Cell-adapted African swine fever virus Pig/HLJ/18 is highly attenuated but fails to induce immune protection against a challenge with its parental virus

doi:10.1016/j.jia.2025.03.017

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Wan Wang¹^*, Li Yin¹^*, Zhenjiang Zhang¹, Fan Liu¹, Xin Zhang¹, Zhigang Wang¹, Rui Zhao¹^{, 2}, Menglong Cao¹, Ying Zhang¹, Leilei Ding¹, Renqiang Liu¹, Encheng Sun¹, Xiangpeng Sheng¹, Weldu Tesfagaber¹, Fang Li¹, Xijun He¹, Zhigao Bu¹, Yuanmao Zhu¹^#, Dongming Zhao¹^{, 2#}

¹State Key Laboratory for Animal Disease Control and Prevention/National African Swine Fever Para-reference Laboratory/National High Containment Facilities for Animal Diseases Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China

²College of Veterinary Medicine, Xinjiang Agricultural University, Urumqi 830052, China

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l A cell-adapted virus, named HLJ18/BK33, was successfully obtained by serially passaging the ASFV Pig/HLJ/18 in BK2258 cells.

l ASFV HLJ18/BK33 exhibited clear CPE and efficient replication in BK2258 cells.

l ASFV HLJ18/BK33 was highly attenuated with significantly reduced biosafety risks.

Abstract
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摘要

非洲猪瘟（African swine fever，ASF）是由非洲猪瘟病毒（African swine fever virus，ASFV）引起的一种急性、出血性疾病，死亡率高达100%。ASF严重危害全球养猪业的健康发展，除越南外仍无可用的商品化疫苗和抗病毒药物。传代致弱是ASF疫苗研究的重要技术路径。本研究将ASFV Pig/HLJ/18株在野猪肾细胞（Boar kidney cells，BK2258）中连续传代成功获得了细胞适应株，命名为HLJ18/BK33株。该适应株在BK2258细胞中形成明显的细胞病变效应（CPE），并能稳定且高效地复制，同时在猪肺泡巨噬细胞（PAM）中保留高效复制能力。全基因组序列分析显示，与Pig/HLJ/18相比，HLJ18/BK33株在181,027-187,188位存在6162 bp的大片段缺失，同时存在4个单核苷酸缺失，导致移码突变，使3个开放阅读框（ORF）（ASFV_G_ACD_00120、ASFV_G_ACD_00350和A179L）截短表达，2个ORF（MGF_110-14L与MGF_110-11L）融合表达。此外，还有4个基因发生错义突变，导致单个氨基酸的改变。将HLJ18/BK33株以10⁶TCID₅₀/头的剂量肌肉注射接种5头6-8周龄的SPF猪，28天后所有猪均健康存活，体温正常，未见异常临床症状，表明其毒力完全丧失。用亲本毒株Pig/HLJ/18进行攻毒，结果4头猪出现持续性发热和ASF相关的临床症状，并在攻毒后13天内死亡；1头猪出现一过性体温升高，在攻毒后28天观察期仍健康存活。以上结果证明，HLJ18/BK33株高度减毒，但不能提供针对亲本强毒株的免疫保护。本研究获得的细胞适应株HLJ18/BK33不具备开发疫苗的潜力，但其在BK2258细胞中稳定复制并形成典型的CPE且高度减毒，大大降低了生物安全风险，是病毒与宿主相互作用、抗病毒药物筛选、诊断方法的研制和病毒生物学特性研究的理想毒株。

Abstract

African swine fever (ASF) is an acute, hemorrhagic disease caused by the African swine fever virus (ASFV), with a mortality up to 100%. The disease poses a seriously threat to the global swine industry, yet no commercial vaccines or antiviral drugs are available other than in Vietnam. ASFV attenuation through serial passages is a key approach for vaccine development. In this study, a cell-adapted virus, named HLJ18/BK33, was successfully generated by serially passaging the ASFV Pig/HLJ/18 in wild boar kidney cells (BK2258). This adapted virus exhibited clear cytopathic effects (CPE) and replicated stably and efficiently in BK2258 cells and porcine alveolar macrophages. Whole-genome sequence analysis revealed that, compared with the Pig/HLJ/18 virus, HLJ18/BK33 had a large deletion of 6162 bp from sites 181,027 to 187,188, and four single nucleotide deletions that led to frameshift mutations, resulting in the truncated expression of three open reading frames (ORFs) (ASFV_G_ACD_00120, ASFV_G_ACD_00350, and A179L), and the fusion expression of two ORFs (MGF_110-14L and MGF_110-11L). Additionally, four genes exhibited missense mutations, leading to single amino acid changes. Five pigs intramuscularly inoculated with 10⁶ TCID₅₀ of HLJ18/BK33 remained healthy with normal body temperatures and no clinical signs, indicating a high attenuation of virulence for HLJ18/BK33 in pigs. Upon challenge with the parental Pig/HLJ/18 virus, four of the five inoculated pigs developed persistent high fever and ASF-related clinical signs and died within 13 days of the challenge; the remaining pig developed transient fever but survived until the end of the observation period. These results indicate that the HLJ18/BK33 virus is highly attenuated but cannot induce protection against the parental virulent virus. Even though the HLJ18/BK33 virus is not a good vaccine candidate, its stable replication and distinct CPE in BK2258 cells as well as its low biosafety risk make it a valuable resource for studies on virus-host interactions, antiviral drug screening, diagnostic methods, and biological characteristics.

Keywords: African swine fever virus cell-adapted virus attenuation protection

Online: 22 March 2025

Fund:

This work was supported by Central public-interest Scientific Institution Basal Research Fund (CAAS-ZDRW202409), Innovation Program of Chinese Academy of Agricultural Sciences (CAAS-CSLPDCP-202301), the National Key Research and Development Program of China (2022YFD1800604, 2021YFD1800101), and the Heilongjiang Provincial Natural Science Foundation of China (JQ2023C005).

About author: #Correspondence Yuanmao Zhu, E-mail: zhuyuanmao@caas.cn; Dongming Zhao, E-mail: zhaodongming@caas.cn *Wan Wang and Li Yin equally contributed to this study.

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Wan Wang, Li Yin, Zhenjiang Zhang, Fan Liu, Xin Zhang, Zhigang Wang, Rui Zhao, Menglong Cao, Ying Zhang, Leilei Ding, Renqiang Liu, Encheng Sun, Xiangpeng Sheng, Weldu Tesfagaber, Fang Li, Xijun He, Zhigao Bu, Yuanmao Zhu, Dongming Zhao. 2025. Cell-adapted African swine fever virus Pig/HLJ/18 is highly attenuated but fails to induce immune protection against a challenge with its parental virus. Journal of Integrative Agriculture, Doi:10.1016/j.jia.2025.03.017

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