Dynamic evolution of the sofosbuvir-associated variant A1343V in HEV-infected patients under concomitant sofosbuvir-ribavirin treatment.

Background & Aims: In the absence of a hepatitis E virus (HEV)-specific antiviral treatment, sofosbuvir has recently been shown to have antiviral activity against HEV in vivo. However, a variant, A1343V, that is strongly associated with viral relapse impedes treatment success. In this study, we investigated the occurrence of variants during sofosbuvir and ribavirin treatment in vivo and assessed the sensitivity of resistance-associated variants to concurrent treatment in cell culture. Methods: Two patients with chronic HEV infection that did not clear infection under ribavirin treatment were subsequently treated with a combination of sofosbuvir and ribavirin. We determined response to treatment by measuring liver enzymes and viral load in blood and stool. Moreover, we analyzed viral evolution using polymerase-targeted high-throughput sequencing and assessed replication fitness of resistance-associated variants using a HEV replicon system. Results: Combination treatment was successful in decreasing viral load towards the limit of quantification. However, during treatment sustained virological response was not achieved. Variants associated with sofosbuvir or ribavirin treatment emerged during treatment, including A1343V and G1634R. Moreover, A1343V, as a single or double mutation with G1634R, was associated with sofosbuvir resistance during concomitant treatment in vitro. Conclusions: These results highlight the importance of variant profiling during antiviral treatment of patients with chronic infection. Understanding how intra-host viral evolution impedes treatment success will help guide the design of next-generation antivirals. Impact and implications: The lack of hepatitis E virus (HEV)-specific antivirals to treat chronic infection remains a serious health burden. Although ribavirin, interferon and sofosbuvir have been reported as anti-HEV drugs, not all patients are eligible for treatment or clear infection, since resistant-associated variants can rapidly emerge. In this study, we analyzed the efficacy of sofosbuvir and ribavirin combination treatment in terms of HEV suppression, the emergence of resistance-associated variants and their ability to escape treatment inhibition in vitro. Our results provide novel insights into evolutionary dynamics of HEV during treatment and thus will help guide the design of next-generation antivirals.

Enhanced monitoring and detection of recent genotype 3 hepatitis E virus infection through urine antigen testing.

Hepatitis E virus (HEV) is the leading cause of acute viral hepatitis. Numerous studies have investigated the dynamics of HEV infection markers, but the most suitable marker for diagnosing ongoing or recent HEV infection remains to be determined. Recent evidence suggests that serum antigen testing is superior to serum IgM and RNA quantification. Moreover, it has been found that infected individuals excrete HEV antigen in significant quantities through urine. To address this question, we conducted a longitudinal analysis involving 16 patients with acute or chronic HEV infection in an area where genotype 3 HEV is prevalent. Our findings indicate that the diagnostic and monitoring capabilities of antigen testing for HEV infection can be further enhanced by measuring it in urine. Additionally, we were able to demonstrate that this enhancement is likely due to the presence of HEV-reactive IgG in blood plasma, which hampers efficient detection of HEV antigen through sandwich ELISA. In conclusion, urine-based antigen testing appears to be superior to measuring anti-HEV antibodies or viral RNA for diagnosing suspected HEV infection and monitoring ongoing infections.

Janus kinase-inhibition modulates hepatitis E virus infection.

Hepatitis E virus (HEV) usually causes a self-limiting disease, but especially immunocompromised individuals are at risk to develop a chronic and severe course of infection. Janus kinase (JAK) inhibitors (JAKi) are a novel drug class for the treatment of autoimmune inflammatory rheumatic disease (AIRD). As JAKs play a key role in innate immunity, viral infections and reactivations are frequently reported during JAKi treatment in AIRD patients. The aim of this study was to characterize the influence of JAKis on HEV replication. To this end, we evaluated liver enzymes of an AIRD patient under JAKi therapy with hepatitis E. Further, experiments with HEV (Kernow-C1 p6) were performed by infection of primary human hepatocytes (PHHs) followed by immunofluorescence staining of viral markers and transcriptomic analysis. Infection experiments in PHHs displayed an up to 50-fold increase of progeny virus production during JAKi treatment and transcriptomic analysis revealed induction of antiviral programs during infection. Upregulation of interferon-stimulated genes (ISG) was perturbed in the presence of JAKis, concomitant with elevated HEV RNA levels. The obtained results suggest that therapeutic JAK inhibition increases HEV replication by modulating the HEV-triggered immune response. Therefore, JAKi treatment and the occurrence of elevated liver enzymes requires a monitoring of potential HEV infections.

Emergence of resistance-associated variants during sofosbuvir treatment in chronically infected hepatitis E patients.

BACKGROUND AND AIMS: Chronic HEV infections remain a serious problem in immunocompromised patients, as specifically approved antiviral drugs are unavailable. In 2020, a 24-week multicenter phase II pilot trial was carried out, evaluating the nucleotide analog sofosbuvir by treating nine chronically HEV-infected patients with sofosbuvir (Trial Number NCT03282474). During the study, antiviral therapy reduced virus RNA levels initially but did not lead to a sustained virologic response. Here, we characterize the changes in HEV intrahost populations during sofosbuvir treatment to identify the emergence of treatment-associated variants. APPROACH AND RESULTS: We performed high-throughput sequencing on RNA-dependent RNA polymerase sequences to characterize viral population dynamics in study participants. Subsequently, we used an HEV-based reporter replicon system to investigate sofosbuvir sensitivity in high-frequency variants. Most patients had heterogenous HEV populations, suggesting high adaptability to treatment-related selection pressures. We identified numerous amino acid alterations emerging during treatment and found that the EC 50 of patient-derived replicon constructs was up to ~12-fold higher than the wild-type control, suggesting that variants associated with lower drug sensitivity were selected during sofosbuvir treatment. In particular, a single amino acid substitution (A1343V) in the finger domain of ORF1 could reduce susceptibility to sofosbuvir significantly in 8 of 9 patients. CONCLUSIONS: In conclusion, viral population dynamics played a critical role during antiviral treatment. High population diversity during sofosbuvir treatment led to the selection of variants (especially A1343V) with lower sensitivity to the drug, uncovering a novel mechanism of resistance-associated variants during sofosbuvir treatment.

EGF receptor modulates HEV entry in human hepatocytes.

BACKGROUND AND AIMS: Being the most common cause of acute viral hepatitis with >20 million cases per year and 70,000 deaths annually, HEV presents a long-neglected and underinvestigated health burden. Although the entry process of viral particles is an attractive target for pharmacological intervention, druggable host factors to restrict HEV entry have not been identified so far. APPROACH AND RESULTS: Here we identify the EGF receptor (EGFR) as a novel host factor for HEV and reveal the significance of EGFR for the HEV entry process. By utilizing RNAi, chemical modulation with Food and Drug Administration-approved drugs, and ectopic expression of EGFR, we revealed that EGFR is critical for HEV infection without affecting HEV RNA replication or assembly of progeny virus. We further unveiled that EGFR itself and its ligand-binding domain, rather than its signaling function, is responsible for the proviral effect. Modulation of EGF expression in HepaRG cells and primary human hepatocytes affected HEV infection. CONCLUSIONS: Taken together, our study provides novel insights into the life cycle of HEV and identified EGFR as a possible target for future antiviral strategies against HEV.

Application of CT contrast medium is not associated with an increased risk for acute kidney injury in patients with decompensated cirrhosis.

BACKGROUND: Acute kidney injury (AKI) is a frequent complication in patients with decompensated cirrhosis. Studies reported conflicting results regarding the nephrotoxic potential of iodinated contrast medium (CM) for computer tomography (CT). AIM: To investigate the impact of diagnostic CM application on kidney function in patients with decompensated cirrhosis. METHODS: First, we evaluated the impact of diagnostic CM-CT on AKI incidence in a cross-sectional approach. Second, we analysed 28-day AKI incidence post-CM-CT in patients with impaired kidney function (i.e., creatinine >133 µmoL/L). Third, we excluded all patients with relevant interventions besides CM-CT. All remaining patients were matched via propensity score matching (PPSM) and further analysed. Last, we validated the results in an independent dataset of prospectively collected registry data of 118 patients with decompensated cirrhosis. Here, plasma samples were analysed regarding neutrophil-gelatinase-associated-lipocalin (NGAL). RESULTS: Of the 611 included patients, 98 (16%) received CM-CT. CM-CT was not associated with AKI in the cross-sectional approach (CM-CT:8% vs. no CM-CT:15%; p = 0.08). Furthermore, CM-CT was not associated with higher 28-day AKI incidence among patients with impaired kidney function (HR:0.79; 95% CI 0.45-1.38; p = 0.40). The PPSM cohort revealed no association between CM-CT and AKI or severe AKI (HR:1.28, p = 0.45 and HR:1.62; p = 0.43). Moreover, CM-CT did not result in worsening of kidney function after CM application. In the validation cohort, CM-CT was also not linked to AKI (p = 0.85) and NGAL levels were not increased in those with CM-CT (CM-CT:309 ng/ml vs. No CM-CT:266 ng/ml, p = 0.35). CONCLUSION: Decompensated cirrhosis per se should not preclude diagnostic CM-CT.

Hepatitis E virus is effectively inactivated by methylene blue plus light treatment.

BACKGROUND AND OBJECTIVES: Photodynamic treatment with methylene blue (MB) and visible light is a well-established pathogen inactivation system for human plasma. This technique is routinely used in different countries. MB/light treatment was shown to inactivate several transfusion-transmittable viruses, but its efficiency for the inactivation of the quasi-enveloped hepatitis E virus (HEV) has not yet been investigated. MATERIALS AND METHODS: Plasma units were spiked with cell culture-derived HEV and treated with the THERAFLEX MB-Plasma system using various light doses (30, 60, 90, and 120 J/cm2 ). HEV titers in pre- and post-treatment samples were determined by virus titration and a large-volume plating assay to improve the detection limit of the virus assay. RESULTS: THERAFLEX MB-Plasma efficiently inactivated HEV in human plasma. Even the lowest light dose of 30 J/cm2 inactivated HEV down to the limit of detection, with a mean log reduction factor of greater than 2.4 for the total process. CONCLUSION: Our study demonstrates that the THERAFLEX MB-Plasma system effectively inactivates HEV in human plasma.

Metamizole-Associated Risks in Decompensated Hepatic Cirrhosis.

BACKGROUND: Because of the increased risk of acute renal failure (ARF), the use of cyclooxygenase (COX) inhibitors is not recommended in patients with decompensated hepatic cirrhosis. Metamizole is not a classic COX inhibitor, but there are insufficient data to support its safe use. In this study, we investigate the effect of metamizole on the risk of ARF in these patients. METHODS: Metamizole use, ARF incidence, and patient mortality were examined in a large, retrospective, exploratory cohort and validated with data from a prospective registry. RESULTS: 523 patients were evaluated in the exploratory cohort. Metamizole use at baseline was documented in 110 cases (21%) and was independently associated with the development of ARF, severe (grade 3) ARF, and lower survival without liver transplantation at follow-up on day 28 (HR: 2.2, p < 0.001; HR: 2.8, p < 0.001; and HR: 2.6, p < 0.001, respectively). Interestingly, the risk of ARF depended on the dose of metamizole administered (HR: 1.038, p < 0.001). Compared to patients who were treated with opioids, the rate of ARF was higher in the metamizole group (49% vs. 79%, p = 0.014). An increased risk of ARF with metamizole use was also demonstrated in the independent validation cohort (p < 0.001). CONCLUSION: Metamizole therapy, especially at high doses, should only be used with a high level of caution in patients with decompensated cirrhosis.

Viral Interference of Hepatitis C and E Virus Replication in Novel Experimental Co-Infection Systems.

BACKGROUND: Hepatitis C virus (HCV) constitutes a global health problem, while hepatitis E virus (HEV) is the major cause of acute viral hepatitis globally. HCV/HEV co-infections have been poorly characterized, as they are hampered by the lack of robust HEV cell culture systems. This study developed experimental models to study HCV/HEV co-infections and investigate viral interference in cells and humanized mice. METHODS: We used state-of-the art human hepatocytes tissue culture models to assess HEV and HCV replication in co- or super-transfection settings. Findings were confirmed by co- and super-infection experiments in human hepatocytes and in vivo in human liver chimeric mice. RESULTS: HEV was inhibited by concurrent HCV replication in human hepatocytes. This exclusion phenotype was linked to the protease activity of HCV. These findings were corroborated by the fact that in HEV on HCV super-infected mice, HEV viral loads were reduced in individual mice. Similarly, HCV on HEV super-infected mice showed reduced HCV viral loads. CONCLUSION: Direct interference of both viruses with HCV NS3/4A as the determinant was observed. In vivo, we detected reduced replication of both viruses after super-infection in individual mice. These findings provide new insights into the pathogenesis of HCV-HEV co-infections and should contribute to its clinical management in the future.

The Human Liver-Expressed Lectin CD302 Restricts Hepatitis C Virus Infection.

C-type lectin domain-containing proteins (CTLDcps) shape host responses to pathogens and infectious disease outcomes. Previously, we identified the murine CTLDcp Cd302 as restriction factor, limiting hepatitis C virus (HCV) infection of murine hepatocytes. In this study, we investigated in detail the human orthologue's ability to restrict HCV infection in human liver cells. CD302 overexpression in Huh-7.5 cells potently inhibited infection of diverse HCV chimeras representing seven genotypes. Transcriptional profiling revealed abundant CD302 mRNA expression in human hepatocytes, the natural cellular target of HCV. Knockdown of endogenously expressed CD302 modestly enhanced HCV infection of Huh-7.5 cells and primary human hepatocytes. Functional analysis of naturally occurring CD302 transcript variants and engineered CD302 mutants showed that the C-type lectin-like domain (CTLD) is essential for HCV restriction, whereas the cytoplasmic domain (CPD) is dispensable. Coding single nucleotide polymorphisms occurring in human populations and mapping to different domains of CD302 did not influence the capacity of CD302 to restrict HCV. Assessment of the anti-HCV phenotype at different life cycle stages indicated that CD302 preferentially targets the viral entry step. In contrast to the murine orthologue, overexpression of human CD302 did not modulate downstream expression of nuclear receptor-controlled genes. Ectopic CD302 expression restricted infection of liver tropic hepatitis E virus (HEV), while it did not affect infection rates of two respiratory viruses, including respiratory syncytial virus (RSV) and the alpha coronavirus HVCoV-229E. Together, these findings suggest that CD302 contributes to liver cell-intrinsic defense against HCV and might mediate broader antiviral defenses against additional hepatotropic viruses. IMPORTANCE The liver represents an immunoprivileged organ characterized by enhanced resistance to immune responses. However, the importance of liver cell-endogenous, noncytolytic innate immune responses in pathogen control is not well defined. Although the role of myeloid cell-expressed CTLDcps in host responses to viruses has been characterized in detail, we have little information about their potential functions in the liver and their relevance for immune responses in this organ. Human hepatocytes endogenously express the CTLDcp CD302. Here, we provide evidence that CD302 limits HCV infection of human liver cells, likely by inhibiting a viral cell entry step. We confirm that the dominant liver-expressed transcript variant, as well as naturally occurring coding variants of CD302, maintain the capacity to restrict HCV. We further show that the CTLD of the protein is critical for the anti-HCV activity and that overexpressed CD302 limits HEV infection. Thus, CD302 likely contributes to human liver-intrinsic antiviral defenses.

[Vaccines against hepatitis E virus: state of development]. / Impfstoffe gegen Hepatitis E: Wo stehen wir?

No vaccine against the hepatitis E virus (HEV) is currently licensed in Europe. In contrast, HEV-239 (Hecolin®, Xiamen Innovax Biotech Co., Xiamen, China), a vaccine against HEV genotype 4, has been available in China for 10 years. Challenges for the development of vaccines arise mainly from the differences between the genotypes with regard to distribution, transmission routes and risk groups. Other obstacles include the envelopment of HEV in blood by host membranes, replication in various organs outside the liver and weaker immune responses in vulnerable groups. This article reviews the current status of vaccines against HEV that are available and in advanced preclinical evaluation, with a focus on vaccine development strategies. Challenges and limitations are described.Current vaccine candidates focus on protein-based immunisation with the aim of inducing protective, neutralising antibody responses. The goal of the HEV-239 pivotal trial with more than 100,000 study participants was to prevent acute symptomatic infections. However, it is unclear to what extent asymptomatic infections were prevented by the vaccine and whether it is effective enough in patients at risk for a complicated course, such as patients with liver cirrhosis, immunosuppressed individuals and pregnant women. Efficient in vitro models are increasingly enabling the development of monoclonal neutralising antibodies for passive immunisation or therapy.Future vaccines should demonstrate clear protection against all genotypes in addition to a very good safety profile. The development of an efficient passive immunisation strategy, especially for immunosuppressed individuals, is desirable.

Hepatitis E virus is highly resistant to alcohol-based disinfectants.

BACKGROUND & AIMS: Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis worldwide and is mainly transmitted via the fecal-oral route or through consumption of contaminated food products. Due to the lack of efficient cell culture systems for the propagation of HEV, limited data regarding its sensitivity to chemical disinfectants are available. Consequently, preventive and evidence-based hygienic guidelines on HEV disinfection are lacking. METHODS: We used a robust HEV genotype 3 cell culture model which enables quantification of viral infection of quasi-enveloped and naked HEV particles. For HEV genotype 1 infections, we used the primary isolate Sar55 in a fecal suspension. Standardized quantitative suspension tests using end point dilution and large-volume plating were performed for the determination of virucidal activity of alcohols (1-propanol, 2-propanol, ethanol), WHO disinfectant formulations and 5 different commercial hand disinfectants against HEV. Iodixanol gradients were conducted to elucidate the influence of ethanol on quasi-enveloped viral particles. RESULTS: Naked and quasi-enveloped HEV was resistant to alcohols as well as alcohol-based formulations recommended by the WHO. Of the tested commercial hand disinfectants only 1 product displayed virucidal activity against HEV. This activity could be linked to phosphoric acid as an essential ingredient. Finally, we observed that ethanol and possibly non-active alcohol-based disinfectants disrupt the quasi-envelope structure of HEV particles, while leaving the highly transmissible and infectious naked virions intact. CONCLUSIONS: Different alcohols and alcohol-based hand disinfectants were insufficient to eliminate HEV infectivity with the exception of 1 commercial ethanol-based product that included phosphoric acid. These findings have major implications for the development of measures to reduce viral transmission in clinical practice. LAY SUMMARY: Hepatitis E virus (HEV) showed a high level of resistance to alcohols and alcohol-based hand disinfectants. The addition of phosphoric acid to alcohol was essential for virucidal activity against HEV. This information should be used to guide improved hygiene measures for the prevention of HEV transmission.

Risk of transfusion-transmitted hepatitis E virus infection from pool-tested platelets and plasma.

BACKGROUND AND AIMS: Immunocompromised patients are at risk of chronic hepatitis E which can be acquired by blood transfusions. Currently, screening of blood donors (BDs) for HEV RNA with a limit of detection (LOD) of 2,000 IU/ml is required in Germany. However, this may result in up to 440,000 IU of HEV RNA in blood products depending on their plasma volume. We studied the residual risk of transfusion-transmitted (tt) HEV infection when an LOD of 2,000 IU/ml is applied. METHODS: Highly sensitive individual donor testing for HEV RNA on the Grifols Procleix Panther system (LOD 7.89 IU/ml) was performed. HEV loads were quantified by real-time PCR. RESULTS: Of 16,236 donors, 31 (0.19%) were HEV RNA positive. Three BDs had viral loads between 710 and 2,000 IU/ml, which pose a significant risk of tt hepatitis E with any type of blood product. Eight BDs had viral loads of >32 to 710 IU/ml, which pose a risk of tt hepatitis E with platelet or plasma transfusions because of their higher plasma volume compared to red blood cell concentrates. Eight of these 11 potentially infectious BDs were seronegative for HEV, indicating a recent infection. Only 8 of 31 donors had viral loads >2,000 IU/ml that would also have been detected by the required screening procedure and 12 had very low HEV loads (<32 IU/ml). CONCLUSIONS: Screening of BDs with an LOD of 2,000 IU/ml reduced the risk of tt HEV infection by about 73% for red blood cell concentrates but by just 42% for platelet and fresh frozen plasma transfusions. Single donor screening (LOD <32 IU/ml) should lead to an almost 100% risk reduction. LAY SUMMARY: Immunocompromised patients, such as solid organ or hematopoietic stem cell recipients, are at risk of chronic hepatitis E, which can be acquired via blood transfusions. The risk of transfusion-transmitted hepatitis E in these patients may not be sufficiently controlled by (mini-)pool hepatitis E virus RNA screening of blood donors. Single donor screening should be considered to improve the safety of blood products.

Quantification of polyreactive immunoglobulin G facilitates the diagnosis of autoimmune hepatitis.

BACKGROUND AND AIMS: Detection of autoantibodies is a mainstay of diagnosing autoimmune hepatitis (AIH). However, conventional autoantibodies for the workup of AIH lack either sensitivity or specificity, leading to substantial diagnostic uncertainty. We aimed to identify more accurate serological markers of AIH with a protein macroarray. APPROACH AND RESULTS: During the search for more-precise autoantibodies to distinguish AIH from non-AIH liver diseases (non-AIH-LD), IgG antibodies with binding capacities to many human and foreign proteins were identified with a protein macroarray and confirmed with solid-phase ELISAs in AIH patients. Subsequently, polyreactive IgG (pIgG) was exemplarily quantified by reactivity against human huntingtin-interacting protein 1-related protein in bovine serum albumin blocked ELISA (HIP1R/BSA). The diagnostic fidelity of HIP1R/BSA binding pIgG to diagnose AIH was assessed in a retrospective training, a retrospective multicenter validation, and a prospective validation cohort in cryoconserved samples from 1,568 adults from 10 centers from eight countries. Reactivity against HIP1R/BSA had a 25% and 14% higher specificity to diagnose AIH than conventional antinuclear and antismooth muscle antibodies, a significantly higher sensitivity than liver kidney microsomal antibodies and antisoluble liver antigen/liver pancreas antigen, and a 12%-20% higher accuracy than conventional autoantibodies. Importantly, HIP1R/BSA reactivity was present in up to 88% of patients with seronegative AIH and in up to 71% of AIH patients with normal IgG levels. Under therapy, pIgG returns to background levels of non-AIH-LD. CONCLUSIONS: pIgG could be used as a promising marker to improve the diagnostic workup of liver diseases with a higher specificity for AIH compared to conventional autoantibodies and a utility in autoantibody-negative AIH. Likewise, pIgG could be a major source of assay interference in untreated AIH.

HEV-Associated Neuralgic Amyotrophy: A Multicentric Case Series.

BACKGROUND: Neuralgic amyotrophy (NA) has been described as a possible extrahepatic manifestation of hepatitis E virus (HEV) infection. Usually, HEV-associated NA occurs bilaterally. The clinical characteristics determining the course of HEV-associated NA have still not been defined. METHODS: In this retrospective multicentric case series, 16 patients with HEV-associated NA were studied and compared to 176 HEV patients without NA in terms of their age, sex, and ALT levels. RESULTS: Neither gender distribution (75% vs. 67% male) nor age (47 vs. 48 years median) differed significantly between the NA patients and controls. Eight NA patients (50%) presented with bilateral involvement-seven of these had right-side dominance and one had left-side dominance. Thirteen cases (81%) were hospitalized. Eight of these patients stayed in hospital for five to seven days, and five patients stayed for up to two weeks. The time from the onset of NA to the HEV diagnosis, as well as the diagnostic and therapeutic proceedings, showed a large variability. In total, 13 (81%) patients received treatment: 1/13 (8%) received intravenous immunoglobulins, 8/13 (62%) received glucocorticoids, 3/13 (23%) received ribavirin, and 6/13 (46%) received pregabalin/gabapentin. Patients with ages above the median (47 years) were more likely to be treated (p = 0.001). CONCLUSION: HEV-associated NA causes a relevant morbidity. In our case series neither the type of treatment nor the time of initiation of therapy had a significant effect on the duration of hospitalization or the course of the disease. The clinical presentation, the common diagnostic and therapeutic procedures, and the patients' characteristics showed large variability, demonstrating the necessity of standardized protocols for this rare but relevant disease.

Significant compartment-specific impact of different RNA extraction methods and PCR assays on the sensitivity of hepatitis E virus detection.

BACKGROUND: RNA detection in plasma/stool is the gold-standard for diagnosis of hepatitis E virus (HEV) infection. The impact of viral extraction methods on HEV RNA detection is poorly investigated. METHODS: We determined the limit of detection of the RealStar HEV RT-PCR V2.0 Kit (altona Diagnostics, RS) utilizing 3 RNA extraction methods (COBAS® AmpliPrep Total Nucleic Acid Isolation Kit, TNAi Roche; MagNA Pure 96 DNA, Viral NA SV Kit, MgP; QIAamp Viral RNA mini Kit Qiagen; VRK) in plasma and stool. The most sensitive method was evaluated in a total of 307 longitudinal samples of patients with HEV infection (acute = 18/chronic = 36) and compared to results with the former diagnostic standard of our centre (TNAi/FastTrack Diagnostic; FTD). RESULTS: The plasma-LOD was 49, 94 and 329 IU/mL for extraction with MgP, VRK and TNAi respectively. In stool, the LOD was 21 IU/mL, 528 IU/mL and indefinable for extraction with TNAi, VRK and MgP respectively. Utilizing longitudinal patient plasma samples, MgP/RS revealed 56 HEV RNA-positive samples in 158 negative samples as determined by TNAi/FTD. In stool, from 37 HEV negative samples (TNAi/FTD), 15 were positive with TNAi/RS. At end of treatment, 8 out of 27 chronically infected patients were RNA positive with MgP/RS, while classified negative with TNAi/FTD. A relapse occurred in 3 of these patients. CONCLUSION: Different methods for RNA extraction and quantification have a significant, compartment-specific impact on the sensitivity of HEV detection. Knowledge about the favourable combinations of extraction and quantification has important implications for diagnosis and patients receiving antiviral therapy.

Hepatitis C reference viruses highlight potent antibody responses and diverse viral functional interactions with neutralising antibodies.

OBJECTIVE: Neutralising antibodies are key effectors of infection-induced and vaccine-induced immunity. Quantification of antibodies' breadth and potency is critical for understanding the mechanisms of protection and for prioritisation of vaccines. Here, we used a unique collection of human specimens and HCV strains to develop HCV reference viruses for quantification of neutralising antibodies, and to investigate viral functional diversity. DESIGN: We profiled neutralisation potency of polyclonal immunoglobulins from 104 patients infected with HCV genotype (GT) 1-6 across 13 HCV strains representing five viral GTs. Using metric multidimensional scaling, we plotted HCV neutralisation onto neutralisation maps. We employed K-means clustering to guide virus clustering and selecting representative strains. RESULTS: Viruses differed greatly in neutralisation sensitivity, with J6 (GT2a) being most resistant and SA13 (GT5a) being most sensitive. They mapped to six distinct neutralisation clusters, in part composed of viruses from different GTs. There was no correlation between viral neutralisation and genetic distance, indicating functional neutralisation clustering differs from sequence-based clustering. Calibrating reference viruses representing these clusters against purified antibodies from 496 patients infected by GT1 to GT6 viruses readily identified individuals with extraordinary potent and broadly neutralising antibodies. It revealed comparable antibody cross-neutralisation and diversity between specimens from diverse viral GTs, confirming well-balanced reporting of HCV cross-neutralisation across highly diverse human samples. CONCLUSION: Representative isolates from six neutralisation clusters broadly reconstruct the functional HCV neutralisation space. They enable high resolution profiling of HCV neutralisation and they may reflect viral functional and antigenic properties important to consider in HCV vaccine design.