Abstract: 【Objective】Porcine Deltacoronavirus (PDCoV) is a newly emerged coronavirus, which has caused diarrhea in pigs, especially in newborn piglets leading to high morbidity and mortality, such that it has become one of the most important causes of death in newborn piglets. In this study, we attempted to establish a reverse transcription-polymerase chain reaction (RT-PCR) assay for detection of newly emerged PDCoV in diarrheal pigs, and investigate the infection situation of PDCoV in diarrheal samples of pigs.【Method】Based on the result of multialignment analysis on the published PDCoV genome sequences on GenBank database, a pair of primers specific to the conserved nucleocapsid (N) gene of PDCoV was designed by using the online software Primer 3.0, and then a RT-PCR for detection of PDCoV was established by utilizing the designed primers. Diarrheal samples were tested by the established RT-PCR assay, and several positive PCR products were selected for cloning and sequencing. To analyze the phylogenetic relationships between PDCoV and porcine epidemic diarrhea virus (PEDV) as well as transmissible gastroenteritis virus (TGEV), a phylogenetic tree was constructed based on the partial N gene sequences of PDCoV obtained in this study and 18 reference PDCoV sequences from other countries/areas, and corresponding N gene sequences of representative PEDV and TGEV strains. To determine the specificity of the RT-PCR assay established, RNAs of PEDV, TGEV, porcine kobuvirus (PKoV), porcine astrovirus (PAstV), porcine reproductive and respiratory syndrome virus (PRRSV) and classical swine fever virus (CSFV) were tested. To evaluate the detection limit of the assay, a recombinant plasmid containing the N gene fragment was constructed and served as templates with 10-fold serial dilution starting from 1.0×10⁶ copies/μL to 1.0×10¹ copies/μL. Clinic diarrheal samples of pigs (n=249) from sows and piglets collected in Jiangxi Province from 2012 to 2015 were tested for the presence of PDCoV by the established RT-PCR, and some PDCoV positive amplicons were cloned and then sequenced for the specificity confirmation of the assay.【Result】A single expected DNA fragment band of 329 bp in size was amplified by using the designed primers. Sequences of the amplicons selected were subjected to a BLAST search against the GenBank database, and the results indicated that the sequences hit PDCoV with a nucleotide identity as high as 99.1%, demonstrating that the amplified targets were PDCoV. A phylogenetic tree was generated based on eight partial N gene of PDCoV obtained in this study, 18 reference PDCoVs from other countries/areas, and two representative PEDV and two selected TGEV sequences with corresponding fragments. All of the PDCoV strains were classified into a cluster, distinct from PEDV and TGEV strains. The eight PDCoV sequences obtained in this study showed that the highest similarity with a Korea PDCoV strain KNU14-04, but less similarity to two Hong Kong strains HKU 15-44 and HKU 15-155. No cross amplification for PEDV, TGEV, PKoV, PAstV, PRRSV and CSFV was observed, indicating the established RT-PCR assay was highly specific. The detection limit of 1.0×103 copies/µL of the assay was determined based on the templates of the recombinant plasmid with 10-fold serial dilution (from 1.0×10⁶ to 1.0×10¹ copies/µL), indicating the assay was sensitive. A total of 249 diarrheal fecal/intestinal samples of sows and piglets collected in Jiangxi Province, China from 2012 to 2015 were examined by the established assay, and the positive rate of PDCoV was 31.3% (78/249), and the samples from sows showed somewhat lower positive rate (27.8%) when compared with that from piglets (31.9%). PDCoV could be detected in samples as early as 2012.【Conclusion】In this study, a RT-PCR assay for detection of newly emerged PDCoV in swine was established and evaluated. The results from this study indicated that PDCoV is a frequently detected virus in diarrheal pigs in Jiangxi, China. The RT-PCR assay established in this study is valuable in terms of clinic diagnosis and epidemiological investigation of PDCoV.

Key words: porcine deltacoronavirus (PDCoV), RT-PCR, diarrhea, phylogenetic tree, application