Innate Immune Response Induced By ALV-J And The Mechanism Of Immune Escape In Chicken Macrophages

Avian leukosis virus subgroup J(ALV-J)infection causes enormous economic losses in the global poultry industry due to inducing tumors and immunosuppression.Natural transmission of ALV-J occurs through two modes including horizontal and vertical transmission.As one of the diseases of avian embryo,ALV-J causes great trouble for poultry breeding.Macrophages are important innate immune cells and play a complicated role in the course of retrovirus infection.Up to now,the interaction between ALV-J and macrophage has not yet been reported.In the present study,we investigated the antiviral innate immune responses induced by ALV-J in chicks and chickens that had developed tumors.Spleen levels ofinterleukin-6(IL-6),IL-10,IL-1?,and interferon-?(IFN-?)were not significantly different between the infected chick groups and the control groups from 1 d post hatch to 7 d post hatch.However,IL-6,IL-1?,and IFN-?protein levels in the three clinical samples with hemangiomas were dramatically increased compared to the healthy samples.In addition,the anti-inflammatory cytokine IL-10 increased sharply in two of three clinical samples.We also found a more than 20-fold up-regulation of ISG12-1 mRNA at 1 d post infection(d.p.i.)and a two fold up-regulation of ZC3HAV1 mRNA at 4 d.p.i.However,there were no statistical differences in ISG12-1 and ZC3HAV1 mRNA expression levels in the tumorigenesis phase.ALV-J infection induced a significant increase of Toll-like receptor 7(TLR-7)at 1 d.p.i.and dramatically increased the mRNA levels of melanoma differentiation-associated gene 5(MDA5)in the tumorigenesis phase.Moreover,the protein levels of interferon regulatory factor 1(IRF-1)and signal transducer and activator of transcription 1(STAT1)were decreased in chickens with tumors.These results suggest that ALV-J was primarily recognized by chicken TLR7 and MDA5 at early and late in vivo infection stages,respectively.ALV-J strain SCAU-HN06 did not induce any significant antiviral innate immune response in 1 w old chicks.However,interferon-stimulated genes were not induced normally during the late phase of ALV-J infection due to a reduction of IRF1 and STAT1 expression.To further clarify the interaction between ALV-J and the host,we mainly discussed the interaction mechanism between ALV-J and chicken macrophages.Chicken primary monocyte-derived macrophages(MDM)were isolated from SPF chicken blood and cultured for 6 d,and identified by microscopy and flow cytometry.Chicken MDM cells were susceptible to ALV-J strain SCAU-HN06.The virus significantly increased the NO production of MDM cells,but had no effect on phagocytic capacity.The course of ALV-J infection in MDM cells is that the active stage is within 3 h of virus infection,latency stage is 6 h to 72 h post infection and ALV-J was reactivated at 96 h post infection.Within 3h of ALV-J infection in MDM cells,innate immune response was strong,JAK-STAT pathway was activated,the expression of STAT1 increased and 729 serine phosphorylation.And then,the STAT1 expression was returned to normal due to the degradation of STAT1 phosphoprotein.Further study showed that the immune escape of ALV-J in MDM cells wasmediated by inhibiting the activity of ISRE and the expression of ISGs with the envelope protein.IFN-?,poly I:C and LPS can't inhibit the replication of ALV-J in MDM cells.In contrast,they have the potential to activate the ALV-J.Interestingly,ALV-J latency can be reactivated by LHRHA3.We found that luteinizing hormone(LH)peak may be associated with ALV-J latency by monitoring the dynamic change of ALV-J viremia and reproductive hormones.Further study about host factors which affect the course of ALV-J infection was screened by high-throughput sequencing.Results showed that after ALV-J infected MDM cells for 3h,the differentially expressed genes were significantly enriched in innate immune pathway and progesterone mediated oocyte maturation pathway.In total,43 down-regulated and 85 up-regulated lncRNA were screened.These differentially expressed lncRNA which cis targeted to the differential expression mRNA significantly were enriched in immune-related biological processes and immune-related pathways.The differential expression mRNA that targeted by lncRNA with ceRNA model significantly were enriched to the pathways including progesterone mediated oocyte maturation pathway,JAK-STAT signaling pathway and so on.Meanwhile,there are 13 up-regulated but only 2 down-regulated mi RNA,and their targets were mainly involved in innate immune response processes,and significantly enriched in progesterone mediated oocyte maturation pathway,JAK-STAT signaling pathway,apoptosis pathway as well.Besides,when ALV-J infected MDM cells for 36 h,the major biological processes that the differentially expressed genes referred included defense response,immunoreaction,antiapoptotic,angiogenesis etc.Only 13 differentially expressed genes were significantly enriched in the cell adhesion molecule pathway and peroxisome proliferator-activated receptor pathway;8 pairs of lncRNA-mRNA cis combinations,and 8 differentially expressed miRNA and 23 differentially expressed genes involved in lncRNA-miRNA-mRNA ceRNA model.In summary,we found that ALV-J induced weak innate immune responses in 1 w old chicks,and may escape innate immune attack with many strategies in chickens that had developed tumors.As an important agent of the interaction between ALV-J and host,the interaction of ALV-J and macrophages including(1)the course of ALV-J in MDM cells is active phase-latency-reactivation;(2)the strong innate immune response induced by ALV-J in MDM cells at the early infection stages,however,the strategy of ALV-J escape innate immune response is that ALV-J inhibit the activity of ISRE and the expression of ISGs by virus envelope protein;(3)the ALV-J latency can be activated by many factors,such as reproductive hormone,and reproductive hormone may be an important factor of ALV-J latency in vivo;(4)lncRNA and miRNA are involved in the course of ALV-J infection in chicken MDM cells.