Within-host dynamics of the hepatitis C virus quasispecies population in HIV-1/HCV coinfected patients.

HIV/HCV coinfected individuals under highly active antiretroviral therapy (HAART) represent an interesting model for the investigation of the role played by the immune system in driving the evolution of the HCV quasispecies. We prospectively studied the intra-host evolution of the HCV heterogeneity in 8 coinfected subjects, selected from a cohort of 32 patients initiating HAART: 5 immunological responders (group A) and 3 immunological non-responders (group B), and in two HCV singly infected controls not assuming drugs (group C). For all these subjects at least two serial samples obtained at the first observation (before HAART) and more than 1 year later, underwent clonal sequence analysis of partial E1/E2 sequences, encompassing the whole HVR1. Evolutionary rates, dated phylogenies and population dynamics were co-estimated by using a Bayesian Markov Chain Monte Carlo approach, and site specific selection pressures were estimated by maximum likelihood-based methods. The intra-host evolutionary rates of HCV quasispecies was 10 times higher in subjects treated with HAART than in controls without immunodeficiency (1.9 and 2.3 × 10(-3) sub/site/month in group A and B and 0.29 × 10(-3) sub/site/month in group C individuals). The within-host Bayesian Skyline plot analysis showed an exponential growth of the quasispecies populations in immunological responders, coinciding with a peak in CD4 cell counts. On the contrary, quasispecies population remained constant in group B and in group C controls. A significant positive selection pressure was detected in a half of the patients under HAART and in none of the group C controls. Several sites under significant positive selection were described, mainly included in the HVR1. Our data indicate that different forces, in addition to the selection pressure, drive an exceptionally fast evolution of HCV during HAART immune restoration. We hypothesize that an important role is played by the enlargement of the viral replicative space.

Phylogeography and epidemiological history of West Nile virus genotype 1a in Europe and the Mediterranean basin.

Aim of this study was to reconstruct the temporal and spatial phylodynamics of WNV-1a, the genotype to which the majority of European/Mediterranean viral strains belongs, by using sequences retrieved from public databases. WNV-1a isolates segregated into two major clades: the recent West Mediterranean sequences formed a single monophyletic group within clade A. Clade B included sequences from East Mediterranean and America. Phylogeographic analysis suggested that WNV-1a probably originated in sub-Saharan Africa in the early XXth century, and then spread northwards since the late 1970s, via two routes: one crossing Eastern Mediterranean and the other the Western Mediterranean countries. Our data suggest that the circulation of the virus in a given geographical area usually precedes the onset of the outbreak by one year or more, and underline the importance of the spatial-temporal phylodynamics reconstruction in clarifying the recent epidemiology and in setting up an efficient surveillance system for emerging/reemerging zoonosis.

Bayesian skyline plot inference of the Toscana virus epidemic: a decline in the effective number of infections over the last 30 years.

Toscana virus (TosV), a sandfly fever virus, is one of the main causes of the aseptic meningitis that occurs during the summer in some Mediterranean regions, and whose epidemiology is largely unknown. We used a Bayesian Markov Chain Monte Carlo approach and a relaxed molecular clock to estimate the demographic history of the TosV infection in a series of isolates sampled between 1980 and 2003. The estimated mean evolutionary rate was 2.5 x 10(-4) substitutions per site per year (95% HPD: 0.31-5.44 x 10(-4)subs/site/year). Bayesian skyline plot revealed a sharp decline in the effective number of infections over the last 30 years. In conclusion, our data suggest that continuous and prolonged perturbations of vector/phlebovirus interactions due to the relatively recent climate changes may have contributed to gradually reducing the viral population in endemic areas.

Phylogeny of human T cell lymphotropic virus type 1 in Peru: a high degree of evolutionary relatedness with South African isolates.

We investigated the prevalence and molecular epidemiology of human T cell lymphotropic virus type 1 in Peruvian HIV-1-positive subjects, and found a 10.1% prevalence in a consecutive series of 318 HIV-1-positive patients living in Lima. Phylogenetic analysis of the long terminal repeat of 10 patient isolates showed that all of them belonged to the HTLV-1aA (Transcontinental) subgroup. Although the majority of the Peruvian sequences included in the analysis formed a clade with other Latin American sequences, the isolates of three patients clustered significantly with South African strains. These data show a high prevalence of HTLV-1 infection in HIV-1-positive subjects living in Lima and confirm the presence in Latin America of HTLV-1 strains probably arising from South Africa.

High level of genetic heterogeneity in S and P genes of genotype D hepatitis B virus.

The genetic heterogeneity of hepatitis B virus (HBV) genotypes and subgenotypes was investigated by directly sequencing amplified PreS, S and P genes of HBV isolates obtained from the plasma of 99 subjects with chronic HBV infection. Genotype D showed the greatest intragenotypic and intrasubgenotypic divergence: in particular, the a determinant was mutated in 58.2% of the genotype D patients, two of whom showed prototypic vaccine-induced escape mutants at codon 145. Moreover, five sites under significant positive selection were found in the S protein of the D isolates: one in the a determinant and four in the highly hydrophobic C terminal. Our results suggest that careful surveillance of vaccine-induced escape mutants should be considered in populations with highly frequent genotype D infections, and raise questions concerning the possible relationship between the genetic heterogeneity, host immunity and pathogenicity of this HBV genotype.

Compartmentalization of hepatitis C virus quasispecies in blood mononuclear cells of patients with mixed cryoglobulinemic syndrome.

The aim of this study was to investigate the quasispecies heterogeneity of hepatitis C virus (HCV) in the plasma, cryoprecipitate, and peripheral lymphocytes of chronically infected HCV patients with mixed cryoglobulinemia (MC). We studied 360 clones from 10 HCV-positive patients with MC and 8 age-, gender- and HCV genotype-matched subjects with chronic HCV infection but without MC. A partial nucleotide sequence encompassing the E1/E2 region, including hypervariable region 1 (HVR1), was amplified and cloned from plasma, cryoprecipitates, and peripheral blood mononuclear cells (PBMC), and the genetic diversity and complexity and synonymous and nonsynonymous substitution rates were determined. Heterogeneous selection pressure at codon sites was evaluated. Compartmentalization was estimated by phylogenetic and phenetic (Mantel's test) approaches. The patients with MC had 3.3 times lower nonsynonymous substitution rates (1.7 versus 5.7 substitutions/100 sites). Among the subjects with HCV genotype 1, the MC patients had significantly less complexity than the controls, whereas the diversity and complexity were similar in the genotype 2 patients and controls. Site-specific selection analysis confirmed the low frequency of MC patients showing positive selection. There was a significant correlation between positive selection and the infecting HCV genotype. The quasispecies were less heterogeneous in PBMC than in plasma. Significant compartmentalization of HCV quasispecies was observed in the PBMC of four of nine subjects (three with MC) and seven of nine cryoprecipitates. In one subject with MC, we detected a 5-amino-acid insertion at codons 385 to 389 of HVR1. Our results suggest reduced quasispecies heterogeneity in MC patients that is related to a low selection pressure which is probably due to an impaired immune response, the HCV genotype, and/or the duration of the infection. The frequent HCV quasispecies compartmentalization in patients' PBMC suggests a possible pathogenetic significance.

High prevalence of human T-lymphotropic virus type 1 (HTLV-1) in immigrant male-to-female transsexual sex workers with HIV-1 infection.

Human T-lymphotropic virus type 1 and 2 (HTLV-1 and HTLV-2) infections in Europe are limited to intravenous drug users and migrants coming from areas in which they are endemic. A survey was undertaken of HTLV-1 and HTLV-2 infections in 393 recent immigrants: 167 HIV-1 positive subjects (including 52 male-to-female transsexual sex workers) and 226 pregnant HIV-1 negative women. The prevalence of HTLV-1 was 3.6% in the HIV-1 positive group and 0.9% in the HIV-1 negative group. The highest HTLV-1 prevalence in both groups was found in persons from Latin America, particularly those born in Peru (up to 26% in the HIV-1 positive group). All of the HIV-1/HTLV-1 co-infected individuals were male-to-female transsexual sex workers in whom the overall prevalence of HTLV-1 infection was 11.5%. HTLV-2 was only found in the HIV-1 positive group (prevalence 1.2%); all of the infected subjects were transsexual sex workers from Brazil (overall prevalence 6.4%). Phylogenetic analysis showed that all of the HTLV-1 isolates were of the cosmopolitan type, clustering with other strains circulating in the patients' birthplaces; the HTLV-2 isolates were of subtype 2a, and clustered significantly with other Brazilian strains. These results suggest the independent origin of each infection in the patient's birthplace. The data raise concerns about the further spread of HTLV infections mainly through the sexual route.