Avian leukosis virus subgroup J (ALV-J) infection can cause tumors and immunosuppression in infected chickens. Macrophages play a central role in host defense against invading pathogens. In this study, we discovered an interesting phenomenon: ALV-J replication is weakened from 3 hours post-infection (hpi) to 36 hpi, which was verified using Western blotting and RT-PCR. To further investigate the interaction between ALV-J and macrophages, transcriptome analysis was performed to analyze the host genes’ function in chicken primary monocyte-derived macrophages (MDM). Compared to the uninfected control, 624 up-regulated differentially expressed genes (DEG) and 341 down-regulated DEG at 3 hpi, and 174 up-regulated DEG and 87 down-regulated DEG at 36 hpi were identified in chicken MDM, respectively. ALV-J infection induced strong innate immune responses in chicken MDM at 3 hpi, instead of 36 hpi, according to the analysis results of Gene Ontology and KEGG pathway. Importantly, the host factors, such as up-regulated MIP-3α, IL-1β, iNOS, K60, IRG1, CH25H, NFKBIZ, lysozyme and OASL were involved in the host defense response during the course of ALV-J infection. On the contrary, up-regulated EX-FABP, IL4I1, COX-2, NFKBIA, TNFAIP3 and the Jak STAT pathway inhibitors including CISH, SOCS1 and SOCS3 are beneficial to ALV-J survival in chicken macrophages. We speculated that ALV-J tropism for macrophages helps to establish a latent infection in chicken MDM from 6 to 36 hpi. The present study provides a comprehensive view of the interactions between macrophages and ALV-J. It suggests the mechanisms of defense of chicken macrophages against ALV-J invasion and how ALV-J escape the host innate immune responses.

Research on Piwi proteins and piRNAs in insects has focused on three experimental models: oogenesis and spermatogenesis in Drosophila melanogaster, the antiviral response in Aedes mosquitoes and the molecular analysis of primary and secondary piRNA biogenesis in Bombyx mori-derived BmN4 cells. Significant unique and complementary information has been acquired and has led to a greater appreciation of the complexity of piRNA biogenesis and Piwi protein function. Studies performed in other insect species are emerging and promise to add to the current state of the art on the roles of piRNAs and Piwi proteins. Although the primary role of the piRNA pathway is genome defense against transposons, particularly in the germline, recent findings also indicate an expansion of its functions. In this review, an extensive overview is presented of the knowledge of the piRNA pathway that so far has accumulated in insects. Following a presentation of the three major models, data from other insects were also discussed. Finally, the mechanisms for the expansion of the function of the piRNA pathway from transposon control to gene regulation were considered.

Subgroup A, B, and J ALVs are the most prevalent avian leukosis virus (ALV). Our study attempted to develop two SYBR Green I-based real-time PCR (RT-PCR) assays for specific detection of ALV subgroup J (ALV-J) and multiplex detection of ALV subgroups A and B (ALV-A/B), respectively. The two assays showed high specificity for ALV-J and ALV-A/B and the sensitivity of the two assays was at least 100 times higher than that of the routine PCR assay. The minimum virus detection limit of virus culture, routine PCR and real-time PCR for detection of ALV-A strain was 103 TCID50 units, 102 TCID50 units and fewer than 10 TCID50 units, respectively. In addition, the coefficients of variation for intra- and inter-assay were both less than 5%. Forty clinical plasma samples were evaluated by real-time PCR, routine PCR, and virus culture with positive rates of 80% (32/40), 72.5% (29/40) and 62.5% (25/40), respectively. When the assay for detection of ALV-J was used to quantify the viral load of various organ tissues in chicken inoculated by ALV-J strains CHN06 and NX0101, the results exhibited that ALV-J genes could be detected in all organ tissues examined and the highest copies of ALV-J were mainly in heart and kidney samples at 30 weeks post-infection. Except in lung, the virus copies of CHN06 group were higher than that of NX0101 group in various organ tissues. The SYBR Green I-based real-time RT-PCR assay provides a powerful tool for the detection of ALV and study of virus replication and infection.

Within the hemolymph, insect hemocytes constitute a heterogeneous population of macrophage-like cells that play important roles in innate immunity, homeostasis and development. Classification of hemocytes in different subtypes by size, morphology and biochemical or immunological markers has been difficult and only in Drosophila extensive genetic analysis allowed the construction of a coherent picture of hemocyte differentiation from pro-hemocytes to granulocytes, crystal cells and plasmatocytes. However, the advent of high-throughput single cell technologies, such as single cell RNA sequencing (scRNA-seq), is bound to have a high impact on the study of hemocytes subtypes and their phenotypes in other insects for which a sophisticated genetic toolbox is not available. Instead of averaging gene expression across all cells as occurs in bulk-RNA-seq, scRNA-seq allows high-throughput and specific visualization of the differentiation status of individual cells. With scRNA-seq, interesting cell types can be identified in heterogeneous populations and direct analysis of rare cell types is possible. Next to its ability to profile the transcriptomes of individual cells in tissue samples, scRNA-seq can be used to propose marker genes that are characteristic of different hemocyte subtypes and predict their functions. In this perspective, the identities of the different marker genes that were identified by scRNA-seq analysis to define 13 distinct cell clusters of hemocytes in larvae of the silkworm, Bombyx mori, are discussed in detail. The analysis confirms the broad division of hemocytes in granulocytes, plasmatocytes, oenocytoids and perhaps spherulocytes but also reveals considerable complexity at the molecular level and highly specialized functions. In addition, predicted hemocyte marker genes in Bombyx generally show only limited convergence with the genes that are considered characteristic for hemocyte subtypes in Drosophila.

The avian leukosis virus subgroup J (ALV-J) belongs to the chicken retrovirus that causes enormous economic losses in the poultry industry. Interferon-stimulated genes (ISGs) are critical for controlling virus infections. Here, we identified 897 type I ISGs induced by interferon-α (IFN-α) in chicken peripheral blood mononuclear cell (PBMC) by RNA-Seq. In addition, we further identified 152 potential anti-ALV-J chicken type I ISGs. Among these potential anti-ALV-J ISGs, chicken cholesterol 25-hydroxylase (chCH25H) was selected for further antiviral mechanism studies in chicken embryo fibroblast cell lines (DF1). The gene chCH25H is located on chromosome 6 and clustered in a distinct group with mammals CH25H in the phylogenetic tree. The core promoter region of chCH25H was located within −75/−1 sequence. We found that chCH25H was induced by chicken IFN-α and ALV-J in DF1 cells. The overexpression of chCH25H significantly inhibited ALV-J replication in DF1 cells at 48 h post infection (hpi). In addition, ALV-J replication was significantly enhanced in the chCH25H- knockout DF1 cells. Furthermore, we demonstrated that chCH25H restricted ALV-J infection through the production of 25-hydroxycholesterol (25HC), rather than type I and II interferon. Our results identified 152 potential anti-ALV-J chicken type I ISGs and revealed that 25HC, the product of chCH25H, could be used as a natural antiviral agent to control ALV-J infection.

Interferon-stimulated genes (ISGs) play an important role in antiviral innate immune responses. Although many ISGs have been identified in mammals, researchers commonly recognize that many more ISGs are yet to be discovered. Current information is still very limited particularly for the systematic identification of type III ISGs. Similarly, current research on ISGs in birds is still in its infancy. The aim of this study was to systematically identify chicken type I (IFN-α), II (IFN-γ) and III (IFN-λ) ISGs and analyze their respective response elements. RNA sequencing (RNA-Seq) was employed to identify those genes with up-regulated expression following chicken IFN-α, IFN-γ and IFN-λ treatment. Two hundred and five type I ISGs, 299 type II ISGs, and 421 type III ISGs were identified in the chicken. We further searched for IFN-stimulated response elements (ISRE) and gamma-activated sequences (GAS) elements in the promoters region of ISGs. The GAS elements were common in the promoter of type II ISGs and were even detected in type I and III ISGs. However, ISRE were not commonly found in the promoters of chicken ISGs. Furthermore, we demonstrated that ISRE in chicken cells were significantly activated by IFN-α or IFN-λ treatment, and expectedly, that GAS elements were also significantly activated by IFN-γ treatment. Interestingly, we also found that GAS elements were significantly activated by IFN-λ. Our study provides a systematic library of ISGs in the chicken together with preliminary information about the transcriptional regulation of the identified ISGs.