Neutralizing activity induced by the attenuated coxsackievirus B3 Sabin3-like strain against CVB3 infection.

Coxsackievirus B3 (CVB3) causes viral myocarditis, and can ultimately result in dilated cardiomyopathy. There is no vaccine available for clinical use. In the present work, we assessed whether the Sabin3-like mutant of CVB3 could induce a protective immunity against virulent CVB3 Nancy and CVB4 E2 strains in mice by both oral and intraperitoneal (IP) routes. Serum samples, taken from mice inoculated with Sabin3-like, were assayed in vitro for their anti-CVB3 neutralizing activity. CVB3 Sabin3-like was highly attenuated in vivo and was able to induce an anti-CVB3 activity of the serum. However, at 4 days post-CVB3 challenge, significant increased titers of CVB3 neutralizing antibodies were detectable in the sera of immunized mice over the next 6 days. Non-immunized mice challenged with CVB3 Nancy had no anti-CVB3 activity in their sera until 10 days post-infection. CVB3 Nancy induced higher viral titers than did the mutant strain. There was no variation of the neutralizing activity of serum taken from mice immunized with CVB3 Sabin3-like and challenged with CVB4 E2, compared to non-immunized mice. Despite the fact that CVB3 and CVB4 are closely related viruses, virus-neutralizing activity clearly distinguish between these viruses. A variable and limited amount of pancreatic inflammation was seen in some mice 10 days after Sabin3-like inoculation by IP route, whereas there was no evidence of pancreatic damage in mice inoculated by oral route. All immunized mice were protected from myocarditis and pancreatitis at 8 days post-challenge with CVB3 or CVB4 E2. These findings strongly suggest that the mutant strain could be considered a candidate for an attenuated CVB3 vaccine.

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.

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 (U473 → 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.

Role of RNA structure motifs in IRES-dependent translation initiation of the coxsackievirus B3: new insights for developing live-attenuated strains for vaccines and gene therapy.

Internal ribosome entry site (IRES) elements are highly structured RNA sequences that function to recruit ribosomes for the initiation of translation. In contrast to the canonical cap-binding, the mechanism of IRES-mediated translation initiation is still poorly understood. Translation initiation of the coxsackievirus B3 (CVB3), a causative agent of viral myocarditis, has been shown to be mediated by a highly ordered structure of the 5' untranslated region (5'UTR), which harbors an IRES. Taking into account that efficient initiation of mRNA translation depends on temporally and spatially orchestrated sequence of RNA-protein and RNA-RNA interactions, and that, at present, little is known about these interactions, we aimed to describe recent advances in our understanding of molecular structures and biochemical functions of the translation initiation process. Thus, this review will explore the IRES elements as important RNA structures and the significance of these structures in providing an alternative mechanism of translation initiation of the CVB3 RNA. Since translation initiation is the first intracellular step during the CVB3 infection cycle, the IRES region provides an ideal target for antiviral therapies. Interestingly, the 5' and 3'UTRs represent promising candidates for the study of CVB3 cardiovirulence and provide new insights for developing live-attenuated vaccines.

Enteroviral central nervous system infections in children of the region of monastir, Tunisia: diagnosis, laboratory findings of cerebrospinal fluid and clinical manifestations.

Human enteroviruses (HEV) are one of the major causes of central nervous system (CNS) infections in pediatrics. A prospective study was conducted to assess the epidemiological, clinical, and laboratory characteristics of enterovirus (EV) infections of the CNS in children under 15-years-old, suspected of having viral CNS infections and admitted to the Pediatric Department of Monastir University Hospital, Tunisia. Enteroviral RNA was detected by 5' NCR nested RT-PCR assay in 33 % (20 out of 60) of cerebrospinal fluid specimens, whereas only six samples (10 %) were EV positive in cell culture. EV-positive patients were clustered according to their clinical manifestations, predominantly diagnosed as aseptic meningitis (65 %) and meningoencephalitis (20 %). Fever, headache, vomiting, and neck stiffness were the most pronounced symptoms. Pleocytosis with the predominance of lymphocytes was observed in 60 % of EV positive specimens. Although patients suffering from EV infections were encountered throughout the year, most occurred during spring and summer months. Using VP1-2A nested RT-PCR and sequence analysis, three of the 20 positive HEV were identified as Echovirus (E)-9. This is the first report of a cluster of aseptic meningitis cases caused by E-9 in Monastir.

Effects of the Sabin-like mutations in domain V of the internal ribosome entry segment on translational efficiency of the Coxsackievirus B3.

The domain V within the internal ribosome entry segment (IRES) of poliovirus (PV) is expected to be important in its own neurovirulence because it contains an attenuating mutation in each of the Sabin vaccine strains. In this study, we try to find out if the results observed in the case of Sabin vaccine strains of PV can be extrapolated to another virus belonging to the same genus of enteroviruses but with a different tropism. To test this hypothesis, we used the coxsackievirus B3 (CVB3), known to be the most common causal agent of viral myocarditis. The introduction of the three PV Sabin-like mutations in the equivalent positions (nucleotides 484, 485, and 473) to the domain V of the CVB3 IRES results in significant reduced viral titer of the Sabin3-like mutant (Sab3-like) but not on those of Sab1- and Sab2-like mutants. This low titer was correlated with poor translation efficiency in vitro when all mutants were translated in rabbit reticulocyte lysates. However, elucidation by biochemical probing of the secondary structure of the entire domain V of the IRES of Sabin-like mutants reveals no distinct profiles in comparison with the wild-type counterpart. Prediction of secondary structure by MFOLD program indicates a structural perturbation of the stem containing the Sab3-like mutation, suggesting that specific protein-viral RNA interactions are disrupted, preventing efficient viral translation.