Identification and evolutionary analysis of papillomavirus sequences in New World monkeys (genera Sapajus and Alouatta) from Argentina.

OBJECTIVE: In this study, we investigated the occurrence of papillomavirus (PV) infection in non-human primates (NHPs) in northeastern Argentina. We also explored their evolutionary history and evaluated the co-speciation hypothesis in the context of primate evolution. METHODS: We obtained DNA samples from 57 individuals belonging to wild and captive populations of Alouatta caraya, Sapajus nigritus, and Sapajus cay. We assessed PV infection by PCR amplification with the CUT primer system and sequencing of 337 bp (112 amino acids) of the L1 gene. The viral sequences were analyzed by phylogenetic and Bayesian coalescence methods to estimate the time to the most common recent ancestor (tMRCA) using BEAST, v1.4.8 software. We evaluated viral/host tree congruence with TreeMap v3.0. RESULTS: We identified two novel putative PV sequences of the genus Gammapapillomavirus in Sapajus spp. and Alouatta caraya (SPV1 and AcPV1, respectively). The tMRCA of SPV1 was estimated to be 11,941,682 years before present (ybp), and that of AcPV1 was 46,638,071 ybp, both before the coalescence times of their hosts (6.4 million years ago [MYA] and 6.8 MYA, respectively). Based on the comparison of primate and viral phylogenies, we found that the PV tree was no more congruent with the host tree than a random tree would be (P > 0.05), thus allowing us to reject the model of virus-host coevolution. CONCLUSION: This study presents the first evidence of PV infection in platyrrhine species from Argentina, expands the range of described hosts for these viruses, and suggests new scenarios for their origin and dispersal.

The RNA world of human ageing.

Ageing is one of the most complex processes in nature; how could we prevent the associated biological changes and chronic diseases that string along with this process, is a challenge in healthcare around the world. Recent advances in next-generation sequencing have reached a stage where it is possible to know from a specific tissue the most abundant transcripts, alternative splicing process and, non-coding RNA molecules (microRNA's, long non-coding RNA's, and circular RNAs). Moreover, our knowledge of several biological processes related to ageing such as senescence and autophagy have dramatically increased in the last years. In the present review, we attempt to summarise the latest scientific advances from the most critical studies performed in human clinical samples, specific to the RNA studies about ageing. Overall, human transcriptomics research indicates that although there are common alterations of the regular expression patterns of the energetic and oxidative metabolism, extracellular matrix regulation and inflammation pathways, ageing seems to be gender and tissue-specific in general. Additionally, there is an age-related implication in several numbers of impaired events on the normal alternative splicing process. On the other hand, there is a direct relation of several non-coding RNA molecules with age-related changes which indicates its possible use as biomarkers for diagnostics and therapeutically purposes. Together, these findings highlight the importance to continue focusing research on RNA studies to improve our knowledge in the pathophysiology of age-related diseases.

Prevalence of hepatitis C virus infection according to the year of birth: identification of risk groups.

Hepatitis C virus (HCV) screening according to the year of birth is recommended is some countries based on epidemiological data. The aim of this study was to analyze anti-HCV prevalence among people born between 1905 and 2015 in Argentina. Patients attending a tertiary care hospital in Buenos Aires, Argentina, from 2001 to 2015, who had a determination of anti-HCV, were included. Of 22,079 patients analyzed, 1,152 (5.2%; 95% confidence interval [CI]: 4.9%-5.5%) patients showed positive anti-HCV and 729 (3.3%; 95% CI: 3.1%-3.5%) patients showed detectable viremia. Three risk groups were identified (HCV prevalence): low-risk group-outpatient clinics/emergencies (2.8%); intermediate-risk group-in-patients (8%); and high-risk group-dialysis/transplants (27.2%). In the low-risk group, being born in 1973 or before was identified as a cut-off value for the risk of anti-HCV acquisition (area under the receiver-operator characteristic curve: 75.1 [95% asymptotic CI: 0.732-0.770; p < 0.001]). Ninety-one patients born after 1973 (0.8%) showed positive anti-HCV versus 457 individuals born in 1973 or before (5.8%), p < 0.001. In this group, positive anti-HCV was observed in 252 females (2.1%) and 296 males (4.1%), p < 0.001. In a multivariate analysis adjusted for gender, alanine-aminotransferase levels and HIV coinfection, being born in 1973 or before was independently identified as a risk for positive anti-HCV (adjusted odds ratio: 14.234 [95% CI: 9.993-20.277]; p < 0.001). People born in 1973 or before without other risk factors should be included in screening programs to link the highest possible number of HCV-infected patients to appropriate care and treatment.

Hepatitis B virus depicts a high degree of conservation during the immune-tolerant phase in familiarly transmitted chronic hepatitis B infection: deep-sequencing and phylogenetic analysis.

When intrafamilial transmission of hepatitis B virus (HBV) occurs, a virus with the same characteristics interacts with diverse hosts' immune systems and may thus result in different mutations to escape immune pressure. In this study, the HBV genomic characterization was assessed longitudinally after intrafamilial transmission using nucleotide sequence data of phylogenetic and mutational analyses, including those obtained by deep-sequencing for the first time. Furthermore, HBeAg-anti-HBe profile and variability of HBV core-derived epitopes were also evaluated. Strong evidence was obtained from intrafamilial transmission of HBV genotype D1 by phylogenetic inferences. HBV isolates exhibited high degree (~99%) of genomic conservation for almost 20 years, when patients were persistently HBeAg positive with normal amino transferase levels. This identity remained high among immune-tolerant siblings. In contrast, it diminished significantly (P = 0.02) when the mother cleared HBeAg (immune clearance phase). By deep-sequencing, the quantitative analysis of the dynamics of basal core promoter (BCP) (A1762T, G1764A; A1766C; T1773C; 8-bp deletion; and other) and precore (G1896A) variants among HBV isolates from family members exhibited differences during the follow-up. However, only those from the mother showed amino acid variations at core protein that would impair their MHC-II binding. Hence, when intrafamilial transmission occurs, HBV was highly conserved under the immune-tolerant phase, but it exhibited mutations more frequently during the immune clearance phase. The analysis of the HBV BCP and precore mutants after intrafamilial HBV transmission contributes to a better understanding of how they evolve over time.

TNF promoter SNP variation in Amerindians and white-admixed women from Misiones, Argentina.

The aim of this study is to describe genetic variation in the TNF promoter in the ethnically diverse population of Misiones, north-eastern Argentina. We analysed 210 women including 66 Amerindians of the Mbya-Guarani ethnic group and 144 white-admixed individuals from urban and rural areas of Misiones. Their DNA samples were surveyed for TNF polymorphisms -376 A/G, -308 A/G -244 A/G and -238 A/G by PCR amplification and direct sequencing and for the Amerindian marker -857 C/T by real-time PCR. Our main findings are as follows:(i) a distinctive pattern of Single Nucleotide Polymorphism (SNP) distribution among these groups, (ii) genetic differentiation between the Mbya-Guarani and the white-admixed populations (P < 0.05), (iii) lower gene diversity (~0.05) in Mbya-Guarani compared with the white-admixed group (~0.21); and (iv) linkage disequilibrium between the -376A and -238A SNPs in white-admixed populations. These data highlight the principal role of population history in establishing present-day genetic variation at the TNF locus and provide a framework for undertaking ethnographic and disease association studies in Misiones.

Comparative analysis of hepatitis B virus genotype a molecular evolution in patients infected with HBV and in patients co-infected with HBV and HIV.

HIV infection has a significant impact on the natural progression of liver disease caused by infection with hepatitis B virus (HBV), but its role in the molecular evolution of HBV is unknown. It is difficult to study the molecular evolution of HBV longitudinally considering its genomic complexity, which implies the analysis of paired samples. This study aimed to analyze the difference in the evolutionary dynamics of HBV among patients with HIV and uninfected individuals. In this study, 17 patients infected chronically with HBV were recruited, 9 of them were co-infected with HIV. Patients were HBe antigen-positive and infected with HBV genotype A. Paired plasma samples were collected from each patient 3 years apart, and they were compared subsequently to each other. The HBV phylogenetic inference among isolates from patients infected with HBV and co-infected with HBV and HIV tends to cluster separately. Likewise, when comparing the HBV evolutionary rate and genetic distances, values were higher in the former in both preC/C and S genomic regions. Intra-host analyses of HBV isolates revealed high diversity and complexity of quasispecies among patients infected with HBV exhibiting high numbers of viral variants and genetic distance. In summary, after studying the HBV molecular evolution among isolates ascribed to genotype A at inter- and intra-host levels, HBV exhibited low quasispecies complexity and diversity as well as low evolutionary rates in the presence of HIV co-infection, suggesting that the co-infection may have an impact on the HBV molecular evolution most likely from the weakened cellular immune response.

Intrafamilial transmission of hepatitis C virus in patients with severe haemophilia A.

The history behind the production of clotting factor concentrates produced differences in the prevalence of Hepatitis C Virus (HCV) and other blood-borne infections in haemophilic patients. Prevalence rates of HCV infection up to 100% were reported in patients treated with concentrates before 1985. Conversely, nowadays, viral inactivation and recombinant technologies have effectively prevented transfusion-transmitted viral pathogens. Recently, new HCV infections in three young brothers were observed. In the absence of any other risk of transmission, their HIV/HCV coinfected uncle, who was living in the same house, was subject to study. Plasma samples of the four relatives were investigated in order to test whether the infections have a common source. A phylogenetic approach using the most variable (E2) viral sequences was carried out using samples from the four family members. The HCV sequences from the study resulted highly related, being those obtained from the uncle the most ancestral ones. Because of the chronological order in which the infections occurred and the relatedness of the sequences, an infection from the uncle to his nephews is the most likely explanation. Special cares must be applied in the case of household contact among members of a family with inherited bleeding disorders.

Evolution of hepatitis C virus hypervariable region 1 in immunocompetent children born to HCV-infected mothers.

Hepatitis C virus (HCV) hypervariable region 1 (HVR1) is the most variable region of the viral genome and its heterogeneity reflects the virus-host interplay during chronicity. Paediatric HCV-infected patients develop liver disease with typical clinical features. Here, the evolution of HVR1 and its adjacent regions were ascertained in plasma samples of two HCV-positive children during a 5-year follow-up period. We report an almost complete conservation of the HVR1 amino acid sequence over time, with underlying nucleotide variability both within and outside HVR1, suggesting some kind of constraint on virus evolution, particularly within HVR1. Although overall d(N)/d(S) rates [rates of nonsynonymous nucleotide substitutions per nonsynonymous site (d(N)) and synonymous nucleotide substitutions per synonymous site (d(S))] were <1 in both patients, a high resolution analysis of selection pressures exerted at the codon level revealed few sites subject to selection and an absolute predominance of invariable positions within HVR1. The HVR1 amino acid sequences showed the antigenic properties expected for this region. Taken together, these data suggest peculiar evolutionary dynamics in our patients, which could be attributed to a mechanism of nucleotide invariability along with purifying selection operating on the HVR1. The lack of HVR1 variability may reflect the adaptation of the virus to a particular environment within each patient or a phenomenon of immune tolerance generated in these immunocompetent patients earlier in life.

Role of viral load in Hepatitis B virus evolution in persistently normal ALT chronically infected patients.

Chronic HBV infection has been associated with severe liver disease although most of them do not progress to this stage. Even though low replicative carriers form the largest group of HBV chronically infected patients, there is a paucity of longitudinal studies to evaluate the molecular evolution of the whole genome in this subset of patients. In this study, longitudinal samples from 10 patients with persistently normal ALT levels were collected. HBV full-length genome sequences were obtained from 3 samples per patient (baseline, 5 and 10-years of follow-up). Patients were grouped according to HBV-DNA level into <103 IU/ml (group A) or > 103 IU/ml (group B). The substitution rate was inversely related with HBV-DNA levels. Moreover, the rate in the 10-year follow-up was significantly higher in group A (6.9 × 10-4 ±â€¯1.3 × 10-4) than group B (2.7 × 10-4 ±â€¯7.4 × 10-5 substitution/site/year, p < .001). Most of the substitutions were in the Core region and the majority were non-synonymous changes. The rate of nucleotide substitution was inversely related to HBV-DNA levels, highlighting the role of viral load in the HBV intra-host dynamics, even in low replicative state patients. Moreover, the difference in the substitution rate between the analysed groups was mainly consequence of substitutions restricted to the Core region, particularly in the simple coding region and antigenic epitopes, which suggest that the immune pressure drives the different evolutionary behaviour of groups.

Dynamics of a hepatitis B virus e antigen minus population ascribed to genotype F during the course of a chronic infection despite the presence of anti-HBs antibodies.

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.

Further characterization of a morphogenetic mutant of the foot-and-mouth disease virus.

In this paper we describe further characterization of a foot-and-mouth disease virus (FMDV) temperature-sensitive mutant, ts 139. This mutant was very sensitive to heat inactivation, suggesting that its viral particles are somehow altered. The electrophoretic analysis of ts 139 structural proteins indicated that VP2 has an altered mobility. Furthermore, two known protein precursors of VP2, VP0 and p88, were shown to be altered, as was p64, which supports a VP2 precursor role for p64. The ts 139 viral particle assembly pathway was analyzed during viral replication. The empty-capsid to complete-viral-particle ratio was clearly increased compared to that found for the wt strain, indicating an alteration in the morphogenetic process. This encouraged us to search for the presence of possible viral precursors of low sedimentation coefficient which have not been described previously in aphthovirus-infected cells. Analysis of the viral morphogenetic process in ts 139-infected cells demonstrated the presence of viral complexes formed by VP0, VP1 and VP3 which sedimented slightly faster than a 12 S marker. The protein composition, the sedimentation coefficient of this complex and the pulse-chase results strongly suggest that it is a morphogenetic precursor of the mature viral particle.

Further characterization of an RNA defective mutant of the foot-and-mouth disease virus.

In this paper a further characterization of a foot-and-mouth disease virus (FMDV) temperature-sensitive mutant, ts 6, is described. This mutant presents a defective RNA synthesis at non-permissive temperature (NPT) by comparison to the wt capacity. However, a low level of viral RNA synthesis (below 10%) was sufficient to achieve an almost normal protein synthesis including a normal pattern of protein cleavage. In addition, morphogenetic precursor particles, 14S and 75S, are formed, indicating that the structural proteins VP0, VP1 and VP3 provide the necessary signal for self-assembly, and that the RNA is not necessary for such assemblage. Finally, although an almost normal synthesis of viral proteins and morphogenetic precursors (14S and 75S) occurs, the induction of cellular protein shut-off was not evident, indicating that this viral effect appears late in the viral cycle once complete viral replication has occurred.

Cytotoxic effect of Argentine medicinal plant extracts on human hepatocellular carcinoma cell line.

Methanolic extracts from Achyrocline satureioides (Dc.) Lam, Aristolochia macroura Gomez, Lithraea molleoides (Vell.) Engl., Schinus molle L., unlike those from Celtis spinosa Spreng, Chenopodium ambrosioides L., Petiveria alliacea L., and Plantago major L. showed cytotoxic activity against a human hepatocellular carcinoma cell line, Hep G2. Schinus molle L. was the most active (IC50=50+/-7 microg/ml). These results call for further studies of these extracts.

Intra-host evolution of multiple genotypes of hepatitis C virus in a chronically infected patient with HIV along a 13-year follow-up period.

The intra-host evolutionary process of hepatitis C virus (HCV) was analyzed by phylogenetic and coalescent methodologies in a patient co-infected with HCV-1a, HCV-2a, HCV-3a and human immunodeficiency virus (HIV) along a 13-year period. Direct sequence analysis of the E2 and NS5A regions showed diverse evolutionary dynamics, in agreement with different relationships between these regions and the host factors. The Bayesian Skyline Plot analyses of the E2 sequences (cloned) yielded different intra-host evolutionary patterns for each genotype: a steady state of a "consensus" sequence for HCV-1a; a pattern of lineage splitting and extinction for HCV-2a; and a two-phase (drift/diversification) process for HCV-3a. Each genotype evolving in the same patient and at the same time presents a different pattern apparently modulated by the immune pressure of the host. This study provides useful information for the management of co-infected patients and provides insights into the mechanisms behind the intra-host evolution of HCV.

Frailty Is Associated with Disability and Recent Hospitalization in Community-Dwelling Elderly: The Coyoacan Cohort.

BACKGROUND: The phenotype of frailty proposed by Fried et al has shown to predict several adverse health-related outcomes in elderly populations worldwide; however, the description of such associations in Latin America is still scarce. OBJECTIVE: To describe the association between frailty and recent hospitalization, disability for basic (ADL) and instrumental activities of daily living (IADL). DESIGN, SETTING AND PARTICIPANTS: Cross-sectional study of 1,124 community-dwelling adults aged 70 and older participating in the Coyoacán cohort. MEASUREMENTS: Frailty was defined by the presence of at least three of the following criteria: weight loss, exhaustion, low physical activity, slowness, and weakness. Multiple regression analyses were used to test the association between frailty and the outcomes of interest, adjusting for potential confounders. RESULTS: Mean age was 78.2 (SD ±6.1) years. Prevalence of frailty was 14.1%. Adjusted multivariate models showed that frail status was associated with ADL disability (OR 3.06, 95%CI 1.52-6.17), IADL disability (OR 17.02, 95%CI 6.16-47.01), and recent hospitalization (OR 3.21, 95%CI 1.31-7.8). CONCLUSION: Among Mexican community-dwelling elderly, frailty is associated with ADL and IADL disability as well as with recent hospitalizations. Moreover, frailty's prevalence in this population appears to be greater compared to what has been reported elsewhere. Social and cultural traits should be further studied as correlates of frailty in diverse populations.

Analysis of the circulation of hepatitis A virus in Argentina since vaccine introduction.

Hepatitis A virus (HAV) has shown intermediate endemicity in Argentina, but its incidence has decreased since vaccine introduction in 2005. Environmental surveillance was conducted in five rivers from Argentina from 2005 to 2012, complementing clinical information. HAV detection decreased since 2005, although its circulation continues, maintaining viral diversity but not undergoing antigenic drift. Most sequences belonged to subgenotype IA, closely related to Argentinean clinical sequences, but one belonged to proposed subgenotype IC, previously undetected in the country. Environmental surveillance might contribute to monitoring the single-dose vaccination schedule, representing not only strains causing disease but also the circulating population and the viral introductions.

Hepatitis B virus in Buenos Aires, Argentina: genotypes, virological characteristics and clinical outcomes.

Hepatitis B virus (HBV) is classified into eight major genotypes, A-H, which are geographically distributed worldwide. The aim of this work was to describe the clinical characteristics associated with the HBV genotypes circulating in Buenos Aires city. The study included 139 patients infected with HBV, whose clinical courses were classified as acute symptomatic self-limiting hepatitis, inactive carrier state and chronic active hepatitis (HBV e-antigen (HBeAg)-positive and HBeAg-negative). The HBV genotypes were determined in 128 patients by PCR-restriction fragment length polymorphism and phylogenetic analysis. Biochemical, virological, clinical and histological features were analysed. A differential distribution of genotypes between acute symptomatic and chronic infections was found. Among the acute cases, genotype F was predominant (65.2%, 30/46) and genotype D was rare (4.3%, 2/46), whereas among the chronic infections, a homogeneous distribution of genotypes A (26.8%, 22/82), D (31.7%, 26/82) and F (36.6%, 30/82), with an unusual presence of genotypes B (1.2%, 1/82) and C (3.7%, 3/82), was observed. Regarding the liver histology of chronically infected patients, genotype F tended to display higher histological activity indexes. Mutations related to HBV surface antigen immunoreactivity, antiviral resistance and HBeAg-negative status were studied. This work constitutes, to our knowledge, the first description of the clinical characteristics related to HBV genotypes in Argentina, where the distribution of genotypes in patients with acute infection has not been reported previously. Finally, it was established that genotype F is the prevalent genotype among the acute symptomatic infections in Buenos Aires city, and that it shows a tendency to cause an adverse disease outcome among the chronic cases.

Molecular identification of adenoviruses in clinical samples by analyzing a partial hexon genomic region.

Here we present a system for adenovirus detection and genotyping based on PCR amplification and phylogenetic analysis of a conserved hexon gene fragment. The system was validated using 157 sequences (86 previously typed and 71 clinical samples) and correctly identified species and serotype in 100% and 84% of sequences, respectively. Known associations between specific serotypes and clinical syndromes are verified. Possible new associations are described to allow further independent testing.

Molecular identification of mumps virus genotypes from clinical samples: standardized method of analysis.

A sensitive nested reverse transcription-PCR assay, targeting a short fragment of the gene encoding the small hydrophobic protein (SH gene), was developed to allow rapid characterization of mumps virus in clinical samples. The sensitivity and specificity of the assay were established using representative genotypes A, B, C, D, E, and F. Mumps virus RNA was characterized directly from cerebrospinal fluid (CSF) samples and in extracts of mumps virus isolates from patients with various clinical syndromes. Direct sequencing of products and subsequent phylogenetic analysis enabled genetic classification. A simple web-based system of sequence analysis was established. The study also allowed characterization of mumps virus strains from Argentina as part of a new subgenotype. This PCR assay for characterization of mumps infections coupled to a web-based analytical program provides a rapid method for identification of known and novel strains.

Effect of lysosomotropic agents on the foot-and-mouth disease virus replication.

The effect of two lysosomotropic agents, NH4Cl and chloroquine, on the foot-and-mouth disease virus (FMDV) replicative cycle was studied. When the drugs were present throughout the viral replicative cycle, an important inhibition of viral RNA synthesis and virus production was detected. The inhibition of viral RNA synthesis was maximal when the drugs were present from 30 min before virus infection up to 30 min after that. Otherwise, if the agents were added once the viral synthesis has started (150 min p.i.) the effect was not evident. The agents neither exerted direct virucidal effects, nor did they affect viral adsorption. The results indicate that the lysosomotropic agents affect preferentially an early intracellular event during the viral infective cycle.