【摘要】 为优化大口黑鲈弹状病毒（MSRV）弱毒株MSRV 0509在鳜鱼脑（CPB）细胞中的最佳增殖条件，本研究首先将MSRV 0509株分别通过同步、异步接种CPB细胞后测定病毒滴度，结果显示MSRV 0509株同步接种病毒滴度可达10^8.20TCID₅₀/mL以上，且具有更高的操作效率和经济性，为最佳接种方式。进一步将MSRV 0509株分别以不同MOI（0.01、0.001、0.0001、0.00001、0.000001）同步和异步接种含有不同血清浓度（2%、4%、6%、8%FBS）的L15培养基和不同密度的CPB细胞[（1.5±0.3）×10⁵个/mL、（3±0.3）×10⁵个/mL、（4.5±0.3）×10⁵个/mL]，经培养后检测病毒滴度，结果显示MSRV 0509株以MOI 0.00001同步接种含4%FBS，细胞密度为（3±0.3）×10⁵个/mL时的病毒滴度最高，为最优的增殖条件。将MSRV 0509株以上述最优条件分别接种第150代和350代的CPB细胞后检测病毒滴度，结果显示病毒滴度均在10^8.30TCID₅₀/mL左右，代次对细胞增殖影响不明显。按上述筛选的最佳条件将MSRV 0509株接种CPB细胞后培养至不同时间检测病毒滴度，绘制同步接种时的病毒生长曲线，结果显示接种病毒后96 h病毒滴度达到峰值。在上述优化的基础上分别添加不同浓度Na₂CO₃-NaHCO₃缓冲液和NaHCO₃-Na₂CO₃缓冲液调整病毒增殖环境的pH值后接种病毒并培养，观察细胞病变并检测病毒滴度，结果显示添加2%0.1 mol/L Na₂CO₃-NaHCO₃缓冲液时病毒的增殖效率最高，病毒滴度可达10^9.18TCID₅₀/mL。综上所述，于含2%Na₂CO₃-NaHCO₃碳酸盐缓冲液和4%血清的L15培养基中将MSRV 0509株以MOI 0.00001同步接种CPB细胞，培养96 h后收获病毒，可使病毒滴度从10^8.5TCID₅₀/mL升高到10^9.18TCID₅₀/mL，实现了该病毒的高效增殖。本研究为MSRV弱毒疫苗的开发与应用奠定了重要基础。更多还原

【Abstract】 To optimize the optimal proliferation conditions of the Micropterus salmoides rhabdovirus（MSRV） attenuated strain0509 in CPB cells, this study first compared synchronous inoculation and asynchronous inoculation of MSRV 0509 onto CPB cells.The results demonstrated that the virus titer of MSRV 0509 under synchronous inoculation reached 10^8.20TCID₅₀/mL, indicating that synchronous inoculation was the optimal inoculation strategy. Subsequently, MSRV 0509 was inoculated under different conditions:multiplicity of infection（MOI）（0.01, 0.001, 0.0001, 0.00001, and 0.000001）, L15 medium with different FBS concentrations（2%,4%, 6%, and 8%）, and different CPB cell seeding densities（（1.5±0.3）×10⁵/mL,（3±0.3）×10⁵/mL,（4.5±0.3）×10⁵/mL）. The results revealed that the highest virus titer was achieved under the conditions of MOI 0.00001, 4% FBS, and cell seeding density of（3±0.3）×10⁵/mL with synchronous inoculation, which represented the optimal proliferation conditions. In addition, the titers of MSRV 0509 inoculated into 150 th-and 350 th-generation CPB cells were both approximately 10^8.30TCID₅₀/mL, indicating that MSRV 0509 exhibited strong cell adaptability. The virus growth curve further determined that the optimal virus harvest time for synchronous inoculation was 96 hours post-inoculation（hpi）. Finally, to optimize the pH environment for virus proliferation, different concentrations of Na₂ CO₃-NaHCO₃ buffer were added to L15 medium. The results showed that virus proliferation efficiency was the highest when 2% 0.1 mol/L Na₂ CO₃-NaHCO₃ buffer was supplemented, with the virus titer reaching 10^9.18TCID₅₀/mL. In conclusion,the efficient proliferation conditions of MSRV 0509 in CPB cells were determined as follows: L15 medium supplemented with 2%Na₂ CO₃-NaHCO₃ buffer and 4% FBS, inoculated synchronously at MOI 0.00001, and virus harvested at 96 hpi. These conditions not only enable efficient virus proliferation but also significantly reduce production costs. This study provides technical guidance for the development of vaccines based on the MSRV attenuated strain MSRV 0509.更多还原