2'-5' Oligoadenylate Synthetase-Like 1- (OASL1-) deficient mice promote antiviral protection against Pseudorabies Virus Infection through robust production of type I interferons

Figure 1. Upregulation of OASL1 following PRV infection, observed both in vivo and in vitro. (A) WT mice were injected with either PBS or varying doses of PRV. Survival rates and body weight changes were monitored for up to 7 dpi. (B) WT mice were infected with PRV, and the expression levels of OASL1, IFN-α, and IFN-β were quantified using quantitative real time- polymerase chain reaction (qRT-PCR). (C) Expression levels of OASL1 and type I IFN in primary myeloid cells infected with PRV at varying multiplicities of infection (MOIs) for 24 h.

Figure 2. Enhanced resistance to PRV and reduced viral burden in Oasl1⁻/⁻ mice. (A) Schematic illustration of the experimental setup. (B) The image shows the clinical symptoms. (C) The survival rate, body weight changes, and clinical scores were monitored for up to 12 days. (D) Virus titers in spleens, lungs, and brains of infected mice were determined using qRT-PCR. (E) Type I IFN levels in the serum were measured by ELISA.

Figure 3. Effects of Oasl1⁻/⁻ on pro-inflammatory mediators in PRV infection as determined by qRT-PCR. mRNA expression of pro-inflammatory mediators in spleens, lungs, and brains of infected mice was assessed.

Figure 4. Oasl1⁻/⁻ mice exhibit reduced inflammation following PRV infection. Sandwich ELISA determined protein levels of pro-inflammatory mediators in the spleens, lungs, brains, and serum of PRV-infected mice.

Figure 5. Oasl1⁻/⁻ mice exhibit less severe pathological alterations following PRV infection. Representative hematoxylin and eosin-stained (H&E) tissue sections of PRV-infected Oasl1⁻/⁻ and WT mice are shown. The arrows indicate diminished white pulp and depletion of immune cells in the spleen. Asterisks mark peri bronchial infiltration of inflammatory cells in the lung. Neurodegeneration was significantly observed in the hippocampus neurons of the brain.

Figure 6. Increased Type I IFN Secretion via IRF7 Signaling in PRV-infected Oasl1⁻/⁻ Mice. (A) mRNA expression patterns of IRF7 and IRF3 in spleen, lung, and brain tissues of WT and Oasl1⁻/⁻ mice post-PRV infection evaluated by qRT-PCR at 2, 3, and 4 dpi. (B) Western blot analysis of tissue lysates from the above tissues of WT and Oasl1⁻/⁻ mice illustrating relative protein levels of IRF7 and IRF3.

Figure 7. OASL1-ablated myeloid-derived cells produce elevated levels of type I IFNs after PRV infection. Primary bone marrow-derived macrophages (BMDMs) isolated from WT and Oasl1⁻/⁻mice were infected with PRV at MOIs of 0.1, 0.5, 1, and 5 at the indicated times. (A-B) The mRNA expression of cytokines, chemokines, and IFN-α/β was evaluated via real-time qRT-PCR. (C) The mRNA expression levels of IRF7 and IRF3 in BMDM following PRV infection. (D) The protein expression levels of IRF7 and IRF3 in BMDMs infected with PRV at MOI 0.5 at 24 dpi.

图1. PRV感染后OASL1的上调表达：体内与体外实验均观测到该现象。(A) 野生型（wild type，WT）小鼠分别注射磷酸盐缓冲液（phosphate buffered saline，PBS）或不同剂量的PRV，监测感染后7天（days post infection，dpi）内的存活率与体重变化。(B) 野生型小鼠感染PRV后，采用实时定量聚合酶链反应（quantitative real time-polymerase chain reaction，qRT-PCR）定量检测OASL1、IFN-α及IFN-β的表达水平。(C) 不同感染复数（multiplicity of infection，MOI）的PRV感染原代髓系细胞24小时后，检测OASL1与I型干扰素的表达水平。

图2. Oasl1基因敲除（Oasl1⁻/⁻）小鼠对PRV的抵抗能力增强且病毒载量降低。(A) 实验流程示意图。(B) 展示临床症状图像。(C) 监测感染后12天内的存活率、体重变化及临床评分。(D) 采用qRT-PCR检测感染小鼠脾脏、肺脏及脑组织中的病毒滴度。(E) 采用酶联免疫吸附测定（enzyme-linked immunosorbent assay，ELISA）检测血清中的I型干扰素水平。

图3. 采用qRT-PCR检测Oasl1基因敲除对PRV感染过程中促炎介质的影响。评估感染小鼠脾脏、肺脏及脑组织中促炎介质的mRNA表达水平。

图4. Oasl1基因敲除小鼠在PRV感染后炎症反应减弱。采用夹心ELISA检测PRV感染小鼠脾脏、肺脏、脑组织及血清中促炎介质的蛋白表达水平。

图5. Oasl1基因敲除小鼠在PRV感染后病理损伤更轻微。展示PRV感染的Oasl1⁻/⁻小鼠与野生型小鼠的代表性苏木精-伊红染色（hematoxylin and eosin-stained，H&E）组织切片。箭头指示脾脏白髓萎缩及免疫细胞耗竭；星号标记肺脏支气管周围炎性细胞浸润；脑组织海马神经元可见明显的神经退行性病变。

图6. PRV感染的Oasl1⁻/⁻小鼠通过IRF7信号通路增加I型干扰素分泌。(A) 分别于感染后2、3、4天采用qRT-PCR检测野生型与Oasl1⁻/⁻小鼠脾脏、肺脏及脑组织中IRF7与IRF3的mRNA表达模式。(B) 对上述组织的蛋白裂解物进行蛋白质印迹（Western blot）分析，展示IRF7与IRF3的相对蛋白水平。

图7. OASL1敲除的髓系来源细胞在PRV感染后产生更多I型干扰素。从野生型与Oasl1⁻/⁻小鼠中分离原代骨髓源巨噬细胞（bone marrow-derived macrophages，BMDMs），分别以感染复数（MOI）为0.1、0.5、1及5的PRV在指定时间点感染。(A-B) 采用实时qRT-PCR检测细胞因子、趋化因子及IFN-α/β的mRNA表达水平。(C) 检测PRV感染的BMDMs中IRF7与IRF3的mRNA表达水平。(D) 以MOI=0.5的PRV感染BMDMs 24天后，检测IRF7与IRF3的蛋白表达水平。