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BK virus–associated nephropathy (BKVAN) causes renal allograft dysfunction. The current management of BKVAN relies on pre-emptive adaptation of immunosuppression according to viral load monitoring. However, this empiric strategy is not always successful. Therefore, pretransplant predictive markers are needed. In a prospective longitudinal study, we enrolled 168 kidney transplant recipients and 69 matched donors. To assess the value of BKV genotype–specific neutralizing antibody (NAb) titers as a predictive marker for BKV replication, we measured BKV DNA load and NAb titers at transplant and followed patients for 24 months. After transplant, 52 (31%) patients displayed BKV replication: 24 (46%) patients were viruric and 28 (54%) patients were viremic, including 13 with biopsy-confirmed BKVAN. At any time, patients with high NAb titers against the replicating strain had a lower risk of developing BKV viremia (hazard ratio [HR], 0.44; 95% confidence interval [95% CI], 0.26 to 0.73; P=0.002). Each log10 increase in NAb titer decreased the risk of developing viremia by 56%. Replicating strains were consistent with donor transmission in 95% of cases of early BKV replication. Genotype mismatch between recipients’ neutralization profiles before transplant and their subsequently replicating strain significantly increased the risk of developing viremia (HR, 2.27; 95% CI, 1.06 to 4.88; P=0.04). A NAb titer against the donor’s strain

Hepatitis C Virus belongs to the Flaviviridae family. One proposed mechanism of HCV persistence in the ability to infect hematopoietic cells, including Dendritic cells (DCs). HCV infection of DCs could impair their functions that represent one of the mechanisms, thus hampering viral clearance by the host immune system. Among HCV-encoded proteins, the highly conserved Core protein has been suggested to be responsible for the immunomodulatory properties of this Hepacivirus. Recombinant viral vectors expressing the HCV Core protein and allowing its transduction and therefore the expression of the protein into DCs could be useful tools for the analysis of the properties of the Core protein. Vaccinia Virus and retrovirus have been used to transduce human DCs. Likewise, gene transfer into DCs using Semliki Forest Virus has been reported. This study aimed to express the HCV Core protein in human monocyte-derived DCs using an SFV vector, in which the subgenomic RNA encoding the structural proteins was replaced by the HCV Core sequence and then analyze the effects of its expression on DCs functions.

Summary Objective This work evaluated seasonal variations and spatio-temporal pattern of respiratory tract infections (RTI) in geriatric nursing homes in order to improve effective surveillance, prevention, control and management of RTI. Methods Prospective surveillance of RTI (Low Respiratory Tract infections and Influenza Like Illnesses) was conducted in 11 sites in Alsace over a 10-year period with clinical case definitions and rapid tests (Immunoassay) to identify influenza virus. Results Influenza national epidemic was a period at high risk of RTI in nursing homes with variable impacts depending on the seasonal period. 2004–2005, 2008–2009, 2011–2012 and 2012–2013 were the periods with the highest impacts. The higher risk was not well understood during these four influenza epidemics and outbreaks occurred in numerous nursing homes despite the alerts and surveillance. Conclusion Information about seasonal variability and spatio-temporal patterns of the RTI during the national epidemic periods is essential for the nursing homes in order to help the health care workers and the visitors to understand the risk for the residents and then to improve the implementation of the control and prevention measures.

In patients with hepatitis C virus (HCV) infection, enhanced activity of indoleamine-2,3-dioxygenase 1 (IDO) has been reported. IDO - a tryptophan-catabolizing enzyme - has been considered as both an innate defence mechanism and an important regulator of the immune response. The molecular mechanism of IDO induction in HCV infection and its role in the antiviral immune response remain unknown. Using primary human hepatocytes, we show that HCV infection stimulates IDO expression. IDO gene induction was transient and coincided with the expression of types I and III interferons (IFNs) and IFN-stimulated genes in HCV-infected hepatocytes. Overexpression of hepatic IDO prior to HCV infection markedly impaired HCV replication in hepatocytes, suggesting that IDO limits the spread of HCV within the liver. siRNA-mediated IDO knock-down revealed that IDO functions as an IFN-mediated anti-HCV effector. Hepatic IDO was most potently induced by IFN-γ, and ongoing HCV replication could significantly upregulate IDO expression. IRF1 (IFN-regulatory factor 1) and STAT1 (signal transducer and activator of transcription 1) regulated hepatic IDO expression. Hepatic IDO expression also had a significant inhibitory effect on CD4+ T-cell proliferation. Our data suggest that hepatic IDO plays a dual role during HCV infection by slowing down viral replication and also regulating host immune responses.

Screening of BK virus (BKV) replication is recommended to identify patients at increased risk of BKV-associated diseases. However, the heterogeneity of molecular techniques hinders the establishment of universal guidelines for BKV monitoring. Here we aimed to compare the performance of the CE-marked BK virus R-gene kit (R-gene) to the performance of our in-house assay for quantification of BKV DNA loads (BKVL). A 12-specimen panel from the Quality Control for Molecular Diagnostics (QCMD) organization, 163 urine samples, and 88 paired specimens of plasma and whole blood (WB) from transplant recipients were tested. Both the R-gene and in-house assays showed a good correlation within the QCMD panel ( r = 0.995 and r = 0.989, respectively). BKVL were highly correlated between assays, although positive biases were observed with the in-house assay in analysis of urine (0.72 ± 0.83 log 10 copies/ml), plasma (1.17 ± 0.63 log 10 copies/ml), and WB (1.28 ± 0.37 log 10 copies/ml). Recalibration with a common calibrator significantly reduced the bias in comparisons between assays. In contrast, BKVL was underestimated with the in-house PCR in eight samples containing BKV genotype II, presenting point mutations at primer-annealing sites. Using the R-gene assay, plasma and WB specimens were found to be equally suitable for quantification of BKVL, as indicated by the high correlation coefficient ( r = 0.965, P < 0.0001). In conclusion, the R-gene assay demonstrated reliable performance and higher accuracy than the in-house assay for quantification of BKVL in urine and blood specimens. Screening of BKV replication by a well-validated commercial kit may enable clinical laboratories to assess viral loads with greater reproducibility and precision.

Respiratory viruses are the leading cause of acute infections in humans. However, the burden of certain respiratory viruses, such as coronaviruses, and the relevance of viral coinfections remain unclear. In this study, we investigated the distribution and seasonal occurrences of respiratory viruses detected by multiplex molecular assay in 6,014 samples from 2008 to 2011 in a French hospital. We assessed the detection frequencies of 14 respiratory viruses and their clinical impact in immunosuppressed and nonimmunosuppressed patients. Furthermore, we explored the preferential association patterns between respiratory viruses in multiple infections. Our results indicated that human rhinovirus/enterovirus (HRV/EV) and coronavirus (HCoV) were frequently detected in respiratory samples (48.81% and 11.74% of infected samples, respectively), and the detection frequencies of these viruses were further increased in immunosuppressed patients. The most common subtypes of HCoV were HCoV-229E (33.80%) and HCoV-HKU1 (32.39%). A sharp increase in the detection frequencies of HCoV-229E and HCoV-HKU1 over several months suggested that these subtypes were epidemic in our population. In immunosuppressed patients, HCoV contributed to upper respiratory tract infections (52%). Evidence did not support lower respiratory tract infections exclusive to a unique HCoV infection. In multiply infected individuals, determined in 6.3% of samples, HRV/EV and HCoV were detected in 33.29% and 22.90% of samples, respectively. Interestingly, nearly 50% of HCoV infections were detected in association with another virus. Since the distributions of respiratory viruses in multiply infected patients were subject to preferential association patterns between viruses, we propose complex interactions between different respiratory viruses and host factors.

Nearly 45 years after the discovery of the first two human polyomaviruses BK and JC, their life-long persistence and mechanisms of pathogenesis remain poorly understood and efficient antiviral treatments are severely lacking. In this review, we sought to provide an update on recent advances in understanding the life cycle of these two viruses, particularly focusing on their interaction with the host immune system and pathogenesis. We have also discussed novel treatment approaches and highlighted areas of future research.

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. The interplay between the virus and host innate and adaptive immune responses determines the outcome of infection. There is increasing evidence that host neutralizing responses play a relevant role in the resulting pathogenesis. Furthermore, viral evasion from host neutralizing antibodies has been revealed to be an important contributor in leading both to viral persistence in acute liver graft infection following liver transplantation, and to chronic viral infection. The development of novel model systems to study HCV entry and neutralization has allowed a detailed understanding of the molecular mechanisms of virus-host interactions during antibody-mediated neutralization. The understanding of these mechanisms will ultimately contribute to the development of novel antiviral preventive strategies for liver graft infection and an urgently needed vaccine. This review summarizes recent concepts of the role of neutralizing antibodies in viral clearance and protection, and highlights consequences of viral escape from neutralizing antibodies in the pathogenesis of HCV infection.

It is estimated that 4-5 million HIV-infected patients are coinfected with HCV. The impact of HIV on the natural course of HCV infection is deleterious. This includes a higher rate of HCV persistence and a faster rate of fibrosis progression. Coinfected patients show poor treatment outcome following standard HCV therapy. Although direct antiviral agents offer new therapeutic options, their use is hindered by potential drug interactions and toxicity in HIV-infected patients under HAART. Overtime, a large reservoir of HCV genotype 1 patients will accumulate in resource poor countries where the hepatitis C treatment is not easily affordable and HIV therapy remains the primary health issue for coinfected individuals. HCV vaccines represent a promising strategy as an adjunct or alternative to current HCV therapy. Here, the authors review the pathogenesis of hepatitis C in HIV-infected patients, with a focus on the impact of HIV on HCV-specific immune responses and discuss the challenges for vaccine development in HIV-HCV coinfection.

Background Human HEV infections reported in Europe without previous travel to endemic regions are linked to exposure to genotype 3 Hepatitis E virus (HEV).Genotype 3 is widely distributed through human cases and zoonotic reservoir. The geographical distribution of genotype 4 is limited to Asian countries. Objectives The first human case of autochthonous genotype 4 hepatitis E infectionwasreported in France. Study design The HEV infection was described in an immunosuppressed patient, presenting an acute myeloblastic leukemia. Investigation of the case was performed on detection of HEV markers in the patient and in the environment. Results Hepatitis E infection was diagnosed on the basis of HEV RNA viremia, and detection of anti-HEV IgM. The prognostic of leukemia was favorable and HEV was cleared without relapsing. HEV isolate was classified into genotype 4. Conclusions The recent characterization of genotype 4 HEV through swine surveillance in Europe and the description of the first human case in France open interesting questions about the circulation of this genotype: health risks in human population, transmission patterns, and zoonotic reservoir.

The ability of hepatitis C virus (HCV) to infect leukocytes could favour HCV pathogenesis. Although viral infection of these immunocompetent cells is poorly (or not) productive, the impact on their immunomodulatory functions could be important. Viral envelope glycoproteins E1 and E2, because of their crucial role in the recognition of viral receptors on permissive cells, could contribute to viral leukocytic tropism and, as a consequence, to the pathophysiology of HCV chronic infection.

The precise role of adaptive immune responses in the clinical outcome of HCV infection is still only partially defined. Recent studies suggest that viral-host cell interactions during the acute phase of infection are essential for viral clearance or progression into chronic HCV infection. This review focuses on different aspects of the adaptive immune responses as determinants of the different outcomes of HCV infection, clearance or persistent infection, and outlines current concepts of HCV evasion strategies. Unravelling these important mechanisms of virus-host interaction will contribute to the development of novel strategies to prevent and control HCV infection.

Gas emission by humans will change climate, warming by 1.4-5.8°C as predicted at the end of the current cen- tury. Climate oscillations between warm and cold phases (El Nino) add complexity in the field. The effects on health could be thermal stress, extreme weather events, and subsequently emerging infectious diseases. Consequences on food yields, social, demographic and economic imbalances, could also favour contagious diseases. Increasing vector-borne infections could represent a major health concern. Additionally, numerous floods and massive movements of people could facilitate the transmission of water-borne infections. Moreover, decrease in food supply could disorganise populations with crowding and concomitant spreading of transmissible infectious pathogens such as viruses. This short review aims to present the potential viral impact on human health in case of climate change, i.e. increased ar- boviruses, "tropical" viruses, and viral infections related to overcrowding in poor healthy context.

Besides hepatocytes, representing the main replication site of hepatitis C virus, peripheral blood mononuclear cells also represent a crucial target for viral infection. Hepatitis C virus compartmentalization (i.e., non-random distribution) of viral variants between plasma and peripheral blood mononuclear cells, more frequently observed in liver transplant patients compared to non-transplanted patients, makes liver transplantation an interesting model for the analysis of hepatitis C leukotropism. This article aims to present, firstly, in clinical and biological features arguing favour of hepatitis C virus infection leukotropism and, secondly, to review current knowledge about compartmentalization between plasma and peripheral blood mononuclear cells, especially in the liver transplantation setting.

Hepatitis C virus (HCV) is a major cause of hepatitis world-wide. The majority of infected individuals develop chronic hepatitis which can then progress to liver cirrhosis and hepatocellular carcinoma. Spontaneous viral clearance occurs in about 20%-30% of acutely infected individuals and results in resolution of infection without sequaelae. Both viral and host factors appear to play an important role for resolution of acute infection. A large body of evidence suggests that a strong, multispecific and long-lasting cellular immune response appears to be important for control of viral infection in acute hepatitis C. Due too the lack of convenient neutralization assays, the impact of neutralizing responses for control of viral infection had been less defined. In recent years, the development of robust tissue culture model systems for HCV entry and infection has finally allowed study of antibody-mediated neutralization and to gain further insights into viral targets of host neutralizing responses. In addition, detailed analysis of antibody-mediated neutralization in individual patients as well as cohorts with well defined viral isolates has enabled the study of neutralizing responses in the course of HCV infection and characterization of the impact of neutralizing antibodies for control of viral infection. This review will summarize recent progress in the understanding of the molecular mechanisms of antibody-mediated neutralization and its impact for HCV pathogenesis.

Hepatitis C virus (HCV) is a member of the Flaviviridae family and causes acute and chronic hepatitis. Chronic HCV infection may result in severe liver damage including liver cirrhosis and hepatocellular carcinoma. The liver is the primary target organ of HCV, and the hepatocyte is its primary target cell. Attachment of the virus to the cell surface followed by viral entry is the first step in a cascade of interactions between the virus and the target cell that is required for successful entry into the cell and initiation of infection. This step is an important determinant of tissue tropism and pathogenesis; it thus represents a major target for antiviral host cell responses, such as antibody-mediated virus neutralization. Following the development of novel cell culture models for HCV infection our understanding of the HCV entry process and mechanisms of virus neutralization has been markedly advanced. In this review we summarize recent developments in the molecular biology of viral entry and its impact on pathogenesis of HCV infection, development of novel preventive and therapeutic antiviral strategies.

Summary Aim Infection with hepatitis C virus (HCV) results in chronic hepatitis in more than 70% of cases. Alterations in the maturation of dendritic cells (DC) might play a role in the immune system's inability to eliminate the virus, although viral factors that could be involved have not been identified. This study in vitro investigated whether HCV structural proteins affect maturation of monocyte-derived DC. Methods HCV proteins (core, E1, E2) were expressed by transduction with recombinant adenoviruses of immature DC. The ability of these transduced DC to respond to a maturation stimulus was evaluated by measuring cell surface markers, allogenic lymphocyte stimulation and interleukin (IL)-12 production. Results Expression of HCV structural proteins did not modify DC maturation in the presence of lipopolysaccharide, as determined by their phenotype and stimulatory functioning. IL-12 secretion was not affected by HCV protein expression in mature DC. Conclusion Our results suggest that HCV structural proteins do not affect maturation of monocyte-derived DC by lipopolysaccharide. These findings are important for further studies to clarify the pathogenesis of chronic HCV infection and towards the rational design of cellular vaccine approaches for immunotherapy against hepatitis C.

Biobanking or collection and storage of specimens for future research purposes have become an essential tool in many fields of biomedical research and aims to provide a better understanding of disease mechanisms as well as the identification of disease-specific biomarkers that can navigate in complex diseases. In this study, we assessed the use of Flinders Technology Associates (FTA) cards as a long-term storage device for cervical specimens with suspected human papillomavirus (HPV) infections. HPV detection and genotyping results in liquid-based transport media were compared to HPV results from FTA cards. The overall agreement for the presence of any HPV infection between liquid-based medium and FTA cards stored for 1 year at ambient temperature was 100%. Reproducibility analysis of HPV detection and genotyping from FTA cards demonstrated that FTA cards are a reliable medium to store and preserve viral nucleic acids. Biobanking of cervical cells on FTA cards may provide a key resource for epidemiological and retrospective HPV studies.

Background: Most studies evaluating antibody detection assays are conducted on samples from healthy blood donors but not on samples of hospitalized patients which can show non-specific reactions. Objectives: To compare the performance of three commercial automated assays for the detection of hepatitis C virus (HCV) antibodies, Monolisa anti-HCV Plus version 2, Axsym anti-HCV 3.0 and Vitros anti-HCV, on a population of hospitalized patients. Study design: The specificity of the assays was prospectively evaluated in 2020 routine serum samples. In order to assign the serostatus of each sample, those giving positive or discordant results were further tested by three immunoblots and by RT-PCR (Roche). Moreover, the sensitivity was evaluated on eight commercial HCV seroconversion panels. Results: The Monolisa, Axsym and Vitros assays showed specificities of 99.64%, 99.12% and 99.33%, respectively. Concerning the sensitivity, among 49 samples, the number of positive results was 21, 24 and 24 for the Monolisa, Axsym and Vitros kits, respectively. The differences were not statistically significant at an alpha risk of 5%. Conclusions: All assays appeared to be reliable for routine screening, but there were a surprising number of indeterminate samples that could not be resolved by confirmatory tests.

Until recently, human cytomegalovirus (hCMV) infection and anti-CMV treatment in transplanted patients have been monitored essentially by pp65 antigenemia, which is time-consuming and requires experienced operators. For the last two years, pp65 antigenemia levels have tended to be lower than previously in our laboratory, which could be due to better monitoring of CMV-related risk. Results obtained by real-time PCR with a LightCycler instrument or by pp65 antigen assay were compared on 145 serial samples from bone marrow or kidney transplant recipients under the usual conditions of our laboratory. CMV DNA was extracted from plasma and quantified by using primers and probes directed to HXFL4 gene. The plasma CMV DNA load was measured by using a standard curve constructed with a commercially available quantified CMV DNA suspension. Among the 145 samples, 139 showed a pp65 antigen which was negative or lower than 20 positively stained cells per 200,000 leukocytes. In the patients with positive pp65 antigenemia, the corresponding values of CMV DNA copy number/ml were significantly higher than those observed in patients without antigenemia (P < 0.001). CMV DNA was detected from 4 up to 52 days before pp65 antigen. Elsewhere, between two dates at which pp65 antigen was positive, intermediate PCR results could be positive while the pp65 antigen was negative. This real-time quantitative PCR assay is a rapid technique adapted to monitor plasma CMV DNA in transplant setting, even for low viremia.

Resume But de l’etude. – Le diagnostic biologique de premiere intention de l’infection par le virus de l’hepatite C (VHC) fait appel a la recherche des anticorps specifiques par des tests serologiques de type Elisa. La plupart des etudes visant a evaluer les performances de ces reactifs se font sur des serums de donneurs de sang, c’est-a-dire des individus a priori en bonne sante. Par rapport aux serums des sujets sains, les serums preleves chez des patients peuvent presenter des reactions non specifiques conduisant a une difficulte d’interpretation des tests serologiques. Materiel et methode. – Dans un premier temps, nous avons compare prospectivement la specificite de deux coffrets commerciaux de recherche d’anticorps anti-VHC, le Monolisa anti-HCV Plus automatise sur l’Evolis et l’Axsym anti-HCV 3.0 sur 2020 serums collectes a l’institut de virologie de Strasbourg. Puis, ces memes coffrets ont ete testes sur huit panels commerciaux de serums de seroconversions afin d’evaluer leur sensibilite. Resultats. – Le Monolisa et l’Axsym montrent, respectivement, une specificite de 99,64 et 99,12 %. Sur les huit panels correspondant a 49 serums, le nombre d’echantillons positifs a ete, respectivement, de 21 et 24. Conclusion. – Il n’existe pas de differences statistiquement significatives entre les deux coffrets aussi bien en termes de sensibilite que de specificite. Neanmoins, le Monolisa pourrait etre choisi en raison d’une specificite et d’une sensibilite plus homogene que celle de l’Axsym. Les criteres de praticabilite et d’interpretation dans la routine du laboratoire sont egalement a prendre en compte lorsque les performances des tests sont proches.

The objective of the study was to assess three immunoblot assays, the Deciscan HCV Plus, the Riba and the Inno-Lia, on 44 discordant samples with three EIA kits. These immunoblots were considered as confirmation reagents. A result was considered as a false positive by anti-HCV antibody assay if the three immunoblots were negative or if two immunoblots were negative with the third being indeterminate and a negative virological genomic diagnosis observed on all the samples. The result was positive if at least two immunoblots out of three were positive. Thus, 34 samples were considered as false positive and ten samples were excluded because it was impossible to conclude between true or false positive result. The 44 discordant results were never confirmed as positive by the use immunoblot or PCR. The three immunoblots were negative for half of the samples and two immunoblots and one indeterminate were observed for 77% of the samples. The false positive results by the Monolisa assay were more often found indeterminate with the Deciscan assay than with the other immunoblots. That was also checked for Vitros/Riba pair. One of the explanations could be the use of common antigens for the reagents from the same manufacturer. The Inno-Lia test is the most specific immunoblot according to the results obtained in our study.

Interleukin-12 and -18 (IL-12 and IL-18) are known to enhance the cytotoxic T-lymphocyte response synergistically, but their precise involvement in hepatitis C virus (HCV) infection is not well known, especially for IL-18. Enzyme-linked immunosorbent assay (ELISA) was used to study the production of these cytokines in plasma and peripheral blood mononuclear cells (PBMCs) stimulated by lipopolysaccharide (LPS) of patients infected chronically with HCV before initiation of antiviral therapy. Fifty-six patients and 40 healthy controls were evaluated. Patients infected with genotype 1 or with genotype other than genotype 1 HCV had significantly a high production of plasma IL-12 compared with controls (P < 0.05). However, patients infected with genotype 1 HCV had lower levels of PBMC IL-18 than were founded in the controls (P < 0.05); plasma IL-18 also tended to be lower in this group of patients than in the controls, although nonsignificantly. Plasma IL-18 was related to hepatic histological activity (P < 0.05). The data suggest a relationship between these two cytokines and some features of HCV infection, so that their respective production in relation to the outcome of the infection deserves further study.

BACKGROUND: Assisted reproduction techniques can minimize the risk of infection and treat possible sterility associated with serodiscordant couples. METHODS: We assessed the efficacy of these techniques in 57 couples in which at least one partner had human immunodeficiency virus (HIV-1) infection that was currently under control (47 men and 10 women). The semen of seropositive men was prepared and tested for viruses. Assisted reproduction techniques included intrauterine insemination (IUI), IVF and especially ICSI, with ovarian stimulation that used a long agonist protocol and recombinant FSH. Embryos were transferred on day 3 after oocyte retrieval. RESULTS: For couples with seropositive men, five IUI and 49 IVF or ICSI attempts were perfomed, whilst for seropositive women these numbers were three IUI and 12 IVF or ICSI. No pregnancy occurred following the eight IUI trials. Seroconversion was not observed in any partners of seropositive men. Efficacy of treatment for these couples with ICSI was good, the clinical pregnancy rate per embryo transfer was 48.8%. The results for seropositive women were disappointing, with a clinical pregnancy rate per embryo transfer of 9.1%. Fourteen babies from 47 treated couples have so far been born and no pregnancies from IUI. CONCLUSIONS: Assisted reproduction techniques and particularly ICSI provide HIV-1-seropositive men with a safe and highly effective means of fathering children. These techniques may be less effective for seropositive women.

In France, HCV seroprevalence is estimated to be 1%. Most cases are related to illicit parenteral drug use. About 80% of patients infected by HCV will develop chronic infection with HCV RNA detectable in their serum. Factors involved in viral persistence are nor yet clearly identified. Patients who develop a chronic infection show a predominant Th2 response, but a weak Th1 response. This immune response imbalance could result from HCV interaction with dendritic cells functions. Several experimental and clinical studies support this hypothesis. New data on HCV strategy to establish chronic infection suggest that immmunomodulatory molecules could be useful to improve efficacy of therapy.

Background/Aims : Histological examination of a liver-biopsy from a patient with chronic hepatitis C shows activated Kupffer cells. In vitro infection of human Kupffer cells (KC) was performed to study their interaction with hepatitis C virus (HCV). Methods : KC, isolated by collagenase perfusion and centrifugal elutriation, were infected with various HCV positive sera. The presence of the viral genome was followed, at different times, quantitatively by a branched-DNA assay and qualitatively by reverse transcriptase-nested polymerase chain reaction. A strand-specific assay performed with the thermostable enzyme r Tth was used to detect the synthesis of a negative replicative intermediate. Cytopathic effect was examined by electron microscopy. Production of cytokines and inducible nitric oxide synthase was evaluated in the supernatants. Results : Quantification of HCV-RNA showed that the level of viral RNA associated with KC after adsorption decreased rapidly. Genomic viral RNA disappeared within 5 days of infection. Negative-strand RNA was never detected in any of these experiments. No cytopathic effects could be detected at any time. KC did not produce inflammatory nor antiviral cytokines. Conclusions : Our results strongly suggest that primary cultures of KC are not permissive for HCV in vitro.

International audience; Background The NS5A protein of the hepatitis C virus has been shown to be involved in the development of hepatocellular carcinoma. Objectives In a French multicenter study, we investigated the clinical and epidemiological features of a new HCV genotype 1b strain bearing a wide insertion into the V3 domain. Study Design We studied NS5A gene sequences in 821 French patients infected with genotype 1b HCV. Results We identified an uncharacterized V3 insertion without ORF disruption in 3.05% of the HCV sequences. The insertion comprised 31 amino-acids for the majority of patients; 3 patients had 27 amino-acids insertions and 1 had a 12 amino-acids insertion. Sequence identity between the 31 amino-acids insertions and the V3 domain ranged from 48 to 96% with E-values above 4e-5, thus illustrating sequence homology and a partial gene duplication event that to our knowledge has never been reported in HCV. Moreover we showed the presence of the duplication at the time of infection and its persistence at least during 12 years in the entire quasispecies. No association was found with extrahepatic diseases. Conversely, patients with cirrhosis were two times more likely to have HCV with this genetic characteristic (p=0.04). Moreover, its prevalence increased with liver disease severity (from 3.0% in patients without cirrhosis to 9.4% in patients with both cirrhosis and HCC, p for trend=0.045). Conclusions We identified a duplicated V3 domain in the HCV-1b NS5A protein for the first time. The duplication may be associated with unfavorable evolution of liver disease including a possible involvement in liver carcinogenesis

In vitro infection of human monocyte-derived dendritic cells was carried out to study their susceptibility to hepatitis C virus (HCV) infection. Immature dendritic cells and mature dendritic cells were incubated overnight at 37 degrees C with HCV-positive (genotype 1) serum samples; the presence of the viral genome associated with the production of its replicative intermediate was used as evidence of infection. In immature dendritic cells, HCV RNA was detectable from days 1-10 post-infection (p.i.), and de novo synthesis of negative-strand HCV RNA could be demonstrated by a strand-specific rTth reverse transcription-polymerase chain reaction at day 2. In mature dendritic cells, the positive-strand form was detectable from days 1-5 p.i., while the negative-strand HCV RNA appeared at days 1 and 2 p.i. Quasispecies present in the inoculum and 6 days p.i. were analyzed by sequencing hypervariable region 1 of the E2 protein. Only two of seven HVR variants present in the inoculum were found in HCV-infected immature dendritic cells. Another two HVR variants not found in the inoculum were recovered from infected immature dendritic cells, suggesting serum minor variants selection or virus evolution during in vitro replication. Analysis by single-strand conformation polymorphism assay of 5' untranslated region of HCV sequences showed that the patterns obtained from the inoculum and infected immature dendritic cells and mature dendritic cells differed slightly. These findings indicate that both immature dendritic cells and mature dendritic cells are susceptible to HCV genotype 1 infection, supporting at least HCV RNA replication. This model should be a valuable tool for the study of modulation of dendritic cell functions in HCV infection.

It is currently unclear whether the hepatocellular damage in chronic hepatitis C virus (HCV) infection is produced through the intrahepatic action of the anti-HCV immune response or through a direct cytopathic effect. In order to investigate the features of HCV replication (morphogenesis and cytopathic effect), we studied the infection of a permissive lymphocytic B cell line, Daudi cells, which were infected with sera of HCV-positive patients, and were examined after various time points under electron microscope. Viral genomic RNA was detected by in situ hybridization, and apoptosis with the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method. The amount of viral genomic RNA was observed to increase during infection. HCV replicated rapidly, since characteristics of viral morphogenesis resembling those of yellow fever virus in a hepatoma cell line could be found 2 days after infection. These included the following: a) several viral particles identical in size (about 42 nm) and structure (a spherical 30-nm-sized electron-dense nucleocapsid surrounded by a membrane) to yellow fever virus were present in the cytoplasm of cells displaying already typical signs of the early stage of apoptosis; b) numerous membrane-bound organelles and in particular the endoplasmic reticulum and vacuoles were observed; c) proliferation of membranes was apparent; and d) intracytoplasmic electron-dense inclusion bodies which have been demonstrated to correspond to nucleocapsids for other flaviviruses were detected. Several cells presented electron-dense areas in the endoplasmic reticulum displaying 30-nm circular structures lying among an amorphous material. Striking cytopathic features with ballooning, extremely enlarged vacuoles and signs of apoptosis were found in cells often containing sequestered aggregates of virus-like particles. By in situ hybridization we found that such enlarged cells contained HCV RNA. Our results thus indicate that the ultrastructural features of HCV viral particles and their morphogenesis resemble that of yellow fever virus and dengue virus. In Daudi cells, HCV infection seems to rapidly trigger apoptotic cell death, and efficient release of viral particles does not seem to take place.

Hepatitis C virus (HCV) is essentially hepatotropic, but clinical observations based on quasispecies composition in different compartments or on viral RNA detection in cells suggest that the virus is able to infect and persist in cells other than liver cells. It was shown previously that peripheral blood mononuclear cells (PBMCs) are permissive to HCV replication in vitro but at a very low rate. Since different viruses associated with chronic infections are known to persist in monocyte/macrophages, it is important to determine whether these mononuclear blood cells are susceptible preferentially to HCV. In order to study HCV interaction with monocytes/macrophages, these cells were isolated from the blood of healthy donors and incubated with HCV genotype 1b positive sera. The detection by RT-PCR of the positive- and negative-strand RNA in the cells at different times and the increase in the amount of intracellular viral RNA measured by the branched DNA assay suggest that monocyte/macrophages can support HCV RNA replication. The rate, however, is very low. The analysis of hypervariable region 1 (HVR-1) nucleotide sequences indicated that some minor variant present in the inoculum might display a specific tropism for the monocytes/macrophages. These results provide evidence that human monocytes/macrophages might represent a reservoir for HCV. This cell tropism may be a crucial factor in the pathogenesis of hepatitis C.

The clinical and radiological presentations of Epstein-Barr virus (EBV) encephalitis are pleomorphic, but a common and characteristic finding is an increased T2-weighted signal in the bilateral thalami and basal ganglia. We report here a case of post-transplant acute limbic encephalitis (PALE) syndrome that was possibly related to EBV infection. Six weeks after hematopoietic stem-cell transplantation, the patient developed confusion and anterograde amnesia. Brain magnetic resonance imaging (MRI) was performed and revealed bi-hippocampal and amygdala signal abnormalities. The technetium-99m single-photon emission computed tomography ((99m)Tc SPECT) imaging confirmed bilateral limbic structural involvement. The clinical, biological and radiological presentations were consistent with a diagnosis of EBV-induced PALE syndrome. To our knowledge, this is the first described case of PALE syndrome possibly related to EBV infection.

The risk factors for clinical recurrent hepatitis C in liver transplant recipients are not clearly defined. It has been suggested that the corticosteroids included in the treatments of patients undergoing allograft rejection might induce acute hepatitis by increasing HCV replication. In this study we investigated the effects of corticosteroid boluses on HCV viremia in liver allograft recipients treated for acute rejection. Since we had previously developed a model of HCV replication in peripheral blood mononuclear cells (PBMC) in vitro, we also studied the effects of corticosteroids on HCV replication in vitro. A transient peak of HCV viremia was observed in patients treated with corticosteroid boluses for an acute allograft rejection. In the cell cultures, corticosteroids induced an increase of the total amount of viral RNA detectable. Our results demonstrate that corticosteroids induce an increase of hepatitis C virus replication in vivo and in vitro.

Prevalence of GBV-C/HGV was determined in a cohort of HIV-infected patients, via a reverse transcription-polymerase chain reaction detection of RNA in serum, amplifying the NS5 region of GBV-C/HGV genome. GBV-C/HGV RNA was detected in 143 (37.7%) of 379 patients, with similar results in the different HIV risk groups: 25/56 (44.6%) in intravenous drug users, 66/161 (41%) in homo- and bisexual men, 35/108 (32.4%) in heterosexual patients, 6/20 (30%) in transfusion recipients (P = 0.41). There was no difference according to the presence or absence of hepatitis C virus infection. In univariate analysis, GBV-C/HGV genome prevalence was lower in patients over 50 years old (18.2%), compared to other age groups (20-29 years: 34.2%; 30-39 years: 44.3%; 40-49 years: 36.7%, P = 0.03), as well as in patients with normal CD4 cell count (29.2% vs. 45.4% between 200-500/mm 3 , and 35.3% below 200 CD4/mm 3 , P = 0.012) and individuals with a chronic hepatitis B. However, in the multivariate analysis, the only prognostic factor of GBV-C/HGV RNA positivity was the presence of a chronic hepatitis B, compared to the absence of any HBV marker, or a previous exposition to HBV (presence of anti-HBc and/or anti-HBs, absence of HBsAg), or the presence of anti-HBs alone.

This study, involving 20 laboratories and using currently available assays for hepatitis C virus RNA quantification, demonstrated that differences in viral load values are due not to interlaboratory variations but rather to the nature of the assay itself. This underlines the importance of using the same assay in multicenter studies or when monitoring antiviral therapy.

Human peripheral blood mononuclear cells (PBMC) can be infected in vitro with sera of hepatitis C virus (HCV)-infected patients. We have treated pools of PBMC with dextran sulfate MW 8,000, before infection with various HCV(+) sera. When the cells were treated with dextran sulfate 10-3 M, HCV RNA was no longer detectable after infection with all HCV genotypes tested. When the cells incubated with dextran sulfate 10-3 M and 10-4 M were maintained in culture for 4 weeks, no significant HCV replication could be observed. Our data suggest that dextran sulfate inhibits the attachment of HCV to the cell surface, since the HCV RNA detection was similar to control cells when dextran sulfate was added after infection.

International audience; OBJECTIVE: Influenza epidemics can have consequences in terms of morbidity and mortality for the patients. This work assesses influenza outbreaks in order to validate and optimize alert and control measures in a psychiatric hospital. METHOD: The prospective monitoring of influenza episodes was conducted for 8 years in 19 units of a mental health hospital. Rapid influenza diagnostic tests were used. The study of the episodes with confirmed influenza cases was carried out. RESULTS: Influenza monitoring and alert were essential with information and laboratory-confirmed cases. Influenza was common with a total of 20 episodes for the studied period. A maximum of 25% (5/20) of the units were affected in 2008-2009. Rapid influenza diagnostic tests allowed a quick identification with an average time of 1.5 days. Mainly, control measures limited the spread of the influenza virus in units with patient not at high risk of complications. On the other hand, antiviral curative treatment and chemoprophylaxis are essential in units with patients at high risk of complications. CONCLUSION: In a psychiatric hospital, influenza management has to take into account the exposed patient's risks for influenza complications and to adapt the strategy according to the risks identified.

It has been demonstrated that morphine stimulates the replication of human immunodeficiency virus in peripheral blood mononuclear cells as well as in Kupffer cells. Since the mechanism of action of this drug is still unknown, we have studied its effects on different properties of isolated human blood monocytes. In the presence of morphine, cultured monocytes showed an increase in the fluidity of their membranes as well as an inhibition in their capacity to differentiate into macrophages. Furthermore, the response of the cells to interferon-γ was significantly decreased and the release of superoxide anions was altered. Finally the production of interferon-α and of prostaglandin E2 induced by stimulation of the cells with endotoxin (LPS) was diminished. We conclude that morphine decreases the functions of monocytes that are essential for their antiviral defence and inhibits their response to activating stimuli, which may explain the increased multiplication of HIV in morphine treated monocytes.

BACKGROUND: A major challenge for antiviral treatment of hepatitis C virus (HCV) infection is viral resistance, potentially resulting from the high variability of HCV envelope glycoproteins and subsequent selection of strains with enhanced infectivity and/or immune escape. METHODS: We used a bioinformatics and functional approach to investigate whether E1/E2 envelope glycoprotein structure and function were associated with treatment failure in 92 patients infected with HCV genotype 1. RESULTS: Bioinformatics analysis identified 1 sustain virological response (R)-related residue in E1 (219T) and 2 non-SVR (NR)-related molecular signatures in E2 (431A and 642V) in HCV genotype 1a. Two of these positions also appeared in minimal networks separating NR patients from R patients. HCV pseudoparticles (HCVpp) expressing 431A and 642V resulted in a decrease in antibody-mediated neutralization by pretreatment sera. 431A/HCVpp entry into Huh7.5 cells increased with overexpression of CD81 and SR-BI. Moreover, an association of envelope glycoprotein signatures with treatment failure was confirmed in an independent cohort (Virahep-C). CONCLUSIONS: Combined in silico and functional analyses demonstrate that envelope glycoprotein signatures associated with treatment failure result in an alteration of host cell entry factor use and escape from neutralizing antibodies, suggesting that virus-host interactions during viral entry contribute to treatment failure.

Approximately 170 million individuals, representing 3% of the global population, are infected with hepatitis C virus (HCV). Whereas strategies for antiviral therapies have markedly improved resulting in clinical licensing of direct-acting antivirals, the development of vaccines has been hampered by the high genetic variability of the virus as well as by the lack of suitable animal models for proof-of-concept studies. Nevertheless, there are several promising vaccine candidates in preclinical and clinical development. After a brief summary of the molecular virology and immunology relevant to vaccine development, this review explains the model systems used for preclinical vaccine development, and highlights examples for most recently developed HCV vaccine candidates.

Summary In patients chronically infected by hepatitis C virus (HCV), peripheral blood mononu-clear cells (PBMCs) were shown to be targets for virus replication and in those coinfected with HIV, HCV viraernia was considerably increased. The purpose of this study was to quantify HCV RNA in PBMCs from 25 patients infected by HCV and from 25 patients coinfected by HCV and HIV. We used the branched DNA assay after extraction of total RNA on 5 × 10 6 cells to quantify HCV RNA, and the Inno LiPA TM assay to determine the HCV genotype. HCV RNA in PBMCs could be quantified in 8/25 patients in each group, but the HCV RNA concentration was very low in comparison with viraemia, since the highest result was 8.1 × 10 4 Eq genome/10 6 cells. In 10 ml of total blood, there was approximately 100 to 5,000 times toes HCV RNA in PBMCs than in the plasma. It is therefore likely that PBMCs play only a minor part in the viral load present in the plasma. There was no preferential genotype associated with quantifiable HCV RNA in the PBMCs. In the case of HIV confection, there was no increase in the HCV-RNA concentration in PBMCs that could explain the increased viraemia observed in these patients. On the contrary, HCV RNA could not even be detected by RT-PCR in some of our coinfected patients.

International audience; The poor response to the combined antiviral therapy of pegylated alfa-interferon and ribavarin for hepatitis C virus (HCV) infection may be linked to mutations in the viral envelope gene E1E2 (env), which can result in escape from the immune response and higher efficacy of viral entry. Mutations that result in failure of therapy most likely require compensatory mutations to achieve sufficient change in envelope structure and function. Compensatory mutations were investigated by determining positions in the E1E2 gene where amino acids (aa) covaried across groups of individuals. We assessed networks of covarying positions in E1E2 sequences that differentiated sustained virological response (SVR) from non-response (NR) in 43 genotype 1a (17 SVR), and 49 genotype 1b (25 SVR) chronically HCV-infected individuals. Binary integer programming over covariance networks was used to extract aa combinations that differed between response groups. Genotype 1a E1E2 sequences exhibited higher degrees of covariance and clustered into 3 main groups while 1b sequences exhibited no clustering. Between 5 and 9 aa pairs were required to separate SVR from NR in each genotype. aa in hypervariable region 1 were 6 times more likely than chance to occur in the optimal networks. The pair 531-626 (EI) appeared frequently in the optimal networks and was present in 6 of 9 NR in one of the 1a clusters. The most frequent pairs representing SVR were 431-481 (EE), 500-522 (QA) in 1a, and 407-434 (AQ) in 1b. Optimal networks based on covarying aa pairs in HCV envelope can indicate features that are associated with failure or success to antiviral therapy.

Hepatitis C virus RNA quantification results obtained in 18 laboratories using real-time PCR methods with 10 negative samples and 22 sample dilutions (viral loads of 0.5 to 500 IU/ml) showed a score of correct results of up to 93.5%. However, 55.6% of the laboratories did not follow the recommendations for the interpretation of their results, leading to ambiguous conclusions.

The presence and the quantity of hepatitis C virus (HCV) RNA were investigated in saliva and serum of patients infected with both HCV and human immunodeficiency virus (HIV). Paired serum and saliva samples were collected from 59 HIV-HCV coinfected patients. HCV RNA was detected by nested-PCR, using primers derived from the 5′ non-coding region of HCV, and positive results were quantified using the b-DNA method. HCV RNA was detected in the saliva of 22/59 (37.3%) patients, with a mean level of 1.15 × 106 genome equivalents/ml; there was no correlation of salivary positivity with immune status (CD4 cell count), age or HIV risk group, but there was with gender (19/38 [50%] positive results in male, compared to 3/21 [14.3%] in female, P = 0.007). HCV RNA was detected in the serum of 45/59 (76.3%) patients at a higher level (mean of 2.52 × 107 genome equivalents/ml) compared to saliva. Positivity was not correlated with age, gender or CD4 + cell count. There was a correlation between qualitative saliva and serum results (P = 0.003), but not between quantifications (P = 0.57). This first study reporting significant amounts of HCV RNA in saliva could have important implications for HCV epidemiology. J. Med. Virol. 63:117–119, 2001. © 2001 Wiley-Liss, Inc.

End-stage liver disease caused by chronic hepatitis C virus (HCV) infection is a leading cause for liver transplantation (LT). Due to viral evasion from host immune responses and the absence of preventive antiviral strategies, reinfection of the graft is universal. The mechanisms by which the virus evades host immunity to reinfect the liver graft are unknown. In a longitudinal analysis of six HCV-infected patients undergoing LT, we demonstrate that HCV variants reinfecting the liver graft were characterized by efficient entry and poor neutralization by antibodies present in pretransplant serum compared with variants not detected after transplantation. Monoclonal antibodies directed against HCV envelope glycoproteins or a cellular entry factor efficiently cross-neutralized infection of human hepatocytes by patient-derived viral isolates that were resistant to autologous host-neutralizing responses. These findings provide significant insights into the molecular mechanisms of viral evasion during HCV reinfection and suggest that viral entry is a viable target for prevention of HCV reinfection of the liver graft.

International audience; Persistent infections with HIV, hepatitis B virus and hepatitis C virus are major causes of morbidity and mortality worldwide. As sentinels of the immune system, dendritic cells (DCs) are crucial for the generation of protective antiviral immunity. Recent advances in our understanding of the role of DCs during infection with these viruses provide insights into the mechanisms used by these viruses to exploit DC function and evade innate and adaptive immunity. In this Review we highlight the current knowledge about the interaction between DCs and these viruses and the underlying mechanisms that might influence the outcome of viral infections.

International audience; Toll-like receptors (TLRs) are pathogen recognition molecules activating the innate immune system. Cell surface expressed TLRs, such as TLR2 and TLR4, have been shown to play an important role in human host defenses against viruses through sensing of viral structural proteins. In this study, we aimed to elucidate whether TLR2 and TLR4 participate in inducing antiviral immunity against hepatitis C virus (HCV) by sensing viral structural proteins. We studied TLR2 and TLR4 activation by cell culture-derived infectious virions and serum-derived virions in comparison to purified recombinant HCV structural proteins and enveloped virus-like particles. Incubation of TLR2 or TLR4 transfected cell lines with recombinant core protein resulted in activation of TLR2-dependent signaling. In contrast, neither infectious virions nor enveloped HCV-like particles triggered TLR2 and TLR4 signaling. These findings suggest that monomeric HCV core protein but not intact infectious particles are sensed by TLR2. Impairment of interaction between TLR and the core in infectious viral particles may contribute to escape from innate antiviral immune responses.

International audience; Development of an effective vaccine against the hepatitis C virus (HCV) has long been defined as a difficult challenge due to the considerable variability of this RNA virus and the observation that convalescent humans and chimpanzees could be re-infected after re-exposure. On the other hand, progress in the understanding of antiviral immune responses in patients with viral clearance has elucidated key mechanisms playing a role in the control of viral infection. Studies investigating prophylactic vaccine approaches in chimpanzees have confirmed that the induction and maintenance of strong helper and cytotoxic T-cell immune responses against multiple viral epitopes is necessary for protection against viral clearance and chronic infection. A multispecific B-cell response, resulting in rapid induction of cross-neutralizing antibodies may assist cellular responses. Therapeutic vaccine formulations currently being evaluated in clinical trials are facing the fact that the immune system of chronic carriers is impaired and needs the restoration of T-cell functions to enhance their efficacy.