Rescue of a Porcine Anellovirus (Torque Teno Sus Virus 2) from Cloned Genomic DNA in Pigs

Abstract

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.

Serological Profile of Torque Teno Sus Virus Species 1 (TTSuV1) in Pigs and Antigenic Relationships between Two TTSuV1 Genotypes (1a and 1b), between Two Species (TTSuV1 and -2), and between Porcine and Human Anelloviruses

“…The truncated ORF1 proteins were expressed in both the nucleus and cytoplasm of the transfected cells (Fig. 7), which was different from what we found for cells transfected with full-length TTSuV DNA clones (16), probably due to the lack of most of the putative nuclear localization signals (NLSs) located at the N-terminal part of ORF1 in the truncated genes (by computer analysis) (data not shown). Table 2 summarizes the results of the cross-reactive immunostaining study.…”

“…Samples were screened for anti-TTSuV1a or anti-TTSuV1b IgG seropositivity by serum WB analysis using purified 1a-ORF1 or 1b-ORF1 as the antigen, respectively. A TTSuV2-seropositive and TTSuV1/TTSuV2 DNA-positive porcine serum sample (16) showed reactivity with the 1a-ORF1 and the 1b-ORF1 antigens, as the ϳ40-kDa band was present in the WB analysis ( Fig. 2A, rightmost two lanes).…”

“…TTSuV comprises two species, TTSuV1 and TTSuV2, each with a genomic size of approximately 2.8 kb and with at least four putative open reading frames (ORFs), ORF1, ORF2, ORF1/1, and ORF2/2 (15,16,29). ORF1 of TTSuV is believed to encode a capsid protein with the largest size relative to those of the four predicted viral proteins (5,14,15).…”

Vaccination of pigs reduces Torque teno sus virus viremia during natural infection

“…The predominant transcripts detected in vitro and in vivo of ORF1 are spliced and full-length ORF1 transcripts have not been detected [5,6]. Splicing of TTSuVk2a ORF1 results in three protein isoforms (ORF1-A, -B and -C) and the amino acid composition between them varies, depending on the splicing site used [5,6].…”

“…ORF3 can only be translated after splicing of the mRNA precursor, and splicing of the ORF1 mRNA transcript results in different protein isoforms [5,6], as is also indicated in human TTV [7,8]. The predominant transcripts detected in vitro and in vivo of ORF1 are spliced and full-length ORF1 transcripts have not been detected [5,6]. Splicing of TTSuVk2a ORF1 results in three protein isoforms (ORF1-A, -B and -C) and the amino acid composition between them varies, depending on the splicing site used [5,6].…”