Expression profile analysis of 5-day-old neonatal piglets infected with porcine Deltacoronavirus
Porcine deltacoronavirus (PDCoV) is a novel coronavirus that can cause diarrhea in nursing piglets. This study was aimed to investigate the roles of host differentially expressed genes on metabolic pathways in PDCoV infections.
Twenty thousand six hundred seventy-four differentially expressed mRNAs were identified in 5-day-old piglets responded to PDCoV experimental infections. Many of these genes were correlated to the basic metabolism, such as the peroxisome proliferator-activated receptor (PPAR) signaling pathway which plays a critical role in digestion. At the same time, in the PPAR pathway genes of fatty acid-binding protein (FABP) family members were observed with remarkably differential expressions. The differential expressed genes were associated with appetite decrease and weight loss of PDCoV- affected piglets.
Fatty acid-binding protein 1 (FABP1) and fatty acid-binding protein 3 (FABP3) were found to be regulated by PDCoV. These two genes not only mediate fatty acid transportation to different cell organelles such as mitochondria, peroxisome, endoplasmic reticulum and nucleus, but also modulate fatty acid metabolism and storage as a signaling molecule outside the cell. Therefore, it can be preliminarily concluded that PPAR differential expression caused by PDCoV was mostly associated with weight loss and death from emaciation.
The host differentially expressed genes were associated with infection response, metabolism signaling and organismal systems signaling pathways. The genes of FABP family members in the PPAR signaling pathway were the most highly altered and played important roles in metabolism. Alteration of these genes were most likely the reason of weight loss and other clinical symptoms. Our results provided new insights into the metabolic mechanisms and pathogenesis of PDCoV infection.
Animal experiment, Determination of viral growth by real-time RT-PCR, Histopathology, Immunohistochemical staining, Microarray analysis.
KeywordsPorcine deltacoronavirus mRNA expression profile Metabolism PPAR signaling pathway
Acetyl-CoA acyltransferase 1
Acyl-Coenzyme A dehydrogenase, long-chain
Acyl-CoA synthetase long chain family member 1
Acyl-CoA synthetase long chain family member 3
Beta-carotene oxygenase 1
Carbonic anhydrase 7
Chymotrypsin like elastase family member 2A
Carnitine palmitoyltransferase 1C
Classical swine fever
Cytochrome P450 2B22
Enoyl-Coenzyme A, hydratase/3-hydroxyacyl Coenzyme A dehydrogenase
Fatty acid binding protein 1
Fatty acid binding protein 3
GRB2 associated binding protein 3
oral-facial-digital syndrome 1 protein-like
Matrix metallopeptidase 1
a multiplicity of infection
Porcine circovirus 3
Porcine epidemic diarrhea virus
Porcine epidemic diarrhea virus
Peptidase inhibitor 3
Peroxisome proliferator-activated receptor
Sterol carrier protein 2
Solute carrier family 27 member 4
Sodium/potassium ATPase inhibitor SPAI-2
Swine testicular cells
Porcine transmissible gastroenteritis virus
Transmissible gastroenteritis virus
Upstream binding transcription factor, RNA polymerase I-like 1
Coronaviruses (CoVs) are single-stranded, positive-sense and enveloped RNA viruses. The Coronaviridae contains at least four major genera that includes Alpha, Beta, Gamma and Delta coronavirus . In 2009, Delta coronaviruses was first discovered from a wide range of domestic and wild birds in Hong Kong . And in 2012, Porcine Deltacoronavirus (PDCoV) was reported from rectal swabs from pigs in Hong Kong, represented by strains of HKU15–155 and HKU15–44 . PDCoV identified from pigs can cause severe diarrhea, vomiting and dehydration in piglets, which are similar to the symptoms of porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV), except the mortality rate of PDCoV infection lower than those of PEDV or TGEV [4, 5]. So far, PDCoV has circulated in pigs in Hong Kong, North America, South Korea, Thailand and some provinces of Mainland China, which has resulted in huge economic losses for the global swine industry [6, 7, 8].
There have been numerous studies examining the pathogenesis and host response to PDCoV infections [9, 10, 11]. For example, the recent study showed that binding of PDCoV to an interspecies conserved site on aminopeptidase N (APN) may facilitate the direct transmission of PDCoV to nonreservoir species, including humans . Although there is no relevant report illustrating that PDCoV has the possibility of cross-species transmission between animals and humans, it is significant to improve the awareness about biosecurity and imperative to research and analysis the pathogenesis of PDCoV. So far, there are no relevant studies about the expression profile of PDCoV, especially on animals. In this study, mRNA transcriptome of 5-day-old piglets responded to PDCoV infections was identified. A number of genes associated with the peroxisome proliferator-activated receptor (PPAR) signaling pathway were significantly differentially expressed. The results would provide new information regarding the metabolic mechanisms and pathogenesis of PDCoV infection.
Materials and methods
Cell culture and virus infection
The swine testicular cell (ST) (ATCC-CRL 1746) were kept in our laboratory and cultured in Dulbecco’s modified Eagle’s medium (DMEM, HyClone) in T-flasks (25 cm2) with 10% v/v fetal bovine serum (FBS, HyClone) at 37 °C within 5% CO2 incubator. Monolayer cells were washed three times with DMEM and then were infected with PDCoV CHN-GD16–05 (GenBank accession no.KY363868.1) strain at a MOI of 1.5 . When ST cells developed > 80% cytopathic effects (CPE) at 36 h post-infection (hpi), virus harvested with a titer of 1.0 × 10^8.6 TCID50/mL. Finally, PDCoV was propagated in ST cells until sixth passage.
A total of 35 piglets that were 5-day-old were purchased form a commercial farm. Rectal swabs from these piglets were collected to detect PDCoV, TGEV and PEDV by virus-specific PCRs, which indicating the negative results. Then 35 piglets were divided into two groups, the experimental group containing 20 piglets and the mock-control group including 15 pigs. Piglets in the experimental group were inoculated with PDCoV(CHN-GD16–05) with 5 ml at 1.0 × 10^8.6 TCID50/mL by oral administration, and weighted every day during the experimental period. Fecal samples were collected by cotton swabs from all piglets on days 0, 1, 2, 3, 4, 5, 6, 7, 14 and 21 days post-infection (dpi) and then were put into the EP tubes of 2 mL containing DMEM. The small intestine including duodenum, jejunum and ileum from 18 PDCoV-inoculated piglets were collected and examined for gross lesions at early (2 dpi), mid-onset (4 dpi) and late onset (11 dpi), with six piglets detected for each dpi. As the same time, the gene expression profiling was also analyzed for these 18 infected piglets and 15 piglets in the control group.
After the experiment, piglets was humanely euthanized after intravenous injection of 80 mg/kg body weight sodium pentobar-bital, and a complete necropsy was performed to collect tissues.
Determination of viral growth by real-time RT-PCR
Primers and probe for PDCoV detection
RNA extraction, library construction and sequencing
Samples of the jejunum closed to the ileum were collected from 18 experimental piglets for Microarray analysis. Nucleic acid was extracted by the Trizol Kit (Promega, USA) following by the manufacturer’s instructions and then was treated with RNase-free DNase I (Takara Bio, Japan) for 30 min at 37 °C to remove residual DNA and obtain RNA. The RNA quality was verified using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) and also checked by RNase free agarose gel electrophoresis. Next, Poly (A) mRNA was isolated using oligo-dT beads (Qiagen). All mRNA was broken into short fragments by adding fragmentation buffer. First-strand cDNA was generated using random hexamer-primed reverse transcription, and then primitive fragmented mRNA was digested by RNase H, followed by the synthesis of the second-strand cDNA. The second-strand cDNA was added end reparation poly (A) and ligated to sequencing adapters. The second-strand cDNA with bases “U” was digested by the enzyme UDG. Finally, the first-strand cDNA was enriched by PCR to construct the final cDNA library. The cDNA library was sequenced on the Illumina sequencing platform (IlluminaHiSeq 2000) using the paired-end technology by Personal Biotechnology Co., Ltd. (Shanghai, China). A Perl program was written to select clean reads by removing low quality sequences. These included the samples with quality scores < Q20, and reads containing adaptor sequences. The sequencing data is available from the NCBI Short Read Archive (SRA) (http:// www.ncbi.nlm.nih.gov/sra).
Tissue samples of duodenum, jejunum and ileum from the experiment group and the control group in 2 dpi, 4 dpi and 11 dpi were routinely fixed in 10% formalin and processed by the following steps, which included dehydrating in graded ethanol, embedding in paraffin, sectioning to 5-μm sections, and final staining with hematoxylin and eosin (H&E). Slides were examined by conventional light microscopy.
The study of Chen et al. introduced the preparation method of monoclonal antibodies and the anti-PDCoV-N protein was used as primary antibody in this article . The slides was processed according to previous studies [6, 11]. Slides were then dehydrated, cleared with xylene and sealed with cover slips. Finally, the slides were observed under an Olympus BX40 fluorescence microscopy.
Feature Extraction software (Version10.7.1.1, Agilent) was used to analyze array images to get raw data. Genespring (Version13.1, Agilent) was employed to finish the basic raw data analysis. The raw data was normalized using the quantile algorithm. Probes possessed at least 100% of the values in any one out of all conditions were flagged as “Detected” and chosen for further data analysis. Differentially expressed genes were then identified through fold change as well as P values calculated using the Students t-test. The threshold set for up- and down-regulated genes was a fold change ≥2.0 and a P value≤0.05 compared with data of piglets in control group.
To confirm the microarray data, six genes were selected for the verification by real-time PCR (see Table 1 for primer sequences). One-step real-time qRT-PCR reactions were performed using One Step PrimeScript RT-PCR kit (Takara) using an ABI PRISM 7500 instrument with the following steps: reverse transcription at 42 °C for 10 min, denaturation at 95 °C for 10 min and 40 cycles at 95 °C for 15 s, 60 °C for 30 s, 72 °C for 30 s and melting curve analysis. All samples were assayed in triplicate and relative gene expression was calculated as the previously described method .
The detection of virus distribution by qRT-PCR
Overview of differentially regulated genes after PDCoV infection
Statistics of differential expression genes between the challenge group and the control group
The total of differential expression genes
Horizontal comparisons identified that biological processes enriched in metabolic regulation included single-organism, cellular lipid metabolism, cellular amino acid metabolic processes, organic acid metabolic process and small molecule metabolic processes. However, only 7 Gene Ontology (GO) terms were significantly different between the control and the experimental group in the recovery phase of the infection. The most significant difference was in serine endopeptidase activity.
Detection of differential expression between the challenge group and the control group
Verification of microarray data using real-time qRT-PCR
GO analysis (http://www.geneontology.org) and KEGG pathway analysis (http://www.genome.jp/kegg/) were applied to determine the roles of differentially expressed mRNAs. A p-value< 0.05 and a false discovery rate of < 0.05 were used as thresholds.
GO term analysis
GO functional enrichment analysis of the differentially expressed genes indicated that 126, 67 and 7 GO terms were significantly enriched in the early, middle and late infection stages, respectively. The top 10 GO terms were enriched in biological processes, cellular components and molecular function. A horizontal comparison of these results indicated that the biological processes were enriched in single-organism catabolic processes, cellular lipid metabolic processes, organic acid metabolic processes and small molecule metabolic processes. The cellular component group were primarily concentrated in the extracellular and plasma membrane regions. The molecular function groups during the early stage of infection were primarily involved in iron and oxidoreductase activity. The middle infection stage revealed transmembrane, cation transmembrane transporter activity and enzyme inhibitor activities. However, the late infection stage showed the least number of differences between experimental and control groups with only 7 GO terms. The serine-type endopeptidase activity was the most significant among the latter.
KEGG pathway analysis
Since PDCoV being identified in 2009 in HongKong, many studies had focused locally on the virus characterization and evolution [1, 2, 3, 4]. To fill the gap of interaction between virus and host, particularly the correlation analysis between clinical symptoms and differential expressed host genes, 5-day-old piglets were orally challenged with PDCoV strain, CHN-GD16–05, and used for mRNA expression profile analysis in this study.
Clinical symptoms, diarrhea, vomiting and mental depression, of pigs were observed as early as the third day after the PDCoV challenge. 3/20 pigs died and the others began to recovery after 11 days. The intestine of diarrhea pig after dissection was found to be filled with water and its wall became almost paper-thin. The highest PDCoV load determined by qPCR was located in the ileum, followed by jejunum, duodenum and mesenteric lymph nodes, which differs from PEDV that concentrates in posterior jejunum . But the mechanism of it needs further study. In this study, the differentially expressed FABPs (Fatty acid-binding proteins) genes and diversification of FABPs gene mediated metabolism process were speculated to be responsible for the co-occurrence of the serious destruction of intestinal epithelial and body weight loss in piglets infected with PDCoV. This is somewhat similar to the study of Chen et al. on expression profile analysis of circRNAs in PEDV infected IPEC-J2 cells, a porcine ileum epithelium cell line . The study of Chen et al. demonstrated that PEDV were closely related to the metabolism process of ubiquitin mediated proteolysis . Study of Zhao et al. also indicated that TGEV was responsible for dysfunctional mitochondria in small intestine epithelial cells of piglets via up-regulating ssc-miR-4331 and activating p38 MAPK pathway .
FABPs, which are highly expressed in the cytoplasm of various cells in the small intestine, play prominent roles in the PPAR signaling pathway and are related to lipogenesis, lipid metabolism, inflammation, insulin sensitivity as well as cell growth and differentiation [19, 20]. Herein FABPs were found to be regulated by PDCoV. Fatty acid-binding protein 1 (FABP1) and fatty acid-binding protein 3 (FABP3), which not only mediate fatty acid transportation to different cell organelles such as mitochondria, peroxisome, endoplasmic reticulum and nucleus, but also modulate fatty acid metabolism and storage as a signaling molecule outside the cell, attracted our special attention .
FABP1 was reported to play a key role in fatty acid storage [21, 22]. Furthermore, fatty acids, particularly arachidonic-acid, can be metabolized into a family of lipid mediators such as eicosanoids, which can function as anti-inflammatory such as cyclopentenone prostaglandins (PGA1, PGA2 and PGJ2) and pro-inflammatory factors [20, 21]. Significant down-expression of FABP1 (fold change =0.083) may result in decreased secretion of anti-inflammatory factors. And increased mucosal inflammatory cells may cause intestinal injury. It is reported that down-regulation of FABP1 expression, significantly associated with poor differentiation and high expression of β-catenin and glutamine synthetase (GS), caused phenotypic changes during tumor progression in almost 10% of hepatocellular carcinoma (HCC) [22, 23, 24]. FABP3 was found to participate in mitochondrial energy metabolism by transporting fatty acid during ATP production and promote myocardial cell apoptosis [23, 24, 25]. So in this study, sharply up-regulated expression (fold change = 6.8) of FABP3 was speculated to mediate the mitochondrial dysfunction and cell apoptosis of the intestinal epithelial cells, which may be responsible for the emaciation of PDCoV challenged piglets. Therefore, it can be preliminarily concluded that PPAR differential expression caused by PDCoV was mostly associated with weight loss and death from emaciation. The verification of correlation analysis between weight loss and the target genes described above will be on schedule in the near future. Intestinal epithelial cells, which may be responsible for the emaciation of PDCoV challenged piglets. Therefore, it can be preliminarily concluded that PPAR differential expression caused by PDCoV was mostly associated with weight loss and death from emaciation. The verification of correlation analysis between weight loss and the target genes described above will be on schedule in the near future.
In summary, this is the first report of mRNA expression of 5-day old piglets following PDCoV infection. 20, 675 significant differentially expressed genes, which were associated with infection response, metabolism signaling and organismal systems signaling pathways, were described in this study. Especially, the highly differential expression of FABP genes was speculated to be mostly associated with weight loss and death from emaciation of PDCoV challenged piglets. These results provide new insights into the metabolic mechanisms and pathogenesis of PDCoV infection.
We would like to acknowledge Guangdong Wen’s Foodstuffs Group Co., Ltd. China, for providing us with piglets’ tissue samples.
This work was supported by the National Key Research and Development Program of China (No. 2016YFD0501304). The funder played no role in the design as well as conclusion of the study.
Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
JYM and XBZ conceived and designed the study. QNL critically revised the manuscript. JLW performed the experiments, analyzed the data, and drafted the manuscript. KJM, DL, RTW, XYT and YS helped in experimental implementation. TL and ZXW helped in sample collection. All authors read and approved the final manuscript.
This study was carried out in accordance with the recommendations of National Standards for Laboratory Animals of the People’s Republic of China (GB149258–2010). The protocol was approved by Animal Research Committees of South China Agricultural University. Pigs used for the study were handled in accordance with good animal practices required by the Animal Ethics Procedures and Guidelines of the People’s Republic of China.
Consent for publication
The authors declare that they have no competing interests.
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