Bluetongue is a serious non-contagious vector-borne viral disease in ruminants, causing poor animal welfare and economic consequences globally. Concern has been raised about the development of novel bluetongue virus (BTV) strains and their possibly altered virulence through the process of viral reassortment. Virulence is traditionally estimated in lethal dose 50 (LD₅₀) studies in murine models, but agreement with both in vitro and virulence in ruminants is questionable, and a refined experimental design is needed. Specific reassortants between wild-type and vaccine strains of BTV-1, -6 and -8 have previously been developed by reverse genetics. The aim of the present study was to rank the in vivo virulence of these parental and reassortant BTV strains by calculating LD₅₀ in a murine model by using an experimental design that is new to virology: a between-patient optimised three-level response surface pathway design. The inoculation procedure was intracranial. Fifteen suckling mice were used to establish LD₅₀ for each strain. Three parental and five reassortant virus strains were included. The LD₅₀s varied from of 0.1 (95% confidence interval (CI) 0–0.20) to 3.3 (95% CI 2.96–3.72) tissue culture infectious dose 50/ml. The results support the hypothesis that reassortment in BTV may lead to increased virulence in mice with potential negative consequences for the natural ruminant host. The ranking showed low agreement with in vitro properties and virulence in ruminants according to existing literature. Refined design such as response surface pathway design was found suitable for use in virology, and it introduces significant ethical and scientific improvements.

蓝舌病是反刍动物中一种严重的非传染性媒介传播病毒性疾病，在全球范围内造成不良的动物福利和经济后果。人们对新型蓝舌病毒 (BTV) 毒株的发展及其可能通过病毒重组过程改变的毒力表示关注。毒力传统上是在小鼠模型的致死剂量 50 (LD ₅₀ ) 研究中估计的，但与体外和反刍动物毒力的一致性值得怀疑，需要改进的实验设计。BTV-1、-6 和 -8 的野生型和疫苗株之间的特定重配子先前已通过反向遗传学开发。本研究的目的是对体内通过使用病毒学新的实验设计计算鼠模型中的LD ₅₀来确定这些亲本和重配 BTV 菌株的毒力：患者间优化的三级响应表面途径设计。接种程序是颅内的。使用 15 只乳鼠来确定每个品系的LD ₅₀。包括三个亲本和五个重配病毒株。LD ₅₀ s 从 0.1（95% 置信区间 (CI) 0-0.20）到 3.3（95% CI 2.96-3.72）组织培养感染剂量 50/ml 不等。结果支持了以下假设，即 BTV 中的重排可能会导致小鼠毒力增加，从而对天然反刍动物宿主产生潜在的负面影响。排名显示与体外的低一致性根据现有文献研究反刍动物的特性和毒力。发现诸如响应表面通路设计之类的精细设计适用于病毒学，并且引入了重大的伦理和科学改进。