Immune Mediators against Toxoplasma Gondii during Reactivation of Toxoplasmic Retinochoroiditis.

Purpose: The purpose of this article is to analyze possible associations between systemic and ocular cytokine levels and specific clinical ophthalmologic signs from patients with a reactivation of toxoplasmic retinochoroiditis (RTR). Methods: A total of 18 patients with an active RTR episode, 8 patients with inactive scars, and 14 control patients were included in the study. Serum samples and aqueous humor (AH) samples were analyzed for IFN (interferon)-γ, interleukin (IL)-10, and IL-6 levels by ELISA. Inflammation grade, location, and size of the retinochoroidal active lesion, sampling time, and time to resolution were recorded. Results: A significantly negative correlation between AH and serum levels of IFN-γ was detected (p < 0.05). Patients with an AH IFN-γ/IL-10 ratio lower than 1 were associated with the longest time to resolution and/or severe complications. Conclusion: Serum IFN-γ levels may be used as a prognostic marker for both time to resolution and the development of possible severe complications during a given RTR episode.

In vitro replication competence of a hepatitis B genotype D/A recombinant virus: dissimilar biological behaviour regarding its parental genotypes.

Hepatitis B virus (HBV) DNA recombinants contribute to ~30% of the overall full-length sequences already deposited in GenBank. However, their biological behaviour has not been analysed so far. In this study, the in vitro replication kinetics of the first D/A recombinant from the American continent differed from its parental genotypes, exhibiting higher extracellular levels of HBV DNA and hepatitis B e antigen. Southern blots of intracellular core-associated HBV DNA were in agreement with such results. Because this recombinant was obtained from an Argentinian injecting drug user belonging to a vulnerable community, these results are of singular relevance for regional public health. Further in vivo studies are urgently needed to determine the pathogenicity of these replicative competent clones.

Antiviral activity against the hepatitis C virus (HCV) of 1-indanone thiosemicarbazones and their inclusion complexes with hydroxypropyl-ß-cyclodextrin.

The hepatitis C virus (HCV) is a major cause of acute and chronic hepatitis in humans. Approximately 5% of the infected people die from cirrhosis or hepatocellular carcinoma. The current standard therapy comprises a combination of pegylated-interferon alpha and ribavirin. Due to the relatively low effectiveness, the prohibitive costs and the extensive side effects of the treatment, an intense research for new direct-acting anti-HCV agents is taking place. Furthermore, NS3 protease inhibitors recently introduced into the market are not effective against all HCV subgenotypes. Thiosemicarbazones (TSCs) have shown antiviral activity against a wide range of DNA and RNA viruses. However, their extremely low aqueous solubility and high self-aggregation tendency often preclude their reliable biological evaluation in vitro. In this work, we investigated and compared for the first time the anti-HCV activity of two 1-indanone TSCs, namely 5,6-dimethoxy-1-indanone TSC and 5,6-dimethoxy-1-indanone N4-allyl TSC, and their inclusion complexes with hydroxypropyl-ß-cyclodextrin (HPß-CD) in Huh-7.5 cells containing the full-length and the subgenomic subgenotype 1b HCV replicon system. Studies of physical stability in culture medium showed that free TSCs precipitated rapidly and formed submicron aggregates. Conversely, TSC complexation with HPß-CD led to more stable systems with minimal size growth and drug concentration loss. More importantly, both TSCs and their inclusion complexes displayed a potent suppression of the HCV replication in both cell lines with no cytotoxic effects. The mechanism likely involves the inhibition of non-structural proteins of the virus. In addition, findings suggested that the cyclodextrin released the drug to the culture medium over time. This platform could be exploited for the study of the drug toxicity and pharmacokinetics animal models.

HDAg-L variants in covert hepatitis D and HBV occult infection among Amerindians of Argentina: new insights.

BACKGROUND: Guidelines suggest that all HBsAg-positive patients should be tested for anti-HDV IgG antibodies and to confirm active hepatitis D virus (HDV) infection by detection of HDV RNA by reverse transcriptase (RT) polymerase chain reaction (PCR). OBJECTIVES: The aim of this study was to determine the serological prevalence and molecular features of HDV within an Amerindian community from Argentina exhibiting positivity for HBsAg and/or anti-HBc total Ig. STUDY DESIGN: Forty-six plasma samples were tested for the detection of total anti-HDV antibodies by ELISA. Concomitantly, a partial RNA region coding for the delta antigen (HDAg) was amplified by RT-nested PCR (RT-nPCR). In silica translation of DNA sequences into the amino acid (aa) sequence of HDAg-S (aa110-195) and HDAg-L (aa110-214) was performed. RESULTS: Out of 46 HDV non-reactive samples by ELISA, 3 were HDV RNA positive by RT-nPCR. These samples were anti-HBc-only positive, 2 of them identified as cases of occult hepatitis B infection (OBI). The 3 cases were HBeAg-negative and showed normal ALT/AST levels. All sequences were ascribed to HDV genotype 1, but exhibited nucleotide differences in HDAg-L coding region, among which, mutations at codons 197 and 201 - reportedly known to promote in vitro an unsuitable interaction with HBsAg - were observed. CONCLUSIONS: These results provide evidence of covert HDV infection even among OBI, highlighting the need to reevaluate the currently applied guidelines for HDV diagnostic algorithms, as well as to explore if the observed mutations promote any effect on HDV pathogenesis.

New natural variants of hepatitis B virus among Amerindians from Argentina with mainly occult infections.

BACKGROUND: Hepatitis B virus infection is frequent among Amerindians. In Argentina HBV genotypes A, B, C, D, E, F and H were described in different populations, while some cases of occult hepatitis B infection (OBI) were reported in human immunodeficiency virus (HIV) coinfected patients. OBJECTIVE: To determine the prevalence, genetic diversity of HBV and to analyze the deduced amino acid sequence of both S and viral polymerase (P) genes among Amerindians of Argentina. STUDY DESIGN: A cross-sectional study including 561 individuals belonging to distinct ethnic groups, the Mbyá-guaraní (MG), the Kolla (K), the Sagua-Huarpe (SH) and the Wichi (W) was performed. RESULTS: The prevalence of HBsAg was 1.7% and 1.4% for the MG and SH, respectively, while anti-HBc was detected in all communities. HBV DNA of S/P and preCore-Core genomic regions were amplified by nested polymerase chain reaction in 59 reactive samples for anti-HBc total Ig and/or HBsAg. Of them, thirteen exhibited detectable HBV DNA, eleven of which were identified as OBI. Genotype F was predominant in the MG community with co-circulation of subgenotypes F4, F1b, A2 and D3, while subgenotype C2 prevailed within the SH community. All cases exhibited the polymorphism rtL217R within the RT domain associated to resistance to adefovir. Mutations rtD206E and rtV207I associated with lamivudine resistance were found in two MG and three SH respectively. Other new substitutions were described within the P sequence. CONCLUSIONS: This study shows for the first time the predominance of OBI, HBV subgenotypes and natural variants in Amerindians from Argentina.

Naturally occurring hepatitis B virus (HBV) variants with primary resistance to antiviral therapy and S-mutants with potential primary resistance to adefovir in Argentina.

Hepatitis B virus (HBV) variants may either emerge in patients with chronic hepatitis B (CHB) as a result of positive selection pressure exerted by their own immune response, or during therapy with nucleos(t)ide analogues (NAs). Naturally occurring HBV variants with primary antiviral resistance are rarely observed. The aim of this study was to retrospectively analyze the (eventual) circulation of HBV variants with natural resistance to NAs currently used as therapy for CHB in Argentina. This study reports 13 cases of CHB-infected patients with natural antiviral resistance to at least one NA. Five of them were also carriers of S-variants that might escape the humoral immune system recognition with potential resistance to adefovir. In addition to the already reported A2 HBV subgenotype association to NAs natural resistance, E and F genotypes association to such resistance is described for the first time. These findings suggest that sequence analysis of the HBV reverse transcriptase might be an essential tool before starting antiviral therapy, in order to choose the proper NAs for optimizing the therapeutic management of chronically infected patients. Moreover, the circulation and transmission of S-mutants with resistance to such antiviral drugs should be of public health concern as they may represent an additional risk for the community.

Drug delivery systems and liver targeting for the improved pharmacotherapy of the hepatitis B virus (HBV) infection.

In spite of the progress made in vaccine and antiviral therapy development, hepatitis B virus (HBV) infection is still the most common cause of liver cirrhosis and hepatocellular carcinoma, with more than 400 million people chronically infected worldwide. Antiviral therapy with nucleos(t)ide analogues and/or immunomodulating peptides is the only option to control and prevent the progression of the disease in chronic hepatitis B (CHB)-infected patients. So far, the current antiviral monotherapy remains unsatisfactory because of the low efficacy and the development of drug resistance mutants. Moreover, viral rebound is frequently observed following therapy cessation, since covalent closed circular DNA (cccDNA) is not removed from hepatocytes by antiviral therapy. First, this review describes the current pharmacotherapy for the management of CHB and the new drug candidates being investigated. Then, the challenges in the development of drug delivery systems for the targeting of antiviral drugs to the liver parenchyma are discussed. Finally, perspectives in the design of a more efficient pharmacotherapy to eradicate the virus from the host are addressed.

Single and multiple mutations in the human cytomegalovirus UL97 gene and their relationship to the enzymatic activity of UL97 kinase for ganciclovir phosphorylation.

In this study we determined that the double mutant M460V/D605E in the UL97 gene of an HCMV isolate from an immunocompromised patient (MMT isolate) is related to resistance to ganciclovir (GCV) therapy. Our results suggest that the aspartic acid-to-glutamic acid substitution at codon 605 may be associated with a natural polymorphism of the UL97 gene, and not with positive selection pressure exerted by the antiviral drug. We also determined that GCV resistance due to the M460V mutation in the HCMV UL97 gene is not offset by a second mutation (D605E) at codon 605. Furthermore, we showed that when the two mutations related to GCV resistance were simultaneously detected in the same HCMV construct, virus-drug resistance might be enhanced in comparison to that of the single mutants studied separately. To our knowledge for the first time, seven of 12 amino acid changes (F102L, D118V, M330T, T400A, R507P and C511R and I533V) in the UL97 gene of an isolate are herein reported.

Successful interferon treatment in a patient chronically infected with hepatitis B virus carrying unusual S- (and P-) mutants in the presence of anti-HBs antibodies.

BACKGROUND: Hepatitis B virus (HBV) immune escape mutants with point mutations within the S gene may arise during the natural course of HBV infection, due to a positive selection pressure exerted by the host immune response. Mutations within the immunodominant B and T cell epitopes of hepatitis B surface antigen (HBsAg) allow the resulting S-mutants to propagate even in the presence of neutralizing anti-HBs antibodies and the HBV-specific T-cell immune response. AIM: To study the antiviral effect of Pegylated-interferon (Peg-IFN) in a patient with chronic hepatitis B carrying unusual S-(and P-) mutants in the presence of anti-HBs antibodies. PATIENTS, METHODS AND RESULTS: We report on a 43-year-old male chronically infected with a genotype A HBV strain, with cocirculation of both HBsAg and anti-HBs antibodies, who received treatment with 120 mug of Peg-IFN for 24 weeks. HBeAg seroconversion and clearance of both HBV DNA by polymerase chain reaction and HBsAg were successfully achieved. Improved histology was observed in a biopsy performed 44 weeks after Peg-IFN therapy was completed. It seems plausible that the ascribed genotype A could have contributed to the effective response to Peg-IFN, even though the treatment was provided only throughout a 24-week period. CONCLUSION: To our knowledge, this is the first report regarding the successful result obtained by using Peg-IFN as a treatment for a chronically HBV-infected patient carrying HBsAg immune escape mutants.

Detection of hepatitis B virus (HBV) genotype E carried--even in the presence of high titers of anti-HBs antibodies--by an Argentinean patient of African descent who had received vaccination against HBV.

Genotype E hepatitis B virus (HBV) was detected in two Argentine sisters exhibiting an African mitochondrial lineage. One of them (who had been vaccinated against HBV) exhibited anti-HBs cocirculating antibodies without HBsAg escape mutants, while her unvaccinated sister showed a D144A HBsAg escape mutant without anti-HBs antibodies. Both sisters carried an unusual L209V substitution within HBsAg.

Unusual naturally occurring humoral and cellular mutated epitopes of hepatitis B virus in a chronically infected argentine patient with anti-HBs antibodies.

Serum hepatitis B virus (HBV) DNA was extracted from a chronically infected patient with cocirculation of hepatitis B surface antigen (HBsAg) and anti-HBs antibodies. Direct PCR and clone-derived sequences of the S and overlapped P genes were obtained. DNA sequences and phylogenetic analysis ascribed this isolate to genotype A (serotype adw2). Five of six HBV DNA clones exhibited point mutations inside and outside the major hydrophilic region, while the sixth clone exhibited a genotype A "wild-type" amino acid sequence. Observed replacements included both humoral and/or cellular (major histocompatibility complex class I [MHC-I] and MHC-II) HBV mutated epitopes, such as S45A, P46H, L49H, C107R, T125A, M133K, I152F, P153T, T161S, G185E, A194T, G202R, and I213L. None of these mutants were individually present within a given clone. The I213L replacement was the only one observed in the five clones carrying nonsynonymous mutations in the S gene. Some of the amino acid substitutions are reportedly known to be responsible for the emergence of immune escape mutants. C107R replacement prevents disulfide bonding, thus disrupting the first loop of the HBsAg. Circulation of some of these mutants may represent a potential risk for the community, since neither current hepatitis B vaccines nor hyperimmune hepatitis B immune globulin are effectively prevent the liver disease thereto associated. Moreover, some of the recorded HBsAg variants may influence the accuracy of the results obtained with currently used diagnostic tests.

In vitro detection of dissimilar amounts of hepatitis C virus (HCV) subtype-specific RNA genomes in mixes prepared from sera of persons infected with a single HCV genotype.

The level of in vitro detection of viral genomes in mixes with two different hepatitis C virus (HCV) subtypes was investigated by artificially mixing previously measured subtype-specific HCV RNA genomes. The RNAs in these mixtures were reverse transcribed and then PCR amplified by using two sets of primers corresponding to the 5' untranslated region and digested with endonucleases to analyze the restriction fragment length polymorphism patterns. This approach facilitated detection of a wider range of type-specific HCV genomes than originally described, beyond equimolar concentrations of contributing HCV subtypes. Moreover, by using computerized image analysis, this study also demonstrated that the true contribution of each virus type-and consequently of mixed infections-may be underestimated when only visual observation is carried out. These results may be useful for comparing data obtained from this and other currently used methodologies.

Estudio de factores genético-moleculares del virus de la hepatitis C asociados a la actividad de telomerasa hepática en el hepatocarcinoma / Study of genetic-molecular factors of the hepatitis C virus associated to the hepatic telomerase activity in hepatocarcinoma

El virus de la hepatitis C (HCV) constituye en el mundo el agente etiológico asociado a la mayor incidencia de hepatitis crónica que puede conducir la cirrosis hepática y, eventualmente al hepatocarcinoma. Más de 300 millones de personas padecen infección crónica por este virus, representando una causa significativa de morbi-mortalidad, frente a la cual urgen medidas de control efectivas. Originalmente, la magnitud de la heterogeneidad genética del virus no fue claramente advertida. Sin embargo, esta característica irrumpió con sus inherentes implicancias en la patogenia, el diagnóstico, la terapéutica y la inmunoprofilaxis. En cada paciente infectado, el HCV circula como una población heterogénea de variantes virales genéticamente relacionadas que se denominan cuasiespecies. Las mismas se exhiben dinámicas en el curso de la infección cambiando en forma progresiva, alentando una estrategia capaz de evadir los mecanismos de inmunidad humoral y celular desarrollados por el hospedador, y consecuentemente, estableciendo una infección persistente. Existen múltiples evidencias que demuestran que la caracterización genómica de HCV es relevante desde el punto de vista clínico. Importantes diferencias han surgido respecto del curso natural y la severidad de la infección, la evolución post-transplante hepático, el diagnóstico molecular e inmunoserológico, la vía de infección y aún la eficacia de la terapéutica con interferón alfa. El desarrollo de herramientas terapéuticas y profilácticas capaces de limitar la infección por HCV, debe estar sustentado por minuciosos estudios que consideren la heterogeneidad genética exhibida por este agente. En diversas neoplasias -incluídos los hepatocarcinomas (HCC)- se ha documentado la actividad de la oncoenzima telomerasa. Esta ribonucleoproteína con actividad telómero. La hipótesis de la senescencia celular asume que el acortamiento progresivo del telómero de células somáticas de organismos multicelulares, genera una señal que las inhabilita para el ingreso a sucesivos ciclos celulares. De este modo, la pérdida del telómero está asociada in vivo con el envejecimiento de modo tal que con posterioridad a un cierto número de duplicaciones, la célula detendrá su división, entrando en senescencia. Diferencialmente, en las células inmortalizadas, hematopoyéticas y reproductivas, la longitud del telómero está estabilizada, por la actividad de la telomerasa...(TRUNCADO)(AU)

Estudio de factores genético-moleculares del virus de la hepatitis C asociados a la actividad de telomerasa hepática en el hepatocarcinoma / Study of genetic-molecular factors of the hepatitis C virus associated to the hepatic telomerase activity in hepatocarcinoma

El virus de la hepatitis C (HCV) constituye en el mundo el agente etiológico asociado a la mayor incidencia de hepatitis crónica que puede conducir la cirrosis hepática y, eventualmente al hepatocarcinoma. Más de 300 millones de personas padecen infección crónica por este virus, representando una causa significativa de morbi-mortalidad, frente a la cual urgen medidas de control efectivas. Originalmente, la magnitud de la heterogeneidad genética del virus no fue claramente advertida. Sin embargo, esta característica irrumpió con sus inherentes implicancias en la patogenia, el diagnóstico, la terapéutica y la inmunoprofilaxis. En cada paciente infectado, el HCV circula como una población heterogénea de variantes virales genéticamente relacionadas que se denominan cuasiespecies. Las mismas se exhiben dinámicas en el curso de la infección cambiando en forma progresiva, alentando una estrategia capaz de evadir los mecanismos de inmunidad humoral y celular desarrollados por el hospedador, y consecuentemente, estableciendo una infección persistente. Existen múltiples evidencias que demuestran que la caracterización genómica de HCV es relevante desde el punto de vista clínico. Importantes diferencias han surgido respecto del curso natural y la severidad de la infección, la evolución post-transplante hepático, el diagnóstico molecular e inmunoserológico, la vía de infección y aún la eficacia de la terapéutica con interferón alfa. El desarrollo de herramientas terapéuticas y profilácticas capaces de limitar la infección por HCV, debe estar sustentado por minuciosos estudios que consideren la heterogeneidad genética exhibida por este agente. En diversas neoplasias -incluídos los hepatocarcinomas (HCC)- se ha documentado la actividad de la oncoenzima telomerasa. Esta ribonucleoproteína con actividad telómero. La hipótesis de la senescencia celular asume que el acortamiento progresivo del telómero de células somáticas de organismos multicelulares, genera una señal que las inhabilita para el ingreso a sucesivos ciclos celulares. De este modo, la pérdida del telómero está asociada in vivo con el envejecimiento de modo tal que con posterioridad a un cierto número de duplicaciones, la célula detendrá su división, entrando en senescencia. Diferencialmente, en las células inmortalizadas, hematopoyéticas y reproductivas, la longitud del telómero está estabilizada, por la actividad de la telomerasa...(TRUNCADO)

Genomic and phylogenetic analysis of hapatitis C virus strains from Argentina

HCV genomic characterization was performed by nucleotide sequence analysis (n=50) combined with restriction fragment length polymorphism (RFLP) of the 5'UTR region in 82 isolates coresponding to different Argentine groups. Genotype 1 was detected in 70.7 percent of the samples (58 out of 82), genotype 2 in 21.9 percent (18 of 82) and genotypes 3 in the remaining 6 sera (7.3 percent). HCV ib subtype contributed with 35.3 percent to the whole population studied (29 of 82) and was detected in 6 out of 21 sporadic cases. Besides their epidemiological significance, these results should be taken into account when future vaccines are considered on the basis of geographical HCV genotypic prevalence.

Bases genómicas y filogenéticas para la propuesta de un nuevo método de caracterización molecular del virus de la hepatitis G / Genomic and phylogenetic bases for the proposal of a new method of molecular characterization of the hepatitis G virus

El agente GBV-C / virus de hepatitis G (HGV) ha sido identificado como infeccioso para el ser humano, aunque su potencial participación como virus patógeno es aún motivo de controversia. Basándose en estudios clínicos y epidemiológicos en pacientes politransfundidos, receptores de hemoderivados de este agente por vía parenteral. La clasificación taxonómica de GBV-C/HGV como miembro de la familia Flaviviridae, lo relaciona, aunque en forma distante, con el virus de hepatitis C (HCV), demostrándose similitudes en la organización genómica y en el mecanismo propuesto de replicación viral. En ésta se enfatiza la participación de una RNA polimerasa viral RNA dependiente carente de lectura de prueba (actividad de proofreading) lo cual constituye el principal sustento de la heterogeneidad nucleotídica observada entre los aislamietos. Hasta el presente, se ha demostrado la existencia de tres genotipos mediante secuenciación nucleotídica (a partir del DNA copia obtenido por retrotranscripción [RT] y amplificación por PCR del RNA viral) o a través del análisis del polimorfismo de la longitud de los fragmentos de restricción (RFLP) mediante uso de endonucleasas sobre amplímeros obtenidos por PCR. Existe una asociación entre el genotipo involucrado y el origen geográfico de los mismos, según se desprende de estudios de filogenia entre los aislamientos caracterizados del virus... (TRUNCADO)(AU)

Genomic and phylogenetic analysis of hapatitis C virus strains from Argentina

HCV genomic characterization was performed by nucleotide sequence analysis (n=50) combined with restriction fragment length polymorphism (RFLP) of the 5UTR region in 82 isolates coresponding to different Argentine groups. Genotype 1 was detected in 70.7 percent of the samples (58 out of 82), genotype 2 in 21.9 percent (18 of 82) and genotypes 3 in the remaining 6 sera (7.3 percent). HCV ib subtype contributed with 35.3 percent to the whole population studied (29 of 82) and was detected in 6 out of 21 sporadic cases. Besides their epidemiological significance, these results should be taken into account when future vaccines are considered on the basis of geographical HCV genotypic prevalence. (AU)

Bases genómicas y filogenéticas para la propuesta de un nuevo método de caracterización molecular del virus de la hepatitis G / Genomic and phylogenetic bases for the proposal of a new method of molecular characterization of the hepatitis G virus

El agente GBV-C / virus de hepatitis G (HGV) ha sido identificado como infeccioso para el ser humano, aunque su potencial participación como virus patógeno es aún motivo de controversia. Basándose en estudios clínicos y epidemiológicos en pacientes politransfundidos, receptores de hemoderivados de este agente por vía parenteral. La clasificación taxonómica de GBV-C/HGV como miembro de la familia Flaviviridae, lo relaciona, aunque en forma distante, con el virus de hepatitis C (HCV), demostrándose similitudes en la organización genómica y en el mecanismo propuesto de replicación viral. En ésta se enfatiza la participación de una RNA polimerasa viral RNA dependiente carente de lectura de prueba (actividad de proofreading) lo cual constituye el principal sustento de la heterogeneidad nucleotídica observada entre los aislamietos. Hasta el presente, se ha demostrado la existencia de tres genotipos mediante secuenciación nucleotídica (a partir del DNA copia obtenido por retrotranscripción [RT] y amplificación por PCR del RNA viral) o a través del análisis del polimorfismo de la longitud de los fragmentos de restricción (RFLP) mediante uso de endonucleasas sobre amplímeros obtenidos por PCR. Existe una asociación entre el genotipo involucrado y el origen geográfico de los mismos, según se desprende de estudios de filogenia entre los aislamientos caracterizados del virus... (TRUNCADO)

A proposed rapid method for genomic characterization of GBV-C/hepatitis G virus (HGV)

GBV-C/Hepatitis G virus (HGV) has been identified as an infectious agent for humans although its potential involvement as a pathogenic virus is still controversial. Hitherto, 3 genotypes have been identified worldwide by c-DNA sequencing. This method allows genomic viral RNA clustering according to the geographical source of the strains, but its potential value in type- (or even strain-) specific pathogenesis has only started to be explored. Since this method requires highly specialized laboratories and is rather expensive, we propose a rapid method based on differential restriction fragment lenght polymorphism (RFLP) of 5'NCR amplicons. Using Hinf I, Dra I and Mae II endonucleases, it is possible to obtain different restriction patterns to discriminate among 1a, 1b, 2a, 2b and 3 subtypes/types. This methodology could be useful for large scale molecular epidemiology as well as for studies on viral pathogenesis.