C) and IFN-λ3-induced STAT1 phosphorylation and nuclear localization. Western blotting revealed that PEDV and its N protein promote STAT1 acetylation via downregulation of HDAC1. Enhanced STAT1 acetylation due to HDAC1 inhibition by PEDV or MS-275 (an HDAC1 inhibitor) impaired STAT1 phosphorylation, indicating that STAT1 acetylation negatively regulated its activation. These results, together with our recent report on PEDV N-mediated inhibition of Sp1, clearly indicate that PEDV manipulates the Sp1-HDAC1-STAT1 signaling axis to inhibit transcription of OAS1 and ISG15 in favor of its replication. This novel immune evasion mechanism is realized by suppression of STAT1 activation through preferential modulation of STAT1 acetylation over phosphorylation as a result of HDAC1 expression inhibition. IMPORTANCE PEDV has developed sophisticated evasion mechanisms to escape host IFN signaling via its structural and nonstructural proteins. STAT1 is one of the key transcription factors in regulating expression of ISGs. We found that PEDV and its N protein inhibit STAT1 phosphorylation and nuclear localization via inducing STAT1 acetylation as a result of HDAC1 downregulation, which, in turn, dampens the host IFN signaling activation. Our study demonstrates a novel mechanism that PEDV evades host antiviral innate immunity through manipulating the reciprocal relationship of STAT1 acetylation and phosphorylation. This provides new insights into the pathogenetic mechanisms of PEDV and even other coronaviruses.

DNAJA3 Interacts with PEDV S1 Protein and Inhibits Virus Replication by Affecting Virus Adsorption to Host Cells.

Porcine epidemic diarrhea virus (PEDV) infection causes huge economic losses to the pig industry worldwide. DNAJA3, a member of the Hsp40 family proteins, is known to play an important role in the replication of several viruses. However, it remains unknown if it interacts with PEDV. We found that DNAJA3 interacted with PEDV S1, initially with yeast two-hybrid screening and later with Co-IP, GST pull-down, and confocal imaging. Further experiments showed the functional relationship between DNAJA3 and PEDV in the infected IPEC-J2 cells. DNAJA3 overexpression significantly inhibited PEDV replication while its knockdown had the opposite effect, suggesting that it is a negative regulator of PEDV replication. In addition, DNAJA3 expression could be downregulated by PEDV infection possibly as the viral strategy to evade the suppressive role of DNAJA3. By gene silencing and overexpression, we were able to show that DNAJA3 inhibited PEDV adsorption to IPEC-J2 cells but did not affect virus invasion. In conclusion, our study provides clear evidence that DNAJA3 mediates PEDV adsorption to host cells and plays an antiviral role in IPEC-J2 cells.

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.

Utility Evaluation of Porcine Enteroids as PDCoV Infection Model in vitro.

Porcine deltacoronavirus (PDCoV) is a novel emerging enteric coronavirus found in pigs. Intestinal enteroids, which partially recreate the structure and function of intestinal villi-crypts, have many physiological similarities to the intestinal tissues in vivo. Enteroids exhibit advantages in studying the interactions between intestines and enteric pathogens. To create a novel infection model for PDCoV, we developed an in vitro system to generate porcine intestinal enteroids from crypts of duodenum, jejunum, and ileum of pigs. Enterocytes, enteroendocrine cells, Paneth cells, stem cells, proliferating cells, and goblet cells were found in the differentiated enteroids. Replication of PDCoV was detected in the cultured enteroids by immunofluorescence and quantitative RT-PCR. Double immunofluorescence labeling demonstrated that PDCoV was present in Sox9-positive intestinal cells and Villin1-positive enterocytes. There were multiple cellular responses shown as changes of transcription of genes related to mucosal immunity, antiviral genes, and marker genes of stem cells and other cells in the enteroids infected with PDCoV. We conclude that the 2-D enteroids derived from porcine jejunum can be used as an in vitro multicellular model for the investigation of pathogenesis and host immune responses to porcine enteric pathogens, such as PDCoV.

Effect of route of inoculation on innate and adaptive immune responses to porcine epidemic diarrhea virus infection in suckling pigs.

The effects of route of administration on systemic and gut mucosal immune responses induced by porcine epidemic diarrhea virus (PEDV) infection in suckling pigs were investigated. Twenty-four conventional 5-day-old suckling piglets were randomly divided into four groups and were inoculated orally, intranasally (I.N.), intramuscularly (I.M.) with PEDV or DMEM (mock). Pigs were monitored daily for clinical signs and fecal viral load. Blood samples were collected at 7, 14, 21 days post infection (dpi) and subjected for the analyses of serum antibody production, T cell and natural killer (NK) cell frequencies, NK cytotoxicity and serum cytokine levels. Oral inoculation led to higher levels of PEDV-specific IgA antibodies in both serum and gut mucosal sites than did other routes of inoculation. Intranasal inoculation elicited significantly higher titers of virus-specific IgG antibodies in serum. PEDV-infected pigs regardless of inoculation routes had significantly lower NK cell frequencies than those of the control pigs at 14 dpi. The orally inoculated pigs had significantly higher CD3+CD8+ T cell frequencies as compared to I.N. or I.M. inoculated pigs at 14 dpi, while there was no significant difference among orally, I.N. or I.M. inoculated pigs and control pigs in CD3+CD4+ T cell frequencies in peripheral blood. PEDV-infected and control pigs had low, but detectable NK cell activities at 14 and 21 dpi, however, NK cell activities were barely detectable at 7 dpi whether the pigs were infected or not. Serum IL-10 levels were induced drastically in orally infected pigs at 7 dpi and then gradually declined. Serum IL-12 levels followed a similar pattern while the fold-change was much lower. In conclusion, oral inoculation may generate more comprehensive immune responses.

The potential role of tubeimosides in cancer prevention and treatment.

Natural compounds are important sources of anticancer drugs. Rhizoma Bolbostemmatis (Chinese name "Tu Bei Mu") is the dry tuber of Bolbostemma paniculatum (Maxim.) Franquet (Cucurbitaceae). It has long been widely used for treating various ailments including cancer in traditional Chinese medicine. Its major pharmacologically active components are the triterpenoid saponins tubeimosides (TBMs) including tubeimoside (TBM) I, II and III. Extensive researches have provided evidences of the anticancer activities of TBMs in different stages of carcinogenesis both in vitro and in vivo model. TBMs could inhibit cell growth and proliferation, induce cell differentiation, apoptosis, autophagy and, inhibit inflammation, and suppress angiogenesis, invasion and metastasis via various signaling pathways. They are effective in combination therapies, particularly at targeting drug-resistant cancer cells. This mini-review aims to summarize and analyze the current knowledge on the pre-clinical studies of anti-tumor effects, the underlying molecular mechanisms and discuss the prospects of the application of TBMs in cancer prevention and treatment. The potential of TBMs as pertinent candidates could be appropriately developed and designed into an efficacious anticancer drug.