Study On The Association Between Lumpy Skin Disease Virus Infection And Endoplasmic Reticulum Stress

Poxviruses have been closely associated with human and animal health for centuries.Since 2019,lumpy skin disease virus(LSDV),a member of the Capripoxvirus genus(Ca PVs),has been widely prevalent in more than 10 provinces and autonomous regions in China.And an outbreak of monkey pox(Mpox)in the UK in 2022.These events raised concerns about poxviruses.LSDV primarily infects cattle,causing distinct nodules on the skin along with symptoms such as fever,swollen lymph nodes,bull infertility,and reduced milk production in dairy cows.These symptoms have significant economic implications.Poxviruses replicate in the cytoplasm,requiring extensive protein synthesis and modification,which may interfere with the endoplasmic reticulum(ER)homeostasis.Although existing evidence suggests that Ca PVs infection can lead to structural abnormalities in the ER,which may be related to the disruption of ER homeostasis and induced ER stress,there is currently no evidence to support this.This paper intends to explore the correlation between LSDV infection and ER stress,aiming to reveal the replication and pathogenic mechanism of LSDV infection.To investigate the correlation between ER stress and LSDV infection,we initially selected cow calves at around 6 months old to establish an in vivo LSDV infection model.The successful establishment of the model was confirmed through the observation of characteristic skin nodules,histopathological diagnosis,and enzyme-linked immunosorbent assay(ELISA).Subsequently,we conducted indirect immunofluorescence assay(IFA),immunohistochemistry(IHC),and western-blot assay to demonstrate that LSDV infection induces the ER stress phenotype in the skin tissue of cows.Furthermore,we conducted in vitro experiments involving time gradient infection with LSDV,employing western-blot assay and fluorescence quantitative PCR methods.These experiments provided additional evidence that LSDV infection also leads to ER stress in vitro.Both in vivo and in vitro results showed that LSDV infection was closely related to ER stress.Moreover,our investigation into the activation of unfolded protein response(UPR)elements in relation to LSDV infection revealed that both in vivo and in vitro infection activate all three UPR signaling pathways,namely protein kinase RNA-like ER kinase(PERK),inositol-requiring enzyme 1(IRE1),and activating transcription factor 6(ATF6)signaling.To explore the relationship between the activation of UPR signaling pathways and LSDV replication,si RNAs with high knockdown efficiency were used to target the UPR signaling pathways for knockdown.The results showed that knockdown of PERK and IRE1 signals led to a reduction in the expression of LSDV viral protein,as well as a decrease in virus titer and copy number.However,knockdown of ATF6 had no effect on these outcomes.To validate the findings of the si RNA knockdown experiment,compound inhibitors were employed to inhibit the UPR signaling pathways.The inhibitors PERKi,4μ8C,and Ceapin-A7 effectively inhibited the PERK,IRE1,and ATF6 signaling pathways,respectively,thus confirming the results obtained from the si RNA experiments.Overall,these results suggest that LSDV replication relies on the activation of the PERK-e IF2α and IRE1-XBP1 rather than the ATF6 signaling cascade.This implies that inhibiting the protein global translation pathway and the splicing pathway of X-box binding protein 1(XBP1)may impede LSDV replication.In summary,LSDV infection in vivo and in vitro collectively characterized the occurrence of ER stress and activated all three UPR signaling pathways.Although UPR signaling activation is beneficial for cells to restore the unbalanced environment,activation of PERK and IRE1 signaling pathways also facilitates the replication of LSDV in vitro.Together,these findings characterize for the first time the association between LSDV and ER stress,suggesting a potential therapeutic approach to target UPR elements in response to LSDV and even other poxvirus invasion and providing scientific basis for the research of blocking technology of other poxvirus infection including Mpox.