| Cold stress is a prevalent form of stress that poultry experience and can result in significant economic losses for the poultry industry.Glucocorticoids（GCs）,which are a type of steroid hormone,are known to rapidly respond to both environmental and physiological stimuli and are closely associated with poultry stress.Studies have demonstrated that cold stress can rapidly activate the hypothalamus-pituitary-adrenal axis in chickens,leading to the secretion of excessive amounts of GCs,which serve as a marker of stress.Dexamethasone（Dex）is a type of GC that is produced by animals under stress conditions,which is often widely used as a glucocorticoid analogue in the establishment of stress models in poultry.This study assesses the impact of both cold stress and Dex treatment on gene expression and virus replication in chicken embryonic fibroblasts（DF-1）.It seeks to elucidate the molecular mechanism underlying Dex’s ability to inhibit IBV replication by regulating NHE3 activity.Furthermore,the study investigates the effects of cold stress on plasma steroid hormone levels in chickens,as well as the impact of Dex-induced stress on growth and development,body metabolism,and IBV replication in chickens.Main contents are as follows:1.Effects of cold stress on cell gene expression and virus replicationIn this study,we investigated the impact of various temperatures on DF-1cells,which were incubated at 37℃（control）,32℃,25℃,18℃and4℃for 4 hours.Using RNA-seq technology,gene expression profiling of cells under cold stress was obtained.The effect of cold stress on the replication of infectious bronchitis virus（IBV）,Newcastle disease virus（NDV）and influenza virus（H9N2）was evaluated through Western Blot and q PCR.Our results suggest that cold stress significantly inhibits the functions of cell ribosome and mitochondrial respiratory chain at the cellular level.Furthermore,it strongly inhibits the replication of IBV,NDV and H9N2.2.Effect of cold stress on plasma steroid hormones in chickensIn this study,10-day-old SPF chickens were subjected to cold stress at15℃for 4 h,and 30℃was used as a negative control.Subsequently,42types of steroid hormones in plasma were quantitatively detected by LC-MS/MS.The results showed that cold stress induced significant changes in plasma steroid hormones,including dihydrotestosterone,corticosterone,testosteroneβ-Sitosterol,androstenedione,7a,27-dihydroxycholesterol,and streptosterol are involved in various metabolic pathways,including Gn RH secretion,endocrine resistance,androstenedione metabolism,androgen,and estrogen metabolism.3.Effect of Dex on virus replication and cell transcription levelIn order to comprehensively evaluate the effect of steroid hormones on the replication of avian respiratory viruses IBV,NDV and H9N2,DF-1 cells were pretreated with Dex and CORT for 12 hours,and then infected with IBV,NDV and H9N2.The level of viral protein was detected by Western Blot.The results showed that the replication of IBV was strongly inhibited by CORT and Dex,but did not affect the replication of NDV and H9N2.To investigate the effect of Dex on the transcription level of DF-1 cells,this study conducted RNA-seq on cell samples treated with Dex for 0h,6h,12h,and 24h,respectively.The results showed that the differential genes induced by Dex were significantly enriched in steroid biosynthesis,endoplasmic reticulum protein processing,ribosome,spliceosome and cell cycle signaling pathways.4.Mechanism of Dex activating NHE3 inhibiting IBV replicationTo assess the effect of Dex on the life cycle of IBV at the cellular level,the m RNA of IBV-N gene,negative chain genomic RNA,and viral proteins were detected in the early stage of infection.The results showed that Dex significantly inhibited the levels of IBV-N gene m RNA and viral proteins（N,S,S1,S2,Nsp3 and Nsp15）after 3hpi,and caused a 1-hour delay in the generation of IBV negative chain genomic RNA.These results suggest that Dex mainly affects the vesicular transport of IBV,resulting in delayed production of negative stranded genomic RNA.Furthermore,the patch clamp technique and BCECF-AM probe detection results demonstrated that Dex activated NHE3 to induce an upregulation of intracellular p H,resulting in an antiviral effect.This effect was inhibited by RU486（a GR antagonist）and Tenaparor（a NHE3 selective inhibitor）,suggesting that the antiviral effect of Dex is dependent on GR and NHE3 activity.In this study,a NHE3（SLC9A3）knockout cell line was constructed,and it was discovered that IBV proliferation in NHE3-/-cells was significantly inhibited when compared to that of NHE3-WT cells.Therefore,it has been established that NHE3 is essential in the replication of IBV.Moreover,the effects of NHE3 mutant plasmids NHE3-S663A（simulated dephosphorylation）and NHE3-S663D（simulated phosphorylation）on IBV replication were assessed.The results showed that NHE3-S663D drastically diminished IBV replication,implying that phosphorylation of NHE3-Sr663 is strongly associated with IBV replication.5.Effects of DEX-induced stress on IBV replication in chickensThis study established a poultry stress model by subcutaneous injection of Dex（2.0 mg/kg,once a day）into chickens for 7 days to clarify the effects of stress on chicken weight,immune status,IBV replication,and metabolic characteristics.The results showed that the body weight,peripheral blood lymphocytes and plasma IL-6 of chickens were significantly inhibited by Dex,while the IBV viral load and plasma cholesterol concentration were significantly increased by Dex.LC-MS/MS results showed that Dex caused significant changes in the abundance of 113 metabolites,most of which belonged to lipid and lipid like molecules and were closely related to amino acid and fatty acid metabolism.In addition,before and after IBV infection,Dex treatment can cause significant changes in body metabolism.These differentially enriched metabolites participate in a variety of metabolic pathways,including amino acid biosynthesis,histidine metabolism,cysteine and methionine metabolism,and the synthesis and degradation of leucine and isoleucine.In summary,Dex causes growth inhibition,metabolic disorders,and immune suppression in chickens,which is beneficial for the replication of IBV in the body.In conclusion,we present the first report of the transcriptional profile of poultry DF-1 cells under cold stress,and explore the correlation between cold stress and plasma steroid hormone levels in chickens.Furthermore,our findings have revealed that Dex primarily suppresses IBV replication at the cellular level by regulating NHE3 activity via GR,while at the animal level,it can cause detrimental effects such as growth inhibition,metabolic disorders,and immune suppression in chickens,which could be beneficial for IBV replication.Our findings provide a valuable reference for further research on poultry stress and target studies of anti-IBV infection drugs. |
