• CVB4;
  • IFNα;
  • primary cultures;
  • splenic and thymic cells


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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.

List of Abbreviations: 

cytopathic effect


coxsackievirus B


fetal calf serum






sendai virus


vesicular stomatitis virus

Type B coxsackieviruses, members of the enterovirus genus of the Picornaviridae family, are small non-enveloped positive-strand RNA viruses frequently implicated in a broad spectrum of acute and chronic human diseases, such as aseptic meningitis, myocarditis, dilated cardiomyopathy and type 1 diabetes. CVB4, being the serotype most frequently detected in patients with type 1 diabetes (1–4), has been used in murine model based investigations aiming to understand the pathophysiological mechanisms of the infection, and those leading to type 1 diabetes and has been evidenced in lymphoid organs, in particular spleen and thymus, and in blood (5–9). It has been suggested that the infection of those key organs by CVB4 can disturb their functions and lead to immunological disorders (6), which in turn can highly influence the subsequent pathophysiology of the infection. Thymus infection can facilitate immune tolerance towards viral antigens and thus favor infection of other tissues (10, 11) or, by contrast, can lead to a defect in self-tolerance which can play a role in the pathogenesis of auto-immunity. Furthermore, the spleen may play a role as a reservoir from which the virus can spread via the bloodstream.

In order to investigate this hypothesis, it is essential to have a better knowledge of interactions between CVB4 and these tissues, with regard to both the pattern of infection and the production of IFNα, a cytokine that has a critical role in both immune response and antiviral protection (12, 13). It has been reported by our team that cultures of human peripheral blood mononuclear cells are difficult to infect with CVB4 and do not produce IFNα, whereas CVB4 infection and CVB4-induced production of IFNα can be enhanced through an antibody-dependent mechanism (14, 15). The infection of splenic or thymic cells with CVB4 remains poorly understood in both human and animal models.

Because access to human spleen and thymus for experimentations is limited, in the present study we decided to use murine organs to explore the interaction of CVB4 with thymus and spleen. The aim was to investigate infection of primary cultures of total murine splenic and thymic cells with both CVB4 E2 (diabetogenic) and CVB4 JVB (prototype) strains, and to determine whether CVB4 could induce the production of IFNα by these cells.

Seven week old female BALB/c (Centre d'Elevage Janvier, Le Genest St Isle, France) and C3H/HeN mice (CDTA, CNRS, Orléans, France), treated according to general ethical rules and maintained under specific pathogen-free conditions with unlimited access to food and water, were killed by cervical dislocation, and their spleens and thymi aseptically removed and used to prepare primary cultures of splenic and thymic cells as described previously (16). Cells were prepared on ice, depleted of erythrocytes by hypotonic shock, then suspended in RPMI-1640 (Eurobio, Paris, France) supplemented with 10% FCS (Sigma, St Louis, MO, USA), 1% L-glutamine (Gibco BRL, Invitrogen, Gaithersburg, MD, USA), 50 μg/ml streptomycin, 50 IU/ml penicillin (BioWhittaker, Walkersville, MD, USA) and 10−5 M β-mercaptoethanol (Sigma, St Louis, MO, USA). Then cells were plated onto 24-well culture plates (Falcon, Oxnard, CA, USA) at 106 cells per well. with 1.05 × 104 TCID50 per well of either the diabetogenic CVB4 E2 virus (kindly provided by J.W. Yoon, Julia McFarlane Diabetes Research Center, Calgary, Alberta, Canada) or the prototype CVB4 JVB virus (kindly provided by J. Almond, Aventis Pasteur, Marcy-L'Etoile, France) in a final volume of 500 μl of RPMI-1640 culture medium, and then incubated at 37 C in a humidified atmosphere with 5% CO2. The next day 500 μl of RPMI-1640 were added to each well. Some wells containing splenic cells from BALB/c mice were inoculated with 100 pfu/cell of Sendai virus (SV) in a final volume of 500 μl per well. SV (kindly provided by D. Garcin, Department of Genetics and Microbiology, University of Geneva, Switzerland) was cultivated as described previously (17). Mock-infected cultures were inoculated with RPMI-1640. The viral inoculum was maintained in the culture medium of cells during the entire incubation period (maximum duration five days).

Total RNA was extracted from murine splenic and thymic cells harvested at one, two, three and five days post infection (p.i.). by using the acid-guanidium thiocyanate-phenol-chloroform (Tri-Reagent, Sigma) method. DNase treated, extracted RNA was submitted to a strand-specific two-step RT-PCR for CVB4 RNA detection by using the Durascript RT-PCR kit (Sigma) according to the manufacturer's instructions. Either the anti-sense (007) or the sense (008) primer (Sigma-Proligo, St Louis, MO, USA), selected within the 5′ untranslated region of the CVB4 genome (7), was used as a template in the synthesis of cDNA for positive- or negative-strand–CVB4 RNA respectively. All results being negative, the first PCR was followed by a semi-nested amplification by using the same Durascript RT-PCR kit with each anti-sense (007) and internal sense (006) primer (Sigma-Proligo) generating a 157 bp fragment (7). For each sample, glyceraldehyde-3-phosphate dehydrogenase mRNA was amplified in the same way, using the primers described by Simpson et al. to generate a 191 bp fragment (18), and used as a positive control to demonstrate the integrity of the extract and the absence of RT-PCR inhibitors. The amplified products were analyzed by electrophoresis on a 2% agarose gel containing 0.5 mg/ml of ethidium bromide (Sigma) and visualized by using the Gel Doc 2000 system (Bio-Rad, Hercules, CA, USA).

As shown by gels in Figure 1A, both CVB4 E2 and CVB4 JVB intracellular RNA can be detected in primary cultures of murine splenic and thymic cells. Positive-strand CVB4 RNA was found in both CVB4 E2- and CVB4 JVB-infected primary cultures of total splenic cells (from days one through five p.i.) whatever the source of cells: BALB/c or C3H/HeN mice (see Fig 1A). Positive-strand CVB4 RNA was also found in CVB4 E2 and CVB4 JVB infected primary cultures of total thymic cells (from days one through five p.i. and on days two and three p.i., respectively) from BALB/c mice, and in CVB4 JVB-infected primary cultures of thymic cells from C3H/HeN mice (on days three and five p.i.), whereas it was not detected in CVB4 E2-infected primary cultures of thymic cells from these mice (see Fig. 1A). As far as negative-strand CVB4 RNA is concerned, it was found in primary cultures of both total splenic and thymic cells from BALB/c mice (from days one through five p.i. in both CVB4 E2- and CVB4 JVB-infected splenic cells and in CVB4 E2-infected thymic cells, and on days two and three p.i. in CVB4 JVB-infected thymic cells), whereas it was only found in CVB4 JVB-infected primary cultures of total splenic cells from C3H/HeN mice (from days one through three p.i.) but not under other conditions (see Fig 1A). Mock-infected cultures, which were submitted to the same sampling, extraction and amplification procedures, did not show any CVB4 RNA specific band.


Figure 1. Kinetics of CVB4 infection of murine splenic and thymic cells. (a) Agarose gel electrophoresis of amplicons specific to the positive and negative strands of CVB4 genome. A strand-specific RT-PCR followed by a semi-nested amplification of a 157 bp fragment, were carried out on total RNA taken from CVB4 E2- and CVB4 JVB-infected splenic and thymic cells. Samples were taken at different days p.i. as shown by the lanes numbers. Lane L corresponds to a 100 bp DNA molecular size ladder. Lanes NC and PC correspond to negative and positive controls, respectively. (b) Release of infectious particles by murine splenic and thymic cells infected with CVB4 E2- and CVB4 JVB. Viral titers in culture supernatant were determined on Vero cells by using the Reed-Muench's method and are expressed as TCID50/ml. Similar results were obtained in two separate experiments.

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The production of infectious particles by CVB4-infected murine splenic and thymic cells was assayed at one, two and three days p.i by determining virus titers in culture supernatants inoculated to Vero cell (BioWhittaker) monolayers and by using the Reed-Muench's method (19). Results are presented in Figure 1B, expressed as TCID50/ml. The value given on day zero represents the inoculum which was maintained in the culture during the entire follow-up period. Between day zero and day one, a marked increase in virus titers was observed in BALB/c mice-derived primary cultures of total splenic and thymic cells inoculated with either CVB4 E2 or CVB4 JVB. The viral titer values in supernatants collected on days one, two and three were roughly similar as shown by the “plateau” form of the curve (see Fig 1B). In contrast, both primary cultures of total splenic and thymic cells from C3H/HeN mice did not produce detectable amounts of infectious particles, since viral titers were lower or equal to that obtained for residual virus on day zero. In cultures of total thymic cells, a continual decrease in viral titers was observed. No infectious particles were detected in supernatants of mock-infected cultures.

Then, a biological assay, in which the antiviral activity of culture supernatants is evaluated by protection of murine L-929 fibroblasts (kindly provided by T Jouault, Lille, France) against VSV-induced cytopathic effect (CPE), was optimized for evaluating the concentration of mouse IFNα in supernatants from SV-stimulated or CVB4-infected cultures as follows. Briefly, 50 μl of DMEM containing 4.5 g/l glucose and sodium pyruvate (Gibco BRL), and supplemented with 5% FCS, 1% L-glutamine, 50 μg/ml streptomycin and 50 IU/ml penicillin, were first distributed into each well of a 96-well flat-bottomed microtiter plate (Falcon). Fifty microliters of supernatants from virus- or mock-infected cultures were then added into the first wells and then serially two-fold diluted. Versène trypsine (Eurobio) dissociated L-929 cells (3.125 × 104 cells per well in 100 μl of the above described supplemented DMEM) were then added into each well. After 18 hours of incubation at 37°C in a humidified atmosphere with 5% CO2, supernatants were removed and cells were inoculated with 100 μl per well of VSV (kindly provided by P Lebon, Paris) diluted in DMEM at 37 TCID50/ml (titer on L-929 cells corresponding to the highest dilution giving 100% CPE after 18 hr of incubation). Virus-induced CPE was assessed by microscopic examination. The inverse of the highest dilution providing 100% protection of the cells from virus-induced CPE was considered as the endpoint for IFNα activity. IFNα concentrations in IU per milliliter were inferred from the natural murine National Institutes of Health IFNα standard Ga02-901-511. The assay detection limit was 3 ± 0.43 IU of IFNα/ml.

Results presented in Figure 2 show that culture supernatants from SV-stimulated cultures harvested 24 hours p.i. and every day up to five days p.i., were able to protect L-929 cells against the CPE of VSV (see Fig. 2Aa and B). The protective effect was inhibited when the samples were preincubated in the presence of anti-IFNα neutralizing antibodies. High concentrations of IFNα were found in the supernatants of primary cultures of total splenic cells inoculated with SV (165 IU/ml). When those samples were submitted to ultraviolet irradiation (30 minutes) before testing, the protective effect was unchanged. The addition of SV-containing medium to L-929 cell cultures did not protect them against the CPE of VSV, which showed that residual SV contained in culture supernatants was not involved in the protective effect observed in our experiments. By contrast, there was no protective effect of culture supernatants from mock- and CVB4-infected splenic and thymic cells (see Fig. 2Ab and B). In order to determine whether residual CVB4 E2 or CVB4 JVB can interfere in the biological assay used in our experiments for detecting IFNα, culture medium samples containing those viruses were added to L-929 cell layers. CVB4 did not induce any cytopathic effect in L-929 cell cultures and did not alter VSV-induced cytolysis of the cells.


Figure 2. IFNα response in CVB4-infected splenic and thymic cells cultures. (A) Microscopic observation of L-929 cells protected from VSV-induced CPE when inoculated with supernatants from SV-stimulated total splenic cells (a) and not protected when inoculated with supernatants from CVB4-infected total splenic or thymiuc cells (b). Photographs were taken 18 hours after inoculation with VSV. Original magnification, ×400. (B) Biological titration of IFNα released in culture supernatant fluids of mock-, CVB4- and SV-infected splenic and thymic cells from BALB/c and C3H/HeN mice. Results are expressed as IFNα IU/ml of culture supernatant and are from one experiment representative of two that gave similar results.

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In vitro studies are necessary to explore the interaction between viruses and organs. Studies carried out in human cells have shown that CVB can replicate in lymphoid cell lines (20) and primary monocytes (21), whereas the replication of CVB in peripheral blood mononuclear cells has not been found (15). Furthermore, it has been reported by our team that CVB4 can infect human thymic epithelial cells in vitro (22) and thymocytes within fetal thymus organ cultures (23).

Access to human tissues being very limited, it is important to develop alternative models as a tissue source for experimentation. In the current work, we used murine tissues to study infection of the spleen and thymus. To the best of our knowledge, this is the first report of infection of primary cultures of total splenic and thymic cells with an enterovirus.

In this study designed to investigate the interaction between virus and cells, virus infection was carried out on freshly harvested total splenic and thymic cells because they encompass all the cell populations, thus producing more natural conditions than continuous cell lines. In addition, in primary cultures, properties of cells such as IFNα production are not affected.

Detection of viral RNA positive strands in splenic and thymic cells shows that they can be infected with CVB4 in vitro. The data regarding the detection of intracellular CVB4 RNA negative strands together with the production and release of infectious particles in culture supernatants samples, show that CVB4 can replicate in splenic and thymic cells from BALB/c mice, but little or not at all in those from C3H/HeN mice. Cells from C3H/HeN mice were maintained in culture for a longer time (up to ten days) than those from BALB/c mice (five days), however intracellular viral RNA and progenies were still not detected (data not shown). The difference in susceptibility between cells from BALB/c and those from C3H/HeN mice is very likely linked to the different genetic backgrounds of these murine strains. Our results are in agreement with those of a previous study based on infection of suckling mice, which showed that BALB/c mice were the most susceptible and C3H mice the most resistant to CVB (24).

In our experiments, the patterns of infection with CVB4 E2 and CVB4 JVB were similar in cells derived from BALB/c, whereas there were some differences between these strains for cells derived from C3H/HeN mice. Intracellular CVB4 JVB positive-strand RNA was found within thymic cells whereas CVB4 E2 positive-strand RNA was not, and intracellular CVB4 JVB negative-strand RNA was found within splenocytes whereas CVB4 E2 negative-strand RNA was not. Together these data show that the susceptibility of cells from the same host towards two variants of CVB4 can be different.

In previous studies from other researchers it has been reported that SV can stimulate the production of IFNα by murine cells such as a splenic dendritic cell line from BALB/c mice (25), and splenocytes and bone marrow cells derived from mouse embryos (26), whereas this is the first report of IFNα production by adult mouse spleen cells cultured in the presence of SV.

IFNα was not detected in culture supernatants of CVB4-infected splenocytes from BALB/c mice. This is not due to a defect in IFNα production by these cells since we have demonstrated that they produce IFNα in response to SV. The absence of IFNα in supernatants from CVB4-infected cultures of splenic and thymic cells from BALB/c and C3H/HeN mice may be due to the relatively low level of infection (the viral genome was detectable only with the sensitive method of semi-nested RT-PCR), and to the fact that, in contrast with other viruses such as SV and herpes simplex virus 1, CVB4 is known to be a weak IFNα inducer (27).

The absence or low concentration (under the limit of the bioassay) of IFNα in culture supernatants of CVB4-infected splenic and thymic cells explains the successful detection of infectious particles in Vero cells in our experiments. Significant amounts of IFNα in culture supernatants of CVB4-infected splenic and thymic cells would have inhibited the infection of Vero cells and hence would have prevented the detection of infectious particles. Interestingly, the absence of IFNα production by CVB4-infected splenic or thymic cells in-vivo may have important consequences on the pathophysiology of the infection. The absence of that antiviral agent could facilitate virus dissemination to other cells and other tissues.

Previous investigations conducted by our team showed that CVB4 was a poor inducer of IFNα in cultures of human peripheral blood mononuclear cells. A synthesis of IFNα, principally by CD14+ monocytes, was however obtained when CVB4 was incubated beforehand with plasma containing non-neutralizing antibodies able to enhance the IFNα-inducer effect of CVB4 (14, 15). Using splenic cells from BALB/c or C3H/HeN mice, future studies will be conducted in our laboratory to determine whether an antibody-dependent enhancement of the CVB4-induced production of IFNα can be obtained in the murine system.

In conclusion, CVB4 can infect and replicate in murine splenic and thymic cells without inducing IFNα production by these cells, which could facilitate spreading of the virus. Together our data suggest that splenic and thymic cells can play a role in CVB4 dissemination and therefore in the pathophysiology of diseases induced by that virus.


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Hela Jaïdane was supported by the Comité Mixte de Coopération Universitaire Franco-Tunisien (CMCU) with grants from Egide Paris. This work was supported by the Ministry for Scientific Research, Technology and the Development of Competencies (Lab-MDT-01), Tunisia, Ministry for the National Education of Research and Technology, UniversityLille 2 (UPRES EA3610), France, CHRU Lille, France and by the EU FP6 Integrated Project EURO-THYMAIDE (Contract LSHB-CT-2003-503410).


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