Transcriptome Analysis Of Susceptible And Resistant Mice And The Mechanism Of Essential Factors During ECTV Infection

Poxviruses,a kind of DNA virus with large genome size,are among the most complex viruses known.They infect a wide range of vertebrate and invertebrate hosts and some of the infections cause death in human and animal populations.Although the eradication of smallpox laid solid foundation for disease control and the development of immunology,it remains a threat to the human population because of the possibility of using as a bioweapon by terrorist groups.Moreover,the increased frequency of zoonotic poxvirus infections emergeing as sporadic cases,including monkeypox virus(MPXV),cowpox(CPXV)and vaccinia virus(VACV),has increased the attention in recent years.Closely related orthopoxviruses such as ectromelia virus(ECTV)are the ideal surrogate for the study of orthopoxvirus in small animal models,as it also has a restricted host range and,in mice,the resulting disease shares common features with variola virus(VARV).ECTV is a mouse-specific virus and causes high mortality in susceptible mice strains,including BALB/c and C3 H,whereas C57BL/6 and 129 strains are resistant to the disease.To understand the host genetic factors in different mouse strains during the ECTV infection,we carried out a microarray analysis of spleen tissues derived from BALB/c and C57BL/6 mice before or after ECTV infection at 3 and 10 days,respectively.Differential expression of genes(DEGs),molecular function and KEGG pathway analysis revealed that the host gene expression profiles in two mouse strains were very different before infection.However,after infection,the susceptible BALB/c mice generated more DEGs than the resistant C57BL/6 mice and the DEGs of susceptible mice were involved in innate immunity,apoptosis,metabolism,and cancer-related pathways,while the DEGs of resistant mice were largely involved in MAPK signaling and leukocyte transendothelial migration.Furthermore,the BALB/c mice showed a strong induction of immune related molecules.Considering the different viral loads in two mouse strains,the genetic factors,the viral laods in spleens and the immune status of different mice might be the importance of the induced DEGs.In this study,based on the data obtained from the microarray and qRT-PCR analysis,it was found that one of HSP70 isoforms,Hspa1 b,is different expressed in two mouse strains during ECTV infection.To investigate the effect of Hspa1 b on ECTV replication,studies with overexpression of Hspa1 b protein and siRNA-mediated gene silencing of Hspa1 b revealed that Hspa1 b is required for efficient replication of ECTV.Furthermore,we show that ECTV replication can be significantly suppressed by two chemical Hspa1 b inhibitors with different concentrations,quercetin and VER155008,which further confirm the importance of Hspa1 b in ECTV replication.The viral genome copy numbers were examined during overexpression or suppression of Hspa1 b,which suggests that cellular Hspa1 b promotes ECTV DNA replication.However,the role of Hspa1 b in other aspects of viral life cycle need to be further explored.In view of the importance of the cGAS in the immune response and data from microarray analysis,we have verified the induction abilities of type I IFNs production during ECTV infection.The induction of the IFN-? promoter in HEK293 T cells and IFN-? expression showed that both cGAS and STING can trigger the IFN-? response during ECTV infection in L929 cells,but this was not in NIH3T3 cells.Consistently,the ECTV infection triggered TBK1 and IRF3 phosphorylation were found in RAW264.7 and L929 cells,whereas it failed to do so in NIH3T3 cells.Disruption of cGAS or STING expression in mouse macrophages blocked the induction of type I IFN production and facilitated ECTV replication.Additionally,mice deficient in TLR9 or STING exhibited lower type I IFN levels and higher viral loads,and are more susceptible to mousepox caused by an infection with low dose of ECTV,whereas similar results showed in cGAS deficiency mice only at a high dose infection.Collectively,our study indicates that the cGAS-STING pathway is critical for the detection of ECTV infection and controlling ECTV replication.In conclusion,we carried out a microarray analysis of spleen tissues derived from BALB/c and C57BL/6 mice,which showed host genetic background is a critical factor which governs resistance to mousepox.After infection,a strong induction of immune response was showed in susceptible BALB/c mice,however,which was weaker in the C57BL/6 mice.Based on the microarray analysis,we also confirmed the importance of Hspa1 b in ECTV replication and the cGAS-STING pathway in controlling ECTV replication through type I IFN production.Collectively,these results will be crucial for further uncovering the molecular mechanisms of the host-poxvirus interaction.