登革热病毒(Dengue virus, DENV)属于黄病毒科(Flaviviridae)，根据抗原性可分为四个不同血清型(DENV 1、2、3和4)，且进一步细分为基因型。DENV3又可分为四到五个基因型，具体取决于分类方法。通过系统发育完整的多肽分析显示，即使在基因上高度相似，DENV3 MG-20和DENV3 PV_BR在小鼠模型中显示出不同的神经毒性。我们的研究小组在E蛋白中鉴定了6个氨基酸突变，包括E62K和E123Q，这些突变可能影响结构域II上的疏水性团簇的相互作用，从而导致研究病毒之间的差异。 我们通过培养基质技术从一种恶性胶质母细胞瘤中得到的人类胶质母细胞瘤细胞(U251)被DENV3 GIL1分离株DENV3 MG-20和DENV3 PV_BR感染，并通过斑点测定、滴度测定、光学显微镜、免疫荧光和透射电镜进行分析。这两个分离株显示出不同的细胞病变效应（CPE）和融合模式，间接免疫荧光进一步证实这一点。透射电镜显示，DENV3 MG-20感染的U251细胞出现明显的细胞病变效应，显示内质网肥大和充盈蛋白和多个病毒的囊泡，与DENV3 PV_BR感染的细胞明显不同。其中一个假设是DENV3 MG-20分离株中的不同氨基酸与病毒感染中的增强膜融合能力相关，从而促进免疫系统逃避和中枢神经系统细胞感染的机会增加。 这些结果强调了分离株之间的生物学差异，在宿主-病毒相互作用和重症登革热发展方面可能起到关键因素。我们的研究提供了高度相似分离株的比较结果，有助于更深入地理解DENV的发病机制。DENV3 MG-20（属于DENV3 GI L1基因型）感染神经系统细胞（U251）表明其神经性，可能有助于理解神经型登革热疾病。 © 2023. BioMed Central Ltd., Springer Nature子公司。

Dengue virus (DENV) is a Flaviviridae member classified into four antigenically distinct serotypes (DENV 1, 2, 3, and 4) and further subdivided genotypes. DENV3 is subdivided into four or five genotypes, depending on the classification adopted. Despite their high genetic proximity, as revealed by phylogenetic complete polyprotein analysis, DENV3 MG-20 and DENV3 PV_BR showed different neurovirulence in mice models. Our group identified six amino acid mutations in protein E, including the E62K and E123Q, which may affect interactions of hydrophobic clusters on domain II, thus leading to the observed differences in the studied viruses.Human glioblastoma cells (U251) derived from a malignant glioblastoma tumor by explant technique were infected by the DENV3 GIL1 isolates DENV3 MG-20 and DENV3 PV_BR and analyzed by plaque assays and titration, optical, immunofluorescence, and transmission electronic microscopy.The two isolates showed different cytopathic effects (CPE) and fusogenic patterns, further confirmed by indirect immunofluorescence. Transmission electron microscopy revealed intense cytopathic effects in DENV3 MG-20 infected U251 cells, displaying endoplasmic reticulum hypertrophy and turgid vesicles with proteins and multiple viruses, distinct from DENV3 PV_BR infected cells. It is hypothesized that the different amino acids in the DENV3 MG-20 isolate are related to an increased membrane fusion ability in viral infection, thus facilitating immune system evasion and increased chances of central nervous system cell infection.These results emphasize the biological differences between the isolates, which could be a critical factor in host-virus interaction and severe dengue development. Our study presents comparative results of highly similar isolates with the potential to generate more subsidies for a deeper understanding of the DENV pathogenesis. The neurotropism of the isolate DENV3 MG-20 (belonging to the DENV3 GI L1 genotype) showing infection of nervous system cells (U251) could contribute to understanding neurological dengue disease.© 2023. BioMed Central Ltd., part of Springer Nature.