Neutralizing Antibodies Targeting BK Polyomavirus: Clinical Importance and Therapeutic Potential for Kidney Transplant Recipients.

Most of the world's adult population is latently infected by the BK polyomavirus. It causes asymptomatic infection in healthy individuals but emerged as a threat to kidney transplant recipients because of virus-associated nephropathy caused by immunosuppressive therapy. In these conditions, when a functional cellular response is impaired by immunosuppression, neutralizing antibodies may play a major role because they can directly prevent infection of target cells, independently of cell-mediated immunity, by binding to the viral particles. Studying the contribution of anti-BK virus neutralizing antibodies in viral control has long been hampered by the lack of convenient in vitro models, but major progress has been made in the past decade. The four BK virus genotypes have been demonstrated to behave as distinct serotypes. A low recipient neutralizing antibody titer against the donor's serotype before kidney transplant has been significantly associated with BK virus replication after transplant. Different mechanisms exploited by the BK virus to evade neutralizing antibodies have been described. Recent studies also support the potential benefit of administering intravenous Igs or monoclonal neutralizing antibodies as a therapeutic strategy, and more interestingly, this could also be used as preventive or preemptive therapy before advanced kidney damage has occurred. Besides, neutralizing antibodies could be induced by vaccination. In this review, we summarize accumulated knowledge on anti-BK virus neutralizing antibodies as well as their clinical importance and therapeutic potential for kidney transplant recipients.

Molecular epidemiology and risk factors associated with BK and JC polyomavirus urinary shedding after kidney allograft.

Polyomaviruses BK (BKPyV) and JC (JCPyV), belonging to the Polyomaviridae, are responsible for human pathologies. In kidney transplant recipients, BKPyV replication can lead to irreversible nephron damage whereas JCPyV replication remains asymptomatic. Concomitant replication is rare and potential competition between the infections has been described. The aim of this retrospective case-control study was to describe the molecular epidemiology and risk factors associated with BKPyV and JCPyV replication in a cohort of kidney transplant recipients. In total, 655 urine samples from 460 patients were tested for BKPyV and JCPyV DNA. Positive samples were submitted to strain genotyping. Demographic and clinical characteristics were also compared. Isolated JCPyV and BKPyV was found in 16.5% and 23.3% of patients, respectively; co-replication was rare (3.9%). BKPyV strains Ib-2, Ib-1, and IVc-2 were the most prevalent. JCPyV strains mostly belonged to genotypes 4 and 1B. During follow-up, JCPyV shedding significantly reduced the risk of BKPyV DNAuria, with an odds ratio of 0.57 (95% confidence interval: 0.35-0.99), and was associated with better prognosis than BKPyV replication, based on the estimated glomerular filtration rate. Molecular epidemiology of BKPyV and JCPyV strains in our region was similar to previous studies. This study suggests that JCPyV is benign and appears to limit damaging BKPyV replication. JCPyV DNAuria screening could thus be a useful strategy to predict BKPyV-related outcomes.

Impact of pre-graft serology on risk of BKPyV infection post-renal transplantation.

OBJECTIVES: BK polyomavirus-associated nephropathy is a troublesome disease caused by BK polyomavirus (BKPyV) infection in immunocompromised renal graft recipients. There are no effective treatments available, making immunosuppression reduction the only management option. Thus, pre-graft predictive BKPyV replication markers are needed for identification of patients at high risk of viraemia. METHODS: We conducted a retrospective study to assess the correlation between pre-transplantation BKPyV serostatus and post-transplantation incidence of BKPyV infection. Sera from 329 recipients and 222 matched donors were tested for anti-BKPyV antibodies against BKPyV serotypes I and IV by using a virus-like particle-based immunoglobulin G enzyme-linked immunosorbent assay, and BKPyV DNA load was monitored for at least 1 year post-transplantation. RESULTS: Eighty recipients were viruric and 59 recipients were viraemic post-transplantation. In the post-transplantation period, the probability of developing viraemia for serotype I increased from 4.3% for the D-/R+ group to 12.1% for the D+/R+ group, climbing to 37.5% for the D+/R- group (P < 0.05). When calculating recipient mean titres for serotypes I and IV, we observed a clear difference in the proportions of viraemia, decreasing from 50% for mean titres <400 to 13.5% for titres ≥400 (P < 0.001), as well as a higher proportion of presumptive nephropathy (50% versus 23.1%, respectively; P < 0.05). In univariate analysis, this parameter had an odds ratio of 6.41 for the risk of developing post-transplantation BKPyV viraemia (95% confidence interval 3.16-13.07; P < 0.0001). CONCLUSIONS: Determination of both donor and recipient BKPyV seropositivity before transplantation and antibody titre measurements may serve as a predictive tool to manage clinical BKPyV infection by identification of patients at high risk.

BK Polyomavirus bkv-miR-B1-5p: A Stable Micro-RNA to Monitor Active Viral Replication after Kidney Transplantation.

BACKGROUND: Bkv-miR-B1-5p is a viral micro-RNA (miRNA) specifically produced during BK polyomavirus (BKPyV) replication. Recent studies have suggested using bkv-miR-B1-5p as a biomarker to monitor viral infection and predict complications in kidney transplant patients. To identify the technical limitations of this miRNA quantification in biological samples, knowledge of its stability and distribution in the extracellular compartment is necessary. Moreover, a proof of concept for using bkv-miR-B1-5p as a biomarker of active replication in chronic infection is still missing in the published literature. METHODS: The stability of bkv-miR-B1-5p was evaluated in samples derived from cell cultures and in urine from BKPyV-infected kidney transplant recipients. The miRNA was quantified in different fractions of the extracellular compartment, including exosomes, and protein binding was evaluated. Finally, we developed an in vitro model for chronic culture of BKPyV clinical isolates to observe changes in the bkv-miR-B1-5p level during persistent infections. RESULTS: Bkv-miR-B1-5p is a stable biomarker in samples from humans and in vitro experiments. Marginally associated with the exosomes, most of the circulating bkv-miR-B1-5p is bound to proteins, especially Ago2, so the miRNA quantification does not require specific exosome isolation. The bkv-miR-B1-5p level is predictable of viral infectivity, which makes it a potential specific biomarker of active BKPyV replication after kidney transplantation.

BK Polyomavirus Hijacks Extracellular Vesicles for En Bloc Transmission.

Most people are asymptomatic carriers of the BK polyomavirus (BKPyV), but the mechanisms of persistence and immune evasion remain poorly understood. Furthermore, BKPyV is responsible for nephropathies in kidney transplant recipients. Unfortunately, the sole therapeutic option is to modulate immunosuppression, which increases the risk of transplant rejection. Using iodixanol density gradients, we observed that Vero and renal proximal tubular epithelial infected cells release two populations of infectious particles, one of which cosediments with extracellular vesicles (EVs). Electron microscopy confirmed that a single vesicle could traffic tens of viral particles. In contrast to naked virions, the EV-associated particles (eBKPyVs) were not able to agglutinate red blood cells and did not use cell surface sialylated glycans as an attachment factor, demonstrating that different entry pathways were involved for each type of infectious particle. However, we also observed that naked BKPyV and eBKPyV were equally sensitive to neutralization by the serum of a seropositive patient or commercially available polyvalent immunoglobulin preparations, which occurred at a postattachment step, after endocytosis. In conclusion, our work shows a new mechanism that likely plays a critical role during the primary infection and in the persistence, but also the reactivation, of BKPyV.IMPORTANCE Reactivation of BKPyV is responsible for nephropathies in kidney transplant recipients, which frequently lead to graft loss. The mechanisms of persistence and immune evasion used by this virus remain poorly understood, and a therapeutic option for transplant patients is still lacking. Here, we show that BKPyV can be released into EVs, enabling viral particles to infect cells using an alternative entry pathway. This provides a new view of BKPyV pathogenesis. Even though we did not find any decreased sensitivity to neutralizing antibodies when comparing EV-associated particles and naked virions, our study also raises important questions about developing prevention strategies based on the induction or administration of neutralizing antibodies. Deciphering this new release pathway could enable the identification of therapeutic targets to prevent BKPyV nephropathies. It could also lead to a better understanding of the pathophysiology of other polyomaviruses that are associated with human diseases.

[Viruses : vesicle tamers]. / Les virus : des dompteurs de vésicules.

There is a close relationship between viruses and lipid vesicles. The most frequently described concerns enveloped viruses, which acquire their envelope through mechanisms involved in extracellular vesicles (EVs) biogenesis. However, EVs' hijacking is not unique to enveloped viruses. In 2013, a new category of viruses emerged : the quasi-enveloped viruses. These are naked viruses found in vesicles at certain steps of their viral cycle. Actually, several naked viruses, from different families, hijack the production routes of EVs : poliovirus, polyomaviruses, rotavirus, etc. This diversion of EVs confers many advantages : diversification of entry and exit pathways, infectivity improvement and immune evasion. This review will take the reader around this subject.

Hepatitis E Virus Lifecycle and Identification of 3 Forms of the ORF2 Capsid Protein.

BACKGROUND & AIMS: Hepatitis E virus (HEV) infection is a major cause of acute hepatitis worldwide. Approximately 2 billion people live in areas endemic for HEV and are at risk of infection. The HEV genome encodes 3 proteins, including the ORF2 capsid protein. Detailed analyses of the HEV life cycle has been hampered by the lack of an efficient viral culture system. METHODS: We performed studies with gt3 HEV cell culture-produced particles and patient blood and stool samples. Samples were fractionated on iodixanol gradients and cushions. Infectivity assays were performed in vitro and in human liver chimeric mice. Proteins were analyzed by biochemical and proteomic approaches. Infectious particles were analyzed by transmission electron microscopy. HEV antigen levels were measured with the Wantaï enzyme-linked immunosorbent assay. RESULTS: We developed an efficient cell culture system and isolated HEV particles that were infectious in vitro and in vivo. Using transmission electron microscopy, we defined the ultrastructure of HEV cell culture-produced particles and particles from patient sera and stool samples. We also identified the precise sequence of the infectious particle-associated ORF2 capsid protein. In cultured cells and in samples from patients, HEV produced 3 forms of the ORF2 capsid protein: infectious/intracellular ORF2 (ORF2i), glycosylated ORF2 (ORF2g), and cleaved ORF2 (ORF2c). The ORF2i protein associated with infectious particles, whereas the ORF2g and ORF2c proteins were massively secreted glycoproteins not associated with infectious particles. ORF2g and ORF2c were the most abundant antigens detected in sera from patients. CONCLUSIONS: We developed a cell culture system and characterized HEV particles; we identified 3 ORF2 capsid proteins (ORF2i, ORF2g, and ORFc). These findings will advance our understanding of the HEV life cycle and improve diagnosis.

Identification of Piperazinylbenzenesulfonamides as New Inhibitors of Claudin-1 Trafficking and Hepatitis C Virus Entry.

Hepatitis C virus (HCV) infection causes 500,000 deaths annually, in association with end-stage liver diseases. Investigations of the HCV life cycle have widened the knowledge of virology, and here we discovered that two piperazinylbenzenesulfonamides inhibit HCV entry into liver cells. The entry of HCV into host cells is a complex process that is not fully understood but is characterized by multiple spatially and temporally regulated steps involving several known host factors. Through a high-content virus infection screening analysis with a library of 1,120 biologically active chemical compounds, we identified SB258585, an antagonist of serotonin receptor 6 (5-HT6), as a new inhibitor of HCV entry in liver-derived cell lines as well as primary hepatocytes. A functional characterization suggested a role for this compound and the compound SB399885, which share similar structures, as inhibitors of a late HCV entry step, modulating the localization of the coreceptor tight junction protein claudin-1 (CLDN1) in a 5-HT6-independent manner. Both chemical compounds induced an intracellular accumulation of CLDN1, reflecting export impairment. This regulation correlated with the modulation of protein kinase A (PKA) activity. The PKA inhibitor H89 fully reproduced these phenotypes. Furthermore, PKA activation resulted in increased CLDN1 accumulation at the cell surface. Interestingly, an increase of CLDN1 recycling did not correlate with an increased interaction with CD81 or HCV entry. These findings reinforce the hypothesis of a common pathway, shared by several viruses, which involves G-protein-coupled receptor-dependent signaling in late steps of viral entry.IMPORTANCE The HCV entry process is highly complex, and important details of this structured event are poorly understood. By screening a library of biologically active chemical compounds, we identified two piperazinylbenzenesulfonamides as inhibitors of HCV entry. The mechanism of inhibition was not through the previously described activity of these inhibitors as antagonists of serotonin receptor 6 but instead through modulation of PKA activity in a 5-HT6-independent manner, as proven by the lack of 5-HT6 in the liver. We thus highlighted the involvement of the PKA pathway in modulating HCV entry at a postbinding step and in the recycling of the tight junction protein claudin-1 (CLDN1) toward the cell surface. Our work underscores once more the complexity of HCV entry steps and suggests a role for the PKA pathway as a regulator of CLDN1 recycling, with impacts on both cell biology and virology.

Apolipoprotein(a) inhibits hepatitis C virus entry through interaction with infectious particles.

The development of different cell culture models has greatly contributed to increased understanding of the hepatitis C virus (HCV) life cycle. However, it is still challenging to grow HCV clinical isolates in cell culture. If overcome, this would open new perspectives to study HCV biology, including drug-resistant variants emerging with new antiviral therapies. In this study we hypothesized that this hurdle could be due to the presence of inhibitory factors in patient serum. Combining polyethylene glycol precipitation, iodixanol gradient, and size-exclusion chromatography, we obtained from HCV-seronegative sera a purified fraction enriched in inhibitory factors. Mass spectrometric analysis identified apolipoprotein(a) (apo[a]) as a potential inhibitor of HCV entry. Apo(a) consists of 10 kringle IV domains (KIVs), one kringle V domain, and an inactive protease domain. The 10 KIVs are present in a single copy with the exception of KIV type 2 (KIV2 ), which is encoded in a variable number of tandemly repeated copies, giving rise to numerous apo(a) size isoforms. In addition, apo(a) covalently links to the apolipoprotein B component of a low-density lipoprotein through a disulfide bridge to form lipoprotein(a). Using a recombinant virus derived from the JFH1 strain, we confirmed that plasma-derived and recombinant lipoprotein(a) as well as purified recombinant apo(a) variants were able to specifically inhibit HCV by interacting with infectious particles. Our results also suggest that small isoforms are less inhibitory than the large ones. Finally, we observed that the lipoprotein moiety of HCV lipoviroparticles was essential for inhibition, whereas functional lysine-binding sites in KIV7 , KIV8 , and KIV10 were not required. CONCLUSIONS: Our results identify apo(a) as an additional component of the lipid metabolism modulating HCV infection. (Hepatology 2017;65:1851-1864).

Correction to: The expression of HCV-associated host factors is dependent on the hepatoma cell line used in HCV studies.

In this article, a clone of HepG2 stably expressing CD81 (HepG2-CD81) was used. Unfortunately, after cell line authentication.

A Simple and Reliable Strategy for BK Virus Subtyping and Subgrouping.

BK virus (BKV)-associated diseases in transplant recipients are an emerging issue. However, identification of the various BK virus subtypes/subgroups is a long and delicate process on the basis of currently available data. Therefore, we wanted to define a simple and effective one-step strategy for characterizing all BK virus strains from the VP1 gene sequence. Based on the analysis of 199 available complete DNA VP1 sequences, phylogenetic trees, alignments, and isolated polymorphisms were used to define an effective strategy for distinguishing the 12 different BK virus subtypes/subgroups. Based on the 12 subtypes identified from the 199 complete BKV VP1 sequences (1,089 bp), 60 mutations that can be used to differentiate these various subtypes/subgroups were identified. Some genomic areas were more variable and comprised mutational hot spots. From a subregion of only 100 bp in the VP1 region (1977 through 2076), we therefore constructed an algorithm that enabled rapid determination of all BKV subtypes/subgroups with 99% agreement (197/199) relative to the complete VP1 sequence. We called this domain of the BK viral genome the BK typing and grouping region (BKTGR). Finally, we validated our viral subtype identification process in a population of 100 transplant recipients with 100% efficiency. The new simpler method of BKV subtyping/subgrouping reported here constitutes a useful tool for future studies that will help us to more clearly understand the impact of BKV subtypes/subgroups on diagnosis, infection, and BK virus-associated diseases.

High association of T1858-G1896 precore mutations with impaired base pairing and high hepatitis B virus DNA levels in HBeAg-negative chronically infected patients.

The progression of liver disease in hepatitis B virus (HBV) infection is fostered by active virus replication. Mutations in the basal core promoter (BCP) and precore (PC) regions of the HBV genome are known to have an impact on viral replication. The aim of the present study was to assess the correlation of mutation profiles in the BCP and PC regions with the viral load in HBeAg-negative chronically infected patients. The HBV genotype, BCP/PC mutations, serum HBV DNA levels, and associated serological markers were analyzed in 92 HBeAg-negative chronically infected patients. Sequence analysis of the BCP and PC regions revealed variability of 19% and 24.1%, respectively. This variability was primarily associated with five critical positions (1753, 1762, 1764, 1896 and 1899). An elevated HBV viral load (>20,000 IU/ml) was classically correlated with F2-F4 liver fibrosis, elevated serum alanine aminotransferase levels, 1762/1764 and 1753 combination mutations, and surprisingly, with an 1858T-1896G double mutation that impairs base pairing at the base of the bulge in the ε encapsidation signal. An analysis of covariance confirmed the independent nature of the relationship between the 1858T-1896G double mutation and the HBV viral load. In conclusion, independently of conventional parameters, this study demonstrates that a high serum HBV DNA level was also associated with PC 1858-1896 mutations. These BCP/PC mutations may have important clinical implications as predictive factors for HBV DNA increase.

Ribavirin restores IFNα responsiveness in HCV-infected livers by epigenetic remodelling at interferon stimulated genes.

OBJECTIVES: Caveats in the understanding of ribavirin (RBV) mechanisms of action has somehow prevented the development of better analogues able to further improve its therapeutic contribution in interferon (IFN)-based and direct antiviral agent-based regimens for chronic HCV or other indications. Here, we describe a new mechanism by which RBV modulates IFN-stimulated genes (ISGs) and contributes to restore hepatic immune responsiveness. DESIGN: RBV effect on ISG expression was monitored in vitro and in vivo, that is, in non-transformed hepatocytes and in the liver of RBV mono-treated patients, respectively. Modulation of histone modifications and recruitment of histone-modifying enzymes at target promoters was analysed by chromatin immunoprecipitation in RBV-treated primary human hepatocytes and in patients' liver biopsies. RESULTS: RBV decreases the mRNA levels of several abnormally preactivated ISGs in patients with HCV, who are non-responders to IFN therapy. RBV increases G9a histone methyltransferase recruitment and histone-H3 lysine-9 dimethylation/trimethylation at selected ISG promoters in vitro and in vivo. G9a pharmacological blockade abolishes RBV-induced ISG downregulation and severely impairs RBV ability to potentiate IFN antiviral action and induction of ISGs following HCV infection of primary human hepatocytes. CONCLUSIONS: RBV-induced epigenetic changes, leading to decreased ISG expression, restore an IFN-responsive hepatic environment in patients with HCV, which may also prove useful in IFN-free regimens.

Comparative Evaluation of Three Nucleic Acid-Based Assays for BK Virus Quantification.

With the growing importance of BK virus (BKV), effective and efficient screening for BKV replication in plasma and urine samples is very important for monitoring renal transplant and hematopoietic stem cell transplant recipients, who are at increased risk of BKV-associated diseases. However, recent assays proposed by many manufacturers have not been tested, and the available tests have not been standardized. The aim of the present study was to evaluate and compare the performances of three commercially available kits, R-gene, GeneProof, and RealStar, on plasma and urine specimens from patients infected with various genotypes and to determine the correlations with the results from a reference laboratory. A qualitatively excellent global agreement (96.8%) was obtained. RealStar PCR tended to give a higher sensitivity, especially for subtype Ib1 samples. Comparison of 30 plasma samples and 53 urine samples showed a good agreement between the three assays, with Spearman's Rho correlation coefficient values falling between 0.92 and 0.98 (P < 0.001). Moreover, a perfect correlation was obtained for comparison of the assay performances with the AcroMetrix BKV panel (P < 0.001 for all comparisons). According to Bland-Altman analysis, more than 95% (240/249 comparisons) of sample comparisons were situated in the range of the mean ± 2 standard deviations (SD). The greatest variability between assays was observed for 10.2% of subtype Ib2 samples, with differences of >1 log10 copies/ml. In conclusion, this study demonstrated the reliable and comparable performances of the R-gene, GeneProof, and RealStar real-time PCR systems for quantification of BKV in urine and plasma samples. All three real-time PCR assays are appropriate for screening of BKV replication in patients.

Which therapeutic option for hepatitis C virus genotype 1?

OBJECTIVE: Sofosbuvir and simeprevir in combination with standard therapy are now available for the treatment of patients chronically infected with hepatitis C virus (HCV) genotype 1. With boceprevir and telaprevir, four treatment options are, therefore, now available to clinicians. Phase 3 studies conducted with simeprevir and sofosbuvir compared sustained virological response (SVR) data with those obtained with standard combination therapy and did not include a control arm. It is important to quantify the contribution of these molecules compared to the first direct antiviral agents available. MATERIAL AND METHODS: For HCV genotype 1 patients, we performed a literature review and compared all SVR data from phase 3 randomized placebo-controlled trials conducted with these four molecules according to virological characteristics (genotype, viral load) and patient characteristics (IL28B polymorphism, stage of fibrosis). RESULTS: Simeprevir and sofosbuvir provide a net gain in terms of SVR compared to boceprevir and telaprevir except in the case of telaprevir for treatment-naïve HCV genotype 1b patients. Sofosbuvir achieves higher SVR rates than simeprevir except for treatment-naïve IL28B CC patients and naïve HCV genotype 1b patient. Further, simeprevir moderately improve SVR rates compared to telaprevir in treatment-naïve patients with F3-F4 fibrosis and with HCV genotype 1a infection. CONCLUSION: Sofosbuvir and simeprevir greatly improve the virological response rate compared to first-generation protease inhibitors. All of these data may help in guiding the physician's treatment decisions, based on financial constraints and patient characteristics. These data can be easily updated with future treatment and demonstrate the contribution of new treatment regimens to achieve optimal SVR rates.

In vitro infection of primary human hepatocytes by HCV-positive sera: insights on a highly relevant model.

OBJECTIVE: Adult primary human hepatocytes (PHHs) support the complete infection cycle of natural HCV from patients' sera. The molecular details underlying sera infectivity towards these cells remain largely unknown. Therefore, we sought to gain a deeper comprehension of these features in the most physiologically relevant culture system. DESIGN: Using kinetic experiments, we defined the optimal conditions to infect PHH and explored the link between cell organisation and permissivity. Based on their infectivity, about 120 sera were classified in three groups. Concentration of 52 analytes was measured in 79 selected sera using multiplexed immunobead-based analyte profiling. RESULTS: PHH permissivity towards HCV infection negatively correlated with cell polarisation and formation of functional bile canaliculi. PHH supported HCV replication for at least 2 weeks with de novo virus production. Depending on their reactivity, sera could be classified in three groups of high, intermediate or low infectivity toward PHH. Infectivity could not be predicted based on the donors' clinical characteristics, viral load or genotype. Interestingly, highly infectious sera displayed a specific cytokine profile with low levels of most of the 52 tested analytes. Among them, 24 cytokines/growth factors could impact hepatocyte biology and infection efficiency. CONCLUSIONS: We identified critical factors leading to efficient PHH infection by HCV sera in vitro. Overall, we showed that this cellular model provides a useful tool for studying the mechanism of HCV infection in its natural host cell, selecting highly infectious isolates, and determining the potency of drugs towards various HCV strains.

Disulfide bonds in hepatitis C virus glycoprotein E1 control the assembly and entry functions of E2 glycoprotein.

Class II membrane fusion proteins have been described in viruses in which the envelope proteins are derived from a precursor polyprotein containing two transmembrane glycoproteins arranged in tandem. Although the second protein, which carries the membrane fusion function, is in general well characterized, the companion protein, which is a protein chaperone for the folding of the fusion protein, is less well characterized for some viruses, like hepatitis C virus (HCV). To investigate the role of the class II companion glycoprotein E1 of HCV, we chose to target conserved cysteine residues in the protein, and we systematically mutated them in a full-length infectious HCV clone by reverse genetics. All the mutants were infectious, albeit with lower titers than the wild-type virus. The reduced infectivity was in part due to a decrease in viral assembly, as revealed by measurement of intracellular infectivity and by quantification of core protein released from cells transfected with mutant genomes. Analyses of mutated proteins did not show any major defect in folding. However, the mutations reduced virus stability, and they could also affect the density of infectious viral particles. Mutant viruses also showed a defect in cell-to-cell transmission. Finally, our data indicate that HCV glycoprotein E1 can also affect the fusion protein E2 by modulating its recognition by the cellular coreceptor CD81. Therefore, in the context of HCV, our data identify an additional function of a class II companion protein as a molecule that can control the binding capacity of the fusion protein.

The antimalarial ferroquine is an inhibitor of hepatitis C virus.

UNLABELLED: Hepatitis C virus (HCV) is a major cause of chronic liver disease. Despite recent success in improving anti-HCV therapy, additional progress is still needed to develop cheaper and interferon (IFN)-free treatments. Here, we report that ferroquine (FQ), an antimalarial ferrocenic analog of chloroquine, is a novel inhibitor of HCV. FQ potently inhibited HCV infection of hepatoma cell lines by affecting an early step of the viral life cycle. The antiviral activity of FQ on HCV entry was confirmed with pseudoparticles expressing HCV envelope glycoproteins E1 and E2 from six different genotypes. In addition to its effect on HCV entry, FQ also inhibited HCV RNA replication, albeit at a higher concentration. We also showed that FQ has no effect on viral assembly and virion secretion. Using a binding assay at 4°C, we showed that FQ does not prevent attachment of the virus to the cell surface. Furthermore, virus internalization was not affected by FQ, whereas the fusion process was impaired in the presence of FQ as shown in a cell-cell fusion assay. Finally, virus with resistance to FQ was selected by sequential passage in the presence of the drug, and resistance was shown to be conferred by a single mutation in E1 glycoprotein (S327A). By inhibiting cell-free virus transmission using a neutralizing antibody, we also showed that FQ inhibits HCV cell-to-cell spread between neighboring cells. Combinations of FQ with IFN, or an inhibitor of HCV NS3/4A protease, also resulted in additive to synergistic activity. CONCLUSION: FQ is a novel, interesting anti-HCV molecule that could be used in combination with other direct-acting antivirals.

The expression of HCV-associated host factors is dependent on the hepatoma cell line used in HCV studies.

Chronic infection by hepatitis C virus (HCV) is a major cause of liver cirrhosis and hepatocellular carcinoma. At present, the most commonly used in vitro model of HCV infection is based on hepatoma cell lines. However, they were obtained from different patients and different cancers and/or differ in their characteristics and permissiveness to HCV. HCV infection can be modulated by several host factors, so we compared six different hepatoma cell lines that are used as in vitro models for HCV for some of these host factors: the seven known HCV entry factors, the six best-characterized HCV-associated microRNAs, and the two single-nucleotide polymorphisms near the IL28B gene associated with response to pegylated alpha interferon and ribavirin combination therapy, all assessed by quantitative PCR. We showed that the cell lines, including Huh-7 and Huh-7-derived cells, have different microRNA and HCV entry factor expression profiles as well as different IL28B genotypes. In conclusion, some of the observed differences might explain the differences in permissiveness of the cell lines, but, above all, they raise questions about the reliability of in vitro HCV research data gathered to date.

The value and complexity of studying cellular immunity against BK Polyomavirus in kidney transplant recipients.

BK Polyomavirus is of particular concern for kidney transplant recipients, due to their immunosuppression. This problem is exacerbated by the high effectiveness of antirejection therapies, which also compromise the organism's ability to fight viral infections. The long-term risk is loss of graft function through BKPyV-associated nephropathy (BKPyVAN). The assessment of host immunity and its link to the control of viral infections is a major challenge. In terms of humoral immunity, researchers have highlighted the prognostic value of the pre-transplantation anti-BKPyV immunoglobulin G titer. However, humoral immunity alone does not guarantee viral clearance, and the correlation between the humoral response and the time course of the infection remains weak. In contrast, cellular immunity variables appear to be more closely associated with viral clearance, given that the cellular immune response to the kidney transplant is the main target of immunosuppressive treatments in recipients. However, the assessment of the cellular immune response to BK Polyomavirus is complex, and many details still need to be characterized. Here, we review the current state of knowledge about BKPyV cellular immunity, as well as the difficulties that may be encountered in studying it in kidney transplant recipient. This is an essential area of research for optimizing the management of transplant recipients and minimizing the risks associated with insidious BKPyV disease.