Sex-Dependent Intestinal Replication of an Enteric Virus

ABSTRACT

Coxsackievirus is an enteric virus that initiates infection in the gastrointestinal tract before disseminating to peripheral tissues to cause disease, but intestinal factors that influence viral replication are understudied. Furthermore, a sex bias for severe sequelae from coxsackievirus infections has been observed in humans. While mouse models mimicking human pathogenesis have been well characterized, many of these experiments use intraperitoneal injection of coxsackievirus to infect mice, bypassing the intestine. In light of recent studies identifying intestinal factors, such as the microbiota, that alter enteric viral replication, we sought to investigate coxsackievirus replication within the intestine. Here, we orally infected mice with coxsackievirus B3 (CVB3) and found that CVB3 replication in the intestine is sex dependent. CVB3 replicated efficiently in the intestine of male mice but not female mice. Additionally, we found that the type I interferon response and sex hormones can alter both viral replication and lethality. Overall, these data suggest that sex and the immune response play a vital role in CVB3 replication in the intestine and should be considered in light of the sex bias observed in human disease.

IMPORTANCE Sex bias in severe sequelae from enteric viral infections has been observed. Since viruses have evolved to achieve optimal levels of fitness in their environmental niches, it is imperative to study viruses at the site of initial replication. Here, we used an oral inoculation system for CVB3, which follows the natural route of infection in the gastrointestinal tract. We found that sex can influence the replication of CVB3 in the intestine. Additionally, the type I interferon response and sex hormones alter both CVB3 intestinal replication and lethality. Overall this work highlights the fact that sex should be considered in investigations of enteric viral replication and pathogenesis.

INTRODUCTION

Sex contributes to the frequency and severity of many human diseases (1–3). Epidemiological studies show that men are more susceptible to bacterial, viral, and fungal infections (2). Women, while more resistant to pathogens, are at an increased risk for autoimmune disorders. Sexual dimorphism in disease has been attributed to differential gene expression, sex hormones, and variations in immune function (4). However, sex as a variable in disease models is understudied, limiting our understanding of the mechanism of sex bias in various diseases.

Coxsackievirus is a nonenveloped RNA virus from the Picornaviridae family and is implicated in a range of diseases (5–8). In humans, males are twice as likely as females to develop sequelae from coxsackievirus infection (9). While most coxsackievirus infections are self-limiting, they can cause severe disease. In particular, coxsackievirus B3 (CVB3) is a leading cause of viral myocarditis, and extensive cardiac damage can lead to cardiac failure (10, 11). Because there are no current vaccines or treatment for coxsackievirus infections, further understanding of CVB3 pathogenesis is imperative.

Mouse models have served as an important tool in advancing our knowledge of CVB3-induced myocarditis. Following intraperitoneal (i.p.) injection, CVB3 replicates to high titers in tissues and causes disease similar to that observed in humans (12–14), and male mice are more likely to develop severe myocarditis than female mice. Experimental evidence suggests that two mechanisms contribute to CVB3-induced myocarditis in these mouse models: direct viral lysis of infected cardiomyocytes and immune-mediated damage of cardiac tissue (15). The latter is hypothesized to be responsible for the sex bias in disease. This hypothesis is based on the observation that CVB3 replicates equally well in the hearts of male and female mice, yet only male mice develop increased cardiac damage due to sex-specific immune responses (16–18).

While previous mouse experiments have provided vital insights into CVB3 pathogenesis, they are based on systemic infection of CVB3 rather than infection by the natural fecal-oral route. CVB3 is an enteric virus, and i.p. injection models bypass the first stage of infection in the gastrointestinal tract. Relatively few studies have used oral inoculation of mice with coxsackievirus (19–23). Therefore, CVB3 replication and factors that influence viral replication in the intestine remain poorly understood. Here, we describe an oral inoculation mouse model of CVB3 to examine CVB3 replication and lethality following initial intestinal infection. We found a sex bias in CVB3 pathogenesis, similar to previous mouse models and human infection, where only male mice succumbed to CVB3-induced disease. In contrast to results from previous i.p. injection mouse models, we found that CVB3 replicated in the intestine of male mice but not female mice. Immune responses were distinct in male and female mice, and sex hormones impacted CVB3 replication. These data highlight the importance of studying enteric viral replication in the intestine.

RESULTS

CVB3 replication is enhanced in the intestine of male mice.

To investigate replication of CVB3 in the gastrointestinal tract, we orally inoculated male and female C57BL/6 IFNAR^−/− mice (deficient for the alpha/beta-interferon [IFN-α/β] receptor) with 5 × 10⁷ PFU of the Nancy strain of CVB3 (CVB3-Nancy). We selected IFNAR^−/− mice based on previous work showing that lack of the type I interferon response enhances susceptibility of mice to infection with human enteric viruses by the oral route (24, 25). Following oral inoculation, feces were collected at 24, 48, and 72 h postinoculation (hpi), and CVB3 titers were quantified by plaque assay. At 48 and 72 hpi, we observed 100- to 1,000-fold higher fecal titers in male mice than in female mice (Fig. 1A). The fecal titers that we observed in male mice suggested that viral replication had occurred, but due to potential flow through of inoculum virus, viral fecal shedding is not always indicative of viral replication in the intestine (26). Therefore, we differentiated between inoculum and replicated CVB3 in feces of male mice using light-sensitive CVB3. Virus propagated in the presence of neutral red dye is light sensitive due to RNA cross-linking leading to inactivation. Upon viral replication in the dark (e.g., intestine), replicated virions lose light sensitivity, which facilitates the assessment of replication by differentiating inoculum virus from replicated virus (27–29). Following oral inoculation of mice with light-sensitive CVB3 in the dark, feces were collected at 24, 48, and 72 hpi in the dark. Following collection, CVB3 replication was quantified by determining the ratio of the titers of light- to dark-exposed virus in fecal samples. Similar to previous data from our laboratory (29), at 48 and 72 hpi, we found that most virus shed from male mice was replicated virus (Fig. 1B). In contrast, undetectable or limited CVB3 replication was observed in feces collected from female mice (Fig. 1B). Overall, these data suggest that CVB3 replicates more efficiently in the intestine of male IFNAR^−/− mice than in female IFNAR^−/− mice.

FIG 1.

Sex-dependent intestinal replication of CVB3. (A) CVB3-Nancy fecal titers in male and female IFNAR^−/− mice orally inoculated with 5 × 10⁷ PFU of CVB3-Nancy (n = 14 to 15 per sex). (B) CVB3-Nancy replication in male and female IFNAR^−/− mice using light-sensitive, neutral-red-labeled virus. Replication status was determined by dividing the number of PFU/milliliter of light-exposed samples by the number of PFU/milliliter of dark-exposed samples and multiplying the result by 100% (n = 4 to 7 per sex). (C) CVB3-Nancy fecal titers in orally inoculated male and female immunocompetent C57BL/6 mice (n = 7 to 12 per sex). (D) CVB3-H3 fecal titers in orally inoculated male and female immunocompetent C57BL/6 mice (n = 10 to 15 per sex). All data are means ± standard errors of the means. *, P < 0.05 (Mann-Whitney test). WT, wild type.

Next, to determine if the loss of the type I IFN response contributed to sex-dependent CVB3 replication in the intestine, we quantified CVB3 fecal titers in wild-type, immunocompetent C57BL/6 mice. Following oral inoculation with 5 × 10⁷ PFU of CVB3-Nancy, limited fecal shedding was observed in both male and female wild-type mice (Fig. 1C). Since male CVB3-Nancy fecal titers at 72 hpi were higher but not statically significant from female fecal titers, we hypothesized that lack of viral replication in wild-type mice masked any replication difference between male and female mice. To overcome this limitation, we quantified the fecal titers of wild-type mice orally inoculated with a more virulent CVB3 strain, CVB3-H3. The H3 strain is a CVB3-Nancy variant that causes severe myocarditis in mice following i.p. injection (30). Indeed, we found that immunocompetent male mice shed significantly more CVB3-H3 than female mice at 3 and 7 days postinoculation (dpi) (Fig. 1D), similar to our results in IFNAR^−/− mice. These data indicate that sex-dependent replication of CVB3 in the intestine is independent of the type I IFN response.

CVB3-induced lethality in IFNAR^−/− mice is sex dependent.

As in humans, previous mouse models have demonstrated a sex bias in CVB3 pathogenesis (16, 18, 31, 32). To examine if orally infected mice display sex-dependent CVB3-induced lethality, we inoculated IFNAR^−/− mice and examined survival following oral infection. We found that only male IFNAR^−/− mice succumbed to disease following CVB3-Nancy infection (Fig. 2A). Next, to determine whether CVB3-induced lethality was dependent on the loss of the type I IFN response, we orally inoculated wild-type immunocompetent mice with CVB3-Nancy. In contrast to IFNAR^−/− mice, all immunocompetent male and female mice survived CVB3 infection (Fig. 2B). Furthermore, oral inoculation with CVB3-H3 was unable to induce lethality in male and female immunocompetent mice (Fig. 2C). Overall, these data suggest that survival following oral inoculation of CVB3 is sex dependent in IFNAR^−/− mice.

FIG 2.

Sex-dependent CVB3-induced lethality in orally inoculated mice. (A) Survival of male and female C57BL/6 IFNAR^−/− mice following oral inoculation with 5 × 10⁷ PFU of CVB3-Nancy. *, P < 0.05 (log rank test; n = 14 to 15 per sex). (B) Survival of C57BL/6 immunocompetent male and female mice following oral inoculation with 5 × 10⁷ PFU of CVB3-Nancy (n = 11 to 12 per sex). (C) Survival of C57BL/6 immunocompetent male and female mice following oral inoculation with 5 × 10⁷ PFU of CVB3-H3 (n = 16 per sex).

The type I IFN response affects sex-dependent replication of CVB3 in peripheral tissues following i.p. inoculation.

Previous mouse models for CVB3 pathogenesis have demonstrated that the sex bias in disease is independent of differences in viral replication. Specifically, while male mice develop more severe myocarditis, CVB3 replication is equivalent in the heart of male and female mice following i.p. inoculation (16, 17). However, we found that in orally inoculated IFNAR^−/− mice, CVB3 replication in the intestine is sex dependent. To determine if sex-dependent CVB3 replication was specific to the intestine or specific to the IFNAR^−/− model, we i.p. inoculated male and female IFNAR^−/− mice with 1 × 10⁴ PFU of CVB3-Nancy. At 3 dpi, the heart, liver, spleen, and kidney were harvested, and CVB3 titers were quantified by plaque assay. Similar to previous reports (16), we found no significant difference in CVB3 titers in the heart and kidney between male and female mice (Fig. 3A). However, we found that viral titers in the liver and spleen of male mice were significantly higher than those in female mice (Fig. 3A), suggesting that replication of CVB3 in the liver and spleen of IFNAR^−/− mice is also sex dependent. Next, to determine if the loss of the type I IFN response plays a role in sex-dependent replication in the liver and spleen, we i.p. inoculated wild-type, immunocompetent mice with 1 × 10⁴ PFU of CVB3-H3. Following i.p. inoculation with CVB3-H3, we found no significant differences in the viral titers of heart, liver, spleen, and kidney from male and female mice (Fig. 3B). These data suggest that CVB3 replication in most tissues of i.p. inoculated mice is independent of sex but that loss of the type I IFN response facilitates sex-dependent replication in the liver and spleen. In contrast, CVB3 replication in the intestine of orally inoculated mice is sex dependent (Fig. 1).

FIG 3.

CVB3 replication in peripheral tissues following i.p. inoculation. Male and female mice were i.p. inoculated with 1 × 10⁴ PFU of CVB3-Nancy or CVB3-H3. Mice were euthanized at 3 dpi, and tissues were collected. (A) CVB3-Nancy tissue titers in male (blue) and female (red) IFNAR^−/− mice (n = 13 to 15 per sex). (B) CVB3-H3 tissue titers in male (blue) and female (red) immunocompetent C57BL/6 mice (n = 6 per sex). All data are means ± standard errors of the means. *, P < 0.05; ns, not significant (Mann-Whitney test).

CVB3-Nancy induces an immune response in orally inoculated male IFNAR^−/− mice but not in female IFNAR^−/− mice.

Since CVB3-Nancy failed to replicate in orally inoculated female IFNAR^−/− mice, we hypothesized that a heightened immune response in female mice may limit CVB3 replication in the intestine. To investigate this hypothesis, we examined serum cytokine levels in male and female IFNAR^−/− mice at 72 hpi since this time point correlated with peak viral shedding in males. Contrary to our hypothesis, we observed increased cytokine levels compared to those in uninfected control mice in CVB3-infected male mice but not in female mice. Male IFNAR^−/− mice had increased serum cytokine levels of IFN-γ, interleukin-1β (IL-1β), IL-6, interferon gamma-induced protein 10 (IP-10), keratinocyte-derived chemokine (KC), monocyte chemoattractant protein 1 (MCP-1), RANTES, and tumor necrosis factor alpha (TNF-α) after CVB3 infection compared with those of uninfected control male IFNAR^−/− mice (Fig. 4). Furthermore, serum cytokine levels in CVB3-infected female IFNAR^−/− mice were low and similar to those of uninfected female mice. These data indicate that orally inoculated female IFNAR^−/− mice do not elicit a strong immune response at 72 hpi.

FIG 4.

Sex-dependent immune response to intestinal CVB3-Nancy replication. Serum cytokine quantification in CVB3-Nancy-infected male (blue) and female (red) IFNAR^−/− mice at 72 hpi or in uninfected (white) mice. Infected mice were orally inoculated with 5 × 10⁷ PFU of CVB3-Nancy. Serum cytokine levels were determined for gamma interferon (IFN-γ), interleukin-1β (IL-1β), interleukin-6 (IL-6), interferon gamma-induced protein 10 (IP-10), mouse keratinocyte-derived cytokine (KC), monocyte chemoattractant protein-1 (MCP-1), RANTES, and tumor necrosis factor alpha (TNF-α) (n = 3 to 6 per sex). n.d., not detectable.

Next, we investigated whether CVB3 infection of male and female IFNAR^−/− mice by the oral route induces an adaptive immune response to CVB3. Previous studies have shown detectable CVB3 neutralizing antibodies 7 days following i.p. inoculation (14, 33). Therefore, to evaluate the adaptive immune response to CVB3 following oral inoculation, serum was collected from surviving male and female mice at 7 days post-oral inoculation and tested for the ability to neutralize CVB3-Nancy in vitro using plaque assays. We found that sera from male mice significantly reduced CVB3-Nancy plaque formation by 10- to 100-fold, while sera from female mice had no effect (Fig. 5). As a control, CVB3-Nancy was incubated with serum from uninfected mice, which was unable to neutralize CVB3-Nancy (data not shown). These data suggest that male, but not female, IFNAR^−/− mice produce neutralizing serum against CVB3 following oral inoculation.

FIG 5.

Serum neutralization of CVB3-Nancy. Male and female IFNAR^−/− mice were orally inoculated with 5 × 10⁷ PFU of CVB3-Nancy, and serum was collected at 7 dpi. Serum was incubated with 1 × 10⁵ PFU of input CVB3-Nancy used for oral inoculations. Following incubation of the serum and virus at 37°C for 1 h, CVB3-Nancy infectivity was quantified by plaque assay. Data are means ± standard errors of the means. *, P < 0.05 (n = 3 to 5 per sex).

Sex hormones alter intestinal CVB3 shedding and lethality.

Previous studies have shown that sex hormones such as testosterone, estradiol, and progesterone can alter CVB3 replication and pathogenesis in mice following i.p. injection (31, 34, 35). To determine if sex hormones contribute to sex-dependent viral shedding in our oral infection model, we examined CVB3 replication in the intestine of castrated and ovariectomized IFNAR^−/− mice. Male mice were surgically castrated to deplete testosterone or subjected to mock castration surgery at 4 weeks of age. Female mice were surgically ovariectomized to deplete estrogen and progesterone or subjected to mock ovariectomy surgery at 4 weeks of age. Six weeks postsurgery, the mice were orally inoculated with CVB3-Nancy. We found that castration of male mice protected against CVB3-induced lethality and significantly reduced CVB3 shedding at 48 hpi (Fig. 6A and B). While ovariectomy did not enhance CVB3 lethality, ovariectomized female mice shed significantly more CVB3 than surgery-control female mice at 24 hpi (Fig. 6C and D). These data indicate that sex hormones can alter both CVB3 shedding and lethality following oral inoculation.

FIG 6.

Sex hormones alter intestinal CVB3 replication and lethality. Four-week-old male and female IFNAR^−/− mice underwent surgery to remove testes and ovaries, respectively. At 10 to 12 weeks of age, castrated and ovariectomized mice were orally inoculated with 5 × 10⁷ PFU of CVB3-Nancy. (A) Survival of mock-castrated and castrated male mice following CVB3-Nancy infection. *, P < 0.05 (log rank test; n = 10 mice per group). (B) Fecal CVB3-Nancy titers in mock-castrated and castrated male mice. Data are means ± standard errors of the means. *, P < 0.05 (Mann-Whitney test; n = 10 mice per group). (C) Survival of mock-ovariectomized and ovariectomized female mice following CVB3-Nancy infection (n = 9 to 10 mice per group). (D) Fecal CVB3-Nancy titers in mock-ovariectomized and ovariectomized female mice. Data are means ± standard errors of the means. *, P < 0.05 (Mann-Whitney test; n = 9 to 10 mice per group).

DISCUSSION

Enteric viruses are spread through the fecal-oral route and initiate infection in the gastrointestinal tract. While previous mouse models for coxsackievirus recapitulate systemic viral infection and pathogenesis of the heart, factors that affect initial CVB3 replication in the intestine are unclear. Here, we used an oral inoculation model for CVB3 to study viral replication in the intestine. In contrast to previous work in i.p. injected mice, we observed sex-dependent replication of CVB3 within the intestine. CVB3 replicated efficiently and with enhanced lethality in male IFNAR^−/− mice but not female IFNAR^−/− mice. To our knowledge, this study represents the first time that sex has been demonstrated to influence the replication of an enteric virus in the intestine.

Our data indicate that sex hormones likely contribute to the replication and pathogenesis differences between male and female mice. We observed that sex hormones affect the ability of CVB3 to replicate in the host gastrointestinal tract, as well as influence lethality (Fig. 6B and D). It is possible that hormones may alter availability of viral receptors. Estrogen has been shown to modulate the expression of the viral entry coreceptor decay-accelerating factor (DAF) (36). Additionally, estrogen and testosterone enhance CVB3 attachment to cardiomyocytes (31). Interestingly, the primary receptor for coxsackievirus, the coxsackievirus and adenovirus receptor (CAR), is found at the tight junction of intestinal epithelial cells. Recent evidence shows that sex hormones regulate tight junction proteins; therefore, it is possible that hormones regulate the availability of CAR in the intestine and therefore modulate CVB3 replication. However, the mechanism for sex hormone modulation of CVB3 intestinal replication is unclear.

Previous studies have shown that susceptibility of mice to several human enteric viruses requires the loss of the type I IFN response (26, 37, 38). Similarly, we found that CVB3-induced lethality was enhanced in IFNAR^−/− male mice compared to that in wild-type immunocompetent male mice. IFN-α/β plays an important role in viral infection by limiting viral replication. Indeed, both in vitro and in vivo studies have demonstrated that IFN-α/β reduces CVB3 replication and pathogenesis (39–42). In agreement with these data, we found that an intact type I IFN response reduced CVB3-Nancy intestinal replication (Fig. 1C). Interestingly, the CVB3-H3 strain was able to partially overcome the type I IFN response and replicate in immunocompetent wild-type male mice. The H3 strain is a CVB3-Nancy variant that causes severe myocarditis in mice (30). Sequence differences in the VP2 region of the viral capsid enhance the ability of the H3 strain to induce myocarditis. Additionally, mutations in the VP1 viral capsid protein cluster near the site of the interaction with the host viral receptor (43); however, it is unknown if these amino acid differences are important for intestinal replication.

We also investigated the immune response to CVB3 in our oral inoculation IFNAR^−/− mouse model. Previous studies suggest that women generate cell-mediated immune responses to viral pathogens that are superior to those of men (1). Therefore, we hypothesized that the lack of viral replication in female mice was due to an increased antiviral cytokine response. However, we observed that male IFNAR^−/− mice had increased serum cytokines at 72 h following CVB3 infection, while the levels of serum cytokines in female IFNAR^−/− mice were similar to those of untreated controls. Furthermore, while serum from surviving CVB3-infected male mice was able to neutralize CVB3 in vitro, serum from females was not protective. These data indicate that female mice fail to elicit an immune response 72 h following oral infection of CVB3; however, future experiments are required to determine if the kinetics of cytokine induction and serum neutralization differ between the two sexes. Overall, these data suggest that female mice are resistant to CVB3 intestinal infection and that factors in the intestinal environment may play a key role in sex-dependent CVB3 replication. Previously, our lab has shown that intestinal bacteria enhance poliovirus replication and pathogenesis (27, 28). Poliovirus is an enteric virus from the same viral family as coxsackievirus; therefore, intestinal bacteria may enhance CVB3 replication as well. Interestingly, recent evidence suggests that sex influences the bacterial composition within the intestine (44, 45). Therefore, sex-specific bacteria may contribute to the replication of CVB3 within the intestine.

In conclusion, we found that CVB3 replication and lethality are higher in orally inoculated male mice than in female mice due to differential viral replication in the intestine. Furthermore, sex hormones and the type I IFN response play important roles in intestinal CVB3 replication and pathogenesis. Overall, these data suggest that sex and the natural oral route of infection should be considered in investigating enteric viruses.

MATERIALS AND METHODS

Cells and viruses.

HeLa cells were propagated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum. The CVB3-Nancy and CVB3-H3 infectious clones were obtained from Marco Vignuzzi (Pasteur Institute, Paris, France). Stocks of CVB3 were prepared in HeLa cells by cotransfection of the infectious clone plasmid and a plasmid expressing T7 RNA polymerase. Virus titers were determined by plaque assay with HeLa cells as previously described (29, 46). To determine whether fecal viruses were input/inoculum virus or virus that had undergone replication, we used neutral-red-labeled, light-sensitive CVB3 as described previously (26, 29). The efficacy of light inactivation was determined by exposing neutral-red-labeled-CVB3 stocks to a fluorescent light bulb for 10 min. The ratio of light-insensitive to light-sensitive PFU in the NR-CVB3 stock was 1 in 2.4 × 10⁵. Following oral inoculation, to determine the percentage of replicated virus in the intestine, samples were processed in the dark, and a portion was light exposed. The percentage of replicated virus was calculated by dividing the light-exposed number of PFU/milliliter by the non-light-exposed number of PFU/milliliter and multiplying the result by 100.

Mouse experiments.

All animals were handled according to the Guide for the Care and Use of Laboratory Animals endorsed by the National Institutes of Health (47). All mouse studies were performed at UT Southwestern (Animal Welfare Assurance A3472-01) using protocols approved by the local Institutional Animal Care and Use Committee in a manner designed to minimize pain, and any animals that exhibited severe disease were euthanized immediately with isoflurane. C57BL/6 PVR IFNAR^+/+ and C57BL/6 PVR IFNAR^−/− mice were obtained from S. Koike (Tokyo, Japan) (25). For castration and ovariectomy surgeries, 4-week-old mice were anesthetized with isoflurane, and the testes and ovaries were surgically removed. Mock-castrated males and mock-ovariectomized females were used as a control for the survival surgery. For oral inoculations, 10- to 12-week-old mice were perorally inoculated with 5 × 10⁷ PFU of CVB3. Disease was monitored until day 14 postinoculation for survival experiments. Mice were euthanized upon severe disease onset. For shedding and replication experiments, feces were collected at 24, 48, and 72 hpi and processed as previously described (27). For intraperitoneal inoculation, 10- to 12-week-old mice were inoculated with 1 × 10⁴ PFU of CVB3 as previously described (33). Heart, liver, kidney, and spleen were aseptically removed and homogenized in phosphate-buffered saline using 0.9- to 2.0-mm stainless steel beads in a Bullet Blender (Next Advance). Cellular debris was removed by centrifugation at 12,000 × g for 10 min at 4°C, and CVB3 was quantified by plaque assay on HeLa cells.

Cytokine expression.

Blood was collected by heart puncture from male and female IFNAR^−/− mice either prior to infection (uninfected) or at 72 hpi with CVB3-Nancy. Blood samples were incubated at room temperature for 30 min to induce coagulation. Serum was collected from each sample following centrifugation for 10 min at 1,000 × g. Serum samples were stored at −20°C until analysis. Cytokine levels were measured using a Mouse Cytokine/Chemokine Magnetic Bead Panel (EMD Millipore), and the assay was carried out by the University of Texas Southwestern Medical Center Metabolic Phenotyping Core. This assay measured 25 cytokines to give a broad range of cytokine/chemokine levels. The cytokines shown in Fig. 4 are those that displayed a sex difference.

Serum neutralization assay.

Blood from the submandibular vein was collected at 7 dpi from uninfected or orally infected mice and allowed to coagulate at room temperature for 30 min. Serum from blood samples was collected following centrifugation at 4,000 rpm for 15 min at 4°C. Serial dilutions of each serum sample were prepared in DMEM and heat inactivated at 56°C for 30 min. Serially diluted serum samples were mixed with 10⁵ PFU of CVB3-Nancy and incubated in glass tubes for 1 h at 37°C, and then CVB3 was quantified by plaque assay on HeLa cells.

Funding Statement

J.K.P. is a Howard Hughes Medical Institute Faculty Scholar.