key: cord-0036435-vgqoexe3
authors: Peng, Ju-Yi; Jian, Cai-Zhen; Chang, Chia-Yu; Chang, Hui-Wen
title: Porcine Epidemic Diarrhea
date: 2016-10-13
journal: Emerging and Re-emerging Infectious Diseases of Livestock
DOI: 10.1007/978-3-319-47426-7_11
sha: f199112e7a37fc7bdc15153737817595b0fc9382
doc_id: 36435
cord_uid: vgqoexe3

Porcine epidemic diarrhea (PED) first appeared in England and Belgium in the 1970s. The etiological agent of the disease is porcine epidemic diarrhea virus (PEDV), which belongs to Coronaviridae. The disease has spread globally and became an endemic disease in many Asian and European countries causing transient diarrhea in postweaning pigs with low mortalities for several decades. Since late 2010, field outbreaks of PED, which reemerged in China, spread to Asian and some European countries and emerged in North America; all led to enormous economic losses in porcine industry. New variants of PEDV exhibit not only significant genetic variations as compared to historic PEDV strains but also more virulent causing severe vomiting and watery yellowish diarrhea in suckling piglets under 1 week of age. Factors underlying the potential pathogenesis of the recent PEDV outbreaks include the mutation of the virus, the lacking of maternal antibodies for the protection of piglets, and the slower turnover rate of enterocytes (5–7 days) of the neonatal piglets as compared to postweaning pigs (2–3 days). The emerging and reemerging of the new variants of PEDV highlight the importance of reviewing the etiology, pathogenesis, diagnosis, and epidemiology of the disease.

Porcine epidemic diarrhea (PED) first appeared in England and Belgium in the 1970s. The etiological agent of the disease is porcine epidemic diarrhea virus (PEDV) , which belongs to the order Nidovirales, genus Alphacoronavirus, and family Coronaviridae (EN 1977; Bridgen et al. 1993; Duarte et al. 1993) . It consists of a single-stranded positive-sense RNA genome of approximately 28 kb in size with gene order 5′-replicase (1a/1b)-S-ORF3-E-M-N-3′. The viral genome encodes three nonstructural protein, Pol 1a/1b and ORF3, and four major structural proteins, the spike (S) glycoprotein, nucleocapsid (N) protein, membrane (M) glycoprotein, and envelope (E) protein. The S protein of PEDV is a type 1 transmembrane envelope protein and consists of the S1 and S2 domains. The S protein is responsible for the viral entry via specific binding of the S1 domain with the cellular receptor, fusion and interaction of the S2 domain with host cellular membrane, and for induction of neutralizing antibodies in the host (Bosch et al. 2003) . The M and E proteins are associated with virus assembly via interacting with S and N proteins (Klumperman et al. 1994) . The primary role of N protein is to pack the viral genomic RNA into viral particles (Spaan et al. 1983 ).

PEDV is mainly transmitted via oral-fecal route, though aerosolized PEDV is also infectious (Alonso et al. 2014) . The major transmission source of PEDV may be from the feces or vomitus. Other possible carriers for PEDV may be asymptomatic pigs or persons that carry contaminated fomites from farm to farm (Lowe et al. 2014) . Besides horizontal transmission, potential route for vertical transmission of PEDV via sow milk is also suggested (Li et al. 2012; Sun et al. 2012 ).

PEDV establishes its infection majorly in porcine villous enterocytes, which express the cellular receptor, porcine aminopeptidase N (pAPN; CD13) (Li et al. 2007) . PEDV replicates in the cytoplasm of villous epithelial cells in the small intestine and sometimes in the colon resulting in severe villous atrophy and leading to malabsorptive diarrhea (Straw et al. 2006) .

In the past, the prevalence of PED was low. It only caused endemic infection with very few mini-outbreaks. Suckling piglets are protected from the disease via maternal antibodies and immunity (Bandrick et al. 2014) . The disease majorly appeared in postweaning pigs as the maternal antibody titer drops. Possibly due to the fast turnover time (2-3 days) of enterocytes from crypt stem cells in postweaning pigs as well as the low virulence of traditional PEDV strains, the affected piglets usually show transient diarrhea with low or without mortalities.

Since late 2010, however, new PEDV variants with evidence of increased virulence have been isolated in several countries. The novel PEDV variants attack neonatal piglets regardless of their vaccination status or maternal immunity, which derived from CV777-based vaccination or preexisting historic PEDV infection (Sun et al. 2012; Chiou 2015; Stevenson et al. 2013) . Factors underlying the potential pathogenesis of the PEDV outbreaks and the high mortality in piglets include the mutation of the virus (Chiou et al. 2015; Pasick et al. 2014) , the lacking of maternal antibodies for protection of the piglets, and the slower turnover rate of enterocytes (5-7 days) of the neonatal piglets as compared to postweaning pigs (2-3 days) (Jung and Saif 2015a; Straw et al. 2006 ).

The major clinical signs of PED are watery diarrhea and/or vomiting. Piglets might die from dehydration and electrolyte imbalance due to severe diarrhea and vomiting. At necropsy, gross lesions majorly include distension of small intestine with yellowish fluid, thin and transparent intestinal walls ( Fig. 11 .1), and the stomach filled with curdle milk. Congestion of mesenteric vessels and edema of mesenteric lymph nodes are often seen. Under microscopic examination, an acute, diffuse, severe atrophic enteritis characterized by reduction in the villous height and crypt depth ratio, villous blunting and fusion, and cell exfoliation on the tips of villous enterocytes are often seen (Straw et al. 2006 ; Jung and Saif 2015a).

Several viruses can cause diarrhea in pigs with similar clinical signs and pathologic features to PED. These viruses include porcine deltacoronavirus (PDCoV), transmissible gastroenteritis virus (TGEV), and porcine rotavirus. As listed in Table  11 .1, these viral infections exhibit similar clinical signs, age tropisms, replication sites, gross lesions, and microscopic lesions. Therefore, a definitive diagnosis of PED majorly depends on molecular methods.

Several laboratory diagnostic methods are available for the definitive diagnosis of PEDV infection, such as immunofluorescence assay (IFA) and immunohistochemistry (IHC) by using specific antibodies for the detection of PEDV antigen in paraffin-embedded tissues (Fig. 11.2.) , enzyme-linked immunosorbent assay (ELISA) for detecting virus or serum antibodies (Okda Ren et al. 2011; Carvajal et al. 1995; Knuchel et al. 1992; van Nieuwstadt and Zetstra 1991) , reverse transcription polymerase chain reaction (RT-PCR) for detecting the viral genome (Ben Salem et al. 2010; Ishikawa et al. 1997; Kweon et al. 1997) , electron microscopy (EM) for demonstration of PEDV particles (Fig.11 .3) in the diarrheal feces (Straw et al. 2006; Jung and Saif 2015a) , and viral isolation together with immunocytochemistry (ICC) performed in Vero cells for propagation and in vitro characterization of the virus (Fig.11.4 ). 

Molecular characteristics of the S gene of PEDV strains have been often investigated to better understand the genetic diversity of PEDVs. After the new PEDV 

In Asia, historic PEDs are not uncommon in the field. Most of PEDV infections caused transient diarrhea in postweaning piglets and rarely any disease in suckling piglets. The new variants of PEDV, however, cause severe diseases and serious economic loss of the neonatal piglets in many Asian countries, including China (Tian et al. 2013; Li et al. 2012) , Taiwan (Chiou et al. 2015; Lin et al. 2014) , South Korea (Cho et al. 2014; Choi et al. 2014) , Thailand (Temeeyasen et al. 2014) , and Japan, raising the importance of PED in Asia. It has been demonstrated that new PEDV strains that emerged in Asia are distinct from previous historic PEDV strains. They cause higher morbidity and mortality in neonatal piglets than previous strains (Jung and Saif 2015b) .

In the past, the infection of PEDV was endemic, and the prevalence of PED was low with few mini-outbreaks reported in China. The CV777-based vaccine is frequently used in the field. Since late 2010, PEDV-infected piglets, regardless of vaccination or immune status, showed large-scale outbreaks of PED. These variants caused severe watery diarrhea in pigs of all ages and high death rate in neonatal and suckling piglets. Based on the phylogenetic analysis of full-length sequences of the S gene of PEDVs isolated in China, both G1 and G2 strains are concurrently isolated. While China G2 PEDV strains share 96-99 % homologies to the CV777-based vaccine strain, the China G1 PEDV strains only share 93-95 % similarity to the CV777based vaccine strain as well as China historic PEDV strains. These China G1 PEDV strains present several nucleotides insertions and deletions in the S gene (Li et al. 2012) . Comparing the G1 PEDV strains with the CV777-based vaccine strain, there are two to three amino acid mutations in the neutralizing epitope SS6 and one to eight amino acid differences in the CO-26 K equivalent epitope (COE) domain (499-638 aa) in the S protein. These mutations have been speculated to represent the viral evolution through escaping from the antibodies derived from the CV777-based vaccine or the historic PEDV strains in several studies Hao et al. 2014 ).

Before outbreaks of new variants of PED, historically PED was endemic in Taiwan. PEDV infection only caused mild transient diarrhea in postweaning piglets. PEDV vaccines, such as the CV777-based or DR13-based vaccines, are not routinely used in Taiwan. Nearly at the same time as in North America, new variants of PEDV infection leading to dramatic outbreaks and losses of suckling piglets reemerged in late 2013 in Taiwan. Phylogenetic analysis of the S gene showed that new variants of Taiwan PEDV strains belong to G1 and are closely related to other G1 strains from the USA, Canada, and China. These new Taiwan PEDV strains share 99.2 to 99.3 % nucleotide sequence identity to China CH/ZMDZY/11 and 94.1 to 94.2 % homologies to historical Taiwan PEDV strains. Similar to other new global variants of PEDV G1 strains, three serine amino acid substitutions (A522S, A554S, and G599S) in the COE and two serine amino acid substitutions (L769S and D771S) in SS6 are also observed in the reemerging Taiwan PEDVs as compared with the CV777-based vaccine strain (Chiou et al. 2015) .

In Korea, the endemic PED has been in the field for decades (Yeo et al. 2003; Kim and Chae 2000; Kubota et al. 1999) , and live PEDV vaccine strains are also available (Song et al. 2007 ). In addition to the reemerging of new variants of G1 PEDV strains resulting in severe PED in suckling piglets Park et al. 2013 Park et al. , 2014 Cho et al. 2014 ), a novel G1 PEDV variant, MF3809, is identified containing numerous sequence variations in the S protein, including a large (204-aa) deletion at positions 713-916 and a 2-aa (D/NI) deletion at positions 163-164. The antigenicity/ immunogenicity alteration of MF3809 is still unknown ).

The first PED case in the USA was reported in April-May, 2013. There was no previous description of the disease in the country before that time. No PEDV vaccines were available in the USA as well. After the first outbreak, PEDV quickly spread throughout the USA, then to nearby countries, including Canada (Song et al. 2015; Pasick et al. 2014 ). The US PEDV strains distributed both in G1 and G2 groups. The strains causing severe outbreaks exhibit high similarity to G1 PEDVs and are designed as S non-INDEL strains. The S non-INDEL PEDV strains were most closely related to China PEDV strains of CH/ZMZDY/11 and AH2012 in G1 group. Later after the outbreak, variants of US PEDV strains are isolated and are clustered in G2 in the phylogenetic analysis. As compared with the original US G1 PEDV strains, the US G2 PEDV strains contain insertions and deletions in S protein and are designed as S INDEL strains. Based on the full-length S gene analysis of PEDVs, the US S non-INDEL strains in G1 shared 99.8-100 % homology to each other and shared 96.6-97.1 % homologies to other PEDV G2 strains from North America. The S INDEL and original S non-INDEL strains are suggestive co-circulating and could have been introduced simultaneously in North America (Anastasia et al. 2014 ).

The first PEDV was reported in Belgium in 1978. The virus then spread over Europe until the end of the 1990s. However, there were no well-documented reports for PEDV in Europe during the past decades; most of the countries have not implemented active monitoring for this particular disease (23). Recently, several European PEDVs variants, such as PEDV/GER/L00719/214 and PEDV/GER/ L00721/2014 from Germany, FR/001/2014 from France, BEL/15 V010/2015 from Belgium, and some isolates from Portugal and Italy, have been isolated and studied. They shared high identity (99 %) with the USA/OH851 strain (S INDEL; G2) and the Chinese AH2012 strain, but less similar (97.1 %) to the historic isolate, CV777, which isolated from Europe (Grasland et al. 2015; Theuns et al. 2015; Dennis Hanke et al. 2015) .

The recent global outbreaks of PED in pigs of all ages with the high mortality in neonatal piglets may be contributed by several factors, including the mutation of the virus, the lacking of maternal antibodies, and the slower turnover of enterocytes (5-7 days) of neonatal piglets (Jung and Saif 2015a; Straw et al. 2006) . It had been demonstrated that current available vaccines, CV777 and the attenuated PEDV DR13 vaccines, might not be able to fully protect piglets against the infection or control disease progression due to the high genetic diversity of new variants of global PEDVs. A new generation of PEDV vaccine is therefore urgently in need.

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