Rapid Detection, Complete Genome Sequencing, and Phylogenetic Analysis of Porcine Deltacoronavirus

In February 2014, porcine deltacoronavirus (PDCoV) was identified in the United States. We developed a PDCoV real-time reverse transcription PCR that identified PDCoV in 30% of samples tested. Four additional PDCoV genomes from the United States were sequenced; these had ≈99%–100% nt similarity to the other US PDCoV strains.

From the PDCoV-positive samples, we selected 4 for complete genome sequencing (online Technical Appendix). The 8 US PDCoV complete genome sequences were 99.9%-100% nt identical to each other and 98.9%-100% nt identical to the China PDCoV strains ( Table 2). The envelope and membrane gene segments were the most conserved and had a 100% nt identity, and the nonstructural (NS) 6 accessory gene had the lowest nucleotide identity (98.9%-100%) within the US strains. Compared with segments of the China strains, the envelope gene segment was the most conserved (99.6% nt identity), and the spike gene segment was the most diverse (98.5%-98.8% nt identity). The China PDCoV strain HKU15-155 contained two 3-nt deletions in the spike gene and 3′ untranslated region; the China strain HKU15 44 and US strains lacked this deletion. Inversely, PDCoV China strain HKU15-44 contained a nucleotide deletion in the 3′ untranslated region that was not present in the US PDCoV strains.
In the complete genome phylogenetic tree, the 8 US PDCoV strains clustered with China PDCoV strain HKU15-155 instead of HKU15-44 ( Figure, panel A). With the open reading frame 1, spike, envelope, membrane, and nucleocapsid gene segments and NS7 accessory gene phylogenetic trees, the US PDCoV strains clustered separately from the China PDCoV strains (Figure, panel B). The phylogenetic tree for the NS6 accessory gene had a different clustering pattern from the China strains (Figure, panel C). China PDCoV strain HKU15-44 clustered with Illinois133 and Illinois134, and China PDCoV strain HKU15-155 clustered independently.

Conclusions
The PDCoV rRT-PCR is a fast and accurate detection method that can be used to diagnose PDCoV infection. Identification of PDCoV in 30% of samples tested indicates that PDCoV is a common viral pathogen of pigs in the midwestern United States. We identified positive PD-CoV in 20 (22%) samples that were negative for TGEV, PEDV, RVA, RVB, and RVC, but PDCoV co-infections were more common (69 [78%] samples), especially with RVC (52 [58%]). Although the samples from Canada were negative for PDCoV, the Animal Health Laboratory has confirmed that 6 Ontario farms contain PDCoV. Because we selected samples on the basis of clinical diarrhea and geographic location was limited, the results do not accurately reflect the prevalence of PDCoV in North America. In addition, the presence of PDCoV RNA in feed does not indicate infectivity of the virus. The prevalence of PDCoV in North America is unknown, and the new PDCoV rRT-PCR can be used to access the prevalence in the United States and Canada.
Phylogenetic analysis of the US PDCoV strains indicates a common ancestor with the China PDCoV strains.
The China PDCoV strains are the only available sequences, and we cannot state that the US PDCoV strains originated in China. Because little is known about PDCoV, the US PDCoV parental strain may never be discovered. The NS6 phylogenetic tree branched differently from the other PDCoV gene segments; therefore, the NS6 accessory gene may evolve differently from the other gene segments. Complete genomes of PDCoV from other countries are needed to increase understanding of the origin, phylogenetic relationship, and evolution of the US PD-CoV strains.
The date that PDCoV was introduced into the United States is unknown. Because the sequenced PDCoV samples were from a similar geographic location, the 99.9%-100% nt identity does not correlate with the possible genetic diversity within the United States. An alternate hypothesis would indicate that PDCoV has been an undiagnosed pathogen of pigs in the United States and, like RVB, has been circulating there for an extended period (6) or that PDCoV might be a secondary infection to other enteric pathogens. PDCoV pathogenesis and retrospective surveillance studies are needed to answer these epidemiologic questions in the United States and to determine PDCoV prevalence worldwide.
In conclusion, a PDCoV rRT-PCR was designed to accurately detect PDCoV in a variety of samples. Complete genome analysis of the US PDCoV strains showed that they share 99.9%-100% nt identity and a common ancestor with the only available PDCoV sequences, the China PDCoV strains.
The University of Minnesota Veterinary Diagnostic Laboratory provided funding for this study. Dr Marthaler is a scientist at the University of Minnesota Veterinary Diagnostic Laboratory. His primary research interests are rotavirus and other pathogens of swine.