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.

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.

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.

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.

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.

The genetic diversity and complete genome analysis of two novel porcine deltacoronavirus isolates in Thailand in 2015.

Porcine deltacoronavirus (PDCoV) was identified in intestinal samples collected from piglets with diarrhea in Thailand in 2015. Two Thai PDCoV isolates, P23_15_TT_1115 and P24_15_NT1_1215, were isolated and identified. The full-length genome sequences of the P23_15_TT_1115 and P24_15_NT1_1215 isolates were 25,404 and 25,407 nucleotides in length, respectively, which were relatively shorter than that of US and China PDCoV. The phylogenetic analysis based on the full-length genome demonstrated that Thai PDCoV isolates form a new cluster separated from US and China PDCoV but relatively were more closely related to China PDCoV than US isolates. The genetic analyses demonstrated that Thai PDCoVs have 97.0-97.8 and 92.2-94.0% similarities with China PDCoV at nucleotide and amino acid levels, respectively, but share 97.1-97.3 and 92.5-93.0 similarity with US PDCoV at the nucleotide and amino acid levels, respectively. Thai PDCoV possesses two discontinuous deletions of five amino acids in ORF1a/b region. One additional deletion of one amino acid was identified in P23_15_TT_1115. The variation analyses demonstrated that six regions (nt 1317-1436, 2997-3096, 19,737-19,836, 20,277-20,376, 21,177-21,276, and 22,371-22,416) in ORF1a/b and spike genes exhibit high sequence variation between Thai and other PDCoV. The analyses of amino acid changes suggested that they could potentially be from different lineages.

Humoral immune responses and viral shedding following vaccination with modified live porcine reproductive and respiratory syndrome virus vaccines.

The antibody response and pattern of shedding of vaccine virus following vaccination with modified live genotype I or II porcine reproductive and respiratory syndrome virus (PRRSV) vaccines (MLVs) were investigated. Ninety PRRSV-free pigs were divided randomly seven, groups including the NEG, EU1, EU2, US1, US2, US3 and US4 groups. The NEG group was unvaccinated. The EU1, EU2, US1, US2, US3 and US4 groups were vaccinated with the following MLVs: AMERVAC® PRRS, Porcillis® PRRS, Fostera™ PRRS, Ingelvac® PRRS MLV, Ingelvac® PRRS ATP, and PrimePac™ PRRS+ , respectively. Sera were quantitatively assayed for viral RNA using qPCR. Antibody responses were measured using Idexx ELISA and serum neutralization (SN). Shedding of vaccine virus was investigated using sentinel pigs and by detection of viral RNA in tonsil scrapings. Antibody responses were detected by ELISA at 7-14 days post-vaccination (DPV) and persisted at high titers until 84 DPV in all MLV groups. The SN titers were delayed and isolate-specific. SN titers were higher for the homologous virus than for heterologous viruses. Age-matched sentinel pigs introduced into the EU2, US2 and US3 groups at 60 DPV seroconverted. In contrast, sentinel pigs introduced at 84 DPV remained negative in all of the MLV groups. Vaccine viral RNA was detected in tonsil scrapings from the EU2, US2 and US3 groups at 84-90 DPV. No viral RNA was detected beyond 70 DPV in the EU1, US1 and US4 groups. In conclusion, all MLV genotypes induced rapid antibody responses, which were measured using ELISA. The development of SN antibodies was delayed and isolate-specific. However, the shedding pattern was variable and depended on the by virus isolate used to manufacture the vaccine.

The first detection and full-length genome sequence of porcine deltacoronavirus isolated in Lao PDR.

Porcine deltacoronavirus (PDCoV) has been reported in many countries, including Hong Kong, the United States, South Korea, China and Thailand. In January 2016, clinical diarrhea similar to that of porcine epidemic diarrhea virus (PEDV) with a lower mortality rate was reported on a swine farm in Lao PDR. Intestine samples were collected from 3-day-old pigs with clinical diarrhea and assayed for the presence of swine enteric coronaviruses. The PCR results were positive for PDCoV but negative for PEDV and TGEV. A phylogenetic tree demonstrated that PDCoV from Lao PDR was grouped separately from PDCoV isolates from China and the USA, but was more closely related to the Chinese isolates than to the US isolates. The full-length genome sequence of the novel PDCoV isolate P1_16_BTL_0116 was determined.

Complete Genome Sequence of Porcine Deltacoronavirus Isolated in Thailand in 2015.

In Thailand, porcine deltacoronavirus (PDCoV) was first identified in November 2015. The virus was isolated from piglets experiencing diarrhea outbreak. Herein, the full-length genome sequence of the Thai PDCoV isolate P23_15_TT_1115 is reported. The results provide a clearer understanding of the molecular characteristics of PDCoV in Thailand.