As a result, we tested the immune response in infected ducklings to verify whether the anti-DHAV-1 effect of BLIN was based on its immuno-regulation ability. The T- and B-lymphocyte proliferation abilities of BLIN were tested. 2017). Animals and cells Animals and ethics statement All the animals were purchased from the Tangquan Poultry Farm, Jiangsu province, China. All animal experiments in our work conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication, Eighth edition, 2011) and was approved by the Nanjing Agricultural University Animal Care Committee. Duck embryonic hepatocytes (DEHs) DEHs were prepared according to the method described previously (Chen et?al. 2014a). First, the tissue was collected, minced and washed three times with D-Hanks. Next, the tissue was digested with a solution of 0.20% trypsin. The tissue was washed three times with D-Hanks after removing the redundant trypsin. The cells were then cultured in GM in a humid atmosphere of 5% CO2 at 37?C. When the hepatocytes grew into a monolayer, the GM was removed and the cells were collected Nicodicosapent for standby. B lymphocytes Splenic B lymphocytes were prepared according to the method of Zhao et?al. (2012). A spleen from a non-immunized 30-day-old cherry valley duck Nicodicosapent was gently ground and then diluted with D-Hanks. The diluted spleen sample was carefully and slowly layered on the surface of the lymphocyte separation medium in a centrifuge tube. After 10?min of centrifugation at 1500?for 10?min, the T lymphocytes were collected and washed twice with D-Hanks. Finally, the T lymphocytes were diluted to 2.5??106 cells/mL with RPMI-1640 and collected for standby. Anti-DHAV-1 reproduction effect assay A 24-well cell culture plate containing a DEHs monolayer was treated with 200?L DHAV-1 (100 TCID50) per well, except for CD69 the control wells. In the meantime, 200?L BLIN at 40, 20, 10 and 5?g/mL (the working concentrations were, respectively, 20, 10, 5 and 2.5?g/mL) was added to the BLIN-treated wells. The cell control and virus control wells were made up to 400?L with Nicodicosapent MM. Then, the DHAV-1 was diluted to 50 TCID50 and BLIN was diluted to 20?g/mL (the concentration had no toxicity to DEHs as determined by a pre-experiment cytotoxicity test), 10, 5 and 2.5?g/mL. The plate was incubated at 37?C in a humid atmosphere of 5% CO2 for 24?h. Finally, the qRT-PCR method was applied to measure the DHAV-1 reproduction level. Assay of antiviral process of BLIN during DHAV-1 viral life Adsorption assay The adsorption assay consisted of two sample-adding modes: pre-adding drug and post-adding drug modes (Chen et?al. 2014a). Briefly, in the pre-adding drug mode, the virus control wells and BLIN-treated wells in a 24-well cell culture plate containing a DEHs monolayer were incubated with 400?L MM and 400?L BLIN (20?g/mL), respectively, at 4?C for 4?h. Then, the plate was washed three times with D-Hanks and 400?L DHAV-1 (50 TCID50) was added to all wells. The plates were then incubated at 37?C in a humid atmosphere of 5% CO2 for 1?h. After that, the qRT-PCR method was used to detect virus adsorption. In the post-adding drug mode, all wells of a 24-well cell culture plate containing a DEHs monolayer were incubated with 400?L DHAV-1 (50 TCID50) at 37?C in a humid atmosphere of 5% CO2 for 1?h. Then, the plate was washed three times with D-Hanks and 400?L MM (virus control wells) or 400?L BLIN (BLIN-treated wells) was added. The plates were then incubated at 4?C for 4?h. Similarly, the qRT-PCR method was used to detect virus adsorption. Cell controls were used in these two assays. Replication.