可中和多种病毒变异株的广谱中和抗体（broadly neutralizing antibodies，bNAbs），可阻断病毒传播、清除感染病毒及增强机体免疫力，在RNA病毒感染防治中具有巨大的潜力。成熟的B细胞需经历多轮体细胞高频突变，才能进化出高亲和力bNAbs，但其中影响B细胞高频突变的遗传因素尚不明确。本研究拟建立LCMV-CL13病毒慢性感染小鼠模型，识别B细胞发育的关键调节因子并筛选出影响SHM发生的相关基因，为明确bNAbs抗体的产生机制，探索通用疫苗提供理论基础。

为建立LCMV-CL13病毒慢性感染小鼠模型，6-8周C57BL/6N小鼠被尾静脉接种2×10⁶ FFU LCMV-CL13病毒，观察小鼠模型临床疾病特征、体重变化，发现小鼠感染后出现明显疾病表现，如竖毛、弓背、体重下降，小鼠体重在7天、32天下降最为明显，感染后每10 天取肠、肾、肝、脑等组织，qRT-PCR法检测肠、肾、肝、脑病毒载量，结果发现LCMV-CL13病毒可在小鼠肠、肾、肝、脑等组织保持高水平复制。利用苏木素（Hematoxylin）-伊红（Eosin）染色观察肠、脾、肾、肝、脑组织病理变化，病理学发现LCMV-CL13病毒对肠、脾、肾、肝、脑等组织均有不同程度损伤，因此，病毒感染组与未感染组在病毒复制、病理损伤等方面有较为明显的差异，表明成功建立LCMV-CL13慢性感染小鼠模型。

为分析LCMV-CL13病毒感染后小鼠细胞、体液免疫反应，感染后每10天取外周血及血清，流式检测外周血T、B细胞、脾脏GC B细胞比例，发现随感染时间的增加，小鼠CD4⁺ T、CD8⁺ T、CD19⁺ B细胞及脾脏GC B细胞比例发生显著性变化，其中B细胞、脾脏GC B细胞比例随感染时间逐渐增加。ELISA法检测病毒特异性IgG 抗体，结果显示小鼠血清持续检测到病毒特异性IgG抗体。进一步分析LCMV-CL13感染小鼠模型的B细胞BCR突变情况，对感染后30、60天小鼠脾脏B细胞进行免疫组库测序，发现BCR重链V基因使用频率下降、突变率显著性升高（P < 0.05）。

为筛选B细胞SHM相关基因，LCMV-CL13病毒感染后30、60天取小鼠脾脏制备脾单细胞悬液，通过免疫组库测序分析B细胞高频突变率，RNA-Seq测序分析筛选出差异表达基因。RNA-Seq结果显示，感染后30天组小鼠与NC组相比共有2061个差异表达基因，感染后60天组小鼠与NC组相比共有2466个差异表达基因，感染后60天小鼠与感染后30天小鼠相比共有260个差异表达基因。通过免疫组库（Immune Repertoire，IR）测序分析证明LCMV-CL13慢性感染小鼠B细胞BCR重链V基因发生SHM，RNA-Seq测序分析感染后60天小鼠与感染后30天小鼠相比分别上调下调67、193个基因，GO分析差异表达基因除富集于对外界刺激反应的正向调节功能外，还与生物膜、生物膜组成成分、氧化还原、能量生成、代谢等生物学功能密切相关。KEGG分析差异基因主要富集到补体和凝血级联反应信号通路、细胞因子-细胞因子受体相互作用通路、肿瘤坏死因子（tumor necrosis factor ，TNF）信号通路、氧化磷酸化等信号通路。

综上所述，本研究利用LCMV-CL13慢性感染小鼠结合免疫组库、RNA-Seq等高通量测序技术筛选B细胞高频突变的影响基因及信号通路。本研究不仅为慢性病毒感染中B细胞高频突变、抗体进化机制研究提供了切入点，也为阐明抗体进化机制和探寻抗病毒药物、疫苗的有效靶点提供新思路和基础数据。

Broadly neutralizing antibodies（bNAbs）that can neutralize a variety of virus variants can block the spread of viruses, clear infectious viruses, enhance body immunity, and have great potential in preventing and treating RNA virus infections. Mature B cells need to undergo multiple rounds of somatic hypermutation to evolve high-affinity bNAbs antibodies, but the genetic factors that affect B cell hypermutation remain unclear. This study intends to establish a mouse model of chronic infection with LCMV-CL13 virus, identify the key regulators of B cell development, and screen out the related genes that affect the occurrence of SHM, to provide a theoretical basis for clarifying the mechanism of bNAbs antibody production and exploring universal vaccines.

To establish a mouse model of chronic infection with LCMV-CL13 virus, 6-8 weeks C57BL/6N mice were inoculated with 2 × 10⁶ FFU LCMV-CL13 virus by tail vein, and the clinical disease characteristics and body weight changes of the mouse model were observed. Obvious disease manifestations, such as ruffled fur, a hunched back, weight loss, the most obvious weight loss in mice at 7 days and 32 days, the intestine, kidney, liver, brain, and other tissues were collected every 10 days after infection, and the viral loads in the intestine, kidney, liver, and brain were detected by qRT-PCR, the results found that LCMV-CL13 virus can maintain a high level of replication in mouse intestine, kidney, liver, brain and other tissues. Hematoxylin-Eosin staining was used to observe the pathological changes in the intestine, spleen, kidney, liver, and brain. Pathology found that the LCMV-CL13 virus had different degrees of damage to the intestine, spleen, kidney, liver, brain, and other tissues. Therefore, the virus infection group and the uninfected group had obvious differences in virus replication, pathological damage, etc., indicating that the LCMV-CL13 chronic infection mouse model was successfully established.

To analyze the cellular and humoral immune responses of mice infected with the LCMV-CL13 virus, peripheral blood and serum were collected every 10 days after infection, and the proportions of T, B cells in peripheral blood and GC B cells in the spleen were detected by flow cytometry. The proportions of CD4⁺ T, CD8⁺ T, CD19⁺ B cells, and spleen GC B cells in mice changed significantly, and the proportions of B cells and GC B cells in the spleen gradually increased with the infection time. Virus-specific IgG antibodies were detected by ELISA, and the results showed that virus-specific IgG antibodies were continuously detected in mouse serum. The BCR mutation of B cells in the LCMV-CL13 infection mouse model was further analyzed. The immune repertoire sequencing of the spleen B cells of the mice 30 and 60 days after infection showed that the frequency of BCR heavy chain V gene usage decreased and the mutation rate increased significantly (P < 0.05).

To screen SHM-related genes of B cells, mouse spleen was taken 30 and 60 days after LCMV-CL13 virus infection to prepare spleen single-cell suspension, and the high-frequency mutation rate of B cells was analyzed by immune repertoire sequencing, and differences expressed genes were screened by RNA-Seq sequencing analysis. RNA-Seq results showed that there were 2061 differentially expressed genes in mice in the 30-day post-infection group compared with the NC group, and 2466 differentially expressed genes in the mice in the 60-day post-infection group compared with the NC group. There were a total of 260 differentially expressed genes in mice at 60 days post-infection compared to mice at 30 days post-infection. Immune Repertoire (IR) sequencing analysis demonstrated that the BCR heavy chain V gene of LCMV-CL13 chronic infected mice B cell occur SHM, RNA-Seq sequencing analysis showed that compared with mice 30 days after infection, the mice 60 days after infection were up-regulated and down-regulated by 67 and 193 genes, GO analysis showed that in addition to being enriched in positive regulation of responses to external stimuli, differentially expressed genes were also closely related to biological functions such as biofilm, biofilm composition, redox, energy generation, and metabolism. KEGG analysis showed that the differential genes were mainly enriched in the complement and coagulation cascade signaling pathways, cytokine-cytokine receptor interaction pathway, tumor necrosis factor (TNF) signaling pathway, oxidative phosphorylation, and other signaling pathways.

In summary, this study used LCMV-CL13 chronically infected mice combined with high-throughput sequencing technologies such as immune repertoire and RNA-Seq to screen the genes and signaling pathways that affect B cell somatic hypermutations. This study not only provides an entry point for the study of somatic hypermutations of B cells and antibody evolution mechanism in chronic viral infection but also provides new ideas and basic data for elucidating the mechanism of antibody evolution and searching for effective targets for antiviral drugs and vaccines.