Torque Teno Sus Virus (TTSuV) in Cell Cultures and Trypsin

Abstract

Torque teno sus virus (TTSuV), a member of the family Anelloviridae, is a single-stranded, circular DNA virus, widely distributed in swine populations. Presently, two TTSuV genogroups are recognized: Torque teno sus virus 1 (TTSuV1) and Torque teno sus virus 2 (TTSuV2). TTSuV genomes have been found in commercial vaccines for swine, enzyme preparations and other drugs containing components of porcine origin. However, no studies have been made looking for TTSuV in cell cultures. In the present study, a search for TTSuV genomes was carried out in cell culture lineages, in sera used as supplement for cell culture media as well as in trypsin used for cell disaggregation. DNA obtained from twenty-five cell lineages (ten from cultures in routine multiplication and fifteen from frozen ampoules), nine samples of sera used in cell culture media and five batches of trypsin were examined for the presence of TTSuV DNA. Fifteen cell lineages, originated from thirteen different species contained amplifiable TTSuV genomes, including an ampoule with a cell lineage frozen in 1985. Three cell lineages of swine origin were co-infected with both TTSuV1 and TTSuV2. One batch of trypsin contained two distinct TTSuV1 plus one TTSuV2 genome, suggesting that this might have been the source of contamination, as supported by phylogenetic analyses of sequenced amplicons. Samples of fetal bovine and calf sera used in cell culture media did not contain amplifiable TTSuV DNA. This is the first report on the presence of TTSuV as contaminants in cell lineages. In addition, detection of the viral genome in an ampoule frozen in 1985 provides evidence that TTSuV contamination is not a recent event. These findings highlight the risks of TTSuV contamination in cell cultures, what may be source for contamination of biological products or compromise results of studies involving in vitro multiplied cells.

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

“…Furthermore, screening of seven additional commonly used cell lines also yielded negative results, as determined by IFA detection (data not shown), indicating that TTSuV contamination in cell cultures is probably not as common as we originally thought. Our result was distinct from that of a recent study by a Brazilian group that reported TTSuV DNA contamination in 15 of 25 cell lines (41). In that study, the five cell lines that were also used in our study, i.e., PK-15, ST, BHK-21, Vero, and MA-104 cells (from which the MARC-145 cell line is derived) were shown to have detectable TTSuV1 and/or TTSuV2 sequences by the use of a one-round duplex PCR assay (41).…”

“…Our result was distinct from that of a recent study by a Brazilian group that reported TTSuV DNA contamination in 15 of 25 cell lines (41). In that study, the five cell lines that were also used in our study, i.e., PK-15, ST, BHK-21, Vero, and MA-104 cells (from which the MARC-145 cell line is derived) were shown to have detectable TTSuV1 and/or TTSuV2 sequences by the use of a one-round duplex PCR assay (41). It is unclear why there is such a major discrepancy between the results of our study and those obtained by the Brazilian group.…”

“…Theoretically, the possibility of TTSuV contamination in cell cultures is very low, since anellovirus has been shown to be extremely difficult to propagate in vitro. The present study utilized the (i) more sensitive qPCR assay (compared to the one-round PCR used by Teixeira et al [41]); (ii) the IFA; and (iii) transfection of circular TTSuV genomic DNA into the cells as the positive control (see below) to demonstrate the absence of TTSuV at both the DNA and amino acid levels in 12 representative cell lines, including four of pig origin (PK-15, ST, 3D4/31, and IPEC-J2). Therefore, based on the results of this study, we conclude that, contrary to what some may believe, there is very little, if any, endogenous TTSuV contamination in well-established continuous cell lineages.…”

The Art of Animal Cell Culture for Virus Isolation

“…For example, porcine trypsin has been traced as the source of Torque teno sus virus (TTSuV), a member of the family Anelloviridae that is a contaminant of many cell lines. Indeed, TTSuV was found in fifteen cell lineages, originating from thirteen different species, and its presence in the cell lines probably traced to the use of porcine trypsin during the propagation of those cells [60]. Anelloviruses are small DNA viruses that replicate within the nuclei of infected cells, and CPE due to their presence have not been well described at present.…”

“…However, as typical of these cases, the findings are not published and thus the information not widely disseminated. Reoviruses however can have wide host range and infect many different cell lines [60,64].…”