Transcriptome Analysis Of The Innate Immune Factors In SPF Chicken Thymus Interacting With Recombined ALV

Avian leucosis(AL)is a kind of tumorigenic infectious disease which is caused by avian leukosis virus(ALV).It has been distributed worldwide.Currently,five subgroups of exogenous ALVs(A,B,C,D,and J)and five subgroups of endogenous ALVs,named ALV E-I,have been found.ALV-A,-B,-C,-D,-E and ALV-J can be separated from the chickens.In recent years,some researchers deemed that existence of another subgroup of ALV(ALV-K)in chickens.Summarizing previous research findings,subgroups including ALV-A,ALV-B and ALV-J are most widely popular in chickens.In 2017,a recombinant ALV strain was isolated and identified from "Hetian" chicken,named ALV-FJ15HT0.This strain included ALV-B specificity gp85 gene sequences,ALV-E gp37 and ALV-J LTR sequences.In order to study the molecular character and pathogenicity of this strain,in this study,we set up an animal infection model by simulating vertical transmission.Herein we analyzed the expression difference of genes in different groups by transcriptome sequencing and then the identification at protein level was involved in order to make further explore the genes involved in the interaction between recombinant ALV and host cells.We hope this study can provide scientific data for further research on the pathogenic mechanisms of recombinant ALV.1.Changes of body weight and immune organ index in infected chickensAt 2d,5d,9d,13d,17d,and 21d,the body weight of the blank control group and the infection group were checked.Following the collection of blood,liver,spleen,bursa of Fabricius and thymus,the the immune organ index was then calculated.The quantitative detection of ALV gp85 gene in these tissues was carried out by qRT-PCR.The results showed that chicken embryos in infected group were significantly inhibited after hatching(P<0.01).The bursa organ index was significantly suppressed in the infected group(P<0.05).The spleen,thymus,and liver organ indexes of the infected and control groups showed no significant difference at these times(P>0.05).Correlation analysis showed that the expression of gp85 in infected tissues including bursa of Fabricius(r=-0.546),spleen(r=-0.502)and liver(r=-0.517)was associated with the corresponding body weight difference(P<0.05).The fold change of gp85 in the thymus of the infected group was significantly associated with the difference in bodies weight(r=-0.559,P<0.01).2.Transcription analysis of the response of chicken thymus to recombinant ALVIn this study,ALV-FJ15HT0 was inoculated into the yolk sac of 6-day-old(ED)SPF chick embryos to simulate vertical transmission;meanwhile,a mock control group was set.The thymus of infected and control chickens was aseptically collect for transcriptome sequencing at 5d,13d and 21d.The results showed that a total of 227 differentially expressed genes(DEGs),286 DEGs and 251 DEGs were found at 5d,13d and 21 d,respectively.The DEGs were associated with many important functions,mainly including cell processes,binding,biological regulation,metabolic processing,stimulatory responses,and immune system responses.Found through signal pathway enrichment analysis showed those genes were majorly involved in notable signaling pathway like nicotinate and nicotinamide metabolism,cytokine-cytokine receptor interaction,fatty acid degradation,PPAR signaling pathway,ECM-receptor interaction,cell adhesion molecules(CAMs)and JAK-STAT signaling pathway.We used qRT-PCR to test and verify some DEGs,and the results of qRT-PCR were consistent with transcriptome sequencing results.3.Ciliary neurotrophic factor(CNTF)and its receptor(CNTFRa)participate in the interaction between virus and hostThrough signal pathway analysis,we found an interesting gene,CNTFRa,which up-expressed obviously in infected SPF chickens.We used qRT-PCR for dynamic quantitative detection of CNTFR?.The results showed the significant up-expression of CNTFR? during 13-21d(P<0.01).The serum CNTF titer was detected using ELISA method,and then it was found that the CNTF titer rises significantly in infected chickens(P<0.05).By Pearson correlation analysis,we found the significant positive correlation between the expression level of CNTFRa gene and the corresponding gp85 gene expression level in infected chicken thymus(r=0.656,P<0.01).Further verification found that there was a significant positive correlation between the expression level of CNTFRa gene and the corresponding gp85 gene expression level in infected chicken livers(r=0.525,P<0.05).This suggests that CNTF and CNTFRa may be involved in the interaction between recombinant ALV and host.Chicken CNTFRa protein was obtained by prokaryotic expression technology and after purified the protein concentration is 0.6 mg/mL.The purified protein was immunized subcutaneously in New Zealand white rabbits with 5 times injections to produce antibodies.Western Blot technology was used to detect specific antibody titer in rabbit serum.The results showed that the antibody binding rate is highest and most specifically at 1:2 000.Then we carried out Western Blot to analyze the CNTFRa protein between infected group and mock control group.The results showed that both of two groups CNTFRa protein expression level were decreased with increasing age.During 13-21d,CNTFRa protein expression level in infected chickens thymus were observably higher than mock control group(P<0.05).This result is consistent with the transcript level of CNTFRa.The results further confirmed that CNTFRa participates in the interaction between virus and organism.