Volume 127, Issue 3, Pages 764-776 (September 2004) Analysis of hepatitis C virus quasispecies transmission and evolution in patients infected through blood transfusion  Tomasz Laskus, Jeffrey Wilkinson, Juan F. Gallegos-Orozco, Marek Radkowski, Debra M. Adair, Marek Nowicki, Eva Operskalski, Zelma Buskell, Leonard B. Seeff, Hugo Vargas, Jorge Rakela  Gastroenterology  Volume 127, Issue 3, Pages 764-776 (September 2004) DOI: 10.1053/j.gastro.2004.06.005 Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 1 Representative SSCP and HMA analysis in 2 patients who developed self-limited acute hepatitis after receiving HCV-infected blood. SSCP analysis was conducted on the 5′UTR and E2/HVR1, whereas HMA was limited to the latter region. HMA analysis was used to calculate entropy and MMS values. The band patterns for both regions were stable, indicating the same quasispecies were present throughout the infection. However, in patient 0102, minor changes occurred just before clearance. w, week(s). ♦, Entropy; ■, MMS. Gastroenterology 2004 127, 764-776DOI: (10.1053/j.gastro.2004.06.005) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 2 Representative SSCP and HMA analysis in 2 patients who remained chronically infected for 15 years after receiving HCV-infected blood. SSCP analysis was conducted on the 5′UTR and E2/HVR1, and HMA was performed on the latter region only. HMA was the basis for entropy and MMS calculations. The band pattern for the 5′UTR was stable, however, it changed over time for the E2/HVR1. w, week(s); y, years. ♦, Entropy; ■, MMS. Gastroenterology 2004 127, 764-776DOI: (10.1053/j.gastro.2004.06.005) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 3 Intraquasispecies diversity measured by entropy and MMS values derived from analysis of signal distribution in HMA gels in patients with resolving acute infection and in patients with acute infection progressing to chronicity. Both parameters were relatively stable in patients who cleared infection but changed in those who remained chronically infected. Gastroenterology 2004 127, 764-776DOI: (10.1053/j.gastro.2004.06.005) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 4 Comparison of changes in entropy and MMS values calculated from HMA gels in patients with self-limited acute hepatitis and in patients progressing to chronic infection. □, Resolved; ▩, chronic. Gastroenterology 2004 127, 764-776DOI: (10.1053/j.gastro.2004.06.005) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 5 Comparison of number of SSCP bands (complexity) (A) and changes in complexity (B) in patients with acute self-limited hepatitis and in patients with acute hepatitis progressing to chronicity. □, Resolved; ▩, chronic. Gastroenterology 2004 127, 764-776DOI: (10.1053/j.gastro.2004.06.005) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 6 Phylogenetic reconstructions showing the evolutionary relationship of HVR1/E2 sequences in patients 0102 and 0203. Bootstrap proportions of greater than 50 of 100 bootstrap replicates are shown at appropriate branch points. The taxa are labeled A, B, C, and D, indicating donor and recipient 9, 12, and 15 weeks after transfusion in patient 0102 and transmitting donor A and recipient 15, 49, and 60 weeks after transfusion in patient 0203. In patient 0102 the viral population is relatively monophyletic over the time of infection until the last sample just before viral clearance, whereas in patient 0203 a closely related monophyletic population is followed by 2 sequential shifts corresponding to changes seen in SSCP and HMA gel analysis. MMS and entropy values are calculated from HMA gels and mean distances within quasispecies calculated from cloned sequences according to the Kimura 2-parameter model. ○, Entropy; ●, MMS.40 Gastroenterology 2004 127, 764-776DOI: (10.1053/j.gastro.2004.06.005) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 7 SSCP analysis of cloned sequences. Two different pairs of plasmid vectors containing 5′UTR fragments 251 nucleotides in length and differing by a single (0.4%) nucleotide substitution (pairs a and b, and c and d) were PCR amplified and analyzed by SSCP (A, B). As seen, the sequence differences affected the mobility of PCR products because band patterns within each pair were clearly different. To determine the sensitivity limit with respect to detection of a mixture of different sequences, cloned 5′UTR fragments differing by one nucleotide substitution (B) and HVR1/E2 fragments 169 nucleotides in length and differing by (C) 7 (4%) and (D) 28 (16.6%) substitutions, were amplified by PCR, quantified by OD reading and gel analysis, and mixed at various proportions. As seen, the band representing the minor population was visible when constituting only (B-D) 3%, or even (B) 1.5%, of the total population. Gastroenterology 2004 127, 764-776DOI: (10.1053/j.gastro.2004.06.005) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 8 HMA analysis of cloned HVR1/E2 fragments as compared with sequencing. (A) Two E2/HVR1 fragments differing by 28 nucleotide substitutions (16.6%) were amplified from plasmid vectors by PCR, quantified by OD reading and gel analysis, and mixed at various proportions before running on HMA gel. (B) Adding 5% of minor sequence b to sequence a resulted in the formation of new heteroduplexes at the top of the gel and corresponding changes in MMS and entropy values. Further increasing the proportion of strain b strengthened the heteroduplex bands and caused further shifts in MMS values. ■, Entropy; ♦, MMS. (C) In contrast, mean distances measured by analyzing 10–15 cloned sequences for each mix did not accurately measure the increasing proportion of the minor variant. Consequently, MMS measurement based on HMA gel analysis better reflected expected mean distance changes, calculated by assuming perfect distribution of 100 cloned sequences a and b, than analysis of actual clones. Mean distances within quasispecies were calculated from cloned sequences according to the Kimura 2-parameter model.40 ■, Observed distance; ♦, expected distance. (A) Sequence a only; (B), 95:5; (C), 90:10; (D), 50:50. Gastroenterology 2004 127, 764-776DOI: (10.1053/j.gastro.2004.06.005) Copyright © 2004 American Gastroenterological Association Terms and Conditions