Search

Peripheral blood mononuclear cells (PBMC) from patients with ocular toxoplasmosis were challenged with total antigens from Toxoplasma gondii lysate (TATL) in a cytokine release assay (CRA), run during the inactive period of the disease. Increased interferon gamma (IFN-γ) levels were detected after PBMC stimulation with either ME49 reference strain ( P = 0.0015) or local TgCkAr-11-9 isolate ( P = 0.0012), as compared with those recorded under basal conditions. TATL from TgCkAr11-9 isolate induced a higher release of IFN-γ than ME49 strain in CRA from all tested patients ( P = 0.02). The median value of IFN-γ release on TgCkAr-11-9 stimulation (26.03 pg/mL) allowed the classification of patients into high- or low-/non-IFN-γ releasers. Clinical correlations were established with both groups. The results obtained in this study suggest the need to include local strains when performing CRA with TATL.

Introduction: Hepatocellular carcinoma is the most common liver malignancy and the third leading cause of cancer death worldwide. One crucial limitation in the pharmacotherapy for this tumour is its chemotherapy-resistant nature produced by the overexpression of several members of the ATP-binding cassette protein family that efflux drugs out of cells, as observed with the breast cancer resistant protein (BCRP)., Objectives: This study aimed to assess the ability of Pluronic ® F127 to reverse the multidrug resistance phenotype in two human hepatocellular cell lines., Methods: PLC/PRF/5 and SKHep1 cells were exposed to Pluronic ® F127 at several concentrations. The effect of F127 on BCRP expression (mRNA and protein), mitochondrial transmembrane potential and cell hypodiploidy was assessed. Finally, the effect of this copolymer on cytotoxicity of doxorubicin in both hepatoma cell lines was investigated, as expressed by its reverse resistance index., Key Findings: It was demonstrated that F127 in both cell lines contributes to chemosensitization, as shown by BCRP down-regulation, an altered mitochondrial transmembrane potential and hypodiploidy and reverse resistance index values. A remarkable dependence of these effects significantly correlated with the copolymer concentration., Conclusions: These findings further uncover the potential usefulness of this copolymer as multidrug resistance reversal agent, increasing the efficacy of cancer therapies., (© 2019 Royal Pharmaceutical Society.)

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 association with hepatitis B virus (HBV), hepatitis delta virus (HDV) is a subviral agent that may promote severe acute and chronic forms of liver disease. Based on the percentage of nucleotide identity of the genome, HDV was initially classified into three genotypes. However, since 2006, the original classification has been further expanded into eight clades/genotypes. The intergenotype divergence may be as high as 35%-40% over the entire RNA genome, whereas sequence heterogeneity among the isolates of a given genotype is <20%; furthermore, HDV recombinants have been clearly demonstrated. The genetic diversity of HDV is related to the geographic origin of the isolates. This study shows the first comprehensive bioinformatic analysis of the complete available set of HDV sequences, using both nucleotide and protein phylogenies (based on an evolutionary model selection, gamma distribution estimation, tree inference and phylogenetic distance estimation), protein composition analysis and comparison (based on the presence of invariant residues, molecular signatures, amino acid frequencies and mono- and di-amino acid compositional distances), as well as amino acid changes in sequence evolution. Taking into account the congruent and consistent results of both nucleotide and amino acid analyses of GenBank available sequences (recorded as of January, 2017), we propose that the eight hepatitis D virus genotypes may be grouped into three large genogroups fully supported by their shared characteristics., (© 2018 John Wiley & Sons Ltd.)

Unlabelled: Recent data have shown that synthetic polymers and nanomaterials display phenotypic effects in cells and signal transduction mechanisms involved in inflammation, differentiation, proliferation, and apoptosis., Aim: This article aims to investigate the effect of poly(ethylene oxide)-poly(propylene oxide) (PEO-PPO) block copolymers with a wide range of biomedical and pharmaceutical applications on apoptosis and/or cell immortalization, by flow cytometry and multiplex RT-PCR for bax, bcl-2, and human telomerase reverse transcriptase (hTERT)., Results: PEO-PPO amphiphiles upregulated bax and hTERT and induced apoptosis of two human hepatoma cell lines., Conclusions: PEO-PPO block copolymers-considered safe for human use-can drastically alter gene expression profiles of genes related to apoptosis/cell proliferation., (© 2015 International Union of Biochemistry and Molecular Biology, Inc.)

Hepatitis C virus (HCV) is a small, enveloped RNA virus. The number of HCV-infected individuals worldwide is estimated to be approximately 200 million. The vast majority of HCV infections persist, with up to 80% of all cases leading to chronic hepatitis associated with liver fibrosis, cirrhosis, and hepatocellular carcinoma. The interaction between HCV and the host have a pivotal role in viral fitness, persistence, pathogenicity, and disease progression. The control of HCV infection requires both effective innate and adaptive immune responses. The HCV clearance during acute infection is associated with an early induction of the innate and a delayed initiation of the adaptive immune responses. However, in the vast majority of acute HCV infections, these responses are overcome and the virus persistence almost inexorably occurs. Recently, several host- and virus-related mechanisms responsible for the failure of both the innate and the adaptive immune responses have been recognized. Among the latter, the wide range of escape mutations to evade the specific-T-and B-cell responses as well as the T cell anergy and the CD8+ T cell exhaustion together with the interference with its function after prolonged virus exposure hold a pivotal role. Other HCV strategies include the modification or manipulation of molecules playing key roles in the induction of the interferon response and its induced effector proteins. In this review, we attempt to gain insights on the main T cell immune evasion strategies used by the virus in order to favor its persistence.

Hepatitis B virus (HBV) is a worldwide public health concern. The circulation of strains carrying mutations in the viral proteins implies both clinical and therapeutics challenges. HBV complete genomes (HBV-CGs) were reported from injecting drug users and HBV chronically infected patients from Argentina-including Amerindians-although no studies were conducted in blood donors. Here, we described HBV-CG sequences from the latter population. Some of the HBV sequences classified as B2 and C2 subgenotypes clustering together with Asian isolates, while others, such as D3, F1b, and F4, were homologous to European and Latin America sequences. New substitutions for all analyzed open reading frames and changes in the HBsAg hydrophobicity profiles were detected. Several HBV-CG subgenotypes are described for the first time in this population. Mutations observed in X, PreS, and P proteins have been associated with advanced liver disease, hepatocellular carcinoma, and/or natural resistance to nucleos(t)ide antiviral treatment. It deserves to be highlighted that these substitutions were detected in a population without epidemiological risk factors for viral infection, and most importantly, without any previous antiviral treatment (natural resistance). Regarding the remaining mutations, further research is warranted in order to determine their clinical and therapeutics relevance.

In order to determine the human pegivirus (HPgV) genotypic diversity in Argentina taking into account the potential contribution of human migration from neighboring countries, samples from 130 Argentine injecting drug users, 116 Argentine- and 50 immigrant-pregnant women were analyzed. HPgV RNA prevalence among human immunodeficiency virus (HIV)-positive injecting drug users was similar to HIV-positive pregnant women, as was the case when comparing HIV-negative injecting drug users and HIV-negative pregnant women (P > 0.05). HPgV genotype 2 (HPgV/2) was prevalent among both Argentine injecting drug users and pregnant women, in contrast to HPgV/3 observed among pregnant women from Latin American countries with predominant indigenous populations and who had experienced their initial sexual intercourses--and possibly their source of infection--in those countries (P < 0.01). In addition, HPgV vertical and horizontal transmission was proven by molecular analysis of E2 gene and construction of identity matrixes with epidemiologically non-related isolates. This study shows that human migration from neighboring Latin American countries with predominant indigenous populations might contribute to HPgV/3 circulation in Argentina., (© 2014 Wiley Periodicals, Inc.)

In Argentina, current procedures to ensure the safety of the blood supply for transfusion include the serologic detection of specific blood-borne infections. The aim of this study was to evaluate the prevalence and the genetic diversity of hepatitis B virus (HBV) and hepatitis D virus (HDV) in blood donor populations from two distantly located Argentine regions. Data from 56,983 blood donations from the Favaloro Foundation, in the city of Buenos Aires (Central Region), and the Central Blood Bank of Misiones Province (Northeast Region) were analyzed. Samples that were reactive for HBsAg were analyzed for HBV-DNA characterization and HDV serological and molecular analysis. The HBV prevalence was 0.12 % for HBsAg and 1.68 % for anti-HBc antibodies in Buenos Aires, and 0.73 % and 8.55 %, respectively, in Misiones. Seventy-seven HBsAg-reactive samples were analyzed by polymerase chain reaction for HBV-DNA. Subgenotypes A2, B2, C2, F1b and F4 (Buenos Aires) and F1b and D3 (Misiones) were detected. Several mutations within the major hydrophilic region of HBsAg, the reverse transcriptase, the basal core promoter, and the precore/core were detected. HDV genotype 1 was identified in Buenos Aires. This study confirms the circulation of several HBV subgenotypes, as well as known and newly identified variants, and the presence of HDV1 in this population. A thorough investigation has to be carried out to evaluate the clinical importance of some of the documented mutations as well as those detected in the HDV1 case.

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.

Aim: To study the subtype prevalence and the phylogenetic relatedness of hepatitis C virus (HCV) sequences obtained from the Argentine general population, a large cohort of individuals was analyzed., Methods: Healthy Argentinian volunteers (n = 6251) from 12 provinces representing all geographical regions of the country were studied. All parents or legal guardians of individuals younger than 18 years provided informed written consent for participation. The corresponding written permission from all municipal authorities was obtained from each city or town where subjects were to be included. HCV RNA reverse transcription-polymerase chain reaction products were sequenced and phylogenetically analyzed. The 5' untranslated region (5'UTR) was used for RNA detection and initial genotype classification. The NS5B polymerase region, encompassing nt 8262-8610, was used for subtyping., Results: An unexpectedly low prevalence of HCV infection in the general population (0.32%) was observed. Our data contrasted with previous studies that reported rates ranging from 1.5% to 2.5%, mainly performed in selected populations of blood donors or vulnerable groups. The latter values are in keeping with the prevalence reported by the 2007 Argentinian HCV Consensus (approximately 2%). HCV subtypes were distributed as follows: 1a (25%), 1b (25%), 2c (25%), 3a (5%), and 2j (5%). Two isolates ascribed either to genotype 1 (5%) or to genotype 3 (5%) by 5'UTR phylogenetic analysis could not be subtyped. Subtype 1a sequences comprised a highly homogeneous population and clustered with United States sequences. Genotype 1b sequences represented a heterogeneous population, suggesting that this genotype might have been introduced from different sources. Most subtype 2c sequences clustered close to the 2c reported from Italy and Southern France., Conclusion: HCV has a low prevalence of 0.32% in the studied general population of Argentina. The pattern of HCV introduction and transmission in Argentina appears to be a consequence of multiple events and different for each subtype.

Multidrug resistance associated with the overexpression of ATP-dependent binding cassette (ABC) proteins is widely accepted as an important cause of treatment failure in patients with neoplastic or infectious diseases. Some of them play also a pivotal role in detoxification processes. Herein, we investigated the effect of hepatitis C virus (HCV) replication and nonstructural 5A (NS5A) protein on the expression and functional activity of two ABC transport proteins: MDR1 and BCRP. RT-quantitative real-time polymerase chain reaction (qPCR) was carried out for mdr1 and bcrp mRNAs in both Huh7 cells expressing NS5A and Huh7.5 cells containing either full-length- or subgenomic-HCV replicon systems. The functional activity of these pumps was studied by performing a dye efflux assay with DiOC2 and Rhodamine 123. A dose-dependent down-regulation of mdr1 expression was documented in Huh7 cells expressing the NS5A protein, as well as in both replicon systems. In contrast, a significant increase of bcrp expression in both systems was recorded, which were in full agreement with the dye efflux assay results. These results warrant further in vivo studies in HCV patients with cholestasis and/or patients that are refractive to the pharmacotherapy due to the activity of these pumps., (© 2012 Blackwell Publishing Ltd.)

Prevalence rates of hepatocellular carcinoma (HCC)-associated hepatitis B virus (HBV) pre-S mutants among most genotypes are still lacking. In this study, viral (sub)genotypes of 70 Argentine nucleotide sequences (33 newly obtained) were determined by phylogenetic analysis, and the presence of such mutants was assessed in the American continent for the first time. Nucleotide substitutions of the pre-S2 start codon were observed in 10% of the HBV/A2 sequences. Ten per cent of the HBV/A2 and 12.5% of the HBV/F1b - but none of HBV/F4 - exhibited a deletion in the pre-S1/pre-S2 region. The contribution of these variants to liver cirrhosis (LC) and/or HCC development among HBV/F and HBV/A isolates deserves further prospective clinical studies., (© 2012 Blackwell Publishing Ltd.)

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-β-cyclodextrin (HPβ-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 HPβ-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., (Copyright © 2012 Elsevier B.V. All rights reserved.)

PCR detection of viral genomes has provided new insights into viral diagnosis. Nowadays, it is the most frequently used nucleic acid testing (qualitative and quantitative) technique. The aim of this study was to analyse the major circulating hepatitis B virus (HBV) variants PCR-amplified by three sets of primers in a patient infected with genotype E. The HBV S/Pol overlapping genomic region was amplified from the serum of an infected child using three primer sets previously described. Sequence analysis corresponding to the HBV S/Pol region revealed the presence of different viral populations depending on the set of primers used. D144A S-escape mutant was detected with two of the primer sets, while the rtL217R mutant within the Pol - conferring resistance to Adefovir - could be picked up with a different pair of primer sets. This study undoubtedly implies that the description of viral polymorphisms should be stated together with the sequence of the primers used for PCR amplification when studies of escape and/or antiviral-resistant HBV mutants are carried out., (© 2012 Blackwell Publishing Ltd.)

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., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Genomic heterogeneity and quasispecies composition of GB virus C (GBV-C) within plasma and lymphocyte subsets in a naturally infected blood donor were investigated. For this purpose, fragments from the 5' untranslated region (5' UTR) and the E2 gene recovered from plasma, B and T lymphocytes, were cloned and sequenced. A total of 63 clones was analysed: 95.2 % of them (n=60) - obtained from plasma and cells - were assigned to genotype 2b, while only three derived from plasma corresponded to genotyope 3. The G215A transition within this region was present in 90.9 % of the clones from B lymphocytes, but absent in the remaining cell compartments (P<0.01). Apparently, most of the circulating GBV-C quasispecies in this blood donor were related to the viral population infecting CD8(+) T cells, and B cells to a lesser extent. This is the first report showing the quasispecies nature of GBV-C in lymphocyte subsets within peripheral blood mononuclear cells.

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., (2010 Elsevier B.V. All rights reserved.)

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.

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.

Previous studies have revealed that hepatitis B virus (HBV)/D and HBV/F predominate among blood donors from Buenos Aires, Argentina. In the present study, blood samples from two high-risk groups were analysed: 160 corresponding to street- and hospital-recruited injecting drug users [81.2% showing the 'anti-hepatitis B core antigen (anti-HBc) only' serological pattern] and 20 to hepatitis B surface antigen (HBsAg)(+)/anti-HBc(+) men who have sex with men. HBV genotypes were assigned by polymerase chain reaction amplification followed by restriction fragment length polymorphism and confirmed by nucleotide sequencing of two different coding regions. HBV DNA was detected in 27 injecting drug users (16.9%, occult infection prevalence: 7.7%), and 14 men who have sex with men (70%). HBV/A prevailed among injecting drug users (81.8%) while HBV/F was predominant among men who have sex with men (57.1%). The high predominance of HBV/A among injecting drug users is in sharp contrast to its low prevalence among blood donors (P = 0.0006) and men who have sex with men (P = 0.0137). Interestingly, all HBV/A S gene sequences obtained from street-recruited injecting drug users encoded the rare serotype ayw1 and failed to cluster within any of the known A subgenotypes. Moreover, one of the HBV strains from a hospital-recruited injecting drug user was fully sequenced and found to be the first completely characterized D/A recombinant genome from the American continent. Data suggest that two simultaneous and independent HBV epidemics took place in Buenos Aires: one spreading among injecting drug users and another one sexually transmitted among the homosexual and heterosexual population.

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.

The in vivo evolution of genotype F HBV variants was recorded in a chronically infected patient throughout a 3-year observation period. Fluctuating levels of HBs Ag and anti-HBs antibodies were recorded, both of them cocirculating in peripheral blood samples at given times. Fifty S gene derived clones were sequenced and phylogenetically analyzed. As expected, some amino acid replacements within the S ORF were also observed within the P ORF while others were silent for the former. Such change was statistically significant for both S and P overlapping genes, which clearly indicates the appearance of a positive selection pressure. Supporting this notion, amino acid replacements were documented at both B and T cell epitopes in samples from 1997 and 1998. Several mutations were documented within and outside the "a" determinant in the major hydrophilic region. Such substitutions might have resulted from the attempt of HBV to evade both humoral and/or cellular immune response. To the best of our knowledge this unusual profile of HBV variants in presence of usually "neutralizing" anti-HBs antibodies was examined in vivo for the first time.

The intrahost hepatitis B virus (HBV) genomic evolution process of an HBe antigen (HBeAg)-negative chronic HBV patient (designated RI) was studied. Two nearly full-length direct sequences obtained in 1995 (RI95) and 1998 (RI98) showed: (a) a mutation rate of 2.7x10(-3) nucleotides per site per year; (b) nucleotide changes mainly located at single coding regions (P=0.002); (c) mixed populations; and (d) a predominance of non-synonymous substitutions (P=0.0036). Population heterogeneity was assessed by cloning and sequencing of a fragment spanning nearly half the genome. Two-thirds of the analysed clones exhibited long nucleotide deletions. Pairwise genetic diversity revealed that diversity was higher for RI95 than for RI98 cloned sequences. In conclusion, a highly heterogeneous genomic population circulated within patient RI, which might support the persistence of HBV. Finally, the structure of the deletant genomes suggests that they might serve as intermediates for integration to the host-cell genome.

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.

Although GB virus C (GBV-C) hepatocyte pathogenicity is still controversial, it appears that at least some strains of this virus are lymphotropic. During the past few years, several reports have documented an apparently beneficial role played by GBV-C in the course of HIV-1 infection. At present, a commercial kit for GBV-C RNA quantitation is not available. In this study, a competitive RT-PCR method for GBV-C in serum samples is described. The sensitivity of the assay proved to be 10(4) and 10(3) genomic equivalents for positive and negative sense RNAs, respectively. This method will discriminate specifically between positive and negative strand RNAs with a discrimination index of at least five log10. Out of 60 samples from different hematological disorders (n = 49), HIV-1 positive patients (n = 7), and blood donors (n = 4), 10 proved to be GBV-C RNA positive. Viral load ranged from 1.1 x 10(7) to 2.34 x 10(8) genomic equivalents/ml. Such values correlated linearly (r = 0.986) with those obtained by a 10-fold serial dilution method. In studies exploring the GBV-C pathogenicity, the measurement of viral load may contribute to understand the possible mechanisms involved.

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.

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.

An adult male farmer with chronic active hepatitis and cirrhosis despite previous circulating anti-HBs antibodies was studied. No markers of other hepatotropic viral infection were observed. HBV DNA was detected in serum by PCR and was characterized further by restriction fragment length polymorphism (RFLP) and sequencing of cloned PCR products derived from the S gene. The HBV DNA was ascribed to genotype F, and single-strand conformational polymorphism (SSCP) demonstrated the co-circulation of multiple quasispecies. Some of the variants exhibited changes located within the neutralizing "a" determinant, located between amino acids 124-147 of the S protein. Within this region, two clones showed either C124R or C124Y mutations. Other mutations were Q129R, C138R, C139R, and S140T (one clone each). Outside the "a" determinant several substitutions were documented. The high degree of the quasispecies variability was probably linked to the severity of the infection. Most members of the patient's family were infected with HBV, all with genotype F., (Copyright 2003 Wiley-Liss, Inc.)

To investigate hepatitis C virus (HCV) and GBV-C/hepatitis G virus (HGV) genotype prevalence among HCV-infected porphyria cutanea tarda (PCT) patients, 19 HCV-infected patients with associated PCT were studied. A control group of 53 age-matched HCV-infected patients without associated PCT was selected. Eighteen of the 19 serologically positive HCV-PCT patients showed HCV RNA in serum. Genotype 1b was the most prevalent among both HCV-PCT patients (72.2%; 13/18) and age-matched HCV controls (50.9%; 27/53). Such different genotypic prevalence failed to reach statistical significance (chi(2) with Yates' correction, p = 0.19). The single HCV-PCT patient without detectable HCV RNA was also infected with genogroup 3 GBV-C/HGV. This GBV-C/HGV RNA prevalence (5.3%) among HCV-PCT patients is not statistically different from that observed among Argentine blood donors (5.5%; 11/200). To our knowledge, these results show for the first time the molecular epidemiology of both HCV and GBV-C/HGV associated to PCT in America., (Copyright 2001 S. Karger AG, Basel)

Typing of hepatitis C virus (HCV) isolates from Argentine patients was performed by using different methodologies in a population of 243 patients. HCV subtype was assigned based upon restriction fragment length polymorphism (RFLP). HCV RNA genomes obtained from serum samples were classified as belonging to clade 1 (53.5%), 2 (23. 0%), or 3 (8.6%); 14.8% of samples showed HCV mixed infections, more frequently implying different subtypes within the same clade. In addition to RFLP typing, phylogenetic relatedness among sequences from both 5' untranslated region (n = 50) and nonstructural 5B coding region (n = 15) was established.

GBV-C/HGV RNA was investigated in serum samples from 70 HIV(+) intravenous drug users (IVDU), as well as from 200 blood donors from Buenos Aires, Argentina. Viral RNA was demonstrated in 21 IVDU by reverse transcription-nested PCR of the 5' UTR. c-DNA amplified products were analyzed and their sequences compared with those downloaded from GenBank. A phylogenetic tree based on 171 sequences demonstrated the presence of three major genogroups, including two subgroups, within local samples, i.e. group 1 (n=1), 2a (n=11), 2b (n=4) and 3 (n=5). These results agreed entirely with those obtained by a novel RFLP (J. Clin. Microbiol. 37, 1340-1347, 1999) of the same 5' UTR amplicons. As expected, GBV-C/HGV RNA prevalence was significantly higher among IVDU than among blood donors (P<0.0001), although within the latter group an unexpectedly high rate was also detected, since 11 of 200 sera (5.5%) proved positive. These viral isolates were ascribed either to subgroup 2a (n=5), subgroup 2b (n=5) or genogroup 3 (n=1). Briefly, this partial view of GBV-C/HGV molecular epidemiology in Argentina shows: (i) different rates of GBV-C/HGV infection within both IVDU and blood donors; (ii) a high prevalence of viral RNA among blood donors; and (iii) a predominant circulation of genogroup 2, with minor contribution of groups 3 and 1.

A phylogenetic tree based on 150 5' untranslated region sequences deposited in GenBank database allowed segregation of the sequences into three major groups, including two subgroups, i.e., 1, 2a, 2b, and 3, supported by bootstrap analysis. Restriction site analysis of these sequences predicted that HinfI and either AatII or AciI could be used for genomic typing with 99.4% accuracy. cDNA sequencing and subsequent alignment of 21 Argentine GB virus C/hepatitis G virus strains confirmed restriction fragment length polymorphism patterns theoretically predicted. This method may be useful for a rapid screening of samples when either epidemiological or transmission studies of this agent are carried out.

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 length 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.

Thirty-three Argentinian patients infected with hepatitis C virus (HCV) were studied for viral genotyping. The patients included 10 hemophiliac and 4 polytransfused children and 19 adults: 3 polytransfused, 7 dialyzed and 9 sporadic cases. Core-based genotyping permitted the classification of 31 samples. Genotypes II, I and V were the most frequent: 21 (63.6%), 16 (48.4%) and 10 (30.3%) of the 33 patients, respectively. Only one polytransfused patient carried genotype IV. Genotype II was detected in 7 out of 9 sporadic cases. Thirteen patients (39.3%) were coinfected with two genotypes, and 2 others were coinfected with three genotypes. The remaining 2 samples which could not be typed were characterized following the restriction fragment length polymorphism (RFLP) method, and were classified as type 1. One of these had two consecutive transitional mutations in the 5' untranslated region (5' UTR).

Guinea pig infection with a highly passaged XJ prototype strain of Junín virus by the intramuscular route (IM) was carried out in order to study viral tropism modification in this host. The neurotropism of this strain was demonstrated by viral isolation, meningitis, and by the presence of Junín antigens as shown by immunofluorescence. These events, not previously observed with the same lower-passaged strain, revealed the appearance of neurotropism after multiple passages in guinea pigs.

Four Cebus apella monkeys, to test a recently proposed model for testing neurovirulence of Junín virus (JV) strains, were intracerebrally infected with 10(5) LD50 of the XJ clone 3 strain of JV. There were no significant electrocardiographic abnormalities or gross lesions, but all infected monkeys exhibited a varying degree of histologic myocardial lesions including focal lymphoblastic infiltrates, vascular ruptures, and mild interstitial reactive change. One Cebus showed lymphocytic infiltrates in the caudal portion of the A-V node without specific cell involvement. These preliminary results demonstrate cardiac involvement in experimental infection of the Cebus monkey with Junín virus.

Guinea pigs infected by the peripheral route with the XJ pathogenic strain of Junin virus showed viscerotropism mainly in reticulo-phagocytic rich organs. By immunofluorescence, heavy infection of reticular-phagocytic cells was demonstrated, supporting the leading role of these cell types. Absence of neurotropism was demonstrated by the inability to recover infectious virus, as well as the absence of antigens, immunoglobulins, or 3rd component of complement deposits, in cells, vessels, or meninges. The correlation between infectivity and antigen expression observed in organs, and the absence of evidence of immunopathologic mechanisms, strongly suggest a direct viral effect in these experimental conditions. The results show that infection of guinea pigs by the peripheral route is an adequate model for human Argentine hemorrhagic fever with the exception of central nervous system involvement. Comparisons are made with infections produced in guinea pigs by attenuated strains, as well as with the disease in primates and humans.

In previous morphological studies on bone marrow of guinea pigs infected with Junin virus, the coexistence of viral particles, antigens and cytopathic effects were observed in megakaryocytes. In addition, bone marrow is one of the organs where highest virus titers were obtained. However, although other myeloid cells presented intense cytopathic effect, viral antigens were observed only in reticular cells and virus particles were seen sporadically associated with immature cells. This study was designed to investigate if bone marrow infetivity was due mainly to megakaryocyte infection. Megakaryocyte obtained from infected bone marrow were concentrated in albumin gradients by gravitational sedimentation. Enriched fractions were studied by immunofluorescense /IF) and virus titration. Results showed that fractions containing 8, 6.4 or 15 % of megakaryocytes had titers of 3.81, 4.23 and 2.7 LD50/mñ respectively. On the contrary in depleted fractions (0 % megakaryocytes), titers were 0,5 to 1 log lower (Fig. 1, Table 1). By IF, Junin antigens were located in the cytoplasm of 70 % of megakaryocytes and also in reticular cells, but not in other cell types. These results suggest that megakaryocytes suffer a productive infection that may well account for the thrombocytopenia observed in this experimental infection. However, the detection of infectivity in other cells, in absence of viral antigens and particles remains to be explained.

To assess the usefulness of the South American primate Cebus apella as a model for neurovirulence of Junín virus, eight monkeys were inoculated with 10(5) LD50 of the attenuated XJ-Clone 3 Junín virus strain by the intrathalamic route. After the second week, weight loss and polyadenopathies were observed in most animals, one-half of which had a transient leukothrombocytopenia. Moderate clinical central nervous system (CNS) involvement was present in four of eight monkeys, while the rest had only mild neurologic signs. All recovered except one, which developed a deep coma and was killed in a pre-mortem stage at 18 days post-infection (pi). Junín virus was isolated from the throat from five, from the blood from three, and from the brain from two monkeys. In the most severely ill animal, virus titers higher than viremia were detected in both inoculated and contralateral brain hemispheres, as well as in lung, lymph node, and small intestine. Junín antigens and "in vivo" bound immunoglobulins were detected by immunofluorescence (IF) in the brain of four animals at 18, 21, 40, and 155 days pi. Moderate lymphocytic parenchymal and meningeal infiltration were observed in the brain of four animals, and gliosis was also present in the most affected monkey. Although the clinical response to infection was not uniform, all infected monkeys developed high IF antibodies. Cebus apella cannot be used as a highly sensitive model for Argentine hemorrhagic fever (AHF). However, the results obtained show that the XJ-Clone 3 strain can replicate in the primate CNS and to induce lesions and immunoglobulin deposition. In addition, viral persistence is suggested by the late detection of viral antigens in brain at 40 and 155 days pi.

An association between viral antigens, cytopathic effect (CPE) and viral titers in blood and lymphoid tissues suggests a direct CPE of Junin virus on the lymphopoietic organs of guinea pigs infected with 10(3) 50% lethal doses of the XJ prototype strain. After seven days of infection, all lymphoreticular organs had infectivity titers higher than those for blood. Virus was recovered from bone marrow and lymph nodes at day 5 after infection; peak titers were obtained from bone marrow, spleen, and lymph nodes after day 10. Granular specific fluorescence was detected in the cytoplasm of reticular monocytes after day 7; megakaryocytes showed positive fluorescence, but specific staining of other lymphoid cells was not observed. Necrosis of bone marrow, lymph nodes, and spleen was observed after day 9. CPE consisted of overdevelopment of reticuloendoplasmic cisterne of reticulomonocytes and myeloblasts. Typical Junin virus particles were observed. Reticular cells were gradually destroyed, and simultaneous necrosis of surrounding lymphoid cells was observed.

The frequency of chromosomal aberrations in bone marrow cells of guinea pigs inoculated with the pathogenic XJ strain of Junin virus increased significantly at 6, 9, and 11 days postinoculation (p.i.). Animals inoculated with the attenuated XJ-clone 3 strain only showed significant increments of achromatic lesions (gaps) at 9 days p.i. Guinea pigs inoculated with the XJ-clone 3 strain and then treated with two doses of caffeine 24 and 12 h before killing at 9 days p.i. exhibited a significant increase of chromatid breaks and a parallel decrease of gaps. Because caffeine acts as an inhibitor of repair mechanisms of genetic damage, these results suggest a mutagenic effect of the attenuated strain.