Fig 1.
Trunk location (A) and gross appearance (B) of nodules present on a wild juvenile African elephant in Botswana (BW1 Male NOD 1–4). Samples collected under Elephants Without Borders Botswana Research Permit/ \#WT8/36/4XV(41). Location: Kalwesi Water Hole, Chobe National Park, Botswana. Red arrows indicate nodular position on the trunk; white arrow indicates the nodule from which the DNA was extracted to create the recombinant plasmid.
Fig 2.
PCR analysis of biological specimens.
PCR analysis of elephant trunk nodules in Botswana and South Africa to detect: A) AelPyV-1 large tumor antigen (expected size 730 bp) and B) viral capsid protein 1 (expected size: 587 bp).
Table 1.
Primers used in this study.
Table 2.
Trunk nodule biopsies from wild elephants (collected by Virginia R. Pearson).
Fig 3.
The T antigen-encoding region was amplified from elephant BW1 MaleNOD1, then inserted into the plasmid pEGFP-N1 (accession #U55762). DH5A competent bacteria were transformed and grown on kanamycin-resistant LB and amplified in a PCR reaction. PCR products were analyzed for expected band size of 2265 bp. Appropriate size gel bands were sequenced with standard primers (CMV forward and SV40 reverse to verify the presence and integrity of the AeIPyV-1 early region sequence.
Fig 4.
Predicted amino acid alignment.
PAelPyV-1-Tag clones 2, 4, 7 and 8 partial nucleotide (A) and amino acid (B) alignment compared to the AelPyV-1 index genome (GenBank accession # NC-0225191.9).
Table 3.
FFPE Trunk nodule biopsies (preserved by Elliot Jacobsen, DVM).
Table 4.
Complete set of Cohort 3 specimens (collected by Virginia R. Pearson).
Table 5.
Aggregated percentage of samples positive for AelPyV-1.
Fig 5.
Successful transfection of elephant endothelial cells.
A: Duncan P.1 umbilical cord primary endothelial cells. B: #1DuncanAMAXA P.4 endothelial cells expressing pMaxGFP. C: #1DuncanAMAXA cells at 105 days, immunostained for vonWillebrand Factor. Photos taken with an Olympus BX50, 20x original magnification.
Fig 6.
Detection of PCR products from other elephant primary endothelial cultures.
Amplification of both AelPyV-1 LTag and STag DNA sequences from endothelial cells derived from an African elephant (Fitz, born 2019, Louisville, KY USA).
Fig 7.
AelPyV1-LTag antigen expression in African elephant fibroblasts.
A. African elephant fibroblasts were transfected with pAelPyV-1Tag (pcDNA3.1)+mCherry or PAelPyV-1-Tag (pcDNA3.1)+mCherry-LTag (encoding the entire early region of AeIPyV-1). 18 hours post-transfection, mCherry expression was assessed by immunofluorescence. B. 24 hours post-transfection, cells were harvested and lysed. Western blots were performed using antibody to mCherry. Predicted molecular weights: mCherry alone: 28.8 kDa; mCherry-STag: 48 kDa; mCherry-LTag: 104.6 kDa. The fluorescent fusion construct mCherry-LTA of original pAelPyV-1-Tag appeared to interfere with protein expression of the spliced LTag region, but not with expression of STag.
Fig 8.
Verification of transcription of the LTag and STag antigens in transformed cells.
The PAelPyV-1-Tag transfected cells expressed both LTag (expected 150–250 bp; white dot) and STag (expected 400–500 bp; black dot). Cells transfected with PAelPyV-1-Tag+mCherry-LTag expressed only STag.