Department of Biomedical Sciences and Pathobiology, College of Veterinary Medicine, Virginia Polytechnic Institute and State University (Virginia Tech), Blacksburg, Virginia, USA.
J Virol. 2012 Jun;86(11):6042-54. doi: 10.1128/JVI.00175-12. Epub 2012 Apr 4.
Anelloviruses are a group of single-stranded circular DNA viruses infecting humans and other animal species. Animal models combined with reverse genetic systems of anellovirus have not been developed. We report here the construction and initial characterization of full-length DNA clones of a porcine anellovirus, torque teno sus virus 2 (TTSuV2), in vitro and in vivo. We first demonstrated that five cell lines, including PK-15 cells, are free of TTSuV1 or TTSuV2 contamination, as determined by a real-time PCR and an immunofluorescence assay (IFA) using anti-TTSuV antibodies. Recombinant plasmids harboring monomeric or tandem-dimerized genomic DNA of TTSuV2 from the United States and Germany were constructed. Circular TTSuV2 genomic DNA with or without introduced genetic markers and tandem-dimerized TTSuV2 plasmids were transfected into PK-15 cells, respectively. Splicing of viral mRNAs was identified in transfected cells. Expression of TTSuV2-specific open reading frame 1 (ORF1) in cell nuclei, especially in nucleoli, was detected by IFA. However, evidence of productive TTSuV2 infection was not observed in 12 different cell lines transfected with the TTSuV2 DNA clones. Transfection with circular DNA from a TTSuV2 deletion mutant did not produce ORF1 protein, suggesting that the observed ORF1 expression is driven by TTSuV2 DNA replication in cells. Pigs inoculated with either the tandem-dimerized clones or circular genomic DNA of U.S. TTSuV2 developed viremia, and the introduced genetic markers were retained in viral DNA recovered from the sera of infected pigs. The availability of an infectious DNA clone of TTSuV2 will facilitate future study of porcine anellovirus pathogenesis and biology.
圆环病毒是一组感染人类和其他动物物种的单链环状 DNA 病毒。尚未建立感染圆环病毒的动物模型和反向遗传学系统。我们在此报告了猪圆环病毒，即扭结藤本病毒 2（TTSuV2）全长 DNA 克隆的体外和体内构建及初步特征。我们首先通过实时 PCR 和使用抗 TTSuV 抗体的免疫荧光分析（IFA），证明包括 PK-15 细胞在内的五种细胞系均未受到 TTSuV1 或 TTSuV2 的污染。构建了来自美国和德国的 TTSuV2 单体或串联二聚化基因组 DNA 的重组质粒。分别将带有或不带有引入遗传标记的环状 TTSuV2 基因组 DNA 和串联二聚化 TTSuV2 质粒转染到 PK-15 细胞中。在转染的细胞中鉴定到病毒 mRNA 的剪接。通过 IFA 在细胞核中检测到 TTSuV2 特异性开放阅读框 1（ORF1）的表达，特别是在核仁中。然而，在用 TTSuV2 DNA 克隆转染的 12 种不同细胞系中均未观察到有效的 TTSuV2 感染。用 TTSuV2 缺失突变体的环状 DNA 转染不会产生 ORF1 蛋白，表明观察到的 ORF1 表达是由细胞内 TTSuV2 DNA 复制驱动的。用串联二聚化克隆或美国 TTSuV2 的环状基因组 DNA 接种的猪出现了病毒血症，并且引入的遗传标记保留在感染猪血清中回收的病毒 DNA 中。TTSuV2 感染性 DNA 克隆的可用性将有助于未来研究猪圆环病毒的发病机制和生物学。
