Effect of polymorphisms in porcine guanylate-binding proteins on host resistance to PRRSV infection in experimentally challenged pigs

Abstract

Guanylate-binding proteins (GBP1 and GBP5) are known to be important for host resistance against porcine reproductive and respiratory syndrome virus (PRRSV) infection. In this study, the effects of polymorphisms in GBP1 (GBP1E2 and WUR) and GBP5 on host immune responses against PRRSV were investigated to elucidate the mechanisms governing increased resistance to this disease. Seventy-one pigs [pre-genotyped based on three SNP markers (GBP1E2, WUR, and GBP5)] were assigned to homozygous (n = 36) and heterozygous (n = 35) groups and challenged with the JA142 PRRSV strain. Another group of nineteen pigs was kept separately as a negative control group. Serum and peripheral blood mononuclear cells (PBMCs) were collected at 0, 3, 7, 14, 21 and 28 days post-challenge (dpc). Viremia and weight gain were measured in all pigs at each time point, and a flow cytometry analysis of PBMCs was performed to evaluate T cell activation. In addition, 15 pigs (5 pigs per homozygous, heterozygous and negative groups) were sacrificed at 3, 14 and 28 dpc, and the local T cell responses were evaluated in the lungs, bronchoalveolar lavage cells (BALc), lymph nodes and tonsils. The heterozygous pigs showed lower viral loads in the serum and lungs and higher weight gains than the homozygous pigs based on the area under the curve calculation. Consistently, compared with the homozygous pigs, the heterozygous pigs exhibited significantly higher levels of IFN-α in the serum, proliferation of various T cells (γδT, Th1, and Th17) in PBMCs and tissues, and cytotoxic T cells in the lungs and BALc. These results indicate that the higher resistance in the pigs heterozygous for the GBP1E2, WUR and GBP5 markers could be mediated by increased antiviral cytokine (IFN-α) production and T cell activation.

Figure 1

Design of the animal study.

Figure 2

Correlation between AUC (area under the curve) viremia and ADWG, AUC viremia and PRRSV viral loads in the serum and lungs, and ADWG. A The correlation (Spearman’s, r) between AUC viremia (0–21 dpc) and ADWG (until 28 dpc) was tested in the pigs after infection with JA142. B The viral loads in the serum samples collected from all pigs were quantified (using qRT-PCR) at 0 (before challenge), 3, 7, 14, 21 and 28 dpc. The viral loads are expressed as TCID₅₀ equivalent/mL (log₁₀). C ADWG in all pigs was calculated at every week up to 28 dpc and expressed as kg/day. D The lungs pathology (gross and microscopic lesions), and residual virus titers in the lung tissues collected from pigs at 28 dpc were measured using a microtitration infectivity assay performed in MARC-145 cells and presented as TCID₅₀/mL (log₁₀). The error bars represent the standard error of the mean (SEM). Asterisks indicate significant differences in each parameter among the genotypes (*p < 0.05). The bars showing different letters represent the values that differ significantly from each other (p < 0.05).

Figure 3

Quantification of anti-PRRSV antibodies (IgG) and VNA titers measured in PRRSV-infected pigs. A Anti-PRRSV antibody (IgG) response was measured in serum samples collected weekly until 28 dpc using a nucleocapsid (N) protein-based ELISA kit based on the manufacturer’s instructions, and the result is expressed as the S/P ratio. An S/P ratio ≥ 0.4 is considered positive for PRRSV-specific antibodies (IgG). B The levels of the VNA titers were measured using a fluorescent focus neutralization (FFN) assay. The error bars represent the SEM. Asterisks indicate significant differences in the level of anti-PRRSV IgG among the genotypes (*p < 0.05).

Figure 4

Quantification of the cytokine protein levels in serum collected from PRRSV-infected pigs. Serum samples collected at 0, 3, 7, 14, and 28 dpc were analysed to detect porcine cytokines. A IFN-α by ELISA and a correlation with average viremia at 3 dpc. B TNF-α by ELISA. The error bars represent the SEM. Asterisks indicate significant differences in the levels of cytokines induced by PRRSV among the genotypes (*p < 0.05).

Figure 5

T cell responses in PBMCs isolated from pigs after PRRSV infection. PBMCs were separated at the indicated days, immunostained with the appropriate antibodies, and analysed by flow cytometry to detect gamma-delta (γδ) T cells (γδT), type 1 helper T cells (Th1), T helper 17 cells (Th17), cytotoxic T lymphocytes (CTLs) and regulatory T cells (Tregs). The error bars represent the SEM. Asterisks indicate significant differences in T cell proliferation among the genotypes (*p < 0.05; **p < 0.001). *nt: not tested because of sample shortage.

Figure 6

T cell responses in lungs and BALc collected from pigs after PRRSV infection. Mononuclear cells isolated from the lungs (A) and BALc (B) were immunostained with the appropriate antibodies on the indicated days and then analysed by flow cytometry to detect gamma-delta (γδ) T cells (γδT), type 1 helper T cells (Th1), T helper 17 cells (Th17), cytotoxic T lymphocytes (CTLs) and regulatory T cells (Tregs). The error bars represent the SEM. Asterisks indicate significant differences in T cell proliferation among the genotypes (*p < 0.05). *nt: not tested because of sample shortage.

Figure 7

T cell responses in lymph nodes and tonsils collected from pigs after PRRSV infection. Mononuclear cells isolated from the lymph nodes (A) and tonsils (B) were immunostained with the appropriate antibodies on the indicated days and analysed by flow cytometry to detect gamma-delta (γδ) T cells (γδT), type 1 helper T cells (Th1), T helper 17 cells (Th17), cytotoxic T lymphocytes (CTLs) and regulatory T cells (Tregs). The error bars represent the SEM. *nt: not tested because of sample shortage.

Figure 8

Measurement of weight gain and AUC (area under the curve) viremia in pigs following PRRSV infection. A Weight gain (WG) was calculated between 0 to 28 dpc and expressed in Kg. B AUC (area under the curve) viremia (of log10 transformed viremia) was measured between 0 to 21 dpc.