Volume 139, Issue 5, Pages 1774-1783.e6 (November 2010) Intravascular Transfer Contributes to Postprandial Increase in Numbers of Very-Low- Density Hepatitis C Virus Particles  Daniel J. Felmlee, David A. Sheridan, Simon H. Bridge, Søren U. Nielsen, Ross W. Milne, Chris J. Packard, Muriel J. Caslake, John McLauchlan, Geoffrey L. Toms, R. Dermot G. Neely, Margaret F. Bassendine  Gastroenterology  Volume 139, Issue 5, Pages 1774-1783.e6 (November 2010) DOI: 10.1053/j.gastro.2010.07.047 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 Levels of triglyceride, cholesterol, apoB-100, and apoB-48. Triglyceride levels in (A) plasma and in the (B) VLDF during alimentary lipemia (n = 10) expressed as fold-change relative to baseline. Cholesterol levels in (C) plasma and in the (D) VLDF in the same period (n = 10). (E) ApoB-100 and (F) apoB-48 in the VLDF represented as fold-change relative to baseline (n = 8). Data are presented as comparison of mean ± standard error of the mean with baseline using (A, E, and F) the paired t test or (B–D) the Wilcoxon signed-rank test. *P < .05; **P < .01. Gastroenterology 2010 139, 1774-1783.e6DOI: (10.1053/j.gastro.2010.07.047) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 HCVVLDF transiently increase after consumption of a high-fat meal. (A) HCV-RNA levels in the VLDF harvested from plasma drawn at serial time points after patients ingested a meal expressed as fold-change relative to levels measured from plasma drawn while patients were in the fasted state (n = 10). (B) Plasma HCV-RNA levels expressed as percentage of baseline values (n = 10). (C) Percentage of HCV RNA within VLDF expressed as HCVVLDF/plasma HCV RNA × 100 (n = 10). Data are presented as comparison of mean ± standard error of the mean with baseline using (A) the paired t test or (B and C) the Wilcoxon signed-rank test. *P < .05; **P < .01. Gastroenterology 2010 139, 1774-1783.e6DOI: (10.1053/j.gastro.2010.07.047) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 HCVVLDF divided into VLDL- and chylomicron-associated subpopulations. VLDF material collected from serial time points after patients consumed a meal was separated further into subpopulations at each time point based on affinity to sepharose-conjugated antibodies recognizing apoB-100, but not apoB-48 (n = 8). (A) Percentage of HCV RNA in VLDF relative to plasma levels for this subset (n = 8). Proportion of apoB-100–associated HCVVLDF (bound) and apoB-48–associated HCVVLDF (unbound) × percentage in VLDF from panel A; each sample was used to generate (B) % HCVVLDF:B-100, and (C) % HCVVLDF:B-48. Data are presented as mean ± standard error of the mean using the Wilcoxon signed-rank test. *P < .05. Gastroenterology 2010 139, 1774-1783.e6DOI: (10.1053/j.gastro.2010.07.047) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 Effects of dietary lipid on buoyant density of HCV RNA in density gradients of plasma drawn at serial time points during the 6-hour period after the meal. Plasma from each patient was fractionated into ten 1.0-mL fractions by 12.5% iodixanol density gradient ultracentrifugation. Average density for each fraction is represented on the y-axis and time is represented on the x-axis. Termini and intersecting points of grid lines represent measured values with data between these points being interpolated. HCV-RNA quantity is represented as the heat of the color for 2 individual patients: (A) patient 8 and (B) patient 4. Density gradient measurements were grouped into high-density fractions (d > 1.075 g/mL, α) and low-density fractions excluding HCVVLDF (1.024 > d > 1.075, β). Proportion of distribution in these fractions is expressed as mean percentage ± standard error of the mean (n = 9) for (C) α grouping, (D) β grouping, or (E) HCVVLDF. *P < .05 different relative to baseline using the (C) paired t test or (E) Wilcoxon signed-rank test. Gastroenterology 2010 139, 1774-1783.e6DOI: (10.1053/j.gastro.2010.07.047) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 Pre-incubation of HCVcc with human serum increases infectivity. Supernatant from Huh7.5 cells transfected with in vitro transcribed J6/JFH1 RNA was passed through a 0.45-μm filter and concentrated before incubating in a 1:1 ratio with PBS or human serum drawn from an uninfected volunteer either in the fasted state or 2 hours after a high-fat milkshake. After 2 hours of incubation, mixed samples were added to naive Huh7.5 cells in serial dilution. Tissue culture 50% infective dose (tcid50) values were calculated by NS5A expression in an end point dilution assay. Values represent mean ± standard error of the mean (n = 6). Gastroenterology 2010 139, 1774-1783.e6DOI: (10.1053/j.gastro.2010.07.047) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 1 Lipid profile of human plasma fractionated by iodixanol density gradient ultracentrifugation. A total of 2.0 mL of plasma taken from a volunteer uninfected with HCV in the fasting state were separated by density gradient centrifugation as described in the Materials and Methods section. This gradient was harvested from the top in 1.0-mL (VLDF), then 0.5-mL fractions and triglyceride, cholesterol, and density were measured for each fraction. Gastroenterology 2010 139, 1774-1783.e6DOI: (10.1053/j.gastro.2010.07.047) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 2 Plasma HCV-RNA levels (closed circles, solid line), HCVVLDF RNA levels (open circles, dotted line), and VLDF triglyceride levels (X marks, dashed line) in the period before and after consumption of a high-fat meal for 10 individual patients. Gastroenterology 2010 139, 1774-1783.e6DOI: (10.1053/j.gastro.2010.07.047) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 3 Large HCV particles increase after a high-fat meal. The SEC column was calibrated with flotation isolated chylomicrons (Sf > 400), VLDL1 (Sf 400–60), VLDL2 (Sf 60–20), intermediate density lipoprotein (IDL) (Sf 20–12), and LDL (Sf 0–12). The relative migration patterns of the lipoprotein fractions through the SEC column were observed by optical density (OD) at 280 angstroms and are depicted by shaded area plots. Depicted are the size distributions of HCV RNA from 4 mL of plasma derived from (A) patient 8, (B) patient 4, and (C) patient 3 in the fasted state, and 120 and 180 minutes after the meal. Gastroenterology 2010 139, 1774-1783.e6DOI: (10.1053/j.gastro.2010.07.047) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 4 Effects of dietary lipid on buoyant density of HCV RNA in density gradients of plasma drawn at serial time points during the 6-hour period after the meal. Average density for each fraction is represented on the y-axis, and time is on the x-axis. Termini and intersecting points of grid lines represent measured values with data between these points being interpolated. HCV-RNA quantity is represented as the heat of the color for 7 individual patients. Gastroenterology 2010 139, 1774-1783.e6DOI: (10.1053/j.gastro.2010.07.047) Copyright © 2010 AGA Institute Terms and Conditions