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.

Hepatitis C virus long-term persistence in peripheral blood mononuclear cells in patients with haemophilia. Detection of occult genotype 1.

Peripheral blood mononuclear cells (PBMC) from chronic hepatitis C virus-infected persons can harbour viral variants that are not detected in plasma samples. We explored the presence and persistence of HCV genotypes in plasma and PBMC cultures from 25 HCV-monoinfected and 25 HIV/HCV-coinfected patients with haemophilia. Cell cultures were performed at different time points between 1993 and 2010-2011, and the HCV genome was examined in culture supernatants. Sequential plasma samples were studied during the same time period. Analysing sequential plasma samples, 21% of patients had mixed-genotype infections, while 50% had mixed infections determined from PBMC culture supernatants. HIV coinfection was significantly associated with the presence of mixed infections (OR = 4.57, P = 0.02; 95% CI = 1.38-15.1). In our previous study, genotype 1 was found in 72% of 288 patients of this cohort. Similar results were obtained with the sequential plasma samples included in this study, 69% had genotype 1. However, when taking into account plasma samples and the results from PBMC supernatants, genotype 1 was identified in 94% of the population. The PBMC-associated variants persisted for 10 years in some subjects, emphasizing their role as long-term reservoirs. The presence of genotype 1 in PBMC may be associated with therapeutic failure and should not be disregarded when treating haemophilic patients who have been infected by contaminated factor concentrates. The clinical implications of persistent lymphotropic HCV variants deserve further examination among multiple exposed groups of HCV-infected patients.

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.

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.

Evidence of occult HCV genotypes in haemophilic individuals with unapparent HCV mixed infections.

Individuals with haemophilia who received non heat-treated factor concentrates were likely to undergo multiple exposures to the hepatitis C virus (HCV). Therefore, HCV mixed-genotype infections might be more frequent in these patients than in the general population. Their prevalence is extremely variable in similar groups of patients tested by different assays due to the fact that currently available genotyping techniques are not suitable to detect multiple HCV genotypes in a viral population. As an HCV viral reservoir, the peripheral blood mononuclear cell (PBMC) might harbor viral variants distinct from the genotypes detected in plasma. We investigated the presence of HCV genotypes in a group of chronically infected haemophilic patients in the PBMC compartment using a non-stimulated cell culture system that allows the detection of the HCV genome in culture supernatants. We compared them to the HCV genotypes found in plasma samples. Cell culture experiments performed with PBMC demonstrated the presence of additional HCV genotypes that were undetected in the corresponding plasma samples with the same genotyping technique. Although mixed infections at HCV genotype level became evident in 5.6% of the patients (16/288), the culture methodology increased the number of HCV infections with multiple genotypes to 62.5% (10/16) (P < 0.0001). Once more, the role of mononuclear cells as HCV viral reservoirs is emphasized. Considering minor strains could influence the outcome of treatment, detection of covert HCV mixed-genotype infections might be essential for choosing the adequate therapeutic regimen.

Disruption of mitochondrial function as the basis of the trypanocidal effect of trifluoperazine on Trypanosoma cruzi.

The tricyclic anti-calmodulin drug trifluoperazine (TFP) inhibited growth and motility of epimastigotes of Trypanosoma cruzi, at concentrations lower than 100 microM, and motility and infectivity of the bloodstream trypomastigote form at 200 microM. Electron microscopy of TFP-treated epimastigotes showed that the major effect was at the mitochondrial level, with gross swelling and disorganization. The oligomycin-sensitive, mitochondrial ATPase was completely inhibited by 20 microM TFP, and the same drug concentration caused a 60% decrease in intracellular ATP content. The results suggest that the trypanocidal effect of TFP may be related more to mitochondrial damage than to the well-known anticalmodulin effect of the drug.

Isometric developed tension and histopathology of myocardium of chagasic mice. I.

Cardiac disease is usually the most serious complication of human infection with Trypanosoma cruzi in our country. This report studies the evolution of the chagasic cardiopathy in mice inoculated with low number of parasites during acute and indeterminate stages in different aspects: contractility, histopathology and pharmacological response. From 2-45 days post infection (p.i.) (acute stage) myocardium contractile force reached higher values than in controls, but norepinephrine (NE) response was significantly lower. Acetylcholine (ACh) caused a negative inotropic effect similar to the observed in control group. In this period cardiac damage evolved to an acute interstitial myocarditis. In the indeterminate phase (45-75 days p.i.) of this parasitosis NE produced either small inotropic effect, negative inotropic effect or had no effect on the ventricles tested. A significantly low ACh effect, sub-endocardiac perivascular fibrosis and necrosis in wall bases were also observed. The abnormal pharmacological response described could be ascribed to modifications in cardiac beta and muscarinic receptors number or affinity. The present results showed that in myocardium isolated from T. cruzi infected mice the histopathology, contractility and pharmacological response were altered from 48 h p.i. reaching a maximum disorder at 60 days p.i.

Isometric developed tension and histopathology of myocardium of chagasic mice. I.

Cardiac disease is usually the most serious complication of human infection with Trypanosoma cruzi in our country. This report studies the evolution of the chagasic cardiopathy in mice inoculated with low number of parasites during acute and indeterminate stages in different aspects: contractility, histopathology and pharmacological response. From 2-45 days post infection (p.i.) (acute stage) myocardium contractile force reached higher values than in controls, but norepinephrine (NE) response was significantly lower. Acetylcholine (ACh) caused a negative inotropic effect similar to the observed in control group. In this period cardiac damage evolved to an acute interstitial myocarditis. In the indeterminate phase (45-75 days p.i.) of this parasitosis NE produced either small inotropic effect, negative inotropic effect or had no effect on the ventricles tested. A significantly low ACh effect, sub-endocardiac perivascular fibrosis and necrosis in wall bases were also observed. The abnormal pharmacological response described could be ascribed to modifications in cardiac beta and muscarinic receptors number or affinity. The present results showed that in myocardium isolated from T. cruzi infected mice the histopathology, contractility and pharmacological response were altered from 48 h p.i. reaching a maximum disorder at 60 days p.i.

Isometric developed tension and histopathology of myocardium of chagasic mice. I.

Cardiac disease is usually the most serious complication of human infection with Trypanosoma cruzi in our country. This report studies the evolution of the chagasic cardiopathy in mice inoculated with low number of parasites during acute and indeterminate stages in different aspects: contractility, histopathology and pharmacological response. From 2-45 days post infection (p.i.) (acute stage) myocardium contractile force reached higher values than in controls, but norepinephrine (NE) response was significantly lower. Acetylcholine (ACh) caused a negative inotropic effect similar to the observed in control group. In this period cardiac damage evolved to an acute interstitial myocarditis. In the indeterminate phase (45-75 days p.i.) of this parasitosis NE produced either small inotropic effect, negative inotropic effect or had no effect on the ventricles tested. A significantly low ACh effect, sub-endocardiac perivascular fibrosis and necrosis in wall bases were also observed. The abnormal pharmacological response described could be ascribed to modifications in cardiac beta and muscarinic receptors number or affinity. The present results showed that in myocardium isolated from T. cruzi infected mice the histopathology, contractility and pharmacological response were altered from 48 h p.i. reaching a maximum disorder at 60 days p.i.

Pharmacological and contractile response of myocardium of Chagasic albino Swiss mice.

The isometric developed tension (IDT) and the pharmacological response of isolated myocardium from T. cruzi infected Albino Swiss mice in the acute and chronic Chagas' disease, were studied. The animals were infected with 7 X 10(4) trypomastigotes, form of T. cruzi, Tulahuen strain for the acute infection and with 45 parasites for the chronic stage. The isolated myocardium from acute and chronic chagasic mice reached IDT levels similar to normal hearts. The addition of norepinephrine (NE) to acute ventricles led to lower IDT values than in control group; this hyporeactivity to NE was absent in the chronic stage. On the other hand, epinephrine (EPI) effects on acute and chronic ventricles lead to significantly higher IDT values when compared with their controls. Propranolol blocked the higher effect of EPI in isolated myocardium from the acute stage, but the hyperreactivity described in the chronic stage could not be inhibited by the beta receptors antagonist. The agonist and antagonist tested act on cardiac plasma membrane beta receptors and their actions modify transmembrane calcium fluxes, so that the present study shows that Chagas' disease modifies the normal behavior of adrenergic beta cardiac receptors in different degree depending on the stage of the trypanosomiasis.

Pharmacological and contractile response of myocardium of Chagasic albino Swiss mice.

The isometric developed tension (IDT) and the pharmacological response of isolated myocardium from T. cruzi infected Albino Swiss mice in the acute and chronic Chagas disease, were studied. The animals were infected with 7 X 10(4) trypomastigotes, form of T. cruzi, Tulahuen strain for the acute infection and with 45 parasites for the chronic stage. The isolated myocardium from acute and chronic chagasic mice reached IDT levels similar to normal hearts. The addition of norepinephrine (NE) to acute ventricles led to lower IDT values than in control group; this hyporeactivity to NE was absent in the chronic stage. On the other hand, epinephrine (EPI) effects on acute and chronic ventricles lead to significantly higher IDT values when compared with their controls. Propranolol blocked the higher effect of EPI in isolated myocardium from the acute stage, but the hyperreactivity described in the chronic stage could not be inhibited by the beta receptors antagonist. The agonist and antagonist tested act on cardiac plasma membrane beta receptors and their actions modify transmembrane calcium fluxes, so that the present study shows that Chagas disease modifies the normal behavior of adrenergic beta cardiac receptors in different degree depending on the stage of the trypanosomiasis.