L 15 medium

Blood was collected from the caudal vein of tongue sole. PBL were isolated from the blood with 61% Percoll and collected as described previously (Li et al., 2017 (link)). The cells were cultured in L-15 medium (Thermo Scientific HyClone, Beijing, China) in 96-well culture plates (10⁵ cells/well). E. tarda TX01, TX01ΔtamA, TX01ΔtamB, TX01ΔtamA/tamA, and TX01ΔtamB/tamB were prepared as above and added to PBL (10⁵ CFU/well). The cells were incubated at 28°C for 0.5, 1, or 2 h. After incubation, the plates were washed with PBS (pH 7), and the cells were lysed with 100 μl PBS containing 1% Triton X-100. The cell lysate was diluted and plated in triplicate on LB agar plates. The plates were incubated at 28°C for 48 h, and the colonies that emerged on the plates were counted. The genetic identities of the colonies were verified by PCR with specific primers and sequence analysis of the PCR products. The experiment was performed three times.

Flounder head kidney (HK) macrophages were prepared as described previously [28 (link)]. The macrophages were cultured in L-15 medium (Thermo Scientific HyClone, Beijing, China) in 96-well culture plates (~10⁵ cells/well). TXHfq, TX01, and TXHfqC suspensions in PBS were prepared as described above and added to macrophages (10⁶ CFU/well). The cells were incubated at 25 °C for 0.5 h. After incubation, the cells were washed with PBS for three times and added with fresh L-15 containing 100 U/mL penicillin and streptomycin (Thermo Scientific HyClone, Beijing, China), followed by incubation at 25 °C for 1.5 h to kill extracellular bacteria. The plates were then washed three times with PBS and incubated at 28 °C for 1 h, 2 h, 4 h, and 8 h. After incubation, the plates were washed with PBS, and the cells were lysed with 100 μL 1% Triton X-100. The cell lysate was serially diluted and plated in triplicate on LB agar plates. The plates were incubated at 28 °C for 48 h, and the colonies that emerged on the plates were counted. The identities of the colonies were verified as described above.

PBL prepared above were resuspended in L-15 medium (Thermo Scientific HyClone, Beijing, China) to 1 × 10⁷ cells/ml. Anti-rCsCD46 antibody, anti-rTrx antibody, anti-rCsTLR2 antibody^{55 (link)}, preimmune antibody, all at 2 µg/ml, or PBS was added to PBL. After incubation at 22 °C for 1 h, the cells were centrifuged at 300 g for 10 min. The cells were washed three times with PBS and resuspended in serum, followed by incubation at 22 °C for 1 h. The control cells were untreated with serum or antibody. To measure cellular damage, PBL were treated with annexin V and propidium iodide (PI) (Majorbio Biotech, Shanghai, China) for 15 min in the dark according to the manufacturer’s instructions. The cells were then subjected to flow cytometry using a FACSort Flow Cytometer (BD Biosciences, USA). Data analysis was performed using FlowJo software 7.6.1 (Tree Star Inc, Ashland, OR, USA). To examine the effect of factor I on CsCD46-induced protection of PBL against complement damage, anti-rCsFI antibody or anti-rTrx antibody (2 µg/ml) was mixed with serum and incubated at 22 °C for 1 h. Treatment of PBL with the serum and measure of cellular damage were performed as above.

FG-9307 cells (46 (link)), a Japanese flounder gill cell line, were cultured at 24°C in L-15 medium (Thermo Scientific HyClone, USA) supplemented with 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin as reported previously (19 (link)). HEK293T cells were cultured at 37°C in DMEM medium (Invitrogen, Grand Island, USA) supplemented with 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin under humidified condition with 5% CO₂ as previously reported (19 (link)). FG-9307 cells were used in this study because it is derived from flounder. HEK293T cells were used in luciferase reporter assay, because efficient transfection can be achieved with this cell line.

Eye and brain tissue samples of NNV-positive seahorses were homogenized in 5 mL of L15 medium (Gibco, USA) without foetal bovine serum (FBS). Then, the tissue homogenates were filtered through a 0.22 μm filter membrane, inoculated on grouper spleen (GS) cells, and cultured in L15 medium supplemented with 10% FBS (Gibco, USA). The same volume of L15 was added to the mock infected group. Inoculated cells were cultured at 28 °C and monitored regularly for the development of cytopathic effects (CPEs). The virus isolate was propagated in GS cells until the cell monolayer was destroyed. The cell culture supernatant was then recovered, centrifuged at 1,000 × g for 10 min at 4 °C and stored at − 80 °C until use. Viral titres were determined by the 50% tissue culture infective dose (TCID50) method.

Microglia-like cells were generated with the BMP4-guided protocol, harvested between D40 and D50 from the culture supernatant of a 6-cm dish, and transferred into one well of an 8-well chamber. Microglia-like cells were cultured in 3:1-DMEM/F12-medium for 24 h. Before imaging, cells were washed once with 1x DPBS (Thermo Fisher Scientific, Cat#14190-250) and stained with Tomato-Lectin (Szabo-Scandic, Cat#VECDL-1174, 1:1000 in 1x DPBS) for 20 minutes at 37°C. Then, cells were washed with 1x DBPS, and L15 medium (Thermo Fisher Scientific, Cat#21083027) was added. Images were acquired with a Zeiss LSM880 inverted microscope and a Plan-Apochromat 20x/NA 0.8 Air objective in a temperature-controlled chamber (37°C). Z-stacked images of the 488 and 568 channel were captured simultaneously every minute. After 20 minutes baseline recording, sonicated pH-sensitive fluorescent beads (Thermo Fisher Scientific, Cat#P35361, 1:40) diluted in L15 medium were added, and cells were imaged for the following 60 minutes. For analysis, surface renderings were generated of z-stacks of the entire image using the surface rendering function in Imaris 9.3 with the surface detail setting of 0.2 μm. Next, the intensity mean of the 568 channel was determined within the microglia-like cell created surfaces.