Effects And Mechanism Of Tembusu Virus Infection On Biological Function Of Duck Embryo Fibroblasts

Tembusu virus（TMUV）infection is an emerging avian infectious viral disease in the region of Jiangsu and Zhejiang in China since 2010,the disease mainly caused body temperature rise,loss of appetite,follicular degeneration and hemorrhage,yolk peritonitis,egg-drop significantly for laying ducks,and head and neck tremor,ataxia and tetraplegia for duckings.Besides,the infectious disease also has the characteristics of wide spread,fast spread and high infection rate and so on.To date,the virus has been circulating in duck farms in the vast aquaculture areas of China,which seriously hinders the healthy development of aquaculture in China.Deep dives the changes of host cell biology function induced by virus infection and the relevant regulatory mechanism of signaling network,it will not only help to understand the intracellular and extracellular signal transmission of viruses,regulatory process after transcription,relationship and interaction between virus and other signaling molecules,it can also have a comprehensive understanding of the host cell metabolism and the occurrence and development of related diseases.However,at present,there are few studies focus on the mechanism of TMUV’s interaction with its host cells,especially on the biological function of host cells induced by the virus.To solve the above problems,this study take duck embryo fibroblast（DEF）as the research object,mosquito source TMUV（TMUV-SDMS）strain isolated from an epidemiological investigation was used as the representative of domestic dominant strains of the virus.The effect of TMUV infection on DEF cell biological function was investigated at the cellular and molecular levels by using molecular biology techniques such as transcriptome sequencing（RNA-Seq）,qRT-PCR and siRNA.Besides,we also screen and validate the key genes or proteins that the virus infects and interacts with host cells with the above molecular biology techniques.In this study,the lethal mechanism of DEF apoptosis and necrosis induced by TMUV-SDMS is preliminarily discussed,which provides data support for elucidation of the host defense mechanism of DEF escape and search for new antiviral targets.The research is mainly divided into the following five aspects:1.Screening of TMUV domestic dominant representative strainsIn this study,on the basis of 11 Tembusu virus isolates isolated from the virus-infected ducks,chickens,geese,sparrows,and mosquitoes in the epidemiological investigation,phylogenetic analysis,predictions of protein tertiary structures and glycosylation sites of 67representative strains of Tembusu virus in National Center of Biotechnology Information（NCBI）database were analyzed.The result showed that the TMUV infection has been circulating in 17 provinces of China,and the outbreak of infectious disease has the seasonal characteristics of the high incidence in autumn.According to the nucleotide and amino acid homology and phylogenetic analysis of the above 78 representative TMUV strains,the mean nucleotide and amino acid homologies of NS1 were lower than those of E,NS3,and NS5,and these isolates mainly clustered into five major groups:Malaysia strains I,Malaysia strains II,Thailand strains,Chinese strains I,and Chinese strains II.The analysis of protein tertiary structure and glycosylation sites of the 78 representative TMUV strains indicated that E protein had¹⁴⁸ASTV¹5151 mutation that changed a random crimp into an alpha helix,beta folding fragment extended variation at amino acid site of 380-381 and 393-397,and a mutated glycosylation site mutation was also found at amino acid site 154 from NYSV/NYSA to NYPA.Furthermore,these strains had a ¹⁸⁰TAV¹8282 mutation that changed a random crimp into an alpha helix,and a mutated glycosylation site mutation was also found at amino acid site 175from NTTD to NITD.Here,we can see that current Chinese TMUV strains are mainly divided into two distinct genotypes（termed as Chinese strains I and Chinese strains II）.Mosquito TMUV（TMUV-SDMS）can be used as a representative of the domestic dominant TMUV strains.2.TMUV-SDMS infection in DEF and RNA-Seq analysis of the virus infection samplesIn the present study,we used C6/36 cells to proliferate and culture TMUV-SDMS.Subsequently,the above proliferation-cultured TMUV-SDMS was inoculated into the DEF with a growth density of 70%⁸0%.DEF cells infected with the virus and DEF cells not infected with the virus were set as experimental group and control group respectively.Total RNA of above groups were extracted at 12 hpi and 24 hpi,and its quality were checked using an Agilent 2100 Bioanalyzer（A260/A280>1.5,A260/A230>1.0 and RIN>7）.Illumina HiSeq 2000^TMM platform was used for transcriptome sequencing（RNA-Seq）of the above samples,and RPKM（Reads Per Kilobase of exon model per million mapped sequence reads,RPKM）method was used to calculate Unigene,and the Unique were screened according to the expression fold changes and the significance of expression changes（Fold change>2 and P<0.05）.Finally,the differentially expressed genes were screened for GO functional classification and KEGG signal pathway enrichment analysis by hypergeometric calculation in the context of the whole genome.The results showed that the quality rates of clean data,Q20 and Q30 were all above94.0%,indicating that the transcriptome sequencing data obtained in this study were of high quality.Here,911（764 upregulated and 147 downregulated genes）and 3008（1791upregulated and 1217 downregulated）differentially expressed genes（DEGs）were identified at 12 hpi and 24 hpi,respectively.Gene ontology（GO）functional classification and Kyoto Encyclopedia of Genes and Genomes（KEGG）enrichment analysis revealed that DEGs were considerably enriched in immune system,cell growth and death,signal transduction and signaling molecules and interaction,and so on.The above results indicate that TMUV-SDMS can induce the biological function changes in the immune system,cell growth and necrosis,signal transduction,signaling molecules and interaction.3.The effect of TMUV-SDMS infection on the immune system in DEFIn this study,according to the information provided by RNA-Seq,we can concluded that the immune-related differentially expressed genes were mainly enriched to Toll-like receptor signaling pathway,Cytosolic DNA-sensing pathway,RIG-I-like receptor signaling pathway,Chemokine signaling pathway,NOD-like receptor signaling pathway,and Hematopoietic cell lineage,and so on.According to the further classification and summary of the differentially expressed genes in the above signaling pathways.Upon DTMUV infection,key PRRs（RIG-I,TMEM173 and MDA5）in the immune system were activated,inducing increased production of cytokines（IFN-a,IL-6,IL-8L,IL-12B,CCR7,CCL19,and CCL20）,transcription factors or signaling molecules（IRF7,NF-kB,STAT1,TMEM173/STING and TNFAIP3）,and antigen-presenting proteins（CD44 and CD70）.These findings broaden our understanding of the innate immune responses of the host cell to TMUV infection.4.Effect of TMUV-SDMS infection on apoptosis and necrosis in DEFCombined with the information provided by RNA-Seq,this study qualitatively and quantitatively detected the effect of TMUV-SDMS infection on DEF cell growth and necrosis by laser scanning confocal microscope,flow cytometry and JC-1 probe.The results showed that the virus infection could not only significantly reduce the cell activity of DEF at 12 hpi and 24 hpi（P<0.01）,but also could induce significantly apoptosis and necrosis,S+G2M phase arrest and decrease of mitochondrial membrane potential of DEF（P<0.05 or P<0.01）.According to the summary analysis of the differentially expressed genes related to apoptosis and necrosis in this study,these genes are mainly located in the endogenous mitochondrial pathway and the exogenous death receptor pathway.The above results indicated that TMUV-SDMS infection can induce S+G2/M arrest in DEF,which may be caused by CDC20B overexpression.And the virus infection induces DEF apoptosis and necrosis mainly through endogenous mitochondrial pathway and exogenous death receptor pathway.5.Screening and validation of key genes for cell growth and necrosisIn order to further identify the key genes of TMUV-SDMS interacting with DEF and clarify its biological function in regulating the growth and necrosis of DEF cells induced by the virus infection.This study based on the information provided by the RNA-Seq,choose B lymphocyte tumor 2 A1 gene（BCL2A1）as objective gene for its the highest expression in the apoptosis and necrosis signal pathway,siRNA and other molecular biological techniques were used to explore its regulatory role in cell cycle regulation,apoptosis and necrosis and other cell growth and necrosis processes.The results showed that BCL2A1 could regulate S phase,apoptosis and necrosis of host cells.In addition,the target gene also has a close relationship with BCL2,MCL1 and CDC20B（i.e.,it has a synergistic effect with CDC20B and MCL1,and an antagonistic relationship with BCL2）,but the specific mechanism needs to be further explored in the future.In this study,the effect of TMUV infection on DEF cell biological function was studied at the molecular level,and the specific mechanism of DEF apoptosis and necrosis induced by this virus is revealed in depth.The results are of great significance for enriching the transcription spectrum of TMUV infected hosts,elucidation of the virus escape host defense mechanism,and exploration of targeted mitochondrial antiviral drug development.