Jordi Gómez

Transmission of Hepatitis C Virus by a Cardiac Surgeon

BACKGROUND In the course of a study conducted in 1992 through 1994 of the efficacy of screening blood donors for antibodies to hepatitis C virus (HCV), we found that two patients had acquired hepatitis C after cardiac surgery, with the transmission apparently unrelated to blood transfusions. Because their surgeon had chronic hepatitis C, we sought to determine whether he was transmitting the virus to his patients. METHODS Of 222 of the surgeons patients who participated in studies of post-transfusion hepatitis between 1988 and 1994, 6 contracted postoperative hepatitis C, despite the use of only seronegative blood for transfusions. All six patients had undergone valve-replacement surgery. Analyses were performed to compare nucleotide sequences encompassing the hypervariable region at the junction between the coding regions for envelope glycoproteins E1 and E2 in the surgeon, the patients, and 10 controls infected with the same HCV genotype. RESULTS The surgeon and five of the six patients with hepatitis C unrelated to transfusion were infected with HCV genotype 3; the sixth patient had genotype 1 and was considered to have been infected from another source. Thirteen other patients of the surgeon had transfusion-associated hepatitis C and were also infected with genotype 1. The average net genetic distance between the sequences from the five patients with HCV genotype 3 and those from the surgeon was 2.1 percent (range, 1.1 to 2.5 percent; P < 0.001), as compared with an average distance of 7.6 percent (range, 6.1 to 8.3 percent) between the sequences from the patients and those from the controls. The results of phylogenetic-tree analysis indicated a common epidemiologic origin of the viruses from the surgeon and the five patients. CONCLUSIONS Our findings provide evidence that a cardiac surgeon with chronic hepatitis C may have transmitted HCV to five of his patients during open-heart surgery.

Suppression of chemokine receptor expression by RNA interference allows for inhibition of HIV-1 replication.

Objectives: Duplexes of 21 base pair RNA, known as short-interfering RNA (siRNA), have been shown to inhibit gene expression by a sequence-specific RNA degradation mechanism termed RNA interference (RNAi). The objective of our study was to evaluate the effect of chemokine receptor gene suppression by RNAi on the entry and replication of HIV-1. Methods: A flow cytometry and microscopy evaluation of HIV co-receptor expression of cells transfected with siRNA. An evaluation of the effect of siRNA on HIV entry and replication by intracellular p24 antigen detection, and virus production by infected cells, respectively. Results: siRNA that target CXCR4 and CCR5 could effectively impede cell surface protein expression and their consequent function as HIV co-receptors. The inhibitory effect of RNAi directed to CXCR4 was detected 48 h after transfection of CXCR4+ U87-CD4+ cells. The expression of CXCR4 and CCR5 was blocked in 63 and 48% of positive cells by the corresponding siRNA. However, siRNA directed to CXCR4 or CCR5 did not have an effect on CD4 cells or green fluorescence protein expression. siRNA directed to CXCR4 did not suppress CCR5 expression or vice versa. The suppression of HIV-1 co-receptor expression effectively blocked the acute infection of CXCR4+ or CCR5+ U87-CD4+ cells by X4 (NL4-3) or R5 (BaL) HIV-1 strains. Inhibition of virus replication occurred regardless of the multiplicity of infection employed. Conclusion: Our results demonstrate that RNAi may be used to block HIV entry and replication through the blockade of cellular gene expression. Gene silencing by siRNA may become a valid alternative for HIV intervention.

Quasispecies and its impact on viral hepatitis.

Abstract Quasispecies dynamics mediates adaptability of RNA viruses through a number of mechanisms reviewed in the present article, with emphasis on the medical implications for the hepatitis viruses. We discuss replicative and non-replicative molecular mechanisms of genome variation, modulating effects of mutant spectra, and several modes of viral evolution that can affect viral pathogenesis. Relevant evolutionary events include the generation of minority virus variants with altered functional properties, and alterations of mutant spectrum complexity that can affect disease progression or response to treatment. The widespread occurrence of resistance to antiviral drugs encourages new strategies to control hepatic viral disease such as combination therapies and lethal mutagenesis. In particular, ribavirin may be exerting in some cases its antiviral activity with participation of its mutagenic action. Despite many unanswered questions, here we document that quasispecies dynamics has provided an interpretation of the adaptability of the hepatitis viruses, with features conceptually similar to those observed with other RNA viruses, a reflection of the common underlying Darwinian principles.

Regulation of the abscisic acid-responsive gene rab28 in maize viviparous mutants

SummaryWe have isolated a new maize gene, rab 28, that responds to abscisic acid (ABA) treatment. This gene has been characterized by determining the sequence of the cDNA and corresponding genomic copy, and by mapping the start site of its transcript. The rab 28 gene encodes a protein of predicted molecular weight 27713 Da which shows strong homology with the Lea D-34 protein identified in cotton. The proximal promoter region contains the conserved ABA-response element, CACGTGG, reported in other plant genes to be responsible for ABA induction. rab 28 mRNA has been identified as ABA-inducible in embryos and young leaves. It is also induced by water-stress in leaves of wild-type plants. Regulation of the rab 28 gene was studied in maize viviparous mutants. The results obtained with the ABA-insensitive vp1 mutant show that rab 28 transcripts do not accumulate to a significant level during embryogenesis. Surprisingly, induction of rab 28 mRNA can be achieved in young embryos by exogenous ABA treatment. Moreover, water-stressed or ABA-treated seedlings of vp1 contain significant levels of rab 28 mRNA which is not detectable in well-watered seedlings. Regulation of the rab 28 gene in excised young embryos of ABA-deficient vp2 mutants, in which influences of the maternal environment are absent, closely resembles that found in non-mutant excised young embryos. The significance of these results is discussed.

Unfinished Stories on Viral Quasispecies and Darwinian Views of Evolution

Experimental evidence that RNA virus populations consist of distributions of mutant genomes, termed quasispecies, was first published 31 years ago. This work provided the earliest experimental support for a theory to explain a system that replicated with limited fidelity and to understand the self-organization and adaptability of early life forms on Earth. High mutation rates and quasispecies dynamics of RNA viruses are intimately related to both viral disease and antiviral treatment strategies. Moreover, the quasispecies concept is being applied to other biological systems such as cancer research in which cellular mutant spectra can be also detected. This review addresses some of the unanswered questions regarding viral and theoretical quasispecies concepts as well as more practical aspects concerning resistance to antiviral treatments and pathogenesis.

Differential regulation of ABA-induced 23-25 kDa proteins in embryo and vegetative tissues of the viviparous mutants of maize.

Previous studies have identified a set of highly phosphorylated proteins of 23–25 kDa accumulated during normal embryogenesis of Zea mays L. and which disappear in early germination. They can be induced precociously in embryos by abscisic acid (ABA) treatment. Here the synthesis and accumulation of this group of proteins and their corresponding mRNAs were examined in ABA-deficient viviparous embryos at different developmental stages whether treated or not with ABA, and in water-stressed leaves of both wild-type and viviparous mutants.During embryogenesis and precocious germination of viviparous embryos the pattern of expression of the 23–25 kDa proteins and mRNAs closely resembles that found in non-mutant embryo development. They are also induced in young viviparous embryos by ABA treatment. In contrast, leaves of ABA-deficient mutants fail to accumulate mRNA in water stress, yet do respond to applied ABA. In water-stressed leaves of wild type plants the mRNAs are induced and translated into 4 proteins with a molecular weight and isoelectric point identical to those found in embryos.These results indicate that the 23–25 kDa protein set is a new member of the recently described class or proteins involved in generalized plant ABA responses.The different pattern of expression for the ABA-regulated 23–25 kDa proteins and mRNAs found in embryo and in vegetative tissues of viviparous mutants is discussed.

Nucleotide and Amino Acid Complexity of Hepatitis C Virus Quasispecies in Serum and Liver

ABSTRACT The quasispecies nature of the hepatitis C virus (HCV) is thought to play a central role in maintaining and modulating viral replication. Several studies have tried to unravel, through the parameters that characterize HCV circulating quasispecies, prognostic markers of the disease. In a previous work we demonstrated that the parameters of circulating viral quasispecies do not always reflect those of the intrahepatic virus. Here, we have analyzed paired serum and liver quasispecies from 39 genotype 1b-infected patients with different degrees of liver damage, ranging from minimal changes to cirrhosis. Viral level was quantified by real-time reverse transcription-PCR, and viral heterogeneity was characterized through the cloning and sequencing of 540 HCV variants of a genomic fragment encompassing the E2-NS2 junction. Although in 95% of patients, serum and liver consensus HCV amino acid sequences were identical, quasispecies complexity varied considerably between the viruses isolated from each compartment. Patients with HCV quasispecies in serum more complex (26%) than, less complex (28%) than, or similarly complex (41%) to those in liver were found. Among the last, a significant correlation between fibrosis and all the parameters that measure the viral amino acid complexity was found. Correlation between fibrosis and serum viral load was found as well (R = 0.7). With regard to the origin of the differences in quasispecies complexity between serum and liver populations, sequence analysis argued against extrahepatic replication as a quantitatively important contributing factor and supported the idea of a differential effect or different selective forces on the virus depending on whether it is circulating in serum or replicating in the liver.

In vitro characterization of a miR-122-sensitive double-helical switch element in the 5′ region of hepatitis C virus RNA

It has been proposed that the hepatitis C virus (HCV) internal ribosome entry site (IRES) resides within a locked conformation, owing to annealing of its immediate flanking sequences. In this study, structure probing using Escherichia coli dsRNA-specific RNase III and other classical tools showed that this region switches to an open conformation triggered by the liver-specific microRNA, miR-122. This structural transition, observed in vitro, may be the mechanistic basis for the involvement of downstream IRES structural domain VI in translation, as well as providing a role of liver-specific miR-122 in HCV infection. In addition, the induced RNA switching at the 5′ untranslated region could ultimately represent a new mechanism of action of micro-RNAs.

High-Resolution Hepatitis C Virus Subtyping Using NS5B Deep Sequencing and Phylogeny, an Alternative to Current Methods

ABSTRACT Hepatitis C virus (HCV) is classified into seven major genotypes and 67 subtypes. Recent studies have shown that in HCV genotype 1-infected patients, response rates to regimens containing direct-acting antivirals (DAAs) are subtype dependent. Currently available genotyping methods have limited subtyping accuracy. We have evaluated the performance of a deep-sequencing-based HCV subtyping assay, developed for the 454/GS-Junior platform, in comparison with those of two commercial assays (Versant HCV genotype 2.0 and Abbott Real-time HCV Genotype II) and using direct NS5B sequencing as a gold standard (direct sequencing), in 114 clinical specimens previously tested by first-generation hybridization assay (82 genotype 1 and 32 with uninterpretable results). Phylogenetic analysis of deep-sequencing reads matched subtype 1 calling by population Sanger sequencing (69% 1b, 31% 1a) in 81 specimens and identified a mixed-subtype infection (1b/3a/1a) in one sample. Similarly, among the 32 previously indeterminate specimens, identical genotype and subtype results were obtained by direct and deep sequencing in all but four samples with dual infection. In contrast, both Versant HCV Genotype 2.0 and Abbott Real-time HCV Genotype II failed subtype 1 calling in 13 (16%) samples each and were unable to identify the HCV genotype and/or subtype in more than half of the non-genotype 1 samples. We concluded that deep sequencing is more efficient for HCV subtyping than currently available methods and allows qualitative identification of mixed infections and may be more helpful with respect to informing treatment strategies with new DAA-containing regimens across all HCV subtypes.

Characterizing the function and structural organization of the 5′ tRNA-like motif within the hepatitis C virus quasispecies

Hepatitis C virus (HCV) RNA is recognized and cleaved in vitro by RNase P enzyme near the AUG start codon. Because RNase P identifies transfer RNA (tRNA) precursors, it has been proposed that HCV RNA adopts structural similarities to tRNA. Here, we present experimental evidence of RNase P sensitivity conservation in natural RNA variant sequences, including a mutant sequence (A368–G) selected in vitro because it presented changes in the RNA structure of the relevant motif. The variation did not abrogate the original RNase P cleavage, but instead, it allowed a second cleavage at least 10 times more efficient, 4 nt downstream from the original one. The minimal RNA fragment that confers sensitivity to human RNase P enzyme was located between positions 299 and 408 (110 nt). Therefore, most of the tRNA-like domain resides within the viral internal ribosome entry site (IRES) element. In the variant, in which the mutation stabilizes a 4 nt stem–loop, the second cleavage required a shorter (60 nt) substrate, internal to the minimal fragment substrate, conforming a second tRNA-like structure with similarities to a ‘Russian-doll’ toy. This new structure did not impair IRES activity, albeit slightly reduced the efficiency of translation both in vitro and in transfected cells. Conservation of the original tRNA-like conformation together with preservation of IRES activity points to an essential role for this motif. This conservation is compatible with the presence of RNA structures with different complexity around the AUG start codon within a single viral population (quasispecies).