M Isabel Costafreda

University of Barcelona, Barcelona, Catalonia, Spain

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Publications (12)47.66 Total impact

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    ABSTRACT: Hepatitis A virus (HAV) has a highly biased and deoptimized codon usage compared to the host cell and fails to inhibit host protein synthesis. It has been proposed that an optimal combination of abundant and rare codons controls the translation speed required for the correct capsid folding. The artificial shut-off of host protein synthesis results in the selection of variants containing mutations in the HAV capsid coding region critical for folding, stability, and function. Here we show that these capsid mutations resulted in changes in their antigenicity, in a reduced stability to high temperature, low pH, and biliary salts and in an increased efficacy of cell entry. In conclusion, the adaptation to cellular shut-off resulted in the selection of large plaque-producing virus populations. HAV has a naturally deoptimized codon usage with respect to that of its cell host and is unable to shut down the cellular translation. This fact contributes to the low replication rate of the virus, in addition to other factors such as the highly inefficient IRES, and explains the outstanding physical stability of this pathogen in the environment mediated by a folding-dependent highly cohesive capsid. Adaptation to artificially induced cellular transcription shut-off, resulted in a re-deoptimization of its capsid codon usage, instead of an optimization.These genomic changes are related to an overall change of capsid folding, which in turn induces changes in the cell entry process. Remarkably, the adaptation to cellular shut-off allowed the virus to significantly increase its RNA uncoating efficiency resulting in the selection of large plaque-producing populations. However, these populations produced much debilitated virions.
    Journal of Virology 02/2014; · 5.08 Impact Factor
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    ABSTRACT: Human enteric viruses are shed in extremely high numbers in the feces of infected individuals, becoming environmental contaminants and eventually leading to contamination of a variety of foodstuffs at the pre-harvest stage. Among these foods at risk is fresh produce and irrigation water is a major vehicle for crop contamination. In the present study, a standardized molecular method for quantitative virus assay has been evaluated in different types of fresh produce and in irrigation water for human norovirus (NoV) detection. Two different virus concentration procedures, polyethylene-glycol precipitation (PEG) and organic flocculation (OF), were employed. The procedures were initially assayed in spiked samples and later validated on naturally contaminated samples from the Nile Delta in Egypt. Overall, PEG provided significantly (p<0.05) better virus recoveries than OF for both irrigation water and salad vegetable virus analysis. NoV GI and GII recoveries in spiked irrigation water ranged from 22.0% to 43.3% and from 12.6% to 16.4% with the PEG and OF methods, respectively. In experimentally contaminated salad vegetables, virus recoveries ranged from 28.0% to 48.0% and from 14.0% to 18.8% by PEG precipitation and OF, respectively. Using PEG precipitation, NoV was found in 31.9% of naturally contaminated irrigation water samples. Both NoV GI and GII were detected in these samples with genome copy numbers of around 10(2) per liter. Virus analyses performed in naturally contaminated fresh produce that included green onion, watercress, radish, leek, and lettuce show that NoV GI was present in 20.8%-34.0% of the samples with genome copy numbers of around 10(2) per gram. When OF was employed, NoV was found in 25.0% of the irrigation water samples. Both genogroups could be found in these samples with genome copy numbers of around 10 per liter. In fresh produce, GI was present in 16.0%-25.7% of the samples with genome copy numbers per gram of around 10. Surprisingly, NoV GII was not detected in any salad vegetable despite highly satisfactory virus/nucleic acid extraction and enzyme efficiencies reported in the assays. Available reliable standardized assays for virus detection in food matrices including appropriate quality assurance and quality control measures to assess the efficiency of critical steps in virus analysis open the possibility to produce consistent and accurate exposure data to be used in QMRA (quantitative microbial risk assessment) and at the same time may enable the formulation of guidelines to ensure the virological quality of selected commodities in specific scenarios to reduce the risk of foodborne virus infections.
    International journal of food microbiology 07/2013; · 3.01 Impact Factor
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    ABSTRACT: Hepatitis A virus (HAV) has previously been reported to bind to human red blood cells through interaction with glycophorin A. Residue K221 of VP1 and the surrounding VP3 residues are involved in such an interaction. This capsid region is specifically recognized by the monoclonal antibody H7C27. A monoclonal antibody-resistant mutant with the mutation G1217D has been isolated. In the present study, the G1217D mutant was characterized physically and biologically in comparison with the parental HM175 43c strain. The G1217D mutant is more sensitive to acid pH and binds more efficiently to human and rat erythrocytes than the parental 43c strain. In a rat model, it is eliminated from serum more rapidly and consequently reaches the liver with a certain delay compared to the parental 43c strain. In competition experiments performed in vivo in the rat model, the G1217D mutant was efficiently outcompeted by the parental 43c strain. Only in the presence of antibodies reacting specifically with the parental 43c strain could the G1217D mutant outcompete the parental 43c strain in serum, although the latter still showed a remarkable ability to reach the liver. Altogether, these results indicate that the G1217D mutation induces a low fitness phenotype which could explain the lack of natural antigenic variants of the glycophorin A binding site.
    Journal of Virology 05/2012; 86(15):7887-95. · 5.08 Impact Factor
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    ABSTRACT: Hepatitis A is the most common infection of the liver worldwide and is fecal-orally transmitted. Its incidence tends to decrease with improvements in hygiene conditions but at the same time its severity increases. Hepatitis A virus is the causative agent of acute hepatitis in humans and belongs to the Hepatovirus genus in the Picornaviridae family, and it has very unique characteristics. This article reviews some molecular and biological properties that allow the virus to live in a very quiescent way and to build an extremely stable capsid that is able to persist in and out of the body. Additionally, the relationship between the genomic composition and the structural and antigenic properties of the capsid is discussed, and the potential emergence of antigenic variants is evaluated from an evolutionary perspective.
    Future Microbiology 03/2012; 7(3):331-46. · 4.02 Impact Factor
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    ABSTRACT: In spite of annual vaccination campaigns, hepatitis A cases increased in Catalonia (North-East Spain) in the period 2002-2005 calling for the elucidation of the underlying mechanisms associated to the epidemiological shifts. The molecular characterization of the circulating strains to trace their origin and the study of the effects of vaccination on the incidence of sporadic and outbreak-associated cases. Forty-eight different hepatitis A virus (HAV) strains isolated from sporadic and outbreaks cases during 2005-2009 in Catalonia were molecularly characterized. Seventeen out of 48 strains were imported from endemic areas through traveling, immigration and food trade, 12 were endemic strains circulating in the men having sex with men (MSM) group and 1 was from a Roman child. The remaining 18 could not be associated to any specific origin and thus were considered autochthonous. Forty-eight percent of the strains belonged to subgenotype IA, 40% to subgenotype IB and 2% to subgenotype IIIA. The remaining 10% belonged to an undetermined subgenotype equidistant from IA and IB. During the period 2005-2009, the annual attack rates remained around 3.5 and even increased up to 6.5 in the first half of 2009. This increase with respect to the period 1999-2001, in which vaccination campaigns started to be implemented, is explained by an increase in the number of outbreaks. The predominant subgenotypes were IA and IB. However a considerable amount of strains imported from Peru through consumption of contaminated shellfish belonged to an undeterminded subgenotype that may constitute a new candidate subgenotype IC.
    Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology 07/2011; 52(2):98-102. · 3.12 Impact Factor
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    ABSTRACT: Six hepatitis A virus antigenic variants that likely escaped the protective effect of available vaccines were isolated, mostly from men who have sex with men. The need to complete the proper vaccination schedules is critical, particularly in the immunocompromised population, to prevent the emergence of vaccine-escaping variants.
    Emerging Infectious Diseases 04/2011; 17(4):734-7. · 6.79 Impact Factor
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    Rosa M Pintó, M Isabel Costafreda, Albert Bosch
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    ABSTRACT: In the present work, we aimed at determining the relationship between the hepatitis A virus (HAV) numbers in imported frozen coquina clams involved in two hepatitis outbreaks, as well as the risk for human health. Due to HAV unculturability, a standardized TaqMan real-time reverse transcription-PCR controlling the virus/nucleic acid extraction and enzyme efficiencies was employed to figure the exposure dose for clams responsible for hepatitis cases. HAV numbers were then employed to figure the risk of infection based on a dose-response model for echovirus 12. The estimated risk of infection after consumption of lightly cooked clams matched actual attack rates. Our data show that prospective monitoring of bivalve samples may fail to prevent the occurrence of outbreaks, since HAV was detected in 44% of samples directly associated with cases but was undetectable in samples that were randomly collected from the importers and belonged to the same batches. A correlation was nevertheless observed between the prevalence of hepatitis A cases in the harvesting areas and positive HAV isolation in clams, which points to the need to identify and prevent hazards rather than relying on random sampling of finished products to ensure safety. However, when evidence shows that a critical limit of viral contamination has been exceeded in the potential sources of contamination discharging into the shellfish-growing beds, quantitative virological analysis addressing quality assurance and quality control requirements should be performed with the bivalves. This work provides the first evidence of accurate HAV levels in shellfish involved in outbreaks that could be of use for risk assessment purposes.
    Applied and environmental microbiology 10/2009; 75(23):7350-5. · 3.69 Impact Factor
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    ABSTRACT: Hepatitis A virus (HAV), the prototype of genus Hepatovirus, has many biological characteristics that distinguish it from other members of the Picornaviridae family. Among these it is worth of note the need for an intact eIF4G factor for the initiation of translation and thus the inability to shut down host protein synthesis by a similar mechanism as in other picornaviruses. Consequently, HAV must inefficiently compete for the cellular translational machinery and this may explain its poor growth in cell culture. In this context of virus/cell competition HAV has strategically adopted a naturally highly deoptimized codon usage. Accordingly, a low protein synthesis may be expected with those proteins involved in RNA replication existing at limiting concentrations. Thus, a very low translation rate and a very low RNA replication rate may play a role in escaping to host cell defenses, allowing the virus to grow in a quiescent way. This could explain the high specific infectivity of HAV in spite of its naturally deoptimized codon usage, which would indicate non-abortive infections due to the antiviral cell response. Additionally, the deoptimized codon usage conveys in the use of abundant and rare codons. Many clusters of such rare codons are present in the capsid surface playing a seminal role in the highly cohesive stability of the HAV virion. Thus, the slow translation rate, resulting from the accumulation of rare codons, is likely to contribute to the highly stable viral capsid necessary for a prolonged survival outside the host body.
    Virus Research 09/2007; 127(2):158-63. · 2.75 Impact Factor
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    ABSTRACT: Molecular methods for the detection and typing of hepatitis A virus (HAV) strains in sewage were applied to determine its distribution in Cairo and Barcelona. The study revealed the occurrence of different patterns of hepatitis A endemicity in each city. The circulating strains characterized, whether in Cairo or Barcelona, were genotype IB. The effects of a child vaccination programme and the increase in the immigrant population on the overall hepatitis A occurrence in Barcelona were evaluated. While vaccination contributed to a significant decrease in the number of clinical cases, the huge recent immigration flow has probably been responsible for the re-emergence of the disease in the last year of study, in the form of small outbreaks among the non-vaccinated population.
    Epidemiology and Infection 03/2007; 135(2):270-3. · 2.87 Impact Factor
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    M Isabel Costafreda, Albert Bosch, Rosa M Pintó
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    ABSTRACT: A standardized real-time reverse transcription-PCR (RT-PCR) assay has been developed for an accurate estimation of the number of genome copies of hepatitis A virus (HAV) in clinical and shellfish samples. Real-time procedures were based on the amplification of a fragment of the highly conserved 5' noncoding region and detection through an internal fluorescent probe, including TaqMan and beacon chemistries, in one- and two-step RT-PCR formats. The best performance in terms of sensitivity and reproducibility was achieved by a one-step TaqMan RT-PCR, with a sensitivity enabling the detection of 0.05 infectious unit and 10 copies of a single-stranded RNA (ssRNA) synthetic transcript. Standard reagents, such as a mengovirus strain and an ssRNA transcript, were employed as controls of nucleic acid extraction and RT-PCR, respectively. The test proved to be highly specific after a broad panel of enteric viruses was tested. Sequence alignment of target regions of the primers and probe proved them to be adequate for the quantification of all HAV genotypes. In addition, a quasispecies analysis of the mutant spectrum indicated that these regions are not prone to variability, thus confirming their robustness.
    Applied and Environmental Microbiology 07/2006; 72(6):3846-55. · 3.68 Impact Factor
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    ABSTRACT: Hepatitis A virus (HAV) has previously been reported to agglutinate human red blood cells at acidic pHs. Treatment of erythrocytes with different enzymes and chemical reagents indicated that HAV attachment is mediated through an interaction with sialylglycoproteins. HAV hemagglutination could be blocked by incubating the virus with glycophorin A, indicating that this sialylglycoprotein is the erythrocyte receptor. The number of receptors used was estimated to be around 500 per cell. At the same time, HAV-induced hemagglutination could also be blocked by either monoclonal antibody H7C27 or an anti-VP3(102-121) ascitic fluid, indicating that lysine 221 of VP1 and the surrounding VP3 residues lining the capsid pit are involved in HAV binding to erythrocytes.
    Journal of Virology 10/2004; 78(18):9807-13. · 5.08 Impact Factor
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    ABSTRACT: Hepatitis A is the most common among all hepatitis worldwide in spite of an efficient vaccine and improved hygiene. Shellfish-borne outbreaks are still of major concern causing hundreds of cases and huge economical losses in the present context of global food trade. Hepatitis A virus (HAV) is a unique picornavirus with many differences in its molecular biology including both its incapacity to induce the inhibition of the cellular protein synthesis and a highly biased and deoptimized codon usage with respect the cell. The final goal of this intriguing strategy seems to be the need for a fine-tuning control of the translation kinetics, particularly at the capsid coding region, and the underlying mechanism is the use of a right combination of common and rare codons to allow a regulated ribosome traffic rate thus ensuring the proper protein folding. Capsid folding is critical to warrant a high environmental stability for a virus transmitted through the fecal–oral route with long extracorporeal periods. KeywordsHepatitis A-Picornavirus-Shellfish-Bivalves-Food-borne outbreaks
    Food and Environmental Virology 2(3):127-135. · 2.51 Impact Factor