Intra-host population dynamics of chikungunya virus in humans and naturally infected Aedes mosquitoes reveal transmission-driven diversity

RNA viruses often exhibit a high mutation rate, mainly because their RNA polymerases lack proofreading, which contributes to their genetic diversity. Arboviruses, which alternate between vertebrate and invertebrate hosts, are subjected to host-specific selective pressures and population bottlenecks, mainly within mosquito vectors. Although experimental studies have brought insights about their evolutionary dynamics, data from naturally infected vectors remain limited. Here, we investigated the intra-host genetic diversity of chikungunya virus (CHIKV-ECSA lineage) through whole-genome sequencing of 19 human- and 19 mosquito-derived genomes from the 2024 outbreak in São José do Rio Preto, Brazil. Our principal component analysis revealed a greater mutation number in mosquito-derived genomes, predominantly driven by low-frequency and unique variants. Overall, intra-host genetic diversity was significantly higher in mosquito-derived than in human-derived CHIKV genomes, and protein-coding regions showed host-specific patterns. We identified 303 mutations across all CHIKV genomes. Interestingly, shared mutations were predominantly classified as synonymous, whereas unique mutations were mainly non-synonymous. Gene-wide selection analyses indicated that purifying selection predominates across CHIKV genomes from both humans and mosquitoes, suggesting that most mutations, particularly non-synonymous ones, are deleterious and subject to purifying selection. However, in mosquito-derived CHIKV genomes, evidence of relaxed purifying selection and neutral evolution, in specific proteins, such as E3 and NSP3, respectively, was observed, in contrast to the stronger purifying selection observed in human-derived CHIKV sequences. Site-specific selection analyses corroborated these results, detecting negatively selected sites in human-derived genomes but not in mosquito-derived genomes for these specific proteins. Together, our results show that these host-specific differences enable mosquitoes to act as reservoirs of genetic diversity by maintaining non-synonymous variants, likely driven by genetic drift. At the same time, human hosts may impose stronger selective pressures, contributing to preserving the genome stability. This dynamic balance between diversification in vector populations and selective constraints in vertebrate hosts likely drives CHIKV evolution and adaptation.RNA病毒通常具有较高的突变率，其主要原因在于其RNA聚合酶（RNA polymerase）缺乏校对活性，进而促成了其遗传多样性的产生。虫媒病毒（Arboviruses）可在脊椎动物与无脊椎动物宿主间交替循环传播，因此会受到宿主特异性选择压力与种群瓶颈效应的影响，其中种群瓶颈主要发生在蚊媒载体中。尽管已有实验研究为阐明其进化动态提供了诸多见解，但来自自然感染媒介的相关数据仍较为匮乏。本研究针对2024年巴西圣若泽杜里约普雷图暴发的基孔肯雅疫情，对19株人类来源与19株蚊媒来源的基孔肯雅病毒（CHIKV-ECSA谱系）基因组进行全基因组测序，以此解析其宿主内遗传多样性。主成分分析（Principal Component Analysis）结果显示，蚊媒来源的病毒基因组中突变数量更多，且该差异主要由低频变异体与独特变异体所驱动。总体而言，蚊媒来源的基孔肯雅病毒基因组的宿主内遗传多样性显著高于人类来源的病毒基因组，且其蛋白质编码区呈现出宿主特异性分布模式。本研究在所有基孔肯雅病毒基因组中共鉴定出303个突变位点。值得注意的是，共享突变大多被归类为同义突变，而独特突变则以非同义突变为主。全基因范围的选择分析结果表明，无论是人类来源还是蚊媒来源的基孔肯雅病毒基因组，均以纯化选择（purifying selection）为主导，这提示绝大多数突变（尤其是非同义突变）均具有有害性，并受到纯化选择的作用。但与人类来源的基孔肯雅病毒序列所呈现的更强纯化选择不同，本研究在蚊媒来源的病毒基因组中观察到特定蛋白（如E3和NSP3）分别存在松弛纯化选择与中性进化的相关证据。位点特异性选择分析进一步验证了上述结果：在上述特定蛋白中，人类来源的病毒基因组中检测到了负选择位点，而蚊媒来源的病毒基因组中则未发现此类位点。综合来看，本研究结果表明，上述宿主特异性差异使得蚊媒能够通过保留非同义突变体成为遗传多样性的储存库，这一过程大概率由遗传漂变（genetic drift）所驱动。与此同时，人类宿主则会施加更强的选择压力，有助于维持病毒基因组的稳定性。媒介种群的多样性演化与脊椎动物宿主的选择约束之间的这种动态平衡，可能正是推动基孔肯雅病毒进化与适应的核心动力。