Origin, Evolution, and Virulence of Porcine Deltacoronaviruses in the United States

Ma, Yuanmei; Zhang, Yu; Liang, Xueya; Lou, Fangfei; Oglesbee, Michael; Krakowka, Steven; Li, Jianrong

To determine the causative agent of the diarrhea outbreaks at Ohio pig farms, one live pig with severe diarrhea was euthanized, and fecal, intestinal content, liver, kidney, lung, and blood samples were collected. Total RNA was extracted from these samples and subjected to reverse transcription (RT)-PCR. Results showed that these samples were positive for PdCV but negative for PEDV, TGEV, and PRCV (Fig. 1A) . Feces and intestinal contents were filtered and subsequently analyzed by electron microscopy. Typical coronaviruslike particles with diameters ranging from 60 to 180 nm were observed ( Fig. 1B and C) . No other viral particles were observed. Subsequently, the filtered duodenum contents were subjected to deep sequencing. No other known viral sequences were identified, Complete genome sequence of PdCV Ohio CVM1. The genomic sequence of PdCV Ohio CVM1 is 25,422 nucleotides (nt) in length, excluding the 3= poly(A) tail, which is 1 nt and 6 nt longer than those of the PdCV Hong Kong strains HKU15-44 and HKU15-155, respectively. The genome organization of PdCV Ohio CVM1 is typical of CoVs. Starting from the 5= end, approximately three-fourths of the viral genome encodes two overlapping open reading frames (ORF1a and ORF1b), producing up to 16 nonstructural proteins (NSP). Downstream of ORF1, PdCV contains six additional ORFs (ORF2 to ORF7) that code for the structural proteins spike (S), envelope (E), membrane (M), nonstructural protein 6 (NS6), nucleocapsid (N), and NS7. Similar to avian deltacoronaviruses, NS6 is located between M and N. However, ORF7 (NS7) of PdCV differs from that of avian deltacoronaviruses. NS7 of PdCV overlaps with the N protein, whereas avian deltacoronaviruses contain 2 to 4 additional ORFs that encode NS7a, NS7b, NS7c, and NS7d, which overlap or are downstream of the N protein (15, 17) . Both 5= and 3= ends of PdCV contain short untranslated regions (UTR) with lengths of 539 and 395 nt, respectively. The genome of Ohio CVM1 shares 99% and 99.1% identity with Hong Kong PdCV strains HKU15-44 and HKU15-155, representing 249-and 206-nt differences, respectively. Compared to the HKU15-44 strain, PdCV Ohio CVM1 has a 1-nt (T) insertion at position 25267 located at the 3= UTR. PdCV Ohio CVM1 also differs by having a 3-nt (AAT) insertion at positions 19477 to 19479, which encodes the N terminus of the S protein, and a 3-nt (GTT) insertion at positions 25047 to 25049, located at the 3= UTR.