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.

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.

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.

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.

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.

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.

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.

Therapeutic effects of saponin components on porcine reproductive and respiratory syndrome virus-infected piglets.

The present study aimed to evaluate the potential therapeutic effects of Anemoside B4 (AB4), Panax notoginseng saponins (PNS), Notoginsenoside R1 (SR1), Saikosaponin A (SSA) and Saikosaponin D (SSD) on piglets infected with porcine reproductive and respiratory syndrome virus (PRRSV). A total of 132 completely healthy piglets were randomly divided into 22 groups consisting of six animals each. Control piglets were intramuscularly injected with 2 ml of PRRSV (NJGC strain) solution containing 106  TCID50  virus/ml. For low-, middle- and high-dose saponin treatment groups, the piglets were initially administrated with the same volume of PRRSV solution, followed by intraperitoneal injection with AB4, PNS, SR1, SSA or SSD at 1, 5 or 10 mg/kg b.w. on day 3. The piglets in drug control group were intraperitoneally injected with 10 mg/kg b.w. of each saponin without prior PRRS challenge, while those in blank control group were injected with the same amount of normal saline. The results indicated that all the five saponin components could decrease the incidence and severity of PRRSV-induced immunopathological damages, including the elevated body temperature, weight loss, anaemia and internal inflammation. Moreover, the saponin components could enhance protein absorption and immune responses. Taken together, this study reveals that the saponin components are effective against PRRSV infection and strengthen the immune system and thus may serve as potential antiviral therapeutic agents.

Complete genome sequences of porcine-circovirus-like mini agents in pigs and cattle.

Porcine circovirus (PCV) genomes are single-stranded circular DNAs of about 1770 nucleotides (nt). Here, we present for the first time two small PCV-like agents with circular DNA genomes (258 and 264 nt) in pigs and two (201 and 347 nt) in cattle, with no obvious protein-coding capacity. Sequences of the four PCV-like mini agents differed by 1.5%-18.7% from each other and by 4.5%-56.7% from other reference PCV strains and PCV-like viruses.

Effects of sinomenine in LPS-associated diseases are related to inhibition of LBP, Mac-1, and L-selectin levels.

The aim of the research was to investigate the anti-endotoxin and anti-inflammatory effects of Sinomenine, an agent commonly found in Chinese herbal medicines. Endotoxin (i.e., 1 mg lipopolysaccharide (LPS)/kg)) was administered via intraperitoneal (IP) injection to piglets in high-, middle-, and low-dose sinomenine groups. Piglets were then treated with 1, 5 or 10 mg/kg sinomenine, intramuscularly (i.m.), 3 hr after LPS. Vehicle was administered, as above, to drug control group piglets followed 3 hr later by 10 mg/kg sinomenine i.m.. LPS control group piglets were challenged with 1 mg/kg LPS IP, followed by vehicle i.m., and naïve control piglets were treated with normal saline IP, followed by normal saline i.m., as above. Temperatures were measured, and blood samples were collected from the precaval veins of piglets at 12, 24, and 48 hr post-LPS or vehicle injection. Clinical signs were recorded, and index levels were analyzed via ELISA. Sinomenine was found to reduce the incidence and severity of LPS-induced toxicities, including body temperature elevation, cell adhesion, and systemic inflammation. These data suggest that sinomenine may be effective for regulating inflammatory responses and has the potential for use as an anti-endotoxin therapy.

Screening of Streptococcus Suis serotype 2 resistance genes with GWAS and transcriptomic microarray analysis.

BACKGROUND: Swine streptococcosis has caused great economic loss in the swine industry, and the major pathogen responsible for this disease is Streptococcus Suis serotype 2 (SS2). Disease resistance breeding is a fundamental way of resolving this problem. With the development of GWAS and transcriptomic microarray technology, we now have powerful research tools to identify SS2 resistance genes. RESULTS: In this research, we generated an F2 generation of SS2 resistant C57BL/6 and SS2 susceptive A/J mice. With the F2 generation of these two mice strains and GWAS analysis, we identified 286 significant mouse genome SNPs sites associated with the SS2 resistance trait. Gene expression profiles for C57BL/6 and A/J were analyzed under SS2 infection pressure by microarray. In total, 251 differentially expressed genes were identified between these two mouse strains during SS2 infection. After combining the GWAS and gene expression profile data, we located two genes that were significantly associated with SS2 resistance, which were the UBA domain containing 1 gene (Ubac1) and Epsin 1 gene (Epn 1). GO classification and over-representation analysis revealed nine up-regulated related to immune function, which could potentially be involved in the C57BL/6 SS2 resistance trait. CONCLUSION: This is the first study to use both SNP chip and gene express profile chip for SS2 resistance gene identification in mouse, and these results will contribute to swine SS2 resistance breeding.

Swinepox virus vector-based vaccines: attenuation and biosafety assessments following subcutaneous prick inoculation.

Swinepox virus (SPV) has several advantages as a potential clinical vector for a live vector vaccine. In this study, to obtain a safer and more efficient SPV vector, three SPV mutants, Δ003, Δ010, and ΔTK were successfully constructed. A virus replication experiment showed that these SPV mutants had lower replication abilities compared to wtSPV in 10 different host-derived cell lines. Animal experiments with mouse and rabbit models demonstrate that these three mutants and wtSPV did not cause any clinical signs of dermatitis. No fatalities were observed during a peritoneal challenge assay with these mutants and wtSPV in a mouse model. Additionally, the three mutants and wtSPV were not infectious at 60 h after vaccination in rabbit models. Furthermore, we evaluated biosafety, immunogenicity and effectiveness of the three mutants in 65 1-month-old piglets. The results show that there were no clinical signs of dermatitis in the Δ003 and ΔTK vaccination groups. However, mild signs were observed in the Δ010 vaccination groups when virus titres were high, and apparent clinical signs were observed at the sites of inoculation. Samples from all experimental pig groups were assessed by qPCR, and no SPV genomic DNA was found in five organs, faeces or blood. This suggests that the infectious abilities of wtSPV and the SPV mutants were poor and limited. In summary, this study indicates that two mutants of SPV, Δ003 and ΔTK, may be promising candidates for an attenuated viral vector in veterinary medicine.

Protective efficacy of a DNA vaccine encoding capsid protein of porcine circovirus-like virus P1 against porcine circovirus 2 in mice.

The capsid protein is the major immunogenic protein of porcine circovirus 2 (PCV2). The nucleotide sequence of porcine circovirus-like virus P1 shares high homology with open reading frame (ORF) 2 of PCV2, and ORF1 of P1 encodes its structural protein. Mice were vaccinated twice intramuscularly with a plasmid expressing the P1 ORF1 protein (pcDNA3.1(+)-ORF1) at 2-week intervals. All animals vaccinated with pcDNA3.1(+)-ORF1 developed higher specific anti-P1 antibody levels, and had less PCV2 viremia and milder histopathological changes than PCV2-challenged mice in the control group. Our results show that the P1 DNA vaccine elicited immune responses against PCV2 infection in a mouse model.

Development and application of an indirect ELISA for the detection of antibodies to porcine epidemic diarrhea virus based on a recombinant spike protein.

BACKGROUND: As the major causative agent of swine viral diarrhea, porcine epidemic diarrhea virus (PEDV) has caused massive losses to the economies of swine raising countries. Accordingly, the serological detection of corresponding antibodies would be beneficial to diagnose PEDV indirectly to control the disease. In this study, an indirect enzyme-linked immunosorbent assay (ELISA) based on the recombinant truncated spike (S) protein of PEDV was developed and validated. RESULTS: The reaction conditions of the developed indirect ELISA were optimized. This indirect ELISA was compared to indirect immunoinfluscent assay (IFA), and the overall coincidence rate was 96.74% based on testing 368 clinical serum samples with different PEDV antibody levels. No cross-reactivity with other common swine pathogens was detected for the developed S1 indirect ELISA. Finally, the S1 indirect ELISA was applied to detect serum antibodies of 3304 field samples collected from different pig farms in eastern China, and it presented an overall substantial agreement on the PEDV infection status. CONCLUSIONS: This established S1 indirect ELISA is capable of detecting serum antibodies against PEDV, and due to its high sensitivity and specificity, it could be applied for serological evaluation and indirect diagnosis of PEDV infection.

Differential Protein Analysis of IPEC-J2 Cells Infected with Porcine Epidemic Diarrhea Virus Pandemic and Classical Strains Elucidates the Pathogenesis of Infection.

Porcine epidemic diarrhea (PED) re-emerged in China in late 2010 and has now become widespread. Accumulated evidence indicates that this large-scale outbreak of diarrhea was caused by variants of the highly virulent porcine epidemic diarrhea virus (PEDV). A pandemic PEDV YC2014 strain (YC2014) was isolated from clinical samples. An iTRAQ-based comparative quantitative proteomic study of IPEC-J2 cells infected with YC2014 and a classical CV777 strain (CV777) was performed to determine the differences between pandemic and classical PEDV strain infection. Totals of 353 and 299 differentially expressed proteins were identified upon YC2014 and CV777 infection, respectively. The canonical pathways and functional networks involved in both PEDV infections were analyzed. The results indicated that the PEDV suppressed protein synthesis of IPEC-J2 cells through down-regulation of the PI3K-AKT/mTOR signaling pathways. Infection with YC2014 could activate the JAK-STAT signaling pathway and the NF-κB pathway more intensively than CV777. YC2014 could activate NF-κB pathway more intensively than CV777. On the basis of differentially expressed proteins, we propose that PEDV might disrupt apoptosis and may elicit stronger inflammatory cascades as well. This study might contribute to an understanding of the pathogenesis of PEDV infection and aid in the development of effective preventive and control vaccines.

Protective effects of sinomenine against LPS-induced inflammation in piglets.

The aim of this study was to investigate in piglets, the anti-endotoxin and anti-inflammatory effects of sinomenine, an agent commonly found in Chinese herbal medicines. In high-, middle- and low-dose sinomenine groups, piglets were initially challenged with endotoxin (i.e., 1 mg lipopolysaccharide (LPS)/kg) by intraperitoneal (IP) injection and, 3 h later, intramuscularly (IM) with sinomenine at 1, 5, or 10 mg/kg. In a drug control group, piglets were dosed IP with vehicle and 3 h late IM with 10 mg/kg sinomenine while those in an LPS control group were challenged with 1 mg LPS/kg (IP) and then vehicle 3 h later; naïve control piglets were administered normal saline IP and then IM only. At 12, 24, and 48 h post-LPS/vehicle injection, blood samples were collected from the precaval vein of piglets. Clinical signs were recorded during the trial and index levels were analyzed by ELISA kits. The results revealed sinomenine could reduce the incidence/severity of certain LPS-induced toxicities, e.g., cell adhesion, systemic inflammation, and multiple organ dysfunction. Taken together, the data suggested to us that sinomenine might effectively be useful to regulate inflammatory responses as part of future anti-endotoxin therapies.

Efficacy and immunogenicity of recombinant swinepox virus expressing the truncated S protein of a novel isolate of porcine epidemic diarrhea virus.

Porcine epidemic diarrhea virus (PEDV) causes significant loss to the swine industry. The emergence of novel PEDV strains in recent years has decreased the effectiveness of PEDV vaccines. We have developed a live recombinant vaccine, a swinepox virus vector that expresses a truncated S protein (rSPV-St) from a recent PEDV strain, SQ2014, and evaluated its immunogenicity and effectiveness in a swine model. Vaccination of swine with rSPV-St elicited a robust antibody response specific for the homologous PEDV SQ2014. Serum IgA titers in rSPV-St-vaccinated animals were significantly higher than in those immunized with inactivated vaccines. The effectiveness of antibodies induced by the rSPV-St vaccine in protection against PEDV was tested in a passive-transfer model in which piglets were challenged with the homologous virus SQ2014 and the heterologous strain CV777. When challenged with the homologous virus, sera from rSPV-St vaccination provided complete protection. However, sera from rSPV-St vaccination did not provide any protection against the heterologous virus challenge. Amino acid sequence differences in the S proteins of the two viruses were identified within neutralizing epitopes, which might have contributed to the divergent clinical results. Our data suggest that rSPV-St is potentially an effective vaccine against infection with emerging PEDV strains.

MicroRNA-30a-5p promotes replication of porcine circovirus type 2 through enhancing autophagy by targeting 14-3-3.

Accumulating evidence demonstrates that autophagy and microRNAs (miRNAs) play key roles in regulating virus-host interactions and can restrict or facilitate viral replication. In the present study we examined whether a functional relationship exists between autophagy, miRNA and porcine circovirus type 2 (PCV2) infection, using several approaches. We demonstrated that there was a positive correlation between PCV2 infection and autophagy in 3D4/21 cells and autophagy induced by PCV2 infection triggered PCV2 replication. Four miRNA were selected by real-time PCR and further studied, but only miR-30a-5p mimic had a significant effect on PCV2 replication. Overexpression of miR-30a-5p significantly enhanced PCV2 infection and autophagy in a dose-dependent manner. Blockage of miR-30a-5p significantly decreased PCV2 replication. We provided further evidence that miR-30a-5p regulate the link between PCV2 infection and host immune system. Furthermore, miR-30a-5p targeted and regulated 14-3-3 gene, which is a regulator of autophagy. Flow cytometry data demonstrated that miR-30a-5p promotes cell cycle arrest at the G2 phase to regulate PCV2 replication and autophagy by interacting directly with 14-3-3, but not with the PCV2 genome. These data not only provide new insights into virus-host interactions during PCV2 infection but also suggest a potential new antiviral therapeutic strategy against PCV2 infection.

Genome sequence of a porcine circovirus-like virus P1 mutant in China.

A new porcine circovirus-like virus P1 mutant was isolated and sequenced in China in 2015. This P1 mutant has a mutation in its ORF1-encoded capsid protein that results in its elongation by eight amino acids.