Infectious Porcine Epidemic Diarrhea Virus Research Articles

HDAC-Specific Inhibitors Induce the Release of Porcine Epidemic Diarrhea Virus via the COPII-Coated Vesicles.

Porcine epidemic diarrhea virus (PEDV) is an alpha-coronavirus causing acute diarrhea and high mortality in neonatal suckling piglets, resulting in huge economic losses for the global swine industry. The replication, assembly and cell egression of PEDV, an enveloped RNA virus, are mediated via altered intracellular trafficking. The underlying mechanisms of PEDV secretion are poorly understood. In this study, we found that the histone deacetylase (HDAC)-specific inhibitors, trichostatin A (TSA) and sodium butyrate (NaB), facilitate the secretion of infectious PEDV particles without interfering with its assembly. We found that PEDV N protein and its replicative intermediate dsRNA colocalize with coat protein complex II (COPII)-coated vesicles. We also showed that the colocalization of PEDV and COPII is enhanced by the HDAC-specific inhibitors. In addition, ultrastructural analysis revealed that the HDAC-specific inhibitors promote COPII-coated vesicles carrying PEDV virions and the secretion of COPII-coated vesicles. Consistently, HDAC-specific inhibitors-induced PEDV particle secretion was abolished by Sec24B knockdown, implying that the HDAC-specific inhibitors-mediated COPII-coated vesicles are required for PEDV secretion. Taken together, our findings provide initial evidence suggesting that PEDV virions can assemble in the endoplasmic reticulum (ER) and bud off from the ER in the COPII-coated vesicles. HDAC-specific inhibitors promote PEDV release by hijacking the COPII-coated vesicles.

A Single-Dose Intramuscular Nanoparticle Vaccine With or Without Prior Intrauterine Priming Triggers Specific Uterine and Colostral Mucosal Antibodies and Systemic Immunity in Gilts but Not Passive Protection for Suckling Piglets.

An effective single-dose vaccine that protects the dam and her suckling offspring against infectious disease would be widely beneficial to livestock animals. We assessed whether a single-dose intramuscular (i.m.) porcine epidemic diarrhea virus (PEDV) vaccine administered to the gilt 30 days post-breeding could generate mucosal and systemic immunity and sufficient colostral and mature milk antibodies to protect suckling piglets against infectious challenge. The vaccine was comprised of polymeric poly-(lactide-co-glycolide) (PGLA)-nanoparticle (NP) encapsulating recombinant PEDV spike protein 1 (PEDVS1) associated with ARC4 and ARC7 adjuvants, a muramyl dipeptide analog and a monophosphoryl lipid A (MPLA) analog, respectively (NP-PEDVS1). To establish whether prior mucosal exposure could augment the i.m. immune response and/or contribute to mucosal tolerance, gilts were immunized with the NP-PEDVS1 vaccine via the intrauterine route at breeding, followed by the i.m. vaccine 30 days later. Archived colostrum from gilts that were challenged with low-dose PEDV plus alum was used as positive reference samples for neutralizing antibodies and passive protection. On day 100 of gestation (70 days post i.m. immunization), both vaccinated groups showed significant PEDVS1-specific IgG and IgA in the serum, as well as in uterine tissue collected on the day of euthanasia. Anti-PEDVS1 colostral IgG antibody titers collected at farrowing were significantly higher relative to the negative control gilts indicating that the NP vaccine was effective in contributing to the colostral antibodies. The PEDVS1-specific colostral IgA and anti-PEDVS1 IgG and IgA antibodies in the mature milk collected 6 days after farrowing were low for both vaccinated groups. No statistical differences between the vaccinated groups were observed, suggesting that the i.u. priming vaccine did not induce mucosal tolerance. Piglets born to either group of vaccinated gilts did not receive sufficient neutralizing antibodies to protect them against infectious PEDV at 3 days of age. In summary, a single i.m. NP vaccine administered 30 days after breeding and a joint i.u./i.m. vaccine administered at breeding and 30 days post-breeding induced significant anti-PEDVS1 immunity in systemic and mucosal sites but did not provide passive protection in suckling offspring.

Propidium Monoazide Combined With RT-qPCR Detects Infectivity of Porcine Epidemic Diarrhea Virus.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) allows sensitive detection of viral particles and viruses in epidemic samples but it cannot discriminate noninfectious viruses from infectious ones. Propidium monoazide (PMA) coupled with quantitative polymerase chain reaction (qPCR) was assessed to detect infectious viruses. Currently, there is no established test method to detect the infection of the porcine epidemic diarrhea virus (PEDV). In this study, propidium monoazide coupled with qPCR detects infectivity of PEDV. We optimized the method from the selection of primers, the working concentration of PMA, and the inactivation method using heat or ultraviolet (UV). The viruses which were treated with PMA before qPCR were inactivated using heat or UV. However, the addition of PMA alone did not affect the detection of live viruses, which indicates that a viral capsid break may be essential for PMA to bind to the genome. A repetition of the method on naked PEDV RNA suggests that it can be used to detect potentially infectious PEDV. The results indicated that an optimal plan of PMA could be extremely useful for evaluating infectious and noninfectious viruses.

Nucleocapsid proteins from other swine enteric coronaviruses differentially modulate PEDV replication

Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV) and porcine deltacoronavirus (PDCoV) share tropism for swine intestinal epithelial cells. Whether mixing of viral components during co-infection alters pathogenic outcomes or viral replication is not known. In this study, we investigated how different coronavirus nucleocapsid (CoV N) proteins interact and affect PEDV replication. We found that PDCoV N and TGEV N can competitively interact with PEDV N. However, the presence of PDCoV or TGEV N led to very different outcomes on PEDV replication. While PDCoV N significantly suppresses PEDV replication, overexpression of TGEV N, like that of PEDV N, increases production of PEDV RNA and virions. Despite partial interchangeability in nucleocapsid oligomerization and viral RNA synthesis, endogenous PEDV N cannot be replaced in the production of infectious PEDV particles. Results from this study give insights into functional compatibilities and evolutionary relationship between CoV viral proteins during viral co-infection and co-evolution.

Feed batch sequencing to decrease the risk of porcine epidemic diarrhea virus (PEDV) cross-contamination during feed manufacturing.

Feed has been identified as a vector of transmission for porcine epidemic diarrhea virus (PEDV). The objective of this study was to determine if feed batch sequencing methods could minimize PEDV cross-contamination. Porcine epidemic diarrhea virus-free swine feed was manufactured to represent the negative control. A 50 kg feed batch was mixed in a pilot scale feed mill for 5 min, sampled, then discharged for 10 min into a bucket elevator and sampled again upon exit. Next, a pathogenic PEDV isolate was used to inoculate 49.5 kg of PEDV-free feed to form the positive control. The positive control was mixed, conveyed and sampled similar to the negative control. Subsequently, 4 sequence batches (sequence 1 to 4) were formed by adding a 50 kg batch of PEDV-negative feed to the mixer after the prior batch was mixed and conveyed; all sequences were mixed, conveyed, and sampled similar to the negative and positive control batches. None of the equipment was cleaned between batches within a replicate. This entire process was replicated 3 times with cleaning the feed mill between replicates. Feed was then analyzed for PEDV RNA by real-time reverse transcriptase semiquantitative polymerase chain reaction (rRT-PCR) as measured by cycle threshold (Ct) and for infectivity by bioassay. Sequence 1 feed had higher (P ˂ 0.05) rRT-PCR Ct values than the positive batch and sequence 2 feed had higher (P ˂ 0.05) Ct values than sequence 1, regardless of sampled location. Feed sampled from the mixer from sequence 2, 3, and 4 was rRT-PCR negative whereas feed sampled from the bucket elevator was rRT-PCR negative from sequence 3 and 4. Bioassay was conducted using 66 mixed sex 10-d-old pigs confirmed negative for PEDV allocated to 22 different rooms. Pigs were initially 10-d old. Control pigs remained PEDV negative for the study. All pigs from the mixer positive batch (9/9) and bucket elevator positive batch (3/3) were rRT-PCR positive on fecal swabs by the end of the study. One replicate of pigs from mixer sequence 1 was rRT-PCR positive (3/3) by 7 dpi. One replicate of mixer pigs from sequence 2 was rRT-PCR positive (3/3) by 7 dpi although no detectable PEDV RNA was found in the feed. The results demonstrate sequenced batches had reduced quantities of PEDV RNA although sequenced feed without detectible PEDV RNA by rRT-PCR can be infectious. Therefore, a sequencing protocol can reduce but not eliminate the risk of producing infectious PEDV carryover from the first sequenced batch of feed.

Stability of Porcine Epidemic Diarrhea Virus on Fomite Materials at Different Temperatures.

Indirect transmission of porcine epidemic diarrhea virus (PEDV) ensues when susceptible animals contact PEDV-contaminated fomite materials. Although the survival of PEDV under various pHs and temperatures has been studied, virus stability on different fomite surfaces under varying temperature conditions has not been explored. Hence, we evaluated the survival of PEDV on inanimate objects routinely used on swine farms such as styrofoam, rubber, plastic, coveralls, and other equipment. The titer of infectious PEDV at 4 °C decreased by only 1 to 2 log during the first 5 days, and the virus was recoverable for up to 15 days on Styrofoam, aluminum, Tyvek® coverall, cloth, and plastic. However, viral titers decreased precipitously when stored at room temperature; no virus was detectable after one day on all materials tested. A more sensitive immunoplaque assay was able to detect virus from Styrofoam, metal, and plastic at 20 days post application, representing a 3-log loss of input virus on fomite materials. Recovery of infectious PEDV from Tyvek® coverall and rubber was above detection limit at 20 days. Our findings indicate that the type of fomite material and temperatures impact PEDV stability, which is important in understanding the nuances of indirect transmission and epidemiology of PEDV.

Efficacy of inactivated variant porcine epidemic diarrhea virus vaccines in growing pigs.

PurposeThe first aim of this study was to develop a novel inactivated porcine epidemic diarrhea virus (PEDV) vaccine using the recently isolated Korean PEDV QIAP1401 strain and to evaluate its protective efficacy in growing pigs. The second was to determine the optimum adjuvant formulation of the inactivated PEDV vaccine that induces protection against viral challenge.Materials and MethodsTo generate high titers of infectious PEDV, the QIAP1401 isolate was passaged in Vero cells. The experimental vaccines were prepared from a binary ethyleneimine-inactivated QIAP1401 strain passaged sequentially 70 times (QIAP1401-p70), formulated with four commercial adjuvants, and administered twice intramuscularly to growing pigs. Challenge studies using a virulent homologous strain of PEDV QIAP1401-p11, which was passaged 11 times after isolation, were performed to assess protection against disease progression and viral shedding during the 15-day observation period. The vaccine-induced antibody responses were measured in serum samples collected at predetermined time points by indirect enzyme-linked immunosorbent assay and virus neutralization test.ResultsThe QIAP1401-p70 strain had 42 amino acid (aa) mutations, including a 25 aa deletion, and was selected as the inactivated PEDV vaccine candidate. Although none of the pigs that received the experimental vaccines were completely protected against subsequent viral challenge, they exhibited a significantly higher immune response than did non-vaccinated control pigs. Among the vaccine groups, the highest antibody responses were observed in the pigs that received an oil-based multiphasic water/oil/water (W/O/W) emulsion adjuvanted vaccine, which delayed the onset of clinical symptoms and viral shedding.ConclusionA novel inactivated PEDV vaccine formulated with a W/O/W emulsion adjuvant was both immunogenic and protective against viral challenge.

First isolation and genetic characteristics of porcine sapeloviruses in Hunan, China.

Outbreaks of diarrhea in piglets cause serious economic consequences in China. Diarrhetic fecal samples from 20 Hunan farm piglets were tested and found to be positive for porcine epidemic diarrhea virus (PEDV) by RT-PCR, although incubation with porcine kidney (PK-15) cells failed to produce infectious PEDV. Four porcine sapelovirus (PSV) strains (designated as PSV-HuNs) were isolated from four of the samples. Genomic sequence analysis revealed open reading frames encoding polyproteins of 2,331 (HuN1, 2 and 3) and 2,332 (HuN4) amino acids. Homology comparisons of the VP1 gene of the four Hunan strains with previously reported PSV strains revealed nucleotide sequence identities ranging from 74.2 to 98.6%, and deduced amino acid sequence identities from 79.5 to 98%. Phylogenetic analyses based on full-length and partial VP1 gene sequences showed that 3 of the PSV-HuN strains (HuN2, 3 and 4) clustered within a clade distinct from HuN1 as well as from all PSVs previously isolated in China, thereby showing that genetic diversity exists within Chinese PSVs. In addition, recombination analysis among PSVs indicates that a recombinant (HuN2 strain) exist in China.

Effects of disinfection on the molecular detection of porcine epidemic diarrhea virus

Routine detection of porcine epidemic diarrhea virus (PEDV) is currently limited to RT-PCR but this test cannot distinguish between viable and inactivated virus. We evaluated the capability of disinfectants to both inactivate PEDV and sufficiently damage viral RNA beyond RT-PCR detection. Five classes of disinfectants (phenol, quaternary ammonium compound, sodium hypochlorite, oxidizing agent, and quaternary ammonium/glutaraldehyde combination) were evaluated in vitro at varying concentrations, both in the presence and absence of swine feces, and at three different temperatures. No infectious PEDV was recovered after treatment with evaluated disinfectants. Additionally, all tested disinfectants except for 0.17% sodium hypochlorite dramatically reduced qRT-PCR values. However, no disinfectants eliminated RT-PCR detection of PEDV across all replicates; although, 0.52%, 1.03% and 2.06% solutions of sodium hypochlorite and 0.5% oxidizing agent did intermittently produce RT-PCR negatives. To simulate field conditions in a second aim, PEDV was applied to pitted aluminum coupons, which were then treated with either 2.06% sodium hypochlorite or 0.5% oxidizing agent. Post-treatment surface swabs of the coupons tested RT-PCR positive but were not infectious to cultured cells or naïve pigs. Ultimately, viable PEDV was not detected following application of each of the tested disinfectants, however in most cases RT-PCR detection of viral RNA remained. RT-PCR detection of PEDV is likely even after disinfection with many commercially available disinfectants.

Evaluation of porcine epidemic diarrhea virus transmission and the immune response in growing pigs.

Clinical disease associated with porcine epidemic diarrhea virus (PEDV) infection in naïve pigs is well chronicled; however, information on endemic PEDV infection is limited. To characterize chronic PEDV infection, the duration of infectious virus shedding and development of protective immunity was determined. On Day 0 (D0), a growing pig was challenged with PEDV and 13 contacts were commingled. On D7, 9 contact pigs (principal virus group (PG)), were selected, moved to a separate room and commingled with one sentinel pig (S1). This process was repeated weekly with S2, S3 and S4. The PG was PEDV-positive by PCR from D3-11, with some pigs intermittently positive to D42. Pigs S1 and S2 were PEDV-positive within 24 hours of commingling. Antibodies were detected in all PG by D21 and by 7 days post-contact in S1 and S2. Pigs S3 and S4 were PCR and antibody negative following commingling. To evaluate protective immunity, 5 naïve pigs (N) and the PG were challenged (N/C, PG/C) with homologous virus on D49. All N/C pigs were PEDV PCR-positive by D52 with detection out to D62 in 3/5 N/C pigs. All PG/C pigs were PEDV PCR-negative post-challenge. By D63, all N/C seroconverted. Although PEDV RNA was demonstrated in pigs after primary infection until D42, infectious PEDV capable of horizontal transmission to naïve pigs was only shed 14–16 days after infection to age-matched pigs. Homologous re-challenge 49 days post initial PEDV exposure did not result in re-infection of the pigs. This demonstrates potential for an effective PEDV vaccine.

Effect of Thermal Mitigation on Porcine Epidemic Diarrhea Virus (PEDV)- Contaminated Feed

Porcine Epidemic Diarrhea Virus (PEDV) is primarily transmitted by fecal-oral contamination. However, epidemiological evidence has shown that swine feed and ingredients may serve as potential vectors of transmission. Since it is known that PEDV is a heat-sensitive virus, we hypothesized that a conditioner and pellet mill mimicking commercial thermal processing would mitigate PEDV infectivity. To test this hypothesis, two experiments were designed to determine if different pellet mill conditioner retention times or temperatures would impact PEDV infectivity determined by polymerase chain reaction (PCR) analysis and bioassay. For the first study, a 3×3×2 factorial was utilized, with three pelleting temperatures (155, 175, or 195°F), three conditioning times (45, 90, or 180 s), and two levels of virus (low: 1×102 TCID50/g, or high: 1×104 TCID50/g). Non-inoculated and PEDV-inoculated unprocessed mash were used as controls. There was no PEDV RNA detected in the PEDV-free mash. The low-dose PEDV-infected mash was 6.8 ± 1.8 cycle threshold (Ct) greater (P < 0.01) than the high dose mash. Regardless of time or temperature, feed processing increased (P < 0.01) the Ct compared to the PEDV-inoculated unprocessed mash. As expected, fecal shedding of PEDV was not detected in rectal swabs from control pigs for the duration of the study. Fecal swabs from pigs fed the PEDV-inoculated unprocessed mash, regardless of dose, were PEDV-positive from 2 to 7 days post-inoculation, at which time the pigs were sacrificed. However, if either PEDV dose of inoculated feed was pelleted at any of the nine tested conditioning time × temperature combinations, no PEDV RNA was detected in fecal swabs or cecum content. Based on these results, a second experiment was developed to determine the impact of lower processing temperatures on PEDV infectivity. The pellet mill was heated for 1 hour at normal manufacturing conditions prior to simulating a plug by turning off the steam supply. This allowed the temperature of the mash feed to decrease below 100°F. The PEDV-inoculated feed was then pelleted at one of five conditioning temperatures (100, 115, 130, 145, or 160°F) for 30 s. This study was repeated three times on three separate days with complete decontamination between each experiment day. Again, non-inoculated and PEDV-inoculated mash were used as controls. The five increasing temperatures led to feed with respective mean Ct values of 32.5, 34.6, 37.0, 36.5, and 36.7. Even though all samples had detectable PEDV RNA in the feed, infectivity was only detected by bioassay in pigs from the 100 and 115°F conditioning treatments. In each of the other processing temperatures, no PEDV RNA was detected in fecal swabs or cecum contents. Our results suggest that processing feed through a conditioner and pellet mill similar to those used in commercial feed mills will be effective as a point-in-time mitigation step for PEDV as long as conditioning temperatures remain above 130°F. Any time feed is processed at temperatures below that level, such as during start-up or when the pellet mill die becomes plugged and the steam is consequently shut off, there is a risk that the feed can act as a vector for transferring infectious PEDV and lead to cross-contamination of post-pelleting handling equipment.

The spray-drying process is sufficient to inactivate infectious porcine epidemic diarrhea virus in plasma

Porcine epidemic diarrhea virus (PEDV) is considered an emergent pathogen associated with high economic losses in many pig rearing areas. Recently it has been suggested that PEDV could be transmitted to naïve pig populations through inclusion of spray-dried porcine plasma (SDPP) into the nursery diet which led to a ban of SDPP in several areas in North America and Europe. To determine the effect of spray-drying on PEDV infectivity, 3-week-old pigs were intragastrically inoculated with (1) raw porcine plasma spiked with PEDV (RAW-PEDV-CONTROL), (2) porcine plasma spiked with PEDV and then spray dried (SD-PEDV-CONTROL), (3) raw plasma from PEDV infected pigs (RAW-SICK), (4) spray-dried plasma from PEDV infected pigs (SD-SICK), or (5) spray-dried plasma from PEDV negative pigs (SD-NEG-CONTROL). For the spray-drying process, a tabletop spray-dryer with industry-like settings for inlet and outlet temperatures was used. In the RAW-PEDV-CONTROL group, PEDV RNA was present in feces at day post infection (dpi) 3 and the pigs seroconverted by dpi 14. In contrast, PEDV RNA in feces was not detected in any of the pigs in the other groups including the SD-PEDV-CONTROL group and none of the pigs had seroconverted by termination of the project at dpi 28. This work provides direct evidence that the experimental spray-drying process used in this study was effective in inactivating infectious PEDV in the plasma. Additionally, plasma collected from PEDV infected pigs at peak disease did not contain infectious PEDV. These findings suggest that the risk for PEDV transmission through commercially produced SDPP is minimal.

Evidence of infectivity of airborne porcine epidemic diarrhea virus and detection of airborne viral RNA at long distances from infected herds.

Porcine epidemic diarrhea virus (PEDV) spread rapidly after being diagnosed in the USA in April 2013. In this study we assessed whether PEDV could become airborne and if so, whether the virus was infectious. Air samples were collected both from a room containing experimentally infected pigs and at various distances from the outside of swine farms experiencing acute PEDV outbreaks. Results indicated presence of infectious PEDV in the air from experimentally infected pigs and genetic material of PEDV was detected up to 10 miles downwind from naturally infected farms. Airborne transmission should be considered as a potential route for PEDV dissemination.

Evidence of infectivity of airborne porcine epidemic diarrhea virus and detection of airborne viral RNA at long distances from infected herds

Porcine epidemic diarrhea virus (PEDV) spread rapidly after being diagnosed in the USA in April 2013. In this study we assessed whether PEDV could become airborne and if so, whether the virus was infectious. Air samples were collected both from a room containing experimentally infected pigs and at various distances from the outside of swine farms experiencing acute PEDV outbreaks. Results indicated presence of infectious PEDV in the air from experimentally infected pigs and genetic material of PEDV was detected up to 10 miles downwind from naturally infected farms. Airborne transmission should be considered as a potential route for PEDV dissemination.