Rescue of a porcine anellovirus ( Torque teno sus virus 2 ) from cloned genomic 1 DNA in pigs

24 Anellovirus is a group of single-stranded circular DNA viruses infecting human and other animal 25 species. Animal models combined with reverse genetics systems of anellovirus have not been 26 developed. We report here the construction and initial characterization of full-length DNA clones 27 of a porcine anellovirus, Torque teno sus virus 2 (TTSuV2), in vitro and in vivo. We first 28 demonstrated that five cell lines including PK-15 cells are free of TTSuV1 or TTSuV2 29 contamination, as determined by a real-time PCR and an immunofluorescence assay (IFA) using 30 anti-TTSuV antibody. Recombinant plasmids harboring monomeric or tandem-dimerized 31 genomic DNA of TTSuV2 from the USA and Germany were constructed. Circular TTSuV2 32 genomic DNA with or without introduced genetic markers and tandem-dimerized TTSuV2 33 plasmids were transfected into PK-15 cells, respectively. Splicing of viral mRNAs was identified 34 in transfected cells. Expression of TTSuV2-specific ORF1 in cell nuclei, especially in nucleoli, 35 was detected by IFA. However, evidence of productive TTSuV2 infection was not observed in 36 12 different cell lines transfected with the TTSuV2 DNA clones. Transfection with circular DNA 37 from a TTSuV2 deletion mutant did not produce ORF1 protein, suggesting that the observed 38 ORF1 expression is driven by TTSuV2 DNA replication in cells. Pigs inoculated with either the 39 tandem-dimerized clones or circular genomic DNA of the U.S. TTSuV2 developed viremia, and 40 the introduced genetic markers were retained in viral DNA recovered from the sera of infected 41 pigs. The availability of an infectious DNA clone of TTSuV2 will facilitate future study of 42 porcine anellovirus pathogenesis and biology. 43