Expression Profiles Of LncRNA And MRNA In Chicken Infected With ALV-J Virus

Avian Leukosis is a collective name for neoplastic diseases in poultry caused by the Avian Leukosis virus and Avian Sarocma virus. In poultry production, this disease mainly causes the drop of egg production, the increase of mortality rate and the decrease of meat quality. Meanwhile, it can inhibit seriously the function of immune system, damage the central immune organ and decrease the resistance of immune system, which lead to an increase in the chance of infection with other disease. Recently, ALV-J was revealed as a major epidemic of avian leukemia virus which endanger the healthy development of livestock and poultry breeding industry, bringing huge economic losses in China and even the world. Up to date, there is no effective vaccines and drugs to control avian leukemia. Nowadays, the main methods to control this disease is to purify the population. However, this method has the disadvantages of high cost, long cycle and great influence base on the outside environment. Therefore, it is urgent to explore the mechanism of ALV-J infection and to find a way to control. In this study, the high throughput sequencing platform of HiSeq Illumina 2500 was used to acquire the whole transcriptome of ALV-J infected chicken cells and tissue samples both in vivo and vitro levels. Then we screen out the key differentially expressed mRNAs and lncRNAs during the process of virus infection, aiming to explore the possible mechanism of lncRNAs, and provide a theoretical basis for the search of tumor markers of avian leukemia and resistance breeding in poultry. The main results were as following:1. To explore the changes of biological characteristics of host cells and viral replication during the infection of ALV-J in immune cells and somatic cells originated from the chicken. We infected the HD11 and CEF cell lines with viral titer of 100 TCID50 of ALV-J, and then detected the viral copies in cells, cell viability and apoptosis rate at 1dpi,2dpi,3dpi,4dpi,5dpi and 7dpi. The result showed that the copies of viral in CEF cells was significantly increased in 4dpi（P<0.05）, and the HD11 cells was in 5dpi; the cell vitality were enhanced dramatically after 5dpi （P<0.05）, on the contrary, the HD11 increased before 4dpi; the rate of apoptosis of the two cell lines both were increased with the time and reached the vertex at 3dpi, then decreased.2. Based on the results of the detection of cell biology, we selected cells infected with ALV-J after 1day,4day and 7day, using RNA-seq to carry out the whole transcriptome of the samples. 336 and 269 differentially expressed genes were obtained through the analysis of large-scale transcriptome data, respectively. Functional annotation of these differentially expressed genes after the validation of qRT-PCR, several pathways relating to the immune reaction were found, such as innate immunity （OASL, CCL4）, adaptive immunity （LYZ, CD72）, apoptosis and autophagy （WISP2, COMP）, inflammation （JSC, IL8）, and tumorgenesis （PCNA, GPX3）.3. To excavate the lncRNA information in the transcription data, cufflinks and several other files of bioinformatics softwares were used to compare and predict, and 12103 lncRNA were obtained. Through the analysis of Cis and Trans, we obtained several pathway associated with innate immune and inflammatory （ISG15-protein conjugation innate immune response） in CEF cells; but in HD11 cells, the predicted genes were enriched in angiogenesis, extracellular matrix （negative regulation pathway of autophagy, T cell activation）. Afterward, a number of lncRNAs （for instance:NONGGAT007033.2, TCONS₀0143593, TCONS₀0286311） were found to have a significant co-expression with several immune related genes （such as:CNN2, MMP2, CCL4, CD72, IL-1） by the analysis of co-expression network.4. Further research on the change of ALV-J infection in vivo should be performed to explore the relationship between the virus and host.3 chickens infected with ALV-J and 3 uninfected chickens were selected, then using RNA-seq to acquire the transcriptome of the spleen tissues. 2104 differentially expressed genes and 9036 differentially expressed lncRNAs were obtained by screening. On account of the analysis of Cis and co-expression with mRNA, we obtained 12 key lncRNAs （such as:TCONS₀0257530, TCONS₀0234495, TCONS₀0273421, TCONS₀0267624） and 14 differentially expressed mRNA （such as:NFATC2, IL9R, OSMR, CD34） which have tight regulatory relationship with the key lncRNAs. However, we need do more validations for exploring the interaction mechanism between these lncRNAs and mRNAs.