Immune response enhancement by dietary supplementation with Caesalpinia sappan extract in weaned pigs challenged with porcine reproductive and respiratory syndrome virus.

BACKGROUND: At present, porcine reproductive and respiratory syndrome (PRRS) caused by the PRRS virus (PRRSV) is one of the most severe epidemics impacting pig farming globally. Despite the fact that a number of studies have been conducted on potential solutions to this problem, none have proven effective. The focus of problem solving is the use of natural ingredients such as plant extracts. Popular throughout Asia, Caesalpinia sappan (CS) is a therapeutic plant that inhibits PRRSV in vitro. Therefore, this study was performed to determine the efficacy of CS extract dietary supplementation on the productive performance, antibody levels, immunological indicators, and lung pathology of PRRSV-challenged weaned pigs. A total of 32 weaned piglets (28 days old) were randomized into 4 groups and kept separately for 14 days. The treatments were organized in a 2 × 2 factorial design involving two factors: PRRSV challenge and supplementation with 1 mg/kg CS extract. The pigs in the PRRSV-challenged groups were intranasally inoculated with 2 mL of PRRSV (VR2332) containing 104 TCID50/mL, while those in the groups not challenged with PRRSV were inoculated with 2 mL of normal saline. RESULTS: In the PRRSV-challenged group (CS + PRRSV), supplementation with CS extract led to an increase in white blood cells (WBCs) on Day 7 post infection (p < 0.05) and particularly in lymphocytes on Days 7 and 14. The antibody titer was significantly greater in the CS + PRRSV group than in the PRRSV-challenged group not administered CS (PRRSV group) on Day 14 postinfection (S/P = 1.19 vs. 0.78). In addition, CS extract administration decreased the prevalence of pulmonary lesions, which were more prevalent in the PRRSV-challenged pigs that did not receive the CS extract. CONCLUSION: The findings of this study suggest that supplementation with CS extract is beneficial for increasing WBC counts, especially lymphocytes, increasing the levels of antibodies and reducing the prevalence of lung lesions in PRRSV-infected pigs.

Designing Selenium Nanoadjuvant as Universal Agent for Live-Killed Virus-Based Vaccine.

Inactivated virus vaccines with whole antigen spectra and good safety are the commonly used modality for preventing infections. However, the poor immunogenicity greatly limits its clinical applications. Herein, by taking advantages of the crucial roles of Se in the functions of immune cells and its biomineralization property, it successfully in-situ synthesized Se nanoadjuvant on inactivated viruses such as porcine epidemic diarrhea virus (PEDV), pseudorabies virus (PRV), and porcine reproductive and respiratory syndrome virus (PRRSV) in a facile method, which is universal to construct other inactivated virus vaccines. The nanovaccine can highly effectively enhance the uptake of PEDV/PRV/PRRSV into dendritic cells (DCs) and activate DCs via triggering TLR4 signaling pathways and regulating selenoproteins expressions. Furthermore, it exhibited better activities in triggering macrophages and natural killer cells-mediated innate immunity and T cells-mediated cellular immunity compared to PEDV and the commercial inactivated PEDV vaccine on both mice and swine models. This study provides a universal Se nanoadjuvant for developing inactivated viruses-based nanovaccines for preventing virus infections.

Glycyrrhiza polysaccharides inhibits PRRSV replication.

Glycyrrhiza polysaccharide (GCP) is a natural plant active polysaccharide extracted from traditional Chinese medicine licorice. In this research, we studied the antiviral activity of glycyrrhiza polysaccharide against porcine reproductive and respiratory syndrome virus (PRRSV), a virus of the Arteriviridae family, with a high rate of variation and has caused huge economic losses to the pig industry in various countries since its discovery. Our results show that GCP can inhibit PRRSV replication in a dose-dependent manner. Furthermore, GCP could inhibit the mRNA expression of receptor genes CD163 and NF-κB p65 and promote the mRNA expression of the SLA-7 gene. Because of these results, GCP can be used as a candidate drug to prevent and treat PRRS.

Highly pathogenic porcine reproductive and respiratory syndrome virus-induced inflammatory response in porcine pulmonary microvascular endothelial cells and effects of herbal ingredients on main inflammatory molecules.

The role of microvascular endothelial cells (MVECs) in viral infection has received increasing attention. Our previous study demonstrated the susceptibility of porcine pulmonary MVECs to highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV), while their responses to the viral infection remain unclear. This study aimed to understand effects of the HP-PRRSV infection on functions of porcine pulmonary MVECs and the intervention effects of Chinese herbal ingredients on them. Highly purified porcine pulmonary MVECs were separated using CD31-immunomagnetic beads and infected with HP-PRRSV JXA1 and HN strain. The virus particles in cells and the ultrastructural pathological changes of cells were revealed by transmission electron microscopy. High-throughput transcriptome sequencing indicated that 104 and 228 genes were differentially expressed at 36 h post-infection, respectively, including many inflammatory molecules such as interleukins, chemokines, and adhesion molecules. The expression kinetics of HP-PRRSV-induced IL-1α, IL-6, IL-8, and VCAM-1 were characterized at the mRNA and protein levels. Luteolin significantly down-regulated HP-PRRSV-induced increase of the four molecules at both levels, and glycyrrhetinic acid and baicalin reduced that of IL-6 and VCAM-1. Our results suggest that porcine pulmonary MVECs play important roles in the inflammatory lung injury caused by HP-PRRSV infection and that herbal ingredients have potential regulatory effects on the HP-PRRSV-induced dysfunction of MVECs.

IFIT3 and IFIT5 Play Potential Roles in Innate Immune Response of Porcine Pulmonary Microvascular Endothelial Cells to Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus.

Our previous study has demonstrated that porcine pulmonary microvascular endothelial cells (MVECs) are susceptible to highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). The innate immune response of MVECs infected with HP-PRRSV would play important roles in controlling virus proliferation, resisting cellular injury, and preventing the virus from spreading to other tissues and organs. Type I interferon is one of the most effective antiviral cytokines in the innate immune response, and interferon-induced proteins with tetratricopeptide repeats (IFITs) are members of interferon-stimulated genes induced by viruses and other pathogens, which are crucial in inhibiting virus proliferation and regulating the innate immune response. However, their effects on HP-PRRSV-induced innate immunity in porcine pulmonary MVECs remain unclear. Here, the roles of IFITs in porcine pulmonary MVECs infected with the HP-PRRSV HN strain were investigated, and the effects of astragalus polysaccharides (APS), a widely used traditional Chinese herbal ingredient with the immunopotentiating effect, on them were studied. The results showed that more autophagosomes were observed in HP-PRRSV-infected MVECs, and the expression of IFN-α, IFIT3, and IFIT5 decreased or increased at different time points after infection. When silencing the genes of IFIT3 or IFIT5, the HP-PRRSV replication in MVECs was significantly increased. The expression of IFIT3 and IFIT5 could be upregulated by APS, whose inhibitory effects on the HP-PRRSV replication significantly declined when the genes of IFIT3 or IFIT5 were silenced. The results suggest that IFIT3 and IFIT5 play an important role in inhibiting the HP-PRRSV replication in porcine pulmonary MVECs, and APS suppress the multiplication of HP-PRRSV by upregulating their expression.

Oral Supplementation of Houttuynia cordata Extract Reduces Viremia in PRRSV-1 Modified-Live Virus-Vaccinated Pigs in Response to the HP-PRRSV-2 Challenge.

This study evaluated the in vitro antiviral activities and the ex vivo immunomodulatory effects of Houttuynia cordata Thunb. (HC) ethanolic extracts in response to porcine reproductive and respiratory syndrome virus (PRRSV). In addition, this study evaluated the in vivo effects of oral supplementation of HC extract on immune responses to and cross-protective efficacy of PRRSV-1 modified-live virus (MLV) vaccine against the highly pathogenic (HP)-PRRSV-2 challenge. In vitro experiments demonstrated that HC extracted in either 50%, 70%, or 95% ethanol (referred to as HC50, HC70, and HC95, respectively) significantly interfered with PRRSV replication in MARC-145 cells. Ex vivo experiments revealed that all HC extracts significantly enhanced mRNA expressions of type I interferon-regulated genes, type I and II interferon (IFN), and pro- and anti-inflammatory cytokines in HP-PRRSV-2-inoculated monocyte-derived macrophages. An in vivo experiment included four groups of six pigs (4 weeks old; n = 24). Group 1 and group 2 were vaccinated with the PRRSV-1 MLV vaccine at 0 dpv (day post vaccination). Group 2 also received oral administration of HC50 extract at 0-49 dpv. Group 3 received the PRRSV-1 MLV vaccine solvent at 0 dpv, while group 4 served as strict control. Groups 1-3 were challenged intranasally with HP-PRRSV-2 at 28 dpv and immune-related and clinical parameters were monitored weekly until 49 dpv. Compared to group 1, group 2 demonstrated significantly increased IFN regulatory factor 3 mRNA expression of PRRSV-recalled peripheral blood mononuclear cells, and significantly reduced HP-PRRSV-2 viremia. No difference in PRRSV-specific antibody responses, rectal temperature, clinical scores, and average daily weight gain was detected. Our study reports the immunomodulatory and anti-PRRSV potentials of HC extract in PRRSV-1 MLV-vaccinated/HP-PRRSV-2 challenged pigs.

Chitosan-Coated Selenium Nanoparticles Attenuate PRRSV Replication and ROS/JNK-Mediated Apoptosis in vitro.

Introduction: Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly prevalent and endemic swine pathogen that causes significant economic losses to the global swine industry. Selenium nanoparticles (SeNPs) have attracted increasing attention in the biomedical field, given their antiviral effects. This study aimed to investigate the inhibitory effect of chitosan-coated SeNPs (CS-SeNPs) on PRRSV replication. Methods: In this study, CS-SeNPs were synthesized by chemical reduction and characterized by assessing the morphology, size distribution, zeta potential, and element composition. Marc-145 cells were infected with r-PRRSV-EGFP (0.1 MOI) and inoculated with CS-SeNPs (10 µM). Subsequently, the concentrations of hydrogen peroxide (H2O2) and glutathione (GSH), and glutathione peroxidase (GSH-Px) activity were measured using specific commercial assay kits. ORF5 RNA expression, viral titer, and nucleocapsid (N) protein expression were assessed using qRT-PCR, TCID50, and Western blot. ROS generation, apoptosis rates, and JNK /caspase-3/PARP protein expression were evaluated using dihydroethidium staining, flow cytometry, and Western blot. Results: The results showed that CS-SeNPs treatment significantly suppressed oxidative stress induced by r-PRRSV-EGFP infection by increasing GSH-Px activity, promoting GSH production, and inhibiting H2O2 synthesis. CS-SeNPs treatment significantly inhibited ORF5 gene expression, viral titers, and N protein of r-PRRSV-EGFP at 24 and 48 hours post-infection (hpi) in Marc-145 cells. The increase in apoptosis rates induced by r-PRRSV-EGFP infection was significantly decreased by CS-SeNPs inoculation through inhibiting ROS generation, JNK phosphorylation levels, and cleavage of caspase-3 and PARP mainly at 48 hpi. Conclusion: These results demonstrated that CS-SeNPs suppress PRRSV-induced apoptosis in Marc-145 cells via the ROS/JNK signaling pathway, thereby inhibiting PRRSV replication, which suggested the potential antiviral activity of CS-SeNPs that deserves further investigation for clinical applications.

A novel strategy for optimal component formula of anti-PRRSV from natural compounds using tandem mass tag labeled proteomic analyses.

BACKGROUND: Porcine Reproductive and Respiratory Syndrome (PRRS) is one of the most important porcine viral diseases which have been threatening the pig industry in China. At present, most commercial vaccines fail to provide complete protection because of highly genetic diversity of PRRSV strains. This study aimed to optimize a component formula from traditional Chinese medicine(TCM)compounds with defined chemical characteristics and clear mechanism of action against PRRSV. METHODS: A total of 13 natural compounds were screened for the anti-PRRSV activity using porcine alveolar macrophages (PAMs). Three compounds with strong anti-PRRSV activity were selected to identify their potential protein targets by proteomic analysis. The optimal compound formula was determined by orthogonal design based on the results of proteomics. MTT assay was used to determine the maximum non-cytotoxic concentration (MNTC) of each compound using PAMs. QPCR and western blot were used to investigate the PRRSV N gene and protein expression, respectively. The Tandem Mass Tag (TMT) technique of relative quantitative proteomics was used to detect the differential protein expression of PAMs treated with PRRSV, matrine (MT), glycyrrhizic acid (GA) and tea saponin (TS), respectively. The three concentrations of these compounds with anti-PRRSV activity were used for orthogonal design. Four formulas with high safety were screened by MTT assay and their anti-PRRSV effects were evaluated. RESULTS: MT, GA and TS inhibited PRRSV replication in a dose-dependent manner. CCL8, IFIT3, IFIH1 and ISG15 were the top four proteins in expression level change in cells treated with MT, GA or TS. The relative expression of IFIT3, IFIH1, ISG15 and IFN-ß mRNAs were consistent with the results of proteomics. The component formula (0.4 mg/mL MT + 0.25 mg/mL GA + 1.95 µg/mL TS) showed synergistic anti-PRRSV effect. CONCLUSIONS: The component formula possessed anti-PRRSV activity in vitro, in which the optimal dosage on PAMs was 0.4 mg/mL MT + 0.25 mg/mL GA + 1.95 µg/mL TS. Compatibility of the formula was superposition of the same target with GA and TS, while different targets of MT. IFN-ß may be one of the targets of the component formula possessed anti-PRRSV activity.

Maternal immune activation and dietary soy isoflavone supplementation influence pig immune function but not muscle fiber formation.

The goals of this study were to determine the impact of maternal PRRSV infection on offspring muscle and immune development and the potential of dietary soy isoflavones to mitigate those effects. Thirteen first-parity gilts ("gilts") were randomly allotted into one of three treatments: not infected and fed a diet devoid of isoflavones (CON), infected with porcine reproductive and respiratory syndrome virus (PRRSV) and fed the control diet (POS) or that supplemented with 1,500 mg/kg soy-derived isoflavones (ISF). Gilts were inoculated with PRRSV intranasally on gestational day (GD) 70. After farrowing (GD 114 ± 2), 1-2 offspring ("pigs") closest to the average litter weight were selected either at birth (3 ± 2 d of age) or weaning (21 ± 2 d of age) to determine body, muscle, and organ weights as well as muscle cell number and size. Four weaned pigs of average body weight within each litter were selected for postnatal immune challenge. At PND 52, pigs were injected with 5 µg/kg BW lipopolysaccharide (LPS) intraperitoneally. Serum was collected at 0, 4, and 8 h following LPS administration to analyze tumor necrosis factor alpha (TNF-α). At PND 59, pigs were administered a novel vaccine to elicit an adaptive immune response. At PND 59, 66, and 73, peripheral blood mononuclear cells were isolated and T-cell populations determined by flow cytometry. Both POS and ISF pigs exhibited persistent PRRSV infections throughout the study (PND 1-73). At PND 3, whole body, muscle, and organ weights were not different (P > 0.22) between groups, with the exception of relative liver weight, which was increased (P < 0.05) in POS compared with CON pigs. At PND 21, ISF pigs had reduced (P ≤ 0.05) whole body and muscle weights, but greater (P < 0.05) kidney weight compared with CON, and greater (P < 0.05) relative liver weight compared with CON and POS. Muscle fiber number and size were not different (P > 0.39) between groups at birth or weaning. After LPS administration, TNF-α was greatest in ISF pigs (P < 0.05) at both 0 and 8 h post-challenge. At the peak time-point of 4 h post-challenge, ISF pigs had the greatest concentration of TNF-α and CON pigs had the lowest, with POS pigs being intermediate (P = 0.01). After vaccination, ISF offspring had shifts in T-cell populations indicating an impaired immune response. These data indicate that maternal PRRSV infection may impact offspring organ growth and immune function, particularly when the dam is supplemented with isoflavones.

Gestational health challenges may influence growth performance and immunity of offspring pigs during postnatal life. In particular, porcine reproductive and respiratory syndrome virus (PRRSV) is endemic in the U.S. herd, but its effects on surviving offspring pigs are largely unknown. Further, dietary supplementation with soy isoflavones lessened the severity of PRRSV infections in weaning and growing pigs. Therefore, the goals of this study were to determine the impact of maternal PRRSV infection on offspring muscle and immune development and the potential of isoflavones to mitigate those effects. Isoflavone supplementation reduced viral load in dams 21 d after infection, but did not alter clinical illness indicators. Pig mortality was increased by PRRSV infection in dams, and surviving pigs were infected with PRRSV throughout the study. Interestingly, muscle and organ weights were not different among treatments at birth, but infected litters were lighter at weaning, likely due to postnatal infection. Muscle fiber number and size did not differ between treatments. Pigs born to infected dams had slower responses during innate immune stimulation and then failed to mount a proper vaccine response during adaptive immune stimulation. Overall, maternal infection altered offspring immune responses but not muscle fiber development. Isoflavone supplementation did not mitigate these effects.

EGCG Restricts PRRSV Proliferation by Disturbing Lipid Metabolism.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection leads to late-term reproductive failure and respiratory illness that affect the global swine industry. Epigallocatechin gallate (EGCG) is a polyphenolic compound from green tea that exerts antiviral activity against diverse viruses. This study aimed to report an uncharacterized mechanism of how EGCG restricted PRRSV proliferation. EGCG showed no significant effects on cell viability, cell cycle progression, and apoptosis in porcine alveolar macrophages and MARC-145 cells. The treatment of cells with EGCG attenuated the replication of both highly pathogenic and less pathogenic PRRSV in vitro. The viral life cycle analysis demonstrated that EGCG affected PRRSV replication and assembly, but not viral attachment, entry, or release. Interestingly, EGCG treatment abrogated the increased lipid droplets formation and lipid content induced by PRRSV infection. We further demonstrated that EGCG blocked PRRSV-stimulated expression of the key enzymes in lipid synthesis. In addition, EGCG attenuated PRRSV-induced autophagy that is critical for PRRSV proliferation. The supplementation of oleic acid restored PRRSV replication and assembly under EGCG treatment. Together, our results support that EGCG inhibits PRRSV proliferation through disturbing lipid metabolism. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped single-positive-stranded RNA virus that causes acute respiratory distress in piglets and reproductive failure in sows, resulting in huge economic losses to the global swine industry. Several lines of evidence have suggested the crucial roles of lipids in PRRSV proliferation. Our previous report demonstrated that PRRSV activated lipophagy to facilitate viral replication through downregulating the expression of N-Myc downstream-regulated gene 1. The manipulation of lipid metabolism may be a new perspective to prevent PRRSV spread. In the present study, we reported that epigallocatechin-3-gallate (EGCG), the major component of green tea catechins, significantly attenuated PRRSV infection through inhibiting lipid synthesis and autophagy. Given that natural products derived from plants have helped in the prevention and treatment of various infectious diseases, EGCG has a great potential to serve as a safe and environmentally friendly natural compound to treat PRRSV infection.

Matrine exhibits antiviral activity in a PRRSV/PCV2 co-infected mouse model.

BACKGROUND: PRRSV and PCV2 co-infection is very common in swine industry which results in huge economic losses worldwide. Although vaccination is used to prevent viral diseases, immunosuppression induced by PRRSV and PCV2 leads to vaccine failure. PURPOSE: Our previous results have demonstrated that Matrine possess antiviral activities against PRRSV/PCV2 co-infection in vitro. This study aims to establish a PRRSV/PCV2 co-infected KM mouse model and evaluate the antiviral activities of Matrine against PRRSV/PCV2 co-infection. STUDY DESIGN: A total of 144 KM mice were randomly divided into six groups with 24 mice in each group, named as: normal control, PRRSV/PCV2 co-infected group (PRRSV/PCV2 group), Ribavirin treatment positive control (Ribavirin control) and Matrine treatment groups (Matrine 40 mg/kg, Matrine 20 mg/kg and Matrine 10 mg/kg). METHODS: Except normal control group, all mice in other five groups were inoculated with PRRSV, followed by PCV2 at 2 h later. At 7 days post-infection (dpi), mice in the treatment groups were intraperitoneally administered with various doses of Matrine and Ribavirin, twice a day for 5 consecutive days. RESULTS: PRRSV N and PCV2 CAP genes were detected by PCR in multiple tissues including heart, liver, spleen, lungs, kidneys, thymus and inguinal lymph nodes. The viral load of PCV2 was the highest in liver followed by thymus and spleen. Although PRRSV were detected in most of tissues, but the replication of PRRSV was not significantly increased, as shown by qPCR analysis. Comparing with PCV2 infection alone, PRRSV infection significantly elevated PCV2 replication and exacerbated PCV2 induced interstitial pneumonia. qPCR analysis demonstrated 40 mg/kg Matrine significantly attenuated PCV2 replication in liver and alleviated virus induced interstitial pneumonia, suggesting Matrine could directly inhibit virus replication. In addition, Matrine treatment enhanced peritoneal macrophages phagocytosis at 13 and 16 dpi, and 40 mg/kg of Matrine increased the proliferation activity of lymphocytes. Body weight gain was continuously promoted by administrating Matrine at 10 mg/kg. CONCLUSION: Matrine possessed antiviral activities via inhibiting virus replication and regulating immune functions in mice co-infected by PRRSV/PCV2. These data provide new insight into controlling PRRSV and PCV2 infection and support further research for developing Matrine as a new possible veterinary medicine.

Alterations of fecal microbiome characteristics by dietary soy isoflavone ingestion in growing pigs infected with porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important disease, and the ingestion of soy isoflavones (ISF) may benefit PRRSV-infected pigs due to demonstrated anti-inflammatory and antiviral properties. The objective of this study was to quantify the effects of ISF consumption on fecal microbiome characteristics at different timepoints across a disease challenge and determine whether any changes, if present, elude to potential biological mechanisms for previously observed performance benefits. In total, 96 weaned barrows were group-housed in a Biosafety Level-2 containment facility and allotted to one of three experimental treatments that were maintained throughout the study: noninfected pigs receiving an ISF-devoid control diet (NEG, n = 24) and infected pigs receiving either the control diet (POS, n = 36) or that supplemented with total ISF in excess of 1,600 mg/kg (ISF, n = 36). Following a 7-d adaptation, pigs were inoculated intranasally with either a sham-control (phosphate-buffered saline) or live PRRSV (1 × 105 median tissue culture infectious dose[TCID]50/mL, strain NADC20). Fecal samples were collected from 48 individual pigs at pre-infection (-2 d post-inoculation [DPI]), peak-infection (10 DPI), and post-infection (144 DPI) timepoints. Extracted DNA was used to quantify fecal microbiota profiles via 16S bacterial rRNA sequencing. Differences in bacterial communities among diet groups were evaluated with principal coordinate analysis and permutational multivariate analysis of variance using UniFrac distance matrices based on both unweighted and weighted UniFrac distances using QIIME 2. All other data were analyzed by one-way ANOVA performed on square root transformations using R. Across all timepoints, only a few differences were observed due to ISF alone mainly in lowly abundant genera. The most notable differences observed were decreased relative abundance of Actinobacteria at 144 DPI between noninfected and infected treatments (P < 0.05), which is consistent with various dysbioses observed in other disease models. Our findings indicate that the differences present were mainly due to PRRSV-infection alone and not strongly influenced by diet, which implies that previously observed performance benefits conferred by dietary ISF are not likely due to the changes in microbiome composition.

In vitro screening antiviral activity of Thai medicinal plants against porcine reproductive and respiratory syndrome virus.

BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV) results in economic losses in the swine industry globally. Several studies have investigated the use of plant extracts in the prevention and control of PRRS outbreaks. Thai medicinal plants may be useful for treating PRRSV infection in pigs. Therefore, we investigated the in vitro anti-PRRSV and antioxidant properties of seven Thai medicinal plants: Caesalpinia sappan Linn., Garcinia mangostana Linn., Houttuynia cordata, Perilla frutescens, Clinacanthus nutans, Phyllanthus emblica, and Tiliacora triandra. RESULTS: Using antiviral screening, we observed that T. triandra extract strongly inhibited PRRSV infectivity in MARC-145 cells [virus titer 3.5 median tissue culture infective dose (TCID50)/ml (log10)] at 24 h post-infection, whereas C. sappan extract strongly inhibited PRRSV replication [virus titer 2.5 TCID50/ml (log10)] at 72 h post-infection. C. sappan extract had the highest total phenolic content [220.52 mM gallic acid equivalent/g] and lowest half-maximal inhibitory concentration [1.17 mg/ml in 2,2-diphenyl-1-picrylhydrazyl and 2.58 mg/ml in 2,2-azino-bis (3-ethylbenzothiazo-line-6-sulfonic acid) diammonium salt]. CONCLUSION: T. triandra extract could inhibit PRRSV infectivity, whereas C. sappan extract was the most effective in inhibiting PRRSV replication in MARC-145 cells. This study elucidates the antiviral activities of Thai medicinal plant extracts in vivo. The results promise that Thai medicinal plant extracts, particularly T. triandra and C. sappan extracts, can be developed into pharmaceutical drugs for the prevention of PRRS in pigs.

Oral supplementation of quercetin in PRRSV-1 modified-live virus vaccinated pigs in response to HP-PRRSV-2 challenge.

This study evaluated the immunomodulatory effect of quercetin on improving cross protection of porcine reproductive and respiratory syndrome virus-1 (PRRSV-1) modified-live virus (MLV) vaccine against highly pathogenic (HP)-PRRSV-2 challenge. Ex vivo experiments demonstrated that quercetin significantly enhanced type I interferon-regulated genes (IRGs) and type I and II interferon (IFN), and significantly decreased pro- and anti-inflammatory cytokine expressions in HP-PRRSV-inoculated monocyte-derived macrophages. In vivo experiments divided pigs (4-week-old; n = 24) into four groups of six pigs. Group 1 and group 2 were immunized with PRRSV-1 MLV vaccine at 0 dpv (day post vaccination). Group 2 also received oral administration of quercetin at 0-49 dpv. Group 3 was injected with PRRSV-1 MLV vaccine solvent at 0 dpv. Group 4 served as strict control. Group 1-3 were challenged intranasally with HP-PRRSV at 28 dpv and immune and clinical parameters were monitored weekly until 49 dpv. Group 1 demonstrated significantly reduced HP-PRRSV viremia, rectal temperature and clinical scores, and significantly improved average daily weight gain (ADWG), compared to group 3. Group 2 demonstrated significantly increased IFN regulatory factor 3, stimulator of IFN genes, IFNα, and significantly decreased transforming growth factor beta (TGFß) mRNA expressions, compared to group 1. The animals demonstrated significantly reduced HP-PRRSV viremia, but did not demonstrate any further improved PRRSV-specific antibody responses, rectal temperature, clinical scores, and ADWG as compared to group 1. Our findings suggest that quercetin up-regulates IRGs, IFNα, and down-regulates TGFß mRNA expressions which may contribute to further reducing number of viremic pigs and HP-PRRSV viremia which were conferred by PRRSV-1 MLV vaccine. Our findings also suggest that quercetin may serve as an effective oral immunomodulator for improving cell-mediated immune defense to HP-PRRSV.

Effect of porcine reproductive and respiratory syndrome virus infection and soy isoflavone supplementation on carcass cutability and meat quality of pigs.

The objective was to evaluate the effects of porcine reproductive and respiratory syndrome virus (PRRSV) infection and dietary soy isoflavone (ISF) supplementation on carcass cutability and meat quality of commercial pigs. Barrows (21 d of age) were randomly allotted to experimental treatments that were maintained throughout the study: noninfected pigs received an ISF-devoid control diet (CON, n = 22) and infected pigs received either the control diet (PRRSV-CON, n = 20) or that supplemented with total ISF in excess of 1,500 mg/kg (PRRSV-ISF, n = 25). Pigs were penned by treatment, with six pigs within a pen. Following a 7-d adaptation, weanling pigs were inoculated once intranasally with either a sham-control (phosphate buffered saline [PBS]) or live PRRSV (1 × 105 tissue culture infective dose [TCID]50/mL, strain NADC20). Pigs were maintained on experimental diets for 166 d after inoculation and then slaughtered (192 or 194 d of age; approximately 120 kg body weight [BW]). At 1-d postmortem, left sides were separated between the 10th and 11th rib for the determination of loin eye area (LEA), backfat (BF) thickness, and loin quality (ultimate pH, instrumental color, drip loss, visual color, marbling, and firmness). Loin chops were aged 14 d postmortem prior to Warner-Bratzler shear force (WBSF) determination. Belly width, length, thickness, and flop distance were determined. Data were analyzed as a one-way ANOVA with pig as the experimental unit. Carcass yield, LEA, BF, and estimated lean percentage did not differ (P > 0.26) among treatments. Loins from CON pigs had increased ultimate pH (P = 0.01), reduced L* scores (P = 0.005) coupled with darker visual color scores (P = 0.004), were firmer (P < 0.0001), and exhibited reduced drip loss (P = 0.01) compared with PRRSV-CON and PRRSV-ISF pigs. However, WBSF did not differ (P = 0.51) among treatments after 14 d of aging. Bellies from CON pigs were more firm compared with bellies from PRRSV-CON and ISF pigs (P < 0.01). These data suggest PRRSV infection did not alter carcass characteristics but may have marginally reduced loin and belly quality. Supplementation with dietary soy isoflavones did nothing to mitigate the detrimental effects of PRRSV infection.

Glycyrrhizic-Acid-Based Carbon Dots with High Antiviral Activity by Multisite Inhibition Mechanisms.

With the gradual usage of carbon dots (CDs) in the area of antiviral research, attempts have been stepped up to develop new antiviral CDs with high biocompatibility and antiviral effects. In this study, a kind of highly biocompatible CDs (Gly-CDs) is synthesized from active ingredient (glycyrrhizic acid) of Chinese herbal medicine by a hydrothermal method. Using the porcine reproductive and respiratory syndrome virus (PRRSV) as a model, it is found that the Gly-CDs inhibit PRRSV proliferation by up to 5 orders of viral titers. Detailed investigations reveal that Gly-CDs can inhibit PRRSV invasion and replication, stimulate antiviral innate immune responses, and inhibit the accumulation of intracellular reactive oxygen species (ROS) caused by PRRSV infection. Proteomics analysis demonstrates that Gly-CDs can stimulate cells to regulate the expression of some host restriction factors, including DDX53 and NOS3, which are directly related to PRRSV proliferation. Moreover, it is found that Gly-CDs also remarkably suppress the propagation of other viruses, such as pseudorabies virus (PRV) and porcine epidemic diarrhea virus (PEDV), suggesting the broad antiviral activity of Gly-CDs. The integrated results demonstrate that Gly-CDs possess extraordinary antiviral activity with multisite inhibition mechanisms, providing a promising candidate for alternative therapy for PRRSV infection.

Dietary soy isoflavones reduce pathogen-related mortality in growing pigs under porcine reproductive and respiratory syndrome viral challenge.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important disease, and ingestion of soy isoflavones (ISF) may benefit PRRSV-infected pigs due to demonstrated anti-inflammatory and antiviral properties. The objective of this experiment was to recreate immunological effects previously observed in young pigs infected with PRRSV receiving ISF and determine how those effects influence growth performance during the entire growth period from weaning to market. In total, 96 weaned barrows were group housed in a biosafety level-2 containment facility and allotted to 1 of 3 experimental treatments that were maintained throughout the study: noninfected pigs received an ISF-devoid control diet (NEG, n = 24), and infected pigs received either the control diet (POS, n = 36) or that supplemented with total ISF in excess of 1,600 mg/kg (ISF, n = 36). Following a 7-d adaptation, weanling pigs were inoculated intranasally with either a sham-control (PBS) or live PRRSV (1 × 105 TCID50/mL, strain NADC20). After inoculation, individual blood samples (n = 8 to 12/treatment) were routinely collected to monitor viral clearance and hematological parameters, including serum neutralizing anti-PRRSV antibody production. Pen-based oral fluids were used to monitor PRRSV clearance at later growth stages. A 1- or 2-way ANOVA was performed to compare experimental treatments depending on whether the outcome was repeatedly measured. In general, PRRSV infection decreased performance during early growth phases, resulting in 5.4% lower final BW for POS vs. NEG pigs (P < 0.05). Dietary ISF elicited inconsistent effects on growth performance, increased (P < 0.05) neutrophil cell counts and the relative proportion of memory T-cells, and decreased (P < 0.05) the time to full PRRSV clearance from oral fluids. Dietary ISF also elicited earlier, more robust anti-PRRSV neutralizing antibody production when compared with POS pigs. Additionally, and most notably, POS pigs experienced ~50% greater infection-related mortality rate vs. ISF pigs (P < 0.05), which may have significant economic implications for producers. Overall, dietary ISF ingestion supported immune responses and reduced mortality in PRRSV-infected pigs when fed to growing pigs though the biological mechanism of these effects remains unclear.

Therapeutic effects of saponin components on porcine reproductive and respiratory syndrome virus-infected piglets.

The present study aimed to evaluate the potential therapeutic effects of Anemoside B4 (AB4), Panax notoginseng saponins (PNS), Notoginsenoside R1 (SR1), Saikosaponin A (SSA) and Saikosaponin D (SSD) on piglets infected with porcine reproductive and respiratory syndrome virus (PRRSV). A total of 132 completely healthy piglets were randomly divided into 22 groups consisting of six animals each. Control piglets were intramuscularly injected with 2 ml of PRRSV (NJGC strain) solution containing 106  TCID50  virus/ml. For low-, middle- and high-dose saponin treatment groups, the piglets were initially administrated with the same volume of PRRSV solution, followed by intraperitoneal injection with AB4, PNS, SR1, SSA or SSD at 1, 5 or 10 mg/kg b.w. on day 3. The piglets in drug control group were intraperitoneally injected with 10 mg/kg b.w. of each saponin without prior PRRS challenge, while those in blank control group were injected with the same amount of normal saline. The results indicated that all the five saponin components could decrease the incidence and severity of PRRSV-induced immunopathological damages, including the elevated body temperature, weight loss, anaemia and internal inflammation. Moreover, the saponin components could enhance protein absorption and immune responses. Taken together, this study reveals that the saponin components are effective against PRRSV infection and strengthen the immune system and thus may serve as potential antiviral therapeutic agents.

Evaluation of PRRSv specific, maternally derived and induced immune response in Ingelvac PRRSFLEX EU vaccinated piglets in the presence of maternally transferred immunity.

In this study, we analyzed PRRS virus (PRRSv) specific lymphocyte function in piglets vaccinated with Ingelvac PRRSFLEX EU® at two and three weeks of age in the presence of homologous maternal immunity. Complete analysis of maternal immunity to PRRSv was evaluated postpartum, as well as passive transfer of antibodies and T cells to the piglet through colostrum intake and before and after challenge with a heterologous PRRSv at ten weeks of age. Maternal-derived antibodies were detected in piglets but declined quickly after weaning. However, vaccinated animals restored PRRSv-specific antibody levels by anamnestic response to vaccination. Cell analysis in colostrum and milk revealed presence of PRRSv-specific immune cells at suckling with higher concentrations found in colostrum than in milk. In addition, colostrum and milk contained PRRSv-specific IgA and IgG that may contribute to protection of newborn piglets. Despite the presence of PRRSv-specific Peripheral Blood Mononuclear cells (PBMCs) in colostrum and milk, no PRRSv-specific cells could be detected from blood of the piglets at one or two weeks of life. Nevertheless, cellular immunity was detectable in pre-challenged piglets up to 7 weeks after vaccination while the non-vaccinated control group showed no interferon (IFN) γ response to PRRSv stimulation. After challenge, all piglets developed a PRRSv-specific IFNγ-response, which was more robust at significantly higher levels in vaccinated animals compared to the primary response to PRRSv in non-vaccinated animals. Cytokine analysis in the lung lumen showed a reduction of pro-inflammatory responses to PRRSv challenge in vaccinated animals, especially reduced interferon (IFN) α levels. In conclusion, vaccination of maternally positive piglets at 2 and 3 weeks of age with Ingelvac PRRSFLEX EU induced a humoral and cellular immune response to PRRSv and provided protection against virulent, heterologous PRRSv challenge.

Xanthohumol inhibits PRRSV proliferation and alleviates oxidative stress induced by PRRSV via the Nrf2-HMOX1 axis.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a prevalent and endemic swine pathogen that causes significant economic losses in the global swine industry. Commercial vaccines provide limited protection against this virus, and no highly effective therapeutic drugs are yet available. In this study, we first screened a library of 386 natural products and found that xanthohumol (Xn), a prenylated flavonoid found in hops, displayed high anti-PRRSV activity by inhibiting PRRSV adsorption onto and internalization into cells. Transcriptome sequencing revealed that Xn treatment stimulates genes associated with the antioxidant response in the nuclear factor-erythroid 2-related factor 2 (Nrf2) signalling pathway. Xn causes increased expression of Nrf2, HMOX1, GCLC, GCLM, and NQO1 in Marc-145 cells. The action of Xn against PRRSV proliferation depends on Nrf2 in Marc-145 cells and porcine alveolar macrophages (PAMs). This finding suggests that Xn significantly inhibits PRRSV proliferation and decreases viral-induced oxidative stress by activating the Nrf2-HMOX1 pathway. This information should be helpful for developing a novel prophylactic and therapeutic strategy against PRRSV infection.