Department of Aqualife Medicine, Chonnam National University, San 96-1, Dunduck Dong, Chonnam 550-749, Republic of Korea.
Fish Shellfish Immunol. 2013 May;34(5):1236-43. doi: 10.1016/j.fsi.2013.02.012. Epub 2013 Feb 24.
The olive flounder (Paralichthys olivaceus) is susceptible to viral haemorrhagic septicaemia virus (VHSV) at 15 °C but no mortality is observed at 20 °C even though the virus can grow profusely in vitro. Thus, we designed an experiment to better understand the immune response of olive flounder to VHSV when the host reared at 15 °C or 20 °C and infected with the virus. Olive flounder (18-22 g) reared at 15 ± 0.5 °C or 20 ± 0.5 °C were intra-peritoneally injected with VHSV (10(7.8) TCID50/fish) and sampled (n = 5) for head kidney at 3, 6, 12 hpi, 1, 2, 4 and 7 dpi; similarly, mock injected control groups (n = 5). Real-time PCR-based absolute quantification method was followed to quantify copies of VHSV gRNA and mRNA, while the immune gene expression of the olive flounder was quantified relative to internal control, β-actin. Viral infection resulted in a cumulative mortality of 24% in olive flounder reared at 15 °C, but no mortality was recorded in the 20 °C group or control groups. TLR2 and TLR7 expression at 15 °C was enhanced during early-infection phase (3-6 hpi) and recovery phase (4-7 dpi) when viral transcription was low, but expression was significantly reduced (12 hpi-1 dpi) at peak-infection period. However, the 20 °C group showed low viral transcription and expressed high level of TLR7 and a moderately higher unchanged level of TLR2. In both the groups, TLR3 expression was unaffected. Nevertheless, expression of MDA5 and LGP2 increased significantly irrespective of rearing temperature at the time of peak infection, hence at 15 °C VHSV down-regulated expression of TLR2 and TLR7 but not MDA5 or LGP2. Comparatively, at 15 °C IRF3 expressed high but IRF7 remained very low. Interleukins (IL-1β, IL-6 and IL-8) were significantly elevated in both the groups, but quicker and for a shorter period at 20 °C. In the 15 °C group, an extended period of expression of ILs could create an unsafe prolonged inflammatory condition. The olive flounders expressed high ISGs at 15 °C but were lagging by 12 h than 20 °C group. Based on these findings, we concluded that viral-mediated disruption of TLR2 and TLR7 expression in the 15 °C group could have delayed the host interferon response and provided a window for high viral growth. However, an effective host immune response at 20 °C contained VHSV from reaching the critical limit.
橄榄石斑鱼（Paralichthys olivaceus）在 15°C 时易感染病毒性出血性败血症病毒（VHSV），但在 20°C 时即使病毒在体外大量生长，也不会观察到死亡。因此，我们设计了一项实验，以更好地了解在 15°C 或 20°C 饲养的宿主感染病毒时，橄榄石斑鱼对 VHSV 的免疫反应。15±0.5°C 或 20±0.5°C 饲养的橄榄石斑鱼（18-22g）经腹腔注射 VHSV（10(7.8)TCID50/鱼）并取样（n=5），分别在 3、6、12 hpi、1、2、4 和 7 dpi；同样，模拟注射对照组（n=5）。采用基于实时 PCR 的绝对定量方法来定量 VHSV gRNA 和 mRNA 的拷贝数，同时定量橄榄石斑鱼的免疫基因表达相对于内部对照β-肌动蛋白。病毒感染导致在 15°C 饲养的橄榄石斑鱼累积死亡率为 24%，但 20°C 组或对照组均未记录到死亡。在病毒转录水平较低的早期感染阶段（3-6 hpi）和恢复期（4-7 dpi），TLR2 和 TLR7 的表达在 15°C 下增强，但在感染高峰期（12 hpi-1 dpi）表达显著降低。然而，20°C 组显示低病毒转录，并表达高水平的 TLR7 和适度较高的不变水平的 TLR2。在两组中，TLR3 的表达均不受影响。然而，在感染高峰期，无论饲养温度如何，MDA5 和 LGP2 的表达均显著增加，因此，在 15°C 时，VHSV 下调了 TLR2 和 TLR7 的表达，但未下调 MDA5 或 LGP2 的表达。相比之下，在 15°C 时，IRF3 表达较高，但 IRF7 仍然非常低。两组中白细胞介素（IL-1β、IL-6 和 IL-8）均显著升高，但在 20°C 时更快且持续时间更短。在 15°C 组中，ILs 的延长表达可能会导致不安全的炎症延长。橄榄石斑鱼在 15°C 时表达高水平的 ISGs，但比 20°C 组滞后 12 小时。基于这些发现，我们得出结论，15°C 组中病毒介导的 TLR2 和 TLR7 表达的破坏可能延迟了宿主干扰素反应，并为病毒的大量生长提供了一个窗口。然而，20°C 时有效的宿主免疫反应阻止了 VHSV 达到临界水平。
