Jawhar Gharbi

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

Environmental 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. Copyright

Prolonged viral RNA detection in blood and lymphoid tissues from Coxsackievirus B4 E2 orally-inoculated Swiss mice

The spreading of viral RNA within Swiss Albino mice orally inoculated with coxsackievirus B4 E2 strain (CVB4 E2) was studied by using RT‐PCR and semi‐nested‐RT‐PCR methods. Viral RNA was detected in various organs: pancreas, heart, small intestine, spleen, thymus, and blood at various postinfectious (p.i.) times ranging from 8 hr to 150 days. Our results show that (i) outbred mice can be infected with CVB4 E2 following an oral inoculation, which results in systemic spreading of viral RNA, (ii) CVB4 E2 infection can be associated with a prolonged detection of viral RNA in spleen, thymus and blood, up to 70 days p.i. and further in other organ tissues.

Immunology in the clinic review series; focus on type 1 diabetes and viruses: enterovirus, thymus and type 1 diabetes pathogenesis

OTHER THEMES PUBLISHED IN THIS IMMUNOLOGY IN THE CLINIC REVIEW SERIES

Comparison of a rapid culture method combining an immunoperoxidase test and a group specific anti-VP1 monoclonal antibody with conventional virus isolation techniques for routine detection of enteroviruses in stools

In order to shorten the time required for the detection of enteroviruses in stool specimens, an 18‐h immunoperoxidase test combining low‐speed centrifugation and the use of a group specific anti‐VP1 monoclonal antibody (5‐D8/1, Dako) was developed. This rapid culture assay (RCA) was compared blindly to a conventional culture assay (CCA) on a panel of 180 childrens stool specimens received for routine diagnosis of enterovirus infection. The same cell lines (human embryonic fibroblasts and KB continuous cell line) were used in both tests. Discrepancies in results were analysed by a PCR technique with primers located in a conserved part of the 5′ non‐coding region of the enterovirus genome. Fourteen specimens were positive and 158 were negative with both tests. Four samples were positive with the RCA yet negative with the CCA and 3 others showed the opposite pattern; an additional sample positive by RCA was uninterpretable by CCA due to bacterial contamination. Subsequent PCR testing of these 8 samples showed no discrepancies; all were positive. Using CCA as the reference, the sensitivity and specificity of RCA were 77.8 and 98% respectively. Kinetic studies using enterovirus isolates demonstrated that RCA was much more sensitive than CCA during the first three days of culture. These results further suggested that RCA sensitivity could be improved by a factor of at least 10 times by prolonging the incubation period by 24 hr. With this change, the RCA assay described below is suggested as a rapid alternative to CCA for the routine diagnosis of enterovirus infection in stool specimens. When an identification at the serotype level is required, samples found positive using RCA could then be subjected to CCA. J. Med. Virol. 54:204–209, 1998.

Persistent Infection of Thymic Epithelial Cells with Coxsackievirus B4 Results in Decreased Expression of Type 2 Insulin-Like Growth Factor

ABSTRACT It has been hypothesized that a disturbance of central self-tolerance to islet β cells may play a role in the enteroviral pathogenesis of type 1 diabetes. Whether enteroviruses can induce an impaired expression of β-cell self-antigens in thymic epithelial cells has been investigated in a murine thymic epithelial (MTE) cell line. This cell line was permissive to the diabetogenic group B4 coxsackievirus (CV-B4) strain CV-B4 E2 and spontaneously expressed type 2 insulin-like growth factor (Igf2), the dominant self-antigen of the insulin family. In this model, a persistent replication of CV-B4 E2 was obtained, as attested to by the prolonged detection of intracellular positive- and negative-strand viral RNA by reverse transcription-PCR (RT-PCR) and capsid protein VP1 by immunofluorescent staining and by the release of infectious particles in culture supernatants. The chronic stage of the infection was characterized by a low proportion of VP1-positive cells (1 to 2%), whereas many cells harbored enteroviral RNA, as displayed by RT-PCR without extraction applied directly to a few cells. Igf2 mRNA and IGF-2 protein were dramatically decreased in CV-B4 E2-infected MTE cell cultures compared with mock-infected cultures, whereas housekeeping and interleukin-6 (Il6) gene expression was maintained and Igf1 mRNA was decreased, but to a lower extent. Inoculation of CV-B3, CV-B4 JVB, or echovirus 1 resulted in a low level of IGF-2 in culture supernatants as well, whereas herpes simplex virus 1 stimulated the production of the protein. Thus, a persistent infection of a thymic epithelial cell line with enteroviruses like CV-B4 E2 can result in a disturbed production of IGF-2, a protein involved in central self-tolerance toward islet β cells.

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.

Infection of primary cultures of murine splenic and thymic cells with coxsackievirus B4.

Infection of primary cultures of total splenic and thymic cells from BALB/c and C3H/HeN mice with CVB4 E2 and JVB strains has been investigated. The presence of positive‐strand viral RNA within cells was determined by semi‐nested RT‐PCR, and viral replication was attested by detection of intracellular negative‐strand viral RNA and by release of infectious particles in culture supernatants. Viral replication occurred with both CVB4 strains to an extent dependent on the genetic background of the host. No interferon‐α production was detected in the supernatants of CVB4‐infected cultures using biological titration. Together these results suggest that infection of splenic and thymic cells can play a role in virus dissemination, and therefore in the pathophysiology of CVB4 infections.

Immunoglobulin G-dependent enhancement of the infection with Coxsackievirus B4 in a murine system

ABSTRACT It was demonstrated that specific IgG can enhance the infection with CV-B4, in vitro, in the human system. This enhancement could be involved in the pathophysiology of CV-B4 induced diseases. To investigate further the role of enhancing IgG in the infection with CV-B4 E2 in vivo, animal models are needed. Therefore, it was decided to assess whether inoculation of CV-B4 E2 to mice results in the appearance of IgG able to enhance the infection with this virus. Swiss albino mice were inoculated with CV-B4 E2 intraperitoneally. Serum samples were obtained from tail vein blood collected from day 0 to day 80 p.i. IgG were isolated by Protein G affinity chromatography. Seroneutralisation assays were carried out. In total murine spleen cells cultures inoculated with CV-B4 E2 mixed with various dilutions of serum or IgG samples, the enhancing activity was assayed through i) the antiviral activity titer of supernatants ii) the detection of intracellular viral RNA by RT-PCR iii) the level of infectious particles in supernatants. In most serum samples (76/105), neutralizing and enhancing activities were detected peaking between days 14 and 30 p.i and were higher in sera from mice inoculated with 2.106 TCID50 units than with lower doses. The enhancing activity was due to the IgG-enriched fraction of serum from CV-B4 E2 infected animals but not from control animals. These data show that IgG from immune mice can enhance the infection of splenocytes with CV-B4 E2 in vitro and open the way to explore whether such an enhancing activity can play a role in vivo.

Nucleotide sequences of IRES domains IV and V of natural ECHO virus type 11 isolates with different replicative capacity phenotypes.

ECHO viruses (ECV) belong to the enterovirus genus of the Picornaviridae family and are the most frequently isolated from clinical and environmental samples. They are responsible for a wide variety of clinical syndromes involving most organs of the human body. We previously postulated that some of the variations in the recognition of ECHO virus type 11 (ECV 11) strains by a group specific monoclonal antibody (Mab) which we have studied could be explained by variations in their replicative capacity in cell culture and variations within the 5′ nontranslated region (5′ NTR) of their genomes. To support this hypothesis, the replicative capacity in cell culture and the nucleotide sequences of domains IV and V of the IRES of the genome of five ECV11 strains (the Gregory reference strain and four wild isolates) were determined, and analysed. Our results indicate that the replicative capacity of wild ECV11 isolates studied by one-step growth cycle in both HEp-2 and Vero cell cultures showed variations among strains in comparison with the Gregory reference strain. The clinical ECV11 strains replicated as well as the reference strain, however environmental strains displayed a phenotype with a significant reduction of replication. The sequences of ECV 11 strains showed significant conservation with that of the poliovirus (PV1) Mahoney strain The comparative examination of the predicted secondary structures revealed, that the nucleotide variations did not affect the secondary structure of stem-loop structure IV and V in the IRES element, however differences were especially observed in the apical stem region (nucleotides 483 to 509) of the domain V of the ECV11 strains and resulted in modification of the central stem structure.

Role of GNRA Motif Mutations within Stem-Loop V of Internal Ribosome Entry Segment in Coxsackievirus B3 Molecular Attenuation

The lengthy 5′ nontranslated region of coxsackievirus B3 (CVB3) forms a highly ordered secondary structure containing an internal ribosome entry segment (IRES), which plays an important role in controlling viral translation and pathogenesis. The stem-loop V (SL-V) of this IRES contains a large lateral bulge loop which encompasses two conserved GNRA motifs. In this study, we analyzed the effects of point mutations within the GNRA motifs of the CVB3 IRES. We characterized in vitro virus production and translation efficiency and we tested in vivo virulence of two CVB3 mutants produced by site-directed mutagenesis. The GNAA1 and GNAA2 RNAs displayed decreased translation initiation efficiency when translated in rabbit reticulocyte lysates. This translation defect was correlated with reduced yields of infectious virus particles in HeLa cells in comparison with the wild type.When inoculated orally into Swiss mice, both mutant viruses were avirulent and caused neither inflammation nor necrosis in hearts. These results highlight the important role of the GNRA motifs within the SL-V of the IRES of CVB3, in directing translation initiation.