A nucleic acid detection assay combining reverse transcription recombinase-aided amplification with a lateral flow dipstick for the rapid visual detection of porcine deltacoronavirus.

Porcine deltacoronavirus (PDCoV) is an emerging enteropathogen causing severe diarrhoea, dehydration, and death in nursing piglets and enormous economic losses for the global swine industry. Furthermore, it can infect multiple animal species including humans. Therefore, a rapid, definitive diagnostic assay is required for the effective control of this zoonotic pathogen. To identify PDCoV, we developed a nucleic acid detection assay combining reverse transcription recombinase-aided amplification (RT-RAA) with a lateral flow dipstick (LFD) targeting the highly conserved genomic region in the ORF1b gene. The RT-RAA-LFD assay exhibited good PDCoV detection reproducibility and repeatability and could be completed within 11 min. Ten minutes at 40 °C was required for nucleic acid amplification and 1 min at room temperature was needed for the visual LFD readout. The assay specifically detected PDCoV and did not cross-react with any other major swine pathogens. The 95% limit of detection (LOD) was 3.97 median tissue culture infectious dose PDCoV RNA per reaction. This performance was comparable to that of a reference TaqMan-based real-time RT-PCR (trRT-PCR) assay for PDCoV. Of 149 swine small intestine, rectal swab, and serum samples, 71 and 75 tested positive for PDCoV according to RT-RAA-LFD and trRT-PCR, respectively. The diagnostic coincidence rate for both assays was 97.32% (145/149) and the kappa value was 0.946 (p < 0.001). Overall, the RT-RAA-LFD assay is a user-friendly diagnostic tool that can rapidly and visually detect PDCoV.

Development of a Nucleocapsid Protein-Based Blocking ELISA for the Detection of Porcine Deltacoronavirus Antibodies.

Porcine deltacoronavirus (PDCoV) is an emerging enteropathogen which mainly causes diarrhea, dehydration and death in nursing piglets, threatening the global swine industry. Moreover, it can infect multiple animal species and humans. Hence, reliable diagnostic assays are needed to better control this zoonotic pathogen. Here, a blocking ELISA was developed using a recombinant nucleocapsid (N) protein as the coating antigen paired with an N-specific monoclonal antibody (mAb) as the detection antibody. The percent inhibition (PI) of the ELISA was determined using 384 swine serum samples, with an indirect immunofluorescence assay (IFA) as the reference method. Through receiver operating characteristic analysis in conjunction with Youden's index, the optimal PI cut-off value was determined to be 51.65%, which corresponded to a diagnostic sensitivity of 98.79% and a diagnostic specificity of 100%. Of the 330 serum samples tested positive via IFA, 326 and 4 were tested positive and negative via the ELISA, respectively, while the 54 serum samples tested negative via IFA were all negative via the ELISA. The overall coincidence rate between the two assays was 98.96% (380/384). The ELISA exhibited good repeatability and did not cross-react with antisera against other swine pathogens. Overall, this is the first report on developing a blocking ELISA for PDCoV serodiagnosis.

Quantitative Proteomic Analysis of Porcine Intestinal Epithelial Cells Infected with Porcine Deltacoronavirus Using iTRAQ-Coupled LC-MS/MS.

Porcine deltacoronavirus (PDCoV) is an emergent enteropathogenic coronavirus associated with swine diarrhea. Porcine small intestinal epithelial cells (IPEC) are the primary target cells of PDCoV infection in vivo. Here, isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to quantitatively identify differentially expressed proteins (DEPs) in PDCoV-infected IPEC-J2 cells. A total of 78 DEPs, including 23 upregulated and 55 downregulated proteins, were identified at 24 h postinfection. The data are available via ProteomeXchange with identifier PXD019975. To ensure reliability of the proteomics data, two randomly selected DEPs, the downregulated anaphase-promoting complex subunit 7 (ANAPC7) and upregulated interferon-induced protein with tetratricopeptide repeats 1 (IFIT1), were verified by real-time PCR and Western blot, and the results of which indicate that the proteomics data were reliable and valid. Bioinformatics analyses, including GO, COG, KEGG, and STRING, further demonstrated that a majority of the DEPs are involved in numerous crucial biological processes and signaling pathways, such as immune system, digestive system, signal transduction, RIG-I-like receptor, mTOR, PI3K-AKT, autophagy, and cell cycle signaling pathways. Altogether, this is the first study on proteomes of PDCoV-infected host cells, which shall provide valuable clues for further investigation of PDCoV pathogenesis.

A strain of porcine deltacoronavirus: Genomic characterization, pathogenicity and its full-length cDNA infectious clone.

As a novel enteropathogenic coronavirus, porcine deltacoronavirus (PDCoV) warrants further investigation. In this study, a Chinese PDCoV strain, designated CHN-HN-1601, was isolated from the faeces of a diarrhoeic piglet. After plaque purification, the genome was determined which shared 97.5%-99.5% nucleotide identities with 71 representative PDCoV strains available in the GenBank. The pathogenic properties of CHN-HN-1601 were evaluated using 5-day-old piglets. All inoculated piglets developed severe diarrhoea from 2 days post-infection (dpi) onwards. To our surprise, two periods of diarrhoea starting from 2 to 7 dpi and from 13 to 19 dpi were observed in affected piglets during the experiment. Faecal viral shedding of the inoculated piglets was detected by real-time RT-PCR, with viral shedding peaked at 4 and 16 dpi, respectively. At necropsy at 5 dpi, the main gross lesions included transparent, thin-walled and gas-distended intestines containing yellow watery contents. Further histopathological examinations, including haematoxylin and eosin staining, immunohistochemistry and RNAscope in situ hybridization, revealed that the virus infection caused severe villous atrophy of the small intestines, with PDCoV antigen and RNA mainly distributed in the cytoplasm of the villous epithelial cells of jejunum and ileum in piglets. The dynamic production of PDCoV-specific IgG and neutralizing antibodies in serum of the affected piglets was also assessed using a whole virus-based ELISA and an immunofluorescence assay-based neutralization test, respectively. Furthermore, a full-length cDNA infectious clone of CHN-HN-1601 was constructed using a bacterial artificial chromosome system. The rescued virus exhibited in vitro growth and pathogenic properties similar to the parental virus. Taken together, our study not only enriches the information of PDCoV, but also provides a useful reverse genetics platform for further pathogenesis exploration of the virus.