Abstract: [Objective] This study was conducted to assess the mechanisms underlying the protective role of honeysuckle extract against Newcastle disease virus （NDV） infection in pigeon. [Method] A total of 40 healthy 28-day-old American king pigeons without NDV vaccination were selected and subjected to a 7-day adaptive rearing. At the age of 35 days, the experimental pigeons were randomly assigned into a honeysuckle extract protective （KO） group （n=10） or a virus challenge control （OE） group （n=10）. The pigeons in KO group drank the water containing 1.2% honeysuckle extract for 7 consecutive days, and at the age of 42 days, they were intramuscularly injected with 0.5 mL NDV Pi/NJ/CH/4416/2016 strain of pigeon origin with HA potency of 5log2 . The pigeons in OE group drank the normal water and were challenged with the same NDV strain at the same dosage at the age of 42 days. Seven days after challenge, 3 pigeons were chosen from both KO and OE groups. The spleen samples were collected for paraffin-embedded tissue section preparation and for total RNA extraction to construct the transcriptome libraries. RNA-Seq was used to identify differentially expressed genes （DEGs） and to perform GO functional enrichment analysis as well as KEGG pathway enrichment analysis. Four randomly selected significant DEGs identified by RNA-Seq were verified by using qRT-PCR. [Result] The splenocytes of the pigeons in OE group had abnormally enlarged nuclei and white vacuoles, accompanied by a large number of granulocyte infiltration in splenic tissues, while those of the pigeons in KO group had many white vacuoles and minor granulocyte infiltration alone, and no abnormally enlarged splenocyte nuclei in splenic tissues were observed. A total of 374 DEGs were identified in the spleens of the pigeons in KO group by RNA-Seq, of which 112 were significantly up-regulated and 262 were significantly down-regulated. GO functional enrichment analysis demonstrated that 875 DEGs were enriched in 312 GO entries, of which 149 belonged to biological process, 33 belonged to cellular component, and 130 belonged to molecular function. KEGG pathway enrichment analysis revealed that 226 DEGs were enriched in 93 KEGG signaling pathways, including information transmission pathway, metabolic pathway, natural immunity related pathway, and apoptosis pathway. The qRT-PCR validation test showed that the relative mRNA abundance of the 4 randomly selected significant DEGs （LOC110360702, LOC110360688, SFRP5 and CHAD） had the same change trend as the RNA-Seq results. [Conclusion] Honeysuckle extract exerts protective role against NDV infection in pigeon by activating important physiological processes of host such as information transmission, metabolic regulation and immune response. The results obtained in this study provide a scientific basis for further investigation on the molecular mechanism of immune response to NDV infection in pigeon.

Key words: pigeon, Newcastle disease virus, honeysuckle, RNA-Seq, differentially expressed genes, natural immunity