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

Abstract

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.

Construction, Characterization and Application of Recombinant Porcine Deltacoronavirus Expressing Nanoluciferase

“…On the basis of reverse genetic systems, many studies have constructed recombinant virus bearing reporter genes, such as Green Fluorescent Protein (GFP), red fluorescent protein [RFP], or luciferase, with extensive advantages, including the analysis of viral characteristics, screening of antiviral drugs, and the detection of the viral host spectrum [ 34 , 35 , 36 , 37 ]. Recently, PDCoV infectious cDNA clone and reporter virus expressing GFP have been successfully constructed by RNA-launched and DNA-launched reverse genetic system [ 38 , 39 ]. However, the construction, characterization and application of PDCoV reporter virus expressing other reporter genes with better performance has not been reported until now.…”

Attenuation of porcine deltacoronavirus disease severity by porcine reproductive and respiratory syndrome virus coinfection in a weaning pig model

“…A Chinese PDCoV strain CHN-HN-1601 (GenBank accession no. MG832584) [ 27 ] and a moderate pathogenic PRRSV strain CHsx1401 (GenBank accession no. KP861625) [ 28 ], representing the current predominant Lineage 1 virus in both China and the United States [ 11 , 29 ], were used at the 8 th passage, as previous study [ 26 ].…”

“…Paraformaldehyde (4%) fixed cells were incubated at 37 °C for 1 h with monoclonal antibodies (mAb) SDOW-17 (RTI) (against PRRSV) [ 30 ] or 1A3 (against PDCoV) made by our lab [ 27 ]. Alexa-fluor-488-conjugated goat anti-mouse polyclonal antibody (dilution 1:1000) (Invitrogen) was used as the secondary antibody.…”

“…Once severe diarrhea was observed, three pigs in each group were randomly selected for euthanasia and necropsy. The gross lesions of the lungs and intestines were observed and scored as the previous description [ 27 , 35 ]. The lung and intestine samples were fixed for histopathological and immunohistochemistry examination.…”

“…RT-qPCR primers (PDCoV: forward 5´-AGGGTTCGGGAGCTGACACTTCT-3´, reverse 5´-GGTCGCGTTTCCTGGGCTGATT-3´; PRRSV: forward 5´-ATGATGRGCTGGCATTCT-3´, reverse 5´-ACACGGTCGCCCTAATTG-3´) were designed and the viral RNA load in each sample was tested as previous description [ 27 , 32 ].…”