Ma Boli, Jiang Yiqian, Li Nan, Wang Fengjie, Li Qian, Yue Huixian, Zhang Yanyan, Hu Rongliang, Miao Faming
College of Life Sciences, Ningxia University, Yinchuan 750021, China.
State Key Laboratory of Pathogen and Biosecurity, Key Laboratory of Prevention & Control for African Swine Fever and Other Major Pig Diseases, Ministry of Agriculture and Rural Affairs, Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Changchun 130122, China.
Emerg Microbes Infect. 2025 Sep 3:2555722. doi: 10.1080/22221751.2025.2555722.
African swine fever virus (ASFV) causes a lethal hemorrhagic disease in domestic pigs and represents a major threat to the global swine industry. Until now, no effective commercial vaccines or antiviral drugs are available for ASF control. In this study, we constructed a recombinant E120R gene-deleted virus, ASFV-ΔE120R, based on the highly virulent genotype II strain SY18, to investigate the biological role of the E120R gene. ASFV-ΔE120R exhibited impaired virion release and formed aberrant tubular structures, rendering viral particles more susceptible to neutralization by convalescent pig sera. RNA sequencing and RT-qPCR analyses revealed that ASFV-ΔE120R infection of porcine alveolar macrophages (PAMs) significantly upregulated the expression of chemokines (CCL5, , ), interferon-related genes (IRF7, , , , ), and the proinflammatory cytokine compared with ASFV-WT. In vivo safety evaluation demonstrated that piglets immunized with a single dose of 5 × 10⁶ TCID₅₀ of ASFV-ΔE120R exhibited no clinical signs of ASF and detectable viral nucleic acid in any organ upon necropsy at days 4, 7, 10, and 14 post-immunization. Notably, even after two rounds of blind cell passage using filtered tissue homogenates, no viral genome was detected. Furthermore, two immunizations at the same dose, administered 21 days apart, did not induce clinical signs or viral shedding during a 28-day observation period. Immunogenicity analysis showed that ASFV-ΔE120R elicited both p54-specific antibody production and IFN-γ-secreting peripheral blood mononuclear cell (PBMC) responses. Upon intramuscular challenge with a lethal dose (10 TCID₅₀) of parental ASFV SY18, two out of five immunized pigs (40%) survived. These survivors displayed increased levels of p54-specific antibodies and IFN-γ-secreting PBMCs. No viral nucleic acid or histopathological lesions were detected in their tissues, and tissue immunofluorescence revealed elevated frequencies of CD8⁺ IFN-γ⁺ T cells. Moreover, mRNA levels of antiviral genes (, , , , , and MX1) in the liver and bone marrow of surviving pigs were significantly upregulated compared with unvaccinated controls. In summary, ASFV-ΔE120R is fully attenuated and safe, and it induces both humoral and cellular immune responses. This study provides further insights into the role of pE120R in viral egress and immune evasion, and highlights its potential as a rational target for the development of live attenuated ASF vaccines.
非洲猪瘟病毒（ASFV）在家猪中引发致命的出血性疾病，对全球养猪业构成重大威胁。截至目前，尚无有效的商业疫苗或抗病毒药物可用于防控非洲猪瘟。在本研究中，我们基于高致病性II型毒株SY18构建了一种重组E120R基因缺失病毒ASFV-ΔE120R，以研究E120R基因的生物学作用。ASFV-ΔE120R的病毒粒子释放受损，并形成异常的管状结构，使病毒颗粒更容易被康复猪血清中和。RNA测序和RT-qPCR分析表明，与ASFV-WT相比，ASFV-ΔE120R感染猪肺泡巨噬细胞（PAM）显著上调了趋化因子（CCL5等等）、干扰素相关基因（IRF7等等）以及促炎细胞因子的表达。体内安全性评估表明，用单剂量5×10⁶ TCID₅₀的ASFV-ΔE120R免疫的仔猪在免疫后第4、7、10和14天进行尸检时，未表现出非洲猪瘟的临床症状，且任何器官中均未检测到可检测的病毒核酸。值得注意的是，即使使用过滤后的组织匀浆进行两轮盲传代，也未检测到病毒基因组。此外，相隔21天以相同剂量进行两次免疫接种后，在28天的观察期内未诱导出临床症状或病毒脱落。免疫原性分析表明，ASFV-ΔE120R引发了p54特异性抗体产生以及分泌IFN-γ的外周血单核细胞（PBMC）反应。在用致死剂量（10 TCID₅₀）的亲本ASFV SY18进行肌肉内攻毒后，五只免疫猪中有两只（40%）存活。这些存活猪的p54特异性抗体水平和分泌IFN-γ的PBMCs增加。在其组织中未检测到病毒核酸或组织病理学病变，组织免疫荧光显示CD8⁺ IFN-γ⁺ T细胞频率升高。此外，与未接种疫苗的对照组相比，存活猪肝脏和骨髓中抗病毒基因（等等）的mRNA水平显著上调。总之，ASFV-ΔE120R完全减毒且安全，可诱导体液免疫和细胞免疫反应。本研究进一步深入了解了pE120R在病毒释放和免疫逃逸中的作用，并突出了其作为减毒活疫苗开发合理靶点的潜力。
