Evolution and virulence of porcine epidemic diarrhea virus following in vitro and in vivo propagation.

Practice of inoculating porcine epidemic diarrhea virus (PEDV) in piglets generating feedback material might influence the genetic evolution and attenuation of PEDV. The study was conducted to evaluate evolutionary rate and attenuation following serial in vitro and in vivo propagation. In the study, PED-JPFP0-PJ, Passage 0 (P0), was isolated from infected pigs and serially passaged in Vero cells for 5 consecutive times, P1-P5. P0, P2 and P5 were then subjected to orally inoculate 3-day-old piglets. At 24 h post inoculation, intestines of each passage (F1), were collected, and subsequently sub-passaged in piglets for 2 additional passages (F2-F3). Virus titration, PEDV genomic copies number, VH:CD ratios, and immunohistochemistry were evaluated. S and ORF3 genes were characterized. The results of the study demonstrated that virus titer and virulence were negatively correlated with increased passages, both in vitro and in vivo. Increased substitution rate was observed in higher passages. The evolutionary rate of S gene was higher than that of ORF3. Seven aa changes at positions 223, 291, 317, 607, 694, 1114 and 1199, with reduced N-linked glycan were observed in P5F3. In conclusion, serial passage of PEDV, both in vitro and in vivo, influence the genetic development and the attenuation of PEDV.

Intradermal needle-free injection prevents African Swine Fever transmission, while intramuscular needle injection does not.

Shared needles are a possible iatrogenic and hematogenous inanimate vector of African Swine Fever virus (ASFV) in farm conditions. To evaluate that possible transmission, sixty, 4-week-old pigs were procured from an ASF free herd free. Upon arrival, pigs were randomly divided into two sets. Set 1 served as seeder pigs, and were randomly allocated to 4 groups. The other pigs were divided into 8 groups, and served as sentinels. Seeder pigs were oronasally challenged with ASFV at high (108 copy numbers/mL), moderate (106 copy numbers/mL) or low (101 copy numbers/mL) challenge titer, except a subgroup that remained unchallenged (negative control). At 7 days post challenge (peak viremia), all four seeder groups were intradermally and intramuscularly (IM) injected with a vaccine adjuvant (Diluvac Forte, MSD Animal Health, The Netherlands) using a needle-free device (IDAL 3G, MSD Animal Health, The Netherlands) and conventional needles, respectively. The same needle or needle-free device was then used to inject the same volume of adjuvant into set 2 (n = 48) pigs. All pigs were observed for clinical disease daily and assayed for the presence of ASFV DNA by quantitative PCR. All seeder groups developed viremia (except the control pigs). ASFV viremia was detected in all sentinel groups injected via the intramuscular route. Transmission rate from the IM route via conventional needles was positively correlated with virus titer in blood circulation of seeders. Sentinels intramuscularly exposed to needles from high titer challenged seeders displayed more severe and acute clinical disease compared to that of exposed to low titer challenged seeders. No viremia nor clinical signs were observed in the sentinel groups injected via the intradermal route. This study confirmed the hematogenous transmission of ASFV between pigs through needle-sharing.

Co-infection of porcine deltacoronavirus and porcine epidemic diarrhea virus induces early TRAF6-mediated NF-κB and IRF7 signaling pathways through TLRs.

Porcine deltacoronavirus (PDCoV) and porcine epidemic diarrhea virus (PEDV) infect the small intestine and cause swine enteric coronavirus disease. The mucosal innate immune system is the first line of defense against viral infection. The modulatory effect of PDCoV and PEDV coinfection on antiviral signaling cascades of the intestinal mucosa has not been reported. Here, we investigate the gene expression levels of pattern recognition receptors, downstream inflammatory signaling pathway molecules, and associated cytokines on the intestinal mucosa of neonatal piglets either infected with a single- or co-infected with PDCoV and PEDV using real-time PCR. The results demonstrate that single-PEDV regulates the noncanonical NF-κB signaling pathway through RIG-I regulation. In contrast, single-PDCoV and PDCoV/PEDV coinfection regulate proinflammatory and regulatory cytokines through TRAF6-mediated canonical NF-κB and IRF7 signaling pathways through TLRs. Although PDCoV/PEDV coinfection demonstrated an earlier modulatory effect in these signaling pathways, the regulation of proinflammatory and regulatory cytokines was observed simultaneously during single viral infection. These results suggested that PDCoV/PEDV coinfection may have synergistic effects that lead to enhanced viral evasion of the mucosal innate immune response.

Using a concurrent challenge with porcine circovirus 2 and porcine reproductive and respiratory syndrome virus to compare swine vaccination programs.

The objectives of the present study were to evaluate the immune response of six commercial vaccines against PRRSV-2 and PCV2, administered as monovalent or combined products via intramuscular (IM) or intradermal (ID) routes. Seventy-two, 3-week-old pigs were randomly allocated into 8 treatments with 9 pigs each: IMPP0/PCVMH7, IDPP0/PCVMH7, IMING0/PCVMH7, IMPP0/PCVMH0, IDPP0/PCVMH0, IMTRF0, NV/CH, and NV/NC. IMPP0/PCVMH0 and IMPP0/PCVMH7 groups were IM vaccinated once with Prime Pac PRRS (MSD Animal Health, The Netherlands) at 0 days post-vaccination (DPV), followed by single IM vaccination with Porcilis PCV M Hyo (MSD Animal Health, The Netherlands) either at 0 or 7 DPV, respectively. IDPP0/PCVMH0 and IDPP0/PCVMH7 groups were ID vaccinated once with Prime Pac PRRS (MSD Animal Health, The Netherlands) at 0 DPV, followed by a single concurrent ID injection of Porcilis PCV ID (MSD Animal Health, The Netherlands) and Porcilis M Hyo ID ONCE (MSD Animal Health, The Netherlands) either at 0 or 7 DPV, respectively. The IMING0/PCVMH7 group was IM vaccinated once with Ingelvac PRRS MLV (Boehringer Ingelheim, Germany) at 0 DPV, and subsequently IM vaccinated with Ingelvac CircoFLEX (Boehringer Ingelheim, Germany) and Ingelvac MycoFLEX (Boehringer Ingelheim, Germany) at 7 DPV. The IMTRF0 group was IM vaccinated once with combined products of Ingelvac PRRS MLV (Boehringer Ingelheim, Germany), Ingelvac CircoFLEX (Boehringer Ingelheim, Germany), and Ingelvac MycoFLEX (Boehringer Ingelheim, Germany) at 0 DPV. The NV/CH and NV/NC groups were left unvaccinated. At 28 DPV (0 days post-challenge, DPC), pigs were intranasally inoculated with a 4 ml of mixed cell culture inoculum containing HP-PRRSV-2 (105.6 TCID50/ml) and PCV2d (105.0 TCID50/ml). Antibody response, IFN-γ-secreting cells (SC), and IL-10 secretion in supernatants of stimulated PBMC were monitored. Sera were collected and quantified for the PRRSV RNA and PCV2 DNA using qPCR. Three pigs from each group were necropsied at 7 DPC, lung lesions were evaluated. Tissues were collected and performed immunohistochemistry (IHC). Our study demonstrated that concurrent vaccination via the ID or the IM route did not introduce additional reactogenicity. We found no interference with the induction of immune response between vaccination timing. In terms of an immune response, ID vaccination resulted in significantly lower IL-10 levels and higher IFN-γ-SC values compared to the IM-vaccinated groups. In terms of clinical outcomes, only one IM-vaccinated group showed significantly better efficacy when antigens were injected separately compared with concurrently. While the vaccines were ID delivered, these effects disappeared. Our findings confirm that concurrent vaccination of PRRSV-2 MLV and PCV2 via either the IM or the ID routes could be a viable immunization strategy to assist with the control of PRDC. In situations where maximal efficacy is required, over all other factors, concurrent vaccination is possible with the ID route but might not be an ideal strategy if using the IM route.

Intranasal delivery of inactivated PRRSV loaded cationic nanoparticles coupled with enterotoxin subunit B induces PRRSV-specific immune responses in pigs.

This study was conducted to evaluate the induction of systemic and mucosal immune responses and protective efficacy following the intranasal administration of inactivated porcine reproductive and respiratory syndrome virus (PRRSV) loaded in polylactic acid (PLA) nanoparticles coupled with heat-labile enterotoxin subunit B (LTB) and dimethyldioctadecylammonium bromide (DDA). Here, 42- to 3-week-old PRRSV-free pigs were randomly allocated into 7 groups of 6 pigs each. Two groups represented the negative (nonvaccinated pigs/nonchallenged pigs, NoVacNoChal) and challenge (nonvaccinated/challenged, NoVacChal) controls. The pigs in the other 5 groups, namely, PLA nanoparticles/challenged (blank NPs), LTB-DDA coupled with PLA nanoparticles/challenged (adjuvant-blank NPs), PLA nanoparticles-encapsulating inactivated PRRSV/challenged (KNPs), LTB-DDA coupled with PLA nanoparticles loaded with inactivated PRRSV/challenged pigs (adjuvant-KNPs) and inactivated PRRSV/challenged pigs (inactivated PRRSV), were intranasally vaccinated with previously described vaccines at 0, 7 and 14 days post-vaccination (DPV). Serum and nasal swab samples were collected weekly and assayed by ELISA to detect the presence of IgG and IgA, respectively. Viral neutralizing titer (VNT) in sera, IFN-γ-producing cells and IL-10 secretion in stimulated peripheral blood mononuclear cells (PBMCs) were also measured. The pigs were intranasally challenged with PRRSV-2 at 28 DPV and necropsied at 35 DPV, and then macro- and microscopic lung lesions were evaluated. The results demonstrated that following vaccination, adjuvant-KNP-vaccinated pigs had significantly higher levels of IFN-γ-producing cells, VNT and IgG in sera, and IgA in nasal swab samples and significantly lower IL-10 levels than the other vaccinated groups. Following challenge, the adjuvant-KNP-vaccinated pigs had significantly lower PRRSV RNA and macro- and microscopic lung lesions than the other vaccinated groups. In conclusion, the results of the study demonstrated that adjuvant-KNPs are effective in eliciting immune responses against PRRSV and protecting against PRRSV infections over KNPs and inactivated PRRSV and can be used as an adjuvant for intranasal PRRSV vaccines.

The phylodynamics of emerging porcine deltacoronavirus in Southeast Asia.

Porcine deltacoronavirus (PDCoV), a recently emerging pathogen, causes diarrhoea in pigs. A previous phylogenetic analysis based on spike genes suggested that PDCoV was divided into three different groups, including China, the United States, and Southeast Asia (SEA). SEA PDCoV, however, is genetically separated from China and the United States but shares a common ancestor. Its origin and evolution have yet been identified. Herein, phylodynamic analyses based on the full-length genome were performed to investigate the origin and evolution of SEA PDCoV. In the study, 18 full-length genome sequences of SEA PDCoV identified in 2013-2016 together with PDCoV from other regions were used in analyses. The results demonstrated that PDCoV was classified into two genogroups including G1 and G2. G1 is further evolved into G1a (China) and G1b (US). G2 (SEA) group is further evolved into three clades, including SEA-1 (Thailand), SEA-2 (Vietnam) and SEA-2r (Vietnam recombinant) clades. The time to the most recent common ancestor (MRCA) of global PDCoV was estimated to be approximately 1989-1990 and possibly have been circulated in SEA more than a decade. SEA PDCoV is genetically diverse compared to China and U.S. PDCoV. The substitution rate of SEA PDCoV was lower than those of China and the United States, but the recombination rate of SEA was higher. Recombination analyses revealed four potential recombinant events in SEA PDCoV, suggesting that they were derived from the same ancestor of China PDCoV. The SEA-2r subgroup was potentially recombinant between SEA-2 and U.S. strains. In conclusion, the major mechanisms driving the complex evolution and genetic diversity of SEA PDCoV were multiple introductions of exotic PDCoV strains followed by recombination.

Safety of PRRSV-2 MLV vaccines administrated via the intramuscular or intradermal route and evaluation of PRRSV transmission upon needle-free and needle delivery.

Two distinct experiments (Exp) were conducted to evaluate the shedding and efficacy of 2 modified live porcine reproductive and respiratory syndrome virus (PRRSV) type 2 vaccines (MLV) when administered intramuscularly (IM) or intradermally (ID) (Exp A), and the potential of PRRSV transmission using a needle-free device (Exp B). One-hundred fifty-four, 3-week-old castrated-male, pigs were procured from a PRRSV-free herd. In Exp A, 112 pigs were randomly allocated into 4 groups of 21 pigs including IM/Ingelvac MLV (G1), IM/Prime Pac (G2), ID/Prime Pac (G3), and non-vaccination (G4). Twenty-eight remaining pigs were served as non-vaccination, age-matched sentinel pigs. G1 was IM vaccinated once with Ingelvac PRRS MLV (Ing) (Boehringer Ingelheim, Germany). G2 and G3 were IM and ID vaccinated once with a different MLV, Prime Pac PRRS (PP) (MSD Animal Health, The Netherlands), respectively. Following vaccination, an antibody response, IFN-γ-SC, and IL-10 secretion in supernatants of stimulated PBMC were monitored. Sera, tonsils, nasal swabs, bronchoalveolar lavage, urines, and feces were collected from 3 vaccinated pigs each week to 42 days post-vaccination (DPV) and assayed for the presence of PRRSV using virus isolation and qPCR. Age-matched sentinel pigs were used to evaluate the transmission of vaccine viruses and were introduced into vaccinated groups from 0 to 42 DPV. Seroconversion was monitored. In Exp B, 42 pigs were randomly allocated into 5 groups of 3 pigs each including IM/High (T1), ID/High (T2), IM/Low (T3), ID/Low (T4), and NoChal. Twenty-seven remaining pigs were left as non-challenge, age-matched sentinel pigs. The T1 and T2, and T3 and T4 groups were intranasally challenged at approximately 26 days of age with HP-PRRSV-2 at high (106) and low (103 TCID50/ml) doses, respectively. At 7 days post-challenge, at the time of the highest viremia levels of HP-PRRSV-2, T1 and T2, and T3 and T4 groups were IM and ID injected with Diluvac Forte using needles and a need-less device (IDAL 3G, MSD Animal Health, The Netherlands), respectively. Same needles or needle-less devices were used to inject the same volume of Diluvac Forte into sentinel pigs. Seroconversion of sentinels was evaluated. The results demonstrated that PP vaccinated groups (G2 and G3), regardless of the route of vaccination, had ELISA response significantly lower than G1 at 7 and 14 DPV. PP-vaccinated groups (G2 and G3) had significantly higher IFN-γ-SC and lower IL-10 secretion compared to the Ing-vaccinated group (G1). The two different MLV when administered intramuscularly demonstrated the difference in virus distribution and shedding patterns. PP-vaccinated pigs had significantly shortened viremia than the Ing-vaccinated pigs. However, ID-vaccinated pigs had lower virus distribution in organs and body fluids without virus shedding to sentinel pigs. In Exp B, regardless of the challenge dose, sentinel pigs intradermally injected with the same needle-less device used to inject challenged pigs displayed no seroconversion. In contrast, sentinel pigs intramuscularly injected with the same needle used to inject challenged pigs displayed seroconversion. The results demonstrated the transmission of PRRSV by using a needle, but not by using a needle-less device. In conclusion, our results demonstrated that ID vaccination might represent an alternative to improve vaccine efficacy and safety, and may be able to reduce the shedding of vaccine viruses and reduce the iatrogenic transfer of pathogens between animals with shared needles.

Development and validation of indirect ELISA for antibody detection against different protein antigens of porcine epidemic diarrhea virus in the colostrum and milk of sows.

The objectives of this study are to develop and optimize indirect ELISA based on three coating antigens of porcine epidemic diarrhea virus (PEDV), recombinant spike (S12), nucleocapsid (N), and whole viral (WV) proteins, for the detection of IgG and IgA antibodies in colostrum and milk and to evaluate the diagnostic sensitivity (DSe) and diagnostic specificity (DSp) of the assay as a diagnostic method. Colostrum (n = 347) and milk (n = 272) samples from sows were employed in this assay. Indirect ELISA based on three coating antigens was assessed by receiver operating characteristic (ROC) curve analysis with a virus neutralization (VN) test as a reference method, and the cutoff value for calculating DSe and DSp was determined. S12-ELISA showed higher DSe and DSp of IgG and IgA detection compared to N- and WV-ELISA in both colostrum and milk samples. Moreover, S12-ELISA showed perfect agreement and a high correlation with the VN test, which was better than the N- and WV-ELISA for both IgG and IgA detection in colostrum and milk. In contrast, N-ELISA showed lower DSe and DSp compared to S12- and WV-ELISA, along with a correlation with VN and substantial agreement with the VN test. Nevertheless, our developed ELISAs have accuracy for repeatability in both inter- and intra-assay variation. Overall, this research demonstrates that S12-ELISA is more suitable than WV- and N-ELISA to detect IgG and IgA antibodies against PEDV from both colostrum and milk samples.

Coinfection of porcine deltacoronavirus and porcine epidemic diarrhea virus increases disease severity, cell trophism and earlier upregulation of IFN-α and IL12.

Porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) cause an enteric disease characterized by diarrhea clinically indistinguishable. Both viruses are simultaneously detected in clinical cases, but a study involving the co-infection has not been reported. The study was therefore conducted to investigate the disease severity following a co-infection with PEDV and PDCoV. In the study, 4-day-old pigs were orally inoculated with PEDV and PDCoV, either alone or in combination. Following challenge, fecal score was monitored on a daily basis. Fecal swabs were collected and assayed for the presence of viruses. Three pigs per group were necropsied at 3 and 5 days post inoculation (dpi). Microscopic lesions and villous height to crypt depth (VH:CD) ratio, together with the presence of PEDV and PDCoV antigens, were evaluated in small intestinal tissues. Expressions of interferon alpha (IFN-α) and interleukin 12 (IL12) were investigated in small intestinal mucosa. The findings indicated that coinoculation increased the disease severity, demonstrated by significantly prolonged fecal score and virus shedding and decreasing VH:CD ratio in the jejunum compared with pigs inoculated with either PEDV or PDCoV alone. Notably, in single-inoculated groups, PEDV and PDCoV antigens were detected only in villous enterocytes wile in the coinoculated group, PDCoV antigen was detected in both villous enterocytes and crypts. IFN-α and IL12 were significantly up-regulated in coinoculated groups in comparison with single-inoculated groups. In conclusion, co-infection with PEDV and PDCoV exacerbate clinical signs and have a synergetic on the regulatory effect inflammatory cytokines compared to a single infection with either virus.

Development of novel cationic microemulsion as parenteral adjuvant for influenza vaccine.

Squalene-based oil-in-water (O/W) emulsions have been used as effective and safe adjuvants in approved influenza vaccines. However, there are concerns regarding the safety and side effects of increasing risk of narcolepsy. In present study, novel O/W microemulsions (MEs) containing wheat germ oil, D-alpha tocopheryl polyethylene glycol 1000 succinate (TPGS) and Cremophor EL (CreEL) or Solutol HS15 were formulated with/without a cationic surfactant, cetyltrimethylammonium bromide (CTAB) and then sterilized by autoclaving. Their physical properties and biological efficacies were evaluated. The results demonstrated that autoclaving reduced the droplet size to ∼20 nm with narrow size distributions resulting in monodisperse systems with good stability up to 3 years. Hemolytic activity, viscosity, pH, and osmolality were appropriate for parenteral use. Bovine serum albumin (BSA), a model antigen, after mixing with MEs retained the protein integrity, assessed by SDS-PAGE and CD spectroscopy. Greater percentages of 28SC cell viability were observed from CreEL-based MEs. Uptake of FITC-BSA-MEs increased with the increasing concentration of CTAB confirmed by CLSM images. Furthermore, cationic CreEL-based MEs could induce Th1 cytokine synthesis with an increase in TNF-α and IL-12 levels and a decrease in IL-10 level. In vivo immunization study in mice of adjuvants admixed with influenza virus solution revealed that nonionic and selected cationic CreEL-MEs enhanced immune responses as measured by influenza-specific serum antibody titers and hemagglutination inhibition titers. Particularly, cationic CreEL-based ME showed better humoral and cellular immunity with higher IgG2a titer than nonionic CreEL-based ME and antigen alone. No differences in immune responses were observed between mice immunized with selected cationic CreEL-based ME and marketed adjuvant. In addition, the selected ME induced antigen-sparing while retained immune stimulating effects compared to antigen alone. No inflammatory change in muscle fiber structure was observed. Accordingly, the developed cationic CreEL-based ME had potential as novel adjuvant for parenteral influenza vaccine.

Immune response and protective efficacy of intramuscular and intradermal vaccination with porcine reproductive and respiratory syndrome virus 1 (PRRSV-1) modified live vaccine against highly pathogenic PRRSV-2 (HP-PRRSV-2) challenge, either alone or in combination with of PRRSV-1.

The study was conducted to evaluate the immune response of pigs vaccinated intramuscularly (IM) or intradermally (ID) with porcine reproductive and respiratory syndrome virus 1 (PRRSV-1) modified live vaccine (MLV). The protective efficacy was evaluated upon challenge with highly pathogenic (HP)-PRRSV-2, either alone or in combination with PRRSV-1. Forty-two, castrated male, PRRSV-free pigs were randomly allocated into 7 groups of 6 pig each. IM/HPPRRSV2, IM/CoChallenge, ID/HPPRRSV2 and ID/CoChallenge groups were vaccinated IM or ID with PRRSV-1 MLV (UNISTRAIN® PRRS, Laboratorios Hipra S.A., Amer, Spain) in accordance to the manufacturer's directions. NV/HPPRRSV2 and NoVac/CoChallenge groups were nonvaccinated/challenged controls. NoVac/NoChallenge group was left as the control. Antibody response, IFN-γ-secreting cells (IFN-γ-SC) and IL-10 production were evaluated following vaccination. At 35 days post vaccination (DPV), all challenged groups were intranasally inoculated with HP-PRRSV-2, either alone or in combination with PRRSV-1. PRRSV viremia and lung lesion scores were evaluated following challenge. The results demonstrated that ID vaccinated pigs had significantly lower IL-10 levels and higher IFN-γ-SC than that of IM vaccinated pigs. Following challenge with HP-PRRSV-2 either alone or with PRRSV-1, PRRSV viremia and lung lesions, both macroscopically and microscopically, were significantly reduced in vaccinated pigs than that of nonvaccinated pigs, regardless to the route of vaccine administration. ID vaccinated pigs had significantly lower levels of PRRSV viremia and lung lesion scores than that of IM vaccinated pigs. The results of the study suggested that the administration of PRRSV-1 MLV, either IM or ID, provided partial protection against HP-PRRSV-2, either alone or when cochallenged with PRRSV-1, as demonstrated by the reduction in lung lesions and viremia. The ID route might represent an alternative to improve vaccine efficacy, as it resulted in lower IL-10 levels and higher IFN-γ-SC levels.

Cationic Polylactic Acid-Based Nanoparticles Improve BSA-FITC Transport Across M Cells and Engulfment by Porcine Alveolar Macrophages.

This work described the development of a cationic polylactic acid (PLA)-based nanoparticles (NPs) as an antigen delivery system using dimethyldioctadecylammonium bromide (DDA) to facilitate the engulfment of BSA-FITC by porcine alveolar macrophages (3D4/2 cells) and heat-labile enterotoxin subunit B (LTB) to enhance the transport of BSA-FITC across M cells. The experimental design methodology was employed to study the influence of PLA, polyvinyl alcohol (PVA), DDA, and LTB on the physical properties of the PLA-based NPs. The size of selected cationic PLA NPs comprising 5% PLA, 5% PVA, and 0.6% DDA with or without LTB absorption was range from 367 to 390 nm with a polydispersity index of 0.26, a zeta potential of + 26.00 to + 30.55 mV, and entrapment efficiency of 41.43%. Electron micrographs revealed NPs with spherical shape. The release kinetic of BSA from the NPs followed the Korsmeyer-Peppas kinetics. The cationic PLA NPs with LTB surface absorption showed 3-fold increase in BSA-FITC transported across M cells compared with the NPs without LTB absorption. The uptake studies demonstrated 2-fold increase in BSA-FITC intensity in 3D4/2 cells with cationic NPs as compared with anionic NPs. Overall, the results suggested that LTB decreased the retention time of BSA-FITC loaded in the cationic PLA NPs within the M cells, thus promoting the transport of BSA-FITC across the M cells, and cationic NPs composed of DDA help facilitate the uptake of BSA-FITC in the 3D4/2 cells. Further studies in pigs with respiratory antigens will provide information on the efficacy of cationic PLA NPs as a nasal antigen carrier system.

Cell-mediated immune response and protective efficacy of porcine reproductive and respiratory syndrome virus modified-live vaccines against co-challenge with PRRSV-1 and PRRSV-2.

Cell-mediated immunity (CMI), IL-10, and the protective efficacy of modified-live porcine reproductive and respiratory syndrome virus (PRRSV) vaccines (MLV) against co-challenge with PRRSV-1 and PRRSV-2 (HP-PRRSV) were investigated. Seventy, PRRSV-free, 3-week old, pigs were allocated into 7 groups. Six groups were intramuscularly vaccinated with MLV, including Porcilis (PRRSV-1 MLV, MSD Animal Health, The Netherlands), Amervac (PRRSV-1 MLV, Laboratorios Hipra, Spain), Fostera (PRRSV-2 MLV, Zoetis, USA), Ingelvac PRRS MLV and Ingelvac PRRS ATP (PRRSV-2, Boehringer Ingelheim, USA), and Prime Pac PRRS (PRRSV-2 MLV, MSD Animal Health, The Netherlands). Unvaccinated pigs were left as control. Lymphocyte proliferative response, IL-10 and IFN-γ production were determined. At 35 days post-vaccination (DPV), all pigs were inoculated intranasally with 2 ml of each PRRSV-1 (105.4 TCID50/ml) and PRRSV-2 (105.2 TCID50/ml, HP-PRRSV). Following challenge, sera were quantitatively assayed for PRRSV RNA. Pigs were necropsied at 7 days post-challenge. Viremia, macro- and microscopic lung lesion together with PRRSV antigen presence were evaluated in lung tissues. The results demonstrated that, regardless of vaccine genotype, CMI induced by all MLVs was relatively slow. Increased production of IL-10 in all vaccinated groups was observed at 7 and 14 DPV. Pigs in Amervac, Ingelvac MLV and Ingelvac ATP groups had significantly higher levels of IL-10 compared to Porcilis, Fostera and Prime Pac groups at 7 and 14 DPV. Following challenge, regardless to vaccine genotype, vaccinated pigs had significantly lower lung lesion scores and PRRSV antigens than those in the control group. Both PRRSV-1 and PRRSV-2 RNA were significantly reduced. Prime Pac pigs had lowest PRRSV-1 and PRRSV-2 RNA in serum, and micro- and macroscopic lung lesion scores (p < 0.05) compared to other vaccinated groups. In conclusion, PRRSV MLVs, regardless of vaccine genotype, can reduce viremia and lung lesions following co-challenge with PRRSV-1 and PRRSV-2 (HP-PRRSV). The main difference between PRRSV MLV is the production of IL-10 following vaccination.

Retrospective study, full-length genome characterization and evaluation of viral infectivity and pathogenicity of chimeric porcine deltacoronavirus detected in Vietnam.

Increased evidence of porcine deltacoronavirus (PDCoV) causing diarrhoea in pigs has been reported in several countries worldwide. The virus has currently evolved into three separated groups including US, China and Southeast Asia (SEA) groups. In Vietnam, PDCoV was first reported in 2015. Based on phylogenetic analyses of spike, membrane and nucleocapsid genes, it is suggested that Vietnam PDCoV is chimeric virus. In the present study, we retrospectively investigated the presence of PDCoV in Vietnam and the full-length genomes of six PDCoV isolates identified in 2014-2016 were further characterized. The results demonstrated that Vietnam PDCoV was first detected as early as 2014. All six Vietnam PDCoV are in the SEA group and further divided into two separated subgroups including SEA-1 and SEA-2. Vietnam PDCoV in SEA-2 was closely related to Thai and Lao PDCoV. Recombination analysis demonstrated that three isolates in SEA-1 were a chimeric virus of which P12_14_VN_0814, the first Vietnam isolate, and US PDCoV isolates were major and minor parents, respectively. The recombination was further evaluated by phylogenetic construction based on 3 recombinant fragments. The first and third fragments, closely related to P12_14_VN_0814, were associated with ORF1a/1b and N genes, respectively. The second fragment, associated with S, E, and M genes, was closely related to US PDCoV isolates. High antigenic and hydrophobic variations were detected in S1 protein. Three-day-old pigs challenged with the chimeric virus displayed clinical diseases and villus atrophy. In conclusion, Vietnam PDCoV is genetically diverse influenced by an external introduction from neighbouring countries. The chimeric Vietnam PDCoV can induce a disease similar to Thai PDCoV.

Rapid Transient Production of a Monoclonal Antibody Neutralizing the Porcine Epidemic Diarrhea Virus (PEDV) in Nicotiana benthamiana and Lactuca sativa.

Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea, vomiting, dehydration, weight loss, and high mortality rate in neonatal piglets. Porcine epidemic diarrhea (PED) has been reported in Europe, America, and Asia including Thailand. The disease causes substantial losses to the swine industry in many countries. Presently, there is no effective PEDV vaccine available. In this study, we developed a plant-produced monoclonal antibody (mAb) 2C10 as a prophylactic candidate to prevent the PEDV infection. Recently, plant expression systems have gained interest as an alternative for the production of antibodies because of many advantages, such as low production cost, lack of human and animal pathogen, large scalability, etc. The 2C10 mAb was transiently expressed in Nicotiana benthamiana and lettuce using geminiviral vector. After purification by protein A affinity chromatography, the antibody was tested for the binding and neutralizing activity against PEDV. Our result showed that the plant produced 2C10 mAb can bind to the virus and also inhibit PEDV infection in vitro. These results show excellent potential for a plant-expressed 2C10 as a PEDV prophylaxis and a diagnostic for PEDV infection.

A new cell-to-cell interaction model for epithelial microfold cell formation and the enhancing effect of epidermal growth factor.

The formation of epithelial microfold (M) cells is mediated through cell-to-cell interactions between enterocytes and lymphocytes. Based on this concept, we developed a cell-to-cell model by encouraging interactions between enterocyte C2BBe1 and Raji B cells through a preincubation approach. Raji B cells and C2BBe1 cells were allowed to interact in detached condition for 2h at ratios of 1:1, 1:2 and 1:4 and then plated in culture plates. Monocultured C2BBe1 cells were used as the control. Flow cytometric analysis of the M cell-specific marker clusterin revealed that the optimum ratio of Raji B to C2BBe1 cells to obtain the maximum number of M cells was 1:1. Scanning electron micrographs exhibiting the lack of microvilli with complete tight junctions and Western blot analysis showing intense expression of clusterin confirmed the unique phenotypes of the formed M cells. Fluosphere® transport studies showed a 7-fold increase in the cell-to-cell model compared to the monoculture control. Importantly, we found that the induction of M cells could be enhanced by the effect of epithelial growth factor (EGF). C2BBe1 cells were pretreated with EGF at 10, 25 and 50ng/mL before co-culturing with Raji B cells. Flow cytometric analysis of clusterin revealed that EGF significantly increased the formation of M cells. From mechanistic studies, we found an increase in the number of M cells involved the induction of stemness by EGF indicated by a dramatic increase in ß-catenin, Nanog, and Oct-4, which in turn up-regulated the cell-to-cell interacting protein Integrin ß-1. Furthermore, we confirmed the transport functions of the conventional, cell-to-cell, and cell-to-cell with EGF models using a Fluosphere® transport assay. Overall, we demonstrated an effective novel protocol for the formation of M cells as well as the effect of EGF on enhancing cell-to-cell interaction, which may benefit transport studies in M cells and promote better understanding of the biology of M cells.

Immune response of gilts to single and double infection with porcine epidemic diarrhea virus.

Immune response of gilts following single and double infection with porcine epidemic diarrhea virus (PEDV) at gilt acclimatization and prepartum were investigated. One hundred PEDV-naïve gilts were divided into two groups: negative (Neg) and feedback (FB) groups. Antibody responses in serum, colostrum, and milk samples were measured by IgG/IgA ELISA and virus neutralization assay (VN). Fecal shedding was investigated using RT-PCR. In summary, a single infection at gilt acclimatization resulted in slightly increased serum antibody titers as determined by VN assay and IgG ELISA, but not by IgA ELISA. Viral RNA was detected in fecal samples up to 6 days post-exposure. A double infection at prepartum resulted in significantly increased IgA and VN titers in milk samples compared to the single-infection group. No fecal shedding was detected following the double infection.

Retrospective investigation and evolutionary analysis of a novel porcine deltacoronavirus strain detected in Thailand from 2008 to 2015.

Porcine deltacoronavirus (PDCoV) in Thailand was first detected in 2015. We performed a retrospective investigation of the presence of PDCoV in intestinal samples collected from piglets with diarrhea in Thailand from 2008 to 2015 using RT-PCR. PDCoV was found to be present as early as February 2013. Phylogenetic analysis demonstrated that all PDCoV variants from Thailand differ from those from other countries and belong to a novel group of PDCoV that is separate from the US and Chinese PDCoV variants. Evolutionary analysis suggested that the Thai PDCoV isolates probably diverged from a different ancestor from that of the Chinese and US PDCoV isolates and that this separation occurred after 1994.

Evolutionary and epidemiological analyses based on spike genes of porcine epidemic diarrhea virus circulating in Thailand in 2008-2015.

Porcine epidemic diarrhea (PED) has been endemic causing sporadic outbreaks in Thailand since 2007. In 2014-2015, several herds had experienced severe PED outbreaks and the reason of the re-current outbreaks was unknown. Whether or not the introduction of exotic strains or continual evolution of existing PEDV, genetic analyses would provide a more understanding in its evolutionary pattern. In the study, 117 complete spike gene sequences of Thai PED virus (PEDV) collected from 2008 to 2015 were clustered along with 95 references of PEDV spike sequences, and analyzed with the US sequences dataset (n=99). The phylogenetic analysis demonstrated that Thai PEDV spike sequences were genetically diverse and had been influenced by multiple introduction of exotic strains. Although Thai PEDV have been evolved into 6 subgroups (TH1-6), Subgroup TH1 strains with the unique 9 nucleotides (CAA GGG AAT) insertion between 688th-689th position of spike (changing amino acid from N to TREY) insertion has become the dominant subgroup since 2014. Thai PEDV spike gene have higher evolutionary rate compare to that of the US sequences. One contributing factor would be the intra-recombination between subgroups. Thailand endemic strain should be assigned into new subclade of G2 (Thai pandemic variant).