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.

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.

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.

How Does Bio-Organic Fertilizer Combined with Biochar Affect Chinese Small Cabbage's Growth and Quality on Newly Reclaimed Land?

The cultivated land area in China is approaching the red line for farmland protection. Newly reclaimed land possesses a large exploratory potential to become a reserved land resource. Identifying a fertilization strategy is vital for improving the poor properties and weak fertility of newly reclaimed land. An experiment was conducted to study the effects of traditional compound fertilizer (Fc) or bio-organic fertilizer (Ft), alone or in combination with biochar addition (6.85 t·ha-1 and 13.7 t·ha-1) on the growth, photosynthesis, yield and quality of Chinese small cabbage (CSC) plant. The results showed that compared to single compound fertilizer application, bio-organic fertilizer application promoted the plant's growth, indicated by the plant height, stem diameter and leaf area index (LAI), and significantly enhanced the yield and dry matter accumulation of CSC. In terms of the combination with biochar, the promoting effects were positively related to the biochar addition rate in the compound fertilizer group, while it was better to apply bio-organic fertilizer alone or in combination with biochar at a low rate of 6.85 t·ha-1. The highest yield was obtained under B2Fc and B1Ft with 29.41 and 37.93 t·ha-1, respectively, and the yield under B1Ft was significantly higher than that under B2Fc. The water productivity (WP) significantly improved in response to both kinds of fertilizer combined with biochar at 6.85 t·ha-1. There was a significant difference between the photosynthetic characteristics of plants treated with single-compound fertilizer and those treated with bio-organic fertilizer. The photosynthetic characteristics increased under compound fertilizer combined with biochar, while they regressed under bio-organic fertilizer combined with biochar. The quality of CSC, especially that of soluble sugars and total phenolics, improved under single bio-organic fertilizer application compared with that under single-compound fertilizer. The nitrite content of the plants increased with increasing biochar addition rate in both fertilizer groups. In conclusion, there is a significant promoting effect of applying bio-organic fertilizer to replace chemical fertilizer alone or combining compound fertilizer with low-rate biochar addition on newly reclaimed land. It is a recommended fertilization strategy to substitute or partially substitute chemical fertilizer with bio-organic fertilizer combined with biochar in newly reclaimed land, and it is of great significance to achieve fertilizer reduction.

Effects of Common Biochar and Acid-Modified Biochar on Growth and Quality of Spinach in Coastal Saline Soils.

The rational development and efficient utilization of saline soils can alleviate the problem of insufficient arable land faced by agricultural production in China. A prominent problem is improving soil salt and water conditions for promoting land resources' productivity in coastal areas. Biochar is widely used for soil improvement, as it has remarkable properties. A pot experiment was conducted to study the effects of two kinds of biochar (common biochar and acid-modified biochar) with three addition rates (2%, 4%, and 8%) on the growth, yield, photosynthetic characteristics, and quality of spinach. The results revealed that 2% and 4% common biochar increased the plant height, stem diameter, and leaf area index, effectively improving the yield of spinach and water productivity, while 8% common biochar was detrimental to the growth of spinach to some extent. Acid-modified biochar significantly benefited the growth and increased the water productivity of spinach, ensuring high yields, while also improved quality. Similarly, acid-modified biochar was less effective at high additions than at low-to-medium additions. The integrated biological response version 2 (IBRV2) values under acid-modified biochar treatments were all significantly higher than those under common biochar, but there is no significant difference among three treatments in the same biochar group, which suggested a pronounced amelioration in spinach growth within saline-alkali soil upon the incorporation of acid-modified biochar. Overall, applying acid-modified biochar at the rate of 4% exhibited enormous potential for increasing the yield and quality of spinach in saline soils.

LDH nanoparticle adjuvant subunit vaccine induces an effective immune response for porcine epidemic diarrhea virus.

Porcine Epidemic Diarrhea (PED) is a highly contagious intestinal disease which mostly caused by Porcine Epidemic Diarrhea Virus (PEDV). The PED has caused huge economic losses to the pig industry all over the world and a valid PEDV vaccine is needed to prevent the infection. In this study, we constructed expression plasmid based on the spike (S) gene of the epidemic PEDV strain. The recombinant eukaryotic S (Se) and prokaryotic S (Sp) subunit proteins were expressed and purified as vaccine antigens. We designed a new subunit vaccine based on S proteins, adjuvanted with layered double hydroxide (LDH). The results indicated that the LDH adjuvanted subunit vaccines induced a better immune effect in terms of antibody level and cellular immune response. In conclusion, this study showed a new design of a PEDV subunit vaccine with nanotechnology and demonstrated the potential for its clinical application.

Characterization and evaluation of the pathogenicity of a natural recombinant transmissible gastroenteritis virus in China.

Porcine transmissible gastroenteritis virus (TGEV) is one of the major etiological agents of viral enteritis and fetal diarrhea in suckling piglets. In this study, a TGEV JS2012 strain was isolated from the feces of piglets in Jiangsu Province, China. The phylogenetic analysis showed that TGEV JS2012 was placed between the Purdue and the Miller clusters. Analysis of recombination confirmed that TGEV JS2012 is a natural recombinant strain between Miller M6 and Purdue 115. Similar to Miller M6, virulent Purdue and China strain TS, in S gene the JS2012 maintained genetic integrity and the characteristics of the TGEV virulent strains. In vivo, TGEV JS2012 caused 100% mortality in newborn piglets, indicating the strong pathogenicity of this isolate. These results reveal that the JS2012 is a novel natural recombinant TGEV with high virulence. Our findings provide valuable information about genetic diversity and infection mechanism of the coronavirus family.

Origin and epidemic status of porcine epidemic diarrhea virus variants in China.

From 2010, porcine epidemic diarrhea virus (PEDV) variants caused sequential outbreaks of disease in Asia and the United States. In this retrospective study, 49 complete spike (S) gene sequences were obtained from PEDV strains collected in China from 2014 to 2016. We observed that variant PEDV strains with novel insertions, deletions, and multiple S gene recombination types were present in China. In addition, mixed infections involving different variant strains were observed in some areas. Based on phylogenetic and recombination analyses, we determined that the newly emerged PEDV variants potentially originated via recombination between the earliest Chinese G1 genogroup strain, JS-2004-2 and earlier Korean pandemic strains. These findings provide important information for understanding ongoing PEDV outbreaks and suggest that novel variants make it more difficult to prevent PEDV infection.

Mortalin restricts porcine epidemic diarrhea virus entry by downregulating clathrin-mediated endocytosis.

Clathrin-mediated endocytosis is a mechanism used for the invasion of cells by a variety of viruses. Mortalin protein is involved in a variety of cellular functions and plays a role in viral infection. In this study, we found that mortalin significantly inhibited the replication of porcine epidemic diarrhea virus (PEDV) through restricting virus entry. Mechanistically, a biochemical interaction between the carboxyl terminus of mortalin and clathrin heavy chain (CLTC) was been found, and mortalin could induce CLTC degradation through the proteasomal pathway, thereby inhibiting the clathrin-mediated endocytosis of PEDV into host cells. In addition, artificial changes in mortalin expression affected the cell entry of transferrin, further confirming the above results. Finally, we confirmed that this host-mounted antiviral mechanism was broadly applicable to other viruses, such as vesicular stomatitis virus (VSV), rotavirus (RV), and transmissible gastroenteritis virus (TGEV), which use the same clathrin-mediated endocytic to entry. These results reveal a new function of mortalin in inhibiting endocytosis, and provide a novel strategy for treating PEDV infections.