The Synergistic Mechanism Of Co-infection Of ALV-J And REV Via Exosome

In recent years, the synergistic infection of avian leukosis virus subgroup J(ALV-J) and reticuloendotheliosis virus(REV) is widespread, causing serious losses for poultry industry.However, little is known about the synergistic mechanism of ALV-J and REV.Exosomes are small membranous vesicles secreted by a number of cell types. It involved in many physiological and pathological processes by adjusting the specific secretion of proteins, lipids, nucleic acids. Exosome provides a new idea for us to study the synergistic mechanism of co-infection of ALV-J and REV.This study proves ALV-J and REV synergistic infection based on the analysis co-infection through ALV-J and REV cell secretion of the exosome in proteomics and transcriptomics, revealing the cellular pathways of ALV-J and REV in common use, so as to clarify the infection mechanism of ALV-J conjunction with REV, provide theoretical basis for joint prevention and control and mechanism of other virus co-infection.ALV-J-NX0101 and REV-SNV strains of virus were inoculated into CEF isolated from a SPF chicken embryos. CEF cells were divided into 4 experimental groups: ALV-J+REV co-infected group, ALV-J infected group, REV infected group, and virus-free control group.By PCR, IFA and avian leukemia Antigen(p27) detection, ensure the CEF cells infected with ALV-J and/or REV. RT-PCR proved ALV-J and REV collaborative infection from viral transcription level, results displayed the co-infection group both ALV-J and REV RNA transcription level increased than single infection group before 72 h. By confocal laser scanning microscopy(CLSM) observation, REV fluorescent in ALV-J+REV group was present at 48 h, earlier than REV single infection group 24 h. ALV-J fluorescent in ALV-J+REV group was brighter than ALV-J single infection group.For research the synergistic immunosuppressive role for the exosome that secreted by co-infected CEF by ALV-J and REV, we isolated spleen cells from 42-day-old white SPF chicken, after adding the appropriate dose of exosomes co-culture time, by flow cytometry detection CD4+, CD8+ T cells, by ELISA detection of the secretion of IL-2, IL-10, IL-12.Results showed that the number of CD4+ and CD8+ T cells of group Exo-RJ were lower than the control group before 12 hours. Secretion of IL-2,Exo-RJ was higher than the control group in 4h, and there was significant difference with Exo-R; Secretion of IL-10, Exo-RJ was significantly lower than the control group in 8h; Secretion of IL-12, Exo-RJ was significantly lower than the control group in 0.5h. It indicated that exosomes from ALV-J+REV co-infection group immune suppression enhanced.To reveal the synergistic pathways during co-infection of REV and ALV-J, we use i TRAQ analysis in proteomics, and mi RNA whole-genome sequencing to screening synergistic pathways. Results showed that we got about 1154 species of proteins, 932 known,785 unknown mi RNA from exosomes. The four group of exosomes are all have symbols of exosome proteins, such as four transmembrane proteins, Hsp70, Hsp90, 14-3-3, and so on. We found ALV-J gag and env proteins, REV gag, pol and env proteins in exosomes, indicating that the exosome secreted from virus infected CEF cells carried the structrual protein of virus.By means of analyze the GO and KEGG pathway of target proteins of differential expression protein and differential expression mi RNA, found different express protein NCKAP1 and the target protein of differences mi RNA-ITGA1 at the same signaling pathway: regulation of actin cytoskeleton, result in ARP2/3 protein down-regulation, inhibite actin polymerization and lamellipodia formation, found ALV-J and REV launched the regulation of actin cytoskeleton pathway, while ALV-J or REV infection alone did not start this path. Actin cytoskeleton, is a network of fibrous actin in cells, its wide variety of biological functions, it has to maintain cell shape, cell adhesion and cell movement and other functions. Experiments show that this pathway abnormalities can cause the occurrence of many diseases, including cancer, and there are many cancer prevention, therapy is realized through the change of actin cytoskeletal structure.The discovery of regulation of actin cytoskeleton pathway involed in co-infection of ALV-J and REV not only for the study of synergistic mechanism of ALV-J+REV is significant,but also provides a theoretical basis for avian tumors or other poultry diseases, and a new idea for the prevention and treatment of avian tumors. However, the validation of this path also needs further experiments.