【摘要】 【背景】新型鸭呼肠孤病毒(novel duck reovirus, NDRV)是鸭出血性坏死性肝炎的病原。本团队前期将NDRV强毒株NP03在番鸭胚成纤维细胞(Muscovy duck embryo fibroblast cells,MDEFs)上连续传代，选育出弱毒株。弱毒株与亲本强毒株NP03比对，弱毒株p18基因第381和382位核苷酸发生了T381A、C382T突变，特别是弱毒株在382位点稳定突变为T，导致产生了一个终止密码子TAA，造成弱毒p18蛋白比亲本强毒少编码35个氨基酸。【目的】建立一种可以快速检测新型鸭呼肠孤病毒弱毒株的RT-qPCR检测方法，并对各传代毒进行检测和测序验证，明确p18基因在致弱过程中的突变规律，为致弱机理研究奠定基础。【方法】根据NDRV弱毒株与亲本强毒株NP03在p18基因第381-382位核苷酸差异，设计一对特异性引物和探针，构建重组质粒作为阳性质粒标准品，建立了一种特异检测NDRV弱毒株的TaqMan RT-qPCR方法，并对该方法的特异性、敏感性、重复性进行评估，同时绘制标准曲线，最后采用该方法对传代致弱过程中的不同代次毒进行检测，确定弱毒p18基因出现位点突变的规律。【结果】本研究建立的RT-qPCR方法仅特异检测NDRV弱毒株，不能检测亲本强毒株NP03，具有良好的特异性，最低检测下限为1×10~1 copies/μL,R~2值为0.99，组内、组间变异系数均小于2%。应用该方法对NP03在MDEF细胞中传代的第10-200代次传代毒进行检测，50代以内的传代毒p18基因检测为阴性，第51代以后传代毒检测为阳性；进一步测序分析表明，p18基因第381-382位点在98代以后由强毒U-C稳定突变为A-U，对应的氨基酸位点由强毒的GAU-CAA突变为弱毒的GAA-UAA，导致弱毒p18蛋白提早产生一个终止密码子，造成弱毒p18蛋白比亲本强毒少编码35个氨基酸。【结论】综上所述，本研究建立了一种特异检测NDRV弱毒株的RT-qPCR检测方法，解析了p18基因在致弱过程中的突变规律，在强弱毒快速鉴别、活疫苗研制及疾病净化等方面具有重要应用价值。更多还原

【Abstract】 [Background] The novel duck reovirus(NDRV) is the causative agent of hemorrhagic necrotic hepatitis in ducks. Our team previously passaged the virulent strain NP03 of NDRV in Muscovy duck embryo fibroblast cells and successfully selected an attenuated strain. Comparative analysis revealed that in the p18 gene of the attenuated strain, nucleotide mutations occurred at positions 381 and 382(T381A and C382T). Notably, the stable mutation C382T resulted in the generation of a termination codon, which caused the p18 protein of the attenuated strain to be 35 amino acids shorter than that of the parental virulent strain. [Objective] To establish a rapid RT-qPCR method for detecting the attenuated strain of NDRV, apply this detection method and sequencing to clarify the site mutation patterns of the p18 gene, and lay a foundation for deciphering the mechanism of attenuation. [Methods] According to the nucleotide differences at positions 381-382 of the p18 gene between the attenuated strain and the parental virulent strain NP03 of NDRV, a pair of specific primers and a probe were designed. A recombinant plasmid was constructed as a positive standard, and a TaqMan RT-qPCR method specific for detecting the attenuated strain was established. The specificity, sensitivity, and repeatability of the established method were evaluated, and a standard curve was generated. Finally, the method was used to detect viruses from different passages during the attenuation process to determine the site mutation pattern of p18 gene in the attenuated strain. [Results] The RT-qPCR method established in this study specifically detected the attenuated strain and did not detect the parental virulent strain NP03, demonstrating strong specificity. The minimum detection limit was 1×10~1 copies/μL, with an R~2 value of 0.99. Both the intra-assay and inter-assay coefficients of variation were less than 2%. When this method was employed to detect viruses from passages 10 to 200 of the NP03 strain in MDEF cells, the p18 gene tested negative before passage 50 and positive after passage 51. Further sequencing analysis revealed that the nucleotide sites 381-382 of the p18 gene stably mutated from U-C(virulent) to A-U(attenuated) after passage 98. The corresponding amino acid codons changed from GAU-CAA in the virulent strain to GAA-UAA in the attenuated strain, resulting in the premature generation of a termination codon. This caused the p18 protein of the attenuated strain to be 35 amino acids shorter than that of the parental virulent strain. [Conclusion] In summary, this study established a RT-qPCR method for specific detection of the attenuated strain of NDRV and elucidated the mutation pattern of the p18 gene during viral attenuation. This method holds significant application value for rapid differentiation between virulent and attenuated strains, development of live vaccines, and disease eradication efforts.更多还原