Molecular analysis of HBV genotypes and subgenotypes in the Central-East region of Tunisia

<p>Abstract</p> <p>Background</p> <p>In Tunisia, country of intermediate endemicity for Hepatitis B virus (HBV) infection, most molecular studies on the virus have been carried out in the North of the country and little is known about other regions. The aim of this study was to determine HBV genotype and subgenotypes in Central-East Tunisia. A total of 217 HBs antigen positive patients were enrolled and determination of genotype was investigated in 130 patients with detectable HBV DNA. HBV genotyping methods were: PCR-RFLP on the pre-S region, a PCR using type-specific primers in the S region (TSP-PCR) and partial sequencing in the pre-S region.</p> <p>Results</p> <p>Three genotypes (D, B and A) were detected by the PCR-RFLP method and two (D and A) with the TSP-PCR method, the concordance between the two methods was 93%. Sequencing and phylogenetic analysis of 32 strains, retrieved the same genotype (D and A) for samples with concordant results and genotype D for samples with discordant results. The sequences of discordant genotypes had a restriction site in the pre-S gene which led to erroneous result by the PCR-RFLP method. Thus, prevalence of genotype D and A was 96% and 4%, respectively. Phylogenetic analysis showed the predominance of two subgenotypes D1 (55%) and D7 (41%). Only one strain clustered with D3 subgenotype (3%).</p> <p>Conclusions</p> <p>Predominance of subgenotype D7 appears to occur in northern regions of Africa with transition to subgenotype D1 in the East of the continent. HBV genetic variability may lead to wrong results in rapid genotyping methods and sequence analysis is needed to clarify atypical results.</p

Impaired binding of standard initiation factors eIF3b, eIF4G and eIF4B to domain V of the live-attenuated coxsackievirus B3 Sabin3-like IRES - alternatives for 5′UTR-related cardiovirulence mechanisms

ABSTRACT: Internal ribosome entry site (IRES) elements fold into highly organized conserved secondary and probably tertiary structures that guide the ribosome to an internal site of the RNA at the IRES 3′end. The composition of the cellular proteome is under the control of multiple processes, one of the most important being translation initiation. In each poliovirus Sabin vaccine strain, a single point mutation in the IRES secondary-structure domain V is a major determinant of neurovirulence and translation attenuation. Here we are extrapolating poliovirus findings to a genomic related virus named coxsackievirus B3 CVB3); a causative agent of viral myocarditis. We have previously reported that Sabin3-like mutation (U(473) → C) introduced in the domain V sequence of the CVB3 IRES led to a defective mutant with a serious reduction in translation efficiency and ribosomal initiation complex assembly, besides an impaired RNA-protein binding pattern. With the aim to identify proteins interacting with both CVB3 wild-type and Sabin3-like domain V RNAs and to assess the effect of the Sabin3-like mutation on these potential interactions, we have used a proteomic approach. This procedure allowed the identification of three RNA-binding proteins interacting with the domain V: eIF4G (p220), eIF3b (p116) and eIF4B (p80). Moreover, we report that this single-nucleotide exchange impairs the interaction pattern and the binding affinity of these standard translation initiation factors within the IRES domain V of the mutant strain. Taken together, these data indicate how this decisive Sabin3-like mutation mediates viral translation attenuation; playing a key role in the understanding of the cardiovirulence attenuation within this construct. Hence, these data provide further evidence for the crucial role of RNA structure for the IRES activity, and reinforce the idea of a distribution of function between the different IRES structural domains. VIRTUAL SLIDE: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/6160165131045880

Molecular Analysis of RNA-RNA Interactions between 5’ and 3’ Untranslated Regions during the Initiation of Translation of a Cardiovirulent and a Live-Attenuated Coxsackievirus B3 Strains

Coxsackievirus B3 (CVB3) is a causative agent of viral myocarditis, meningitis and pancreatitis. CVB3 overcome their host cells by usurping the translation machinery to benefit viral gene expression. This is accomplished through alternative translation initiation in a cap independent manner at the viral internal ribosomal entry site. The 5’ untranslated region (5’UTR) of CVB3 genomic RNA is highly structured. It is the site of multiple RNA-protein and RNA-RNA interactions and it plays a critical role during translation initiation. Similar to the 5’UTR, CVB3 3’ untranslated region (3’UTR) also contains secondary structural elements consisting of three stem-loops followed by a poly (A) tail sequence. Long-range RNA-RNA interactions between 5’ and 3’ ends of some viral genomes have been observed. Because of their dual role in translation and replication, the 5’ and 3’UTRs represent promising candidates for the study of CVB3 cardiovirulence. Taking into account that efficient initiation of mRNA translation depends on a temporally and spatially orchestrated sequence of protein-protein, protein-RNA and RNA-RNA interactions, and that, at present, little is known about RNA-RNA interactions between CVB3 5’ and 3’UTRs, we aimed in the present study, to assess a possible RNA-RNA interaction between 5’ and 3’UTRs during the initiation of translation of a wild-type and a previously characterized mutant (Sabin3-like) CVB3 strains and to investigate the effect of the Sabin3-like mutation on these potential interactions. For this purpose, “Electrophoretic Mobility Shift” assays were carried out. Data obtained did not show any RNA-RNA direct interactions between the 5’- and 3’- ends. Therefore, we can suggest that the possible mechanism by which 3’UTR enhances CVB3 IRES activity may be by bridging the 5’ to the 3’ end through RNA-protein interaction and not through RNA-RNA direct contact. However, these findings need to be confirmed by carrying out further experiments

Etudes des mécanismes cellulaires et moléculaires de l'infection à coxsackievirus B4 dans un modèle animal

Nombreuses sont les études épidémiologiques ayant associé les infections à entérovirus, et notamment à coxsackievirus B4 (CV-B4), à l'émergence du diabète de type 1 (DT1) chez les sujets génétiquement prédisposés. Les investigations expérimentales suggèrent que plusieurs mécanismes pathogéniques de l'infection à CV-B4 sont en mesure de participer au processus de destruction des cellules b du pancréas. Il ne peut être exclu que l'infection d'autres tissus que le pancréas puisse contribuer à la genèse de la maladie. Le DT1 étant une pathologie auto-immune, il est important de mieux connaître l'impact de l'infection des organes lymphoïdes, et notamment du thymus, siège central d'établissement de la tolérance vis-à-vis des antigènes du soi, dans le développement de la maladie. Pour commencer, il est nécessaire de connaître le devenir de CV-B4 suite à son introduction dans l'organisme par voie naturelle. Ainsi nous avons mis au point un modèle d'infection de souris Swiss par la souche diabétogène CV-B4 E2 inoculée par la voie orale. Le génome viral, recherché par RT-PCR et RT-PCR semi-nichée, a été retrouvé jusqu'à plus de 70 jours post-infection (p.i.) dans divers tissus : intestin, ganglions mésentériques, coeur, pancréas, mais aussi rate, thymus et sang. Ces résultats suggèrent que CV-B4 E2 peut provoquer une infection systémique avec atteinte des organes lymphoïdes. Afin de mieux comprendre l'interaction entre CV-B4 et le tissu lymphoïde, nous avons infecté des cultures primaires de cellules spléniques et thymiques totales dérivées de souris BALB/c et C3H/HeN par la souche diabétogène CV-B4 E2 et la souche prototype CV-B4 JVB. L'infection est mise en évidence en faisant appel à une approche sensible de RT-PCR semi-nichée. La réplication virale, prouvée par la détection de brins négatifs du génome de CV-B4 dans les cellules et de progénies virales dans les surnageants de culture, semble dépendre du patrimoine génétique de l'hôte puisqu'elle n'a été obtenue qu'avec les cellules dérivées des souris BALB/c. Par ailleurs l'infection ne s'est pas traduite par une réponse IFNa puisque ce dernier, recherché à l'aide d'une méthode biologique, n'a été retrouvé que dans les cultures infectées par le virus de Sendai (SV). L'infection de cellules thymiques par CV-B4 E2 et par CV-B4 JVB, et ses conséquences sur l'expression d'IGF2 ont été étudiées dans un modèle in vitro. IGF2 est une protéine qui participerait à l'éducation des lymphocytes T à tolérer les principaux auto-antigènes des cellules ß. Une lignée murine de cellules épithéliales thymiques, MTE4-14, a été infectée par CV-B4 E2 et CV-B4 JVB. Ces deux souches virales peuvent infecter les MTE de manière persistante avec des conséquences cytologiques différentes : effet cytopathique et cytolyse d'une partie des cellules dans le cas de CV-B4 E2 et pas d'effet notable dans le cas de CV-B4 JVB. L'infection est caractérisée par une production continue de particules infectieuses, des quantités relatives des brins positifs et négatifs d'ARN viral intracellulaire similaires, et dans le cas de CV-B4 E2, par l'infection d'une importante proportion de cellules (attestée par l'immunomarquage de la protéine virale VP1). Par ailleurs, l'infection à CV-B4 E2 s'est traduite par une diminution significative (mise en évidence par une approche semi-quantitative) des transcrits d'IGF2. L'infection de cultures organotypiques de thymus foetal de souris CD-1 a également été étudiée. Nous avons observé que CV-B4 E2, mais pas CV-B4 JVB, peut se répliquer dans ce système comme attesté par la détection de brins négatifs d'ARN viral intracellulaire par RT-PCR quantitative et de particules infectieuses dans le surnageant de culture. CV-B4 E2 ne provoque pas d'altération cytologique ou histologique apparente dans les cultures organotypiques de thymus foetal de souris, mais il est capable d'y entraîner des anomalies des populations thymocytaires (étudiées par cytométrie en flux) suggérant une perturbation du processus de maturation/différenciation des thymocytes. Au total, nos résultats montrent que CV-B4 est capable d'atteindre la rate et le thymus lors d'une infection systémique et que l'infection du thymus peut perturber les fonctions de cet organe. Nos travaux ont permis d'obtenir des données en faveur de l'hypothèse de l' atteinte par CV-B4 du thymus, dont la fonction altérée est susceptible de jouer un rôle dans la pathogenèse du DT1.LILLE2-BU Santé-Recherche (593502101) / SudocSudocFranceF

Ribosomal Initiation Complex Assembly within the Wild-Strain of Coxsackievirus B3 and Live-Attenuated Sabin3-like IRESes during the Initiation of Translation

Abstract: Coxsackievirus B3 (CVB3) is an enterovirus of the family of Picornaviridae. The Group B coxsackieviruses include six serotypes (B1 to B6) that cause a variety of human diseases, including myocarditis, meningitis, and diabetes. Among the group B, the B3 strain is mostly studied for its cardiovirulence and its ability to cause acute and persistent infections. Translation initiation of CVB3 RNA has been shown to be mediated by a highly ordered structure of the 5’-untranslated region (5’UTR), which harbors an internal ribosome entry site (IRES). Translation initiation is a complex process in which initiator tRNA, 40S and 60S ribosomal subunits are assembled by eukaryotic initiation factors (eIFs) into an 80S ribosome at the initiation codon of the mRNA. We have previously addressed the question of whether the attenuating mutations of domain V of the poliovirus IRES were specific for a given genomic context or whether they could be transposed and extrapolated to a genomic related virus, i.e., CVB3 wild-type strain. In thisInt. J. Mol. Sci. 2013, 14 440

Histology of hearts and pancreas of Swiss mice at day 10 after oral inoculation with CVB3 wild type and mutant viruses as described previously [26].

Shown are murine heart (a) and pancreas (b) tissues stained with hematoxylin and eosin from mice inoculated with CVB3 wild type, (c, d) heart and pancreas from a control uninfected mice and (e, f) heart and pancreas from mice inoculated with M3 virus (all 4 mutant viruses induced a similar lack of pathology in mice, S1 Fig). Lymphocyte infiltrations are shown in each organ tissues. Experiments were performed on three mice for each mutant and for each day post inoculation.</p

Enteroviruses and type 1 diabetes: towards a better understanding of the relationship

International audienceEnvironmental factors, especially viruses, are involved in the initiation or the acceleration of type 1 diabetes (T1D) pathogenesis. Epidemiological data strongly suggest that enteroviruses, such as coxsackievirus B4 (CV‐B4), can be associated with T1D. It has been demonstrated that enterovirus infections were significantly more prevalent in at risk individuals, such as siblings of diabetic patients, when they developed anti‐β‐cell autoantibodies or T1D, and in recently diagnosed diabetic patients, compared with control subjects. The isolation of CV‐B4 from the pancreas of diabetic patients strengthened the hypothesis of a relationship between the virus and the disease. Studies performed in vitro and in vivo in animal models helped to discover mechanisms of the infection of pancreas and other tissues, potentially able to play a role in the pathogenesis of T1D. Interestingly, it cannot be excluded that enteroviruses behave as half‐devil half‐angel since experimental studies suggest that, in certain conditions, these agents would be able to protect individuals against the disease. All of the plausible mechanisms by which enterovirus may be related to T1D will be reviewed here