Hepatitis C virus quasispecies in chronically infected children subjected to interferon–ribavirin therapy

Accumulating evidence suggests that certain features of hepatitis C virus (HCV), especially its high genetic variability, might be responsible for the low efficiency of anti-HCV treatment. Here, we present a bioinformatic analysis of HCV-1a populations isolated from 23 children with chronic hepatitis C (CHC) subjected to interferon–ribavirin therapy. The structures of the viral quasispecies were established based on a 132-amino-acid sequence derived from E1/E2 protein, including hypervariable region 1 (HVR1). Two types of HCV populations were identified. The first type, found in non-responders, contained a small number of closely related variants. The second type, characteristic for sustained responders, was composed of a large number of distantly associated equal-rank variants. Comparison of 445 HVR1 sequences showed that a significant number of variants present in non-responding patients are closely related, suggesting that certain, still unidentified properties of the pathogen may be key factors determining the result of CHC treatment

2′-O Methylation of Internal Adenosine by Flavivirus NS5 Methyltransferase

RNA modification plays an important role in modulating host-pathogen interaction. Flavivirus NS5 protein encodes N-7 and 2′-O methyltransferase activities that are required for the formation of 5′ type I cap (m7GpppAm) of viral RNA genome. Here we reported, for the first time, that flavivirus NS5 has a novel internal RNA methylation activity. Recombinant NS5 proteins of West Nile virus and Dengue virus (serotype 4; DENV-4) specifically methylates polyA, but not polyG, polyC, or polyU, indicating that the methylation occurs at adenosine residue. RNAs with internal adenosines substituted with 2′-O-methyladenosines are not active substrates for internal methylation, whereas RNAs with adenosines substituted with N6-methyladenosines can be efficiently methylated, suggesting that the internal methylation occurs at the 2′-OH position of adenosine. Mass spectroscopic analysis further demonstrated that the internal methylation product is 2′-O-methyladenosine. Importantly, genomic RNA purified from DENV virion contains 2′-O-methyladenosine. The 2′-O methylation of internal adenosine does not require specific RNA sequence since recombinant methyltransferase of DENV-4 can efficiently methylate RNAs spanning different regions of viral genome, host ribosomal RNAs, and polyA. Structure-based mutagenesis results indicate that K61-D146-K181-E217 tetrad of DENV-4 methyltransferase forms the active site of internal methylation activity; in addition, distinct residues within the methyl donor (S-adenosyl-L-methionine) pocket, GTP pocket, and RNA-binding site are critical for the internal methylation activity. Functional analysis using flavivirus replicon and genome-length RNAs showed that internal methylation attenuated viral RNA translation and replication. Polymerase assay revealed that internal 2′-O-methyladenosine reduces the efficiency of RNA elongation. Collectively, our results demonstrate that flavivirus NS5 performs 2′-O methylation of internal adenosine of viral RNA in vivo and host ribosomal RNAs in vitro

Genetic variability of hepatitis C virus before and after combined therapy of interferon plus ribavirin

We present an analysis of the selective forces acting on two hepatitis C virus genome regions previously postulated to be involved in the viral response to combined antiviral therapy. One includes the three hypervariable regions in the envelope E2 glycoprotein, and the other encompasses the PKR binding domain and the V3 domain in the NS5A region. We used a cohort of 22 non-responder patients to combined therapy (interferon alpha-2a plus ribavirin) for which samples were obtained before initiation of therapy and after 6 or/and 12 months of treatment. A range of 25-100 clones per patient, genome region and time sample were sequenced. These were used to detect general patterns of adaptation, to identify particular adaptation mechanisms and to analyze the patterns of evolutionary change in both genome regions. These analyses failed to detect a common adaptive mechanism for the lack of response to antiviral treatment in these patients. On the contrary, a wide range of situations were observed, from patients showing no positively selected sites to others with many, and with completely different topologies in the reconstructed phylogenetic trees. Altogether, these results suggest that viral strategies to evade selection pressure from the immune system and antiviral therapies do not result from a single mechanism and they are likely based on a range of different alternatives, in which several different changes, or their combination, along the HCV genome confer viruses the ability to overcome strong selective [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]

Identification and characterization of antibacterial compound(s) of cockroaches (Periplaneta americana)

Infectious diseases remain a significant threat to human health, contributing to more than 17 million deaths, annually. With the worsening trends of drug resistance, there is a need for newer and more powerful antimicrobial agents. We hypothesized that animals living in polluted environments are potential source of antimicrobials. Under polluted milieus, organisms such as cockroaches encounter different types of microbes, including superbugs. Such creatures survive the onslaught of superbugs and are able to ward off disease by producing antimicrobial substances. Here, we characterized antibacterial properties in extracts of various body organs of cockroaches (Periplaneta americana) and showed potent antibacterial activity in crude brain extract against methicillin-resistant Staphylococcus aureus and neuropathogenic E. coli K1. The size-exclusion spin columns revealed that the active compound(s) are less than 10 kDa in molecular mass. Using cytotoxicity assays, it was observed that pre-treatment of bacteria with lysates inhibited bacteria-mediated host cell cytotoxicity. Using spectra obtained with LC-MS on Agilent 1290 infinity liquid chromatograph, coupled with an Agilent 6460 triple quadruple mass spectrometer, tissues lysates were analyzed. Among hundreds of compounds, only a few homologous compounds were identified that contained isoquinoline group, chromene derivatives, thiazine groups, imidazoles, pyrrole containing analogs, sulfonamides, furanones, flavanones, and known to possess broad-spectrum antimicrobial properties, and possess anti-inflammatory, anti-tumour, and analgesic properties. Further identification, characterization and functional studies using individual compounds can act as a breakthrough in developing novel therapeutics against various pathogens including superbugs

Dynamics of hepatitis C virus NS5A quasispecies during interferon and ribavirin therapy in responder and non-responder patients with genotype 1b chronic hepatitis C

The quasispecies nature of hepatitis C virus (HCV) may have important implications concerning resistance to antiviral agents. To determine whether HCV NS5A quasispecies composition and dynamics are related to responsiveness to combined interferon (IFN) and ribavirin therapy, extensive sequence analyses of cloned RT-PCR amplification products of HCV-1b NS5A quasispecies of sequential isolates from 15 treated (nine sustained responders and six non-responders) and three untreated patients were performed. Accumulation of mutations in NS5A during therapy was relatively frequent in the V3 domain, but unusual elsewhere. Amino acid changes were the result of the imposition of minor variants that were already present before treatment and always occurred within the first week of therapy. Before treatment, the complexity and diversity of quasispecies were lower in isolates from responders than in those from non-responders, particularly in the V3 domain, where differences in nucleotide entropy (0.35 vs 0.64, P=0.003), genetic distance (0.0145 vs 0.0302, P=0.05) and non-synonymous substitutions (0.0102 vs 0.0203, P=0.036) were statistically significant. These differences became more apparent during treatment, because complexity and diversity remained stable or tended to increase in non-responders, whereas they tended to decrease in responders. These observations suggest that the composition and dynamics of HCV NS5A quasispecies, particularly in the V3 domain, may play a role in the response to combined IFN/ribavirin therapy. © 2005 SGM