Host Avian Beta-Defensin and Toll-Like Receptor Responses of Pigeons following Infection with Pigeon Paramyxovirus Type 1

Abstract

The high morbidity and mortality in pigeons caused by pigeon paramyxovirus type 1 (PPMV-1) highlights the need for new insights into the host immune response and novel treatment approaches. Host defense peptides (HDPs) are key components of the innate immune system. In this study, three novel avian β-defensins (AvBDs 2, 7, and 10) were characterized in pigeons and shown to possess direct antiviral activity against PPMV-1 in vitro. In addition, we evaluated the mRNA expression of these AvBDs and other immune-related genes in tissues of 2-month-old infected pigeons at 3 and 7 days postinfection. We observed that the expression of AvBD2 in the cecal tonsil, lungs, and proventriculus, as well as the expression of AvBD10 in the spleen, lungs, proventriculus, and kidneys, was upregulated in infected pigeons. Similarly, the expression of both Toll-like receptor 3 (TLR3) and TLR7 was increased in the spleen, trachea, and proventriculus, while TLR15 expression was increased only in the lungs of infected pigeons. In addition, inducible nitric oxide synthase (iNOS) expression was upregulated in the spleen, the bursa of Fabricius, the trachea, and the proventriculus of infected pigeons. Furthermore, we observed a high correlation between the expression of AvBD2 and the expression of either TLR7 or TLR15, as well as between AvBD10 expression and either TLR3 or TLR7 expression in respective tissues. The results suggest that PPMV-1 infection can induce innate host responses characterized by the activation of TLRs, particularly TLR3 and TLR7, AvBDs (2 and 10), and iNOS in pigeons.

FIG 1

Deduced amino acid sequence alignment of three novel avian β-defensins (AvBDs) from pigeons and SDS-PAGE analysis of His-tagged recombinant AvBD (His-rAvBD) proteins expressed in E. coli BL21(DE3) cells. (A) Deduced amino acid sequence alignment of three novel AvBDs from pigeons. The signal sequences of the AvBDs are italicized. The six conserved cysteines (C) are underlined. Dashes indicate that no identical or conserved residues were observed. (B) SDS-PAGE analysis of His-tagged recombinant AvBD (His-rAvBD) proteins expressed in E. coli BL21(DE3) cells. (Left panel) Lanes from left to right are as follows: total proteins from BL21 containing AvBD2, -7, and -10 (without IPTG induction), respectively; total proteins from BL21 containing AvBD2, -7, and -10 (with IPTG induction), respectively; protein molecular weight marker. (Right panel) Lanes from left to right are as follows: inclusion bodies of AvBD2, -7, and -10, respectively; purified proteins of AvBD2, -7, and -10, respectively; protein molecular weight marker. IPTG, isopropyl-β-d-thiogalactoside.

FIG 2

Antiviral activities of His-rAvBDs against PPMV-1 in pigeon embryos and hemolytic activities of His-rAvBDs. (A) Antiviral activities of His-rAvBDs. The virus titers were tested by hemagglutinin (HA) at 24, 36, and 48 hpi. All assays were performed in three independent experiments, with five replicates per experiment, and each bar is the mean ± SD. (B) Hemolytic activities of His-rAvBDs. Freshly isolated pigeon red blood cells were incubated with different concentrations of AvBDs (0 to 500 μg/ml). The release of hemoglobin, as a measure of hemolysis, was measured at 405 nm. The release of hemoglobin upon the addition of 1% Triton X-100 was set at 100%. The percentage of hemolysis was calculated as [(A_{405 nm, peptide} − A_{405 nm, PBS})/(A_{405 nm, 1% Triton X-100} − A_{405 nm, PBS})] × 100%. All assays were performed in three independent experiments, with three replicates per experiment, and each point is the mean ± SD. Different letters above columns indicate values that are significantly different (P < 0.05).

FIG 3

Relative gene expression of avian β-defensins (AvBDs) in the tissues of pigeons in response to PPMV-1 infection. cDNA copy numbers in the tissue samples from five pigeons of each group were measured by quantitative PCR (qPCR) at 3 and 7 dpi. AvBD levels were normalized to the levels of β-actin in the same samples. All assays were performed in triplicate, with five replicates per experiment, and each bar is the means ± SD. Different letters above columns indicate values that are significantly different (P < 0.05).

FIG 4

Relative gene expression of Toll-like receptors (TLRs) in the tissue samples of pigeons in response to PPMV-1 infection. cDNA copy numbers in the tissue samples from five pigeons of each group were measured by quantitative PCR (qPCR) at 3 and 7 dpi. TLR levels were normalized to the levels of β-actin in the same samples. All assays were performed in triplicate, with five replicates per experiment, and each bar is the mean ± SD. Different letters above columns indicate values that are significantly different (P < 0.05).

FIG 5

Analysis of relative gene expression of iNOS in the tissue samples of pigeons in response to PPMV-1 infection. cDNA copy numbers in the tissue samples from five pigeons of each group were measured by qPCR at 3 and 7 dpi. iNOS levels were normalized to the levels of β-actin in the same samples. All assays were performed in triplicate, with five replicates per experiment, and each bar is the mean ± SD. Different letters above columns indicate values that are significantly different (P < 0.05).

FIG 6

Correlation between AvBD and TLR expression in tissues of pigeons in response to PPMV-1 infection. cDNA copy numbers in tissue samples from five pigeons of each group were measured by qPCR at 3 and 7 dpi. Gene levels were normalized to the levels of β-actin in the same samples. All assays were performed in triplicate, with five replicates per experiment, and each bar is the mean ± SD.