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Background Pathogenesis of Ebola virus disease remains poorly understood. We used concomitant determination of routine laboratory biomarkers and Ebola viremia to explore the potential role of viral replication in specific organ damage. Methods We recruited patients with detectable Ebola viremia admitted to the EMERGENCY Organizzazione Non Governativa Organizzazione Non Lucrativa di Utilita Sociale (ONG ONLUS) Ebola Treatment Center in Sierra Leone. Repeated measure of Ebola viremia, alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin, creatine phosphokinase (CPK), lactate dehydrogenase (LDH), activated prothrombin time (aPTT), international normalized ratio (INR), creatinine, and blood urea nitrogen (BUN) were recorded. Patients were followed up from admission until death or discharge. Results One hundred patients (49 survivors and 51 nonsurvivors) were included in the analysis. Unadjusted analysis to compare survivors and nonsurvivors provided evidence that all biomarkers were significantly above the normal range and that the extent of these abnormalities was generally higher in nonsurvivors than in survivors. Multivariable mixed-effects models provided strong evidence for a biological gradient (suggestive of a direct role in organ damage) between the viremia levels and either ALT, AST, CPK LDH, aPTT, and INR. In contrast, no direct linear association was found between viremia and either creatinine, BUN, or bilirubin. Conclusions This study provides evidence to support that Ebola virus may have a direct role in muscular damage and imbalance of the coagulation system. We did not find strong evidence suggestive of a direct role of Ebola virus in kidney damage. The role of the virus in liver damage remains unclear, but our evidence suggests that acute severe liver injury is not a typical feature of Ebola virus disease.

Acute hepatitis B infection (AHB) is still a common viral acute hepatitis worldwide. As vaccination, antiviral treatment, and immigration are bound to affect the epidemiological landscape of HBV infections, and some of its aspects need to be investigated: (1) the circulation of vaccine escape mutants and of primary drug resistant strains, (2) the change in HBV genotype prevalence, and (3) the clinical implications of AHB and the probability of chronification. The serological, virological, and clinical parameters of 75 patients, acutely infected by HBV, were gathered for a retrospective study. Long-term follow up, either to complete seroconversion or for up to five years, was possible for 44 patients. Sequence analysis of the reverse transcriptase/HBsAg and precore regions was performed to investigate the molecular epidemiology and pathogenesis of recent infections by HBV. Genotype distribution in AHB in Italian patients was radically different from that of chronic infections, with a dramatic increase of extra-European genotypes (A1, F), suggesting that a proportion of AHBs are currently related to imported strains. None of the documented infections occurred in vaccinated individuals, while HBsAg variants (potentially vaccine escape variants) were rare and less prevalent than in chronic infections. No drug resistant strains were observed. Spontaneous viral clearance occurred in all but three cases. Time to viral clearance was inversely proportional to liver damage, but HBsAg titer on day 28 and, better still, HBsAg decay from day 0 to day 28 after admission, were the best predictors of chronification. They are, thus, potentially useful to guide antiviral treatment to prevent chronic evolution.

HCV quasispecies variability represents the background for the selection of mutations and for the development of drug resistance. Natural aminoacid changes in NS3, associated with reduced protease inhibitor susceptibility, have been observed in treatment-naïve patients. Massively parallel sequencing has been used to analyze NS3 quasispecies in patients infected with HCV genotype 1, naive to anti-HCV treatment, with/without HIV-coinfection, to establish the genetic heterogeneity and the presence of amino acid substitutions at positions responsible for drug resistance. Genomes carrying substitutions represented either predominant or minority components of viral quasispecies, and were observed in 85.7% of patients. Multiple substitutions, frequently associated on the same haplotype, were observed in 46.4% of patients. High resistance combinations were not detected, neither on the same genome, nor in the whole quasispecies. Heterogeneity of HCV NS3 was lower in HIV-coinfected as compared to HCV-monoinfected patients, but factors underlying this difference remain to be established. Although the relevance of naturally occurring mutations with respect of resistance development and probability of success of direct acting antivirals is questioned, UDPS may be beneficial to help understanding viral dynamics, providing high resolution view of viral diversity.

In March/April 2009, a new pandemic influenza A virus (A(H1N1)pdm09) emerged and spread rapidly via human-to-human transmission, giving rise to the first pandemic of the 21th century. Influenza virus may be present in the infected host as a mixture of variants, referred to as quasi-species, on which natural and immune-driven selection operates. Since hemagglutinin (HA) and non-structural 1 (NS1) proteins are relevant in respect of adaptive and innate immune responses, the present study was aimed at establishing the intra-host genetic heterogeneity of HA and NS1 genes, applying ultra-deep pyrosequencing (UDPS) to nasopharyngeal swabs (NPS) from patients with confirmed influenza A(H1N1)pdm09 infection. The intra-patient nucleotide diversity of HA was significantly higher than that of NS1 (median (IQR): 37.9 (32.8-42.3) X 10(-4) vs 30.6 (27.4-33.6) X 10(-4) substitutions/site, p = 0.024); no significant correlation for nucleotide diversity of NS1 and HA was observed (r = 0.319, p = 0.29). Furthermore, a strong inverse correlation between nucleotide diversity of NS1 and viral load was observed (r = - 0.74, p = 0.004). For both HA and NS1, the variants appeared scattered along the genes, thus indicating no privileged mutation site. Known polymorphisms, S203T (HA) and I123V (NS1), were observed as dominant variants (>98%) in almost all patients; three HA and two NS1 further variants were observed at frequency >40%; a number of additional variants were detected at frequency

We compared two commercial assays for HBV DNA quantitation, Versant HBV 3.0, System 340 (bDNA; Bayer Diagnostics) and COBAS AmpliPrep-COBAS TaqMan HBV Test (TaqMan; Roche Diagnostics). Analytical sensitivity, calculated on WHO International Standard, predicted 95% detection rate at 11.4 and 520.2IU/ml for TaqMan and bDNA, respectively. Specificity, established on 50 blood donor samples, was 100% and 84% for TaqMan and bDNA, respectively. When using clinical samples, HBV DNA was detected by TaqMan in 21/55 samples negative to bDNA. Mean values of HBV DNA obtained with bDNA were higher than those obtained with TaqMan (4.09log(10)+/-1.90 versus 3.39log(10)+/-2.41, p0.001), and 24.4% of samples showed differences in viral load values0.5log(10), without association with HBV genotype. There was a good correlation for HBV DNA concentrations measured by the two assays (r=0.94; p0.001) within the overlapping range, and the distribution of results with respect to relevant clinical threshold recently confirmed (20,000 and 2000IU/ml) was similar. Approximately 50% of samples with low HBV DNA, appreciated by TaqMan but not by bDNA, were successfully sequenced in pol region, where drug resistance mutations are located.

To the Editor: The biologic role of amino acid variants at position 222 of the hemagglutinin (HA) gene of pandemic (H1N1) 2009 virus in severe infections has been extensively discussed. A recent series of studies (1–3) confirm the initial suggestions that G or N in this position might confer greater pathogenic potential to the virus than to the wild type. In contrast, their data suggest that no particular pathogenicity is associated with the 222E variant because it occurs at the same frequency in severe and mild infections. Most authors also seem to agree that D222G or N appears sporadically in phylogenetically distant viruses, with limited transmissibility. However, Puzelli et al. (4) reported transmission of a 222G mutant from son to father (with the appearance of an additional G155E mutation). In Italy, the pattern of D222 variants has been peculiar, with extremely rare appearances of 222G and high diffusion of 222E isolates. At the National Institute for Infectious Diseases in Rome, 82 isolates (GenBank accession nos. {"type":"entrez-nucleotide-range","attrs":{"text":"CY063455-CY063469","start_term":"CY063455","end_term":"CY063469","start_term_id":"296238920","end_term_id":"296238948"}}CY063455-CY063469 for new sequences in this study) were monitored for D222 variants. No 222G or N variants were detected, even in 24 severe infections, nor was the G155E mutation detected. This finding was not surprising, given the worldwide low frequency of this mutation, even in severe infections. Conversely, D222E was detected in 12 of the 82 cases, peaking in September 2009, when it was present in most of the infections, with no overrepresentation in severe cases. Subsequently, it was substituted by different 222D viruses during the autumn–winter outbreak. The analysis of publicly available sequences from other centers in Italy confirmed the trend: 222E was the dominant variant during the summer, 222G was detected only in 4 cases, and 222N was never detected. As we previously reported (5), phylogenetic analysis of 222E variants allowed identification of them as an authentic circulating subclade of clade 7 (6) or cluster 2 (7), rather than as sporadically occurring variants. To further investigate the origin and the evolution of 222 variants, we have extended the phylogenetic analysis (neighbor-joining) to 2,492 complete HA sequences from the Global Initiative on Sharing All Influenza Data database (expanded Figure online, www.cdc.gov/EID/content/17/4/749-F.htm), confirming the clustering of all the worldwide 222E variants in a well-defined subclade (Figure; expanded Figure online). The same analysis showed that D222G variants could be reconciled with 2 different phylogenetic patterns. The first less frequent pattern includes sequences appearing sparsely throughout the tree, confirming the mentioned hypothesis of sporadic mutation. In contrast, the second pattern (the majority) relates to small groups of sequences appearing in monophyletic microclusters. Among these microclusters, 2 are particularly interesting because they include only 222G sequences, isolated in different parts of the world (Figure). This finding is still compatible with sporadic mutation; bootstrap values are low because of the low general variability of these sequences. However, the possibility that D222G variants are transmissible and might sustain small epidemics of their own or that they might arise more easily from specific, phylogenetically related backgrounds, is intriguing. In a few countries, such as Italy, Norway, or Sweden, where the 222E virus has been circulating as a substantial proportion of the total virus, the 222G variants appeared more frequently in the genetic context of the 222E virus (1,4), as demonstrated by phylogenetic analysis and confirmed by the analysis of codon 239 (the codon determining the 222 residue specificity): GAA to GGA (E to G), instead of GAT to GGT (D to G). In these cases, the correct definition of 222G variants would therefore be E222G rather than D222G. From this point of view, the 222N variant would have a higher genetic barrier to change from E because it would require 2 mutations (GAA to AAT) instead of 1 (GAT to AAT), and indeed none of the 16 available (worldwide) 222N full-length variants clustered with the 222E virus. On the basis of these findings, 2 different amino acids, D and E, might be considered polymorphic variants at position 222, and the potentially more pathogenic mutants or circulating variants would be G or N. Figure Phylogenetic relationships among 2,492 complete hemagglutinin (HA) genes of pandemic (H1N1) 2009. At the center, the whole neighbor-joining tree. On the left, enlargement of 2 regions of the tree including pure monophyletic D222G clusters, indicated in ...

Reduced circulation of hepatitis A virus lead to an increase of susceptible individuals, and outbreaks occurred recently. In Northern Italy an outbreak is ongoing, attributed to a monophyletic genotype IA strain, with mixed frozen berries as probable source. From 01/01/2013 to 07/15/2013, 30 cases were diagnosed at National Institute for Infectious Diseases, Rome, Italy, representing about twice the number of cases in whole 2012. Phylogenetic analysis indicated that most, although not all, infections were attributable to the same monophyletic genotype IA strain identified in the contemporary Northern Italy outbreak. This strain is also very similar to previous isolates from Venezuela.

Influenza A( H1N1)v has spread rapidly in all parts of the globe in 2009 as a true pandemic, although fortunately a clinically mild one. The relevant evolutionary steps for the new virus to adapt to human populations occurred very early during the pandemic, before the end of April. Of the several resulting clades or clusters, clade 7 appeared later and proved more successful, substituting all other early clades before the bulk of the worldwide infections occurred.

HIV quasispecies was analysed in plasma and proviral genomes hosted by duodenal mucosa and peripheral blood cells (PBMC) from patients with early or chronic infection, with respect to viral heterogeneity, tropism compartmentalization and extent of immune activation. Seventeen HIV-1-infected combined antiretroviral therapy naive patients were enrolled (11 early infection and six chronic infection). V3 and nef genomic regions were analysed by ultra-deep pyrosequencing. Sequences were used to infer co-receptor usage and to construct phylogenetic trees. As markers of immune activation, plasma sCD14 and soluble tumour necrosis factor receptor II (sTNFRII) levels were measured. Median diversity of HIV RNA was lower in patients with early infection versus chronic infection patients. Overall, direct correlation was observed between V3 diversity and X4 frequency; V3 diversity of HIV RNA was inversely correlated with CD4 T-cell count; median sCD14 and sTNFRII values were similar in early and chronic patients, but X4 frequency of HIV RNA was directly correlated with plasma sCD14. The proportion of patients harbouring X4 variants and median intra-patient X4 frequency of proviral genomes tended to be higher in chronic infection than early infection patients. More pronounced compartmentalization of proviral quasispecies in gut compared with PBMC samples was observed in patients with early infection compared with chronic patients. The loss of gut/PBMC compartmentalization in more advanced stages of HIV infection was confirmed by longitudinal observation. More studies are needed to understand the pathogenetic significance of early HIV quasispecies compartmentalization and progressive intermixing of viral variants in subsequent phases of the infection, as well as the role of immune activation in tropism switch.