Porcine Epidemic Diarrhea Virus Strains in the United Origin

Coronaviruses are known to infect humans and other animals and cause respiratory and gastrointestinal diseases. Here we report the emergence of porcine epidemic diarrhea virus (PEDV) in the United States and determination of its origin, evolution, and genotypes based on temporal and geographical evidence. Histological lesions in small intestine sections of affected pigs and the complete genomic sequences of three emergent strains of PEDV isolated from outbreaks in Minnesota and Iowa were characterized. Genetic and phylogenetic analyses of the three U.S. strains revealed a close relationship with Chinese PEDV strains and their likely Chinese origin. The U.S. PEDV strains underwent evolutionary divergence, which can be classified into two sublineages. The three emergent U.S. strains are most closely related to a strain isolated in 2012 from Anhui Province in China, which might be the result of multiple recombination events between different genetic lineages or sublineages of PEDV. Molecular clock analysis of the divergent time based on the complete genomic sequences is consistent with the actual time difference, approximately 2 to 3 years, of the PED outbreaks between China (December 2010) and the United States (May 2013). The finding that the emergent U.S. PEDV strains share unique genetic features at the 5=-untranslated region with a bat coronavirus provided further support of the evolutionary origin of PEDV from bats and potential cross-species transmission. The data from this study have important implications for understanding the ongoing PEDV outbreaks in the United States and will guide future efforts to develop effective preventive and control measures against PEDV. IMPORTANCE The sudden emergence of porcine epidemic diarrhea virus (PEDV), a coronavirus, for the first time in the United States causes significant economic and public health concerns. Since its recognition in May 2013, PEDV has rapidly spread across the United States, resulting in high mortality in piglets in more than 17 States now. The ongoing outbreaks of Middle East respiratory syndrome coronavirus in humans from countries in or near the Arabian Peninsula and the historical deadly nature of the 2002 outbreaks of severe acute respiratory syndrome coronavirus create further anxiety over the emergence of PEDV in the United States due to the lack of scientific information about the origin and evolution of this emerging coronavirus. Here we report the detailed genetic characterization, origin, and evolution of emergent PEDV strains in the United States. The results provide much needed information to devise effective preventive and control strategies against PEDV in the United States. Received 2 September 2013 Accepted 19 September 2013 Published 15 October 2013 Citation Huang YW, Dickerman AW, Piñeyro P, Li L, Fang L, Kiehne R, Opriessnig T, Meng XJ. 2013. Origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the United States. mBio 4(5):00737-13. doi:10.1128/mBio.00737-13. Editor Diane Griffin, Johns Hopkins University School of Public Health Copyright © 2013 Huang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited. Address correspondence to Xiang-Jin Meng, xjmeng@vt.edu, and Yao-Wei Huang, yhuang@vt.edu. Coronaviruses in the subfamily Coronavirinae of the family Coronaviridae of the order Nidovirales are single-stranded, positive-sense RNA viruses with the largest genome that are known to infect humans, other mammals, and birds, usually causing subclinical or respiratory and gastrointestinal diseases (1, 2). Currently, the subfamily Coronavirinae is classified into four genera, including Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus (1). The first two genera are believed to originate from bats, whereas the last two are believed to have derived from birds (3, 4). The emergences of two highly pathogenic human betacoronaviruses, the severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002 and the Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012, have posed serious public health concerns over pandemic diseases associated with novel coronaviruses (4–6). While the MERS-CoV currently continues to spread in countries in or near the Arabian Peninsula, most recently, an alphacoronavirus, porcine epidemic diarrhea virus (PEDV) has suddenly emerged in the United States and rapidly spread across the country, resulting in high mortality in infected newborn piglets in more than 17 states in less than 3 months (7). Porcine epidemic diarrhea (PED) was first recognized as a devastating enteric disease in feeder and fattening pigs, resembling transmissible gastroenteritis (TGE) in pigs in the United Kingdom in 1971. The etiological agent was identified as a coronavirus, PEDV (strain CV777), in Belgium in 1978 (8). The full-length OBSERVATION September/October 2013 Volume 4 Issue 5 e00737-13 ® mbio.asm.org 1 genomic sequence of the prototype Belgian CV777 strain was determined in 2001 (9), which was more closely related to a Scotophilus bat coronavirus (BtCoV) 512/2005 than to other known alphacoronaviruses, such as TGE virus (TGEV) and human coronaviruses 229E and NL63, in phylogeny as well as genome organization (10), suggesting that PEDV and BtCoV/512/2005 had a common evolutionary precursor and that cross-species transmission of coronavirus might have occurred between bats and pigs. Outbreaks of PED have been documented in many European and Asian countries in the past (11). PED has been documented in China since the 1980s; however, variant strains of PEDV associated with large-scale outbreaks of diarrhea with 80 to 100% morbidity and 50 to 90% mortality in suckling piglets have emerged in China since 2010 and pose a serious concern for the swine industry of China (12–14). Emergence of PEDV in the United States. PEDV has been exotic in the United States until May 2013. Currently PEDV is spreading rapidly in swine farms in the United States, posing significant economic and public health concerns. To determine the origin and evolution of the PEDV outbreaks in the United States, we characterized four PED cases from Minnesota and two cases from Iowa. Clinical signs were characterized by acute vomiting, anorexia, and watery diarrhea, with high mortality in pigs less than 10 days old. For the four dead neonatal piglets from Minnesota, upon microscopic examination, the piglets had signs of emaciation and dehydration. The gross pathological lesions were confined to the small intestine and were characterized by thin translucent intestinal walls that contained moderate amounts of yellow watery feces without macroscopic traces of blood (see Fig. S1A in the supplemental material). No other gross abnormalities were noticed. Histological evaluation revealed regions of small intestines with villus blunting and fusion and minimal lymphoplasmacytic infiltration of the villi of the lamina propria (see Fig. S1B). The gross and histological lesions from the PEDV outbreaks in the United States are similar to those observed in China (14). PEDV RNA was detected in porcine small intestine samples from all four dead piglets from Minnesota by reverse transcription-PCR (RTPCR) with primers targeting the N gene (data not shown). Additionally, we also tested fecal and small intestinal samples from diseased suckling pigs from two different Iowa farms. The complete genomes of three representative strains of PEDV from the ongoing outbreaks in the United States— one from Minnesota and two from Iowa, designated strains MN and IA1 and IA2, respectively—were amplified by RT-PCR from total RNAs extracted from the fecal or small intestine sample and genetically characterized. Determination of the full-length genomic sequences of the emergent PEDV strains in the United States. To amplify the complete genomic sequence of the emergent U.S. strains of PEDV, the extreme 5= and 3= termini of the MN strain were first determined by rapid amplification of cDNA ends (RACE). Subsequently, a total of eight overlapping fragments covering the entire PEDV genome for each of the three emergent U.S. PEDV strains were amplified by RT-PCR using PfuUltra II high-fidelity DNA polymerase (Agilent, Santa Clara, CA), with primers based upon the conserved regions among the CV777 and the Chinese PEDV sequences (Fig. 1A; see Table S1 in the supplemental material). The RT-PCR products were individually excised from the agarose gel, purified, and subsequently cloned into a pSC-B-amp/kan vector (Agilent) or a pCR-Blunt vector (Invitrogen). For each amplicon, three to five individual clones were sequenced to determine the consensus sequence of any given genomic region. Sequence contigs with the consensus sequence were assembled into the fulllength genome for each of the three emergent U.S. PEDV strains using the Lasergene package (DNAStar, Inc., Madison, WI). Nucleotide sequence accession numbers. The complete genome sequences of the three U.S. PEDV strains have been deposited in GenBank under accession no. KF468752 (MN), KF468753 (IA1), and KF468754 (IA2). Unique genetic features of the emergent PEDV strains in the United States. The three emergent U.S. PEDV genomic sequences have the same size of 28,038 nucleotides (nt), excluding the polyadenosine tail, and share the genome organization with the prototype PEDV CV777 strain characterized by a gene order of 5=open reading frame 1a/1b (ORF1a/1b)-S-ORF3-E-M-N-3= (Fig. 1B.) These three U.S. PEDV sequences shared 99.8 to 99.9% nucleotide identities. In particular, strains MN and IA2 had only 11 nucleotide differences across the entire genome. To date, there are a total of 23 complete genomic sequences of PEDV available in the GenBank database, and 19 of