Fig 2: Immunoregulation of the brain tissue during HEV infection. HEV-RNA positive brain tissues collected on 14, 21, and 28 dpi were used for immunohistochemistry study of SP (rabbit polyclonal IgG) and VIP (rabbit polyclonal IgG). Goat anti-rabbit IgG was chosen as secondary antibody. The positive signal was measured via the Motic Med 6.0 CMIAS Image Analysis System. (A,B) A positive signal of SP was found in the cytoplasm of neuronal cells of brain tissue in both groups and was significantly induced in HEV infected animals compared with animals from mock group (**p < 0.01). (C,D) Expression of VIP was observed in vascular endothelial cells, astrocytes and oligodendrocytes in both groups, with significantly increased levels in HEV infected tissues compared with tissues from mock group (*p < 0.05).

Fig 3: (A) The multiple alignments sequence of VIP (a) and VIPR1 (b) from several species. The signal peptide domain was marked in the pink dotted box. The GLUCA domain sequence and VIP peptides are marked in red and green dotted boxes, and the hormone receptors and seven-transmembrane G protein-coupled receptors are marked in yellow and purple dotted boxes. (B) The phylogenetic trees of VIP and VIPR1 were created using the MEGA 6.0 software. On-VIP (a) and On-VIPR1 (b) are marked in blue.

Fig 4: The transcriptional levels of On-VIP and On-VIPR1 in the liver, head kidney, brain, spleen, and intestine of tilapia infected with S. agalactiae at various time points via qRT-PCR. The transcriptional levels of On-VIP and On-VIPR1 at 0 h were set as 1. Every value was presented as the mean and standard deviation; n = 3. The different letters were applied to reveal the significant difference (p < 0.05).

Fig 5: (A) By using qRT-PCR, the transcription level of On-VIP and On-VIPR1 in various organs of unchallenged and healthy tilapia was identified. Every value is presented as a mean and standard deviation; n = 3. The transcriptional levels of On-VIP and On-VIPR1 in the spleen and skin were set as 1, respectively. (B) By using Western blot, the protein level of On-VIP and On-VIPR1 in various organs of unchallenged and healthy tilapia were assessed. (C) The location of On-VIP and On-VIPR1 in the intestine of unchallenged and healthy tilapia. The results were observed using the ZEISS Axioscope 5 microscope with × 200 and × 400 magnifications (Zeiss, Jena, Germany).