SHORT COMMUNICATION Gambato et al., Journal of General Virology 2019;100:63–68 DOI 10.1099/jgv.0.001175 Hepatitis C virus intrinsic molecular determinants may contribute to the development of cholestatic hepatitis after liver transplantation Martina Gambato,1,2† Josep Gregori,3,4 Josep Quer,3 George Koutsoudakis,1 Patricia Gonza lez,1 Noelia Caro-Perez,1‡ Damir García-Cehic,3 Neris García-Gonz alez,5 Fernando Gonz alez-Candelas,5 Juan Ignacio Esteban,3 Gonzalo Crespo,1 Miquel Navasa,1 Xavier Forns1 and Sofía Pe rez-del-Pulgar1,* Abstract Cholestatic hepatitis C (CHC) is a severe form of hepatitis C virus (HCV) infection recurrence that leads to high graft loss rates early after liver transplantation (LT). To investigate the pathogenic mechanisms of CHC, we analysed HCV quasispecies in CHC patients compared to a control group (mild hepatitis C recurrence) by deep pyrosequencing. At the time of LT, NS5B quasispecies complexity was similar between the two groups but, after LT, it decreased more sharply in CHC patients than in the control group. Interestingly, the major variant before LT propagated efficiently and remained as the dominant sequence after LT in 62 % of CHC patients versus 11 % of controls (P=0.031). Sequence analysis of the complete non- structural region in a limited number of patients revealed a potential 12 aa signature specific to the CHC group. These data suggest that intrinsic molecular determinants in the circulating HCV quasispecies may provide a fitness advantage, contributing to the development of CHC. Cirrhosis secondary to chronic hepatitis C infection was the and rapid progression to graft failure within the first year leading indication for liver transplantation (LT) worldwide after LT. Patients who develop CHC have among the before the extended use of direct acting antivirals (DAAs). highest viral loads in serum and liver, with levels of viral In patients who do not receive DAAs therapy, recurrent replication significantly higher than in the non-immuno- hepatitis C virus (HCV) infection of the allograft is univer- suppressed state [7]. In CHC patients receiving IFN and sal if the virus is detectable at the moment of transplant sur- ribavirin, the virological response was very low and associ- gery. Due to the accelerated course of the disease after LT, if ated with adverse effects, but introduction of DAAs changed patients are left untreated, almost one-third of liver recipi- the outcome in these patients. Clinical trials and case ents will progress to cirrhosis within 5 years after LT. repor ts have reported high virological responses with com- Indeed, before the use of DAAs, hepatitis C recurrence after plete recovery of liver function in these patients [8–13]. LT led to a significantly reduced graft and patient survival However, there is still limited understanding of the mecha- [1–3]. nisms leading to the development of CHC [14]. In general, Cholestatic hepatitis C (CHC) is an uncommon and severe HCV itself is not considered to be cytopathic, so immune variant of HCV infection recurrence that usually occurs responses against HCV would be the main cause of liver early after LT, between 3 and 6 months, in 2–14 % of cases damage in chronic hepatitis C infection [15]. In contrast, [4–6]. CHC is typically characterized by marked cholestasis hepatocellular injury in CHC might be mediated by a Received 3 August 2018; Accepted 22 October 2018; Published 19 November 2018 Author affiliations: 1Liver Unit, Hospital Clínic, IDIBAPS, CIBERehd, University of Barcelona, Barcelona, Spain; 2Multivisceral Transplant Unit and Gastroenterology, Padova University Hospital, Padova, Italy; 3Liver Unit, Vall d’Hebron Institut de Recerca-Hospital Universitari Vall d’Hebron, CIBERehd, Barcelona, Spain; 4Roche Diagnostics SL. Sant Cugat del Vall es, Barcelona, Spain; 5Joint Research Unit Infección y Salud Pública, FISABIO-Universitat de Valencia, I2SysBio, CIBERESP, Valencia, Spain. *Correspondence: Sofía Perez-del-Pulgar, sofiapp@clinic.cat Keywords: hepatitis C virus; liver transplantation; cholestatic hepatitis C; quasispecies; deep sequencing. Abbreviations: CHC, cholestatic hepatitis C; HCV, hepatitis C virus; LT, liver transplantation; VESPA, Viral Epidemiology Signature Pattern Analysis; DAAs, Direct-acting antivirals. †Present address: Multivisceral Transplant Unit and Gastroenterology, Padova University Hospital, Padova, Italy. ‡Present address: Department of Laboratory Medicine, Division of Clinical Microbiology, Karolinska Institutet, Stockholm, Sweden. NS5B deep sequencing data have been deposited in GenBank under accession numbers SAMN10256206–SAMN10256249 (SRA database Bioproject PRJNA493793). N2-NS5B direct sequences have been deposited under accession numbers MK092096–MK092111. Supplementary material is available with the online version of this article. 001175 ã 2019 The Authors 63 Gambato et al., Journal of General Virology 2019;100:63–68 Table 1. Clinical and virological characteristics of the patients included in the study Categorical data are expressed as number (%) and numerical data as median (interquartile range). ALP, alkaline phosphatase; CsA, cyclosporine A; GGT, g-glutamyltransferase; IS, immunosuppressive. Cholestatic HCV (cases), n=13 Mild recurrence (controls), n=9 P Recipient gender (male) 9 (69 %) 7 (78 %) 0.627 Recipient age (years) 61 (51–66) 50 (47–56) 0.056 Donor age (years) 62 (45–65) 35 (28–49) 0.003 HCV genotype (1b) 12 (92 %) 7 (78 %) 0.368 Recipient IL28B (CC genotype) 1 (7 %) 3 (33 %) 0.091 Main IS drug 0.420 Tacrolimus 9 (70 %) 4 (45 %) CsA 2 (15 %) 3 (33 %) Everolimus 2 (15 %) 1 (11 %) Azathioprine 0 (0 %) 1 (11 %) Induction IS therapy 7 (53 %) 1 (11 %) 0.074 Acute rejection 0 (0 %) 3 (33 %) 0.050 Viral load before LT (log IU ml 1) 5.65 (4.86–6.17) 5.54 (5.07–5.77) 0.695 Viral load after LT* (log IU ml 1) 8.00 (7.63–8.34) 5.69 (5.53–6.40) 0.000 GGT (IU l 1) 1513 (765–2708) 216 (23–520) <0.001 ALP (IU l 1) 860 (553–1141) 255 (153–358) <0.001 Bilirubin (mg dl 1) 6.4 (3.5–16) 1.1 (0.6–1.4) 0.001 *Viral load at the time of cholestatic hepatitis C – sample obtained between 1 and 3 months after LT. cytopathic effect of HCV which, replicating at a high level, approved by the Ethics Committee of the Hospital Clínic of could directly induce cellular degeneration in a short period Barcelona and was conducted in compliance with the Decla- of time, causing progressive and rapid liver failure [16–20]. ration of Helsinki, Good Clinical Practice guidelines and local regulatory requirements. All patients gave written The analysis of multiple HVR1 clones has shown that the informed consent to participate. composition of the HCV quasispecies changes after LT [21, 22]. The role of the complexity of viral quasispecies in the All CHC cases were diagnosed with a liver biopsy after pathogenesis of HCV infection recurrence after LT has also excluding other conditions such as arterial or biliary com- been investigated in previous studies, providing controver- plications. The diagnosis of CHC was made according to sial results [23–26]. Most of these studies focused on the previous published criteria [7]: bilirubin >6 mg dl 1, g-glu- analysis of the glycoprotein E2, mainly the hypervariable tamyl transferase (GGT) and alkaline phosphatase (ALP) region 1 (HVR1), and used techniques with poor sensitivity, 5 ULN (Upper Limit of Normal), very high serum HCV- hindering the detection of low-frequency viral populations RNA, and typical histology of CHC in the absence of bili- and limiting the identification of mutations potentially ary/arterial complications. The characteristic histological related to the severity of hepatitis C recurrence. Therefore, state included ballooning of hepatocytes predominantly in the analysis of HCV quasispecies composition with more the perivenular zone (not necrosis or fallout), paucity of sensitive methodologies may provide a better understanding inflammation and variable degrees of cholangiolar prolifera- of CHC. tion without bile duct loss. Mild hepatitis C recurrence was defined by absent or minimal fibrosis (F0–F1) [27] or liver We hypothesized that genetic evolution of the HCV region stiffness measurement below 8.7 kPa, during a follow-up of coding for the NS5B polymerase, and the selection of spe- 5 years after LT. The clinical and virological characteristics cific variants might be implicated in the pathogenesis of of these patients are shown in Table 1. All patients included CHC, which would explain the high virus replication levels in the study were infected by HCV genotype 1. In agree- in CHC patients. The aim of our study was to analyse HCV ment with previous reports [28], CHC patients received quasispecies in patients with CHC (compared to a control organs from older donors than patients with mild recurrent group) by deep pyrosequencing. To this end, we included hepatitis C (62 versus 35 years, respectively, P=0.003). 22 HCV-infected patients undergoing LT: 13 patients with a Recipient age also tended to be higher in CHC patients than diagnosis of CHC and nine patients with mild hepatitis C in the control group, but it did not reach statistical signifi- recurrence (control group). Patients were followed by a cance (61 versus 50 years, P=0.056). Standard immunosup- standard protocol and relevant variables were collected pro- pressive therapy consisted of steroids plus tacrolimus spectively and included in a database. This study was (TAC) or cyclosporine A (CsA). Although more CHC 64 Gambato et al., Journal of General Virology 2019;100:63–68 Fig. 1. HCV quasispecies complexity according to the severity of hepatitis C recurrence after LT. NS5B genetic diversity and mutation frequency in CHC patients and in the control group before LT (a), and before and after LT (b). Closed and open circles represent CHC patients and controls, respectively. Solid lines in (a) indicate median values±interquartile range (IQR). patients (n=7, 53 %) received anti-CD25 compared to con- with an average coverage of 8076 reads per sample (Supple- trols (n=1, 11 %), its relevance is difficult to determine due mentary Material and Table S1, available in the online ver- to the small number of patients included in the study. Even sion of this article) [34, 35]. Viral quasispecies complexity so, the few data available on the effects of monoclonal anti- of each sample was computed from these filtered haplotypes body preparations against IL-2 receptor on recurrent hepa- by means of nucleotide diversity (Pi, defined as the average titis C after LT are still contradictory [29–31]. number of differences between all possible haplotype pairs corrected based on their frequencies in the population) and Two serum samples were collected from each patient: one the mutation frequency (Mf, the sum of the differences sample obtained at the time of LT and another sample between each haplotype and the dominant haplotype, cor- obtained between 1 and 3 months after LT at the time of rected based on frequency). diagnosis of acute hepatitis. We amplified a 340 nt fragment of the NS5B region (nt 8279–8618, according to isolate H77, At the time of LT, quasispecies diversity and mutation fre- accession number AF009606). Deep pyrosequencing was quency were similar between patients with CHC and performed using the 454/GS-Junior platform (Roche) as patients with mild hepatitis C recurrence (Fig. 1a). After LT, previously described [32, 33]. Deep sequencing of the sam- HCV genetic diversity and mutation frequency decreased ples yielded a total of 1 163 930 raw reads, 427 881 (37 %) of significantly in CHC patients, showing a marked homogeni- which accounted for haplotypes that passed the quality fil- zation of the quasispecies (Pi 0.00976 versus 0.00476, ters. Of these, 355 324 reads (31 %) belonged to haplotypes P=0.033; and Mf 0.00703 versus 0.00270, P=0.039, respec- with abundance above 1 % in the respective population, tively). In contrast, in the control group, mutation 65 Gambato et al., Journal of General Virology 2019;100:63–68 Fig. 2. Identification of specific amino acid motifs associated with the severity of hepatitis C recurrence after LT. Sequence logo illus- trating a 12-residue signature that differentiated viral sequences obtained from CHC patients from those derived from the controls. Signature amino acids along the HCV non-structural proteins were identified with VESPA (threshold 0.8). Non-structural proteins are depicted in turquoise and the numbers in the yellow boxes below indicate amino acid positions relative to the Con-1 polyprotein (Gen- Bank accession no. AJ238799). frequency did not change after LT (Fig. 1b). When we ana- NS5B analysis, we did not find any signature pattern differ- lysed nucleotide diversity among transitions, transversions, entiating viral sequences obtained before or after LT, for and synonymous and non-synonymous sites, we observed either of the two groups of patients. Interestingly, by apply- that this parameter always decreased after LT for CHC ing VESPA with a threshold of 0.8, we found a 12-residue patients but not for controls (data not shown). However, signature that differentiated viral sequences obtained from due to the small sample size, this decrease was only statisti- CHC patients from those derived from the controls. As cally significant for transitions (Pi 0.00925 versus 0.00431, shown in Fig. 2, variants carrying D1581 (helicase domain P=0.007). We did not find any differences between the of NS3), H1805 (NS4B), D2143 (NS5A) and I2843 (NS5B number of haplotypes before and after LT in the CHC polymerase) were detected in the five CHC patients ana- patients (74 versus 73, P=1.000), but a slight increase in the lysed. This suggests that some of these positions may confer control group (62 versus 80, P=0.066). a fitness advantage which would account for the high viral Regarding the propagation of HCV quasispecies after trans- loads observed in these patients. Nonetheless, we cannot plantation, the percentage of the master sequence remained rule out that there may be variations in the frequency of constant in the control group (26 versus 28 %, P=0.214), amino acid residues in other positions in the same variant whereas it showed a tendency to increase in the CHC group as a result of hitchhiking selection. (27 versus 37 %, P=0.064). Interestingly, in eight of 13 Before the use of DAAs, CHC was the most severe form patients with CHC (62 %), the master sequence present at of HCV infection recurrence after LT. Because of its rela- the moment of LT propagated efficiently and remained as tively low frequency, limited clinical data are available, the dominant sequence after LT. By contrast, this behaviour and the risk factors for CHC development remain unre- was seen in only one of nine patients included in the control solved [14]. Previous reports have shown that older group (11 %, P=0.031). donors [28], corticosteroid treatment for acute cellular To determine whether specific amino acid motifs were asso- rejection [37], high levels of HCV RNA after LT [38] and ciated with disease outcomes, amino acid master sequences IL28B genotype [39] may be implicated in the develop- of NS5B were compared between the CHC and control ment of CHC. To our knowledge, this is the first study groups by using the Viral Epidemiology Signature Pattern that has investigated HCV quasispecies evolution in Analysis (VESPA) program with a threshold of 0.8 [36]. We patients with CHC after LT based on deep sequencing. did not find any amino acid signature that differentiated We have shown that the diversity of HCV quasispecies at HCV NS5B variants obtained after LT from those in the the time of LT did not correlate with disease progression corresponding pre-LT sample, either for the CHC cases or after LT. On the contrary, after LT, patients with CHC for the controls. Since there is a high degree of conservation showed a pronounced homogenization of the HCV quasis- in the NS5B region at the amino acid level, we hypothesized pecies that was not observed in patients with mild hepati- that there might be some molecular determinants related to tis C recurrence. Our data are in agreement with previous the severity of the hepatitis C recurrence in other regions, studies suggesting an inverse correlation between HCV apart from the NS5B polymerase. Thus, we amplified and genetic diversification and the severity of hepatitis C analysed by direct sequencing a fragment of 6321 bp (2107 recurrence during the first year after LT [24, 25, 40]. Sev- aa) encompassing almost the complete non-structural eral authors have previously reported that quasispecies region, from NS2 to NS5B (2916–9236 bp) in a selected complexity decreases in the absence of an effective group of genotype 1b infected patients: five CHC and three immune response early after LT [21, 26, 41, 42]. However, controls (Supplementary Material). Similarly to the previous this has not been confirmed by others [23, 25]. These 66 Gambato et al., Journal of General Virology 2019;100:63–68 conflicting results may be due, in part, to the HCV region present in pathogenic variants are able to suppress or evade analysed (envelope glycoprotein E2, mainly HVR1), the immune control remain to be elucidated. different time points at which the post-LT serum samples were obtained, the characteristics of the study populations Funding information (sometimes poorly defined) and the techniques available at This study was sponsored by the Instituto de Salud Carlos III through that time, such as single-strand conformation polymor- the Plan Estatal de Investigación Científica y T ecnica y de Innovación phism (SSCP), heteroduplex mobility assay (HMA) and 2013–2016, grants PI13/00155 (SPP), PI15/00151 (XF) and PI16/ sequencing of a limited number of clones, which are far 00337 (JQ), co-funded by the European Regional Development Fund (ERDF), and by Plan Estrat egico Nacional contra la hepatitis C (Spanish less sensitive methods than deep sequencing. Health Ministry). XF received support from Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement (grant Our results suggest that among the divergent genomes pres- 2017_SGR_1753) and CERCA Programme/Generalitat de Catalunya. ent in the viral quasispecies before LT, one or a few viruses FGC and NGG received funding by Ministerio de Economía y Competiti- may infect the graft, overgrow the others and generate a rel- vidad (MINECO, grant BFU2017-89594R) and Generalitat Valenciana (PROMETEO/2016/122). GK was supported by Fundación IBEROSTAR atively uniform quasispecies after LT. One could argue that and MG was funded by EASL Sheila Sherlock Entry-level Research the low-grade genetic evolution of NS5B observed in CHC Fellowship. might be related to the weak immune pressure against HCV, resulting from the immunosuppressive therapy. How- Conflicts of interest XF received unrestricted gran support from Abbvie and has acted as ever, we did not observe any significant difference between advisor for Abbvie and Gilead. the two groups in terms of immunosuppression regimens. On the other hand, the fact that lymphocytes from patients Ethical statement This study was approved by the Ethics Committee of the Hospital Clínic with severe hepatitis C recurrence fail to mount virus- of Barcelona and was conducted in compliance with the Declaration of specific T-cell responses [43] suggests that some intrinsic Helsinki, Good Clinical Practice guidelines and local regulatory features in the viral genome might play a role in the patho- requirements. All patients signed a written informed consent to genesis of HCV-related liver injury after LT. Supporting the participate. latter suggestion, we found that the master sequence present References before LT propagated in most of our CHC patients after LT, 1. Gane EJ. 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