Investigation Of The Interaction Between Infectious Laryngotracheitis Virus And Host Cellular Metabolism Using Transcriptomics And Metabolomics Approaches

Infectious laryngotracheitis virus(ILTV)is an Iltovirus within the Herpesviridae family that causes severe respiratory tract disease in chickens throughout the world and causes heavy economic losses in the poultry industry worldwide every year.LTV can evade the host's immune surveillance through latent infection,and then repeatedly trigger the epidemic when the host's immunity is low or suppressed.Studying of the endogenous mechanism for host cell resistance to ILTV infection is expected to establish a prevention and control method that does not depend on the immune response of the host and complement existing prevention and control measures.ILTV has different sensitivity to host cells.This research used ILTV-susceptible LMH cells and ILTV-insensitive DF1 cells as a comparative model.Detection of viral envelope and host cell membrane fusion and transmission electron microscope,confirms that ILTV can enter DF1 cells.However,different from LMH cells,only the immediately early gene ICP4 and early gene ICP27 of the virus could be expressed in DF1 cells by RT-qPCR assay.The transcription of ICP4 and ICP27 genes could not be maintained after 3 hours in DF1 cells.In addition,ILTV cannot proliferate and produce CPE in DF1 cells,but it can transiently inhibit the proliferation of DF1 cells.In order to explore the host regulatory mechanisms of ILTV gene expression,we examined the cellular transcriptional profile of ILTV infected LMH and DF1 cells at 3 hours.Analysis of the KEGG pathway revealed that ILTV infection extensively inhibits metabolic-related signaling pathways in DF1 cells,including central carbon metabolism,pyruvate metabolism,glycolysis,tricarboxylic acid cycle,amino acid synthesis and propionic acid metabolism,etc,however,there is no significant change in LMH cells.Based on the results of transcriptomic analysis,this study regulated the glucose metabolism,glutamine metabolism,and fatty acid synthesis metabolism in LMH cells,respectively.Inhibition of glucose and glutamine metabolic pathways can both significantly inhibit the transcription of the very early ILTV gene ICP4 and ILTV genome replication and the maturation of progeny virions.Inhibition of fatty acid synthesis has no effect on the transcription of the very early ILTV gene ICP4 and ILTV genome replication,but it can inhibit the assembly and release of progeny virions.Further transcription factor analysis combined with protein interaction analysis predicts that the host transcription factors Jun and Fos may play an important role in the regulation of gene expression in ILTV infecting cell lines.Given that Jun and Fos are highly expressed in LMH cells and hardly expressed in DF1 cells.,we specifically knocked down the Jun or Fos genes in LMH cells using small interfering RNA and found that inhibiting Fos expression can significantly inhibit ILTV the transcription of the very early ILTV gene ICP4 and ILTV genome replication and the maturation of progeny virions.However,neither overexpression of Jun or Fos in DF1 cells had no significant effect on ILTV transcription and replication.In order to further detect changes of specific metabolites,LC-MS was used to explore the metabolomes of LMH and DF1 cells at 3 h and 24 h of infection with ILTV.Differences between LMH and DF1.cells in the infection group and the control.group were compared.Pathway analysis of differential metabolites found that ILTV affects host cell metabolism in diametrically opposite patterns in DF1 cells and LMH cells.ILTV infection results in the extensive depletion of various amino acid and nucleotide metabolic precursors in LMH,but it accumulates in DF1 cells.In addition,ILTV infection of LMH and DF1 cells,Changes were induced in many metabolic pathways such as amino acid synthesis,aminoacyl-tRNA synthesis,phospholipid metabolism,nucleotide metabolism,and fatty acid metabolism in host cells.The most prominent changes are the metabolism of glutamine pathway,phenylalanine and tyrosine and tryptophan biosynthetic pathway,glutathione(GSH)metabolic pathway,alanine and glutamate metabolism.In this research,the interaction between ILTV infection and the metabolic response of host cells was initially explored using a combination of transcriptome and metabolomics methods,and some of the molecular mechanisms in the process were initially revealed,broaden understanding of the current interaction between ILTV and host cells.