Gripe do tomate / Tomato flu

A Gripe do Tomate é uma doença cuja etiologia ainda não está bem definida, podendo ser causada por uma variante do vírus Coxsackie, responsável pela doença mão-pé-boca ou ainda, por um quadro pós-viral de Chikungunya ou Dengue (FERREIRA, 2022; GZH SAÚDE, 2022). Embora seja conhecida por Gripe ou Febre do Tomate, a doença não possui nenhuma relação com o consumo do fruto, mas refere-se a ele pela semelhança das erupções de bolhas vermelhas e dolorosas que acometem todo o corpo e aumentam gradualmente (CAMAÇARI NOTÍCIAS, 2022; FOLHA VITÓRIA, 2022)

Tomato Flu is a disease whose etiology is not yet well defined, and may be caused by a variant of the Coxsackie virus, responsible for hand-foot-and-mouth disease, or by a post-viral condition of Chikungunya or Dengue (FERREIRA, 2022). ; GZH HEALTH, 2022). Although it is known as Influenza or Tomato Fever, the disease does not have any relationship with the consumption of the fruit, but refers to it by the similarity of the eruptions of red and painful blisters that affect the whole body and gradually increase (CAMAÇARI NOTÍCIAS, 2022; FOLHA VITÓRIA, 2022)

Attenuated strain of CVB3 with a mutation in the CAR-interacting region protects against both myocarditis and pancreatitis.

Coxsackievirus B3 (CVB3), is commonly implicated in myocarditis, which can lead to dilated cardiomyopathy, in addition to causing acute pancreatitis and meningitis. Yet, no vaccines are currently available to prevent this infection. Here, we describe the derivation of a live attenuated vaccine virus, termed mutant (Mt) 10, encoding a single amino acid substitution H790A within the viral protein 1, that prevents CVB3 infection in mice and protects from both myocarditis and pancreatitis in challenge studies. We noted that animals vaccinated with Mt 10 developed virus-neutralizing antibodies, predominantly containing IgG2a and IgG2b, and to a lesser extent IgG3 and IgG1. Furthermore, by using major histocompatibility complex class II dextramers and tetramers, we demonstrated that Mt 10 induces antigen-specific T cell responses that preferentially produce interferon-γ. Finally, neither vaccine recipients nor those challenged with the wild-type virus revealed evidence of autoimmunity or cardiac injury as determined by T cell response to cardiac myosin and measurement of circulating cardiac troponin I levels, respectively. Together, our data suggest that Mt 10 is a vaccine candidate that prevents CVB3 infection through the induction of neutralizing antibodies and antigen-specific T cell responses, the two critical components needed for complete protection against virus infections in vaccine studies.

VP1 of Enterovirus 71 Protects Mice Against Enterovirus 71 and Coxsackievirus B3 in Lethal Challenge Experiment.

Enterovirus and Coxsackievirus are the major viruses that cause hand, foot, and mouth disease (HFMD) outbreaks worldwide. Several studies have shown the potential of viral envelope protein 1 (VP1) on providing protective effects from viral strains of different genotypes. However, whether VP1 has the cross-protection in Enteroviruses or Coxsackievirus has not been studied in-depth. In this study, the vp1 gene of Enterovirus 71 (EV71) and Coxsackievirus B3 (CB3) was inserted into the vector pET22b (+) to form the respective expression plasmids pEVP1 or pCVP1, and then transformed into Escherichia coli strain BL21 (DE3). The recombinant EVP1 or CVP1 protein was overexpressed successfully and effectively purified to homogeneity. Then, we identified that EVP1 and CVP1 protein could generate effectively specific humoral immunity and cellular immunity in mice, what's more, we determined the cross-protection of VP1 between EV71 and CB3 in a murine model. The results showed that immunization with EVP1 could effectively induce specific IgG and secretory IgA against CVP1 and the sera from EVP1-immunized mice could neutralize CB3 with mean titers 1:440. In contrast, no measurable neutralizing antibodies to EV71 were detected in CVP1-immunized mice. Then, newborn BALB/C mice, whose mother was immunized with EVP1 or CVP1, were administered with different lethal doses of EV71 or CB3. The EVP1 immunized group showed a 90% protective efficacy for a CB3 dosage of 120 LD50, but the CVP1 immunized group showed no significantly different protective efficacy against 15 LD50 of EV71 compared with the BSA immunized group. Hence, EVP1 is a promising subunit vaccine candidate against Enterovirus 71 and Coxsackievirus B3 caused HFMD.

A comparative study of the effect of UV and formalin inactivation on the stability and immunogenicity of a Coxsackievirus B1 vaccine.

Type B Coxsackieviruses (CVBs) belong to the enterovirus genus, and they cause both acute and chronic diseases in humans. CVB infections usually lead to flu-like symptoms but can also result in more serious diseases such as myocarditis, aseptic meningitis and life-threatening multi-organ infections in young infants. Thus, CVBs have long been considered as important targets of future vaccines. We have previously observed CVB1 capsid disintegration and virus concentration decrease with 12-day long formalin inactivation protocol. Here a scalable ion exchange chromatography purification method was developed, and purified CVB1 was inactivated with UV-C or formalin. Virus morphology and concentration remained unchanged, when the UV (2 min) or formalin (5 days) inactivation were performed in the presence of tween80 detergent. The concentration of the native and UV inactivated CVB1 remained constant at 4 °C during a six months stability study, whereas the concentration of the formalin inactivated vaccine decreased 29% during this time. UV treatment decreased, whereas formalin treatment increased the thermal stability of the capsid. The formalin inactivated CVB1 vaccine was more immunogenic than the UV inactivated vaccine; the protective neutralizing antibody levels were higher in mice immunized with formalin inactivated vaccine. High levels of CVB1 neutralizing antibodies as well as IgG1 antibodies were detected in mice that were protected against viremia induced by experimental CVB1 infection. In conclusion, this study describes a scalable ion exchange chromatography purification method and optimized 5-day long formalin inactivation method that preserves CVB1 capsid structure and immunogenicity. Formalin treatment stabilizes the virus particle at elevated temperatures, and the formalin inactivated vaccine induces high levels of serum IgG1 antibodies (Th2 type response) and protective levels of neutralizing antibodies. Formalin inactivated CVB vaccines are promising candidates for human clinical trials.

FTY720 alleviates coxsackievirus B3-induced myocarditis and inhibits viral replication through regulating sphingosine 1-phosphate receptors and AKT/caspase-3 pathways.

Fingolimod (FTY720) after phosphorylation, as the ligand of sphingosine 1-phosphate receptors (S1PRs), plays an important role in cell proliferation and differentiation. In this article, FTY720 in the treatment of coxsackievirus B3 (CVB3)-induced viral myocarditis was closely related to apoptosis and AKT/caspase-3 apoptotic pathways. We found that CVB3 inhibited myocardial apoptosis at the early stage with upregulating p-AKT level and downregulating activated caspase-3 level for replication of virus progeny, whereas it promoted apoptosis at a late stage with downregulating p-AKT and upregulating activated caspase-3 for releasing the newly synthesized virus to spread. Interestingly, FTY720 could reverse this trend; it promoted apoptosis at an early stage and inhibited apoptosis at the late stage in vivo and vitro, which proved the antiviral effect. We also found that S1PR1, S1PR4, and S1PR5, rather than S1PR2 and S1PR3, were regulated by FTY720 in this process. The results confirmed that FTY720 alleviates CVB3-induced myocarditis and inhibits viral replication through regulating S1PRs and AKT/caspase-3 pathways with a bidirectional regulation of apoptosis.

The cholinergic anti-inflammatory pathway ameliorates acute viral myocarditis in mice by regulating CD4+ T cell differentiation.

Many studies have found that abnormalities in the proportion and differentiation of CD4+ T cells (Th cells) are closely related to the pathogenesis of viral myocarditis (VMC). Our previous research indicates that the cholinergic anti-inflammatory pathway (CAP) attenuates the inflammatory response of VMC and downregulates the expression of cytokines in Th1 and Th17 cells. This suggests that the cholinergic anti-inflammatory pathway likely attenuates the inflammatory response in VMC by altering Th cell differentiation. The aim of this study is to investigate the effect of CAP on CD4+ T cell differentiation in VMC mice. CD4+ T cells in the spleen of VMC mice were obtained and cultured in the presence of nicotine or methyllycaconitine (MLA). Cells were harvested and analyzed for the percentage of each Th cell subset by flow cytometry and transcription factor release by Western blot. Then, we detected the effect of CAP on the differentiation of Th cells in vivo. Nicotine or MLA was used to activate and block CAP, respectively, in acute virus-induced myocarditis. Nicotine treatment increased the proportion of Th2 and Treg cells, decreased the proportion of Th1 and Th17 cells in the spleen, reduced the level of proinflammatory cytokines, and attenuated the severity of myocardium lesions and cellular infiltration in viral myocarditis. MLA administration had the opposite effect. Our result demonstrated that CAP effectively protects the myocardium from virus infection, which may be attributable to the regulation of Th cell differentiation.

NOD2 (Nucleotide-Binding Oligomerization Domain 2) Is a Major Pathogenic Mediator of Coxsackievirus B3-Induced Myocarditis.

BACKGROUND: The cytoplasmatic pattern recognition receptor, NOD2 (nucleotide-binding oligomerization domain 2), belongs to the innate immune system and is among others responsible for the recognition of single-stranded RNA. With Coxsackievirus B3 (CVB3) being a single-stranded RNA virus, and the recent evidence that the NOD2 target, NLRP3 (NOD-like receptor family, pyrin domain containing 3) is of importance in the pathogenesis of CVB3-induced myocarditis, we aimed to unravel the role of NOD2 in CVB3-induced myocarditis. METHODS AND RESULTS: Endomyocardial biopsy NOD2 mRNA expression was higher in CVB3-positive patients compared with patients with myocarditis but without evidence of persistent CVB3 infection. Left ventricular NOD2 mRNA expression was also induced in CVB3-induced myocarditis versus healthy control mice. NOD2 knockdown(-/-) mice were rescued from the detrimental CVB3-mediated effects as shown by a reduced cardiac inflammation (less cardiac infiltrates and suppression of proinflammatory cytokines), cardiac fibrosis, apoptosis, lower CAR (Coxsackievirus and adenovirus receptor) expression and CVB3 copy number, and an improved left ventricular function in NOD2-/- CVB3 mice compared with wild-type CVB3 mice. In agreement, NOD2-/- decreased the CVB3-induced inflammatory response, CVB3 copy number, and apoptosis in vitro. NOD2-/- was further associated with a reduction in CVB3-induced NLRP3 expression and activity as evidenced by lower ASC (apoptosis-associated speck-like protein containing a CARD) expression, caspase 1 activity, or IL-1ß (interleukin-1ß) protein expression under in vivo and in vitro CVB3 conditions. CONCLUSIONS: NOD2 is an important mediator in the viral uptake and inflammatory response during the pathogenesis of CVB3 myocarditis.

AIM2 Co-immunization with VP1 Is Associated with Increased Memory CD8 T Cells and Mounts Long Lasting Protection against Coxsackievirus B3 Challenge.

The recurrent Coxsackievirus B3 (CVB3) infection is the most important cause of intractable myocarditis which often leads to chronic myocarditis and even dilated cardiomyopathy. Therefore, enhanced DNA vaccines capable of memory CD8 T cells are essential for long-lasting immunological protection against CVB3 infection. In this study, absent in melanoma 2 (AIM2) was used as an adjuvant to enhance the induction of memory CD8 T cells elicited by VP1 (viral capsid protein 1) vaccine. Mice were intramuscularly injected with 50 µg AIM2 plasmid and equal amount of VP1 plasmid (pAIM2/pVP1) vaccine 4 times at 2 week-intervals. We observed that the protection of pAIM2/pVP1 vaccine against CVB3 challenge was evidenced by significantly improved cardiac function, reduced myocardial injuries, and increased survival rate when compared with immunization with pVP1. Co-immunization with pAIM2/pVP1 robustly augmented T lymphocytes proliferation and CVB3-specific cytotoxic T lymphocyte responses. Importantly, 16 weeks after the last immunization, pAIM2/pVP1 co-immunization significantly enhanced the expression of Bcl-6, SOCS3, and Sca-1 which are critical for memory CD8 T cells as compared with pVP1 immunization. Notably, CD8 T cells that are likely vaccine-induced memory T cells were responsible for the protective efficacy of pAIM2/pVP1 vaccine by abolition of a CD8 T cell immune response following a lethal dose of CVB3 infection. Our results indicate that AIM2-adjuvanted vaccine could be a potential and promising approach to promote a long-lasting protection against CVB3-induced myocarditis.

Bifidobacteria-derived lipoproteins inhibit infection with coxsackievirus B4 in vitro.

The aim of the present study was to investigate the potential of bifidobacteria in protecting cells from coxsackievirus B4 (CV-B4) infection. Bifidobacterial screening identified two of five strains that protected human epithelial type 2 (HEp-2) cell viability when bifidobacteria were incubated with viral particles prior to inoculation. In contrast, no effect was shown by incubating HEp-2 cells with bifidobacteria prior to CV-B4 inoculation. Cell wall lipoprotein aggregates (LpAs) secreted by the selected strains were assayed for their antiviral activity. The two LpAs exhibited antiviral activity when they were incubated with viral particles prior to inoculation of HEp-2 cells. Recombinant LpA-derived protein exhibited identical antiviral activity. To identify the peptide sequences interacting with the virus particles, LpA proteins were aligned with the peptide sequences of the north canyon rim and puff footprint onto coxsackievirus and adenovirus receptor (CAR). The in silico molecular docking study using CV-B3 as template showed low-energy binding, indicating a stable system for the selected peptides and consequently a likely binding interaction with CV-B. Bifidobacterium longum and Bifidobacterium breve peptides homologous to the viral north rim footprint onto CAR sequence formed hydrogen bonds with several viral residues in the north rim of the canyon, which were already predicted as interacting with CAR. In conclusion, proteins from bifidobacterial LpAs can inhibit infection with CV-B4, likely through binding to the capsid amino acids that interact with CAR.

Protective Efficacies of Formaldehyde-Inactivated Whole-Virus Vaccine and Antivirals in a Murine Model of Coxsackievirus A10 Infection.

Coxsackievirus A10 (CVA10) is one of the major pathogens associated with hand, foot, and mouth disease (HFMD). CVA10 infection can cause herpangina and viral pneumonia, which can be complicated by severe neurological sequelae. The morbidity and mortality of CVA10-associated HFMD have been increasing in recent years, particularly in the pan-Pacific region. There are limited studies, however, on the pathogenesis and immunology of CVA10-associated HFMD infections, and few antiviral drugs or vaccines have been reported. In the present study, a cell-adapted CVA10 strain was employed to inoculate intramuscularly 5-day-old ICR mice, which developed significant clinical signs, including reduced mobility, lower weight gain, and quadriplegia, with significant pathology in the brain, hind limb skeletal muscles, and lungs of infected mice in the moribund state. The severity of illness was associated with abnormally high expression of the proinflammatory cytokine interleukin 6 (IL-6). Antiviral assays demonstrated that ribavirin and gamma interferon administration could significantly inhibit CVA10 replication both in vitro and in vivo In addition, formaldehyde-inactivated CVA10 whole-virus vaccines induced immune responses in adult mice, and maternal neutralizing antibodies could be transmitted to neonatal mice, providing protection against CVA10 clinical strains. Furthermore, high-titer antisera were effective against CVA10 and could relieve early clinical symptoms and improve the survival rates of CVA10-challenged neonatal mice. In summary, we present a novel murine model to study CVA10 pathology that will be extremely useful in developing effective antivirals and vaccines to diminish the burden of HFMD-associated disease.IMPORTANCE Hand, foot, and mouth disease cases in infancy, arising from coxsackievirus A10 (CVA10) infections, are typically benign, resolving without any significant adverse events. Severe disease and fatalities, however, can occur in some children, necessitating the development of vaccines and antiviral therapies. The present study has established a newborn-mouse model of CVA10 that, importantly, recapitulates many aspects of human disease with respect to the neuropathology and skeletal muscle pathology. We found that high levels of the proinflammatory cytokine interleukin 6 correlated with disease severity and that ribavirin and gamma interferon could decrease viral titers in vitro and in vivo Whole-virus vaccines produced immune responses in adult mice, and immunized mothers conferred protection on neonates against challenge from CVA10 clinical strains. Passive immunization with high-titer antisera could also improve survival rates in newborn animals.

Interleukin-37 Ameliorates Coxsackievirus B3-induced Viral Myocarditis by Modulating the Th17/Regulatory T cell Immune Response.

Myocarditis is a heterogeneous group of disorders defined by inflammation of the heart muscle with an excessively activated immune response. Numerous interventions have been investigated for the treatment of myocarditis while success is limited. Interleukin-37 (IL-37), a novel member of the IL-1 cytokine family, is a natural inhibitor of innate immunity associated with autoimmune diseases. However, the modulatory effect of IL-37 in myocarditis is unknown. In this study, we investigated the immunological regulation of IL-37 in the coxsackievirus B3-induced model of murine viral myocarditis. The results show that IL-37 significantly ameliorates the signs of myocarditis with increased survival rate and bodyweight, improved histological changes, reduced activities of MB isoenzyme of creatine kinase and cardiac troponin I, and a suppressed response of Th17 cells and enhanced response of regulatory T cells (Tregs) in the spleen. Moreover, IL-37 down-regulates the expression of Th17-related cytokines IL-6 and IL-17A, while promoting Treg-related cytokine IL-10 levels in the heart. Therefore, IL-37 may exhibit anti-inflammatory activity in the murine model of myocarditis by regulating the balance between Th17 and Treg cells, thereby providing a possible novel therapeutic target in myocarditis.

Yeast-produced recombinant virus-like particles of coxsackievirus A6 elicited protective antibodies in mice.

Coxsackievirus A6 (CA6) has recently emerged as the predominant pathogen of hand, foot and mouth disease (HFMD), causing significant morbidity in children and adults. The increasing prevalence of CA6 infection and its associated disease burden underscore the need for effective CA6 vaccines. However, CA6 grows poorly in cultured cells, making it difficult to develop inactivated whole-virus or live attenuated vaccines. Here we report the development of a recombinant virus-like particle (VLP) based CA6 vaccine. CA6 VLPs were produced in Pichia pastoris yeast transformed with a vector encoding both P1 and 3CD proteins of CA6. Immunization with CA6 VLPs elicited CA6-specific serum antibodies in mice. Passive transfer of anti-VLP antisera protected recipient mice against lethal CA6 challenge. Collectively, these results demonstrate that CA6 VLPs represent a viable CA6 vaccine candidate which warrants further preclinical and clinical development.

Involvement of Endoplasmic Reticulum Stress-Mediated C/EBP Homologous Protein Activation in Coxsackievirus B3-Induced Acute Viral Myocarditis.

BACKGROUND: This study tested the hypothesis whether endoplasmic reticulum (ER) stress/C/EBP homologous protein (CHOP) signaling is linked with coxsackievirus B3 (CVB3)-induced acute viral myocarditis (AVMC) in vivo. METHODS AND RESULTS: AVMC was induced by intraperitoneal injection of 1000 tissue culture infectious dose (TCID50) of CVB3 virus in mice. In AVMC mouse hearts (n=11), ER stress and CHOP were significantly activated, and were linked to the induction of proapoptotic signaling including reduction of Bcl-2, activation of Bax and caspase 3, compared with the controls (n=10), whereas these could be markedly blocked by ER stress inhibitor tauroursodeoxycholic acid administration (n=11). Moreover, chemical inhibition of ER stress significantly attenuated cardiomyocytes apoptosis, and prevented cardiac troponin I elevation, ameliorated cardiac dysfunction assessed by both hemodynamic and echocardiographic analysis, reduced viral replication, and increased survival rate after CVB3 inoculation. We further discovered that genetic ablation of CHOP (n=10) suppressed cardiac Bcl-2/Bax ratio reduction and caspase 3 activation, and prevented cardiomyotes apoptosis in vivo, compared with wild-type receiving CVB3 inoculation (n=10). Strikingly, CHOP deficiency exhibited dramatic protective effects on cardiac damage, cardiac dysfunction, viral replication, and promoted survival in CVB3-caused AVMC. CONCLUSIONS: Our data imply the involvement of ER stress/CHOP signaling in CVB3-induced AVMC via proapoptotic pathways, and provide a novel strategy for AVMC treatment.

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.

Soluble coxsackievirus B3 3C protease inhibitor prevents cardiomyopathy in an experimental chronic myocarditis murine model.

BACKGROUND: Coxsackievirus B3 (CVB3) is a common cause of myocarditis and dilated cardiomyopathy. CVB3 3C protease (3CP) cleaves the viral polyprotein during replication. We tested whether a water soluble 3CP inhibitor (3CPI) had antiviral effects in a chronic myocarditis model. METHODS: Chronic myocarditis was established using DBA/2 strain mice. Starting on post-infection (p.i) day 3, CVB3-infected mice (n=41) were treated with 3CPI by daily intraperitoneal (i.p.) injection at a concentration of 50 µM (1.7 mg/kg/day) per day for 3 consecutive days. Additional mice (n=49) were injected with PBS as a control. RESULTS: The 5-week survival rate was significantly higher with 3CPI treatment (82.3% versus 47.9%; P<0.05). Organ virus titers at day 3 and 7 and myocardial damage were significantly lower in 3CPI-treated mice. Echocardiography at day 31 indicated strong protection of heart function by 3CPI (FS, 51.2±1.5 versus 26.1±1.5%; P<0.001). Hemodynamic measurements indicated that 3CPI treatment markedly reduced CVB3-induced LV dysfunction on day 31 (dP/dTmax, 5302±352 versus 4103±408 mmHg/s, P<0.05; dP/dTmin, -3798±212 versus -2814±206 mmHg/s, P<0.01). CONCLUSIONS: Water soluble 3CPI was delivered through i.p. injection after CVB3 infection. This agent preserved heart function and decreased organ viral titers and myocardial damage. Soluble 3CPI may be beneficial in the treatment of cardiomyopathy associated with enterovirus infection.

[Protective effect of hydrogen sulfide on mice with experimental viral myocarditis and its mechanism].

OBJECTIVE: To explore the protective effect of hydrogen sulfide (H2S) on viral myocarditis (VMC) mice and its mechanism. METHODS: Seventy BALB/c mice were randomly divided into 4 groups: normal control group (n=10), myocarditis group (n=20), DL-propargylglycine (PAG) group (n=20) and sodium hydrosulfide (NaHS) group (n=20). Mice in the latter three groups were inoculated with 0.1 mL Eagle's solution containing Coxsackievirus B3 (CVB3) intraperitoneally; whereas those in normal control group were treated with 0.1 mL Eagle's solution. On the day of inoculation, mice in PAG and NaHS groups received intraperitoneal administration with 40 mg/kg PAG and 50 µmol/kg NaHS, respectively; however, those in the other two groups were treated with PBS instead. PAG, NaHS or PBS were given one time for one day and persisted for 14 days. On day 15, all mice were killed after weighing body mass (BM). The mortality was compared among groups. Serum was separated and serum cardiac troponin I (cTnI) levels were detected using ELISA. The heart was removed and weighed to calculate heart mass (HM). Histological cross sections of heart were stained with hematoxylin-eosin, and myocardial histopathologic scores were counted under optical microscope. The activities of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione-peroxidase (GSH-Px) and catalase (CAT) in the homogenate of myocardium were examined. Myocardial interleukin (IL)-1ß, IL-6 and tumor necrosis factor-α (TNF-α) contents were detected by ELISA. The expression level of myocardial nuclear factor-κB (NF-κB) p65 in the nucleus was examined using Western blotting. RESULTS: Compared with normal control group, the HM/BM, serum cTnI levels, myocardial MDA activity, NF-κB p65 expression level in the nucleus and myocardial contents of IL-1ß, IL-6 and TNF-α were elevated, but myocardial SOD, GSH-Px and CAT activities were reduced in myocarditis group (P<0.05 or P<0.01). In comparison with myocarditis group, HM/BM, serum cTnI levels, myocardial histopathologic scores, myocardial MDA activity, NF-κB p65 expression level in the nucleus and myocardial contents of IL-1ß, IL-6 and TNF-α significantly increased while myocardial SOD, GSH-Px and CAT activities significantly decreased in PAG groups (P<0.05). There was no significant difference in the mortality between PAG group and myocarditis group (P>0.05). The mortality, HM/BM, serum cTnI levels, myocardial histopathologic scores, myocardial MDA activity, NF-κB p65 expression level in the nucleus and myocardial contents of IL-1ß, IL-6 and TNF-α were lower, while myocardial SOD, GSH-Px and CAT activities were higher in NaHS group than in myocarditis group (P<0.05). CONCLUSION: H2S can produce effective protection against VMC in mice. The mechanism may be associated with enhancing antioxidative ability and inhibiting inflammatory response.

A vesicular stomatitis virus-based mucosal vaccine promotes dendritic cell maturation and elicits preferable immune response against coxsackievirus B3 induced viral myocarditis.

Recombinant vesicular stomatitis virus (VSV) is widely used as a vaccine platform. However, the capacity of VSV-based vaccines to induce mucosal immunity has not been fully investigated. In the present study, a recombinant VSV expressing coxsackievirus B3 (CVB3) major immunogen VP1 has been generated and the immune protection elicited by VSV-VP1 was evaluated. We demonstrated that intranasal delivery of VSV-VP1 can induce a potent antigen-specific mucosal immune response as well as a systemic immune response, particularly the induction of polyfunctional T cells. Importantly, mice immunized with VSV-VP1 were better protected against CVB3-induced viral myocarditis than those receiving a chitosan-formulated DNA vaccine. Increased dendritic cell (DC) maturation in the mesenteric lymph node (MLN) was observed in the mice vaccinated with VSV-VP1, which could be a potential mechanism for the protective immune response. These findings support VSV as a viral delivery vector that can induce robust mucosal immunity that should be considered for further vaccine development.

[Protective effect and mechanism of IL-17 monoclonal antibody on mice with viral myocarditis].

OBJECTIVE: To explore the protective effects of interleukin-17 monoclonal antibody (IL-17 mAb) on viral myocarditis (VMC) mice and its possible molecular mechanisms. METHODS: Ninety BALB/c mice were randomly divided into 4 groups: normal control group (n=15), model group (n=25), isotype control group (n=25) and IL-17 mAb group (n=25). Mice in model, isotype control and IL-17 mAb groups were inoculated with 0.1 mL Eagle's solution containing Coxsackievirus B3 (CVB3) intraperitoneally; and those in normal control group were treated with 0.1 mL Eagle's solution without CVB3. On the day 3 and 5 after inoculation, mice in isotype control and IL-17 mAb groups received intragastric administration of 100 µg non-specific IgG antibody and IL-17 mAb, respectively. On day 7 postinoculation, 5 mice were killed in each group, and the hearts were removed. Virus titer was detected using Reed-Muench method, and CVB3 mRNA copy number was measured by real-time quantitative PCR. All mice were killed on day 14 after weighing body mass (BM). The mortality was compared among groups. Serum was separated and serum cardiac troponin I (cTnI) concentration was detected using ELISA. The heart was removed and weighed to calculate heart index (HM/BM). Histological sections of heart were stained with hematoxylin-eosin and myocardial histopathologic scores were counted under optical microscope. The expression of nuclear factor-κB (NF-κB) p65 was examined by Western blotting. Myocardial interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) levels were detected by ELISA. RESULTS: The HM/BM, serum cTnI concentration, NF-κB p65 expression level and myocardial IL-6 and TNF-α contents in model group were higher than those in normal control group (P<0.01). In comparison with model and isotype control groups, mortality, HM/BM, serum cTnI concentration, myocardial histopathologic scores, virus titer, CVB3 mRNA copy number, NF-κB p65 expression level, and myocardial IL-6 and TNF-α contents in IL-17 mAb group were significantly reduced (P<0.05 or 0.01). There was no difference in the above indicators between isotype control group and model group(P>0.05). CONCLUSION: IL-17 mAb can improve myocardial injury in VMC mice, and the mechanisms are associated with the inhibition of viral replication and NF-κB activation.

Interspecies differences in virus uptake versus cardiac function of the coxsackievirus and adenovirus receptor.

UNLABELLED: The coxsackievirus and adenovirus receptor (CAR) is a cell contact protein with an important role in virus uptake. Its extracellular immunoglobulin domains mediate the binding to coxsackievirus and adenovirus as well as homophilic and heterophilic interactions between cells. The cytoplasmic tail links CAR to the cytoskeleton and intracellular signaling cascades. In the heart, CAR is crucial for embryonic development, electrophysiology, and coxsackievirus B infection. Noncardiac functions are less well understood, in part due to the lack of suitable animal models. Here, we generated a transgenic mouse that rescued the otherwise embryonic-lethal CAR knockout (KO) phenotype by expressing chicken CAR exclusively in the heart. Using this rescue model, we addressed interspecies differences in coxsackievirus uptake and noncardiac functions of CAR. Survival of the noncardiac CAR KO (ncKO) mouse indicates an essential role for CAR in the developing heart but not in other tissues. In adult animals, cardiac activity was normal, suggesting that chicken CAR can replace the physiological functions of mouse CAR in the cardiomyocyte. However, chicken CAR did not mediate virus entry in vivo, so that hearts expressing chicken instead of mouse CAR were protected from infection and myocarditis. Comparison of sequence homology and modeling of the D1 domain indicate differences between mammalian and chicken CAR that relate to the sites important for virus binding but not those involved in homodimerization. Thus, CAR-directed anticoxsackievirus therapy with only minor adverse effects in noncardiac tissue could be further improved by selectively targeting the virus-host interaction while maintaining cardiac function. IMPORTANCE: Coxsackievirus B3 (CVB3) is one of the most common human pathogens causing myocarditis. Its receptor, the coxsackievirus and adenovirus receptor (CAR), not only mediates virus uptake but also relates to cytoskeletal organization and intracellular signaling. Animals without CAR die prenatally with major cardiac malformations. In the adult heart, CAR is important for virus entry and electrical conduction, but its nonmuscle functions are largely unknown. Here, we show that chicken CAR expression exclusively in the heart can rescue the otherwise embryonic-lethal CAR knockout phenotype but does not support CVB3 infection of adult cardiomyocytes. Our findings have implications for the evolution of virus-host versus physiological interactions involving CAR and could help to improve future coxsackievirus-directed therapies inhibiting virus replication while maintaining CAR's cellular functions.

The adapter protein c-Cbl-associated protein (CAP) protects from acute CVB3-mediated myocarditis through stabilization of type I interferon production and reduced cytotoxicity.

c-Cbl-associated protein (CAP), also called Sorbs1 or ponsin, has been described as an essential adapter protein in the insulin-signalling pathway. Here, we describe for the first time a unique protective role for CAP in viral myocarditis. Mortality and heart failure development were increased in CAP(-/-) mice compared to CAP(+/+) littermates after Coxsackievirus (CVB3) infection. Mechanistically, CAP protected from tissue apoptosis because of reduced CD8(+) T and natural killer cell cytotoxicity. Despite reduced cytotoxic elimination of CVB3-infected cells in CAP(+/+) hearts, however, CAP enhanced interferon regulatory factor 3 (IRF3)-dependent antiviral type I interferon production and decreased viral proliferation in vitro by binding to the cytoplasmic RIG-I-like receptor melanoma differentiation-associated protein 5 (MDA5). Taken together, these findings reveal a novel modulatory role for CAP in the heart as a key protein stabilizing antiviral type I interferon production, while protecting from excessive cytotoxic responses. Our study will help to define future strategies to develop treatments to limit detrimental responses during viral heart inflammation.