Genomic Sequence and Virulence of Clonal Isolates of Vaccinia Virus Tiantan, the Chinese Smallpox Vaccine Strain

Despite the worldwide eradication of smallpox in 1979, the potential bioterrorism threat from variola virus and the ongoing use of vaccinia virus (VACV) as a vector for vaccine development argue for continued research on VACV. In China, the VACV Tiantan strain (TT) was used in the smallpox eradication campaign. Its progeny strain is currently being used to develop a human immunodeficiency virus (HIV) vaccine. Here we sequenced the full genomes of five TT clones isolated by plaque purification from the TT (752-1) viral stock. Phylogenetic analysis with other commonly used VACV strains showed that TT (752-1) and its clones clustered and exhibited higher sequence diversity than that found in Dryvax clones. The ∼190 kbp genomes of TT appeared to encode 273 open reading frames (ORFs). ORFs located in the middle of the genome were more conserved than those located at the two termini, where many virulence and immunomodulation associated genes reside. Several patterns of nucleotide changes including point mutations, insertions and deletions were identified. The polymorphisms in seven virulence-associated proteins and six immunomodulation-related proteins were analyzed. We also investigated the neuro- and skin- virulence of TT clones in mice and rabbits, respectively. The TT clones exhibited significantly less virulence than the New York City Board of Health (NYCBH) strain, as evidenced by less extensive weight loss and morbidity in mice as well as produced smaller skin lesions and lower incidence of putrescence in rabbits. The complete genome sequences, ORF annotations, and phenotypic diversity yielded from this study aid our understanding of the Chinese historic TT strain and are useful for HIV vaccine projects employing TT as a vector.

尽管1979年全球已实现天花根除，但天花病毒（variola virus）带来的潜在生物恐怖威胁，以及痘苗病毒（vaccinia virus, VACV）作为疫苗开发载体的持续应用，均要求持续开展针对VACV的相关研究。在中国，天坛株痘苗病毒（VACV Tiantan strain, TT）曾被用于天花根除运动。其后代毒株目前正被用于开发人类免疫缺陷病毒（human immunodeficiency virus, HIV）疫苗。本研究对从TT (752-1)病毒毒种中经噬斑纯化分离得到的5个TT克隆的全基因组进行了测序。通过与其他常用VACV毒株开展系统发育分析，结果显示TT (752-1)及其克隆聚为同一分支，且序列多样性高于Dryvax克隆。TT的基因组全长约190 kbp，共编码273个开放阅读框（open reading frames, ORFs）。位于基因组中部的开放阅读框相较于基因组两端的序列更为保守，而基因组两端存在大量与毒力及免疫调节相关的基因。本研究鉴定出多种核苷酸变异模式，包括点突变、插入与缺失。对7个毒力相关蛋白及6个免疫调节相关蛋白的多态性进行了分析。此外，本研究分别在小鼠与家兔体内评估了TT克隆的神经毒力与皮肤毒力。结果表明，TT克隆的毒力显著低于纽约市卫生局（New York City Board of Health, NYCBH）毒株：小鼠实验中表现为体重下降幅度更小、发病率更低；家兔实验中则表现为皮肤损伤更小、坏疽发生率更低。本研究获得的完整基因组序列、开放阅读框注释信息及表型多样性数据，有助于深入理解中国经典天坛株VACV，同时对以TT为载体的HIV疫苗开发项目具有重要应用价值。