Systematic Identification of Host Immune Key Factors Influencing Viral Infection in PBL of ALV-J Infected SPF Chicken

Abstract

Although research related to avian leukosis virus subgroup J (ALV-J) has lasted for more than a century, the systematic identification of host immune key factors against ALV-J infection has not been reported. In this study, we establish an infection model in which four-week-old SPF chickens are infected with ALV-J strain CHN06, after which the host immune response is detected. We found that the expression of two antiviral interferon-stimulated genes (ISGs) (Mx1 and IFIT5) were increased in ALV-J infected peripheral blood lymphocytes (PBL). A significant CD8⁺ T cell response induced by ALV-J appeared as early as seven days post-infection (DPI), and humoral immunity starting from 21 DPI differed greatly in the time scale of induction level. Meanwhile, the ALV-J viremia was significantly decreased before antibody production at 14 DPI, and eliminated at 21 DPI under a very low antibody level. The up-regulated CD8⁺ T cell in the thymus (14DPI) and PBL (7 DPI and 21 DPI) was detected, indicating that the thymus may provide the output of CD8⁺ T cell to PBL, which was related to virus clearance. Besides, up-regulated chemokine CXCLi1 at 7 DPI in PBL was observed, which may be related to the migration of the CD8⁺ T cell from the thymus to PBL. More importantly, the CD8 ^high+ T cell response of the CD8αβ phenotype may produce granzyme K, NK lysin, or IFN-γ for clearing viruses. These findings provide novel insights and direction for developing effective ALV-J vaccines.

Keywords: ALV-J; CD8+ T cell response; PBL; chicken; phenotype.

Figure 1

Dynamic detection of the avian leukosis virus subgroup J (ALV-J) shedding, ALV-J viremia, and ALV-J antibody. Five chickens were randomly selected for sampling every seven days post-infection (DPI). (A) ALV-J shedding was monitored via detecting the p27 expression levels in cloacal swabs. S/P value below 0.2 (red) indicated negative ALV-J shedding. (B) ALV-J viremia was monitored via virus isolation with the routine method. An S/P value above 0.2 (red) indicated positive ALV-J viremia. (C) The ALV-J antibody level in the serum was monitored using the commercial ALV-J antibody test kit. An S/P value above 0.6 (red) was considered ALV-J antibody positive. (D) ALV-J viremia and antibody level of chicken number 12 were analyzed from 7 DPI to 63 DPI. The paired t-test was used for statistical comparison. * p < 0.05, ** p < 0.01.

Figure 2

Dynamic detection of T lymphocyte percentage in peripheral blood lymphocytes (PBL). Five days before infection (DBI) and each week after infection, PBL derived from five chickens of infected and control groups were isolated to detect the T lymphocyte percentage, including the percentage of (A) the CD3⁺CD8⁺ T cell, (B) CD3⁺CD4⁺ T cell, and (C) CD3⁺CD4⁺CD8⁺. (D) The ratio of CD3⁺CD4⁺/CD3⁺CD8⁺ was detected. Each sample collected 1 × 10⁵ cells for flow cytometric analysis. The two-way ANOVA was used for statistical comparison. ns p > 0.05, * p < 0.05, ** p < 0.01.

Figure 3

Analysis of T lymphocyte percentage in the thymus. Thymus single-cell suspensions derived from chickens of infected and control groups were isolated to detect the T lymphocyte percentage. Every dot stands for one chicken. The unpaired t-test was used for statistical comparison. ns p > 0.05, * p < 0.05, ** p < 0.01. Each sample collected 2 × 10⁵ cells for flow cytometric analysis.

Figure 4

Analysis of T lymphocyte percentage in spleen. Spleen single-cell suspensions derived from chickens of infected and control groups were isolated to detect the T lymphocyte percentage. Every dot stands for one chicken. The unpaired t-test was used for statistical comparison. ns p > 0.05, * p < 0.05.. Each sample collected 2 × 10⁵ cells for flow cytometric analysis.

Figure 5

Analysis expression of immune-related genes in PBL by qRT-PCR. Expressions of immune-related genes in PBL were detected by qRT-PCR. The total RNA of PBL was extracted from three chickens of the infected and control groups, respectively. The data was collected from three biological samples in each group, each sample performed in triplicate. The results were presented as means ± SEM and the paired t-test was used for statistical comparison. ns p > 0.05, * p < 0.05, ** p < 0.01.

Figure 6

T cell phenotype analysis. (A) Gating of CD8α⁺, CD4⁺, and CD4⁺CD8α⁺ T cells with the CD3⁺(APC), CD4⁺(FITC), and CD8α⁺(PE) antibodies. (B) Analysis of the percentage and phenotype of T cells in PBL of ALV-J infected chicken number 12 and control chicken number 29 at various time points. Histogram of the CD3⁺CD8α⁺ T cells in PBL of (C) ALV-J infected group and (D) control group at 21 DPI. Each sample collected 1 × 10⁵ cells for flow cytometric analysis.

Figure 7

CD8⁺ T cell phenotype identification in PBL of ALV-J infected chicken number 12. (A) Gating strategy for analysis of CD8^highα⁺, CD8^mediumα⁺, CD4⁺, and CD4⁺CD8^lowα⁺ T cells with the CD3⁺(APC), CD4⁺(FITC), and CD8α⁺(PE) antibodies. (B) Gating strategy for analysis of CD8⁺ αα and CD8⁺αβ phenotype in three CD8⁺ populations with the CD4⁺ (APC), CD8β⁺ (FITC), and CD8α⁺ (PE) antibodies. (C) Gating strategy for analysis of CD8⁺ αα and CD8⁺αβ phenotype in three CD8⁺ populations with the CD3⁺ (APC), CD8β⁺ (FITC), and CD8α⁺ (PE) antibodies. Each dot indicated each collected cell. And the circle or fame indicated the gated target cell population labelled by various antibodies. Each sample collected 1 × 10⁵ cells for flow cytometric analysis.

Figure 8

CD8⁺ T cell phenotype analysis. Contour plot of CD8⁺ αα and CD8⁺αβ T cells in PBL of (A) control chicken number 34 and (B) ALV-J infected chicken number 12 with the CD3⁺ (APC), CD8β⁺ (FITC), and CD8α⁺ (PE) antibodies. Each sample collected 1 × 10⁵ cells for flow cytometric analysis. The circle or fame indicated the gated target cell population labelled by various antibodies. And the red arrow indicated that each CD8α⁺T cell population was further subdivided to CD8αα and CD8αβ phenotype.