Water temperature is an important factor for immune responses in poikilothermic fish. Especially, it has been known that adaptive immunity is more sensitive to temperature than innate immunity in fish. The optimal temperature for olive flounder (Paralichthys olivaceus) culture is known between 20 and 25 °C, and there are several papers reporting the low or no effectiveness of inactivated vaccines in olive flounder kept at low water temperatures. Previously, we had reported that a vaccine based on single-cycle viral hemorrhagic septicemia virus (VHSV) that was modified to produce the transmembrane and C-terminal cytoplasmic region-deleted G protein in host cells (rVHSV-GΔTM) induced significantly higher survival rates in olive flounder than a vaccine of rVHSV-ΔG that had no G gene in the genome. In the present study, we evaluated the availability of rVHSV-GΔTM as a protective vaccine that can be used in olive flounder at low water temperature periods. Olive flounder fingerlings were divided into 6 groups: group 1 and 2 were kept at 14 °C, group 3 and 4 were kept at 20 °C, and group 5 and 6 were kept at 14 °C for 1 week and then shifted to 20 °C. Fish in groups 1, 3, and 5 were intramuscularly (i.m.) immunized with 8.5 × 10⁴ PFU/fish of rVHSV-GΔTM, and fish in groups of 2, 4, and 6 were i.m. Injected with L-15 alone. In the challenge test, the survival rates of fish immunized with rVHSV-GΔTM were significantly higher than those of control group fish that were injected with L-15 alone. Among three vaccination groups (group 1, 3, and 5), group 1 showed no mortality. The cumulative mortalities of group 3 and group 5 were both 25%. While fish in control groups (group 2, 4, and 6) showed 90–100% mortalities. The qPCR genome copy number of rVHSV-GΔTM in the kidney of fish immunized at 14 °C was clearly higher than that in fish immunized at 20 °C, which suggests that higher amount of secretory viral glycoprotein would be produced in fish vaccinated at 14 °C than at 20 °C. Olive flounder immunized with rVHSV-GΔTM at 14 °C showed the serum neutralization activity as high as fish immunized at 20 °C, suggesting that the humoral immune response of olive flounder was effectively induced at lower water temperature. These results suggest that VHSV vaccines based on single-cycle viruses can be used as prophylactic vaccines even at low water temperature period.

水温是对Poikilothermic鱼的免疫反应的重要因素。特别是，众所周知，适应性免疫比鱼类的先天免疫对温度更敏感。比目鱼（Paralichthys olivaceus）的最佳温度。）培养在20至25°C之间为人所知，有几篇论文报道在低水温下保存的比目鱼灭活疫苗效果不佳或没有效果。以前，我们曾报道过一种基于单周期病毒性败血性败血病病毒（VHSV）的疫苗，该疫苗经过修饰可在宿主细胞中产生跨膜和C端胞质区缺失的G蛋白（rVHSV-GΔTM），可显着提高存活率比在基因组中没有G基因的rVHSV-ΔG疫苗更容易。在本研究中，我们评估了rVHSV-GΔTM作为保护性疫苗的可用性，该疫苗可在低水温时期用于比目鱼。橄榄比目鱼种分为6组：第1组和第2组保持在14°C，第3组和第4组保持在20°C，第5组和第6组在14°C下保持1周，然后转移到20°C。第1、3和5组中的鱼用8.5×10肌内（im）免疫⁴ 仅用L-15注射rVHSV-GΔTM的PFU /鱼，以及2、4和6组的鱼。在攻击试验中，用rVHSV-GΔTM免疫的鱼的存活率明显高于仅注射L-15的对照组鱼的存活率。在三个疫苗接种组（第1、3和5组）中，第1组未显示死亡率。第3组和第5组的累积死亡率均为25％。对照组（第2、4和6组）中的鱼显示出90–100％的死亡率。在14°C免疫的鱼的肾脏中，rVHSV-GΔTM的qPCR基因组拷贝数明显高于在20°C免疫的鱼的肾脏，这表明在14°C免疫的鱼中会产生更多的分泌型病毒糖蛋白。 C高于20°C。用rVHSV-GΔTM在14°C免疫的比目鱼的血清中和活性与在20°C免疫的鱼一样高，表明在较低水温下能有效诱导比目鱼的体液免疫反应。这些结果表明，即使在低水温时期，基于单周期病毒的VHSV疫苗也可用作预防性疫苗。