Rhinovirus C Infection Induces Type 2 Innate Lymphoid Cell Expansion and Eosinophilic Airway Inflammation.

Rhinovirus C (RV-C) infection is associated with severe asthma exacerbations. Since type 2 inflammation is an important disease mechanism in asthma, we hypothesized that RV-C infection, in contrast to RV-A, preferentially stimulates type 2 inflammation, leading to exacerbated eosinophilic inflammation. To test this, we developed a mouse model of RV-C15 airways disease. RV-C15 was generated from the full-length cDNA clone and grown in HeLa-E8 cells expressing human CDHR3. BALB/c mice were inoculated intranasally with 5 x 106 ePFU RV-C15, RV-A1B or sham. Mice inoculated with RV-C15 showed lung viral titers of 1 x 105 TCID50 units 24 h after infection, with levels declining thereafter. IFN-α, ß, γ and λ2 mRNAs peaked 24-72 hrs post-infection. Immunofluorescence verified colocalization of RV-C15, CDHR3 and acetyl-α-tubulin in mouse ciliated airway epithelial cells. Compared to RV-A1B, mice infected with RV-C15 demonstrated higher bronchoalveolar eosinophils, mRNA expression of IL-5, IL-13, IL-25, Muc5ac and Gob5/Clca, protein production of IL-5, IL-13, IL-25, IL-33 and TSLP, and expansion of type 2 innate lymphoid cells. Analogous results were found in mice treated with house dust mite before infection, including increased airway responsiveness. In contrast to Rorafl/fl littermates, RV-C-infected Rorafl/flIl7rcre mice deficient in ILC2s failed to show eosinophilic inflammation or mRNA expression of IL-13, Muc5ac and Muc5b. We conclude that, compared to RV-A1B, RV-C15 infection induces ILC2-dependent type 2 airway inflammation, providing insight into the mechanism of RV-C-induced asthma exacerbations.

Rapidly developing vesicular eruption.

The patient's previously diagnosed dermatologic condition, paired with her recent exposure history, led to the diagnosis in this case.

Myoglobin for Detection of High-Risk Patients with Acute Myocarditis.

There is an unmet need for accurate and practical screening to detect myocarditis. We sought to test the hypothesis that the extent of acute myocarditis, measured by late gadolinium enhancement (LGE) on cardiac magnetic resonance imaging (CMR), can be estimated based on routine blood markers. A total of 44 patients were diagnosed with acute myocarditis and included in this study. There was strong correlation between myoglobin and LGE (rs = 0.73 [95% CI 0.51; 0.87], p < 0.001), while correlation was weak between LGE and TnT-hs (rs = 0.37 [95% CI 0.09; 0.61], p = 0.01). Receiver operating curve (ROC) analysis determined myoglobin ≥ 87 µg/L as cutoff to identify myocarditis (92% sensitivity, 80% specificity). The data were reproduced in an established model of coxsackievirus B3 myocarditis in mice (n = 26). These data suggest that myoglobin is an accurate marker of acute myocarditis. Graphical Abstract Receiver operating curve analysis determined myoglobin ≥ 87 µg/L as cutoff to identify myocarditis and these data were reproduced in an established model of coxsackievirus B3 myocarditis in mice: CMRI, cardiac magnetic resonance imaging; Mb, myoglobin; LGE, late gadolinium enhancement; ROC, receiver operating curve analysis.

Tanshinone IIA Ameliorate Coxsackie Virus B3-Induced Viral Myocarditis through the Inhibition of Inflammation and Modulation T Helper 1/T Helper 2 Balance in Mice.

To investigate the effect of Tanshinone IIA (TSA) on viral myocarditis (VMC). VMC animal model was established using BALB/c mice by intraperitoneally injecting Coxsackie virus B3 (CVB3). The mice were randomly divided into control group, model group, and TSA group. We detected the survival rate, the heart weight to body weight (HW/BW) ratio and hemodynamic and cardiac function parameters. The pathological features of VMC were measured through H&E staining. The expression of serum enzyme, inflammatory cytokines, and T helper (Th)1/Th2 markers was also investigated. TSA remarkably alleviated CVB3-caused myocardial injury, decreased the HW/BW ratio, and improved survival rate. TSA obviously improved hemodynamic parameters and reversed the damage to the heart pump function. Furthermore, the serum levels of lactate dehydrogenase, creatine kinase, and Th1 cytokines in the TSA group were significantly lower than those in the VMC group, and TSA treatment significantly improved the pathological condition. The interferon-gamma (IFN-γ) and interleukin-2 (IL-2) levels in VMC model group was higher than control group, and lower levels of IL-4 and IL-10 were identified. However, TSA treatment elevated IL-4 and IL-10 levels and decreased IFN-γ and IL-2 levels. TSA could effectively protect the myocardium against CVB3-induced myocarditis by the inhibition of inflammation and modulation Th1/Th2 balance in mice.

A case report of fulminant type 1 diabetes mellitus associated with drug-induced hypersensitivity syndrome in an elderly patient with coxsackie B4 virus infection and human leukocyte antigen-A24 haplotype.

Drug-induced hypersensitivity syndrome (DIHS) is a severe systemic adverse drug reaction. Previous studies showed that DIHS is associated with the onset of fulminant type 1 diabetes mellitus (FT1D). Although genetic background and abnormalities in immune response or viral infection are considered to be associated with pathogenesis of FT1D, it remains unclear whether virus infection and specific human leukocyte antigen (HLA) typing are involved in DIHS-associated FT1D. Here, we report a case of a 78-year-old female patient with FT1D after DIHS treatment. She was diagnosed as DIHS caused by carbamazepine, and treatment with predonisolone was initiated. After 46 days from the occurrence of DIHS, she was admitted to our hospital because of type 1 diabetes mellitus and diabetic ketoacidosis. Although her Hemoglobin A1c (HbA1c) was elevated by predonisolone treatment (HbA1c: 9.2%), we diagnosed her as fulminant type 1 diabetes mellitus considering the abrupt onset of the ketoacidosis. Her general condition was improved by treatment with fluid infusion and insulin administration. During her clinical course, the infection of coxsackie B4 virus was observed. In addition, the examination of HLA typing showed HLA-A24 haplotype. These findings suggest that the coxsackie B4 virus infection may be involved in the pathogenesis of DIHS-induced FT1D, and that HLA-A24 haplotype might relate to DIHS-associated FT1D.

Vitamin D receptor protects glioblastoma A172 cells against Coxsackievirus A16 infection induced cell death in the pathogenesis of hand, foot, and mouth disease.

Hand, foot, and mouth disease (HFMD) was one of the most common children illnesses. Coxsackievirus A16 was one of the major pathogens that cause HFMD. However, the role of vitamin D underlying this common illness has not been elucidated. Our study examined that vitamin D levels was significantly lower in 33 HFMD patients, compared to 36 healthy children. Unexpectedly, both mRNA and protein expression of VDR were significantly decreased in CA16 infected glioblastoma A172 cells. And overexpression of VDR or vitamin D treatment in CA16 infected glioblastoma A172 cells could reverse the CA16 infection induced cell death, apoptosis or mitochondrial membrane rupture. Therefore, our study, for the first time, demonstrated that vitamin D and VDR could associate with the pathogenesis of HFMD. Thus might provide useful information for HFMD prevention and treatments.

Anti-coxsackievirus B4 (CV-B4) enhancing activity of serum associated with increased viral load and pathology in mice reinfected with CV-B4.

In previous studies it was shown that inoculation of Swiss albino mice with CV-B4 E2 resulted in the production of serum IgG capable of enhancing the CV-B4 E2 infection of murine spleen cells cultures. To investigate whether such an enhancing activity of serum can play a role in vivo, we decided to study the CV-B4 E2 infection in mice exposed to successive inoculations of virus. In Swiss albino mice infected with CV-B4 E2 at the age of 21 days, anti-CV-B4 E2 neutralizing and enhancing activities of their serum peaked after 55 d. In contrast, mice inoculated at the age of 55 d expressed much lower activities. Despite the neutralizing activity of serum, CV-B4 E2 inoculated a second time to 55 day-old animals spread into the host. At the age of 72 and 89 d the levels of viral RNA and infectious particles were higher in organs of animals exposed to 2 successive infections compared with animals infected once at the age of 21 d or 55 d. In animals with 2 successive inoculations of CV-B4 E2 there was a relationship between the anti-CV-B4 E2 enhancing activity of serum and the level of viral RNA in organs and an enhancement of pathology was observed as displayed by histological analysis of pancreas and hyperglycaemia. Altogether our data strongly suggest that an anti-CV-B4 E2 enhancing activity in the host can play a role in the outcome of a secondary infection with this virus.

Coxsackievirus B3 Directly Induced Th17 Cell Differentiation by Inhibiting Nup98 Expression in Patients with Acute Viral Myocarditis.

Th17 cells play a key role in the progression of coxsackievirus B3 (CVB3)-induced acute viral myocarditis (AVMC). However, the direct effect of virus on Th17 cell differentiation is still unknown. Recently, nucleoporin (Nup) 98 has been proved to be associated with lymphocyte differentiation. Therefore, we investigated whether Nup98 mediated Th17 cell differentiation in AVMC. In our study, patients with AVMC and healthy controls were recruited. The results showed that CVB3 could enter into the CD4+ T cells in AVMC patients and healthy controls. After transfecting purified CD4+ T cells with CVB3 in vitro, the Th17 cell frequency, IL-17 secretion, and RORγT synthesis were increased while the Nup98 levels were decreased. Furthermore, down-regulating Nup98 expression by siRNA-Nup98 in CD4+ T cells resulted in the elevated Th17 cell frequency and IL-17 secretion, along with enhanced levels of RORγT, dissociative p300/CBP, and acetylated Stat3. Up-regulation of Nup98 expression by pcDNA3.1-Nup98 showed the opposite effects. Our results suggested that CVB3 directly induced CD4+ T cell differentiation into Th17 cells by inhibiting Nup98 expression, representing a therapeutic target in AVMC.

An epidemic of coxsackievirus B3 infection in infants and children in Jiangsu Province, China: a prospective cohort study.

To investigate the epidemiological data on coxsackievirus B3 (CVB3) infection and its incidence in infants and children, a prospective cohort study was carried out from 2012 to 2014 in Jiangsu Province, China. According to the results of seropositive rates and NTAb titers of CVB3, an epidemic of CVB3 infection was found, and a dynamic change in CVB3 neutralizing antibody was also observed. One case was recorded with CVB3-associated hand, foot and mouth disease (HFMD), and the isolates belonged to the CVB3 D2 subtype. Our data help us to better understand the epidemic characteristics of CVB3 infection in infants and children.

Fulminant Type 1 Diabetes Mellitus Associated with Coxsackie Virus Type A2 Infection: A Case Report and Literature Review.

A 65-year-old Japanese man presented to our hospital in June 2013 with a 6-day history of fever and fatigue, a 24-h history of thirst, and polyuria. His temperature was 37.8°C and he was alert. However, laboratory tests revealed severe hyperglycemia, undetectable C-peptide levels, and diabetic ketoacidosis. Serum antibody testing confirmed a Coxsackie virus A2 infection. A variety of viral infections are reported to be involved in the development of fulminant type 1 diabetes mellitus (FT1D). Our patient is the first reported case of FT1D associated with Coxsackie virus A2 infection and supports the etiological role of common viral infections in FT1D.

Comparative pathogenicity of Coxsackievirus A16 circulating and noncirculating strains in vitro and in a neonatal mouse model.

An enterovirus 71 (EV71) vaccine for the prevention of hand, foot, and mouth disease (HMFD) is available, but it is not known whether the EV71 vaccine cross-protects against Coxsackievirus (CV) infection. Furthermore, although an inactivated circulating CVA16 Changchun 024 (CC024) strain vaccine candidate is effective in newborn mice, the CC024 strain causes severe lesions in muscle and lung tissues. Therefore, an effective CV vaccine with improved pathogenic safety is needed. The aim of this study was to evaluate the in vivo safety and in vitro replication capability of a noncirculating CVA16 SHZH05 strain. The replication capacity of circulating CVA16 strains CC024, CC045, CC090 and CC163 and the noncirculating SHZH05 strain was evaluated by cytopathic effect in different cell lines. The replication capacity and pathogenicity of the CC024 and SHZH05 strains were also evaluated in a neonatal mouse model. Histopathological and viral load analyses demonstrated that the SHZH05 strain had an in vitro replication capacity comparable to the four CC strains. The CC024, but not the SHZH05 strain, became distributed in a variety of tissues and caused severe lesions and mortality in neonatal mice. The differences in replication capacity and in vivo pathogenicity of the CC024 and SHZH05 strains may result from differences in the nucleotide and amino acid sequences of viral functional polyproteins P1, P2 and P3. Our findings suggest that the noncirculating SHZH05 strain may be a safer CV vaccine candidate than the CC024 strain.

Platelets interact with Coxsackieviruses B and have a critical role in the pathogenesis of virus-induced myocarditis.

BACKGROUND: To further understand the role of platelets in the pathogenesis of viral infections we explored platelet interaction with Coxsackieviruses B (CVB) 1 and 3. CVB is a group of viruses that cause the majority of human enterovirus-related viral myocarditis; their receptor (CAR) is expressed on the platelet surface and there is a well-characterized CVB3-induced myocarditis murine model. METHODS: Human platelets were infected with CVB1 and 3 and viruses were detected in pellets and in supernatants. C57BL/6J mice with or without platelet depletion were inoculated with CVB3 and peripheral blood and heart samples collected at different times post-infection. RESULTS: CVB1 and 3 RNA and a capsid protein were detected in infected platelets. Despite the fact that titration assays in Vero cells showed increasing infectivity titers over time, supernatants and pellets from infected platelets showed similar levels, suggesting that platelets were not susceptible to a replicative infectivity cycle. CVB binding was CAR-independent and resulted in P-selectin and phosphatidylserine (PS) exposure. CVB3-infected mice showed a rapid thrombocytopenia that correlated with an increase in platelet PS exposure and platelet-leukocyte aggregates without modification of platelet P-selectin expression or von Willebrand factor levels. Mortality, viremia, heart viral titers and myocarditis were significantly higher in platelet-depleted than normal animals. Type I IFN levels were not changed but IgG levels were lower in infected and platelet-depleted mice. CONCLUSIONS: Our data reveal that platelets play a critical role in host survival and immune response against CVB3 infection.

Correlations among persistent viral infection, heart function and Chinese medicine syndromes in dilated cardiomyopathy patients.

OBJECTIVE: To investigate the correlations among persistent viral infection, heart function and Chinese medicine (CM) difined-syndromes in patients with dilated cardiomyopathy (DCM). METHODS: Fifty patients with DCM in the First Affiliated Hospital of Zhejiang Chinese Medical University from October 2009 to December 2011 were selected as the research subjects, and 30 healthy people were simultaneously selected as the normal control group to detect persistent viral infections after admission. The CM syndrome type and grade of heart function were then evaluated. The expression level of Coxsackie adenovirus receptor (CAR) was detected using the flow cytometry (FCM) technique, coxsackie virus RNA (CVB-RNA) using reverse transcription polymerase chain reaction (RTPCR), and the plasma brain natriuretic peptide (BNP) level with a Triage meter plus diagnosis instrument. Finally, the parameters such as left ventricular end diastolic diameter (LVEDd) and left ventricular ejection fraction (LVEF) were measured by ultrasonic cardiogram. Person correlation analysis was used for measured data, Spearman correlation analysis for rating data, and the Chi-square test for numerical data. RESULTS: CVB-RNA was positive in 22 patients (44%) with DCM, while only 6 cases (20%) were CVB-RNA-positive in the normal control group, with a significant difference between the two groups (P<0.01). The expression level of CAR was significantly elevated in the DCM group compared with the normal control group (P<0.01). In CVB-RNA-positive patients (22 cases), the expression level of CAR was significantly higher than in CVB-RNA-negative patients (28 cases; P<0.01). In the DCM patients, there was a positive correlation between the CAR expression and the BNP level (r=0.34, P<0.05), while no significant difference was found between the CAR expression and the LVEF and LVEDd (r=-0.32, 0.30, P>0.05). There was no clear correlation between virus infection and the CM syndrome types in DCM patients (r=-0.22, P>0.05). According to the sequence of syndrome types: phlegm → qi deficiency → blood stasis → hydroretention with asthenic yang (from low to high), a positive correlation was existed between the BNP levels and CM syndrome types (r=0.139, P<0.05). CONCLUSION: The expression of CAR on the surface of white cells could be used to detect persistent viral infection. The expression level of CAR and heart function in DCM patients were highly correlated. The expression level of BNP may serve as an objective index for differentiating CM syndromes for patients with DCM.

Inhibition of coagulation factor Xa improves myocardial function during CVB3-induced myocarditis.

INTRODUCTION: Myocarditis is induced by coxsackievirus B3 (CVB3). Myocardial inflammation is tied to the activation of coagulation. Coagulation factor (F) Xa, a central player in coagulation, activates matrix metalloproteinases (MMP), which modulate the remodeling. AIMS: In this study, we investigated the effects of pharmacological FXa inhibition on myocardial function, inflammation, and remodeling during a CVB3-induced myocarditis. METHODS AND RESULTS: Immune cells and matrix proteins were detected by immunohistochemistry. The expression of cytokines was measured by real-time PCR and the activity of MMP-2 by zymography. Left ventricular function was analyzed using microconductance pressure catheter. Treatment with the FXa inhibitor fondaparinux led to an improved left ventricular function in CVB3-induced mice compared to saline-treated controls (dPdtmax: fondaparinux 4632 ± 499.6 vs. saline 3131 ± 374.0 [mmHg/s], P = 0.0503; SV: fondaparinux 33.19 ± 4.893 vs. saline 19.32 ± 2.236 [µL], P < 0.118; CO: fondaparinux 15124 ± 2183 vs. saline 8088 ± 1035 [µL/min], P < 0.05). Therapy with fondaparinux reduced the activity of MMP-2 (fondaparinux 1.208 ± 0.1247 vs. saline 1.565 ± 0.05476, P < 0.05). The collagen type I/III ratio as well as the expression of TIMP-1 was comparable in both infection groups postinfectionem (p.i.), despite an increased infiltration of macrophages into the hearts of mice treated with fondaparinux 8 days p.i. (CD68+: fondaparinux 494.2 ± 64.73 vs. saline 306.9 ± 43.73 [cells/mm(2) ], P < 0.05). Anti-inflammatory CD206-positive M2-type macrophages were increased in the infected hearts after fondaparinux treatment (CD206+: fondaparinux 182.1 ± 18.18 vs. saline 111.6 ± 21.07 [cells/mm(2) ], P < 0.05), whereas CD80-positive M1-type macrophages were comparable in both groups. CONCLUSION: In conclusion, selective inhibition of FXa improves the left ventricular function during CVB3-induced myocarditis and seems to be associated with an improved myocardial remodeling.

Seroprevalence of coxsackievirus B3 in Yantai, China.

Coxsackievirus B3 (CVB3) is an important human pathogen, which is frequently associated with aseptic meningitis and encephalitis in many parts of the world. However, the seroprevalence of this viral infection in China is not well understood; therefore, in the present study, we conducted a serological survey in which 373 serum samples obtained from healthy people belonging to 10 age groups in Yantai city, China were examined. The results revealed that CVB3 is widely distributed in the population with a seroprevalence of 52.3%. The seroprevalence of CVB3 was lowest (3.7%) in the group aged 8-12 months and >50% in all groups aged >15 years. A significant increase (P < 0.001) was observed among the successive four groups aged 1, 2-4, 5-7, and 8-10 years. Variation of geometric mean titer related to age showed similar tendencies. Our results indicated that CVB3 infections occurred mostly in preschool and early-elementary school settings, and children <5 years of age are the most susceptible populations due to their low CVB3 antibody levels.

Coxsackievirus B3 infects the bone marrow and diminishes the restorative capacity of erythroid and lymphoid progenitors.

Coxsackievirus B3 (CVB3) is known to infect stem cells in the neonatal central nervous system. Here, we evaluated the effects of CVB3 infection on the major source and repository of stem cells, the bone marrow (BM). Viral genome was detectable in BM within 24 h of infection, and productive infection of BM cells was evident, peaking at 48 h postinfection (p.i.), when ∼1 to 2% of BM cells produced infectious virus particles. Beginning at 2 to 3 days p.i., a dramatic and persistent loss of immature erythroid cells, B and T lymphocytes, and neutrophils was observed in BM and, by day 3 to 4 p.i., the femoral BM stroma was largely destroyed. Analysis of peripheral blood revealed a modest neutrophilia, a loss of reticulocytes, and a massive lymphopenia. The abundance of multipotent progenitor cells (Lin(-)/c-kit(+)/Flt3(+)) in BM declined ∼10-fold during CVB3 infection and, consistent with a deficiency of primitive hematopoietic progenitors, serum levels of the hematopoietic growth factor Flt3 ligand were dramatically elevated. Therefore, we analyzed the regenerative capacity of BM from CVB3-infected mice. Granulocyte/macrophage progenitors displayed a relatively normal proliferative ability, consistent with the fact that the peripheral blood level of neutrophils-which are very short-lived cells-remained high throughout infection. However, erythroid and lymphoid hematopoietic progenitors in BM from CVB3-infected mice showed a markedly reduced colony-forming capacity, consonant with the observed loss of both lymphocytes and immature erythroid cells/reticulocytes from the BM and peripheral blood. In summary, CVB3 infects the BM and exerts differential effects on the various hematopoietic progenitor populations.

Viral kinetics are associated with changes in cytokines and chemokines in serum and target organs of SSM-CVB3-infected macaques.

OBJECTIVE: To determine the relationship between viral kinetics and the expression patterns for different cytokines and chemokines in the serum and organs of coxsackievirus B3 (SSM-CVB3)-infected macaques over the course of infection. METHODS: SSM-CVB3 levels in serum and organs were measured using the Spearman-Karber 50% tissue culture infectious dose (TCID(50)) method. Cytokine and chemokine levels in the serum and organs were measured by indirect-ELISA. RESULTS: Low viral titers were detected in the serum samples on the first day post-inoculation (p.i.) and peaked at 6 to 10 days p.i. in the serum samples from five macaques. Serum levels of IL-1ß, IL-2, IL-6, IL-12p40, IL-17α, IFN-γ, TNF-α, MCP-1 and MIP-1ß were detected each day and, similar to the viral titers, peaked at 6 to 10 days. IL-10 was only detected on days 10 to 14 p.i. Additionally, higher viral titers and relative viral mRNA levels were associated with higher cytokine and chemokine levels in selected tissues from infected macaques including heart, liver, spleen, lung, kidney and brain. CONCLUSION: The results indicate that patterns of cytokine and chemokine response are associated with viral kinetics in the serum and target organs of SSM-CVB3-infected macaques, suggesting that the changes in cytokines and chemokines could help further our understanding of the progress of CVB3 infections in clinical settings.

Outcome of challenge with Coxsackievirus B4 in young mice after maternal infection with the same virus during gestation.

Enteroviral infections go usually unnoticed, even during pregnancy, yet some case histories and mouse experiments indicate that these viruses may be transmitted vertically. More frequently, however, transmission occurs by (fecal) contamination during and shortly after birth. The aim of this study was to investigate the effect of maternal infection in mice (1) on gravidity outcome and (2) on subsequent challenge of the offspring with the same virus. CD1 outbred female mice were infected by the oral route with coxsackievirus B4 strain E2 or mock-infected at days 4, 10, or 17 of gestation. Weight and signs of sickness were noted daily. Pups were infected at day 25 after birth (4 days postweaning). Organs (brain, pancreas, and heart) were analyzed for viral RNA and histopathology. We observed that maternal infection at day 4 or day 17 of gestation had little effect on pregnancy outcome, whereas infection at day 10 affected dams and/or offspring. Infection of pups resulted in severe inflammation of the pancreas, but only when dams were previously infected, especially at day 17. The blood glucose levels were elevated. Because no trace of infection was found at the time of challenge, a role for immunopathology is suggested.