The YMDD and rtA194T mutations result in decreased replication capacity in wild-type HBV as well as in HBV with precore and basal core promoter mutations.

BACKGROUND: A recent study indicated that addition of the hepatitis B e antigen (HBeAg) precore (PC) or basal core promoter (BCP) mutations to wild-type HBV offset the reduced replication of the HBV polymerase rtA194T±rtL180M+rtM204V mutations. rtA194T was reportedly associated with tenofovir resistance. We investigated these findings in genotype D HBV, where both PC and BCP naturally occur in vivo. METHODS: A plasmid containing a wild-type 1.3 genome length genotype D HBV laboratory strain was used as a parent for PC, BCP, rtA194T±rtL180M+rtM204V, rtL180M+rtM204V and rtM204I mutants. Viral replication was evaluated by Southern blot analysis of intracellular HBV core DNA following transient transfection of HepG2 cells. Drug susceptibility was evaluated by quantitative PCR of intracellular HBV DNA. RESULTS: PC and BCP mutations each increased HBV DNA replication by approximately 200% over wild-type. rtA194T reduced replication by <40%, whereas rtL180M+rtM204V, rtL180M+rtA194T+rtM204V or rtM204I each reduced by >75% from their respective wild-type, PC or BCP genome backbone (P<0.05). The enhanced replication by PC or BCP offset the reduction by rtA194T; however, the other reverse transcriptase (RT) mutations in PC or BCP backbones remained significantly lower than wild-type (P<0.05). Regardless of the backbone, rtA194T±rtL180M+rtM204V remained susceptible to tenofovir in vitro. rtA194T alone remained susceptible to lamivudine, while rtL180M+rtM204V and rtL180M+rtA194T+rtM204V were resistant. CONCLUSIONS: PC or BCP mutations increased HBV DNA replication, offset the decreased replication by rtA194T alone, but they did not fully rescue the impaired replication conferred by other RT mutations as compared with wild-type. rtA194T±rtL180M+rtM204V did not confer tenofovir resistance.

No resistance to tenofovir disoproxil fumarate detected after up to 144 weeks of therapy in patients monoinfected with chronic hepatitis B virus.

UNLABELLED: Tenofovir disoproxil fumarate (TDF) is a nucleotide analogue with potent activity against human immunodeficiency virus type 1 and hepatitis B virus (HBV). To date, no reports of HBV clinical resistance to TDF have been confirmed. In two phase 3 studies (GS-US-174-0102 and GS-US-174-0103), 375 hepatitis B e antigen-negative (HBeAg(-) ) patients and 266 HBeAg(+) patients with chronic hepatitis B (some nucleoside-naive and some lamivudine-experienced) were randomized 2:1 to receive TDF (n = 426) or adefovir dipivoxil (ADV; n = 215) for 48 weeks. After week 48, eligible patients received open-label TDF with no interruption. The studies are being continued through week 384/year 8; week 144 data are presented here. Per protocol, viremic patients (HBV DNA level ≥ 400 copies/mL or 69 IU/mL) had the option of adding emtricitabine (FTC) at or after week 72. Resistance analyses of HBV polymerase/reverse transcriptase (pol/RT) were based on population dideoxy sequencing. Phenotypic analyses were conducted in HepG2 cells with recombinant HBV derived from patient serum. Most patients maintained TDF monotherapy treatment across both studies (607/641, 95%). A resistance analysis of HBV pol/RT was performed at the baseline for all patients, for viremic patients at week 144 or at the last time when they were on TDF monotherapy (34 on TDF and 19 on ADV-TDF), and for patients who remained viremic after the addition of FTC (7/20 on TDF and 5/14 on ADV-TDF). No patient developed amino acid substitutions associated with resistance to TDF. Virological breakthrough on TDF monotherapy was infrequent over 144 weeks (13/426, 3%) and was attributed to documented nonadherence in most cases (11/13, 85%). Persistent viremia (≥400 copies/mL) through week 144 was rare (5/641, 0.8%) and was not associated with virological resistance to TDF by population or clonal analyses. CONCLUSION: No nucleoside-naive or nucleoside-experienced patient developed HBV pol/RT mutations associated with TDF resistance after up to 144 weeks of exposure to TDF monotherapy.

HBV DNA replication mediated by cloned patient HBV reverse transcriptase genes from HBV genotypes A-H and its use in antiviral phenotyping assays.

The aim of this study is to establish a phenotyping assay to analyze patient HBV polymerase/reverse transcriptase (RT) sequences for potential drug resistance against RT inhibitors. HBV RT (pol aa 304-715, including the entire RT) from clinical isolates were amplified and ligated into a plasmid vector (pRTAN) expressing a genotype A HBV genome lacking the RT region. HBV DNA replication of the recombinants and their drug susceptibilities were assessed by transient transfection into HepG2 cells and intracellular core DNA was analyzed either by Southern blot or using a 96-well format and quantification by qPCR. Cloning of the HBV RT gene from clinical isolates representing genotypes A-H was successful and led to virus DNA replication. Recombinants expressing patient derived RT genes containing the rtL180M+M204V lamivudine resistance (LAM-R) mutations demonstrated a LAM-R phenotype. Similarly, patient derived RT genes containing the adefovir resistance (ADV-R) mutations rtA181V or rtN236T demonstrated an ADV-R phenotype. Recombinants containing HBV RT from paired patient samples without genotypic changes had similar EC(50) values. In conclusion, a phenotyping assay for HBV RT gene was developed, allowing evaluation of patient-derived HBV RT from genotypes A-H, and confirmed the drug resistance phenotype in samples containing LAM-R or ADV-R mutations.

Anti-hepatitis B virus activity in vitro of combinations of tenofovir with nucleoside/nucleotide analogues.

BACKGROUND: Long-term management of some chronic hepatitis B patients might require combination therapy using drugs with distinct resistance profiles to sustain viral suppression and to reduce the resistance-associated failure. Tenofovir disoproxil fumarate (TDF), approved for hepatitis B virus (HBV) and HIV-1 treatment, is active against wildtype HBV and HBV containing YMDD mutations, which confer resistance to emtricitabine (FTC), lamivudine (3TC) and telbivudine (LdT) and contribute to entecavir (ETV) resistance. We therefore evaluated the in vitro anti-HBV activity of tenofovir (TFV), the active parent drug of TDF, combined with FTC, 3TC, ETV, LdT and adefovir (AFV). METHODS: The anti-HBV activities of the compounds were tested using the AD38 cell line that expresses wild-type HBV from a tetracycline-controllable promoter. Intracellular HBV DNA levels were quantified using real-time PCR assay and cytotoxicities were assessed with XTT assays. The antiviral data of the drug combinations were evaluated using MacSynergy analyses on the basis of the Bliss independence model as well as isobologram analyses on the basis of the Loewe additivity theory. RESULTS: All drug combinations tested, FTC+TFV, 3TC+TFV, ETV+TFV, LdT+TFV and AFV+TFV, showed additive antiviral interactions as analysed by MacSynergy. Isobologram analyses revealed that these combination pairs were additive, with the exception of FTC+TFV, which demonstrated slight synergistic activity. No cytotoxic or antagonistic effects were observed with any of the combinations tested. CONCLUSIONS: The combination of TFV with FTC, 3TC, ETV, LdT or AFV had additive to slightly synergistic anti-HBV effects in vitro. These results support the use of TDF as a component in combination regimens with currently available anti-HBV nucleoside analogues.

Hepatitis B virus containing the I233V mutation in the polymerase reverse-transcriptase domain remains sensitive to inhibition by adefovir.

An isoleucine-to-valine change at position 233 (rtI233V) of hepatitis B virus (HBV) polymerase was recently reported to cause decreased in vitro susceptibility to, and treatment failure of, adefovir dipivoxil (ADV). To further evaluate these findings, we screened our ADV clinical-study sequence database of 853 patients and identified 4 who, at baseline, had HBV with this mutation. All 4 patients responded to treatment with ADV, with a median change in HBV DNA levels of 4.0 log(10) copies/mL after 48 weeks of treatment. Phenotypic evaluation of clinical isolates and of a laboratory strain with the rtI233V mutation demonstrated their full susceptibility to adefovir in vitro, and HBV with the rtI233V mutation developed in none of the patients.

In vitro drug susceptibility analysis of hepatitis B virus clinical quasispecies populations.

Analysis of the replication and drug resistance of patient serum hepatitis B virus (HBV) populations can contribute to the therapeutic management of chronic hepatitis B. We developed a procedure for cloning serum HBV quasispecies populations and for phenotypic analysis of the cloned populations for in vitro drug susceptibility. Equivalent sequences were compared to the respective serum HBV DNAs of the cloned quasispecies by population sequencing. Analysis of individual clones revealed that each population contained a diversity of HBV quasispecies. Furthermore, secreted HBV in the supernatant following transfection of the quasispecies populations remained mostly unchanged from the respective input populations. HBV obtained from patients who had developed resistance to adefovir or lamivudine, as demonstrated by development of the rtA181V or rtL180M/M204V mutations in HBV polymerase, respectively, were tested. Phenotypic analysis demonstrated that a population containing the HBV rtA181V mutation showed a 2.9-fold increase in the 50% effective concentration (EC(50)) for adefovir compared to the wild-type baseline isolate, while the lamivudine-resistant HBV quasispecies population showed a >1,000-fold increase in the lamivudine EC(50). In summary, a strategy of cloning full genome HBV quasispecies populations from patient sera was developed, which could provide a useful tool in clinical HBV drug resistance phenotyping and studies of the evolution of clinical viral species.

Altered NGF response but not release in the aged septo-hippocampal cholinergic system.

An important aspect of aging and Alzheimer's disease (AD) pathology includes the degeneration of basal forebrain cholinergic neurons (BFCNs), possibly due to disrupted nerve growth factor (NGF) signaling. Previous studies on disrupted NGF signaling have focused on changes in retrograde transport. This study focuses on two other possible mechanisms for loss of trophic support: diminished release of NGF from hippocampal neurons or diminished TrkA receptor response of BFCNs to NGF. We measured NGF levels in the effluent of hippocampal slices from young and aged rats in response to potassium chloride and glutamate. We found that release of NGF was not altered in aged hippocampal slices compared to slices from young controls. To measure the in situ response of the BFCNs to NGF, we injected NGF intraparenchymally into the right hippocampus of young and aged rats. Injections of cytochrome C served as controls. Fifteen minutes post-administration, a dramatic increase in TrkA immunoreactivity was found in the cell bodies of medial septal neurons. We found that this rapid response was blunted in aged rats compared to young adult controls. To determine whether retrograde transport was necessary for this rapid response, we injected colchicine prior to NGF injection. The NGF-induced upregulation was not blocked by colchicine, suggesting that this acute response was not dependent on classical retrograde transport. Since cholinergic degeneration coupled with altered levels of NGF and TrkA receptors are also seen in human aging and AD, the loss of acute responsivity to NGF in the BFCNs may also play a role in these processes.

[Listeria monocytogenes in vegetables minimally processed]. / Listeria monocytogenes en vegetales mínimamente procesados.

The demand of vegetables minimally processed (ready-to-use) has increased partly due to the frequent use of the food services, where the salads are always included in the daily menus. The use of new technologies for processing and packaging has made possible to obtain a product ready to serve. Nevertheless the associated risk of the presence of emergent pathogens, such as Listeria monocytogenes seems to be involved. The aim of this work was to assess the microbiological quality of this kind of food. 120 samples of vegetables minimally processed ready-to-use were analyzed for their content of aerobic mesophilic bacteria, total and fecal coliforms and E. coli, and the presence of Shigella spp. Vibrio cholerae and Listeria monocytogenes. The TECRA UNIQUE LISTERIA, the BCM Listeria monocytogenes and the API LISTERIA systems, and the methods of molecular detection AccuProbe and GENE-TRAK were used for isolation and identification. E. coli was detected in approximately 30.3% of the vegetables used in this study. The genus Listeria was evidenced in 25% of the samples; 30% corresponded to L. monocytogenes. Shigella spp and Vibrio cholerae were not isolated. The findings of this study suggest the need of the microbiological control of the vegetables minimally processed ready-to-use to assure their quality and safety.

Listeria monocytogenes en vegetales mínimamente procesados / Listeria monocytogenes in vegetables minimally processed

The demand of vegetables minimally processed (ready-to-use) has increased partly due to the frequent use of the food services, where the salads are always included in the daily menus. The use of new technologies for processing and packaging has made possible to obtain a product ready to serve. Nevertheless the associated risk of the presence of emergent pathogens, such as Listeria monocytogenes seems to be involved. The aim of this work was to assess the microbiological quality of this kind of food. 120 samples of vegetables minimally processed ready-to-use were analyzed for their content of aerobic mesophilic bacteria, total and fecal coliforms and E. coli, and the presence of Shigella spp. Vibrio cholerae and Listeria monocytogenes. The TECRA UNIQUE LISTERIA, the BCM Listeria monocytogenes and the API LISTERIA systems, and the methods of molecular detection AccuProbe and GENE-TRAK were used for isolation and identification. E. coli was detected in approximately 30.3 of the vegetables used in this study. The genus Listeria was evidenced in 25 of the samples; 30 corresponded to L. monocytogenes. Shigella spp and Vibrio cholerae were not isolated. The findings of this study suggest the need of the microbiological control of the vegetables minimally processed ready-to-use to assure their quality and safety.

Determinación de la calidad microbiológica de alimentos servidos en comedores de empresas privadas / Assessment of foods microbiological quality in food-service establishments

El objetivo del presente trabajo ha sido determinar la calidad microbiológica de los alimentos servidos en comedores de empresas privadas. Se analizó un total de 620 muestras de alimentos en los que se determinó recuento de aerobios de mesófilos (AM), mohos y levaduras, Escherichia coli y Staphylococcus aureus e investigación de Salmonella; se realizó el análisis microbiológico del agua, de los equipos, utensilios, ambientes, superficies, y personal. Se dan los resultados de los análisis realizados; en general se observa una elevada contaminación por E.coli. En vegetales crudos 76,2 por ciento, en cocidos 15,2 por ciento, en carnes de res y cerdo 15,9 por ciento, en aves 16,7 por ciento, en pescados 11,8 por ciento, en postres 27,3 por ciento, en equipos y utensilios 57,9 por ciento, en superficies y ambientes 53,6 por ciento y en operarios 21,9 por ciento. Estos resultados se evaluaron de acuerdo a criterios o límites de aceptación fijados. Los resultados obtenidos permiten concluir que estos alimentos deben estar sujetos a controles microbiológicos continuos y se considera que siguen siendo un factor de riesgo tanto el personal como las superficies y equipos