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Article

Anti-Coxsackievirus B4 Activity of Serum and Saliva from Mice Exposed to the Virus via the Mucosal Route

by
Chaldam Jespère Mbani
1,2,
Magloire Pandoua Nekoua
1,
Laurine Couture
1,
Arthur Dechaumes
1,
Cyril Debuysschere
1,
Famara Sane
1,
Enagnon Kazali Alidjinou
1,
Donatien Moukassa
2 and
Didier Hober
1,*
1
Laboratoire de Virologie ULR3610, Université de Lille, CHU Lille, 59000 Lille, France
2
Laboratoire de Biologie Cellulaire et Moléculaire, Faculté des Sciences et Technique, Université Marien Ngouabi, Brazzaville BP 69, Congo
*
Author to whom correspondence should be addressed.
Microorganisms 2026, 14(2), 289; https://doi.org/10.3390/microorganisms14020289
Submission received: 17 December 2025 / Revised: 22 January 2026 / Accepted: 23 January 2026 / Published: 27 January 2026
(This article belongs to the Section Virology)
Browse Figures
Versions Notes

Abstract

Coxsackieviruses B are single-stranded RNA viruses belonging to the Enterovirus genus and are associated with various clinical outcomes, ranging from acute infections to chronic diseases, such as type 1 diabetes (T1D). It was previously shown that inoculation of Swiss albino mice with CVB4 by the intraperitoneal route induced both anti-CVB4 neutralizing and enhancing activities of serum. This study aimed to investigate the humoral immune response of mice inoculated with CVB4 by the mucosal route. Mice were inoculated orally or intranasally with CVB4, and the anti-CVB4 neutralizing activity of serum and saliva was assessed by a cell culture neutralization assay. Anti-enterovirus (EV) IgG and IgA antibodies were detected in serum and saliva, respectively, by ELISA. The serum-dependent enhancement of CVB4 infection in cultures of murine splenocytes was evaluated by detecting intracellular viral RNA using RT-qPCR. At day 45 post-inoculation, an anti-CVB4 neutralizing activity, the extent of which depends on the amount of inoculated infectious particles, was detected in the serum of mice exposed orally or intranasally. An increase in anti-CVB4 neutralizing activity was observed in the saliva of mice inoculated orally or intranasally during the follow-up. Oral or intranasal inoculation of CVB4 induced a systemic IgG and mucosal IgA response. In addition, serum from these mice harbored an anti-CVB4 enhancing activity in vitro. These data indicate that Swiss albino mice exposed to CVB4 via the mucosal route constitute a potentially useful model for testing strategies to promote the production of protective mucosal and systemic anti-CVB4 antibodies and for verifying whether or not enhanced antibodies are produced.

1. Introduction

Group B coxsackieviruses (CVB1–6), members of the Enterovirus genus of the Picornaviridae family, are non-enveloped, positive-sense single-stranded RNA viruses [1]. Their genome is contained in an icosahedral capsid consisting of sixty capsomeres, each of which is composed of four structural proteins designated VP1, VP2, VP3, and VP4 [2]. These viruses are ubiquitous and are mainly transmitted by the faecal–oral route. They cause infections in human beings that are often asymptomatic, but they can also cause acute diseases such as pancreatitis, encephalitis, bronchiolitis, pneumonia, myocarditis, and meningitis [3,4,5,6]. Epidemiological data have shown a strong association between enterovirus infections, particularly CVB4, and type 1 diabetes (T1D) [7]. Indeed, markers of enterovirus infection (proteins and RNA) were observed more often in patients with T1D than in controls [8,9,10,11,12,13].
Antibodies are an essential component of the antiviral immune response. Neutralizing antibodies can inhibit viral infection by binding to specific viral surface proteins. However, non-neutralizing antibodies or sub-neutralizing concentrations of antibodies can enhance the viral infection of cells in vitro and in vivo [14,15]. The enhancement of viral infection by antibodies has been reported for several viruses, including dengue virus [16,17], and has also been observed in the case of CVB4 in vitro in a human system [14]. Previous studies have shown that intraperitoneal inoculation of CVB4 to Swiss albino mice induces neutralizing activity but also enhancing activity of serum, which is capable of increasing the infection of murine splenocytes with CVB4 in vitro [18]. Furthermore, inoculation of mice with CVB4 resulted in the production of serum IgG with an anti-CVB4 enhancing activity; in these animals, a second infection with the same virus resulted in high viral load in organs, particularly in the pancreas, pancreatic tissue damage, and hyperglycemia [18]. These observations suggest that the virulence of CVB4 may be enhanced by antibodies.
Mucosal humoral immunity is crucial for preventing enteroviral infections [14]. It has been reported that mouse intranasal and oral immunization can induce neutralizing antibodies against enteroviruses such as enterovirus A71 (EV-A71), CVB3, and CVA6 [19,20,21]. However, the antibody response to mucosal exposure to CVB4 has not been studied. Most in vivo models of CVB4 infection are based on intraperitoneal inoculation of the virus to mice, whereas the natural mode of infection by this virus is the mucosal route.
The study aimed to determine whether exposure of mice to CVB4 through the mucosal route results in anti-CVB4 neutralizing and enhancing activity of serum and whether specific IgG antibodies in serum and specific IgA antibodies in saliva are produced.

2. Materials and Methods

2.1. Virus and Cell Line

The diabetogenic CVB4 E2 strain (kindly provided by J. W. Yoon, Julia McFarlane Diabetes Research Center, Calgary, AB, Canada) was propagated in HEp-2 cells (BioWhittaker, Vervier, Belgium) cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco®, Waltham, MA, USA) supplemented with 10% heat-inactivated fetal calf serum (FCS) at 37 °C in a humid atmosphere with 5% CO2. As soon as 100% cytopathic effect (CPE) was reached, the cells were frozen and thawed three times. After centrifugation at 2000× g and 4 °C for 10 min, the culture supernatant was recovered, aliquoted, and stored at −80 °C. The viral titer of the culture supernatant was then determined on HEp-2 cells using a final dilution assay. Viral titer expressed as TCID50 (50% Tissue Culture Infectious Dose) was determined using the Reed–Muench method [22].

2.2. Inoculation of Mice with CVB4

Three-week-old male Swiss albino mice (Janvier labs, Le Genest-Saint-Isle, France) were inoculated orally with 200 μL of CVB4 at different infectious doses, 2 × 104, 2 × 105, 2 × 106, or 2 × 107 TCID50, using an FTP-20-30 cannula (Phymep SARL, Paris, France) or were inoculated intranasally with a drop deposited directly in each nostril of the mice. Saliva samples were collected in tubes containing protease inhibitors (Sigma-Aldrich, St. Louis, MO, USA) on days 0 (before virus inoculation), 15, 30, and 45 post-inoculation from mice anesthetized with isoflurane and injected with pilocarpine hydrochloride (Sigma-Aldrich), a cholinergic agent that activates the salivary glands. On day 45 post-infection, the mice were sacrificed by cervical dislocation, and their blood was collected by cardiac puncture. Serum samples obtained following centrifugation of the blood were stored at −80 °C. Another group of control mice receiving culture supernatant from uninfected HEp-2 cells either orally or intranasally was also followed for 45 days. Mice were housed in a specific pathogen-free facility and had free access to sterile food and water. Experiments were conducted in accordance with French and European animal welfare guidelines and were approved by the Nord-Pas-de-Calais Animal Experimentation Ethics Committee (CEEA 75, Lille, France).

2.3. Neutralization Test

A neutralization assay was used to assess the anti-CVB4 activities of serum and saliva samples as previously described [23]. Briefly, serial (twofold) dilutions of 15 µL of each sample in 15 µL of DMEM were prepared in 96-well microtiter plates, then 15 µL of a viral suspension containing 25 TCID50 of CVB4 was added to each well. The plates were then incubated for 2 h at 37 °C in a humid atmosphere containing 5% CO2. Then, 50 µL of DMEM containing 2 × 104 HEp-2 cells were added to each well, and the plates were returned to the incubator at 37 °C in a humid atmosphere containing 5% CO2. The cell layers were examined 48 h later under an inverted microscope (Olympus CKX41, Rungis, Fance) at a magnification of 400×. The neutralizing titer is expressed as the inverse of the last dilution inhibiting the virus-induced cytopathic effect.

2.4. Detection of Anti-Enterovirus (EV) IgG and IgA by ELISA

ELISA tests (Euroimmun, Nice, France) for the detection of anti-enterovirus IgG and IgA in human serum samples were adapted for the detection of these antibodies in murine serum and saliva samples. The ELISA plates were coated by the manufacturer with VP1 antigens, validated for the detection of cross-reactive enteroviral VP1 antibodies. Briefly, 100 μL of mouse serum and saliva samples diluted at 1:50 and 1:4, respectively, in dilution buffer (PBS + 0.05% Tween 20 + 0.1% BSA) were added to each well of 96-well ELISA plates coated with purified VP1 of ECHO 6 and CVB5. After 2 h of incubation at room temperature, the wells were washed 4 times with washing buffer (PBS + 0.05% Tween 20). Then, 100 μL of a secondary antibody (goat anti-mouse IgG biotinylated [RRID. AB_2534743] or goat anti-mouse IgA HRP-conjugated [RRID. AB_2533951], Thermo Fisher) diluted at 1:5000 in dilution buffer was added to each well. After 1 h of incubation at room temperature, the wells were washed 4 times, and for IgG wells only, 100 μL of ExtrAvidin®-peroxidase (Sigma-Aldrich, St. Louis, MO, USA) diluted at 1:2000 was added to each well. The plates were incubated at room temperature for 30 min, and the wells were then washed 4 times. For color development, 100 μL of TMB/H2O2 was added to each well, and the plates were incubated at room temperature in the dark for 15 min. Then, 100 μL of stop solution (0.5 M sulfuric acid) were added to each well. Absorbance was read at 450 nm using an ELISA reader (Multiskan Go, Thermo Scientific, Waltham, MA, USA).

2.5. Spleen Cell Cultures

The enhancing activity of mouse serum was evaluated using primary splenic cell cultures. Briefly, three-week-old male Swiss albino mice were sacrificed by cervical dislocation, and their spleens were aseptically collected to prepare total splenic cell cultures, as previously described [18]. Murine splenic cells were cultured for 24 h at 37 °C under a humid atmosphere at 5% CO2 in 96-well plates at 5 × 105 cells/well in RPMI-1640 medium (Gibco®) supplemented with 10% FCS, 1% L-Glutamine, 50 μg/mL Streptomycin, 50 IU/mL Penicillin, and 10−5 M β-mercaptoethanol (Sigma-Aldrich). CVB4 (2 × 104 TCID50/mL) was incubated for 2 h at 37 °C in the presence of culture medium or in the presence of serum from controls or from CVB4-exposed mice diluted at 1:500, 1:1000, and 1:10,000. Then, 100 μL of these mixtures was inoculated into splenic cell cultures. After 48 h of incubation, spleen cells were washed 5 times with PBS and stored at −20 °C for RNA extraction.

2.6. RNA Extraction and RT-qPCR

Total RNA was extracted from washed spleen cells using the Tri-Reagent/chloroform extraction protocol (Sigma-Aldrich). Extracted RNA was dissolved in 30 μL of nuclease-free water (Promega, Madison, WI, USA) and quantified by reading absorbance at 260 nm and 280 nm using a nanodrop plate (Thermo Fisher Scientific, Waltham, MA, USA) and a Multiskan GO spectrophotometer (Thermo Fisher Scientific).
RT-qPCR was performed using the TaqMan® Fast Virus 1-Step kit (Applied Biosystems/Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s recommendations, on the Mx3000p® thermal cycler (Stratagene/Thermo Fisher Scientific, Waltham, MA, USA). Briefly, total RNA samples extracted from spleen cells were normalized to 100 ng/µL and subjected to an initial reverse transcription step at 50 °C for 5 min, followed by initial denaturation at 95 °C for 10 s. The cDNA was then amplified over 40 cycles, including a denaturation step at 95 °C for 5 s, followed by a hybridization/elongation step at 60 °C for 30 s. The primers used to detect enteroviral RNA were as follows: forward, 5′-CCC TGA ATG CGG CTA ATC-3′, and reverse, 5′-ATT GTC ACC ATA AGC AGC-3′. The probe had the sequence FAM-AAC CGA CTA CTT TGG GTG TCC GTG TTT-TAMRA. CVB4 RNA, extracted from a culture supernatant of infected-HEp-2 cells with a titer of 108.5 TCID50/mL (or 105.5 TCID50/µL), was used as the standard for quantification. RNA extracted from this supernatant was quantified at 152 ng/µL, and since 1 µL of viral suspension contained 105.5 TCID50, it was determined that 1 µg of viral RNA corresponded to approximately 2.08 × 106 TCID50. The standard range was obtained by successive decimal dilutions of this viral RNA with 5 µL of viral RNA as the first range point, corresponding to 760 ng of RNA or 1.58 × 106 TCID50, which gave a Ct (cycle threshold) of 20. Quantification of viral RNA in the samples was performed using the standard curve of Ct as a function of TCID50. The results were expressed as TCID50 per µg of total RNA.

2.7. Statistical Analysis

Data are presented as mean ± standard deviation. Statistical analyses were performed using Student’s t-test or the nonparametric Mann–Whitney test, when appropriate. Differences were considered statistically significant at p < 0.05. Graphs and data analyses were carried out using GraphPad Prism 8 (GraphPad Inc., San Diego, CA, USA).

3. Results

3.1. Neutralizing Activity of Serum and Saliva from Mice Inoculated with CVB4 by Mucosal Route

The levels of anti-CVB4 neutralizing activity of serum from mice inoculated orally or intranasally were higher than or equal to 16 on day 45 post-infection (p.i.). The mean level in serum was higher in intranasally exposed mice than in orally exposed mice, but the difference was not statistically significant (222.9 vs. 140.4, p = 0.28) (Figure 1A). The anti-CVB4 neutralizing activity of serum was dependent on the amount of inoculated infectious particles. The values in serum were higher in mice inoculated with 2 × 105, 2 × 106, and 2 × 107 TCID50 (mean values ranging from 156 to up to 463) compared with mice inoculated with 2 × 104 TCID50 (mean values 39 or less, p < 0.05), regardless of the route of administration (Figure 1B,C).
Saliva was collected on days 0, 15, 30, and 45 p.i. No anti-CVB4 neutralizing activity was detected in samples collected on day 0. On days 15, 30, and 45 p.i., the mean levels of titers were 12.1, 24.2, and 42.2, respectively, in saliva from orally exposed mice, and they were 18.3, 36.7, and 48.5, respectively, in saliva from intranasally exposed mice. The neutralizing activity titers obtained after oral or intranasal exposure were not statistically different throughout the follow-up (Figure 2).

3.2. Anti EV IgA and IgG Detected by ELISA in Saliva and Serum from Mice Inoculated with CVB4 by Mucosal Route

Saliva was obtained from orally or intranasally exposed mice and controls at 0, 15, 30, and 45 days p.i. The levels of anti-EV IgA in saliva were determined by ELISA. In saliva from controls, OD values remained low throughout the experiment (≤0.16). In contrast, oral and intranasal inoculation induced a gradual increase in salivary anti-EV IgA levels over time, with values reaching similar levels at day 45 p.i. (1.12 ± 0.07 and 1.11 ± 0.06, respectively; Figure 3A). Salivary anti-EV IgA levels in orally and intranasally inoculated mice were significantly higher than those observed in control animals at day 15, 30, and day 45 p.i. (p < 0.05 for both routes versus controls). No significant difference was observed between the oral and intranasal groups throughout the follow-up (p > 0.05). Serum samples from these animals were collected on day 45 p.i. after intranasal or oral infection. No significant difference was observed between the intranasal (OD 1.23 ± 0.37) and oral (OD 1.10 ± 0.28) groups (p > 0.05) (Figure 3B).

3.3. Serum from Mice Inoculated with CVB4 by Mucosal Route Enhances the Infection of Spleen Cells with the Virus In Vitro

The enhancing activity of serum samples from mice inoculated with CVB4 by the mucosal route was investigated in vitro. Serum dilutions of 1:500, 1:1000, and 1:10,000 were incubated with CVB4, and then the mixtures were added to murine splenic cell cultures. After 48 h of culture, total RNA was extracted, and intracellular viral RNA levels were determined by RT-qPCR, and the results were expressed as TCID50/µg of total RNA, as described in Section 2.
In spleen cell cultures infected with CVB4 mixed with serum from mice inoculated via the intranasal or oral route, the levels of intracellular viral RNA were higher (p < 0.05) compared with cultures incubated with serum from control mice at serum dilutions of 1:500 and 1:1000 (Figure 4).

4. Discussion

This study is the first to investigate the neutralizing activity of serum and saliva, and the enhancing activity of serum, from mice inoculated orally or intranasally with CVB4.
In contrast to previous studies, which used the intraperitoneal route [18], this study focuses on mucosal infection, which better reflects the conditions of transmission of enteroviruses in humans [24]. This model opens the possibility to study the interactions between the virus and the host following a physiological route of infection.
The production of neutralizing antibodies is a crucial component of the adaptive immune response and is often correlated with effective protection against enterovirus infections [25]. Day 45 was chosen as the endpoint in order to observe the immune response. After infection with an enterovirus, IgA antibodies in pharyngeal samples peak after approximately 4 weeks [26], while IgG antibodies in serum, which reflect systemic immunity, peak between 2 and 3 weeks and can persist for life [27]. In previous studies, it was observed that inoculation of Swiss albino mice with CVB4 intraperitoneally, a non-physiological route, induced an anti-CVB4 neutralizing activity of serum [18]. To assess the neutralizing activity of serum and saliva from mice in the present study, we used cell culture neutralization assays as described previously [18]. This is the first report of the neutralizing activity of saliva following mucosal infection of mice with CVB4. Furthermore, the present study shows that mucosal infection of mice with CVB4 results in neutralizing activity of serum. This is reminiscent of other studies showing that mucosal immunization via intranasal or oral inoculation of mice with strains of enteroviruses (EV-A71, CVA16, or CVB3) elicits a specific neutralizing antibody response detected in serum [21,22]. The higher levels of neutralizing antibodies observed in serum compared with saliva in this study may be explained by the fact that total antibody concentrations are generally lower in saliva than in serum [28].
Saliva sampling offers advantages for studies monitoring mucosal immune responses. It allows repeated, non-invasive sampling of the same animals, without having to resort to more restrictive methods, such as blood sampling from the retro-orbital sinus or caudal vein. In this study, saliva was collected from mice anaesthetized with isoflurane and stimulated by pilocarpine injection on days 0, 15, 30, and 45. On day 45, the fourth dose of isoflurane was administered just before sacrifice. Isoflurane was well tolerated by the animals, with no clinical signs of distress and no significant behavioral changes. A study of the impact on well-being of repeated isoflurane administration in mice showed that mice receiving up to six iterative doses of isoflurane, every 3 to 4 days over a three-week period, showed no major effects compared with mice anesthetized only once [29]. In saliva samples, IgA concentrations may fluctuate according to the volume collected after pharmacological stimulation [30]. However, despite these variations, virus-specific antibodies remained consistently detectable, indicating that CVB4 exposure elicited a measurable mucosal immune response.
All mice exposed to infectious CVB4 developed detectable anti-CVB4 neutralizing antibodies, indicating that oral or intranasal exposure effectively elicited a humoral response. Although the same inoculum dose was administered within each group, interindividual variation in neutralizing antibody titers was observed. Such variability is expected in outbred Swiss mice and most likely reflects natural heterogeneity in immune responsiveness rather than differences in initial infection efficiency. Moreover, mucosal exposure routes inherently introduce variability in the effective contact between infectious particles and epithelial surfaces, which can influence the magnitude of the antibody response.
The mucosal adaptive immune response is mainly characterized by the production of IgA antibodies, often in dimeric or multimeric form [31]. In order to determine whether CVB4 infection of mice by the mucosal route can induce humoral responses, the levels of IgA and IgG antibodies in saliva and serum, respectively, were investigated by ELISA. In our study, purified VP1 (from ECHO 6/CVB5) coated in microwells allowed for the detection of IgA and IgG in saliva and in serum, respectively, obtained from mice inoculated with CVB4 either by the oral or nasal route. VP1 is known to contain conserved N-terminal antigenic regions (first 11 amino acids) shared across Enterovirus B serotypes, including CVB4 and CVB5 [32,33], which explains why antibodies induced by CVB4 infection react with VP1 derived from CVB5 or ECHO6. Our results show that mucosal infection with CVB4 results in the production of IgA in saliva and IgG in serum, indicating activation of local and systemic immune responses. These results corroborate those of previous studies in which nasal and serum IgA responses were observed after intranasal immunization with enterovirus strains [20,21]. However, unlike those earlier studies that focused exclusively on nasal administration, our data demonstrate that oral exposure to CVB4 also elicits a mucosal immune response. Our system may be useful for conducting future studies of the detailed kinetics of the immune response to CVB4, including the early stages of the acute phase of infection.
Antibodies can enhance viral infection of Fcγ receptor-bearing cells [34]. In the present study, serum-dependent enhancement of CVB4 infection was assessed using murine splenocyte cultures, containing Fcγ receptor-expressing mononuclear cells, which are a suitable model to evaluate serum-dependent enhancement of CVB4 infection in vitro, as previously described [18]. The enhancing activity of serum has been evaluated in vitro using real-time RT-PCR, and the results were expressed as TCID50 equivalents per microgram of total RNA. This quantification method is based on the prior establishment of a standard curve, generated from serial dilutions of total RNA extracted from supernatants of CVB4-infected HEp-2 cell cultures, for which the infectious titer (expressed as TCID50) was known. This curve correlates the Ct values obtained by RT-PCR with equivalent amounts of infectious virus based on the correspondence between the amount of RNA and the infectious titer initially determined. Thus, Ct values obtained from total RNA extracted from spleen cells exposed to CVB4 (in the presence or absence of serum) can be interpolated on this standard curve to estimate the amount of infectious virus, expressed in TCID50 equivalents. The results are then related to the total amount of RNA extracted (TCID50 equivalents per microgram of total RNA), ensuring a standardized comparison between samples. This approach provides a quantitative method for estimating the viral load in cells and allows for the assessment of enhancing activity. In previous studies, it was observed that serum from mice inoculated intraperitoneally with CVB4 was able to enhance the infection of spleen cells with CVB4 in vitro [18]. The method reported in the present study to evaluate the level of intracellular viral RNA enabled us to demonstrate that serum from mice inoculated by the mucosal route can enhance the infection of spleen cells with CVB4 in vitro.

5. Conclusions

In conclusion, this study shows that mucosal CVB4 infection of mice leads to the production of salivary IgA and serum IgG antibodies, which can be detected by ELISA. Anti-CVB4 neutralizing activity of saliva and serum and anti-CVB4 enhancing activity of serum from these animals can be detected by biological assays. Thus, our model, based on Swiss albino mice, may be useful for testing strategies aimed at promoting the production of mucosal and systemic protective anti-CVB4 antibodies while avoiding those likely to promote CVB4 infection. Future studies will be directed along this line in our laboratory.

Author Contributions

Methodology, C.J.M. and L.C.; writing—original draft preparation, C.J.M.; M.P.N. and D.H.; writing—review and editing, C.J.M., L.C., M.P.N., F.S., A.D., C.D., D.M. and E.K.A.; supervision, D.H.; project administration, D.H.; funding acquisition, D.H. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Ministère de l’Education Nationale de la Recherche et de la Technologie, Université de Lille (ULR3610), and the Centre Hospitalier et Universitaire de Lille. C.J.M. was supported by an “SSHN JEUNE CHERCHEUR” scholarship from the Ministère des Affaires étrangères et du Développement International de la République Française.

Institutional Review Board Statement

The animal study protocol was approved by the Ethical committee for animal experimentation of Nord-Pas-de-Calais, CEEA 75, France (approval code APAFIS#11873-2017101908475765v6; approval date: 4 February 2019).

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Acknowledgments

The authors thank Delphine Lobert and the team of the Laboratoire de Virologie ULR3610 and all their collaborators.

Conflicts of Interest

The authors declare no conflicts of interest.

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Microorganisms 14 00289 g001
Figure 1. Individual representation of the anti-CVB4 neutralizing activity of serum from mice inoculated orally or intranasally with CVB4. (A) Mice were exposed to CVB4 at 2 × 105 TCID50 orally (white circles) or intranasally (white triangles). The anti-CVB4 neutralizing activity of serum collected 45 days post-inoculation (p.i.) was assessed. (B,C) Mice were inoculated with CVB4 at 2 × 104, 2 × 105, 2 × 106, or 2 × 107 TCID50 intranasally (B) or orally (C) (n = 7 in each group). The Anti-CVB4 neutralizing activity of serum collected 45 days p.i. was assessed. Neutralizing titers are defined as the inverse of the last serum dilution that completely inhibits the cytopathogenic effect of CVB4 in HEp-2 cell cultures. The horizontal bars represent geometric mean values. The dotted lines indicate the detection limit of the assay. Black circles: control; * p < 0.05.
Figure 1. Individual representation of the anti-CVB4 neutralizing activity of serum from mice inoculated orally or intranasally with CVB4. (A) Mice were exposed to CVB4 at 2 × 105 TCID50 orally (white circles) or intranasally (white triangles). The anti-CVB4 neutralizing activity of serum collected 45 days post-inoculation (p.i.) was assessed. (B,C) Mice were inoculated with CVB4 at 2 × 104, 2 × 105, 2 × 106, or 2 × 107 TCID50 intranasally (B) or orally (C) (n = 7 in each group). The Anti-CVB4 neutralizing activity of serum collected 45 days p.i. was assessed. Neutralizing titers are defined as the inverse of the last serum dilution that completely inhibits the cytopathogenic effect of CVB4 in HEp-2 cell cultures. The horizontal bars represent geometric mean values. The dotted lines indicate the detection limit of the assay. Black circles: control; * p < 0.05.
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Figure 2. Individual representation of the anti-CVB4 neutralizing activity of saliva from mice inoculated orally or intranasally with CVB4. Mice were inoculated with CVB4 at 2 × 105 TCID50. Anti-CVB4 neutralizing activity of saliva collected on days 0, 15, 30, and 45 post-inoculation was assessed. Neutralizing titers are defined as the inverse of the last saliva dilution that completely inhibits the cytopathogenic effect of CVB4 in HEp-2 cell cultures. The horizontal bars represent the geometric mean values. The dotted line indicates the detection limit of the assay.
Figure 2. Individual representation of the anti-CVB4 neutralizing activity of saliva from mice inoculated orally or intranasally with CVB4. Mice were inoculated with CVB4 at 2 × 105 TCID50. Anti-CVB4 neutralizing activity of saliva collected on days 0, 15, 30, and 45 post-inoculation was assessed. Neutralizing titers are defined as the inverse of the last saliva dilution that completely inhibits the cytopathogenic effect of CVB4 in HEp-2 cell cultures. The horizontal bars represent the geometric mean values. The dotted line indicates the detection limit of the assay.
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Figure 3. Detection of anti-enterovirus (EV) IgA in saliva and anti-EV IgG in serum from mice inoculated with CVB4 by the mucosal route. Mice were inoculated orally or intranasally with CVB4 (2 × 105 TCID50). (A) Anti-EV IgA were detected by ELISA in saliva from these animals and controls collected on days 0, 15, 30, and 45 post-inoculation. (B) Detection of anti-EV IgG by ELISA in serum from exposed and control mice collected on day 45 post-inoculation. The results expressed as optical density (OD) are mean ± standard deviation.
Figure 3. Detection of anti-enterovirus (EV) IgA in saliva and anti-EV IgG in serum from mice inoculated with CVB4 by the mucosal route. Mice were inoculated orally or intranasally with CVB4 (2 × 105 TCID50). (A) Anti-EV IgA were detected by ELISA in saliva from these animals and controls collected on days 0, 15, 30, and 45 post-inoculation. (B) Detection of anti-EV IgG by ELISA in serum from exposed and control mice collected on day 45 post-inoculation. The results expressed as optical density (OD) are mean ± standard deviation.
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Microorganisms 14 00289 g004
Figure 4. Enhancing activity of serum from mice inoculated with CVB4 by the mucosal route. Murine spleen cell cultures were inoculated with CVB4 (2 × 104 TCID50) mixed with various dilutions of serum (1:500, 1:1000, and 1:10,000) collected on day 45 post-inoculation from control mice or mice inoculated with CVB4 orally or intranasally. After 48 h, total RNA was extracted from spleen cells, and the amount of viral RNA was determined by RT-qPCR. The results were expressed as TCID50 per µg of total RNA, as described in Section 2. Data are presented as mean ± standard deviation (* p < 0.05).
Figure 4. Enhancing activity of serum from mice inoculated with CVB4 by the mucosal route. Murine spleen cell cultures were inoculated with CVB4 (2 × 104 TCID50) mixed with various dilutions of serum (1:500, 1:1000, and 1:10,000) collected on day 45 post-inoculation from control mice or mice inoculated with CVB4 orally or intranasally. After 48 h, total RNA was extracted from spleen cells, and the amount of viral RNA was determined by RT-qPCR. The results were expressed as TCID50 per µg of total RNA, as described in Section 2. Data are presented as mean ± standard deviation (* p < 0.05).
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Mbani, C.J.; Nekoua, M.P.; Couture, L.; Dechaumes, A.; Debuysschere, C.; Sane, F.; Alidjinou, E.K.; Moukassa, D.; Hober, D. Anti-Coxsackievirus B4 Activity of Serum and Saliva from Mice Exposed to the Virus via the Mucosal Route. Microorganisms 2026, 14, 289. https://doi.org/10.3390/microorganisms14020289

AMA Style

Mbani CJ, Nekoua MP, Couture L, Dechaumes A, Debuysschere C, Sane F, Alidjinou EK, Moukassa D, Hober D. Anti-Coxsackievirus B4 Activity of Serum and Saliva from Mice Exposed to the Virus via the Mucosal Route. Microorganisms. 2026; 14(2):289. https://doi.org/10.3390/microorganisms14020289

Chicago/Turabian Style

Mbani, Chaldam Jespère, Magloire Pandoua Nekoua, Laurine Couture, Arthur Dechaumes, Cyril Debuysschere, Famara Sane, Enagnon Kazali Alidjinou, Donatien Moukassa, and Didier Hober. 2026. "Anti-Coxsackievirus B4 Activity of Serum and Saliva from Mice Exposed to the Virus via the Mucosal Route" Microorganisms 14, no. 2: 289. https://doi.org/10.3390/microorganisms14020289

APA Style

Mbani, C. J., Nekoua, M. P., Couture, L., Dechaumes, A., Debuysschere, C., Sane, F., Alidjinou, E. K., Moukassa, D., & Hober, D. (2026). Anti-Coxsackievirus B4 Activity of Serum and Saliva from Mice Exposed to the Virus via the Mucosal Route. Microorganisms, 14(2), 289. https://doi.org/10.3390/microorganisms14020289

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