基于新一代测序技术精准确定复制缺陷型痘苗病毒天坛株全基因组序列及其开放阅读框组成

Accurate determination of the whole genome sequencing and open reading frames composition of non-replicating Tiantan strain of vaccinia virus based on novel long read sequencing platform

目的应用新一代长读长测序技术精准确定复制缺陷型痘苗病毒天坛株(non-replicating Tiantan strain of vaccinia virus,NTV)全基因组序列及其开放阅读框(ORFs)组成。方法基于本实验室储备的NTV,在鸡胚成纤维细胞上进行扩增并纯化,提取NTV全长基因组核酸。PacBio HiFi测序从头组装获取NTV全长基因组序列。以同源注释的策略确定其ORF组成,并分析其与已知复制缺陷型痘苗病毒毒株ORF异同。结果NTV全长共171729 bp,GC含量为33%,其独特的倒置末端重复(ITR)区域包含发夹结构、两个串联重复区域及3个非重复区域。NTV共有166个ORFs,与第三代天花病毒疫苗改良安卡拉株(MVA-BN)和复制缺陷型哥本哈根株(NYVAC)株相比,主要差异位于ITR及其周围区域,3个毒株共同享有138个ORFs,NTV特有6个编码与病毒逃避宿主抗病毒反应相关的ORFs。结论通过三代测序技术精准确定NTV全基因组序列及ORFs组成,并揭示其与其他复制缺陷型痘苗病毒毒株的异同,为新一代猴痘疫苗和痘苗病毒载体研发应用提供重要参考。

Objective To accurately ascertain the whole genome sequencing and the composition of open reading frames(ORFs)of non-replicating Tiantan strain of vaccinia virus(NTV)using next-generation long-read sequencing technology.Methods NTV,obtained from our laboratory stock,was amplified and purified on chicken embryo fibroblast cells(CEFs),and the full-length genomic nucleic acid of NTV was extracted.The PacBio HiFi sequencing platform was utilized for de novo assembly to obtain the complete genomic sequence of NTV.Using a homology annotation strategy,we identified its ORF composition and compared it with known non-replicating vaccinia virus strains.Results The total length of NTV′s genome was 171729 bp,with a GC content of 33%.Its unique inverted terminal repeat(ITR)region comprised hairpin structures,two tandem repeat regions,and three non-repeat regions.NTV contained 166 ORFs,with major differences observed in the ITR and its surrounding regions when compared to MVA-BN and NYVAC.These three strains shared a common set of 138 ORFs.NTV encoded six unique ORFs related to virus evasion of host antiviral response.Conclusions This study accurately determines the whole genome sequencing and ORFs composition of NTV,and reveals its similarities and differences with other replication-deficient vaccinia virus strains,which pave a way for the development and application of the next generation of monkeypox vaccines and novel viral vectors.