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

Abstract

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.

Keywords: cross-protection; houttuynia cordata; interferon; modified-live virus vaccine; porcine reproductive and respiratory syndrome virus.

Figure 1

High-performance liquid chromatography (HPLC) chromatograms of rutin, quercetin, HC50, HC70, and HC95.

Figure 2

Determination of non-cytotoxic concentration of HC50, HC70, and HC95. (A) MARC-145 cells were incubated with 2-fold serially diluted HC50 (black bar), HC70 (divot bar), and HC95 (dotted bar), each starting at a final concentration of 100 mg/ml for 96 h. The cultures were then fixed with acetone:methanol solution, and stained with 0.5% crystal violet and Sorenson’s buffer. MARC-145 cells incubated with vehicle solution served as vehicle control (white bar). The O.D. values obtained from MARC-145 cells treated with HC50, HC70, HC95, and vehicle solution were compared with those from MARC-145 cells incubated with only MEM⁺⁺ (untreated control) and expressed as %viability. (B) MDMs (n = 4 pigs) were incubated with 2-fold serially diluted HC50 (black bar), HC70 (divot bar), and HC95 (dotted bar), each starting at a final concentration of 100 mg/ml for 36 h. The cultures were then harvested and stained with trypan blue. MDMs incubated with vehicle solution served as vehicle control (white bar). Error bar indicates the standard deviation (SD).

Figure 3

Heat map illustrating levels of immune-related gene expressions of MDMs (n = 4 pigs) treated with HC50, HC70, and HC95 (0.8 mg/ml final) for 12, 24, and 36 h as determined by real-time PCR. MDMs stimulated with poly IC (for induction of Mx1, IRF3, IRF7, OAS1, STING, OPN, IFNα, IFNβ, IL-10, and TGFβ gene expressions) or LPS (for induction of IFNγ and TNFα gene expressions) served as positive control. mRNA expressions of immune-related genes were normalized to geometric average of two housekeeping genes, i.e., RPL32 and YWHAZ, of the same pigs. Expressions of immune-related genes in all groups were transformed to log2 scale and were presented in fold change, according to the 2^−ΔΔCT method, relative to those in untreated control.

Figure 4

Effects of HC50, HC70, and HC95 on PRRSV-1 and HP-PRRSV-2 infectivity and replication in vitro. (Top panel) HC50 (black bar), HC70 (divot bar), and HC95 (dotted bar) were incubated with 10-fold serially diluted PRRSV-1 (A) and HP-PRRSV-2 (B) for 1 h prior to subsequent inoculation onto adherent MARC-145 cells. The cultures were incubated for 1 h, then the supernatants were removed, and the cells were washed and incubated further in MEM⁺⁺ for 96 h. (Bottom panel) MARC-145 cells were inoculated with 10-fold serially diluted PRRSV-1 (C) and HP-PRRSV-2 (D) for 1 h, then the supernatants were discarded, and the cultures were washed and further incubated with HC50 (black bar), HC70 (divot bar), and HC95 (dotted bar) for 96 h. The virus titers were determined by IPMA. MARC-145 cells receiving only serially diluted PRRSV and vehicle solution served as PRRSV and vehicle controls, respectively. One-way ANOVA, followed by Dunnett’s test was used to determine the significant difference in mean virus titers. Error bar indicates the SEM. Different letters represent a significant mean difference among groups (p < 0.05).

Figure 5

Heat map illustrating levels of immune-related gene expressions of MDMs (n = 4 pigs) inoculated with HP-PRRSV-2 and treated with either HC50, HC70, or HC95 (0.8 mg/ml final) as determined by real-time PCR. MDMs were inoculated with HP-PRRSV-2 for 48 h, then received either HC50, HC70, HC95, or RPMI⁺⁺ for 12 h. Controls were MDMs incubated with mock Ags and received either vehicle solution (vehicle control) or RPMI⁺⁺ (mock control). Levels of mRNA expressions of immune-related genes were normalized to geometric average of RPL32 and YWHAZ of the same pigs. Expressions of immune-related genes in all groups were transformed to log2 scale and were presented in fold change, according to the 2^−ΔΔCT method, relative to those in mock control. I = Vehicle control; II = HP-PRRSV-2-inoculated MDMs; III = HP-PRRSV-2-inoculated/HC50-treated MDMs; IV = HP-PRRSV-2-inoculated/HC70-treated MDMs; and V = HP-PRRSV-2-inoculated/HC95-treated MDMs.

Figure 6

PRRSV-specific Ab response tested over time by ELISA. Pigs (n = 6) were injected i.m. with Amervac^® PRRS MLV (0 dpv) (MLV) and Amervac^® PRRS MLV (0 dpv) and fed p.o. with HC50 (0–49 dpv) (MLV+HC50) or vaccine solvent used for resuspension of lyophilized Amervac^® PRRS MLV (0 dpv) (Challenge CTRL). The animals were challenged i.n. with HP-PRRSV-2 at 28 dpv (dotted vertical line). Strict control pigs (Strict CTRL) received no treatment. Sera were collected weekly from all pigs at 0–49 dpv. The positive Ab response was determined at an s/p ratio of 0.4 or higher. One-way repeated measures ANOVA, followed by Dunnett’s test was used to determine the significant difference in mean s/p ratios (p < 0.05). Error bar indicates the SD.

Figure 7

Immune-related gene expressions in PBMCs re-stimulated ex vivo with HP-PRRSV-2. Pigs (n = 6) were injected i.m. with Amervac^® PRRS MLV (0 dpv) (MLV) and Amervac^® PRRS MLV (0 dpv) and fed p.o. with HC50 (0–49 dpv) (MLV+HC50) or vaccine solvent used for resuspension of lyophilized Amervac^® PRRS MLV (0 dpv) (Challenge CTRL). The animals were challenged i.n. with HP-PRRSV-2 at 28 dpv (dotted vertical line). Strict control pigs (Strict CTRL) received no treatment. PBMCs were collected weekly from all pigs at 0–49 dpv. Harvested PBMCs were incubated with HP-PRRSV-2 for 72 h prior to determination of immune-related gene expressions by real-time PCR. PBMCs receiving mock Ag served as mock control. PBMCs stimulated with poly IC (for induction of Mx1, IRF3, IRF7, OAS1, STING, OPN, IFNα, IFNβ, IL-10, and TGFβ gene expressions) or LPS (for induction of IFNγ and TNFα gene expressions) served as positive control. mRNA expressions of immune-related genes were normalized to a geometric average of two housekeeping genes, i.e., RPL32 and YWHAZ, of the same pigs. Expressions of immune-related genes in all groups were transformed to log2 scale and were presented in fold change, according to the 2^−ΔΔCT method, relative to those in mock control. One-way repeated measures ANOVA, followed by Dunnett’s test, was used to determine the significant difference in mean fold expressions (p < 0.05). Error bar indicates the SD. p-values of immune-related genes that showed significant difference between MLV and MLV+HC50 groups either prior to and after the HP-PRRSV-2 challenge was presented.

Figure 8

Number of viremic pigs and log₁₀ PRRSV ORF7 copy numbers in serum following the HP-PRRSV-2 challenge. Pigs (n = 6) were injected i.m. with Amervac^® PRRS MLV (0 dpv) (MLV) and Amervac^® PRRS MLV (0 dpv) and fed p.o. with HC50 (0–49 dpv) (MLV+HC50) or vaccine solvent used for resuspension of lyophilized Amervac^® PRRS MLV (0 dpv) (Challenge CTRL). The animals were challenged i.n. with HP-PRRSV at 28 dpv (0 dpc) (dotted vertical line). Strict control pigs (Strict CTRL) received no treatment. Serum samples were collected weekly from all pigs at 0–21 dpc and were determined for PRRSV ORF7 copy numbers by real-time PCR. The C_T obtained from each sample was compared with the C_T standard curve generated from 10¹ to 10⁸ copies of recombinant PRRSV ORF7 plasmids. The calculated copy numbers were transformed to log₁₀ scale. Data were presented per ml of serum sample. Poisson regression analysis was used to determine the significant difference in the number of viremic pigs. One-way repeated measures ANOVA, followed by Dunnett’s test, was used to determine the significant difference in mean log₁₀ PRRSV ORF7 copy numbers (p < 0.05). Error bar indicates the SD. p-value of statistical difference between MLV and MLV+HC50 groups after the HP-PRRSV challenge was presented.

Figure 9

Rectal temperature, clinical score, weight gain (WG), and average daily weight gain (ADWG) of pigs following the HP-PRRSV-2 challenge. Pigs (n = 6) were injected i.m. with Amervac^® PRRS MLV (0 dpv) (MLV) and Amervac^® PRRS MLV (0 dpv) and fed p.o. with HC50 (0–49 dpv) (MLV+HC50) or vaccine solvent used for resuspension of lyophilized Amervac^® PRRS MLV (0 dpv) (Challenge CTRL). The animals were challenged i.n. with HP-PRRSV-2 at 28 dpv (0 dpc). Strict control pigs (Strict CTRL) received no treatment. Rectal temperature and clinical score were recorded daily. WG was calculated from 0 to 28 dpv, and from 0 to 21 dpc. ADWG was calculated from 0 to 21 dpc. One-way repeated measures ANOVA, followed by Dunnett’s test was used to determine the significant difference in mean rectal temperature and clinical score. One-way ANOVA, followed by Dunnett’s test, was used to determine the significant difference in mean WG and ADWG. Error bar indicates the SD. Different letters represent a significant mean difference among groups (p < 0.05).