Characterization Of Transcriptional Profiling And Determination Of Polarization Direction Of Host Monocytes/Macrophages Response To Tembusu Virus Infection

Avian Tembusu virus(TMUV)is a newly emerging avian pathogenic flavivirus,belonging to the flavivirus genus in the Flaviviridae family,which spreads rapidly,has an expanding host range and undergoes cross-species transmission.TMUV mainly infects poultry and is the only flavivirus found to cause disease in ducks.After infection,TMUV mainly invades the reproductive system and causes a sharp decline in egg production in affected female ducks.At present,TMUV is one of the important pathogens threatening the duck industry in China.Avian macrophages have been shown to serve as the main target for some avian viruses,such as Marek's disease virus,Newcastle disease virus,Infectious bronchitis virus,and so on.Our previous study proved monocytes/macrophages as the main targets of TMUV infection experimentally and further demonstrated that the infection of monocytes/macrophages is essential for the viral replication,transmission,and pathogenesis of TMUV.However,the mechanism of TMUV infection of host macrophages is still not comprehensive.To explore the effect of TMUV genome replication on macrophages,in this study we use Poly(I:C)and UV-inactivated viruses to mimic the viral genome and non-infectious virus particle proteins respectively.The chicken macrophage line HD11 was stimulated in vitro,and the differences in gene expression profiles of the host cells were compared with those of live virus replication by transcriptome sequencing.The KEGG and GO enrichment analysis of differentially expressed genes showed that live TMUV virus replication at the transcriptional level mainly initiated innate immune signaling pathways,whether at 12 hpi or 24 hpi,and inhibited phagocytosis,lysosomal and other related pathways at 24 hpi.Among them,the Poly(I:C)stimulation group did not initiate innate immune-related pathways,which were only partially intersected with pathways initiated by live virus replication.However,24 hpi did not inhibit phagocytosis,lysosomal and other related pathways.Viral particles with replication defects initiate completely different pathways from the previous two groups.Firstly,ribosome and oxidative phosphorylation pathways are initiated at 12 hpi,while signaling pathways such as steroid synthesis and insulin are triggered at 24 hpi,which can inhibit inflammatory pathways.These results suggest that the innate immune-related pathways at the transcriptional level are mainly initiated by viral replication.The polarization of host macrophages determines the functional phenotypes of macrophages;however,the effect of TMUV infection on macrophage polarization remains unclear.Here,we analysed the expression spectra of the marker genes of macrophage polarization upon TMUV infection in the HD11 chicken macrophage cell line and primary monocytes/macrophages isolated from the peripheral blood of specific pathogen-free(SPF)chickens and ducks.We found that viral replication mainly induced M1 marker genes and triggered nitric oxide(NO)release at different levels,suggesting that TMUV infection led mainly to host macrophages polarizing into the classically activated(M1)type.However,the NO did not inhibit TMUV genome replication in macrophage line HD11 in vitro.Furthermore,upon TMUV infection,polarized HD11 cells exhibited increased migration but reduced phagocytosis,as evidenced by scratch assay and neutral red uptake assay,respectively.Our present study characterized the polarization of host monocytes/macrophages upon TMUV infection,which may lay a foundation for further research on the immune escape mechanism and pathogenic mechanism of TMUV.