Julien Lupo

Assessment of selective real-time PCR for quantitation of lamivudine and adefovir hepatitis B virus-resistant strains and comparison with direct sequencing and line probe assays.

A selective real-time PCR (sPCR) assay has been developed to detect the rtM204V/I and rtN236T mutations of hepatitis B virus (HBV) associated with resistance to lamivudine and adefovir. Using mixtures of mutant and wild-type plasmids, this sPCR was able to detect 0.1% of mutated strain in a total plasmid population of 10(5) copies and was more sensitive in detecting resistant strains than the line probe INNO-LiPA-DR-v2 assay and a direct sequencing assay. The comparison of these methods on 20 clinical specimens from treated patients confirmed the plasmid results: the three methods were concordant for the detection of the mutant strains in 72% of the cases and the discrepant results were caused mainly by the sequencing assays lack of sensitivity. The line probe assay was more sensitive for detecting mutations than sPCR when the viral load was less than 10(4) copies/ml; conversely, the sPCR provided a more sensitive detection when the viral load was greater than 10(4) copies/ml. Although difficult to perform in clinical practice, sPCR appears to be a reliable technique for detecting and quantifying quasi-species resistant to lamivudine (LAM) and adefovir (ADV) and can be useful to gain a better understanding of the natural history of antiviral resistance during the treatment of chronic hepatitis B (CHB).

Identification of new interacting partners of the shuttling protein ubinuclein (Ubn-1)

We have previously characterized ubinuclein (Ubn-1) as a NACos (Nuclear and Adherent junction Complex components) protein which interacts with viral or cellular transcription factors and the tight junction (TJ) protein ZO-1. The purpose of the present study was to get more insights on the binding partners of Ubn-1, notably those present in the epithelial junctions. Using an in vivo assay of fluorescent protein-complementation assay (PCA), we demonstrated that the N-terminal domains of the Ubn-1 and ZO-1 proteins triggered a functional interaction inside the cell. Indeed, expression of both complementary fragments of venus fused to the N-terminal parts of Ubn-1 and ZO-1 was able to reconstitute a fluorescent venus protein. Furthermore, nuclear expression of the chimeric Ubn-1 triggered nuclear localization of the chimeric ZO-1. We could localize this interaction to the PDZ2 domain of ZO-1 using an in vitro pull-down assay. More precisely, a 184-amino acid region (from amino acids 39 to 223) at the N-terminal region of Ubn-1 was responsible for the interaction with the PDZ2 domain of ZO-1. Co-imunoprecipitation and confocal microscopy experiments also revealed the tight junction protein cingulin as a new interacting partner of Ubn-1. A proteomic approach based on mass spectrometry analysis (MS) was then undertaken to identify further binding partners of GST-Ubn-1 fusion protein in different subcellular fractions of human epithelial HT29 cells. LYRIC (Lysine-rich CEACAM1-associated protein) and RACK-1 (receptor for activated C-kinase) proteins were validated as bona fide interacting partners of Ubn-1. Altogether, these results suggest that Ubn-1 is a scaffold protein influencing protein subcellular localization and is involved in several processes such as cell-cell contact signalling or modulation of gene activity.

Evolution of EBV seroprevalence and primary infection age in a French hospital and a city laboratory network, 2000–2016

Background According to rare studies, the age at EBV primary infection (PI) has recently risen in some developed countries. A later age at infection is generally considered a risk factor for severe EBV PI, although few studies exist on this subject. Our investigation aimed to determine whether EBV seroprevalence and EBV PI epidemiology have evolved in France, and to what extent age and infection intensity (regarding biological parameters) are correlated. Methods and findings We conducted a retrospective study of the following EBV serological tests databases: tests carried out at Grenoble University Hospital (2000–2016) (n = 53,553); and tests carried out by a network of city laboratories in Grenoble area (2008–2015) (n = 27,485). The hospital population showed a continuous, significant decrease in EBV seroprevalence over the studied period for patients aged 20 and over (p<0.01). The seroprevalence also decreased for different age classes (<10, 15–19, 20–30, and 30–40 years old) over the periods 2001–2005, 2006–2010, and 2011–2015. Consistently, the age at PI was significantly higher in the years 2008–2015 than in the years 2001–2007 (15.6±12.0 vs. 13.7±11.0; p = 0.03). The city laboratory population showed the same trend of decreasing seroprevalence (p = 0.06); no significant variations in age at PI were observed. The age at PI was positively correlated with ASAT, ALAT, γGT, and bilirubin blood levels (p<0.01) and negatively correlated with platelet counts (p<0.05). Conclusion In the last 15 years, the age at EBV PI has increased, whereas seroprevalence has decreased. Moreover, our findings confirm the positive correlation between age and biological abnormalities. Taken together, these results suggest that the incidence of severe EBV PI will increase in the future.

Fatal measles without rash in immunocompetent adult, France.

To the Editor: The reemergence of measles in Europe is a reminder of the forgotten risk for severe illness and death associated with this disease in industrialized countries. Since 2008, >20,000 measles cases and 9 measles-associated deaths (in 7 immunocompromised and 2 immunocompetent persons) have been reported to the French Institute for Public Health. Among these cases, the reported causes of death were pneumonia and/or acute respiratory distress syndrome (ARDS) (n = 7) and encephalitis (n = 2). All patients except 1, an immunocompromised patient, had the typical morbillous rash. We report another fatal case of measles, with intractable ARDS but no rash, in an apparently immunocompetent adult. n nThe patient was a 29-year-old woman in Grenoble, France, who smoked but had no relevant medical history except an episode of depression. In 2011, she sought care for fever, cough, coryza, diarrhea, and a 10-kg weight loss over 10 days. A general practitioner empirically prescribed pristinamycin and oral prednisone (60 mg/d for 5 d) for sinusitis. Five days later, the patient was admitted to the hospital because of persistent signs and symptoms. Physical examination at admission (day 1) detected fever (38.5°C), dyspnea, and a low body mass index of 17.5 kg/m2. Hematologic tests showed nonregenerative anemia (hemoglobin concentration 9 g/dL) and leukopenia (2.2 × 109 leukocytes/L) with profound lymphopenia (0.2 × 109 lymphocytes/L) and mild thrombocytopenia (135.0 × 109 platelets/L). A chest radiograph showed bilateral diffuse interstitial infiltrates. Antimicrobial therapy with levofloxacin and ceftriaxone was started. n nOn day 2, several examinations were conducted to explore the possibility of underlying immunosuppressive disease. Body scans showed no adenopathy or lesions suggestive of cancer. HIV test result was negative. General immunologic test results were within normal limits (immunoglobulin quantification, autoantibody testing) or consistent only with an acute viral infection (serum protein electrophoresis). A bone marrow biopsy sample indicated isolated erythroblastopenia with no abnormality of other cell lineages (PCR for parvovirus B19 was negative). n nOn day 3, because of severe respiratory failure, the patient was transferred to the intensive care unit, where the diagnosis of ARDS was confirmed and mechanical ventilation was started. Treatment with tazocillin/tazobactam, ciprofloxacin, amphotericin B, and acyclovir was also started. Microbiological findings from bronchoalveolar lavage (BAL) samples were repeatedly negative for bacteria, mycobacteria, fungi, and Pneumocystis jirovecii. Cytology of BAL samples showed an acute inflammatory response with atypical epithelial cells, supporting a diagnosis of viral infection. However, none of 14 respiratory viruses or human herpesviruses type 1, 3, 4, 5, or 6 were recovered from BAL samples by PCR. Blood and urine culture results were repeatedly negative, as were serologic test results for Legionella spp., Mycoplasma pneumoniae, and Chlamydia pneumoniae. n nOn day 5, because of refractory ARDS, venoarterial extracorporal membrane oxygenation was started. On day 6, the results of a broad serologic investigation demonstrated isolated IgM against measles virus. The patient was additionally given ribavirin, corticosteroids, and intravenous immunoglobulin. On day 10, the lymphocyte level had returned to reference range and the anemia had become regenerative. However, the patient’s respiratory condition did not improve, and after 2 weeks of the oxygenation therapy, the patient died of hemorrhagic shock. Her parents declined an autopsy. n nPCR testing of the patient’s saliva by the French National Reference Center confirmed the presence of measles virus. Retrospective testing of serum, bone marrow, and BAL specimens collected during days 2–20 of hospitalization demonstrated measles virus RNA. The strain was identified as genotype D4, which is the epidemic strain circulating in France and elsewhere in Europe (1). The patient had no history of enanthem (Koplik spots) or morbilliform rash before or after symptom onset and no documented history of measles vaccination. n nDeaths from measles with pneumonia or ARDS but without rash have been reported but mostly in patients with deficient cell-mediated immunity (2–6). Despite all our testing, we found no indications of an underlying immunosuppressive disease in this patient; however, we cannot categorically rule out this possibility, especially that of a primary immunodeficiency. The initial therapy with corticosteroids and the patient’s weight loss could also have interfered with her cellular immune response. The diagnosis of ARDS caused by measles was supported by detection of the measles genome in BAL samples and body fluids in the absence of any other pathogen, but pulmonary superinfection with unidentified pathogens could not be ruled out. Detection of the measles genome and isolated erythroblastopenia in the bone marrow biopsy sample is consistent with reports that measles virus can infect erythroid progenitors and interfere indirectly with hematopoiesis (7,8). Although ribavirin and passive immunotherapy have been reported to aid in recovery from severe measles pneumonia, their clinical efficacy is still unproven (9,10); and for the patient reported here, they were probably used too late. n nThis unusual case underscores the need for physicians to consider the diagnosis of measles, even in the absence of classical clinical features, during measles outbreaks. It also reemphasizes the insufficient vaccination coverage against measles in France.

Sustained virological and biochemical responses to lamivudine and adefovir dipivoxil combination in a chronic hepatitis B infection despite mutations conferring resistance to both drugs

BackgroundSequential monotherapies of nucleotide analogs used in chronic hepatitis B treatment can lead to the selection of a resistance mutation to each antiviral drug.Case presentationA patient with chronic hepatitis B was successively treated with lamivudine monotherapy, lamivudine-adefovir dual therapy, adefovir monotherapy and again with an adefovir-lamivudine dual therapy. Lamivudine-associated mutations (rtL180M and rtM204V/I) followed by adefovir-associated mutations (rtN236T and rtA181V) emerged during the two monotherapy regimens. Despite the presence of rtM204V/I, rtA181V, and rtN236T mutations at the beginning of the second dual therapy, sustained biochemical and virological responses have been observed thus far after 23 months.ConclusionThis case illustrates that rtM204V/I, rtA181V, and rtN236T resistance mutations can coexist in a patient but do not preclude the recycling of lamivudine and adefovir in combination therapy, when no other therapeutic choices are available.

Dynamics of Epstein‐Barr viral load after hematopoietic stem cell transplantation and effect of preemptive rituximab therapy

Epstein‐Barr virus (EBV) displays oncogenic properties, particularly in the immunocompromised host. Notably, hematopoietic stem cell transplantation (HSCT) recipients with a detectable blood EBV viral load (BEBVL) are considered at higher risk of post‐transplant lymphoproliferative diseases (PTLD). Therefore, BEBVL is monitored after HSCT, and preemptive rituximab may be used in patients with high values. However, little is known about post‐HSCT BEBVL dynamics, and the threshold that should lead to anti‐CD20 therapy is poorly defined.

Difference in cytokine production and cell cycle progression induced by Epstein-Barr virus Lmp1 deletion variants in Kmh2, a Hodgkin lymphoma cell line

BackgroundEpstein-Barr virus (EBV) is associated with 20-40% of Hodgkin’s Lymphoma (HL) cases. EBV-encoded latent membrane protein 1 (LMP1) is a well-known oncogenic protein and two C-terminal deletion variants, del30-LMP1 and del69-LMP1, have been described in animal models to be more tumorigenic than the wild-type form. This work aims to detail the implication of LMP1 in the development of HL and to characterize the particular effects of these variants.MethodsWe established HL-derived cell lines stably transfected with the pRT-LMP1 vector coding for the EBNA1 gene and allowing expression of the different LMP1 variants under the control of a doxycyclin-inducible promoter. Communication between cells was assessed by measuring the expression of various pro-inflammatory cytokines by flow cytometry after intracellular LMP1 and cytokine double staining. Proliferative properties of LMP1 variants were also compared by studying the repartition of cells in the different phases of the cell cycle after EdU incorporation combined to LMP1 and DAPI staining.ResultsAll LMP1 proteins induced the expression of several pro-inflammatory cytokines such as TNF-α, TNF-β, IL-6, RANTES/CCL5 and IFN-γ. However, the del30-LMP1 variant induced cytokine expression at a lower level than the other variants, especially IFN-γ, while the del69-LMP1 variant stimulated greater cytokine expression. In addition, we measured that all LMP1 proteins greatly impacted the cell cycle progression, triggering a reduction in the number of cells in S-phase and an accumulation of cells in the G2/M phase compared to the HL-non induced cells. Interestingly, the del30-LMP1 variant reduced the number of cells in S-phase in a significantly greater manner and also increased the number of cells in the G0/G1 phase of the cell cycle.ConclusionWeak IFN-γ expression and specific alteration of the cell cycle might be a way for del30-LMP1 infected cells to escape the immune anti-viral response and to promote the development of cancer. The differences observed between the LMP1 variants reflect their own oncogenic properties and eventually impact the development of HL.

Interaction of Ubinuclein-1, a nuclear and adhesion junction protein, with the 14-3-3 epsilon protein in epithelial cells: implication of the PKA pathway.

Ubinuclein-1 is a NACos (Nuclear and Adhesion junction Complex components) protein which shuttles between the nucleus and tight junctions, but its function in the latter is not understood. Here, by co-immunoprecipitation and confocal analysis, we show that Ubinuclein-1 interacts with the 14-3-3ɛ protein both in HT29 colon cells, and AGS gastric cells. This interaction is mediated by an Ubinuclein-1 phosphoserine motif. We show that the arginine residues (R56, R60 and R132) which form the 14-3-3ɛ ligand binding site are responsible for the binding of 14-3-3ɛ to phosphorylated Ubinuclein-1. Furthermore, we demonstrate that in vitro Ubinuclein-1 can be directly phosphorylated by cAMP-dependent protein kinase A. This in vitro phosphorylation allows binding of wildtype 14-3-3ɛ. Moreover, treatment of the cells with inhibitors of the cAMP-dependent protein kinase, KT5720 or H89, modifies the subcellular localization of Ubinuclein-1. Indeed, KT5720 and H89 greatly increase the staining of Ubinuclein-1 at the tight junctions in AGS gastric cells. In the presence of the kinase inhibitor KT5720, the amount of Ubinuclein-1 in the NP40 insoluble fraction is increased, together with actin. Moreover, treatment of the cells with KT5720 or H89 induces the concentration of Ubinuclein-1 at tricellular intersections of MDCK cells. Taken together, our findings demonstrate novel cell signaling trafficking by Ubinuclein-1 via association with 14-3-3ɛ following Ubinuclein-1 phosphorylation by the cAMP-dependent protein kinase-A.

Lytic EBV infection investigated by detection of Soluble Epstein-Barr virus ZEBRA in the serum of patients with PTLD

The ZEBRA protein (encoded by the BZLF1 gene), is the major transcription factor of EBV, expressed upon EBV lytic cycle activation. Several studies highlighted the critical role of EBV lytic infection as a risk factor for lymphoproliferative disorders like post-transplant lymphoproliferative disease (PTLD). Here, we use an antigen-capture ELISA assay specifically designed to detecting the circulating soluble ZEBRA (sZEBRA) in serum samples (threshold value determined at 40ng/mL). We retrospectively investigated a population of 66 transplanted patients comprising 35 PTLD. All the samples from a control population (30 EBV-seronegative subjects and 25 immunocompetent individuals with EBV serological reactivation), classified as sZEBRAu2009<u200940ng/mL were assigned as negative. At PTLD diagnosis, EBV genome (quantified by qPCR with EBV DNA>200 copies/mL) and sZEBRA were detectable in 51% and 60% of cases, respectively. In the patients who developed a pathologically-confirmed PTLD, the mean sZEBRA value in cases, was 399 ng/mL +/− 141 versus 53ng/mL +/− 7 in patients who did not (p u2009<u20090,001). This is the first report relating to the detection of the circulating ZEBRA in serum specimens, as well as the first analysis dealing with the lytic cycle of EBV in PTLD patients with this new biomarker.

Herpes simplex type 2 encephalitis and methotrexate medication: a fortuitous or causative association in a patient with spondyloarthritis?

It is unclear whether immunosuppression is a risk factor for herpes encephalitis. Herein, we describe a rare case of herpes simplex virus type 2 encephalitis in a patient treated with low-dose methotrexate for HLA-B27-associated spondyloarthritis. The patient was successfully treated with acyclovir but presented sequelae of encephalitis. Here we discuss the possible role of low-dose methotrexate therapy as a risk factor of neurological herpes reactivation and severe disease. The host-related and viral risk factors are also addressed.