Porcine epidemic diarrhea Virus (PEDV) is a severe and highly contagious swine disease. While older pigs have a chance of survival, 80 to 100 percent of the PEDV-infected piglets die within 24 hours of being infected. PEDV spreads primarily through fecal-oral contact (Pospischil et al., 2002; Song and Park, 2012). Once internalized it destroys the inner lining of piglets' intestines, making them incapable of digesting and deriving nutrition from milk and feed (Pospischil et al., 2002). The virus causes diarrhea, vomiting and death from severe dehydration and starvation in piglets. Moreover, the infected piglets shed virus for seven to ten days (Song and Park, 2012).
Porcine Epidemic Diarrhea was first reported as a clinical entity in England in 1971 and was determined to be separate from porcine transmissible gastroenteritis virus (TGEV) (Wood, 1977). The infectious agent was further characterized and identified as a coronavirus-like particle in Belgium in 1978 (Pensaert and de Bouck, 1978). Since then, PEDV has been reported in many European and Asian countries including the Czech Republic, Hungary, Italy, Germany, Spain, Korea, the Philippines, China, Thailand and Japan (Song and Park, 2012; Pospischil et al., 2002). In contrast to infections in Asia, severe PEDV outbreaks with high mortality are rare in Europe. Within Asia, China has seen a large increase in outbreaks since 2010, which has been attributed to the emerging of new strains (Li et al., 2012). In contrast, PEDV has not been detected or reported from Central America or South America countries to date.
PEDV was first reported in the United States in May 2013 in Iowa. Since then, the PEDV has spread rapidly nationwide (The Pig Site, 2013; Promed 2013). The number of confirmed cases of PEDV increased by 296 during March, thus bringing the total number reported to 4,757, since the outbreak according to the U.S. Department of Agriculture's National Animal Health Laboratory Network (NAHLN). Twenty seven U.S states have reported PEDV infection as of March, 2014. However, one case can represent an individual animal or an entire herd at a single site. The hog industry analysts estimate that PEDV has killed approximately 5 million U.S. hogs alone since May 2013. Although highly infectious in pigs, PEDV does not affect humans and is not a food safety risk.
PEDV is a member of the Coronavirinae family and belongs to alphacoronavirus genera. These viruses are enveloped, positive-sense, single-stranded RNA and with a nucleocapsid of helical symmetry of 130 nm in diameter (Pensaert and de Bouck, 1978; Spaan et al., 1988; Kocherhans et al., 2001). Their genomic size ranges from an approximately 26 to 32 Kb, relatively large for an RNA virus. Coronavirus are the largest viruses that are known to infect humans, other mammals, and birds, usually causing subclinical respiratory or gastrointestinal diseases. The PEDV subgenomic mRNAs, which are transcribed from the genome, produce viral protein subunits, such as the spike (S, ˜180-220 kDa), envelope (E, ˜8.8 kDa), membrane (M, 27-32 kDa), nucleoprotein (N, 55-58 kDa), and several other proteins of unknown function (Kocherhans et al., 2001; Li et al., 2012).
About two-thirds of the 5′ end of the genome encodes a replicase protein. These proteins are encoded by two slightly overlapping open reading frames (ORF), ORF1a and ORF1b (Bridgen et al., 1988; Kocherhans et al., 2001). These two ORF subunits are connected by a ribosomal frame shift site in all the coronaviruses. This regulates the ratio of the two polypeptides encoded by ORF1a and the read-through product ORF1ab. About 70-80% of the translation products are terminated at the end of ORF1a, and the remaining 20-30% continues to transcribe until the end of ORF1b. The polypeptides are posttranslationally processed by viral encoded proteases (Bridgen et al., 1988; Park et al., 2012; Park et al., 2013). These proteases are encoded within ORF1a and the polymerase-/helicase-function are encoded by ORF1b. The analysis and amino acid alignment of N, M, E, ORF3 and S gene sequences of the highly virulent PEDV strain CV777 shows that PEDV occupies an intermediate position between the two well-characterized members of the group I corona viruses, TGEV and human coronavirus (HCoV-229E) (Pratelli 2011).
The nucleoprotein (N) subunit is a RNA-binding protein, and plays an important role in both virus RNA synthesis and modulating host cell processes. Phosphorylation and dephosphorylation may regulate these processes by exposing various functional motifs (Spencer et al., 2008; Hsieh et al., 2005). The N protein subunit has been implicated in various functions throughout the coronavirus life cycle including encapsulation, packaging, correct folding of the RNA molecule, the deregulation of the host cell cycle (Surjit, et al., 2006; Masters and Sturman, 1990), inhibition of interferon production, up-regulation of COX2 production, up-regulation of AP1 activity, induction of apoptosis, association with host cell proteins, and RNA chaperone activity (Stohlman et al., 1988; Tang et al., 2005; Nelson et al., 2000).
The PEDV E protein subunit is a homooligomer which interacts with the membrane (M) protein subunit in the budding compartment of the host cell, which is located between the endoplasmic reticulum (ER) and the Golgi complex (Duarte et al., 1994; Bridgen et al., 1998). The E protein subunit is a component of the viral envelope that plays a central role in virus morphogenesis and assembly. It also acts as a viroporin, inducing the formation of hydrophilic pores in cellular membranes and is sufficient to form virus-like particles (Madan et al., 2005). The PEDV E protein subunit has no effect on the intestinal epithelial cells (IEC) growth, cell cycle and cyclin-A expression. In contrast, the cells expressing PEDV E protein induce higher levels of IL-8 than control cells (Xu et al., 2013). Studies have shown that PEDV E protein induces ER-stress and activates transcription factor NF-κB, which is responsible for the up-regulation of interleukin 8 (IL-8) and Bcl-2 expression (Liao et al., 2006; Liao et al., 2004; Xu et al., 2013).
The M protein subunit of PEDV is the most abundant component of the viral envelope. In silico analysis of the M protein subunit shows that it consists of a triple-transmembrane segment flanked by a short amino-terminal domain on the exterior of the virion and a long carboxy-tail located inside the virion. The M protein subunit of coronaviruses is indispensable in the assembly process and budding of virions (Zhang et al., 2012). The immune reaction to the M protein of coronaviruses plays an important role in the induction of protection and in mediating the course of the disease (Zhang et al., 2012). Monoclonal antibodies against the M protein subunit of coronaviruses have virus-neutralizing activity in the presence of complement (Qian et al., 2006). Furthermore, the M protein subunit of coronavirus can also stimulate the production of alpha-interferon (α-IFN) which can inhibit viral replication (Xing et al., 2009).
The function of the PEDV ORF3 product subunit remains enigmatic, however computational modeling of PEDV OFR3 protein subunit shows that it may function as an ion channel and regulate virus production (Wang et al., 2012). Small interfering RNA (siRNA) knockdown of ORF3 gene in PEDV infected cells reduces the number of particles released from the cells (Wang et al., 2012). Passing PEDV in cell culture leads to the truncation or loss of ORF3 (Schmitz et al., 1998; Utiger et al., 1995). Homologues of the ORF3 protein subunit are found in all other alphacoronaviruses. The ORF3 protein of hCoV-NL63 was shown to be N-glycosylated at the amino terminus and incorporated into virions. However, deletion of the ORF3 gene from the viral genome had little effect on virus replication in vitro (Donaldson et al., 2008). Similar to other alphacoronaviruses (TGEV and, HCoV-229E) loss of PEDV ORF3 does not affect its replication in vitro (Dijkman et al., 2006; Woods, 2001). Despite a non-essential role in cell culture, the maintenance of the ORF3 gene in alphacoronavirus field strains strongly points to an important role of the ORF3 protein in natural infection in the animal host. Consistently, the loss of virulence of live-attenuated PEDV vaccine strains has been associated with mutations in the ORF3 gene resulting from cell culture adaptation (Song et al., 2007). However, this loss of virulence can also be attributed to concomitant mutations in other genes such as the spike protein gene (Park et al., 2008; Sato et al., 2012). The specific function of the ORF3 protein (and other viral proteins in the 3′ genome region) in PEDV replication and pathogenesis can now be investigated using the reverse genetics system (Li et al., 2013).
The spike protein of the PEDV is a large glycoprotein of ˜180 to 200 kDa, and belongs to the class I fusion proteins (Bosch et al., 2003). The functional S protein subunit forms a homotrimer on the virion surface. The coronavirus S proteins consists of two subunits and are cleaved by host proteases into the N-terminal S1 subunit and the C-terminal membrane-anchored S2 subunit. The S1 subunit binds to its receptor on the host cell, while the S2 subunit is responsible for fusion activity (Park et al., 2007; de Haan et al., 2004). This cleavage initiates the cell-to-cell fusion and virus entry into cells (Spaan et al., 2008; Simmons et al., 2004). Various proteases are known to be utilized for cleavage of the S protein subunit of each coronavirus. For example, in murine coranavirus mouse hepatitis virus (MHV), the basic amino acid cluster in the middle of the S protein is cleaved by a protease, furin, during its biogenesis. The cleaved S protein subunit is retained on the virion and infected-cell surfaces, inducing cell-to-cell fusion (Spaan et al., 2008). In contrast, S proteins of severe acute respiratory syndrome coronavirus (SARS-CoV), nonfusogenic MHV-2, and HCoV-229E, have no furin recognition site, therefore these S proteins are not cleaved during their biogenesis (Simmons et al., 2004; Matsuyama et al., 2004; Yoshikura et al., 1988; Shirato et al., 2011). These S proteins without a furin recognition site are cleaved by endosomal proteases, such as cathepsins, and other proteases activated by the low-pH environment (Shirato et al., 2011). These coronaviruses, once bound to the receptor, are transported to the endosome, where the S protein subunit is cleaved and activated for fusion, which, in turn, results in the release of the virus genome into the cytoplasm from the endosome (Shirato et al., 2011). Thus, these coranavirus fail to induce syncytia in infected cells, and the S protein on the virion is not in a cleaved form (Shirato et al., 2011). Furthermore, the efficiency of infection of these coronavirus is not influenced by exogenous proteases. Similarly, PEDV has uncleaved S protein and PEDV-infected cells produce syncytia only after treatment with an exogenous protease, features similar to those of the coronavirus described above (Duarte et al., 1994; Durante and Laude, 1994). However, without the exogenous protease treatment, PEDV cannot grow efficiently in vitro (Park et al., 2007; Shirato et al., 2011). This explains the need for protease mediated cleavage of PEDV S protein subunit for virulence and in vitro propagation.
The complete genomic sequences of PEDV isolated from outbreaks in Minnesota and Iowa are available in the GenBank (Colorado, USA: USA/Colorado/2013, accession no. KF272920; 13-019349, accession no. KF267450 and ISU13-19338E-IN-homogenate, accession number KF650370). The genetic and phylogenetic analysis of the three U.S. strains reveals a close relationship with Chinese PEDV strains and possible Chinese origin. The U.S. PEDV strains underwent evolutionary divergence, and are 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 PEDV strain-divergence based on the complete genomic sequences shows an approximately 2 to 3 years' time-frame between the Chinese (December 2010) and the U.S (May 2013) outbreaks [US-USDA, Technical note, PED. Fort Collins (Colo.): USDA; 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 (Graham and Baric 2010; Wang et al., 2014).
All the isolates from recent studies have shown that all PEDV strains in the U.S. are clustered together in one clade within the subgenogroup 2a and are closely related to a strain from China, AH2012 (Sun et al., 2012; Park et al. 2012; Park et al., 2013). However, in February 2014, the Animal Disease Diagnostic Laboratory of the Ohio Department of Agriculture announced that it identified a variant PED strain OH851 which showed 99% and 97% nucleotide identity to PEDVs currently circulating in the United States (Colorado, Iowa, Indiana, and Minnesota) for the whole genome and the full-length spike gene, respectively (Wang et al., 2014). By distinct contrast, the strain OH851 showed only 89% or even lower nucleotide identity to PEDVs currently circulating in the United States in the first 1,170 nt of the S1 region. In that region, nucleotide similarity to that of a PEDV strain from China (CH/HBQX/10, JS120103) was 99%, suggesting that strain OH851 is a new PEDV variant. Phylogenetic analysis of the complete genome indicated that the novel OH851 PEDV is clustered with other strains of PEDV currently circulating in the US, including another strain from Ohio, OH1414. However, phylogenetic analysis of the full-length S gene showed that strain OH851 is clustered with other strains of PEDV from China and most closely related to a PEDV strain from China, CH/HBQX/10, but distantly related to other PEDV strains currently circulating in the US and strain AH2012 (Zheng et al., 2013). These finding strongly suggests that strain OH851 is a variant PEDV. In comparison with the S gene of other strains from the US, the S gene of strain OH851 has 3 deletions (a 1-nt deletion at position 167, a 11-nt deletion at position 176, and a 3-nt deletion at position 416), a 6-nt insertion between positions 474 and 475, and several mutations mainly located in the first 1,170 nt of the S1 region (Zheng et al., 2013).
Due to these sequence deletions, insertion, and mutations the strain OH851 may have been attenuated. Since this strain does not cause severe clinical disease, including death, the novel virus is a potential vaccine candidate that could help protect the US swine industry from the infection caused by the virulent strains of PEDV currently circulating in the US. Furthermore, this analysis also indicates that the US PEDV strains are still evolving.
Over the years numerous PEDV vaccines have been developed and tried without much success. Although vaccines for PEDV exist in China, Japan and South Korea, there is no approved vaccine in the US or Europe (USDA 2013). There are two types of vaccines against PEDV that are currently available in the market—killed or live attenuated. Several Japanese, Chinese and South Korean companies manufacture PEDV vaccines, however, the efficacy and protection by PEDV vaccines is not promising or adequate for the global swine industry. On the other hand, piglets can obtain immunity from their mothers if the sow has an adequate amount of antibodies to pass immunity through colostrum (Geiger et al., 2013). Due to the lack of any efficacious vaccine in the US, one of the common practices followed by the veterinarians to protect the herd is via feedback, which is unacceptable. Recently an alphavirus based PEDV vaccine developed, licensed and distributed by a US company has failed to provide adequate protection.
The available evidence clearly indicates that PEDV is still evolving in the US and there is an immediate need to develop a more effective large scale vaccine. To achieve this, Newport Laboratories Inc., sequenced a PEDV strain isolated from the Midwest region to study its genetics, diversity and develop a subunit or attenuated vaccine.