Institute of Preventive Veterinary Medicine, Sichuan Agricultural Universitygrid.80510.3c, Chengdu, Sichuan, China.
Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural Universitygrid.80510.3c, Chengdu, Sichuan, China.
Microbiol Spectr. 2022 Oct 26;10(5):e0244922. doi: 10.1128/spectrum.02449-22. Epub 2022 Oct 10.
Tembusu virus (TMUV), an avian mosquito-borne flavivirus, was first identified from Culex tritaeniorhynchus in 1955. To validate the effects of the 3'-untranslated region (3'UTR) in viral host-specific adaptation, we generated a set of chimeric viruses using CQW1 (duck strain) and MM 1775 (mosquito strain) as backbones with heterogeneous 3'UTRs. Compared with rMM 1775, rMM-CQ3'UTR (recombinant MM 1775 virus carrying the 3'UTR of CQW1) exhibited enhanced proliferation , with peak titers increasing by 5-fold in duck embryonic fibroblast (DEF) cells or 12-fold in baby hamster kidney (BHK-21) cells; however, the neurovirulence of rMM-CQ3'UTR was attenuated in 14-day-old Kunming mice via intracranial injection, with slower weight loss, lower mortality, and reduced viral loads. In contrast, rCQ-MM3'UTR showed similar growth kinetics and neurovirulence in mice compared with those of rCQW1. Then, the Stem-loop I (SLI) structure, which showed the highest variation within the 3'UTR between CQW1 and MM 1775, was further chosen for making chimeric viruses. The peak titers of rMM-CQ3'UTRSLI displayed a 15- or 4-fold increase , and the neurovirulence in mice was attenuated, compared with that of rMM 1775; rCQ-MM3'UTRSLI displayed comparable multiplication ability but was significantly attenuated in mice, in contrast with rCQW1. In conclusion, we demonstrated that the TMUV SLI structure of the 3'UTR was responsible for viral host-specific adaptation of the mosquito-derived strain in DEF and BHK-21 cells and regulated viral pathogenicity in 14-day-old mice, providing a new understanding of the functions of TMUV 3'UTR in viral host switching and the pathogenicity changes in mice. Mosquito-borne flaviviruses (MBFVs) constitute a large number of mosquito-transmitted viruses. The 3'-untranslated region (3'UTR) of MBFV has been suggested to be relevant to viral host-specific adaptation. However, the evolutionary strategies for host-specific fitness among MBFV are different, and the virulence-related structures within the 3'UTR are largely unknown. Here, using Tembusu virus (TMUV), an avian MBFV as models, we observed that the duck-derived SLI of the 3'UTR significantly enhanced the proliferation ability of mosquito-derived TMUV in baby hamster kidney (BHK-21) and duck embryonic fibroblast (DEF) cells, suggesting that the SLI structure was crucial for viral host-specific adaptation of mosquito-derived TMUVs in mammalian and avian cells. In addition, all SLI mutant viruses exhibited reduced viral pathogenicity in mice, indicating that SLI structure was a key factor for the pathogenicity in mice. This study provides a new insight into the functions of the MBFV 3'UTR in viral host switching and pathogenicity changes in mice.
腾布苏病毒（TMUV）是一种禽源蚊媒黄病毒，于 1955 年首次从三带喙库蚊中分离得到。为了验证 3'非翻译区（3'UTR）在病毒宿主特异性适应中的作用，我们使用 CQW1（鸭株）和 MM 1775（蚊株）作为骨架，生成了一组嵌合病毒，其具有异质的 3'UTR。与 rMM 1775 相比，rMM-CQ3'UTR（携带 CQW1 的 3'UTR 的重组 MM 1775 病毒）表现出增强的增殖能力，在鸭胚成纤维细胞（DEF）或仓鼠肾细胞（BHK-21）中的峰值滴度分别增加了 5 倍或 12 倍；然而，rMM-CQ3'UTR 在 14 日龄昆明小鼠中通过颅内注射的神经毒力减弱，体重减轻较慢，死亡率较低，病毒载量降低。相比之下，rCQ-MM3'UTR 在小鼠中的生长动力学和神经毒力与 rCQW1 相似。然后，进一步选择茎环 I（SLI）结构作为在 CQW1 和 MM 1775 之间 3'UTR 中变异最高的结构来构建嵌合病毒。rMM-CQ3'UTRSLI 的峰值滴度增加了 15 倍或 4 倍，神经毒力在小鼠中减弱，与 MM 1775 相比；rCQ-MM3'UTRSLI 表现出相当的增殖能力，但在小鼠中明显减弱，与 rCQW1 相比。总之，我们证明了 TMUV 3'UTR 的 SLI 结构负责蚊源性株在 DEF 和 BHK-21 细胞中的宿主特异性适应，并调节 14 日龄小鼠中的病毒致病性，为 TMUV 3'UTR 在病毒宿主转换和小鼠致病性变化中的功能提供了新的认识。蚊媒黄病毒（MBFV）构成了大量的蚊传播病毒。MBFV 的 3'非翻译区（3'UTR）被认为与病毒宿主特异性适应有关。然而，MBFV 之间的宿主特异性适应性的进化策略不同，而 3'UTR 内的毒力相关结构在很大程度上是未知的。在这里，我们使用禽源 MBFV 腾布苏病毒（TMUV）作为模型，观察到鸭源性 3'UTR 的 SLI 显著增强了蚊源性 TMUV 在仓鼠肾细胞（BHK-21）和鸭胚成纤维细胞（DEF）中的增殖能力，表明 SLI 结构对于蚊源性 TMUV 在哺乳动物和禽类细胞中的宿主特异性适应至关重要。此外，所有 SLI 突变病毒在小鼠中的致病性均降低，表明 SLI 结构是影响小鼠致病性的关键因素。本研究为蚊媒黄病毒 3'UTR 在病毒宿主转换和小鼠致病性变化中的功能提供了新的见解。
