A focus reduction neutralization assay for hepatitis C virus neutralizing antibodies

BACKGROUND/AIM: The role of humoral immunity in hepatitis C virus (HCV) infection is poorly understood. Nevertheless, there is increasing interest in characterizing the neutralizing antibodies in the serum of HCV-infected patients. Focus reduction assays have been widely used to evaluate neutralizing antibody responses against a range of non-cytopathic viruses. Based on the recent development of a HCV cell culture system using the genotype 2 JFH-1-strain, we developed a focus reduction assay for HCV-neutralizing antibodies. METHODS: The focus reduction assay was based on a standard microneutralization assay in which immunostained foci on tissue culture plates are counted. The neutralizing anti-HCV antibodies titers of purified serum immunoglobulin samples from seventy-seven individuals were determined using a 50% focus reduction neutralization assay. Each titer was determined as the log value of the reciprocal antibody dilution that reduced the number of viral foci by 50%. IgG antibodies were first purified from each serum in order to avoid the facilitating effect of HDL on HCV entry. RESULTS: The assay's cut-off using an ELISA and RNA HCV-negative samples was found to be 1.25 log, corresponding to a dilution of 1:18. The assay was compared with a commercial HCV ELISA and exhibited specificity and sensitivity values of 100% and 96.5%, respectively, and good reproducibility (with intra-assay and inter-assay coefficients of variation of 6.7% and 12.6%, respectively). The assay did not show any cross-reactivity with anti-HIV, anti-HBs or heterophile antibody-positive samples. The neutralizing antibodies titers were 2.13 log (1:134) for homologous samples from HCV genotype 2 infected patients harboring the same genotype as JFH-1 and 1.93 log (1:85) for heterologous samples from patients infected by genotypes other than type 2. These results confirm the presence of broadly cross-neutralizing antibodies already reported using the HCV pseudoparticles system. CONCLUSION: This study presents a simple, specific and reproducible cell culture-based assay for determination of HCV-neutralizing antibodies in human sera. The assay should be an important tool for gauging the relationship between the neutralizing antibodies response and viral load kinetics in acutely or chronically infected patients and for investigating the possible eradication or prevention of HCV infection by neutralizing antibodies

Comparison of two RT-qPCR methods targeting BK polyomavirus microRNAs in kidney transplant recipients

BackgroundBK polyomavirus replication leads to progressive tubulointerstitial nephritis and ureteral stenosis, with a considerable risk of subsequent graft failure in kidney transplant recipients. Since specific antiviral therapies are lacking, new tools are required to enhance the biological monitoring of the infection. Viral microRNAs are promising new biomarkers, but the performance of RT-qPCR methods limits the clinical application and the validation of a standard method for quantification.MethodsWe compared TaqMan microRNA Assays and TaqMan Advanced miRNA Assays for bkv-miR-B1-3p and bkv-miR-B1-5p quantification in synthetic microRNA templates and in 44 urine samples belonging to 14 consecutive kidney transplant recipients with BK polyomavirus replication from Amiens University Medical Center in a 1-year span.ResultsCycle threshold values were constantly higher with TaqMan Advanced MicroRNA Assays. TaqMan microRNA Assays showed better performance in predicting the good prognosis of BK polyomavirus nephropathy.ConclusionOverall, TaqMan MicroRNA Assays appeared to be a more sensitive and accurate RT-qPCR method than TaqMan Advanced MicroRNA Assays to quantify bkv-miR-B1-3p and bkv-miR-B1-5p BKPyV miRNAs in patients’ urine samples

Annexin-V positive extracellular vesicles level is increased in severe COVID-19 disease

ObjectivesTo evaluate extracellular vesicles levels in a cohort of SARS-CoV-2’s patients hospitalized in an intensive care unit with and without COVID-19 associated thromboembolic events.MethodsIn this study, we aim to assess endothelial and platelet membrane-derived extracellular vesicles levels in a cohort of SARS-CoV-2 patients with and without COVID-19-associated thromboembolic events who were hospitalized in an intensive care unit. Annexin-V positive extracellular vesicles levels were prospectively assessed by flow cytometry in one hundred twenty-three critically ill adults diagnosed with acute respiratory distress syndrome associated with a SARS-CoV-2 infection, ten adults diagnosed for moderate SARS-CoV-2 infection and 25 healthy volunteers.ResultsOn our critically ill patients, thirty-four patients (27.6%) had a thromboembolic event, Fifty-three (43%) died. Endothelial and platelet membrane-derived extracellular vesicles were drastically increased in SARS-CoV-2 patients hospitalized in the ICU compared to healthy volunteers. Moreover a slighty higher small/large ratio for platelets membrane-derived extracellular vesicles in patients was linked to thrombo-embolic events.ConclusionA comparison between total annexin-V positive extracellular vesicles levels in severe and moderate SARS-CoV-2 infection and healthy controls showed a significant increase in patients with severe infection and their sizes could be considered as biomarkers of SARS-CoV-2 associated thrombo-embolic events

The treatment response of chronically hepatitis C virus-infected patients depends on interferon concentration but not on interferon gene expression in peripheral blood mononuclear cells.

International audienceThe current treatment of chronic hepatitis C is based on pegylated alpha interferon (PEG-IFN-α) and ribavirin. The aim of this study was to identify biological and clinical variables related to IFN therapy that could predict patient outcome. The study enrolled 47 patients treated with PEG-IFN and ribavirin combined therapy. The interferon concentration was measured in serum by a bioassay. The expression of 93 interferon-regulated genes in peripheral blood mononuclear cells was quantified by real-time quantitative reverse transcription-PCR (RT-PCR) before and after 1 month of treatment. The interferon concentration in the serum was significantly lower in nonresponders than in sustained virological responders. Moreover, a significant correlation was identified between interferon concentration and interferon exposition as well as body weight. The analysis of interferon-inducible genes in peripheral blood mononuclear cells among the genes tested did not permit the prediction of treatment outcome. In conclusion, the better option seems to be to treat patients with weight-adjusted PEG-IFN doses, particularly for patients with high weight who are treated with PEG-IFN-α2a. Although the peripheral blood mononuclear cell samples are the easiest to obtain, the measurement of interferon-inducible genes seems not be the best strategy to predict treatment outcome

Seroconversion of Immunoglobulins to SARS-CoV2 in Patients With Inflammatory Bowel Disease Patients Treated by Biologics

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Pre-transplantation assessment of BK virus serostatus: Significance, current methods, and obstacles.

International audienceThe 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)

Intercellular Transmission of Naked Viruses through Extracellular Vesicles: Focus on Polyomaviruses

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

MICA and NKG2D variants as risk factors in spondyloarthritis: a case-control study

International audienceThe 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

Biology of the BKPyV: An Update

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