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Background Coronavirus disease 2019 (COVID-19) is associated with increased morbidity and mortality in older adults. Although the advent of the first vaccines has significantly reduced these rates, data on older adults in clinical trials are scarce. Objectives We quantified and compared the humoral response in individuals with vs. without pre-existing seropositivity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in a cohort of 69 patients living in a nursing home and who had received the recommended two doses of the Comirnaty (Pfizer-BioNTech®) vaccine. Results All 69 patients (100%) tested positive for antibodies against SARS-CoV-2 at 2 months post-vaccination. Residents with a pre-vaccination infection had significantly higher titers of anti-spike 1 IgG than those with no prior infection (median [interquartile range]: 55,726 [14463–78852] vs. 1314 [272–1249] arbitrary units, respectively; p

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.

BackgroundIn the fight against SARS-COV-2, the development of serological assays based on different antigenic domains represent a versatile tool to get a comprehensive picture of the immune response or differentiate infection from vaccination beyond simple diagnosis.ObjectivesHere we use a combination of the Nucleoprotein (NP), the Spike 1 (S1) and Spike 2 (S2) subunits, and the receptor binding domain (RBD) and N-terminal domain (NTD) of the Spike antigens from the Syrius-CoViDiag® multiplex IgG assay, to follow the immune response to SARS-CoV-2 infection over a long time period and depending on disease severity.ResultsUsing a panel of 209 sera collected from 61 patients up to eight months after infection, we observed that most patients develop an immune response against multiple viral epitope, but anti-S2 antibodies seemed to last longer. For all the tested IgGs, we have found higher titers for hospitalized patients than for non-hospitalized ones. Moreover the combination of the five different IgG titers increased the correlation to the neutralizing antibody titers than if considered individually.ConclusionMultiplex immunoassays have the potential to improve diagnostic performances, especially for ancient infection or mild form of the disease presenting weaker antibody titers. Also the combined detection of anti-NP and anti-Spike-derived domains can be useful to differentiate vaccination from viral infection and accurately assess the antibody potential to neutralize the virus.

Since December 2019, an emerging infectious viral disease implicating a coronavirus SARS-CoV-2 has caused a global pandemic. Elderly people, being more fragile, are the most affected by the severity and lethality of this disease. The NH and LTCU of the Amiens University Hospital registered their first Covid-19 cases in February 2020, which lead to the opening of a Covid-19 dedicated unit and of specific protocol for confinement. This descriptive study was analyzing the prevalence of Covid-19 seroconversion within the NH and the LTCU of the Amiens University Hospital. Both this screening test and the nasopharyngeal swab PCRs were in order to assess the impact of the Covid-19 epidemic in NH and LTCU. On June 15th and 16th, the serological tests for Covid-19 were positive for 146 (66.1%) of the residents tested. The seroconversion rate was significantly different (p 0.001) between the NH (88.7%) and the LTCU (45.6%). During the epidemic, there was no excess mortality index within the NH and LTCU services of the Amiens University Hospital. Among frail patients, the role of immunosenescence can be discussed to account for the absence of this inflammatory reaction. This study showed that isolating the infected patient in a dedicated unit significantly reduces the risk of seroconversion and contamination compared to isolating them within their own unit.

Background: Children are underrepresented in the COVID-19 pandemic and often experience milder disease than adolescents and adults. Reduced severity is possibly due to recent and more frequent seasonal human coronaviruses (HCoV) infections. We assessed the seroprevalence of SARS-CoV-2 and seasonal HCoV specific antibodies in a large cohort in north-eastern France. Methods: In this cross-sectional seroprevalence study, serum samples were collected from children and adults requiring hospital admission for non-COVID-19 between February and August 2020. Antibody responses to SARS-CoV-2 and seasonal HCoV (229E, HKU1, NL63, OC43) were assessed using a bead-based multiplex assay, Luciferase-Linked ImmunoSorbent Assay, and a pseudotype neutralisation assay. Findings: In 2,408 individuals, seroprevalence of SARS-CoV-2-specific antibodies was 7-8% with three different immunoassays. Antibody levels to seasonal HCoV increased substantially up to the age of 10. Antibody responses in SARS-CoV-2 seropositive individuals were lowest in adults 18-30 years. In SARS-CoV-2 seronegative individuals, we observed cross-reactivity between antibodies to the four HCoV and SARS-CoV-2 Spike. In contrast to other antibodies to SARS-CoV-2, specific antibodies to sub-unit 2 of Spike (S2) in seronegative samples were highest in children. Upon infection with SARS-CoV-2, antibody levels to Spike of betacoronavirus OC43 increased across the whole age spectrum. No SARS-CoV-2 seropositive individuals with low levels of antibodies to seasonal HCoV were observed. Interpretation: Our findings underline significant cross-reactivity between antibodies to SARS-CoV-2 and seasonal HCoV, but provide no significant evidence for cross-protective immunity to SARS-CoV-2 infection due to a recent seasonal HCoV infection. In particular, across all age groups we did not observe SARS-CoV-2 infected individuals with low levels of antibodies to seasonal HCoV. Funding: This work was supported by the « URGENCE COVID-19 » fundraising campaign of Institut Pasteur, by the French Government's Investissement d'Avenir program, Laboratoire d'Excellence Integrative Biology of Emerging Infectious Diseases (Grant No. ANR-10-LABX-62-IBEID), and by the REACTing (Research & Action Emerging Infectious Diseases), and by the RECOVER project funded by the European Union's Horizon 2020 research and innovation programme under grant agreement No. 101003589, and by a grant from LabEx IBEID (ANR-10-LABX-62-IBEID).

Background Fourth-generation immunoassays (such as the ADVIA Centaur® HIV Ag/Ab Combo (CHIV) assay) have improved the early diagnosis of human immunodeficiency virus (HIV), and their sensitivity and specificity usually exceed 99%. In regions with a low prevalence of HIV infection, however, the regular occurrence of false positives interferes with a medical laboratory’s workflow. The additional reagent and staff costs associated with false positives can nevertheless be avoided or reduced by gaining a better knowledge of the CHIV assay’s performance. Objectives/study design To improve our HIV diagnosis strategy, we retrospectively analyzed all the Centaur® CHIV assays and confirmatory tests performed at Amiens University Medical Center between 2012 and 2018. We used open-source machine learning software to process this large database, develop a predictive model, and identify a new cut-off for Centaur® CHIV index interpretation. Results A total of 56,682 HIV serological assay results were analyzed. The results of the CHIV assay were initially reactive or indeterminate for 449 samples. After p24 antigen and/or immunoblotting, there were 171 (38%) false positives and 278 (62%) confirmed true positives. The application of a cut-off of 2.12 led to reclassification of 130 of the 171 false positives as true negatives. Combining our predictive model with medical record analysis reduced the number of false positive CHIV assay results from 171 to 12. Conclusions The efficiency of the Centaur® CHIV assay can be increased by adjusting its cut-off for positivity. This adjustment may reduce the number of unnecessary confirmatory tests and accelerate the delivery of HIV test results.

In the context of a second wave of SARS-CoV-2 transmission, the use of saliva sampling has become an issue of real importance. SARS-CoV-2 RNA screening was performed on nasopharyngeal and saliva swabs collected from 501 individuals from residential homes for the elderly. The saliva samples were collected at the same time as the nasopharyngeal samples. Nasopharyngeal samples yielded positive results for 26 individuals, only two of whom also tested positive with saliva swabs. In this context, saliva collected by swabbing the fluid is not an ideal sample.

Background: Kidney transplant recipients (KTRs) are exposed to a high risk of BK polyomavirus (BKPyV) replication, which in turn may lead to graft loss. Although the microRNAs (miRNAs) bkv-miR-B1-3p and bkv-miR-B1-5p are produced during the viral cycle, their putative value as markers of viral replication has yet to be established. In KTRs, the clinical relevance of the changes over time in BKPyV miRNA levels has not been determined. Methods: In a retrospective study, we analyzed 186 urine samples and 120 plasma samples collected from 67 KTRs during the first year post-transplantation. Using a reproducible, standardized, quantitative RT-PCR assay, we measured the levels of bkv-miR-B1-3p and bkv-miR-B1-5p (relative to the BKPyV DNA load). Results: Detection of the two miRNAs had low diagnostic value for identifying patients with DNAemia or for predicting DNAuria during follow-up. Seven of the 14 KTRs with a sustained BKPyV infection within the first year post-transplantation showed a progressive reduction in the DNA load and then a rapid disappearance of the miRNAs. DNA and miRNA loads were stable in the other seven KTRs. Conclusions: After the DNA-based diagnosis of BKPyV infection in KTRs, bkv-miR-B1-3p and bkv-miR-B1-5p levels in the urine might be valuable markers for viral replication monitoring and thus might help physicians to avoid an excessive reduction in the immunosuppressive regimen.

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 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.

International audience; No abstract available

ObjectivesHealthcare workers are more likely to be infected by SARS-CoV-2. In order to assess the infectious risk associated with working in a hospital, we sought to estimate the proportion of healthcare professionals infected with SARS-CoV-2 by screening staff in a University Medical Center in France.SettingA hospital-wide screening campaign (comprising a serological test and a questionnaire) ran from 18 May to 26 July 2020.Primary and secondary outcome measuresThe seroprevalence rate was analysed in a multivariate analysis according to sociodemographic variables (age, sex and profession), exposure to SARS-CoV-2 and symptoms.ResultsA total of 4840 professionals were included, corresponding to 74.5% of the centre’s staff. The seroprevalence rate (95% CI) was 9.7% (7.0% to 12.4%). Contact with a confirmed case of COVID-19 was significantly associated with seropositivity (OR (95% CI: 1.43, (1.15 to 1.78)). The seroprevalence rate was significantly higher among nursing assistants (17.6%) than among other healthcare professionals. The following symptoms were predictive of COVID-19: anosmia (OR (95% CI): 1.55, (1.49 to 1.62)), ageusia (1.21, (1.16 to 1.27)), fever (1.15, (1.12 to 1.18)), myalgia (1.03, (1.01 to 1.06)) and headache (1.03, (1.01 to 1.04)).

Background The emergence of the global SARS-CoV-2 pandemic required the rapid and large-scale deployment of PCR and serological tests in different formats. Objectives Real-life evaluation of these tests is needed. Using 168 samples from patients hospitalized for COVID-19, non-hospitalized patients but infected with SARS-CoV-2, patients participating in screening campaigns, and samples from patients with a history of other seasonal coronavirus infections, we evaluated the clinical performance of 5 serological assays widely used worldwide (WANTAI®, BIORAD®, EUROIMMUN®, ABBOTT® and LIAISON®). Results For hospitalized patients, all these assays showed a sensitivity of 100 % from day 9 after the symptoms onset. On the other hand, sensitivity was much lower for patients who did not require hospitalization for COVID-19 confirmed by PCR (from 91.6 % for WANTAI® to 69 % for LIAISON®). These differences do not seem to be due to the antigens chosen by the manufacturers but more to the test formats (IgG detection versus total antibodies). In addition, more than 50 days after a positive PCR for CoV-2-SARS the proportion of positive patients seem to decrease. We did not observe any significant cross-reactions for these techniques with the four other seasonal coronaviruses. Conclusion In conclusion, the evaluation and knowledge of the serological tests used is important and should require an optimized strategy adaptation of the analysis laboratories to best meet patient’s expectations in the face of this health crisis.

Extracellular vesicles have recently emerged as a novel mode of viral transmission exploited by naked viruses to exit host cells through a nonlytic pathway. Extracellular vesicles can allow multiple viral particles to collectively traffic in and out of cells, thus enhancing the viral fitness and diversifying the transmission routes while evading the immune system. This has been shown for several RNA viruses that belong to the Picornaviridae, Hepeviridae, Reoviridae, and Caliciviridae families; however, recent studies also demonstrated that the BK and JC viruses, two DNA viruses that belong to the Polyomaviridae family, use a similar strategy. In this review, we provide an update on recent advances in understanding the mechanisms used by naked viruses to hijack extracellular vesicles, and we discuss the implications for the biology of polyomaviruses.

In order to fight the SARS-CoV-2 pandemic infection, there is a growing need and demand for diagnostic tools that are complementary and different from the RT-PCR currently in use. Multiple serological tests are or will be very soon available but need to be evaluated and validated. We have thus tested 4 immunochromatographic tests for the detection of antibodies to SARS-CoV-2. In addition, we assessed the kinetics of antibody appearance using these assays in 22 patients after they were tested positive by RT-PCR. We observed great heterogeneity in antibody detection post-symptom onset. The median antibody detection time was between 8 and 10 days according to the manufacturers. All the tests showed a sensitivity of 60 to 80% on day 10 and 100% on day 15. In addition, a single cross-reaction was observed with other human coronavirus infections. Thus, immunochromatographic tests for the detection of anti-SARS-CoV-2 antibodies may have their place for the diagnostic panel of COVID-19.

Aims Coronavirus disease 2019 (COVID‐19) is a rapidly progressing pandemic, with four million confirmed cases and 280,000 deaths at the time of writing. Some studies have suggested that diabetes is associated with a greater risk of developing severe forms of COVID‐19. The primary objective of the present study was to compare the clinical features and outcomes in hospitalized COVID‐19 patients with vs. without diabetes. Methods All consecutive adult patients admitted to Amiens University Hospital (Amiens, France) with confirmed COVID‐19 up until April 21st, 2020, were included. The composite primary endpoint comprised admission to the intensive care unit (ICU) and death. Both components were also analyzed separately in a logistic regression analysis and a Cox proportional hazards model. Results A total of 433 patients (median age: 72; 238 (55%) men; diabetes: 115 (26.6%)) were included. Most of the deaths occurred in non‐ICU units and among older adults. Multivariate analyses showed that diabetes was associated neither with the primary endpoint (odds ratio (OR): 1.12; 95% confidence interval (CI): 0.66‐1.90) nor with mortality (hazard ratio: 0.73; 95%CI: 0.40‐1.34) but was associated with ICU admission (OR: 2.06; 95%CI 1.09‐3.92, p=0.027) and a longer length of hospital stay. Age was negatively associated with ICU admission and positively associated with death. Discussion Diabetes was prevalent in a quarter of the patients hospitalized with COVID‐19; it was associated with a greater risk of ICU admission but not with a significant elevation in mortality. Further investigation of the relationship between COVID‐19 severity and diabetes is warranted. This article is protected by copyright. All rights reserved.

Reactivation of BK polyomavirus (BKPyV) infection is frequently increasing in transplant recipients treated with potent immunosuppressants and highlights the importance of immune system components in controlling viral reactivation. However, the immune response to BKPyV in general and the role of antiviral cytokines in infection control in particular are poorly understood. Here, we investigated the efficacy of interferons (IFN) alpha, lambda and gamma with regard to the BKPyV multiplication in Vero cells. Treatment with IFN-gamma inhibited the expression of the viral protein VP1 in a dose-dependent manner and decreased the expression of early and late viral transcripts. Viral inhibition by IFN-gamma was confirmed in human cells (Caki-1 cells and renal proximal tubular epithelial cells). One of the IFN-stimulated genes most strongly induced by IFN-gamma was the coding for the enzyme indoleamine 2,3 dioxygenase (IDO), which is known to limit viral replication and regulates the host immune system. The antiviral activity induced by IFN-gamma could be reversed by the addition of an IDO inhibitor, indicating that IDO has a specific role in anti-BKPyV activity. A better understanding of the action mechanism of these IFN-gamma-induced antiviral proteins might facilitate the development of novel therapeutic strategies.

Aims To describe the characteristics of patients hospitalized with COVID-19 (including their long-term at-home medication use), and compare them with regard to the course of the disease. To assess the association between renin–angiotensin system inhibitors (RASIs) and disease progression and critical outcomes. Methods and results All consecutive hospitalized patients with laboratory-confirmed COVID-19 in a university hospital in Amiens (France) were included in this study. The primary composite endpoint was admission to an intensive care unit (ICU) or death before ICU admission. Univariable and multivariable logistic regression models were used to identify factors associated with the composite endpoint. Between 28 February 2020 and 30 March 2020, a total of 499 local patients tested positive for SARS-CoV-2. Of these, 231 were not hospitalized {males 33%; median [interquartile range (IQR)] age: 44 (32–54)}, and 268 were hospitalized [males 58%; median (IQR) age: 73 (61–84)]. A total of 116 patients met the primary endpoint: 47 died before ICU admission, and 69 were admitted to the ICU. Patients meeting the primary endpoint were more likely than patients not meeting the primary endpoint to have coronary heart disease and to have been taking RASIs; however, the two subsets of patients did not differ with regard to median age. After adjustment for other associated variables, the risk of meeting the composite endpoint was 1.73 times higher (odds ratio 1.73, 95% confidence interval 1.02–2.93) in patients treated at baseline with a RASI than in patients not treated with this drug class. This association was confirmed when the analysis was restricted to patients treated with antihypertensive agents. Conclusions We highlighted a potential safety signal for RASIs, the long-term use of which was independently associated with a higher risk of severe COVID-19 and a poor outcome. Due to the widespread use of this important drug class, formal proof based on clinical trials is needed to better understand the association between RASIs and complications of COVID-19.

In the last 20 years, the management of BK polyomavirus (BKPyV) reactivation in kidney transplant patients has become a true challenge for the transplant community. The only treatment option is based on the early identification of at-risk patients. The number of reported risk factors for BKPyV reactivation has increased markedly in the literature last years, although they are sometimes in an unclear or contradictory manner. Our purpose is to provide a systematic review and meta-analysis of risk factors for BKPyV viremia and nephropathy described in multivariate analyses. The PubMed database was searched for prospective or prospectively-based observational studies on risk factors for BKPyV viremia and/or nephropathy. Our qualitative assessment of risk factors was based on the odds ratios and hazard ratios calculated in multivariate regression analyses. Of the 241 publications screened, 34 were included in the qualitative analysis. In all, 144 and 19 distinct factors were analyzed for BKPyV viremia and for BKPyV nephropathy, respectively. Our evaluation highlighted eight risk factors for BKPyV viremia: a tacrolimus regimen, a deceased donor, a male recipient, a history of previous transplant, age at transplantation, ureteral stent use, delayed graft function, and acute rejection episodes increased the risk of BKV viremia to varying extents. Tacrolimus and acute rejection episodes were also associated with a higher incidence of BKPyV nephropathy. BKPyV reactivation is a serious complication after renal transplantation. With a view to combating this problem, existing data should be published in full, and new prospective international multicenter studies should be performed.

International audience; The major histocompatibility complex class I polypeptide-related sequence A (MICA) glycoprotein mediates the activation of the natural killer group 2D receptor (NKG2D) expressed on NK and CD8+ T cells. A methionine or valine at position 129 in exon 3 results in strong (MICA129 met) or weak (MICA129 val) binding to NKG2D. The MICA A5.1 allele causes a premature stop codon. Various NKG2D polymorphisms are associated with low (NKC3 C/C and NKC4 C/C) or high (NKC3 G/G and NKC4 T/T) levels of NK cell cytotoxic activity. In 162 patients with spondyloarthritis (115 with ankylosing spondyloarthritis, 46 with psoriatic arthritis and 1 with reactive arthritis) compared to 124 healthy controls, MICA-129 with methionine allele was more frequent in patients with spondyloarthritis (odds ratio (OR) (95% confidence interval) = 4.84 (2.75-8.67)), whereas MICA-129 val/val, MICA A5.1 and NKC3 C/C variants were less frequent (OR = 0.20 (0.11-0.37), 0.15 (0.06-0.36) and 0.24 (0.13-0.44), respectively). After adjustment for HLA-B*27 status, only NKC3 C/C remained linked to spondyloarthritis (adjusted OR = 0.14 (0.06-0.33)). Homozygosity for MICA A5.1 is linked to ankylosing spondyloarthritis, and NKC3 C/C and MICA-129 val/val to psoriatic arthritis. MICA and NKC3 polymorphisms (related to a low NK cell cytotoxic activity) constituted a genetic association with spondyloarthritis.

Highlights • The respiratory viral profile varied with age. • The distribution of viruses is variable over the year depending on the species. • Persistence of non-enveloped viruses throughout the year. • Atmospheric temperature was rarely a limiting factor in the circulation of viruses., Background Acute respiratory infections are a principal cause of illness and mortality especially in young children worldwide. Objectives To study the epidemiology and seasonality of viral respiratory infections in hospitalized children (under the age of 16) between September 2012 and August 2016. Study design Nasopharyngeal swabs or aspirates were collected from 3199 symptomatic patients and then screened with a routine multiplex PCR assay. Results Respiratory viruses were detected for 1624 (50.8%) of the 3199 children in the study population. Of these, 210 (13.3%) were positive for two viruses, 28 (1.7%) were positive for three, and 3 (0.2%) were positive for four. The viral profile varied with age. Some viruses were significantly more frequent in children under the age of 1 month (such as human respiratory syncytial virus (p

International audience; Hepatitis C virus (HCV) is a human hepatotropic virus, but many hepatoma cell lines are not permissive to this virus. In a previous study, we observed that SNU‐182, SNU‐398 and SNU‐449 hepatoma cell lines were nonpermissive to HCV. To understand the nonpermissivity, we evaluated the ability of each cell line to support the different steps of HCV life cycle (entry, replication and production of infectious particles). Using retroviral pseudoparticles pseudotyped with HCV envelope proteins and recombinant HCV produced in cell culture, we observed that low level or absence of claudin‐1 (CLDN1) expression limited the viral entry process in SNU‐182 and SNU‐398 cells, respectively. Our results also showed that supplementation of the three cell lines with miR‐122 partly restored the replication of a JFH1 HCV replicon. Finally, we observed that expression of apolipoprotein E (ApoE) was very low or undetectable in the three cell lines and that its ectopic expression permits the production of infectious viral particles in SNU‐182 and SNU‐398 cells but not in SNU‐449 cells. Nevertheless, the supplementation of SNU‐182, SNU‐398 and SNU‐449 cells with CLDN1, miR‐122 and ApoE was not sufficient to render these cells as permissive as HuH‐7 cells. Thus, these cell lines could serve as cell culture models for functional studies on the role of CLDN1, miR‐122 and ApoE in HCV life cycle but also for the identification of new restriction and/or dependency host factors essential for HCV infection.

Assessment of the intensity of immunosuppression in transplant recipients to estimate the risk of rejection and infection is not entirely satisfactory at the present time. Determination of Torque teno virus (TTV) viral load appears to be a promising tool in this setting.We evaluated the level of replication and kinetics of TTV during the first three months after kidney transplantation compared to BK virus replication.In a retrospective cohort of 116 renal transplant recipients, TTV viral load gradually increased during the first three months post-transplantation with no significant difference or discriminatory threshold between patients with and without BK virus replication. However, the level of TTV replication appeared to be indirectly related to the risk of BK virus replication, particularly according to the induction treatment used (antithymocyte globulin: ATG or basiliximab). Among patients receiving ATG, those receiving cyclosporine had significantly lower TTV viral loads (p 0.01) with threefold lower reactivation of BKPyV (13 vs 37%) 3 months post-transplantation. Similarly, among the women in our cohort, TTV viral load was significantly higher in women receiving ATG (6.58 ± 1.57 versus 4.62 ± 2.0 logThe multiparametric variation of TTV viral load does not appear to be individually appropriate for the early detection or monitoring of possible post-transplant BKPyV virus reactivation in renal transplant recipients.

International audience; OBJECTIVES: After kidney transplantation, human BK polyomavirus (BKPyV) can induce a progressive disease, in three stages: viruria, viremia, and then nephropathy after a few months of viral replication. Therapeutic intervention is recommended when BKPyV is detected in the plasma. The objective of our study was to assess urinary BKPyV nucleic acid test as a predictor for developing viremia. METHODS: We first defined a viruria threshold based on 393 time-matched urine and plasma samples collected after kidney transplantation and then to validate this threshold, we followed-up a cohort of 236 kidney transplant patients. RESULTS: A BKPyV viruria threshold of 6.71 log10 copies/mL best discriminated between plasma-positive and plasma-negative patients (sensitivity: 90.9% (95%CI: 86.5-95); specificity: 90.3% (95%CI: 86.3-94.3); area under the curve: 0.953 (95%CI: 0.933-0.974). In the validation cohort, the risk of developing BKPyV viremia at one year was 16.5% (39/236) and rose to 90.7% (39/43) if BKPyV viruria remained above the threshold of 6.71 for more than one month. CONCLUSION: Sustained BKPyV viruria is a reliable, early marker of patients at high risk of developing BKPyV viremia. This marker should alert the clinician early, and thus allow timely therapeutic intervention.

International audience; The immunosuppression required for graft tolerance in kidney transplant patients can trigger latent BK polyomavirus (BKPyV) reactivation, and the infection can progress to nephropathy and graft rejection. It has been suggested that pre-transplantation BKPyV serostatus in donors and recipients is a predictive marker for post-transplantation BKPyV replication. The fact that research laboratories have used many different assay techniques to determine BKPyV serostatus complicates these data analysis. Even studies based on the same technique differed in their standard controls choice, the antigenic structure type used for detection, and the cut-off for seropositivity. Here, we review the different BKPyV VP1 antigens types used for detection and consider the various BKPyV serostatus assay techniques' advantages and disadvantages. Lastly, we highlight the obstacles in the implementation of a consensual BKPyV serologic assay in clinics (e.g., the guidelines absence in this field).

Treatment options for Hepatitis C infection have greatly improved with direct-acting antiviral (DAA) combinations achieving high cure rates. Nevertheless, the cost of this treatment is still high and access to treatment in many countries has been preferentially reserved for patients with more severe fibrosis (F3 and F4). In this French nationwide study, we investigated the epidemiological characteristics and genotype distribution of hepatitis C virus (HCV) in treatment-naive patients with METAVIR fibrosis stages between F0 and F2 in order to identify patient profiles that became eligible for unrestricted treatment in a second period. Between 2015 and 2016 we collected data from nine French university hospitals on a total of 584 HCV positive patients with absent, mild or moderate liver fibrosis. The most represented genotypes were genotype 1b (159/584; 27.2%), followed by genotype 1a (150/584; 25.7%); genotype 3 (87/584: 14.9%); genotype 4 (80/584; 13.7%). Among genotype 4: 4a was predominantly encountered with 22 patients (27.5% of genotype 4). Genotypes 1b and 1a are currently the most frequent virus types present in treatment-naive patients with mild fibrosis in France. They can be readily cured using the available DAA. Nevertheless, non-a/non-d genotype 4 is also frequent in this population and clinical data on the efficacy of DAA on these subtypes is missing. The GEMHEP is the French group for study and evaluation of viral hepatitis on a national scale. Data collection on epidemiological and molecular aspects of viral hepatitis is performed on a regular basis in all main French teaching hospitals and serves as a basis for surveillance of these infections. Analysis and trends are regularly published on behalf of the GEMHEP group. Data collection was performed retrospectively over the 2015–2016 period, covering nine main university hospitals in France. A total of 584 hepatitis C positive patients were included in this study. Genotyping of the circulating viruses showed a high prevalence of genotypes 1b and 1a in our population. The epidemiology of hepatitis C is slowly changing in France, particularly as a consequence of the rise of ‘non-a non-d’ genotype 4 viruses mainly originating from African populations. More data concerning treatment efficacy of these genotypes is needed in order to guide clinical care.

Chronic liver diseases caused by either hepatitis B or C viruses are a major health problem around the world. Despite major advances accomplished in recent years in understanding the physiology of both viruses using in vitro and/or in vivomodels, there is no vaccine for HCV available. Moreover, susceptibility to acute and chronic infection and the response to treatments are different between HBV or HCV infected patients. Crucial information can be collected using a robust cell model that permits the culture of clinical isolates along with the investigation of the virus-host interaction. The recent progress in the field of cell reprogramming and differentiation has opened new opportunities in viral hepatitis research raising the hopes of developing new improved therapeutics. In this review, we discuss current models for hepatitis B and C studies and their limitations, and also the iPSC model, and its relevance to the viral host cell interactions.

Chronic infection with hepatitis B (HBV) and C virus (HCV) is linked with a pro-inflammatory state, predisposing to cirrhosis and liver cancer, particularly hepatocellular carcinoma (HCC). A role for Toll-like receptor (TLR) signalling in hepatocarcinogenesis was recently documented. We hypothesised a link TLR3 and TLR4 polymorphisms and HCC, as surrogates for the significance of TLR signalling in the promotion and initiation of HCC.We recruited 174 HCV-infected patients, 100 HBV-infected patients and 360 healthy control subjects. TLR3 (rs3775290) and TLR4 (rs4986790) genotyping was done by PCR-restriction fragment length polymorphisms (PCR-RFLP), LFTs and AFP by standard routine techniques. Liver fibrosis was assessed clinically by the Fibrotest and Actitest.The TLR3 rs3775290 minor T genotype was linked with increased risk of chronic HBV (P = 0.05) and HCV (P = 0.031) infection. The TLR4 rs4986790 minor G genotype was linked with significantly increased risk for HBV/HCV chronic infection (P0.001). Subgroups analyses indicated decreased risk of HBV-related HCC in relation to TLR3 rs3775290 CC/CT genotype (P = 0.022), with increased risk ascribed to the minor (T) allele (P = 0.04). Likewise, TLR4 rs4985790 minor (GG) genotype was positively associated with HBV-linked HCC (P0.001). Furthermore, a link between TLR3 TT (P0.001) andTLR4 GG (P = 0.04) minor genotypes was noted in relation to increased risk of HCV-related disease.TLR3 and TLR4 polymorphisms are promising biomarkers of liver cirrhosis and cancer associated with HBV and HCV infection.

The present study was undertaken to investigate the association between Hepatitis B Virus (HBV) infection and polymorphisms of tumour necrosis factor alpha TNF-α -308 G>A, TNF-α -238 G>A and TNF RII VNTR (p75) gene promoter in a Tunisian population. Blood samples were collected from 100 Tunisian patients with HBV infection, 45 with Chronic Hepatitis (CH), 36 with Liver Cirrhosis (LC), 15 with Hepatocellular Carcinoma (HCC) and 200 healthy individuals of similar ethnicity. Genomic DNA was extracted from peripheral blood leukocytes. Genotyping of the analysed polymorphisms was performed using Amplified Refractory Mutation System-Polymerase Chain Reaction (ARMS-PCR), Restriction Fragment Length Polymorphism (RFLP) and Variable Number Tandem Repeat PCR (PCR-VNTR). The variant homozygotes -308 GG were associated with 50% decreased risk of HBV chronic infection (GG vs AA+GA; p=0.010; OR=0.50; 95%CI=0.29-0.85). However, the carriers of minor allele -308 A have higher risk (1.5 times) to develop a chronic infection than other patients (p=0.027; OR=1.46; 95%CI=1.04-2.06). The minor allele of -238 polymorphism was positively associated with virus resistance and the development of chronic infection (p=0.043; OR=1.42; 95%CI =1.01 1.99). The distribution of -308, -238 and TNF RII VNTR (p75) among the three groups differed significantly. For HCC groups, there were statistically significant differences in allele distribution in -308, -238 respectively in which A allele remains a risk factor for HBV evolution to HCC (p=0.008 and p=0.026). Haplotype analysis revealed that TNF-α (-308A; -238A) was significantly associated to HBV chronic infection and moreover to disease aggravation to HCC stage. Our findings imply that variations in the genes governing the levels of constitutive and inducible TNF-α and TNF RII might be an important risk factor, which could explain the variable outcomes of HBV infection.

Anemia is a well-known RBV-related event in HCV therapy which is exacerbated by the addition of telaprevir and boceprevir. This retrospective study evaluated and compared ribavirin exposure and parameters able to influence hemoglobin decrease in a large population of patients treated with dual or triple therapy. Patients on triple therapy had higher ribavirin concentrations at week 12 of treatment (3460 ng/mL vs. 1843 ng/mL; P .0001). An association was also observed between week 12 eGFR and ribavirin concentration only for patients on triple therapy (P = .002). The proportion of patients with a 20 mL/min/1.73 m(2) decrease in eGFR at week 12 was higher among patients on triple therapy: 32%, 14%, and 5% for boceprevir, telaprevir, and dual therapy, respectively (P = .025 and .026). No correlation was observed between boceprevir and telaprevir concentrations and hemoglobin or eGFR decrease. Exacerbation of anemia in patients on triple therapy is related to higher ribavirin concentrations. We provide an explanation for this increase in plasma RBV concentration. Triple therapy with PEG-IFN, RBV, and telaprevir or boceprevir will remain the only HCV treatment option for many patients. Our data show that the RBV dose can be decreased while maintaining adequate plasma concentrations and reducing anemia.

This study compared the performance of 3 automated immunoassays, Architect

Objectives TLRs are one of the most studied families of pathogen recognition receptors (PRRs) and play a pivotal role during HCV infection. The binding of ligands to TLRs on antigen presenting cells (APCs) leads to secretion of inflammatory cytokines, such as IL6, and induction of the acquired immunity response. Therefore, it has become necessarily to harness the TLRs properties’ on therapeutically tools to enhance the HCV treatment response. Herein, we investigated the association between TLR3, TLR4 variants and nine IL-6 polymorphisms, and response to anti-viral treatment during HCV infection. Methods Study subjects comprised 120 patients infected with HCV-1b and treated with Peg-IFN/RBV. Genotyping of nine IL-6 variants were done by real –time PCR and genotyping of TLRs polymorphisms were done by RFLP-PCR. Results High frequency of TLR3 rs3775290 C/C genotype and TLR4 rs4986790 A/A genotype were noticed among patients with sustained viral response compared to Non-responder patients. The genetic association of TLR3 and TLR4 variants was evidenced by the improvement in the kinetics of viral load decline, with superiority of TLR3 compared to TLR4. Among, nine polymorphisms studied on IL-6 only rs1800796, rs2069845 and rs1880242 were associated with sustained viral response. Our study reports also that the common favourable IL-28B variant is essential for TLR-activated antiviral protection. Conclusion TLR3 and TLR4 are involved in the pathogenesis of viral infections. TLR3 may be better suited than TLR4 to activate anti-viral program. Moreover, we propose that the Th2 cytokine, IL-6, constitutes a determinant of the outcome of therapy in HCV patients.

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.

Comment in : New Models to Study Hepatitis E Virus Replication and Particular Characteristics of Infection: The Needle Hides in the Hay Stack. [Gastroenterology. 2018]; International audience; 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.

HCV has been associated with a pro-inflammatory state, which predisposes to hepatocellular carcinoma (HCC). However, the different molecular mechanisms underlying the effect of HCV infection on HCC progression remain unclear. Although HCV infection illustrates the potential role of host genetics in the outcome of infectious diseases, there is no clear overview of some single nucleotide polymorphisms (SNPs) influencing spontaneous or treatment-induced HCV eradication. We studied the possible role of HCV infection in the processes of HCC initiation and performed a systematic analysis using data mining approaches to identify host polymorphisms associated with treatment response and HCC development using topological analysis of protein-proteins interactions (PPI) networks. On the basis of our analysis performed, we identified key hub proteins related to HCV-treatment response infection and to HCC development. Host genetic polymorphisms, such as inosine triphosphatase (ITPA), interferon, lambda 3 (IFNL3), Q5 interferon, lambda 4 (IFNL4), toll-like receptors (TLRs) and interferon-stimulated gene 15 (ISG-15), were identified as key genes for treatment prediction and HCC evolution. By comparing unique genes for HCV-treatment response and genes particular to HCV-HCC development, we found a common PPI network that may participate in more extensive signalling processes during anti-HCV treatment, which can play important roles in modulating the immune response to the occurrence of HCC. Data mining is an effective tool for identifying potential regulatory pathways involved in treatment response and HCC development. Our study may contribute to a better understanding of HCV immunopathogenesis and highlights the complex role of host genetics in HCV clearance.

The optimization of combination therapy with ribavirin (RBV) and pegylated interferon alpha has substantially improved sustained virologic response (SVR) rates and lowered virologic relapse rates in patients infected with hepatitis C virus (HCV). In this study, we performed an analysis of the relationship between the end-of-treatment plasma RBV concentration and virologic relapse.Thirty-four patients with HCV treated with pegylated interferon/RBV and with an end-of-treatment response were assayed for plasma RBV concentration using liquid chromatography assay coupled to tandem mass-spectrometric detection on the last day of the treatment. Clinical data and the concentration of RBV were compared between patients classified as either relapsers or nonrelapsers.Eleven patients (32.4%) relapsed and 23 patients (67.6%) achieved an SVR. The mean plasma RBV concentration on the last day of treatment was 1380 ± 312 ng/mL for relapsers and 2278 ± 569 ng/mL for SVR patients (P0.0001). A receiver operating characteristic analysis showed that a threshold of 1960 ng/mL was associated with the greatest sensitivity and specificity (100% and 83%, respectively, with an area under the curve of 0.94; P0.0001) for discriminating between patients who relapsed and those who did not. A univariate logistic regression analysis indicated that a plasma RBV concentration of1960 ng/mL at the end of the treatment was strongly associated with relapse (odds ratio, 55; 95% confidence interval, 7.24-∞; P = 0.0001) independently of age, body weight, RBV dose, baseline viral load, the interleukin-28B genotype, and response to previous courses of treatment.Our study results highlight the relevance of measuring plasma RBV concentrations during and at the end of HCV treatment, with a view to avoiding virologic relapse.

Ribavirin-induced anemia is the major side effect observed during HCV therapy. In an in vitro study, we recently discovered that DHEA can strongly inhibit this adverse event. We also evaluated a possible link between pre-treatment serum DHEA and hemoglobin during HCV therapy. Among the 108 patients of our cohort serum baseline DHEA levels were associated with hemoglobin levels at week 12 of treatment (r = 0.35; P = 0.0021). Patients with low baseline serum DHEA developed severe anemia. A serum level of DHEA less than 1,500 ng/ml had a sensitivity of 94.3% and a positive predictive value of 93.1% for the detection of hemoglobin less than 11 g/dl during the first 12 week of treatment. With pre-treatment DHEA levels below the cutoff, anemia was observed in 24.4% and 60.5% of patients treated with dual therapy and triple therapy, respectively, versus 0% and 15% of patients with higher DHEA levels. At week 12, the mean difference between patients with serum DHEA below and above the cutoff, in terms of absolute hemoglobin for dual and triple therapy groups were 1.2 and 1.7 g/dl, respectively (P = 0.005 and0.001). Pretreatment DHEA levels are associated with hemoglobin levels during treatment. Based on these data, pretreatment assay of DHEA could be considered systematically in order to propose DHEA supplementation to potentiate the efficacy of the current and future use of ribavirin for HCV and HEV therapy. J. Med. Virol. 89:1033-1039, 2017. © 2016 Wiley Periodicals, Inc.

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 e 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.

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)

International audience; Genetic recombination is now a well-established feature of the hepatitis C virus (HCV) variability and evolution, with the recent identification of circulating recombinant forms. In Amiens University Hospital Centre (France), a discrepancy of genotyping results was observed for 9 samples, between their 5' untranslated region assigned to genotype 1b and their NS5B region assigned to genotype 1a, suggesting the existence of a recombinant strain. In the present study, clinical and phylogenetic analyses of these isolates were conducted and a putative relationship with previously identified HCV 1b/1a recombinants was investigated. The results revealed that all 9 strains displayed a breakpoint within the beginning of the core protein, were closely related between each other and with the H23 strain identified in Uruguay (Moreno et al., 2009). Then, the clinical characteristics of the 9 unlinked individuals infected with this 1b/1a genotype were analysed. This is the first report on the circulation, in a French population, of a HCV recombinant strain 1b/1a. The identification of this genotype in other patients and in other geographical zones would allow to further investigate its prevalence in the population and to better understand its molecular epidemiology. (C) 2015 Elsevier B.V. All rights reserved.

Background BK virus is a common opportunistic post-transplantation viral infection. Although some risk factors have been studied in this context, the contribution of NK cells has not been assessed in detail. Methods In a group of kidney transplant recipients, we studied the association between (i) the likelihood of BK virus replication during the two-year period after kidney transplantation and (ii) the genotypes of the killer cell immunoglobulin-like receptor (KIR) repertoire and their human leukocyte antigen (HLA) ligands. Other clinical factors (such as defective organ recovery and immunosuppressive treatment) were also assessed. Results BK virus replication was observed in 43 of the 103 recipients (41%). Patients with BK virus replication in the plasma were more likely to display defective organ recovery in the first seven days post-transplantation. BK virus replication was not associated with Missing KIR ligands. However, BK virus replication was more frequent in patients with responsive NK cells (i.e. when a ligand for activating KIRs was not homozygous in the recipient and present in the donor). Conclusion Our results suggest that defective organ recovery and the recipient's activating KIR repertoire may be related (depending on HLA ligands present in the couple recipient / donor) to the reactivation of BK virus replication after kidney transplantation. This article is protected by copyright. All rights reserved.

International audience; Carbohydrate binding agents (CBAs), including natural lectins, are more and more considered as broad-spectrum antivirals. These molecules are able to directly inhibit many viruses such as Human Immunodeficiency Virus (HIV), Hepatitis C Virus (HCV), Dengue Virus, Ebola Virus or Severe Acute Respiratory Syndrome Coronavirus through binding to envelope protein N-glycans. In the case of HIV, it has been shown that CBAs select for mutant viruses with N-glycosylation site deletions which are more sensitive to neutralizing antibodies. In this study we aimed at evaluating the HCV resistance to CBAs in vitro. HCV was cultivated in the presence of increasing Galanthus nivalis agglutinin (GNA), Cyanovirin-N, Concanavalin-A or Griffithsin concentrations, during more than eight weeks. At the end of lectin exposure, the genome of the isolated strains was sequenced and several potential resistance mutations in the E1E2 envelope glycoproteins were identified. The effect of these mutations on viral fitness as well as on sensitivity to inhibition by lectins, soluble CD81 or the 3/11 neutralizing antibody was assessed. Surprisingly, none of these mutations, alone or in combination, conferred resistance to CBAs. In contrast, we observed that some mutants were more sensitive to 3/11 or CD81-LEL inhibition. Additionally, several mutations were identified in the Core and the non-structural proteins. Thus, our results suggest that in contrast to HIV, HCV resistance to CBAs is not directly conferred by mutations in the envelope protein genes but could occur through an indirect mechanism involving mutations in other viral proteins. Further investigations are needed to completely elucidate the underlying mechanisms.

Background Today, treatment of chronic hepatitis C is based on a synergistic combination of pegylated interferon and ribavirin with antiprotease inhibitors. Haemolytic anaemia, which is the major side effect of ribavirin treatment, disrupts ribavirin treatment compliance and varies significantly from one patient to another. There is an individual susceptibility to ribavirin haemolysis. With a view to studying haemolysis, and thus optimizing the treatment response, we have developed a new in vitro tool for analysing the ribavirin-induced lysis of red blood cells. Methods Resuspended red blood cells were incubated with isotonic buffer and a range of concentrations of ribavirin. Haemolysis was quantified by spectrophotometric measurement of the supernatant at 540 nm. The assay was used to test the effects of various compounds and to investigate the susceptibility of patients to haemolytic anaemia. Results In our assay, the degree of haemolysis is dependent on the ribavirin concentration used and can be inhibited by the addition of dipyridamole (50% inhibitory concentration [IC50] 30 μM), ATP or glutathione (IC50 1.63 mM and 767 μM, respectively). We observed a strong decrease in red blood cell haemolysis in the presence of the ribavirin prodrug viramidine (Taribavirin®). When testing the performance of this assay with blood from 24 patients before treatment, we observed a strong correlation between in vitro haemolysis before treatment and the decrease in haemoglobin levels seen in vivo during subsequent treatment ( PConclusions With this new tool it is possible to better evaluate individual susceptibility to ribavirin-induced haemolysis before the start of treatment. In addition, this model will enable the mechanism of ribavirin-induced anaemia to be further explored and allow molecules that could reduce ribavirin haemolysis to be screened and tested in vitro. This approach could help optimize current and future therapeutic strategies involving ribavirin in the treatment of chronic hepatitis C.

HCV core antigen (Ag) and HCV RNA levels were evaluated in matched liver biopsy samples and sera from 22 patients with hepatitis C infection by using the quantitative Architect HCV Ag immunoassay and a real-time RT-qPCR assay, respectively. The data showed a strong correlation between liver and serum compartments of HCV Ag levels ( r = 0.80) and HCV RNA levels ( r = 0.87). In summary, the serum HCV Ag and RNA levels reflect the intrahepatic values.

To date, few natural intergenotypic recombinant hepatitis C virus (HCV) strains have been characterized. A recombinant strain 2k/1b was detected for one HCV RNA-positive individual who had just completed therapy for HCV 3a genotype infection. In the present report, five serum samples collected over the pre- and post-treatment periods were used to investigate all the present HCV strains and the change over time of the infection pattern. Interestingly, the 2k/1b strain was already present during the genotype 3a infection and persisted during treatment. In the specimen collected three months post-treatment, two distinct strains, 2k/1b and type 1, were found and then one 2k/1b strain in the subsequent ones. A genomic variant of the HCV RF1_2k/1b strain was identified. It was part of a mixed HCV infection and persisted and re-emerge after eradication of the dominant subtype 3a. This case indicates that HCV co-infection screening after relapse should be an alternative to explain the lack of response to treatment and the necessity to carefully study the epidemic spreading of this recombinant strain.

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 log 10 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.

Despite considerable progress in the treatment of chronic hepatitis C, many countries do not have access to these new treatments.Predictive markers of response to treatment are therefore necessary before initiating with historical combination therapy (PEG-IFN+ribavirin) for these populations.We therefore evaluated the influence of IL28B polymorphisms on treatment response and Inosine Triphosphate (ITPA) polymorphisms on the incidence of anaemia in a population of 120 Tunisian patients infected with HCV genotype 1b and treated.The frequencies of favourable IL28B genotypes were 47% (CC for rs12979860) and 63% (TT for rs8099117). Patients in whom favourable IL28B alleles were identified had a higher chance of successful therapy: 82% for CC (rs12979860) and 75% for TT (rs8099117). Viral load decline during the first twelve weeks of treatment was more pronounced in patients with a favourable genotype (p0.0001). For patients with an unfavourable genotype, the second phase of viral decline was more pronounced in patients with SVR. A viral load decline cut-off of 2.68logIU/mL at week 12 was best suited to discriminate responders from non-responders with an odds ratio of 40 (95% CI:11.53-170.3). Analysis of ITPA polymorphisms revealed that 16% of Tunisian patients presented ITPase deficiency. None of these patients experienced a decline of ribavirin doses during treatment versus 67% for patients without ITPase deficiency (p0.001).These data obtained in a Tunisian population should optimize before and during treatment the chances of success for treatments currently available in Tunisia for chronic HCV infection.

Two types of peginterferon alfa (2a and 2b) are available for the treatment of chronic hepatitis C genotype 1 or non-1. Comparative studies of bitherapy suggest equivalent results in terms of sustained virologic response for the two types of interferon possibly with a slight advantage in favor of type 2a. However, these studies report only limited data concerning relapse. We analyzed studies comparing the two types of peginterferon with data concerning relapse. In the 6 studies examined (8538 patients), peginterferon 2a was clearly associated with a much higher rate of end of treatment response but also a higher relapse rate than type 2b (29.3% vs 21.1%; relative risk of relapse with peginterferon 2a:1.54 [1.35-1.76], p=0.0024). These data are highlighted in overweight patients. We tried to explain these differences between these two types of interferon by discussing in terms of pharmacokinetics. Peginterferon remains the cornerstone of HCV treatment and must be carefully chosen on the basis of the patient's history and cofactors. These data are important and must be considered in the era of DAA.

The BK virus (BKPyV) is a member of the Polyomaviridae family first isolated in 1971. BKPyV causes frequent infections during childhood and establishes persistent infections with minimal clinical implications within renal tubular cells and the urothelium. However, reactivation of BKPyV in immunocompromised individuals may cause serious complications. In particular, with the implementation of more potent immunosuppressive drugs in the last decade, BKPyV has become an emerging pathogen in kidney and bone marrow transplant recipients where it often causes associated nephropathy and haemorrhagic cystitis, respectively. Unfortunately, no specific antiviral against BKPyV has been approved yet and the only therapeutic option is a modulation of the immunosuppressive drug regimen to improve immune control though it may increase the risk of rejection. A better understanding of the BKPyV life cycle is thus needed to develop efficient treatment against this virus. In this review, we provide an update on recent advances in understanding the biology of BKPyV.