Hela Jaïdane

Coxsackievirus B4 and type 1 diabetes pathogenesis: contribution of animal models

The role of enteroviruses, in particular type B coxsackieviruses (CV‐B), in type 1 diabetes (T1D) pathogenesis is supported by epidemiological, clinical and experimental observations.

Immunology in the clinic review series; focus on type 1 diabetes and viruses: role of antibodies enhancing the infection with Coxsackievirus-B in the pathogenesis of type 1 diabetes

OTHER THEMES PUBLISHED IN THIS IMMUNOLOGY IN THE CLINIC REVIEW SERIES

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

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.

Coxsackievirus B4 infection of murine foetal thymus organ cultures.

The infection of foetal thymus with coxsackievirus B4 (CV‐B4) E2 has been studied ex vivo by using CD‐1 mice on foetal day 14, as a ready source of organs for experimentation to investigate the hypothesis of the role of thymic viral infections in the pathogenesis of type 1 diabetes. The replication of CV‐B4 E2 in murine foetal thymus organ cultures has been demonstrated by evaluating the levels of positive‐ and negative‐stranded viral RNA in cells by using a real‐time quantitative RT‐PCR method and by determining titres of infectious viral particles in culture supernatants for 7 days post‐infection (p.i.). Staining of tissue sections with an anti‐cytokeratin antibody and haematoxylin–eosin showed that CV‐B4 infection had no visible effect on cell survival and organ integrity. Cell counts in mock‐ and virus‐infected foetal thymus organ cultures increased from day 1 through day 7, and live cell numbers were comparable in both conditions as shown by Trypan blue exclusion test and 7‐amino‐actinomycin D staining of thymocytes. Compared with controls on day 7 p.i., cytofluorometric analyses on cells from CV‐B4 E2‐infected foetal thymus organ cultures displayed a marked increase in the percentage of the most immature CD3−CD4−CD8− thymocytes, and a decrease in the percentage of immature CD3−CD4+CD8+ cells, together with an increase in the percentage of mature CD3+CD4+ and CD3+CD8+ cells. These data show that CV‐B4 E2 disturbs T‐cell maturation and differentiation processes in infected murine foetal thymus organ cultures and provide evidence of a suitable system to investigate the effect of viruses in T‐cell differentiation. J. Med. Virol. 80:659–666, 2008.

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.