Volume 119, Issue 5, Pages 1228-1235 (November 2000) JC virus DNA sequences are frequently present in the human upper and lower gastrointestinal tract  Luigi Ricciardiello, Luigi Laghi, Pradeep Ramamirtham, Christina L. Chang, Dong K. Chang, Ann E. Randolph, C.Richard Boland  Gastroenterology  Volume 119, Issue 5, Pages 1228-1235 (November 2000) DOI: 10.1053/gast.2000.19269 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 PCR strategy. (A) Amplification of JCV-TAg 520-bp fragment using primer T1 or T1D (degenerate) on the sense strand and T2 on the antisense strand at the N-terminus of the translation product (which reads from right to left). The PCR is blotted onto a nylon membrane and hybridized with the oligoprobe 90PRO or 90 PRO-D. (B) Of the first PCR reaction, 2% is amplified with nested primers 90PRO or 90PRO-D (degenerate) on the sense strand and ESPB or ESPB-D on the antisense strand to confirm Southern blot results, obtaining a 429-bp fragment. Primer sequences are shown in the box. Gastroenterology 2000 119, 1228-1235DOI: (10.1053/gast.2000.19269) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 JCV sequences demonstrated in GI mucosal DNA. (Panel 1) Ethidium bromide–stained 2.5% agarose gel showing the amplification of 520-bp DNA corresponding to the N-terminus of the JCV Mad1 TAg. Although sample 17E displayed a band of approximately 520 bp, this result was not confirmed by Southern analysis and nested PCR, indicating that this sample did not harbor the viral sequence. (Panel 2) Southern blot analysis of the 520-bp fragment using a radiolabeled nested primer confirming the amplification shown in panel 1. (Panel 3) Ethidium bromide–stained 2.5% agarose gel showing amplification of a 429-bp fragment. Upper GI tract samples are on the left, and colorectal samples are on the right; bl, blank template control; mw, size marker lane. Gastroenterology 2000 119, 1228-1235DOI: (10.1053/gast.2000.19269) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 SSCP analysis showing sequence heterogeneity. (A) SSCP analysis of the 429-bp JCV-TAg DNA fragment amplified from DNA samples collected from the same patient; 5T is from transverse colon, 5S is from the sigmoid colon, and 5R is from the rectum. The heterogeneity found at SSCP analysis was confirmed by sequencing the amplified fragment. (B) Sequencing of the antisense strand. 5T and 5R had mutations at 4 and 3 nucleotide sites, respectively, and 5S was a perfect match with the corresponding sequence of JCV Mad1 strain. 5T had mutations at nucleotide positions 4757 (A→G), 4769 (A→G), 4832 (T→C), and 4879 (T→C) (data not shown). Although 5R shared mutations at nucleotides 4757 and 4832 with 5T, this sample did not have mutations at 4879 and 4769 but was mutated at 4705 (A→C) (data not shown). Gastroenterology 2000 119, 1228-1235DOI: (10.1053/gast.2000.19269) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 Sequence analysis of the TAg protein. The PCR product covered much of the t/T common exon (residues 23–82) and almost all of the small t coding region (residues 23–175). The top panel depicts the domains and their functions on SV40 T/t exon and small t alone. Residues of particular interest are compared with the corresponding regions in SV40 and BKV. Gastroenterology 2000 119, 1228-1235DOI: (10.1053/gast.2000.19269) Copyright © 2000 American Gastroenterological Association Terms and Conditions