Dissection of key factors correlating with H5N1 avian influenza virus driven inflammatory lung injury of chicken identified by single-cell analysis.

Chicken lung is an important target organ of avian influenza virus (AIV) infection, and different pathogenic virus strains lead to opposite prognosis. Using a single-cell RNA sequencing (scRNA-seq) assay, we systematically and sequentially analyzed the transcriptome of 16 cell types (19 clusters) in the lung tissue of chickens infected with H5N1 highly pathogenic avian influenza virus (HPAIV) and H9N2 low pathogenic avian influenza virus (LPAIV), respectively. Notably, we developed a valuable catalog of marker genes for these cell types. Compared to H9N2 AIV infection, H5N1 AIV infection induced extensive virus replication and the immune reaction across most cell types simultaneously. More importantly, we propose that infiltrating inflammatory macrophages (clusters 0, 1, and 14) with massive viral replication, pro-inflammatory cytokines (IFN-ß, IL1ß, IL6 and IL8), and emerging interaction of various cell populations through CCL4, CCL19 and CXCL13, potentially contributed to the H5N1 AIV driven inflammatory lung injury. Our data revealed complex but distinct immune response landscapes in the lung tissue of chickens after H5N1 and H9N2 AIV infection, and deciphered the potential mechanisms underlying AIV-driven inflammatory reactions in chicken. Furthermore, this article provides a rich database for the molecular basis of different cell-type responses to AIV infection.

Duck CD8+ T Cell Response to H5N1 Highly Pathogenic Avian Influenza Virus Infection In Vivo and In Vitro.

Domestic ducks are the important host for H5N1 highly pathogenic avian influenza virus (HPAIV) infection and epidemiology, but little is known about the duck T cell response to H5N1 AIV infection. In infection experiments of mallard ducks, we detected significantly increased CD8+ cells and augmented expression of cytotoxicity-associated genes, including granzyme A and IFN-γ, in PBMCs from 5 to 9 d postinfection when the virus shedding was clearly decreased, which suggested the importance of the duck cytotoxic T cell response in eliminating H5N1 infection in vivo. Intriguingly, we found that a CD8high+ population of PBMCs was clearly upregulated in infected ducks from 7 to 9 d postinfection compared with uninfected ducks. Next, we used Smart-Seq2 technology to investigate the heterogeneity and transcriptional differences of the duck CD8+ cells. Thus, CD8high+ cells were likely to be more responsive to H5N1 AIV infection, based on the high level of expression of genes involved in T cell responses, activation, and proliferation, including MALT1, ITK, LCK, CD3E, CD247, CFLAR, IL-18R1, and IL-18RAP. More importantly, we have also successfully cultured H5N1 AIV-specific duck T cells in vitro, to our knowledge, for the first time, and demonstrated that the CD8high+ population was increased with the duck T cell activation and response in vitro, which was consistent with results in vivo. Thus, the duck CD8high+ cells represent a potentially effective immune response to H5N1 AIV infection in vivo and in vitro. These findings provide novel insights and direction for developing effective H5N1 AIV vaccines.

The Transcriptional Differences of Avian CD4+CD8+ Double-Positive T Cells and CD8+ T Cells From Peripheral Blood of ALV-J Infected Chickens Revealed by Smart-Seq2.

It is well known that chicken CD8+ T cell response is vital to clearing viral infections. However, the differences between T cell subsets expressing CD8 receptors in chicken peripheral blood mononuclear cells (PBMCs) have not been compared. Herein, we used Smart-Seq2 scRNA-seq technology to characterize the difference of chicken CD8high+, CD8high αα+, CD8high αß+, CD8medium+, and CD4+CD8low+ T cell subsets from PBMCs of avian leukosis virus subgroup J (ALV-J)-infected chickens. Weighted gene co-expression network analysis (WGCNA) and Trend analysis revealed that genes enriched in the "Cytokine-cytokine receptor interaction" pathway were most highly expressed in the CD8high αα+ T cell population, especially T cell activation or response-related genes including CD40LG, IL2RA, IL2RB, IL17A, IL1R1, TNFRSF25, and TNFRSF11, suggesting that CD8high αα+ T cells rather than other CD8 subpopulations were more responsive to ALV-J infections. On the other hand, genes involved in the "FoxO signaling pathway" and "TGF-beta signaling pathway" were most highly expressed in the CD4+CD8low+ (CD8low+) T cell population and the function of CD4+CD8low+ T cells may play roles in negatively regulating the functions of T cells based on the high expression of CCND1, ROCK1, FOXO1, FOXO3, TNFRSF18, and TNFRSF21. The selected gene expressions in CD8+ T cells and CD4+CD8low+ double-positive T cells confirmed by qRT-PCR matched the Smart-Seq2 data, indicating the reliability of the smart-seq results. The high expressions of Granzyme K, Granzyme A, and CCL5 indicated the positive response of CD8+ T cells. Conversely, CD4+CD8+ T cells may have the suppressor activity based on the low expression of activation molecules but high expression of T cell activity suppressor genes. These findings verified the heterogeneity and transcriptional differences of T cells expressing CD8 receptors in chicken PBMCs.