State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, 220 Handan Rd., Shanghai 200433, People's Republic of China.
Vet Res. 2010 May-Jun;41(3):30. doi: 10.1051/vetres/2010002. Epub 2010 Jan 13.
In this study, specific sequences within three genes (3D, VP4 and 2B) of the foot-and-mouth disease virus (FMDV) genome were determined to be effective RNAi targets. These sequences are highly conserved among different serotype viruses based on sequence analysis. Small interfering RNA (siRNA)-expressing plasmids (p3D-NT19, p3D-NT56, pVP4-NT19, pVP4-NT65 and p2B-NT25) were constructed to express siRNA targeting 3D, VP4 and 2B, respectively. The antiviral potential of these siRNA for various FMDV isolates was investigated in baby hamster kidney (BHK-21) cells and suckling mice. The results show that these siRNA inhibited virus yield 10- to 300-fold for different FMDV isolates of serotype O and serotype Asia I at 48 h post infection in BHK-21 cells compared to control cells. In suckling mice, p3D-NT56 and p2B-NT25 delayed the death of mice. Twenty percent to 40% of the animals that received a single siRNA dose survived 5 days post infection with serotype O or serotype Asia I. We used an attenuated Salmonella choleraesuis (C500) vaccine strain, to carry the plasmid that expresses siRNA directed against the polymerase gene 3D (p3D-NT56) of FMDV. We used guinea pigs to evaluate the inhibitory effects of recombinant S. cho (p3D-NT56/S. cho) on FMDV infection. The results show that 80% of guinea pigs inoculated with 10(9) CFU of p3D-NT56/S. cho and challenged 36 h later with 50 ID(50) of homologous FMDV were protected. We also measured the antiviral activity of p3D-NT56/S. cho in swine. The results indicate that 100% of the animals treated with 5 x 10(9) CFU of p3D-NT56/S. cho were protected in 9 days.
在这项研究中，确定了口蹄疫病毒（FMDV）基因组中三个基因（3D、VP4 和 2B）的特定序列是有效的 RNAi 靶标。基于序列分析，这些序列在不同血清型病毒之间高度保守。构建了表达针对 3D、VP4 和 2B 的 siRNA 的质粒（p3D-NT19、p3D-NT56、pVP4-NT19、pVP4-NT65 和 p2B-NT25）。在婴儿仓鼠肾（BHK-21）细胞和乳鼠中研究了这些 siRNA 对各种 FMDV 分离株的抗病毒潜力。结果表明，与对照细胞相比，这些 siRNA 在感染后 48 小时抑制了不同血清型 O 和血清型亚洲 I 的 FMDV 分离株的病毒产量 10-300 倍。在乳鼠中，p3D-NT56 和 p2B-NT25 延迟了小鼠的死亡。接受单次 siRNA 剂量的 20%至 40%的动物在感染血清型 O 或血清型亚洲 I 后 5 天存活。我们使用减毒霍乱弧菌（C500）疫苗株携带针对 FMDV 聚合酶基因 3D（p3D-NT56）的表达 siRNA 的质粒。我们使用豚鼠评估重组 S. cho（p3D-NT56/S. cho）对 FMDV 感染的抑制作用。结果表明，接种 10（9）CFU 的 p3D-NT56/S. cho 并在 36 小时后用同源性 FMDV 50 ID（50）攻毒的 80%的豚鼠得到保护。我们还测量了 p3D-NT56/S. cho 在猪中的抗病毒活性。结果表明，用 5 x 10（9）CFU 的 p3D-NT56/S. cho 处理的 100%的动物在 9 天内得到保护。
