The novel HBx mutation F30V correlates with hepatocellular carcinoma in vivo, reduces hepatitis B virus replicative efficiency and enhances anti-apoptotic activity of HBx N terminus in vitro.

OBJECTIVE: We aimed to investigate HBx genetic elements correlated with hepatitis B virus (HBV) -related hepatocellular carcinoma (HCC) and their impact on (a) HBV replicative efficiency, (b) HBx binding to circular covalently closed DNA (cccDNA), (c) apoptosis and cell-cycle progression, and (d) HBx structural stability. METHODS: This study included 123 individuals chronically infected with HBV: 27 with HCC (77.9% (21/27) genotype D; 22.1% (6/27) genotype A) and 96 without HCC (75% (72/96) genotype D; 25.0% (24/96) genotype A). HepG2 cells were transfected by wild-type or mutated linear HBV genome to assess pre-genomic RNA (pgRNA) and core-associated HBV-DNA levels, HBx-binding onto cccDNA by chromatin immunoprecipitation-based quantitative assay, and rate of apoptosis and cell-cycle progression by cytofluorimetry. RESULTS: F30V was the only HBx mutation correlated with HCC (18.5% (5/27) in HCC patients versus 1.0% (1/96) in non-HCC patients, p 0.002); a result confirmed by multivariate analysis. In vitro, F30V determined a 40% and 60% reduction in pgRNA and core-associated HBV-DNA compared with wild-type (p <0.05), in parallel with a significant decrease of HBx binding to cccDNA and decreased HBx stability. F30V also decreased the percentage of apoptotic cells compared with wild-type (14.8 ± 6.8% versus 19.1 ± 10.1%, p <0.01, without affecting cell-cycle progression) and increased the probability of HBx-Ser-31 being phosphorylated by PI3K-Akt kinase (known to promote anti-apoptotic activity). CONCLUSIONS: F30V was closely correlated with HBV-induced HCC in vivo, reduced HBV replicative efficiency by affecting HBx-binding to cccDNA and increased anti-apoptotic HBx activity in vitro. This suggests that F30V (although hampering HBV's replicative capacity) may promote hepatocyte survival, so potentially allowing persistent production of viral progeny and initiating HBV-driven hepatocarcinogenesis. Investigation of viral genetic markers associated with HCC is crucial to identify those patients at higher risk of HCC, who hence deserve intensive liver monitoring and/or early anti-HBV therapy.

Influenza B burden during seasonal influenza epidemics in France.

CONTEXT: Seasonal flu outbreaks are linked to the circulation of influenza virus type A or B. Special attention has always been paid to influenza A epidemics; but recently, several studies have investigated the impact of influenza B virus epidemics, particularly as, since the 1980s, two antigenically different influenza B lineages co-circulate, raising the issue of vaccine matching. OBJECTIVES: We present the results of influenza B burden during nine influenza seasons (2003-2013) and vaccine matching of the circulating lineages. PATIENTS AND METHODS: Clinical and virological influenza surveillance data, collected by the Regional Groups for Influenza Surveillance Network in France, allows for studying the burden of influenza in the practice of the population of ambulatory care physicians. RESULTS AND CONCLUSION: Our analysis is based on 37,801 samples, of which 12,036 were virologically confirmed influenza cases (31.8%), including 3576 cases of influenza B (29.7% of influenza cases). Influenza B viruses significantly circulated during six seasons. For each season, the influenza B epidemic peaked later than the influenza A epidemic. Influenza B is very common in children of school age but also affects other age groups. Finally, more than one-third of the analyzed influenza B viruses belonged to a different lineage than the one used in the composition of the trivalent vaccine. Our results are comparable to those described in other countries.

Study of the concordance between p16 immunohistochemistry and HPV-PCR genotyping for the viral diagnosis of oropharyngeal squamous cell carcinoma.

OBJECTIVE: The diagnosis of HPV-related oropharyngeal cancer in clinical practice is based on p16 immunohistochemistry and PCR detection of viral DNA (HPV-PCR). The primary objective of this study was to evaluate the concordance between these 2 diagnostic tests. The secondary objective was to study the clinical characteristics of these patients. MATERIALS AND METHODS: This single-centre prospective study was conducted between February 2010 and July 2012. Immunohistochemical analysis of p16 and HPV-PCR were performed on tumour biopsies. Concordance was evaluated according to Cohen's kappa coefficient and was interpreted according to the Landis and Koch scale. The patients' clinical data were analysed as a function of the diagnostic test results. RESULTS: Seventy-one patients were included in this study. The prevalence of HPV was 43.7% according to p16 and 31% according to HPV-PCR. The concordance study revealed a kappa coefficient of 0.615. A tumour of the tonsil or base of the tongue was detected in 100% of p16+/HPV-PCR+ cases. Smoking and alcohol abuse were significantly less frequent among HPV+ patients regardless of the method of detection. These patients were older and presented tumours with a lower grade of histological differentiation. CONCLUSION: p16 immunohistochemistry or HPV-PCR used alone appear to be insufficient. These results confirm the high prevalence of HPV-related oropharyngeal squamous cell carcinoma (OSCC) and the previously reported specific clinical and histological features, apart from age. It appears essential for future clinical trials to be stratified according to smoking and tumour HPV status, defined by means of reliable virological tests targeting E6/E7 mRNA and no longer a simple positive response to the p16 marker, as is frequently the case at the present time. New tests suitable for use in routine practice therefore need to be developed.

[Viral epidemiology and clinical severity during the peak of the influenza A(H1N1) variant epidemic in febrile respiratory diseases of children]. / Epidémiologie virale et sévérité clinique pendant le pic d'épidémie grippale A(H1N1) variant dans les atteintes respiratoires fébriles de l'enfant.

UNLABELLED: In 2009, a new emerging flu virus, A(H1N1), was identified. Its true medical impact on children's health remains widely debated. AIM: To define the prevalence of respiratory disease in children hospitalized with fever during the influenza A(H1N1) epidemic and to determine the clinical, paraclinical, and outcome characteristics according to the viruses identified. MATERIAL AND METHODS: Children hospitalized for a febrile respiratory disease were included in this prospective cohort study conducted at Bordeaux University's Children's Hospital (France) during the influenza epidemic from 2009/11/23 to 2009/12/20. RESULTS: Seventy-three children were included in the study. Viruses were identified by PCR in 52% (38/73) of cases, including 23% (17/73) A(H1N1) virus and 29% (21/73) other viruses, 22% (16/73) of which were syncytial respiratory viruses. There was only one case of co-infection between A(H1N1) virus and another virus from the para-influenza virus or adenovirus or bocavirus pool. No significant difference regarding age, sex, or risk factors in the different viral groups was noted. Regarding the A(H1N1) virus, the most frequent symptoms were deterioration of the overall health status, cough, ENT disease, and rapid breathing, with significantly less increased breathing effort and auscultatory abnormality albeit with more seizures. There was no significant difference between groups regarding laboratory data. Management and outcome were similar. CONCLUSION: The prevalence of A(H1N1) virus during the 2009 epidemic in Aquitaine was low among febrile hospitalized children with breathing symptoms. Clinical and paraclinical signs were non-specific. The tolerance and prognosis of influenza A(H1N1) infection in children was satisfactory.

HIV-1 dynamics and coreceptor usage in Maraviroc-treated patients with ongoing replication.

There is evidence that HIV-1 evolution under maraviroc (MVC) pressure can lead to the selection of either X4-tropic variants and/or R5-tropic, MVC-resistant isolates. However, the viral dynamics of HIV-1 variants in patients with virological failure (VF) on MVC-containing regimens remain poorly studied. Here, we investigated the V3 loop evolution of HIV-1 on MVC in relation to coreceptor usage and the nature of HIV-1 quasispecies before MVC therapy using bulk population sequences and ultradeep sequencing. The majority of patients had no detectable minority X4 variant at baseline. The evolution of tropism was followed up until VF and showed three possibilities for viral evolution in these patients: emergence of preexisting X4 variants, de novo selection of R5 variants presenting V3 loop mutations, or replication of R5 variants without selection of known mutations.

Cervical human papillomavirus and HIV infection in women of child-bearing age in Abidjan, Côte d'Ivoire, 2010.

BACKGROUND: We sought to document the association of Human immunodeficiency Virus (HIV) infection and immunodeficiency with oncogenic Human Papillomavirus (HPV) infection in women with no cervical neoplastic lesions identified through a cervical cancer screening programme in Côte d'Ivoire. METHODS: A consecutive sample of women stratified on their HIV status and attending the national blood donor clinic or the closest HIV clinic was recruited during a cervical cancer screening programme based on the visual inspection. Diagnosis of HPV infection and genotype identification were based on the Linear Array; HPV test. RESULTS: A total of 445 (254 HIV-positive and 191 HIV-negative) women were included. The prevalence of oncogenic HPV infection was 53.9% (95% confidence interval (CI) 47.9-59.9) in HIV-positive women and 33.7% (95% CI 27.1-40.3) in HIV-negative women (odds ratio (OR)=2.3 (95% CI 1.5-3.3)). In multivariate analysis, HIV-positive women with a CD4 count <200 cells mm(3) or between 200 and 499 cells mm(3) were more likely to harbour an oncogenic HPV compared with women with a CD4 count ≥500 cells mm(3) with OR of 2.8 (95% CI 1.1-8.1) and 1.7 (95% CI 1.0-2.9), respectively. CONCLUSION: A high prevalence of oncogenic HPV was found in women with no cervical neoplastic lesions, especially in HIV-positive women. Despite antiretroviral use, immunodeficiency was a main determinant of the presence of oncogenic HPV.

Stimulation of the human RAD51 nucleofilament restricts HIV-1 integration in vitro and in infected cells.

Stable HIV-1 replication requires the DNA repair of the integration locus catalyzed by cellular factors. The human RAD51 (hRAD51) protein plays a major role in homologous recombination (HR) DNA repair and was previously shown to interact with HIV-1 integrase (IN) and inhibit its activity. Here we determined the molecular mechanism of inhibition of IN. Our standard in vitro integration assays performed under various conditions promoting or inhibiting hRAD51 activity demonstrated that the formation of an active hRAD51 nucleofilament is required for optimal inhibition involving an IN-DNA complex dissociation mechanism. Furthermore we show that this inhibition mechanism can be promoted in HIV-1-infected cells by chemical stimulation of the endogenous hRAD51 protein. This hRAD51 stimulation induced both an enhancement of the endogenous DNA repair process and the inhibition of the integration step. Elucidation of this molecular mechanism leading to the restriction of viral proliferation paves the way to a new concept of antiretroviral therapy based on the enhancement of endogenous hRAD51 recombination activity and highlights the functional interaction between HIV-1 IN and hRAD51.

Structure-analysis of the HIV-1 integrase Y143C/R raltegravir resistance mutation in association with the secondary mutation T97A.

The HIV-1 integrase (IN) mutations Y143C/R are known as raltegravir (RAL) primary resistance mutations. In a previous study (S. Reigadas et al., PLoS One 5:e10311, 2010), we investigated the genetic pathway and the dynamics of emergence of the Y143C/R mutations in three patients failing RAL-containing regimens. In these patients, the Y143C/R mutation was associated with the T97A mutation. The aim of the present biochemical and molecular studies in vitro was to evaluate whether the secondary mutation, T97A, associated with the Y143C/R mutation could increase the level of resistance to RAL and impact IN activities. Site-directed mutagenesis experiments were performed with expression vectors harboring the region of the pol gene coding for IN. With a 3'-end processing assay, the 50% inhibitory concentrations (IC(50)) were 1.2 µM, 1.2 µM, 2.4 µM (fold change [FC], 2), and 20 µM (FC, 16.7) for IN wild type (WT), the IN T97A mutation, the IN Y143C/T97A mutation, and the IN Y143R/T97A mutation, respectively. FCs of 18 and 100 were observed with the strand transfer assay for IN Y143C/T97A and Y143R/T97A mutations, with IC(50) of 0.625 µM and 2.5 µM, respectively. In the strand transfer assay, the IN Y143C or R mutation combined with the secondary mutation T97A severely impaired susceptibility to RAL compared to results with the IN Y143C or R mutation alone. Assays without RAL suggested that the T97A mutation could rescue the catalytic activity which was impaired by the presence of the Y143C/R mutation. The combination of the T97A mutation with the primary RAL resistance mutations Y143C/R strongly reduces the susceptibility to RAL and rescues the catalytic defect due to the Y143C/R mutation. This result indicates that the emergence of the Y143C/R/T97A double-mutation pattern in patients is a signature of a high resistance level.

Hepatitis C virus (HCV) protease variability and anti-HCV protease inhibitor resistance in HIV/HCV-coinfected patients.

OBJECTIVES: Data on the natural selection of isolates harbouring mutations within the NS3 protease, conferring resistance to hepatitis C virus (HCV) protease inhibitors (PIs), are limited for HIV/HCV-coinfected patients. The aim of this study was to describe the natural prevalence of mutations conferring resistance to HCV PIs in HIV/HCV-coinfected patients compared with HCV-monoinfected patients. METHODS: The natural prevalences of HCV PI resistance mutations in 120 sequences from HIV/HCV-coinfected patients (58 genotype 1a, 18 genotype 1b and 44 genotype 4) and 501 sequences from HCV-monoinfected patients (476 genotype 1 and 25 genotype 4), retrieved from GenBank as a control group, were compared. RESULTS: Of 76 sequences from HIV/HCV genotype 1-coinfected patients, six (7.9%) showed amino acid substitutions associated with HCV PI resistance (V36L, n=1; V36M, n=2; T54S, n=2; R155K, n=1). In 31 of 476 (6.5%) HCV genotype 1 sequences retrieved from the GenBank database, HCV PI resistance mutations were found. The difference was not statistically significant (P=0.6). All of the sequences from HIV/HCV genotype 4-coinfected patients and those retrieved from the GenBank database had amino acid changes at position 36 (V36L). CONCLUSION: Our study suggests that the natural prevalence of strains resistant to HCV PIs does not differ between HCV-monoinfected and HIV/HCV-coinfected patients. Further studies on larger cohorts are needed to confirm these findings and to evaluate the impact of these mutations in clinical practice.

[Influenza A(H1N1)v virus infection in infants less than 6 months of age in southwestern France]. / Grippe A(H1N1)v chez les nourrissons de moins de 6 mois en Aquitaine.

OBJECTIVE: We evaluated the severity of influenza A(H1N1)v clinical forms among infants less than 6 months of age. This population group was considered a high-risk group, so all people around them should be vaccinated first. PATIENTS AND METHODS: In south-western France in Aquitaine, we collected all infants less than 6 months of age during a period between the 6th September 2009 and the 6th January 2010 with influenza A(H1N1)v confirmed by PCR. For each of them, the risk factors, clinical presentation, hospitalization, and course of, the disease were identified. We compared two groups: children under 3 months and infants aged 3-6 months. RESULTS: We identified 74 infants. The average age was 3 months. Sixteen infants had at least 1 risk factor: 9 respiratory diseases (12%), 8 born prematurely (but there was no preterm baby under 33 weeks); one infant presented a cardiac disease, and another 1 epilepsy. Five infants showed no fever, 73% had cough, and 24% had gastro-intestinal symptoms. Infants under 3 months of age presented less cough (P<0.025) and fewer gastro-intestinal symptoms (P<0.01) than older ones. Only 5 infants needed oxygen and 4 presented pneumonia. Forty-eight infants were hospitalized, including 1 in intensive care, with a median duration of 3 days. Forty-five percent spent 2 days or less in the hospital. Infants under 3 months of age were more often hospitalized (P<0.001). CONCLUSIONS: Infants under 6 months of age did not present a severe form of influenza A(H1N1)v. Infants under 3 months of age were less symptomatic than older infants and were often hospitalized, but hospital stays were short with a good outcome.

Pandemic influenza A(H1N1) 2009: molecular characterisation and duration of viral shedding in intensive care patients in Bordeaux, south-west France, May 2009 to January 2010.

From May 2009 to January 2010, the Virology Laboratory at the University Hospital of Bordeaux received more than 4,000 nasopharyngeal samples from the Aquitaine region (south-west France) for the diagnosis of pandemic influenza A(H1N1)2009. Eighty-three infected patients deteriorated and were admitted to intensive care units. Our study focused on 24 of these patients. Positivity for influenza A(H1N1)2009 was monitored by realtime PCR and duration of viral shedding was determined. The first available sample of each patient was analysed for bacterial, fungal and viral co-infection. We observed six bacterial (or bacterial/fungal) co-infections and one viral co-infection with respiratory syncytial virus. The samples were analysed for the presence of the neuraminidase H275Y (N1 numbering) mutation, which confers resistance to oseltamivir, by realtime PCR of the neuraminidase gene. No H275Y mutation was observed in any of the viral strains screened in this study. In parallel, a fragment of the haemagglutinin gene encoding amino acid residues 173 to 362 was sequenced to detect mutations that had been reported to increase the severity of the disease. Two patients were infected by strains bearing the D222G (H3 numbering) mutation. The viral shedding of A(H1N1)2009 in this study ranged from four to 28 days with a median of 11 days.

[First cases of secondary transmission of a novel swine-origin influenza A (H1N1) virus in France]. / Premiers cas de transmission secondaire en France du nouveau virus grippal d'origine porcine A(H1N1)v.

On April 2009, a new swine-origin A(H1N1) influenza virus, A(H1N1)v, was identified in the United States. Today (June 12, 2009), more than 29,000 cases have been reported in the world, and 73 in France. This is the first report of secondary transmission in France. The three patients presented with common influenza signs including cough, fever, and sore throat. The incubation period could last from two to four days; it should be kept in mind that the first international data mentioned one to seven days. The buildup and maintenance of an infectious focus involve secondary transmissions.

Prolonged shedding of influenza A(H1N1)v virus: two case reports from France 2009.

We observed a prolonged shedding of virus 14 and 28 days after symptom onset in two patients with pandemic H1N1 influenza, who did not have immunodepression and were treated with neuraminidase inhibitor. This prolonged shedding was not associated with the emergence of resistance mutation H275Y in the viral neuraminidase gene.

Oseltamivir susceptibility in south-western France during the 2007-8 and 2008-9 influenza epidemics and the ongoing influenza pandemic 2009.

The recent emergence of seasonal influenza A(H1N1) strains resistant to oseltamivir makes it necessary to monitoring carefully the susceptibility of human influenza viruses to neuraminidase inhibitors. We report the prevalence of the oseltamivir resistance among influenza A viruses circulating in south-western France over the past three years: seasonal influenza A(H1N1), seasonal influenza A(H3N2), and the influenza A(H1N1)v viruses associated with the ongoing 2009 pandemic. The main result of the study is the absence of oseltamivir resistance in the pandemic H1N1 strains studied so far (n=129).

Quantitation of HIV-1 RNA in dried blood and plasma spots.

Dried blood spots (DBSs) and dried plasma spots (DPSs) are an attractive method for serological and molecular diagnosis of HIV infection. Recently, Youngpairoj et al. [Youngpairoj, A.S., Masciotra, S., Garrido, C., Zahonero, N., de Mendoza, C., Garcia-Lerma, J.G., 2008. HIV-1 drug resistance genotyping from dried blood spots stored for 1 year at 4 degrees C. J. Antimicrob. Chemother. 61, 1217-1220] showed that HIV-1 can be genotyped efficiently from DBS specimens stored at 4 degrees C for 1 year. The viral load obtained from DBS and DPS samples was compared with that obtained from plasma samples. A total of 86 samples was prepared from people infected with HIV subtype B or non-B and spotted on 903 filter papers stored with desiccant at 4 degrees C. RNA was extracted using the QIAamp Viral RNA mini kit (Qiagen, Courtaboeuf, France). RNA from DBS or DPS samples was quantified in accordance with the Agence Nationale de Recherche sur le SIDA (ANRS, AC11, Paris, France) assay for HIV-1 quantitation. When the mean viral load of plasma samples and DPS samples or plasma samples and DBS samples were compared, there were no significant differences. The overall data showed that although the sensitivity threshold of the assays was different, there was a correlation between the three specimen types and that DBS and DPS samples can be routinely used for viral load quantification particularly in resource-limited settings.

Improvement of progressive multifocal leukoencephalopathy after cidofovir therapy in a patient with a destructive polyarthritis.

The human neurotropic JC virus (JCV) is responsible for progressive multifocal leukoencephalopathy (PML), an infectious demyelinating brain disease with major morbidity and mortality, usually refractory to treatment. We describe a PML in a 67-year-old woman with a destructive polyarthritis associated with anti-JO1 antibodies treated with corticosteroids. Although glucocorticoid therapy was maintained, administration of cidofovir improved the neurological condition. Our observation demonstrates the expanding clinical importance of JCV in systemic rheumatic diseases, particularly when immunosuppressive agents are used, and neurological symptoms or white matter changes on central nervous system imaging should arouse the suspicion of PML.