key: cord-0751424-9q1i20o6
authors: Jung, Kwonil; Saif, Linda J.; Wang, Qiuhong
title: Porcine epidemic diarrhea virus (PEDV): An update on etiology, transmission, pathogenesis, and prevention and control
date: 2020-06-02
journal: Virus Res
DOI: 10.1016/j.virusres.2020.198045
sha: 90ed73b90d735bb33f08ab033b3e3dfa523549e6
doc_id: 751424
cord_uid: 9q1i20o6

Porcine epidemic diarrhea virus (PEDV), a member of the genus Alphacoronavirus in the family Coronaviridae, causes acute diarrhea and/or vomiting, dehydration and high mortality in neonatal piglets. Two different genogroups of PEDV, S INDEL [PEDV variant containing multiple deletions and insertions in the S1 subunit of the spike (S) protein, G1b] and non-S INDEL (G2b) strains were detected during the diarrheal disease outbreak in US swine in 2013-2014. Similar viruses are also circulating globally. Continuous improvement and update of biosecurity and vaccine strains and protocols are still needed to control and prevent PEDV infections worldwide. Although the non-S INDEL PEDV was highly virulent and the S INDEL PEDV caused milder disease, the latter has the capacity to cause illness in a high number of piglets on farms with low biosecurity and herd immunity. The main PEDV transmission route is fecal–oral, but airborne transmission via the fecal–nasal route may play a role in pig-to-pig and farm-to-farm spread. PEDV infection of neonatal pigs causes fecal virus shedding (alongside frequent detection of PEDV RNA in the nasal cavity), acute viremia, severe atrophic enteritis (mainly jejunum and ileum), and increased pro-inflammatory and innate immune responses. PEDV-specific IgA effector and memory B cells in orally primed sows play a critical role in sow lactogenic immunity and passive protection of piglets. This review focuses on the etiology, transmission, pathogenesis, and prevention and control of PEDV infection.

Porcine epidemic diarrhea virus (PEDV), a member of the genus Alphacoronavirus in the family Coronaviridae of the order Nidovirales, causes acute diarrhea, vomiting, dehydration and high mortality in neonatal piglets. The disease was reported in the European and Asian pig industries over the last 30 years, with the virus first appearing in England (Wood, 1977) and J o u r n a l P r e -p r o o f

One study reported that four sows exposed via feedback to a G1b S INDEL PEDV at approximately seven months pre-farrowing and then re-exposed to a G2b non-S INDEL PEDV at day 109 of gestation provided long-term (7 months), passive immune protection of piglets against challenge with the same G2b non-S INDEL PEDV (Goede et al., 2015) . There was 0% mortality rate of piglets (with 57% reduced incidence of diarrhea), compared with mean 33% mortality and 100% morbidity rates of piglets born to non-immunized sows. However, another study revealed that more than 80% of the piglets inoculated orally with a G1b S INDEL PEDV (Iowa106 strain) at 3-4 days of age had diarrhea after challenging with a G2b non-S INDEL PEDV at 24 days of age, whereas none of the piglets inoculated previously and then challenged with the same non-S INDEL PEDV showed diarrhea (Annamalai et al., 2017; Lin et al., 2015a) .

Antigenic cross-reactivity or cross-neutralization between PEDV and feline infectious peritonitis virus (FIPV) was detected by enzyme linked immunosorbent assay (ELISA), immunoblotting and immune-precipitation (Zhou et al., 1988) , or VN tests (Zhao et al., 2019) . Despite no serological cross-neutralization detected between PEDV and TGEV (Hofmann and Wyler, 1989; Pensaert and de Bouck, 1978) , investigators found some antigenic cross-activity between PEDV and TGEV (Miller, but not Purdue strain) based on at least two conserved epitopes on the N-terminal region of their N proteins, as well as via their M proteins or whole virus particles (Gimenez-Lirola et al., 2017; Lin et al., 2015b; Xie et al., 2019) . Truncation of the N-terminal region of the N protein or avoidance of M-or purified whole virus-based serologic tests may eliminate PEDV and TGEV shared epitope(s) or reduce the possible cross-reactivity for serologic J o u r n a l P r e -p r o o f assays (Gimenez-Lirola et al., 2017; Xie et al., 2019) . Polyclonal hyperimmune antisera against PDCoV did not cross-react with PEDV . Among N, M and E proteins and whole PEDV particles, only the M protein of PEDV showed cross-reactivity in 1/12 convalescent sera from PDCoV-infected pigs (Gimenez-Lirola et al., 2017) . However, one study reported antigenic cross-reactivity between US PDCoV and PEDV strains, possibly due to at least one shared epitope in the N-terminal region of their N proteins . For SADS-CoV, monoclonal antibody against the N protein did not cross-react with PEDV, TGEV or PDCoV (Pan et al., 2017) .

The fecal-oral route is the main means of direct transmission of PEDV via the feces and/or vomitus of infected pigs (Jung and Saif, 2015) . Indirect contact transmission of PEDV is also frequent within and between farms, particularly, with a low biosecurity, via other contaminated fomites including (Jung and Saif, 2015; Kim et al., 2017b) : transport trailers (Lowe et al., 2014) , farm workers' hands, boots and clothes (Kim et al., 2017b) , feed (Bowman et al., 2015a; Dee et al., 2014; Schumacher et al., 2017) , feed ingredients and additives, such as spray-dried porcine plasma (Pasick et al., 2014; Perri et al., 2018) , and feed totes used for transporting bulk feed or feed ingredients (Anon., 2015; Scott et al., 2016) . PEDV remained infectious on tote material for 35 days at room temperature (Scott et al., 2016) . PEDV cross-contamination also occurred during feed manufacturing (Schumacher et al., 2018) .

J o u r n a l P r e -p r o o f 8 The fecal-nasal route is another route of pig-to-pig, or farm-to-farm (up to 10 miles away) airborne transmission of PEDV via aerosolized PEDV particles that are infectious in nursing pigs (Alonso et al., 2014; Alonso et al., 2015; Beam et al., 2015; Gallien et al., 2018a; .

Airborne PEDV transmission occurs within farrowing herds where newborn piglets, highly susceptible to the virus, are raised (Alonso et al., 2015; Niederwerder et al., 2016) . The nasal cavity of naïve pigs housed at a distance from clinical pigs was frequently positive for PEDV RNA (Niederwerder et al., 2016) . Aerosolized PEDV does not infect only the intestine of pigs (Alonso et al., 2015) , but it also infects the epithelium lining the nasal cavity . reported that dendritic cells in the lamina propria of the nasal mucosa or lymphoid tissue carry and transfer PEDV to CD3 + T cells. PEDV-loaded T cells may reach the intestine via unknown specialized endothelial venules during blood circulation. The virus loaded onto T cells had the capacity to infect intestinal epithelial cells via cell-to-cell contact and transfer infection . Compared with neonatal piglets, however, higher doses of aerosolized PEDV may be required to infect weaned and older pigs (Niederwerder et al., 2016) . In the study, none of aerosol contact, 28-day-old pigs (0/5 pigs) was infected, although their nasal cavities (5/5 pigs) were positive for PEDV RNA.

The severity of PEDV infection and the disease, and the transmissibility of PEDV, depends on the overall immunity and health status of the pig population and the levels of biosecurity on farms (Pensaert and Martelli, 2016) . Nevertheless, transmissibility of PEDV via direct or aerosol J o u r n a l P r e -p r o o f contact also varies dependent on the PEDV genogroup. Direct contact or aerosol transmission rates were significantly higher in pigs infected with non-S INDEL PEDV compared with S INDELinfected pigs (Gallien et al., 2018a) . Gallien et al. (2018) revealed that despite the presence of aerosolized S INDEL PEDV, none of aerosol contact pigs (0/10 pigs) was infected, whereas 10/10 aerosol contact pigs were infected by non-S INDEL PEDV (Gallien et al., 2018a) .

Porcine small intestinal villous enterocytes express aminopeptidase N (APN), a 150-kDa glycosylated transmembrane protein, which was tentatively identified as the cellular receptor for PEDV (Li et al., 2007; Liu et al., 2015a; Nam and Lee, 2010) . However, porcine APN may not be the major cell surface receptor for PEDV (Ji et al., 2018; Shirato et al., 2016) .

Concomitantly, APN knockout pigs were susceptible to infection with PEDV, but not with TGEV that uses APN as the cellular receptor (Whitworth et al., 2019; Zhang et al., 2019) . Cell membrane cholesterol or two cell surface molecules, such as sialic acids and occludin expressed on the apical surface of secretary (goblet) or absorptive enterocytes, respectively, were involved partially in binding and entry of PEDV into enterocytes (Deng et al., 2016; Jeon and Lee, 2017; Luo et al., 2017) . The strains of PEDV, such as cell culture-adapted vs. wild-type, may be a factor involved in the cellular entry via sialic acids (Deng et al., 2016; . Intracytoplasmic localization of PEDV antigen in goblet cells of infected pigs and porcine intestinal enteroids in vitro (Jung and Saif, 2017; raises questions on whether or how goblet cells are utilized for PEDV replication, or if they only transfer PEDV to absorptive enterocytes.

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We demonstrated the tissue tropism of PEDV to certain intestinal locations (Jung et al., 2018) . During acute PEDV infection [post-inoculation hour (PIH) 12-24] in nursing pigs, PEDV initially infected mainly the mid-jejunum and ileum and to a lesser extent, the proximal and distal jejunum and duodenum (Jung et al., 2018) (Fig. 1) . The pylorus was not the site of acute PEDV infection (Jung et al., 2018) , whereas the villous enterocytes of the large intestine were frequently infected; however, infected colonic enterocytes did not undergo necrosis (Jung et al., 2015a; Jung et al., 2014) . Unlike colonic enterocytes, PEDV-infected small intestinal villous enterocytes undergo acute necrosis and exfoliation from the lamina propria, leading to marked villous atrophy or fusion in the small intestine (Jung et al., 2018; Jung et al., 2014) . Like TGEV (Kim et al., 2000) and PDCoV (Jung et al., 2016) , PEDV may not induce apoptotic death of intestinal villous enterocytes in vivo (Jung and Saif, 2015) . However, PEDV-infected Vero cells in vitro underwent apoptosis (Kim and Lee, 2014) .

During acute PEDV infection (PIH 12-24) of nursing pigs, early localization of PEDV antigen was evident in the villous-crypt interface of the small intestine, rather than the villous tips (Jung et al., 2018) (Fig. 1) . Iimmature enterocytes were the major site of initial PEDV infection, although the exact reason remains obscure (Jung et al., 2018) . The PEDV antigen-positive regions subsequently expanded to the upper and then the entire villous epithelium of the jejunum to ileum (<24 hours after oral inoculation) (Jung et al., 2018) . The villous-crypt interface is close to blood vessels in the submucosa. Although PEDV-related viremia might be a result of diffusion of replicated PEDV from the acutely infected intestine to blood (Jung et al., 2018) , further studies are J o u r n a l P r e -p r o o f needed to investigate whether PEDV or PEDV-loaded CD3+ T cells circulating in blood reach and infect the villous-crypt interface or other villous regions, and other types of cells of extra-intestinal origin (Jung et al., 2018; .

PEDV antigens were also frequently detected in the intestinal crypt cells or antigen presenting cells, such as macrophages, in the lamina propria or Peyer's patches (Debouck et al., 1981; Jung et al., 2014; Lin et al., 2015a; Madson et al., 2016; Stevenson et al., 2013; Sueyoshi et al., 1995) . PEDV remains infectious in dendritic cells for <24 hours in vitro (Gao et al., 2015) , but dendritic cells may not be a site of PEDV replication . Instead, PEDV appeared to employ dendritic cells to cross the epithelial barrier of the nasal cavity . Lung tissues of oronasally infected pigs were negative for PEDV antigen (Debouck et al., 1981; Jung et al., 2014; Stevenson et al., 2013; Sueyoshi et al., 1995) . However, the upper respiratory tract may be the site of PEDV infection, because the epithelium lining the nasal cavity was positive for PEDV antigens . Although acute viremia was frequently noted in infected pigs during the acute or incubation stage of infection (Jung et al., 2018; Jung et al., 2014; , PEDV antigens were not detected in other major organs, such as liver and kidneys (Debouck et al., 1981; Jung et al., 2014; Stevenson et al., 2013; Sueyoshi et al., 1995) . The reproductive organs of experimentally infected boars, such as Cowpers's glands, were also negative for PEDV RNA, but their semen was transiently positive for PEDV RNA (Gallien et al., 2018b; Gallien et al., 2019) . Nevertheless, whether the PEDV RNA-positive semen contained infectious viral particles is unclear.

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During the incubation period (<PIH 16) in nursing pigs, a few to 50% of the villous epithelial cells positive for PEDV antigen were seen in the villous-crypt interface and upper villous epithelium of the small intestine (Debouck et al., 1981; (Jung et al., 2018) . During this period, infected enterocytes underwent necrosis, but complete villous atrophy was not observed (Jung et al., 2018) . When clinical signs were first seen (PIH 16-24), moderate to large numbers of infected villous epithelial cells were observed throughout the small intestine (Debouck et al., 1981) . During this period, up to 100% of the epithelial cells were positive for PEDV antigen in the mid-jejunum to ileum where moderate to severe villous atrophy was observed (Jung et al., 2018) . During the middle [post-inoculation day (PID) 1-3] to late (PID 4-5) stages of PEDV infection of nursing pigs (5-9-day-old), regardless of the locations (except for the duodenum with milder villous atrophy), all small intestinal segments exhibited similar, extensive villous atrophy (mean 1-2 intestinal villous height to crypt depth ratios, compared with 5-9 of uninfected pigs) (Chen et al., 2016a; Jung et al., 2015a) . During this period, large numbers of PEDV-infected epithelial cells could still be observed (Debouck et al., 1981) .

Diarrhea induced by PEDV is a consequence of malabsorption and maldigestion due to massive loss and functional disorders of infected enterocytes, followed by decreased brush border membrane-bound digestive enzymes (Coussement et al., 1982; Jung et al., 2006) . Dehydration is exacerbated by vomiting and decreased appetite (Jung and Saif, 2015) . Serotonin is involved in induction of vomiting, and increased pro-inflammatory cytokine responses in part led to decreased appetite (Jung et al., 2018) . PEDV-infected piglets also showed hyperkalemia and acidosis due to J o u r n a l P r e -p r o o f the loss of bicarbonate (Jung and Saif, 2015) . The cardiac contractility is impaired by acidosis and dehydration. In the small intestine of clinically infected pigs, the tight or adherens junctions of the villous enterocytes were destroyed (Jung et al., 2015b) . Expression of the junction proteins, such as zonula occludin-1, was also decreased or altered, followed by decreased transepithelial resistance (Curry et al., 2017; Jung et al., 2015b; Luo et al., 2017; Zong et al., 2019) . During the clinical period, there were remarkably reduced numbers of goblet cells in the small intestine (Jung and Saif, 2017) , followed by decreased amounts of mucins (Curry et al., 2017; Jung and Saif, 2017) . The damaged gut integrity may lead to uptake of luminal food and bacteria causing allergic reactions and co-infections. The impaired gut integrity or digestive function usually recovered by the second week after infection in survived pigs (Curry et al., 2017) .

In infected gnotobiotic or conventional nursing piglets (1-10-day-old), a few (<5%) to all of the inoculated pigs began to show diarrhea at PID 1, which mostly coincided with or was detected several hours later than the first detection of viral RNA in serum or feces (Chen et al., 2016a; Gerber et al., 2016; Jung et al., 2015a; Jung et al., 2018; Liu et al., 2015c; Madson et al., 2016; Thomas et al., 2015a) . Fecal viral RNA titers shed peaked by PID 1, or occasionally, 1-3 days later, and the titers decreased remarkably during the middle to late stages of infection and then titers remained low (with at least one more recurrent peak, possibly due to PED re-infection of regenerating enterocytes) during the recovery stage of infection (Chen et al., 2016a; Jung et al., 2015a; Lin et al., 2015a; Madson et al., 2016) . A one-three-day delay in onset of diarrhea was observed in nursing pigs inoculated with S INDEL PEDV compared with the non-S INDEL counterparts (Gallien et al., 2018a; Lin et al., 2015a) , indicating a longer incubation time for S J o u r n a l P r e -p r o o f INDEL PEDV. In infected conventional weaned to feeder pigs (21-56-day-old), a few (<5%) to all of the inoculated pigs began to show diarrhea at PID 1-4, which coincided with or was detected 1-2 days later than the first detection of viral RNA in feces (Gallien et al., 2018a; Gerber et al., 2016; Jung et al., 2015a; Lohse et al., 2017; Madson et al., 2014; Niederwerder et al., 2016; Thomas et al., 2015a) (Table 1) . Fecal viral RNA titers shed peaked by 1-3 days after diarrhea was first detected (Gallien et al., 2018a; Jung et al., 2015a; Lohse et al., 2017; Madson et al., 2014; Niederwerder et al., 2016; Thomas et al., 2015a) . Detailed data from boars or sows are summarized in Table 1 .

Duration of diarrhea in infected nursing pigs varied from 2-8 days, with a higher variability for the US S INDEL strain (2-7 days) compared with the US non-S INDEL (7-8 days) (Lin et al., 2015a) . Prolonged fecal viral RNA shedding (at least PID 14) was evident in 20/33 (61%) US non-S INDEL PEDV-infected nursing (10-day-old) pigs (Gerber et al., 2016) (Table 1) . Diarrhea in weaned to feeder pigs (21-56-day-old) inoculated orally with US non-S INDEL PEDV was observed for approximately 6 days Niederwerder et al., 2016; Thomas et al., 2015a) . Prolonged fecal viral RNA shedding was evident in non-S INDEL or S INDEL PEDVinfected weaned or feeder pigs for 15-26 days (Gallien et al., 2018a; Lohse et al., 2017; Madson et al., 2014; Niederwerder et al., 2016; Thomas et al., 2015a) . Duration of fecal shedding was similar between non-S INDEL-(15-26 days) (Gallien et al., 2018a; Lohse et al., 2017; Niederwerder et al., 2016; Thomas et al., 2015a) and S INDEL-infected weaned or feeder pigs (15-18 days) (Gallien et al., 2018a; Lohse et al., 2017) . Longer fecal viral RNA shedding was observed in weaned pigs infected via direct contact (42-46 days) or aerosol contact (48 days) compared with the orally infected counterparts (18-21 days) (Crawford et al., 2015; Gallien et al., 2018a) . However, infectious PEDV was shed in feces only for the initial 2 weeks tested using sentinel naïve pigs (Crawford et al., 2015) .

The minimum infectious dose [0.56 median tissue culture infectious dose (TCID50)] of non-S INDEL PEDV required to infect 5-day-old nursing pigs was lower than for 3-week-old weaned pigs (56 TCID50) (Thomas et al., 2015a) . Similarly, 3 plaque-forming unit (PFU)/pig of the non-S INDEL PC22A strain caused diarrhea in 100% of 4-day-old Cesarean-derived, colostrum-deprived (CDCD) piglets (Liu et al., 2015c) . Compared with nursing pigs that began to show diarrhea, villous atrophy, and fecal virus shedding at PID 1, a longer incubation time of non-S INDEL PEDV was required for weaned pigs to show fecal virus shedding (1 more day), or diarrhea and lesions (2 more days) (Jung et al., 2015a) . One factor contributing to the higher susceptibility of nursing pigs to non-S INDEL PEDV infection is immaturity of gut innate immunity, as evident by a functional defect in natural killer cells in the ileum and blood (Annamalai et al., 2015) . The lack of diversity and decreased Firmicutes to Bacteroidetes ratio in the gut microbiota of neonatal piglets contributed to the immature gut innate immunity . Compared with grower pigs (gastric pH 2-3), neonatal pigs have a higher gastric pH (4-6) that allows PEDV to survive in the stomach (Mavronmichalis, 2016).

Compared with nursing pigs, several factors that influence a shorter recovery of weaned pigs from PED include: 1) the faster regeneration of enterocytes in weaned pigs compared with J o u r n a l P r e -p r o o f nursing pigs (Moon et al., 1973) ; and 2) the anatomically and physiologically fully developed large intestine of weaned pigs where water reabsorption is greater (Jung et al., 2015a) . There was a lack of LGR5 (leucine-rich repeat-containing G protein-coupled receptor 5, marker for crypt stem cells)-positive crypt base columnar cells (LGR5+ cells) and low proliferation of crypt cells in the small intestine of naïve nursing piglets (9-day-old), causing the slower turnover of enterocytes (Jung et al., 2015a) . Unlike nursing pigs, naïve weaned pigs (3-week-old) exhibited high proliferation of intestinal crypt cells and large numbers of LGR5+ cells in the crypts, leading to the higher turnover rate of enterocytes (Jung et al., 2015a) . After PEDV infection, large numbers of LGR5+ cells and high proliferation of crypt cells were maintained, possibly relating to the rapid recovery from PED in weaned pigs.

Detection of viremia where viral RNA in serum ranged from 4.5-8.6 log10 genomic equivalents (GE)/ml was identified in gnotobiotic neonatal (5/5; 100%), or conventional 9-dayold nursing (16/16; 100%) and 26-day-old weaned pigs (11/20; 55%) infected with a US non-S INDEL PEDV strain at PID 1-5 (Jung et al., 2015a; Jung et al., 2014) . PEDV RNA was detected in all sera from 20 nursing pigs infected with either one of three different US non-S INDEL strains or a US S INDEL strain at PID 3 (Chen et al., 2016a) . The classical PEDV Br1/87 also induced viremia in 5/13 inoculated weaned pigs (38.5%) at PID 2-7 (Lohse et al., 2017) . In experimentally infected nursing pigs, the frequency of viremia and serum viral RNA titers peaked at PID 1, then decreased progressively, and ceased at PID 14-21 (Chen et al., 2016a; Jung et al., 2015a; Opriessnig et al., 2014) . A significantly higher frequency of viremia in weaned pigs infected via direct or aerosol contact, or orally with non-S INDEL PEDV was identified, compared with the S J o u r n a l P r e -p r o o f INDEL counterparts (Gallien et al., 2018a) . The role of viremia in the pathogenesis of PEDV is still obscure, although PEDV identified in the serum could be infectious Pasick et al., 2014; Perri et al., 2018) .

During the clinical period, alteration in the abundance or proportion of gut microbiota (dysbiosis) occurred in nursing pigs and their dams Koh et al., 2015; Liu et al., 2015b; Song et al., 2017; Tan et al., 2019) . The transfer of a healthy gut microbiota from infected sows to their offspring was impaired . The dysbiosis was characterized by reduced numbers of beneficial bacteria (Verrucomicrobia) and increased numbers of harmful bacteria (Fusobacterium and Proteobacteria) (Liu et al., 2015b; Tan et al., 2019) , followed by decreased cellular transport and catabolism . A study showed that feeding Bacillus subtilis to 2-week-old pigs infected with PEDV reduced intestinal pathology (but not clinical disease) (Canning et al., 2017) . Similarly, live or cell-free supernatants of some Lactobacillus plantarum strains had antiviral effects against PEDV infection in Vero cells (Sirichokchatchawan et al., 2017) . However, this feed additive method (1/77 herds surveyed; 1.2 %) was rarely used to mitigate PED on US pig farms during the 2013-2017 epidemic (Niederwerder and Hesse, 2018) .

Early studies verified high enteropathogenicity of the G1a CV777 in nursing pigs (Coussement et al., 1982; Pensaert and Martelli, 2016) . The S1 of the S protein and the rest of the genome of the S INDEL PEDV (G1b) was genetically closest to that of G1a and the highly virulent J o u r n a l P r e -p r o o f G2 PEDV, respectively (Lee, 2015; Pensaert and Martelli, 2016; Vlasova et al., 2014) , the strain was initially thought to be virulent. However, during the initial outbreaks by S INDEL PEDV in the US and Europe, the low to zero mortality rates of pigs in the field were distinct from those of US non-S INDEL infections with up to 100% mortality rates in nursing pigs (Boniotti et al., 2016; Goede et al., 2015; Stevenson et al., 2013; Wang et al., 2014b) . Nevertheless, difference in virulence of the two types of strains in the field was not often observed (Grasland et al., 2015; Mesquita et al., 2015; Stadler et al., 2015; Steinrigl et al., 2015) . A study using a wild-type strain of S INDEL PEDV reproduced as severe PED in infected sows and their piglets as that observed in the field (Leidenberger et al., 2017) . Other studies showed generally milder pathogenicity of S INDEL PEDV in 3-7-day-old conventional pigs, compared with non-S INDEL PEDV (Chen et al., 2016a; Lin et al., 2015a; Yamamoto et al., 2015) . However, the virulence of S INDEL PEDV was increased in the PEDV challenged suckling piglets when the mothers became sick, corresponding to poor immune, nutritional or health conditions (Lin et al., 2015a) . The mortality rates of piglets increased from 0% to 75%, and the peak fecal PEDV RNA titers shed were also as high as those of non-S INDEL strain.

The virulence of PEDV was attenuated by: 1) high cell-culture passage (93 rd -160 th ) in the cell line of non-swine origin, Vero cells (with trypsin in the cell culture medium) (Chen et al., 2015; Kweon et al., 1999; Lin et al., 2017a; Sato et al., 2011; Song et al., 2003) ; 2) modified cell . In addition, PEDV variants of mild virulence, such as the S1 N-terminal domain (NTD)-Del variant, also occurred naturally (Su et al., 2018; Suzuki et al., 2016; Zhang et al., 2018a) .

PEDV strains fully adapted to Vero cells and serially passaged at least 70 times (with trypsin or GCDCA in the cell culture medium) tended to replicate less efficiently in the intestine of pigs (Chen et al., 2015; Kim et al., 2017a; Lin et al., 2017a; Sato et al., 2011; Wu et al., 2019) (Table   2 ) and elicited low serum PEDV-specific antibody titers (Lin et al., 2017a) . Twelve-week-old pigs orally inoculated with 8.2 log10 TCID50 of G1a PEDV 83P-5 (100 th passage in Vero cells) exhibited only intermittent fecal viral RNA shedding in 3 of 4 pigs (Sato et al., 2011) . None of the 10-dayold pigs orally inoculated with 6 log10 TCID50 of G2b PEDV YN (144 th passage in Vero cells)

showed fecal viral RNA shedding; however, PEDV antigen was detected in the small intestine (Chen et al., 2015) . Compared with G2b PEDV YN (144 th ) and PC22A (140-160 th passage in Vero cells) grown with trypsin, G2b PEDV 8aa (70 th -105 th passage in Vero cells) grown with GCDCA and attenuated at lower cell passage numbers, caused no or little fecal viral RNA shedding in 1day-old pigs (6 log10 TCID50/pig) (Kim et al., 2017a) . Four-day-old CDCD pigs orally inoculated with 2 log10 PFU of PC22A (140 th -160 th ) showed low to moderate fecal viral RNA shedding titers, with mild villous atrophy (Lin et al., 2017a) (Table 2) . Six-day-old conventional pigs orally inoculated with 6 log10 TCID50 of G2b PEDV CT (120 th passage in Vero cells) showed mild diarrhea and moderate fecal viral RNA shedding titers, with mild villous atrophy .

On the other hand, pregnant sows orally inoculated with G1a PEDV DR13 (100 th passage in Vero cells) showed no clinical signs but high VN antibody levels in the colostrum (Song et al., 2007) . IL-17) responses in acutely infected neonatal pigs (Annamalai et al., 2015; Jung et al., 2018) . This coincided with increased numbers of PEDV antigen-positive cells (Gao et al., 2015; Madson et al., 2016) or inflammatory cells (lymphocytes or neutrophils) in the gut-associated lymphoid tissue (GALT) (Coussement et al., 1982; de Arriba et al., 2002b; Debouck et al., 1981; Jung and Saif, 2015) , and the onset of necrosis and exfoliation of infected enterocytes (Jung et al., 2018) . Necrosis of enterocytes is in part involved in the innate or pro-inflammatory cytokine responses in infected pigs, as also observed in vitro (Koonpaew et al., 2019; Lin et al., 2017b) . The increased systemic innate and pro-inflammatory cytokine responses that occur during acute PEDV infection are beneficial for differentiation of naïve T cells into cytotoxic or helper T cells, leading to removal of infected cells or production of VN antibodies, respectively (Siegrist, 2013) . However, highly increased, prolonged pro-inflammatory cytokines that can cause decreased appetite, etc. are also detrimental (Jung et al., 2018; Langhans, 2000) . Increased systemic IFNα and IFNγ cytokine responses were also observed in PEDV-infected gilts at PID 13-17 and PID 6-8, respectively (Langel et al., 2019b) .

Clinically infected pigs responded to a primary PEDV infection and seroconverted at PID 7-14 (mean PID 14) (PID 14-21 for serum PEDV-specific IgA antibodies), with peak serum J o u r n a l P r e -p r o o f PEDV-specific IgG or IgA antibody titers at PID 21 (Annamalai et al., 2017; Chen et al., 2016b; de Arriba et al., 2002a; de Arriba et al., 2002c; Gallien et al., 2018a; Gerber et al., 2016; Niederwerder et al., 2016; Opriessnig et al., 2014; Thomas et al., 2015a) . There were lower serum IgG or VN antibodies in S INDEL-infected pigs compared with the non-S INDEL counterpart (Annamalai et al., 2017; Chen et al., 2016b) . Serum VN antibodies were detected 7 days earlier than serum PEDV-specific IgG antibodies (Chen et al., 2016b; Thomas et al., 2015a) . There were low titers of PEDV-specific secretory IgA (sIgA) in the feces of PEDV-infected pigs (10-day or 8-week-old) by PID 14 (Gerber et al., 2016) . Serum PEDV-specific IgA and IgG antibodies in naturally infected sows remained high for 6 months, whereas fecal PEDV-specific sIgA antibodies disappeared 1-2 months after infection (Ouyang et al., 2015) . PEDV-specific IgA or VN antibody titers of sows infected at 3 months pre-farrowing peaked in the colostrum at post-farrowing day (PFD) 1, decreased rapidly in milk by PFD 3, and then declined only gradually during PFD 4-19 . Memory B cell responses were first identified by in vitro secondary stimulation in mononuclear cells from PEDV CV777-infected nursing pigs at PID 21 (de Arriba et al., 2002a) .

The number of PEDV-specific IgA antibody-positive pigs also rose from 30% at the day of reinfection to 70% 3 days later (Gerber et al., 2016) . Similarly, the number of PEDV-specific IgA antibody secreting cells of sows infected at 3-4 months pre-farrowing and then after re-exposure to PEDV at PFD 3-5, increased in the milk at PFD 8-14, compared with PFD 0 (colostrum) or PFD 

Enhanced biosecurity, such as decontamination and disinfection of transport trailers and other potential contaminated fomites, supply of secure feed or feed additives (Gordon et al., 2019; Niederwerder and Hesse, 2018) , and maintenance of strong hygiene measures on swine farms and farm workers (Kim et al., 2017b) , is critical to prevent direct or indirect contact transmission of PEDV. Transport trailers (19/72 herds surveyed; 26.4%) or feed (21/72 herds surveyed; 29.2%) were thought to be the main contaminated fomites (55.6% in total) in US swine farms during the 2013-2017 epidemic (Niederwerder and Hesse, 2018) . Spillover of PEDV from PEDV-infected farms to others or wildlife should also be controlled through high quarantine protocols that should include no movement of pigs until the diarrhea is terminated and the disease is controlled (Niederwerder and Hesse, 2018) . Indeed, 28 of 287 fecal samples ( 

Use of 0.5% Virkon S and 2.06% Clorox that reduced the quantity of PEDV RNA were the best disinfectants for PEDV inactivation in feces (Bowman et al., 2015b) . Super-oxidized water J o u r n a l P r e -p r o o f (pH 6.0) also effectively inactivated PEDV at room temperature after 10-90 min contact . Washing trailers with a high pressure power washer, followed by disinfection and drying, was effective for their sanitation and decontamination (Baker et al., 2018; Thomas et al., 2015b) . Accelerated hydrogen peroxide or peroxygen-based disinfectants effectively inactivated PEDV on contaminated metal (trailer) surfaces at 20 °C after 30 min contact (Holtkamp et al., 2017) and at -10 °C after 40-60 min contact (Baker et al., 2017) , or at -10 to 4 °C after 10-30 min contact (Baker et al., 2018) , respectively. PEDV RNA was still detectable after disinfection, but no infectious virus was detected.

The levels of PEDV-specific sIgA antibody in the colostrum/milk of dams are key to determine the extent of passive immunity of their piglets against PEDV infection (Langel et al., 2019a; Langel et al., 2019b; Langel et al., 2016; Langel et al., 2020; Saif et al., 2012) . The sIgA antibody is resistant to proteolytic enzymes and is highly efficient at neutralizing PEDV in the intestinal mucosa (Langel et al., 2016) . In the mammary glands of female swine, however, there is an absence of lymphoid follicles for antigen processing and IgA or IgG antibody production ( Fig. 2A and B) . The PEDV-specific sIgA antibodies in the colostrum and milk are produced by effector B cells or plasmablasts that migrate from the GALT of the PEDV-infected intestine to the mammary gland (Langel et al., 2016; Langel et al., 2020; Saif et al., 2012) . This is defined as the gut-mammary-sIgA axis, that was first described by Saif and Bohl in studies of immunity to TGEV [reviewed in (Saif et al., 2012) ]. The details were reviewed previously (Langel et al., 2016; Langel et al., 2020; Saif et al., 2012) . The endothelium in the mammary glands contains specialized J o u r n a l P r e -p r o o f endothelial venules that attract and bind to the specific integrins on the surface of the IgAplasmablasts, such as α4β7 and chemokine receptor 9 (CCR9) (Bourges et al., 2008; Jiang et al., 2016; Salmon, 2000) . Mucosal addressin cellular adhesion molecule 1 (MAdCAM-1) and chemokine ligand 25 expressed in the specialized venules ( Fig. 2D and E) interact with α4β7 and CCR9, respectively. Extravasated sIgA antibody-secreting cells reside in the alveoli-supporting fibrous stroma of the mammary glands ( Fig. 2A-C) . The number of PEDV-specific sIgA antibodysecreting cells may positively correlate with the amount and density of these endothelial surface molecules in the mammary glands (Salmon, 2000) , although the specific role of each surface molecule in the gut-mammary-sIgA axis needs to be better characterized.

Immunization of pregnant sows is important in the control of epidemic PED and to reduce the number of deaths of suckling piglets (Jung and Saif, 2015; Langel et al., 2020) . The most common practice used to initiate herd immunity in US pig farms during the 2013-2017 epidemic when no PEDV vaccines were available, was use of whole-herd feedback using a load-closeexpose protocol (59/77 herds surveyed; 76.6%), in which a controlled exposure for gilt acclimation (23/77 herds surveyed; 29.9%) was included (Niederwerder and Hesse, 2018) , whereas 19/77 herds surveyed (24.7%) used vaccination. There is no standard feedback protocol for any type of pig production systems to have the most consistent and highest efficacy (Niederwerder and Hesse, 2018) . Immunization of pregnant sows, or gilts expected to be bred (gilt acclimation), is undertaken by exposure to virulent autogenous virus, such as minced intestines from infected neonatal piglets negative for other infectious agents (Jung and Saif, 2015; Niederwerder and Hesse, 2018) . Feedback stimulated lactogenic immunity via the gut-mammary-sIgA axis in sows (Saif et J o u r n a l P r e -p r o o f al., 2012), as well as prolonged, passive immune protection of piglets against PEDV infection (Goede et al., 2015) . In our study, strong protective immunity (100% survival rate of the piglets challenged with PC22A) was induced in gilts inoculated orally once with virulent non-S INDEL PEDV strain PC22A (10 5 PFU/gilt) during mid-gestation (day 57-59) (Langel et al., 2019a ). Gilts exposed to the virus earlier (day 19-22) or later (day 96-97) in the gestation period showed less protective immunity, as evident by 87.2% or 55.9% survival rates of their piglets, respectively. Sows given feedback or orally inoculated with virulent PEDV during mid-gestation also discontinued fecal PEDV shedding prior to farrowing (Langel et al., 2019a; Leidenberger et al., 2017) . Whether prolonged PEDV shedding post-exposure and during farrowing and lactation may cause infection of piglets, remains unclear. However, the most efficient and safest timing for feedback should be mid-gestation.

Because of the frequent inefficiency or lack of safety, feedback should be replaced by oral immunization with high, consistent doses of live attenuated PEDV that is minimally replicative, but immunogenic in the intestine of pigs (Song et al., 2007) . Pregnant sows orally inoculated with live attenuated PEDV (DR13 strain) vaccine twice at 2 and 4 weeks pre-farrowing exhibited higher PEDV-specific IgA and VN antibody levels in the colostrum compared with the counterpart sows administered the same vaccine IM (Song et al., 2007) . Although the IM route has also been used as an administration option using live PEDV vaccine (Kweon et al., 1999) , to what extent PEDVspecific IgA memory B cells are induced in sows initially primed via an IM route with live PEDV vaccine, has not been confirmed. The efficacy of PEDV IM boost vaccination is dependent on induction of IgA memory B cells in initially field exposed or orally primed sows (Gillespie et al., J o u r n a l P r e -p r o o f 2018), similar to the success of TGEV oral prime/parenteral boost vaccine strategies (Saif et al., 2012) . Live attenuated strains of PEDV have been isolated from the feces of pigs, suggesting that recombinant or mutated strains of PEDV might have emerged (Gerdts and Zakhartchouk, 2017; Guo et al., 2016) .

For maintaining or boosting herd immunity initiated through feedback or oral live vaccines in farrowing herds, IM administration with either live PEDV (Sato et al., 2018) or inactivated, adjuvanted whole-virus PEDV vaccine (Gillespie et al., 2018) once or twice at 4-5 or 2 weeks prefarrowing was effective in boosting lactogenic immunity in sows positive for PEDV-specific IgA memory B cells in GALT. Many vaccine candidates or products have been tested and developed in the US and worldwide. The details were reviewed previously Gerdts and Zakhartchouk, 2017) . IM live vaccine booster plus feedback was more effective in reducing the number of deaths of suckling piglets, compared with either IM live vaccine alone or feedback alone (Sato et al., 2018) . Prime and boost IM vaccination with inactivated PEDV provided incomplete protection of piglets against PEDV infection (Gerdts and Zakhartchouk, 2017; Niederwerder and Hesse, 2018) , because of poor lactogenic immunity induced in their vaccinated dams (Gillespie et al., 2018) . Despite the safety issue, therefore, feedback alone, or prime and boost vaccination via oral and/or IM routes with live PEDV have been frequently used for maternal immunization against PEDV infection.

J o u r n a l P r e -p r o o f PEDV remains a threat to the swine industry in the US and worldwide because no effective vaccines are currently available. Regardless of the genogroup, PEDV can be deadly in pig populations with no immunity or low immunity or health status. The extent of PEDV transmission and PED on farms has a reverse relationship with the levels of biosecurity. High biosecurity to control PED should be practiced prior to seasonal or expected epidemics. Concurrently, pregnant sows are immunized by either feedback or vaccination. However, the current feedback or vaccination protocols are frequently inefficient or unsafe, due to: 1) no standardized protocol for feedback; 2) poor capacities of current IM vaccines (live or killed) to induce lactogenic immunity;

3) antigenic differences of vaccine vs. epidemic strains; and 4) possibility of continuous reinfection by PEDV used for feedback within herds. In addition, current live vaccines, which were generated by conventional cell culture-adaptation methods, may revert to virulent PEDV or recombine with field PEDV strains to generate new strains after they are applied in the field. Therefore, better vaccination protocols and vaccine strains (attenuated oral vaccines) are required for prevention and control of PED. PEDV-specific IgA memory B cells in orally primed sows play a critical role in boosting lactogenic immunity of sows and passive protection of piglets when piglets are exposed to PEDV during the suckling period. Feedback can be replaced by oral immunization with live attenuated PEDV vaccines that are minimally replicative, but immunogenic in the intestine of pregnant sows, thereby inducing PEDV-specific IgA memory B cells in the GALT or other mucosal and systemic lymphoid tissues and lactogenic immunity.

Lastly, ideal live attenuated vaccines should not revert to virulent PEDV or recombine with field PEDV strains to generate new virulent PEDV strains, which remains an obstacle in vaccine development (Hou and Wang, 2019) .

None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. Annamalai, T., Saif, L.J., Lu, Z., Jung, K., 2015. Age-dependent variation 

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Isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the United States

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Fighting a deadly pig disease

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Receptor usage and cell entry of porcine epidemic diarrhea coronavirus

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Characterization of Porcine Epidemic Diarrhea Virus Isolate US/Iowa/18984/2013 Infection in 1-Day-Old Cesarean-Derived Colostrum-Deprived Piglets

Pathogenesis of porcine epidemic diarrhea virus isolate (US/Iowa/18984/2013) in 3-week-old weaned pigs

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Age dependent resistance to transmissible gastroenteritis of swine (TGE). I. Clinical signs and some mucosal dimensions in small intestine

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Swine enteric coronavirus disease: A review of 4 years with porcine epidemic diarrhoea virus and porcine deltacoronavirus in the United States and Canada

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Coronaviruses

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Porcine epidemic diarrhea virus introduction into the United States: Root cause investigation

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Emergence of Porcine epidemic diarrhea virus in the United States: clinical signs, lesions, and viral genomic sequences

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Distinct characteristics and complex evolution of PEDV strains

Porcine epidemic diarrhea virus variants with high pathogenicity

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Detection and genetic characterization of deltacoronavirus in pigs

New variant of porcine epidemic diarrhea virus

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Resistance to coronavirus infection in amino peptidase N-deficient pigs

Discovery of seven novel Mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus

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Histologic (A and B) and immunohistochemical (C) localization of mammary IgAsecreting cells, and expression of mucosal addressin cellular adhesion molecule

Hematoxylin and eosin-stained mammary gland tissue of a gilt at 4 days post-farrowing, showing fully expanded alveoli, with mostly empty lumens. (B) Higher magnification of Panel A (inset), showing clusters of plasma cells characterized by the typical peripheral nuclei in the abundant cytoplasm in the interlobular stroma. Original magnification, 200 (A) and 400 (B). (C) Immunohistochemistry (IHC)-stained mammary gland tissue of a gilt at 5 days post-farrowing, showing a moderate number of IgA-positive cells

We sincerely thank all the authors cited in our review for their efforts and contributions to understand PEDV. We apologize to authors whose contributions to PEDV research may have been 

Up to PID 21 9.7-10.2 log10 RNA copies/ml at PID 4-6 Direct contact PID 4 PID 4-14 Up to PID 49 6.6-9.3 log10 RNA copies/ml at PID 6-21 Indirect aerosol contactNo clinical signs in 10 pigs (0/10) Loose stool in 2/5 pigs (40%) at PID 1-10 or 15Low (with mean peak viral RNA shedding titer of 5.00 log10 GE/ml), as tested at PID 1-10 or 15Mild villous atrophy at PID 9 (mean VH:CD ratio of 5.2 vs. 6.8 in the control)No detection of PEDV antigen at PID 9CT (120 th ) 2b (non-S INDEL) 6 log10 TCID50 6-day 5-6 0 at PID

Loose stool in some pigs at PID 4-5, at monitored at PID 1-10Moderate (with mean peak viral RNA shedding titer of approximate 6-7 log10 GE/ml at PID 4-5), as tested at PID 1-10 Mild villous atrophy in a pig at PID 3No or little detection of PEDV antigen in a pig at PID 3Wu et al., 2019