E2 quasispecies specificity of hepatitis C virus ... - aasld - Wiley

E2 Quasispecies Specificity of Hepatitis C Virus Association With Allografts Immediately After Liver Transplantation Michael G. Hughes, Jr.,1 Christine K. Rudy,1 Tae W. Chong,1 Robert L. Smith,1 Heather L. Evans,1 Julia C. Iezzoni,2 Robert G. Sawyer,1 and Timothy L. Pruett1 It is unknown whether all hepatitis C virus (HCV) quasispecies variants found within patient serum have equal capacity to associate with the liver after transplantation; however, in vitro models of HCV infection suggest that variations in the hypervariable region 1 (HVR1) of the second envelope protein (E2) may be important in infectivity. The hypothesis of the current study is that the two hypervariable regions (HVR1 and HVR2) within E2 are important in the initial virus–liver interaction, and, therefore, certain HCV quasispecies variants will be isolated from the liver after reperfusion. In 8 patients with endstage liver disease secondary to HCV infection, HCV envelope quasispecies were determined from intraoperative serum samples obtained before the anhepatic phase of transplantation and from liver biopsies 1.5 to 2.5 hours after the transplanted liver was perfused. Explanted (native) liver biopsies were taken as a control. Sequence analysis was performed on clones of specific HCV reverse transcriptase-polymerase chain reaction products spanning HVR1 and HVR2 of the E2 protein. HVR1 was more variable than HVR2 for all samples. Quasispecies isolated from postperfusion liver differed more from serum than did explanted liver quasispecies at HVR1 (P ⴝ 0.03) but not at HVR2 (P ⴝ 0.2). Comparison of HVR1 sequences from postperfusion liver versus serum revealed significantly less HVR1 genetic complexity and diversity (P ⴝ 0.02 and P ⴝ 0.04, respectively). Immediately after transplantation but before actual infection, liver allografts

Abbreviations: HCV, hepatitis C virus ; HVR, hypervariable region; E2, second envelope protein; RT-PCR, reverse transcriptasepolymerase chain reaction; RT, reverse transcriptase; SSCP, single strand conformational polymorphism; TBE, tris borate EDTA. From the University of Virginia, 1Department of Surgery, Surgical Infectious Disease Laboratory, Charlottesville, VA (institution at which all research was performed) and 2Department of Pathology, Charlottesville, VA Supported in part by the ASTS Roche Surgical Scientist Award to Michael Hughes, MD, and through a grant from the Falk Foundation. Address reprint requests to Michael Hughes, MD, 1507 Minor Ridge Ct., Charlottesville, VA 22901. Telephone: 434-996-4535; FAX: 434924-1218; E-mail: [email protected] Copyright © 2004 by the American Association for the Study of Liver Diseases Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/lt.20060

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select out from the infecting serum inoculum a less heterogeneous, more closely related population of quasispecies variants. (Liver Transpl 2004;10:208 –216.)

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iver failure induced by hepatitis C (HCV) is the leading indication for liver transplantation in the United States.1 After transplantation, allografts are universally infected with the virus, yet the timing and magnitude of infection differ from patient to patient.2– 4 The study by Garcia-Retortillo et al. of viral kinetics during the transplantation procedure5 demonstrated that allografts clear virus from the circulation immediately upon reperfusion, which led the authors to hypothesize that “after graft reperfusion, massive entrance of HCV into the hepatocytes or HCV uptake by the liver reticuloendothelial system is the cause, at least in part, of HCV clearance.”5, p.685 However, the rate of clearance varied between patients. Although this suggests that allografts differ in their ability to associate with virus or that viral subpopulations differ in their ability to associate with allografts, this concept has never been studied. If the mechanics of these interactions were better understood, interventions targeted at disrupting initial virus–allograft association could be implemented during reperfusion of the allograft to alter subsequent disease progression. The second envelope protein (E2) of the virus is thought to be the primary interface with the host cells.6,7 Within the E2 coding region are two hypervariable regions.8,9 The first, HVR1,10,11 is a stretch of 27 amino acids with conserved basic residues consistent with a role in protein receptor binding.12 Hepatocytes and lymphocytes selectively associate with certain HVR1 sequences over others,13,14 and antibodies directed against HVR1 have prevented infection in vitro7,15–17 and in a chimpanzee model of infection.18,19 The second hypervariable region, HVR2, which consists of 9 amino acids, has a predicted 3-dimensional protein structure also consistent with a role in protein receptor binding,20 and antibodies against this also have prevented attachment to cells in vitro.21 Mutations within these two regions give rise to multiple subpopulations of virus within an individual called quasispe-

Liver Transplantation, Vol 10, No 2 (February), 2004: pp 208 –216

Allograft Selectivity for HVR1 Quasispecies

cies.22–25 These quasispecies are defined by their amino acid sequences at HVR1 or HVR2. We hypothesized that HCV selectively associates with allografts during reperfusion. In 8 patients undergoing liver transplantation for HCV, we compared the quasispecies population of the infectious inoculum (serum sampled during transplantation but before the anhepatic phase) with the quasispecies population in a biopsy of the previously uninfected liver 1.5 to 2.5 hours after perfusion. If our hypothesis that the allograft selectively associates with circulating quasispecies variants is true, then HCV quasispecies population of the allograft after it is perfused, but before it is infected (no detectable viral negative-strand RNA), should be significantly different from that of the serum sampled during transplantation. However, if the serum and allograft have similar quasispecies populations, then either the allograft is associating with circulating HCV in a quasispecies nonspecific manner (a non–HVR1/HVR2 dependent fashion), or the quasispecies population of the postperfusion liver actually represents only circulating, unbound viral particles in the hepatic microcirculation.

Materials and Methods Patients and Samples The study was approved by the Human Investigation Committee at the University of Virginia. All patients signed informed consent for study inclusion before transplantation. Eight patients with end-stage liver disease secondary to HCV injury underwent liver transplantation. Serum samples were drawn after the incision was made but before removal of the diseased organ and were stored at – 80°C. Serum samples rather than whole blood or circulating blood cells were obtained in all patients because serum has previously been shown to be infectious,13,18,19,26 and serum viral load has predicted outcomes after transplantation.27 In 2 patients, heparinized whole blood was obtained at the same time as serum; from these samples, peripheral blood cells were isolated by Histopaque gradient (Sigma Diagnostics, St Louis, MO) with further separation of neutrophils and red blood cells by 3% dextran T500 (Amersham Pharmacia Biotech AB, Uppsala, Sweden). Wedge biopsies of explanted livers were taken as control tissue for the HCV reverse transcriptasepolymerase chain reaction (RT-PCR) technique because the quasispecies population of serum and diseased liver have been shown to have identical consensus sequences.27 Postperfusion allograft biopsies were taken 1.5 to 2.5 hours after reperfusion. Liver biopsies were immediately frozen in liquid nitrogen and then stored at – 80°C. Liver transplantation was performed in a piggyback fashion, leaving portal and arterial inflow until the diseased liver was removed and limiting the

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anhepatic phase to approximately 30 minutes. All blood products that were administered during the surgery underwent nucleic acid testing for HCV before transfusion.

Histologic Grading of Procurement Injury Procurement injury describes allograft damage that results from ischemia and reperfusion. As significant harvest injury could alter cell surface receptor expression and thus possible quasispecies selection, a liver pathologist (JCI) determined the degree of harvest injury for postperfusion liver biopsies as follows: mild injury (mild microvesicular steatosis, mild intrasinusoidal neutrophils, no hepatocyte necrosis), moderate injury (moderate microvesicular steatosis, moderate intrasinusoidal neutrophils, scattered individual necrotic hepatocytes or small clusters of necrotic hepatocytes), and severe injury (moderate or greater microvesicular steatosis, moderate or greater intrasinusoidal neutrophils, zonal hepatocyte necrosis). The extent of hepatocyte necrosis was the most important feature for assessing the degree of harvest injury for postperfusion liver biopsies.

RNA Extraction, RT-PCR, Cloning, and Sequencing HCV RNA was isolated from serum samples (250 ␮L), cell fractions (250 ␮L), and liver biopsies (approximately 5 mg) using monophasic phenol and guanidine isothiocyanate solution (TriReagent LS, Molecular Research Center, Inc., Cincinnati, OH, or Trizol Reagent, Invitrogen Corp., Carlsbad, CA, respectively) following the manufacturers’ instructions. Extracted RNA (4 ␮L) was reverse-transcribed using SuperScript II Reverse Transcriptase (RT) (Invitrogen Corp.) according to manufacturer’s instructions. To ensure that potentially infectious minor quasispecies populations would be detected, the extracted RNA was not diluted before use in the RT reaction. Concurrent negative controls were run with extracted RNA but without SuperScript II RT to assess for genomic and crossover DNA contamination. The resultant cDNA was amplified in a nested-PCR with a high-fidelity proofreading DNA polymerase (PfuTurbo Hotstart DNA Polymerase, Stratagene, La Jolla, CA) and primers flanking HVR1 and HVR2 (outer sense: 5⬘-GCA ATT GCT CTA TCT ATC CCG-3⬘, outer antisense: 5⬘-TCT ATC CAG GTA CAA CCG-3⬘, inner sense: 5⬘-GTC ACC GCA TGG CAT GGG ATA-3⬘, and inner antisense: 5⬘-CAT TTT CAC CCC AGC TGT AGG-3⬘). Five ␮L of RT reaction or 1 ␮L of PCR product was used for the first or second round PCR respectively, following manufacturer’s instructions. The first- and second-round PCR products were 724 and 518 nucleotides in length, respectively. Ethidium bromide gel–purified PCR product was ligated into a linearized, blunt-ended vector (pBlueScript SK⫹, Stratagene) using DNA ligase (T4 DNA Ligase, GibcoBRL Life Technologies, Carlsbad, CA). Super competent Escherichia coli (One Shot INV␣⬘ E. coli cells, Invitrogen Corp.) were transformed with the vector and then cultured on LB

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agar plates with ampicillin and X-gal. Twenty-five white colonies were selected and grown up in Terrific Broth with ampicillin. DNA was purified from those isolates successfully grown in broth (PerfectPrep Plasmid Mini Prep Kits Eppendorf AG, Hamburg, Germany), and DNA insertion confirmed by EcoRI/XhoI restriction digest (Invitrogen Corp.). Purified DNA was sequenced using an Applied Biosystems (Branchburg, NJ) automated sequencer.

RT-PCR for Viral Negative-Strand Viral negative-strand RNA was detected in 5 patients using sense primer (5⬘-TCC CGG GAG AGC CAT AGT GGT CTG CGG AAG C-3⬘) and rTth DNA polymerase (Applied Biosystems) in the RT reaction and AmpliTaq Gold (Applied Biosystems) for the PCR as previously reported.28 For each sample, 2 negative controls were run: one without sense primer during the RT reaction and the other without reverse transcriptase. For each reaction, 3 positive controls (0.1 fg, 0.01 fg, and 0.001 fg synthetic negative-strand RNA) were included. The 345 base pair (bp) PCR product was run on a 1.5% agarose gel to perform a Southern blot using a 32-P end-labeled oligomer probe (5⬘-TGC TCA TGG TGC ACG GTC TAC GAG AC-3⬘) specific for a sequence between the two PCR primers. The blot was hybridized, washed, and then exposed to film.

Single Strand Conformational Polymorphism To determine whether serum HVR1 quasispecies variants change during the transplant procedure, serum HVR1 quasispecies variants from serum obtained immediately after the incision and just before liver reperfusion were compared using single strand conformational polymorphism (SSCP) in 1 patient (patient 2). This technique was chosen for its ability to qualitatively examine the population of quasispecies variants within serum. cDNA made with SuperScript II reverse transcriptase (RT) as described earlier was used in a semi—1-sided PCR to amplify the HVR1 region. For the PCR reaction, a radio-labeled sense primer (5⬘ 32P-TAT TTC TCC ATG GTG GGG AAC TGG-3⬘) and 1/20th concentration of the antisense primer (5⬘- TCC TGT TGA TGT GCC AAC TGC C-3⬘) were used. Two ␮L and 5 ␮L of the PCR products (193 bp) were denatured and separated on a 0.5x MDE gel (BioWhittaker Molecular Applications, Rockland, ME) in 0.6x TBE buffer, as previously described.29 The gel was transfered to filter paper, dried in a gel drier, and exposed to film.

HCV Population Parameters Amino acid sequences were determined from nucleotide sequences using Vector NTI Suite v.7.1 (InforMax, Bethesda, MD). Sequences were aligned with a modified ClustalW algorithm. Distances between sequences were determined using the amino acid substitution matrix BLOSUM62.30 This was used over other available matrices because of the intermediate homology and entropy of HVR1 in patient samples. Consensus sequences were defined as the amino acids most frequently encountered at each position rather than the

most frequently encountered quasispecies. To determine the significance of differences between quasispecies populations obtained at different times or locations, the Hamming distances31 between consensus sequences were compared. Explanted liver was used as a control because consensus sequences between paired serum and diseased liver samples have previously been shown to be identical.27,32,33 Genetic complexity of each quasispecies population was expressed as normalized Shannon entropy.27,34,35 Shannon entropy was calculated as S ⫽–⌺i(pi ln pi) with pi representing the frequency of each variant in the sampled population. The Shannon entropy was then normalized to the number of clones sequenced for a given population to allow for comparisons between populations. The normalized Shannon entropy (Sn ⫽ S/ln N) (N representing the total number of clones in a sample) varied from 0 (no complexity) to 1 (maximal complexity). These values were compared with a paired Student t test. Genetic diversity was determined by excluding redundant sequences and then determining the divergence (%) between quasispecies within a single sample by the amino acid substitution matrix BLOSUM62. This method was used because previously reported methods, such as Pn27 and the two-parameter amino acid substitution matrix of Kimura,35 take into account only the number of polymorphic sites. Values for each sample quasispecies population were expressed as percent divergence between the two least similar quasispecies. This was referred to as the maximum amino acid divergence. These values were then compared using a paired Student t test. Genetic diversity was thus used to describe the degree of relatedness between sequences without regard for quasispecies number or frequency.

Results Patient, Viral, and Sample Characteristics All livers came from donors that were HCV seronegative. In all samples, the preperfusion donor liver had no HCV RNA detectable by RT-PCR. Patient serum viral loads before transplantation were variable, ranging from 106,004 to 2,151,471 IU/ml (Table 1). All patients tested were genotype 1. Harvest injury was mild for all patients, except for patient 6, who had moderate injury. Transfusion requirements ranged from 0 to 6 units, with an average of 2.5 units. Negative-strand HCV RNA (the putative intermediate product of viral replication) could not be detected in any of the postperfusion liver samples tested despite readily demonstrable positive-strand HCV, whereas negative-strand viral RNA could be detected in all explanted liver samples tested. Synthetic negative-strand viral RNA (positive control) was detected to 0.01 fg.

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Table 1. Viral, Operative, and Sample Characteristics Pretransplant Serum Viral Viral Procurement Units of Blood Patient Load* Genotype Injury† Transfused‡ 1 2 3 4 5 6 7 8

1,629,399 2,151,471 397,962 180,980 106,004 NA 232,074 484,558

1a 1 1a NA 1b NA 1 1b

mild mild mild mild mild moderate mild mild

0 1 1 4 3 4 1 6

Positive-Strand Viral RNA Explanted Liver Serum ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹

⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹

Negative-Strand Viral RNA

Postperfusion Liver

Explanted Liver

Postperfusion Liver

⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹

⫹ ⫹ ⫹ ⫹ ⫹

⫺ ⫺ ⫺ ⫺ ⫺ NA NA NA

Abbreviations: ⫹, detectable viral RNA; ⫺, no detectable viral RNA; NA, not available. * IU/mL; †degree of reperfusion injury determined by histology; ‡units of blood transfused during entire operation.

Serum and Liver HVR1 Quasispecies (Patients 1 Through 6)

Serum and Liver HVR2 Quasispecies (Patients 1 Through 6)

Explanted liver and serum sample HVR1 quasispecies populations were similar but not identical, in patients 1– 6 (Fig. 1). The distribution of quasispecies variants retrieved from the postperfusion liver was significantly different from the distribution of quasispecies isolated from the infecting serum inoculum. Two patterns of quasispecies distribution are evident. In patients 4, 5, and 6, all but one postperfusion liver variant was identified in the serum. In all cases where the postperfusion liver quasispecies variants were also recovered from serum, the proportion of the total quasispecies population each represented was different in postperfusion liver compared with serum except for patient 6. In the second pattern (patients 1, 2, and 3), the postperfusion liver had a significant population of variants not identified in the serum. For patient 2, the postperfusion quasispecies variants not found in the serum were recovered from explanted liver; however, for patients 1 and 3, there was a significant portion of the postperfusion quasispecies population that was not recovered from the explanted liver or serum. To demonstrate that these findings could not be attributed to dilution of serum (due to transfusion and fluid resuscitation) or potential sampling error (selection of RTPCR product clones), the quasispecies variants of serum collected before the anhepatic phase were compared with those of serum collected at the same time the postperfusion biopsy was obtained by SSCP in a single patient. The quasispecies variants of these 2 samples were identical.

HVR2 sequencing for the first 6 patients demonstrated that explanted liver, serum, and postperfusion liver samples contained few HCV quasispecies variants. A single amino acid sequence for HVR2 was found in explanted liver, serum, and postperfusion liver for patients 1 and 2. In patients 3 and 6, a predominant sequence was found in all 3 samples. For patient 5, there was a second major sequence in the serum. In patient 4, although the same 3 sequences appeared in all 3 samples, the distribution within samples differed. For those with more than a single HVR2 quasispecies (patients 3, 4, and 5) certain serum HVR2 quasispecies appeared to associate with the postperfusion liver over others. Unlike with HVR1, all HVR2 quasispecies variants recovered from the postperfusion liver also were recovered from the serum. Quasispecies Populations of Liver, Serum, and Peripheral Blood Cells There was concern that the variation in postperfusion liver variants represented HCV from circulating recipient blood cells in the biopsy. Therefore, in addition to serum and liver PCR amplification, peripheral blood cells were isolated in 2 patients to determine whether the previously unseen variants represented HCV in blood cells that were in the allograft microcirculation. In 1 patient (patient 7), positive-strand viral RNA was detected only in the neutrophil fraction, whereas in the other (patient 8), HCV was detected in the red blood cell, neutrophil, and monocyte fractions. In patient 7 (similar to patients 1, 2, and 3), the predominant vari-

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Figure 1. HVR1 quasispecies populations from explanted liver, serum, and postperfusion liver samples. C: consensus sequence. Number previous to sequence identifies the quasispecies for each patient.

ant recovered from the postperfusion liver (variant 13) was not recovered from the inoculating serum. However, this variant also was not recovered from the circulating blood cell fraction. In patient 8 (similar to patients 4, 5, and 6), all the postperfusion liver quasispecies variants recovered from the postperfusion liver also were identified in the inoculating serum. Therefore, HVR1 variants in the blood cell fractions of either recipient did not obviously explain the selection observed in the liver. This is even truer for HVR2, where in patient 8, none of the 7 quasispecies variants seen only in the cells were seen in the postperfusion liver sample.

Hamming Distances Between Consensus Sequences Consensus HVR1 and HVR2 sequences were determined for each sample (Fig. 2). Hamming distances were then calculated between consensus sequences to quantify differences between samples (i.e., are serum and postperfusion liver quasispecies populations less similar than serum and explant liver quasispecies populations?) The Hamming distance between explanted liver and serum consensus sequences was thus considered a baseline for each patient. For HVR1, the Ham-


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Genetic Complexity Genetic complexity (normalized Shannon entropy) was calculated for each sample to determine whether selection occurred to a statistically significant degree. Because genetic complexity represents the degree of heterogeneity for a given sample, a decrease in genetic complexity comparing serum with postperfusion liver represents selection for a subpopulation of quasispecies variants over others. Because only the frequency of each clone and the total number of clones are used to calculate Shannon entropy, this measure does not take into account the physiochemical properties of each variant. HVR1 quasispecies populations were more variable than HVR2 quasispecies; therefore, HVR1 sequences were used to calculate genetic complexity. Quasispecies complexity of explanted liver was as complex or more complex than that of serum (Fig. 4). Conversely, the quasispecies complexity of postperfusion liver was as complex or less complex than that of serum. In all cases, the quasispecies complexity of the postperfusion liver was less than that of explanted liver. Overall, there was no significant difference between explanted liver and serum complexity; however, postperfusion liver complexity was significantly less than that of serum. Genetic Diversity Figure 2. Consensus sequences from explanted liver, serum, and postperfusion liver samples. Hyphen represents conserved amino acids at each position. *, Signifies that two amino acids have equal frequency at that position.

ming distances between serum and postperfusion liver consensus sequences were significantly greater than the distances between serum and explanted liver consensus sequences (Fig. 3). For HVR2, the Hamming distances between samples were not significantly different.

Because a significant decrease in genetic complexity from inoculating serum to postperfusion liver suggested a possible selection for certain quasispecies over others by the allograft, genetic diversity (percent maximal amino acid divergence) was calculated to determine whether those quasispecies retrieved from the allograft were more closely related to one another than those of the inoculating serum. Serum quasispecies populations were less genetically diverse than those of explanted liver (P ⫽ 0.03) and more diverse than those of postperfusion

Figure 3. Hamming distances between consensus sequences of each sample. Hamming distances were calculated as the number of polymorphic sites between consensus sequences. Gray lines represent inidividual patients and black line represents mean values.

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Figure 4. Genetic complexity at HVR1. Sn represents normalized Shannon entropy. Gray lines represent individual patients and black line represents mean values.

liver (P ⫽ 0.04) (Fig. 5). Therefore, postperfusion liver quasispecies were more closely related to one another than those of the inoculating serum. Despite this, there were no significant differences in the following polypeptide characteristics between serum and postperfusion liver as determined by Vector NTI computer software: isoelectric point (8.14 ⫾ 1.57 vs. 8.13 ⫾ 1.91; P ⫽ 1.0), charge at pH 7 (0.84 ⫾ 1.22 vs. 1.03 ⫾ 0.85; P ⫽ 0.4) or the percentage of residues that were charged (21.29 ⫾ 5.42 vs. 21.04 ⫾ 3.60; P ⫽ 0.8), acidic (4.95 ⫾ 3.09 vs. 4.36 ⫾ 2.70; P ⫽ 0.3), basic (10.80 ⫾ 5.50 vs. 11.17 ⫾ 3.06; P ⫽ 0.7), polar (33.27 ⫾ 7.97 vs. 34.61 ⫾ 8.80; P ⫽ 0.1), or hydrophobic (30.67 ⫾ 4.32 vs. 30.17 ⫾ 4.75; P ⫽ 0.4).

Discussion This study demonstrates that HCV quasispecies retrieved from reperfused liver allografts differ significantly from HVR1 variants isolated from the serum. This occurred to a statistically significant degree because (1) the Hamming distance between HVR1 consensus sequences of the serum and postperfusion liver were greater than between serum and explanted liver, and (2) HVR1 genetic complexity was less in postperfusion liver than in the serum inoculum. Because genetic diversity was also lower in postperfusion liver than in the serum inoculum, selection occurred for a more closely related population of quasispecies. This selection was not accounted for by the HVR1 variants of circulating blood cells, which might have been trapped or circulating in the hepatic microcirculation. Platelet-associated HCV was not assessed because of the low numbers and small amount of RNA

in the cirrhotic patient, rapid consumption during surgery, and dilution from platelet transfusion. In addition, even with normal numbers of platelets, HCV has not been isolated from this cell fraction in the HCV infected patient. Several potential criticisms need to be addressed. First, could these results be accounted for by sampling error or some other technical limitation? A major criticism of quasispecies determination by sequence analysis is that only a small number of variants are sampled. Single strand conformation polymorphism and the heteroduplex mobility assay potentially sample “all” variants in a sample. However, we were interested in assessing complexity and diversity, which can only reliably be determined from amino acid sequences. Direct sequencing is the only available technique for determining the amino acid sequences as SSCP, and the heteroduplex mobility assay can define quasispecies variants only by nucleotide sequence. To address the sampling issue, at least 10 clones (the currently accepted standard in the literature) were sequenced. In all but one instance, the number of HVR1 sequences from the serum and transplanted liver was more than 15. Furthermore, sampling error cannot account for the observations of the current study because the differences in Hamming distance, genetic complexity, and genetic diversity between serum and postperfusion liver variants were all statistically significant. The very limited variability of HVR2 also suggests that these results cannot be accounted for by polymerase error. Second, can these results be accounted for by serum dilution during the transplantation procedure? If postperfusion liver variants represented only diluted serum, genetic complexity would have decreased without any

Figure 5. Genetic diversity at HVR1. MAAD represents Maximal Amino Acid Divergence. Gray lines represent individual patients and black line represents mean values.


change in genetic diversity. Analogous to the practice of diluting RNA before RT-PCR for preferential amplification of predominant variants, dilution of serum would have resulted in minor variants from the serum not being detected in the postperfusion liver; however, the predominant serum variants would have remained the predominant postperfusion liver variants. This was not the case in our study; the predominant serum and postperfusion liver variants differed greatly. These findings are consistent with in vivo data demonstrating that those variants that associate with primary hepatocytes were not always recovered from the inoculating serum.13 Even if dilution could account for these findings, the patients in this study were transfused fewer units than in a study of viral kinetics,5 which demonstrated that the viral half-life could be as long as 10.3 hours (less than 5 units transfused during the entire operation [except for 1 patient] for the current study compared with more than 5 units transfused during only the anhepatic phase of the viral kinetics study). Interestingly, in the 1 patient with significant harvest injury, the postperfusion liver quasispecies population did appear to represent diluted serum. This lack of selection by the allograft is consistent with the inability of allografts with significant harvest injury to clear virus during reperfusion at the same rate as uninjured allografts.5 There was concern that the population of HVR1 quasispecies variants in the serum would undergo change during the transplant operation. Our surgical technique minimizes the anhepatic time, leaving that primary source of HCV intact for the majority of the procedure. The report by Garcia-Retortillo5 suggested that if blood transfusion during the anhepatic time is less than 5 units, then the amount of serum HCV does not change significantly. For the current study, this amount of blood was never transfused during the anhepatic phase; in 7 of 8 cases, the total transfusion for the entire transplant was less than 4 units. Finally, in a single patient, we demonstrated by SSCP that the distribution of quasispecies variants was identical between serum sampled before the anhepatic phase and serum sampled postperfusion. Thus, our results cannot be explained by changing populations of HVR1 variants within patient serum. This study is only a snapshot of what occurs during the transplanted liver–HCV association process. The virus retrieved from the liver immediately after reperfusion may not represent the HCV virions that subsequently infect the liver; however, theoretically, for the virus to enter the liver, it needs to first associate. We demonstrate that the initial association with the liver is specific for HVR1 of the viral envelope protein. This

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finding is not readily explained by infected cells trapped within the liver microcirculation. Viral load has been demonstrated to reach a nadir as early as 8 hours after blood flow to the allograft has been reestablished.5 To determine which quasispecies variants were selectively removed from the circulation by the allograft, biopsy needed to occur before this time. If allograft quasispecies were determined after the viral load began to increase (suggesting that virus had begun to replicate in the allograft), our results would have been confounded by viral mutation and evolution. Because the current study did not detect negative-strand viral RNA (the replicative intermediate) in any postperfusion allografts, these potentially confounding variables cannot explain the results. Furthermore, as time progresses after transplantation, the viral quasispecies variants that associate with allografts may evolve with further selection for certain variants. Although different variants may associate at different times, the time point of the current study examined a reasonable, early time point that could be obtained easily and that placed the patient at little risk. Future studies will examine this evolution in quasispecies selection. In conclusion, allografts select out a portion of more closely related HVR1 quasispecies from the inoculating serum. These results support the hypotheses that a liver receptor interacts directly with specific viral envelope proteins or with a blood compartment that has specific HVR1 E2 associations. In either case, there may be a target for disruption of the HCV–liver interaction in the immediate posttransplant period.

Acknowledgments Particular thanks are paid to Tamara Golding, RN, Nan Carroll, RN, and Terry Ryan, RN who were critical in organizing all aspects of this study. Additional thanks is bestowed on Walter Perry, PhD, for discussing the use of normalized Shannon entropy and maximum amino acid divergence to describe genetic complexity and diversity.

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