Identification and Characterization of Novel SPHINX/BMMF-like DNA Sequences Isolated from Non-Bovine Foods

Abstract

Sixteen novel circular rep-encoding DNA sequences with high sequence homologies to previously described SPHINX and BMMF sequences were isolated for the first time from non-bovine foods (pork, wild boar, chicken meat, Alaska pollock, pangasius, black tiger shrimp, apple, carrot, and sprouts from alfalfa, radish, and broccoli). The phylogenetic analysis of the full-length circular genomes grouped these together with previously described representatives of SPHINX/BMMF group 1 and 2 sequences (eight in each group). The characterization of genome lengths, genes present, and conserved structures confirmed their relationship to the known SPHINX/BMMF sequences. Further analysis of iteron-like tandem repeats of SPHINX/BMMF group 1-related genomes revealed a correlation with both full-length sequence tree branches as well as Rep protein sequence tree branches and was able to differentiate subtypes of SPHINX/BMMF group 1 members. For the SPHINX/BMMF group 2 members, a distinct grouping of sequences into two clades (A and B) with subgroups could be detected. A deeper investigation of potential functional regions upstream of the rep gene of the new SPHINX/BMMF group 2 sequences revealed homologies to the dso and sso regions of known plasmid groups that replicate via the rolling circle mechanism. Phylogenetic analyses were accomplished by a Rep protein sequence analysis of different ssDNA viruses, pCRESS, and plasmids with the known replication mechanism, as this yielded deeper insights into the relationship of SPHINX/BMMF group 1 and 2 Rep proteins. A clear relation of these proteins to the Rep proteins of plasmids could be confirmed. Interestingly, for SPHINX/BMMF group 2 members, the relationship to rolling circle replication plasmids could also be verified. Furthermore, a relationship of SPHINX/BMMF group 1 Rep proteins to theta-replicating plasmid Reps is discussed.

Current Research on Small Circular Molecules: A Comprehensive Overview on SPHINX/BMMF

“…Specifically, the relation of SPHINX/BMMF group 2 to rolling circle replicating plasmids fits with the detected conserved dso/sso regions mentioned above, because these dso/sso regions were also found in rolling circle-type plasmids [19]. The question which then remains is if SPHINX/BMMFs are plasmids, in which host are these contained?…”

“…Clearly, these are unsolved questions which should be addressed in further studies. [10,13,14,18,19,30]. Schematic image of the regulatory region (framed), which potentially includes oriT (green) [19], AT-rich region (potential origin of replication, grey) [19,31] and iteron-like tandem repeats (ITRs, orange) [19].…”

“…Then, in 2019, a press conference held by the German Cancer Research Center (DKFZ) attracted attention, in which the BMMFs were presented as “factors” or “novel pathogens” originating from European domestic cattle ( Bos taurus ) and which were stated to be involved in the development of cancer [ 11 ]. As there were considerable doubts regarding the interpretation of existing data by the DKFZ, several further studies were subsequently published by various groups [ 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 ].…”

Bovine Meat and Milk Factor-like Sequences Are Frequently Detected in Renal Cell Carcinoma Tissues

“…All previous studies testing for the presence of BMMF DNA have used a combination of rolling circle amplification (RCA) and subsequent BMMF DNA PCR. Of interest, two recent studies have reported the finding of BMMF DNA in an amazingly broad spectrum of food sources, possibly indicating that BMMFs are not solely of bovine origin and are widespread [8,9]. Although these findings await confirmation by others, the sheer finding of exogenous DNA in human tissues and body fluids, which is capable of replicating in HEK cells, is of significant concern.…”

“…It has been proposed that non-pathogenic persistent infections in animals and their products might be pathogenic if transmitted to humans [5]. BMMF DNAs have also been identified in the milk from water buffaloes, sheep, and goats, among others [6][7][8][9][10].…”