Origin, evolution, and virulence of porcine deltacoronaviruses in the United States

Abstract

A novel porcine deltacoronavirus (PdCV) was first discovered in Ohio and Indiana in February 2014, rapidly spread to other states in the United States and Canada, and caused significant economic loss in the swine industry. The origin and virulence of this novel porcine coronavirus are not known. Here, we characterized U.S. PdCV isolates and determined their virulence in gnotobiotic and conventional piglets. Genome analyses revealed that U.S. PdCV isolates possess unique genetic characteristics and share a close relationship with Hong Kong and South Korean PdCV strains and coronaviruses (CoVs) of Asian leopard cats and Chinese ferret-badgers. The PdCV-positive intestinal content (Ohio CVM1) and the cell culture-adapted PdCV Michigan (MI) strain were orally inoculated into gnotobiotic and/or conventional piglets. Within 1 to 3 days postinfection, profuse watery diarrhea, vomiting, and dehydration were observed. Clinical signs were associated with epithelial necrosis in the gastric pits and small intestine, the latter resulting in severe villous atrophy. Mild interstitial pneumonia was identified in the lungs of PdCV-infected piglets. High levels of viral RNA (8 to 11 log RNA copies/g) were detected in intestinal tissues/luminal contents and feces of infected piglets, whereas moderate RNA levels (2 to 5 log RNA copies/g) were detected in blood, lung, liver, and kidney, indicating multisystemic dissemination of the virus. Polyclonal immune serum against PdCV but not immune serum against porcine epidemic diarrhea virus (PEDV) reacted with PdCV-infected small-intestinal epithelial cells, indicating that PdCV is antigenically distinct from PEDV. Collectively, we demonstrate for the first time that PdCV caused severe gastrointestinal diseases in swine.

Importance: Porcine coronaviruses (CoVs) are major viral infectious diseases of swine. Examples of porcine CoVs include porcine transmissible gastroenteritis coronavirus (TGEV), porcine epidemic diarrhea virus (PEDV), and porcine respiratory coronavirus (PRCV). In February 2014, another porcine CoV, porcine deltacoronavirus (PdCV), emerged in Ohio and Indiana and subsequently spread rapidly across the United States and Canada, causing significant economic losses. Here, we report the detailed genetic characterization, phylogeny, and virulence of emergent PdCV strains in the United States. We found that PdCV caused severe diarrhea, vomiting, and dehydration in gnotobiotic and conventional piglets, signs that were clinically indistinguishable from those caused by PEDV and TGEV. In addition to extensive intestinal lesions, PdCV caused significant lesions in the stomach and mild pulmonary lesions that have not been reported for TGEV and PEDV. The finding that PdCV is a significant enteric disease of swine highlights the need to develop effective measures to control this disease.

FIG 1

Detection of PdCV in a 7-day-old piglet with severe diarrhea and vomiting from an Ohio farm. (A) RT-PCR analysis of PdCV samples. The N gene was amplified by RT-PCR using primers specific for PdCV, PEDV, TGEV, or PRCV. The PCR products were detected on a 1% agarose gel. (B and C) Electron microscopy analysis of PdCV. Filtered intestinal contents were analyzed by electron microscopy.

FIG 2

Phylogenetic analysis of mammalian and avian deltacoronaviruses. (A) Phylogenetic tree of the S protein; (B) phylogenetic tree of the NS7 protein. The phylogenetic tree was constructed by the neighbor-joining method using the MEGA 6 program. The scale bar indicates the estimated number of substitutions per 20 amino acids. Virus strains used for phylogenetic analysis and accession numbers are shown in Table S1 in the supplemental material. ALC denotes Asian leopard cat, and CFB denotes Chinese ferret-badger. The two strains (Ohio CVM1 and Michigan 8977) used in this study are highlighted by red color. The South Korean strain is highlighted by blue color.

FIG 3

Histologic lesions of intestine and stomach caused by PdCV Ohio CVM1. (A) Severe villous atrophy in the duodenum of a PdCV-infected Gn piglet at 24 h p.i. (hematoxylin and eosin, ×300 magnification); (B) duodenum of uninfected Gn piglet; (C) normal gastric fundus (×400), uninfected Gn piglet; (D) syncytial giant cells are present (white arrows) as well as foci of cell necrosis (black arrow) in the fundic region of the stomach, PdCV Ohio CVM1-infected Gn piglet, 24 h p.i.; (E) dilated gastric pits are present by 48 h p.i.; (F) a focus of lymphocytic and monocytic inflammation in the gastric mucosa, 72 h p.i.; (G) illustrates necrosis of mucosal epithelial cells characterized by nuclear changes (pyknosis, karyorrhexis, and karyolysis) indicative of cell necrosis from a Gn piglet infected with PdCV Ohio CVM1, 48 h p.i. (×400); (H) multinucleated giant cell formation in jejunal epithelium from the Gn piglet infected with PdCV Ohio CVM1, 72 h p.i. (×400). A viable PdCV-induced syncytial giant cell in jejunal epithelia (1) lies adjacent to a necrotic syncytial giant cell that is in the process of being sloughed into the intestinal lumen (2). Unaffected jejunal mucosal epithelial cells (3) are also present.

FIG 4

Mild (multifocal) interstitial pneumonia in a PdCV Ohio CVM1-infected Gn piglet, 48 h p.i. (A) Pulmonary tissue from PdCV-infected piglets (×50). The figure illustrates the bronchocentric distribution of the nonexudative histologic change in the lung. (B) Pulmonary tissue from PdCV-infected piglets (×400). The figure illustrates interstitial thickening of the alveolar cell walls. (C) Pulmonary tissue from an uninfected control (×50); (D) pulmonary tissue from an uninfected control (×400).

FIG 5

Immunohistochemistry (IHC) analysis of small-intestine sections from Gn piglets. PEDV VBS2-infected duodenal tissue at 48 h p.i. (A and B), PdCV Ohio CVM1-infected duodenal tissue at 24 h p.i. (D and E), and uninfected controls (C and F). Panels A to C were stained with hyperimmune serum against PEDV. Panels D to F were stained with hyperimmune serum against PdCV. Black arrows indicate positive antigens. Hematoxylin, ×300.

FIG 6

Viral RNA distribution of Gn piglets infected by PdCV Ohio CVM1. Three 19-day-old Gn piglets were orally inoculated with 10⁶ genomic RNA copies of PdCV Ohio CVM1. Gn piglets were terminated at 24, 48, and 72 h p.i. Viral RNA genome copies in pig tissues were quantified by real-time RT-PCR. No PdCV-specific RNA was detected in mock-infected Gn piglets.

FIG 7

Viral RNA distribution of Gn piglets infected by the PdCV MI strain. (A) Location of each piglet in the germfree isolator, showing the enclosures that physically separated each piglet (C1 to C5). Three 10-day-old Gn piglets (C2, C4, and C5) were orally inoculated with 10⁶ PFU of the PdCV MI strain and were terminated on day 3 p.i. Piglets C1 and C3 did not receive virus inoculation and were terminated on day 7 p.i. (B) Viral RNA shedding in feces after virus inoculation. (C) Viral RNA distribution in tissues from three PdCV-inoculated Gn piglets. The number of positive samples was indicated at the top of each column. (D) Viral RNA distribution in tissues from two Gn piglets infected via indirect contact.

FIG 8

Viral RNA distribution in conventional piglets infected by the PdCV Michigan strain. Four 10-day-old conventional piglets were orally inoculated with 10⁶ PFU of the PdCV MI strain and were terminated on day 21 p.i. (A) Viral RNA shedding in feces after virus inoculation; (B) viral RNA distribution in pig tissues.