Coxsackievirus B3 Research Articles

Coxsackievirus Protease 2A Targets Host Protease ATG4A to Impair Autophagy.

Enteroviruses (EVs) are medically important RNA viruses that cause a broad spectrum of human illnesses for which limited therapy exists. Although EVs have been shown to usurp the cellular recycling process of autophagy for pro-viral functions, the precise manner by which this is accomplished remains to be elucidated. In the current manuscript, we sought to address the mechanism by which EVs subvert the autophagy pathway using Coxsackievirus B3 (CVB3) as a model. We showed that CVB3 infection selectively degrades the autophagy cysteine protease ATG4A but not other isoforms. Exogenous expression of an N-terminally Flag-labeled ATG4A demonstrated the emergence of a 43-kDa cleavage fragment following CVB3 infection. Furthermore, bioinformatics analysis coupled with site-directed mutagenesis and in vitro cleavage assays revealed that CVB3 protease 2A cleaves ATG4A before glycine 374. Using a combination of genetic silencing and overexpression studies, we demonstrated a novel pro-viral function for the autophagy protease ATG4A. Additionally, cleavage of ATG4A was associated with a loss of autophagy function of the truncated cleavage fragment. Collectively, our study identified ATG4A as a novel substrate of CVB3 protease, leading to disrupted host cellular function and sheds further light on viral mechanisms of autophagy dysregulation.

Multiple subtypes of coxsackievirus group B can cause congenital heart disease.

Different serotypes of coxsackievirus B (CVB), which is the most common cause of viral myocarditis, target cardiomyocytes through Coxsackie and Adenovirus Receptor and Decay-Accelerating Factor. Both receptors are expressed in the fetal heart. We hypothesized that infection with different serotypes of CVB during early pregnancy plays a role in pathogenesis of congenital heart defect (CHD). In this study, we use a murine model to infect with CVB1, CVB4, and combination of CVB3 + CVB4 during a critical period in gestation. We examined offspring of pregnant mice for fetal death and heart defects following viral infection. Fetuses from uninfected control dams showed normal heart development, while maternal CVB infection precipitates CHD: majorly ventricular septal defects (VSD) and non-compaction of ventricular myocardium (NC), with some infrequent cases of double outlet right ventricle, left ventricle wall rupture, right ventricle hypertrophy, and thickened/dysplastic semilunar valves. Infection of pregnant dams with CVB1 leads to 44% VSD and 41.2% NC cases, while with CVB4 leads to 31.7% VSD and 13.3% NC cases. Co-infection with CVB3 + CVB4 increases fetal pathology to 51.3% VSD and 41% NC cases. Infection can also result in fetal death, with higher incidences with CVB3 + CVB4 with 46.2% cases, compared to 33.3% by CVB1 and 21.7% by CVB4. Male fetuses were more susceptible to all phenotypes. Our report shows that prenatal CVB infections can lead to pathogenesis of certain heart defects in mouse model, particularly exacerbated with co-infections. This data confirms a link between prenatal CVB infection and CHD development and highlights it is not unique to just one serotype of CVB.

Structure-function analysis of enterovirus protease 2A in complex with its essential host factor SETD3

Enteroviruses cause a number of medically relevant and widespread human diseases with no approved antiviral therapies currently available. Host-directed therapies present an enticing option for this diverse genus of viruses. We have previously identified the actin histidine methyltransferase SETD3 as a critical host factor physically interacting with the viral protease 2A. Here, we report the 3.5 Å cryo-EM structure of SETD3 interacting with coxsackievirus B3 2A at two distinct interfaces, including the substrate-binding surface within the SET domain. Structure-function analysis revealed that mutations of key residues in the SET domain resulted in severely reduced binding to 2A and complete protection from enteroviral infection. Our findings provide insight into the molecular basis of the SETD3-2A interaction and a framework for the rational design of host-directed therapeutics against enteroviruses.

Circular RNA circ_0076631 promotes coxsackievirus B3 infection through modulating viral translation by sponging miR-214-3p.

Coxsackievirus B (CVB), a member of Enterovirus genus of Picornaviridae, is the leading pathogen of viral myocarditis and dilated cardiomyopathy. The pathogenesis of CVB-induced myocarditis has not been completely elucidated, and no specific antiviral measurement is available presently. Circular RNAs (circRNAs) have been reported to be able to modulate viral replication and infection through bridging over non-coding RNAs (ncRNAs) and coding messenger RNAs (mRNAs). To date, the role of circRNAs in CVB infection is largely unknown. In this study, we found that hsa_circ_0076631 (circ_0076631) significantly promoted CVB type 3 (CVB3) replication. Further study showed that the underneath mechanism was circ_0076631 indirectly interacting with CVB3 through sponging miR-214-3p, which targeted the 3D-coding region of CVB3 genome to suppress viral translation. Knocking down circ-0076631 caused a suppression of CVB3 infection; thus, circ-0076631 may be a potential target for anti-CVB therapy.

Engineered coxsackievirus B3 containing multiple organ-specific miRNA targets showed attenuated viral tropism and protective immunity.

Coxsackievirus B3 (CVB3) can cause viral myocarditis, pancreatitis, and aseptic meningitis. This study aimed to construct an engineered CVB3 harboring three different tissue-specific miRNA targets (CVB3-miR3*T) to decrease the virulence of CVB3 in muscles, pancreas, and brain. CVB3-miR3*T and CVB3-miR-CON (containing three sequences not found in the human genome) were engineered and replicated in HELA cells. A viral plaque assay was used to determine the titers in HELA cells and TE671 cells (high miRNA-206 expression), MIN-6 cells (high miRNA-29a-3p expression), and mouse astrocytes (high miRNA-124-3p expression). We found that engineered CVB3 showed attenuated replication and reduced cytotoxicity, the variability of each type of cell was also increased in the CVB3-miR3*T group. Male BALB/c mice were infected to determine the LD50 and examine heart, pancreas, and brain titers and injury. Viral replication of the engineered viruses was restricted in infected mouse heart, pancreas, and brain, and viral plaques were about 100 fold lower compared with the control group. Mice immunized using CVB3-miR3*T, UV-inactivated CVB3-WT, and CVB3-miR-CON were infected with 100×LD50 of CVB3-WT to determine neutralization. CVB3-miRT*3-preimmunized mice exhibited complete protection and remained alive after lethal virus infection, while only 5/15 were alive in the UV-inactivated mice, and all 15 mice were dead in the PBS-immunized group. The results demonstrate that miR-206-, miRNA-29a-3p-, and miRNA-124-3p-mediated CVB3 detargeting from the pancreas, heart, and brain might be a highly effective strategy for viral vaccine development.

TBK1 and GABARAP family members suppress Coxsackievirus B infection by limiting viral production and promoting autophagic degradation of viral extracellular vesicles.

Host-pathogen dynamics are constantly at play during enteroviral infection. Coxsackievirus B (CVB) is a common juvenile enterovirus that infects multiple organs and drives inflammatory diseases including acute pancreatitis and myocarditis. Much like other enteroviruses, CVB is capable of manipulating host machinery to hijack and subvert autophagy for its benefit. We have previously reported that CVB triggers the release of infectious extracellular vesicles (EVs) which originate from autophagosomes. These EVs facilitate efficient dissemination of infectious virus. Here, we report that TBK1 (Tank-binding kinase 1) suppresses release of CVB-induced EVs. TBK1 is a multimeric kinase that directly activates autophagy adaptors for efficient cargo recruitment and induces type-1 interferons during viral-mediated STING recruitment. Positioning itself at the nexus of pathogen elimination, we hypothesized that loss of TBK1 could exacerbate CVB infection due to its specific role in autophagosome trafficking. Here we report that infection with CVB during genetic TBK1 knockdown significantly increases viral load and potentiates the bulk release of viral EVs. Similarly, suppressing TBK1 with small interfering RNA (siRNA) caused a marked increase in intracellular virus and EV release, while treatment in vivo with the TBK1-inhibitor Amlexanox exacerbated viral pancreatitis and EV spread. We further demonstrated that viral EV release is mediated by the autophagy modifier proteins GABARAPL1 and GABARAPL2 which facilitate autophagic flux. We observe that CVB infection stimulates autophagy and increases the release of GABARAPL1/2-positive EVs. We conclude that TBK1 plays additional antiviral roles by inducing autophagic flux during CVB infection independent of interferon signaling, and the loss of TBK1 better allows CVB-laden autophagosomes to circumvent lysosomal degradation, increasing the release of virus-laden EVs. This discovery sheds new light on the mechanisms involved in viral spread and EV propagation during acute enteroviral infection and highlights novel intracellular trafficking protein targets for antiviral therapy.

Aerosolized Hydrogen Peroxide Decontamination of N95 Respirators, with Fit-Testing and Viral Inactivation, Demonstrates Feasibility for Reuse during the COVID-19 Pandemic.

ABSTRACTIn response to the demand for N95 respirators by health care workers during the COVID-19 pandemic, we evaluated decontamination of N95 respirators using an aerosolized hydrogen peroxide (aHP) system. This system is designed to dispense a consistent atomized spray of aerosolized, 7% hydrogen peroxide (H2O2) solution over a treatment cycle. Multiple N95 respirator models were subjected to 10 or more cycles of respirator decontamination, with a select number periodically assessed for qualitative and quantitative fit testing. In parallel, we assessed the ability of aHP treatment to inactivate multiple viruses absorbed onto respirators, including phi6 bacteriophage, herpes simplex virus 1 (HSV-1), coxsackievirus B3 (CVB3), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For pathogens transmitted via respiratory droplets and aerosols, it is critical to address respirator safety for reuse. This study provided experimental validation of an aHP treatment process that decontaminates the respirators while maintaining N95 function. External National Institute for Occupational Safety & Health (NIOSH) certification verified respirator structural integrity and filtration efficiency after 10 rounds of aHP treatment. Virus inactivation by aHP was comparable to the decontamination of commercial spore-based biological indicators. These data demonstrate that the aHP process is effective, with successful fit-testing of respirators after multiple aHP cycles, effective decontamination of multiple virus species, including SARS-CoV-2, successful decontamination of bacterial spores, and filtration efficiency maintained at or greater than 95%. While this study did not include extended or clinical use of N95 respirators between aHP cycles, these data provide proof of concept for aHP decontamination of N95 respirators before reuse in a crisis-capacity scenario.IMPORTANCE The COVID-19 pandemic led to unprecedented pressure on health care and research facilities to provide personal protective equipment. The respiratory nature of the SARS-CoV2 pathogen makes respirator facepieces a critical protective measure to limit inhalation of this virus. While respirator facepieces were designed for single use and disposal, the pandemic increased overall demand for N95 respirators, and corresponding manufacturing and supply chain limitations necessitated the safe reuse of respirators when necessary. In this study, we repurposed an aerosolized hydrogen peroxide (aHP) system that is regularly utilized to decontaminate materials in a biosafety level 3 (BSL3) facility, to develop a method for decontamination of N95 respirators. Results from viral inactivation, biological indicators, respirator fit testing, and filtration efficiency testing all indicated that the process was effective at rendering N95 respirators safe for reuse. This proof-of-concept study establishes baseline data for future testing of aHP in crisis-capacity respirator-reuse scenarios.

Testosterone Promotes the Intestinal Replication and Dissemination of Coxsackievirus B3 in an Oral Inoculation Mouse Model.

Enteroviruses initiate infection in the gastrointestinal tract, and sex is often a biological variable that impacts pathogenesis. Previous data suggest that sex hormones can influence the intestinal replication of Coxsackievirus B3 (CVB3), an enterovirus in the Picornaviridae family. However, the specific sex hormone(s) that regulates intestinal CVB3 replication is poorly understood. To determine if testosterone promotes intestinal CVB3 replication, we orally inoculated male and female Ifnar-/- mice that were treated with either placebo or testosterone-filled capsules. Following oral inoculation, we found that the testosterone-treated male and female mice shed significantly more CVB3 in their feces than did the placebo-treated mice, indicating that testosterone enhances intestinal replication. Similarly, testosterone enhanced viral dissemination in both sexes, as we observed higher viral loads in peripheral tissues following infection. Further, the testosterone-treated male mice also had a higher mortality rate than did the testosterone-depleted male mice. Finally, we observed that testosterone significantly affected the immune response to CVB3. We found that testosterone broadly increased proinflammatory cytokines and chemokines while decreasing the number of splenic B cells and dendritic cells following CVB3 infection. Moreover, while testosterone did not affect the early CD4 T cell response to CVB3, testosterone reduced the activation of CD8 T cells. These data indicate that testosterone can promote intestinal CVB3 replication and dissemination while also impacting the subsequent viral immune response. IMPORTANCE Biological sex plays a significant role in the outcomes of various infections and diseases. The impact of sex hormones on the intestinal replication and dissemination of Coxsackievirus B3 remains poorly understood. Using an oral inoculation model, we found that testosterone enhances CVB3 shedding and dissemination in male and female mice. Further, testosterone can alter the immune response to CVB3. This work highlights the role of testosterone in CVB3 pathogenesis and suggests that sex hormones can impact the replication and dissemination of enteric viruses.

Mitochondrial calpain-1 activates NLRP3 inflammasome by cleaving ATP5A1 and inducing mitochondrial ROS in CVB3-induced myocarditis

Treatment options for myocarditis are currently limited. Inhibition of calpains has been shown to prevent Coxsackievirus B3 (CVB3)-induced cardiac injuries, but the underlying mechanism of action of calpains has not been elucidated. We investigated whether NOD-, LRR-, and pyrin domain-containing 3 (NLRP3) inflammasome participated in CVB3-induced myocarditis, and investigated the effects of calpain-1 on CVB3-induced cardiac injury. NLRP3 inflammasome was activated in CVB3-infected hearts, evidenced by elevated protein levels of NLRP3, N-terminal domain of Gasdermin D, and cleaved caspase-1, and the increased co-localization of NLRP3 and apoptosis-associated speck-like protein. The intraperitoneal administration of MCC950, a selective inhibitor of the NLRP3 inflammasome, led to decreased levels of serum creatine kinase-MB, cardiac troponin I, lactate dehydrogenase, interleukin-18, interleukin-1β, prevention of the infiltration of inflammatory cells, and improvement of cardiac function under CVB3 infection. Transgenic mice overexpressing the endogenous calpain inhibitor calpastatin (Tg-CAST mice) exhibited not only decreased apoptosis, inflammation, fibrosis, and enhanced cardiac function but also inhibition of NLRP3 inflammasome and pyroptosis. The selective inhibition of calpain-1 using PD151746 protected cardiomyocytes in vitro from CVB3 infection by downregulating NLRP3 inflammasome and, thus, preserved cell viability. Mechanistically, we showed that mitochondrial dysfunction preceded inflammatory response after CVB3 treatment and elimination of mitochondrial reactive oxygen species (ROS) using mitochondria-targeted antioxidants (mito-TEMPO) recapitalized the phenotype observed in Tg-CAST mice. Furthermore, the promotion or inhibition of calpain-1 activation in vitro regulated the mitochondrial respiration chain. Mito-TEMPO reversed calpain-1-mediated NLRP3 inflammation activation and cell death. We also found that mitochondrial calpain-1, which was increased after CVB3 stimulation, activated the NLRP3 inflammasome and resulted in cell death. Furthermore, ATP synthase-α (ATP5A1) was revealed to be the cleaving target of calpain-1 after CVB3 treatment. Downregulating ATP5A1 using ATP5A1-small interfering RNA impaired mitochondrial function, decreased cell viability, and induced NLRP3 inflammasome activation. In conclusion, CVB3 infection induced calpain-1 accumulation in mitochondria, and led to subsequent ATP5A1 cleavage, mitochondrial ROS overproduction, and impaired mitochondrial function, eventually causing NLRP3 inflammasome activation and inducing pyroptosis. Therefore, our findings established the role of calpain in viral myocarditis and unveiled its underlying mechanism of its action. Calpain appears as a promising target for the treatment of viral myocarditis.

Stem cell factor/mast cell/CCL2/monocyte/macrophage axis promotes Coxsackievirus B3 myocarditis and cardiac fibrosis by increasing Ly6Chigh monocyte influx and fibrogenic mediators production.

Mast cells (MCs), central players in allergy and parasitic infections, play key roles in inflammation and fibrosis. Here, the impact of MCs on the progression of Coxsackievirus B3 (CVB3)-induced viral myocarditis (VMC) and fibrosis was investigated using MC-deficient KitW-sh mice. Viral titres, cellular infiltrates and heart pathologies were evaluated and compared with wild-type (WT) mice during acute CVB3 infection of C57BL/6 mice. CVB3 infection induced an increased accumulation and degranulation of MCs in the hearts of mice during acute infection. MC-deficient KitW-sh mice had slightly higher viral titres, decreased VMC and cardiac fibrosis and improved cardiac dysfunction compared to WT mice via decreasing cardiac influx of Ly6Chigh monocytes/macrophages (Mo/Mφ). While bone marrow-derived MC reconstitution decreased viral titre and worsened improved survival and VMC severity in Wsh mice. MC-fibroblasts co-culture revealed a cardiac MC-fibroblasts crosstalk during early infection: fibroblasts trigger MC degranulation and secretion of CCL2 and tumour necrosis factor alpha (TNF-α) via producing early stem cell factor (SCF); while MCs-fibrogenic mediators (TNF-α) stimulate fibroblasts to increase CCL2, α-smooth muscle actin (SMA), collagen and transforming growth factor beta（TGFβ)expression, thus aggravating cardiac fibrosis. MCs and fibroblast-derived CCL2s are both essential for cardiac Ly6Chigh Mo/Mφ influx. Administration of recombinant mouse SCF to CVB3-infected mice aggravates VMC via accelerating MCs accumulation and cardiac influx of Ly6Chi Mo/Mφ. Collectively, our data highlight an early MC-fibroblast crosstalk and SCF/MC/CCL2/Mo/Mφ axis as important mechanisms required for triggering VMC and myocardial fibrosis. This finding indicates critical roles of MCs in initiating and modulating cardiac innate response to CVB3 and has an implication in developing new and more effective treatments for VMC.

Monoterpenoid indole alkaloids isolated from the stems and twigs of Strychnos cathayensis

Seven undescribed monoterpenoid indole alkaloids, two N(4)-chloromethylation artifacts, and 10 known alkaloids were isolated from the stems and twigs of Strychnos cathayensis. The corresponding structures were elucidated via spectroscopic data interpretation and electronic circular dichroism. The absolute configuration of (17S)-12-hydroxy-11-methoxydiaboline, the major anomer of 12-hydroxy-11-methoxydiaboline, was characterized by X-ray diffraction analysis for the first time. At an intraperitoneal dose of 30 mg/kg, 12-hydroxy-11-methoxy-N(4)-chloromethyldiaboline and (−)-macusine A exhibited potential analgesic effects with prolongation rates of 99% and 47% for the latency time of hind-paw licking, respectively, compared to the blank control. 12-Hydroxy-11-methoxydiaboline, 12-hydroxy-11-methoxydiaboline N(4)-oxide, retuline N-oxide, and (−)-vincosamide exhibited antiviral activity against Coxsackie virus B3 (CVB3) with IC50 values of 33.33 μM.

Structural basis for the synergistic neutralization of coxsackievirus B1 by a triple-antibody cocktail

Coxsackievirus B1 (CVB1) is an emerging pathogen associated with severe neonatal diseases including aseptic meningitis, myocarditis, and pancreatitis and also with the development of type 1 diabetes. We characterize the binding and therapeutic efficacies of three CVB1-specific neutralizing antibodies (nAbs) identified for their ability to inhibit host receptor engagement. High-resolution cryo-EM structures showed that these antibodies recognize different epitopes but with an overlapping region in the capsid VP2 protein and specifically the highly variable EF loop. Moreover, they perturb capsid-receptor interactions by binding various viral particle forms. Antibody combinations achieve synergetic neutralization via a stepwise capsid transition and virion disruption, indicating dynamic changes in the virion in response to multiple nAbs targeting the receptor-binding site. Furthermore, this three-antibody cocktail protects against lethal challenge in neonatal mice and limits pancreatitis and viral replication in a non-obese diabetic mouse model. These results illustrate the utility of nAbs for rational design of therapeutics against picornaviruses such as CVB.

A new miRNA-Modified coxsackievirus B3 inhibits triple negative breast cancer growth with improved safety profile in immunocompetent mice

Coxsackievirus B3 (CVB3) displays great oncolytic activity against various cancer cells. Previously, we demonstrated that adding targeting sequences (TS) of miR-145/143, which are downregulated in cancer compared with normal cells, into CVB3 genome drastically attenuates tissue toxicity, while retaining its oncolytic activity towards lung tumor. Here we extended to assess miR-modified CVB3 in breast cancer therapy. We generated a new miRNA-CVB3 by inserting TS of muscle-specific miR-1 and pancreas-selective miR-216 into the above miR-145/143-modified CVB3. We found that this newly established CVB3 (termed miR-CVB3-1.1) is safe without triggering noticeable pathogenesis when applied to immunocompetent mice. In vitro studies revealed that miR-CVB3-1.1 can infect and lyse a wide range of breast cancer cells. Animal experiments using a syngeneic breast cancer mouse model showed that intratumoral inoculation of miR-CVB3-1.1 significantly suppresses tumor growth and metastasis, associated with productive viral growth and enhanced immune cell infiltration in the tumor microenvironment. Moreover, we observed substantially reduced toxicity and prolonged survival in mice treated with miR-CVB3-1.1 compared with wild-type CVB3. Together, our results support miR-CVB3-1.1 as a promising candidate, which can be further evaluated for clinical treatment of breast cancer.

The relevance of selenium to viral disease with special reference to SARS-CoV-2 and COVID-19.

In this review, the relevance of selenium (Se) to viral disease will be discussed paying particular attention to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and coronavirus disease (COVID-19). Se, the active centre in selenoproteins has an ongoing history of reducing the incidence and severity of viral infections. Host Se deficiency increased the virulence of RNA viruses such as influenza A and coxsackievirus B3, the latter of which is implicated in the development of Keshan disease in north-east China. Significant clinical benefits of Se supplementation have been demonstrated in HIV-1, in liver cancer linked to hepatitis B, and in Chinese patients with hantavirus that was successfully treated with oral sodium selenite. China is of particular interest because it has populations that have both the lowest and the highest Se status in the world. We found a significant association between COVID-19 cure rate and background Se status in Chinese cities; the cure rate continued to rise beyond the Se intake required to optimise selenoproteins, suggesting an additional mechanism. Se status was significantly higher in serum samples from surviving than non-surviving COVID-19 patients. As regards mechanism, SARS-CoV-2 may interfere with the human selenoprotein system; selenoproteins are important in scavenging reactive oxygen species, controlling immunity, reducing inflammation, ferroptosis and endoplasmic reticulum (ER) stress. We found that SARS-CoV-2 significantly suppressed mRNA expression of GPX4, of the ER selenoproteins, SELENOF, SELENOM, SELENOK and SELENOS and down-regulated TXNRD3. Based on the available data, both selenoproteins and redox-active Se species (mimicking ebselen, an inhibitor of the main SARS-CoV-2 protease that enables viral maturation within the host) could employ their separate mechanisms to attenuate virus-triggered oxidative stress, excessive inflammatory responses and immune-system dysfunction, thus improving the outcome of SARS-CoV-2 infection.

A Single Mutation in the Cryptic AUG (cAUG) Affects In Vitro Translation and Replication Efficiencies and In Vivo Virulence of Coxsackievirus B3 (CVB3).

The 5'UTR of the genomic RNA of CVB3, unusually long and rich on highly structured secondary structure, contains a conserved cis acting RNA element named the cryptic AUG (cAUG), where the cellular 48S complex is formed. In this study, we investigate the role of this cAUG in CVB3 translation, replication, and virulence. Mutant viral sub-genomic replicon RNA was constructed by site-directed mutagenesis. We characterize in vitro translation and replication efficiencies and in vivo virulence of a cAUG mutant in comparison with wild-type strain. UV-cross-linking assay and Real-Time PCR were used, respectively, to detect binding host proteins and to quantify viral production. Secondary structures of domain containing the cAUG site were studied and compared. The results suggest that introduced mutation in the CVB3 5'UTR affects in vitro and ex vivo viral translation which cannot be rescued by compensatory mutations. A reduced interaction of the La and PCBP2 translation initiation factors with cAUG residue of mutant was revealed. Decreasing production of viral mutant RNA was also demonstrated. Furthermore, secondary structure prediction reveals changes in the ribosome binding sites of the cAUG moiety of mutant sense strand RNA and no alterations in the structure of wild type, suggesting that cAUG mutation specifically affects the secondary structure of the sense RNA strand. Taken together, AUG integrity influences the efficiency of ribosome recruitment through IRES element and the capacity of replication.

Advances in cell death mechanisms involved in viral myocarditis.

Viral myocarditis is an acute inflammatory disease of the myocardium. Although many etiopathogenic factors exist, coxsackievirus B3 is a the leading cause of viral myocarditis. Abnormal cardiomyocyte death is the underlying problem for most cardiovascular diseases and fatalities. Various types of cell death occur and are regulated to varying degrees. In this review, we discuss the different cell death mechanisms in viral myocarditis and the potential interactions between them. We also explore the role and mechanism of cardiomyocyte death with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Exploring the mechanisms may help in the early identification and the development of effective treatments, thus improving the quality of life of patients with viral myocarditis. We believe that the inhibition of cardiomyocyte death has immense therapeutic potential in increasing the longevity and health of the heart.

Abstract P3061: A Monoclonal Antibody Blocking Interleukin-1 Receptor Accessory Protein Reduces Acute Viral Myocarditis Severity

IL-1, IL-33 and IL-36 have complementary, pro-inflammatory roles during the immune response that promote autoimmune diseases, particularly myocarditis. While antibodies that neutralize circulating cytokines exist, we hypothesized that blockade of their shared co-receptor, IL-1 receptor accessory protein (IL1RAP), would exhibit a more potent and broader anti-inflammatory profile. The CAN10 antibody blocks IL1RAP and is currently under development for treating severe autoimmune and inflammatory diseases. To investigate if IL1RAP blockade is an effective treatment of cardiac inflammation, we induced autoimmune viral myocarditis in 8-week-old Balb/c male mice with Coxsackievirus B3 and treated them with mCAN10, an anti-mouse IL1RAP monoclonal antibody. Mice were euthanized at day 10, the peak of acute inflammation in the heart. IL1RAP blockade reduced the severity of myocarditis at this timepoint when compared to isotype, saline or IL-1 receptor antagonist (IL1RA) treatment (the gold standard in IL-1 receptor blockade), without affecting viral clearance from the heart. Spectral flow cytometry of heart immune populations revealed that mCAN10 significantly reduced the number of infiltrating leukocytes of broad populations, especially effector CD4 + and CD8 + T cells, inflammatory Ly6C + CCR2 + monocytes, neutrophils and eosinophils. We also observed early echocardiographical parameters suggesting improved heart function, including ejection fraction, fractional shortening and interventricular septum diameter. Interestingly, mCAN10-treated mice demonstrated reduced proliferation of bone marrow precursors of inflammatory myeloid and lymphoid lineage leukocytes. Splenic immune cell populations were not affected, indicating that mCAN10 does not cause global immune suppression in these animals. Altogether, our data show that a single monoclonal antibody targeting three inflammatory cytokine systems, IL-1, IL-33 and IL-36, potently reduces acute viral myocarditis severity in a broader fashion that is not recapitulated by IL-1α/β blockade, and shows translational promise for therapeutics of inflammatory disease. Phase I clinical studies of the CAN10 antibody are in preparation.

TRIM18 is a critical regulator of viral myocarditis and organ inflammation

BackgroundInfections by viruses including severe acute respiratory syndrome coronavirus 2 could cause organ inflammations such as myocarditis, pneumonia and encephalitis. Innate immunity to viral nucleic acids mediates antiviral immunity as well as inflammatory organ injury. However, the innate immune mechanisms that control viral induced organ inflammations are unclear.MethodsTo understand the role of the E3 ligase TRIM18 in controlling viral myocarditis and organ inflammation, wild-type and Trim18 knockout mice were infected with coxsackievirus B3 for inducing viral myocarditis, influenza A virus PR8 strain and human adenovirus for inducing viral pneumonia, and herpes simplex virus type I for inducing herpes simplex encephalitis. Mice survivals were monitored, and heart, lung and brain were harvested for histology and immunohistochemistry analysis. Real-time PCR, co-immunoprecipitation, immunoblot, enzyme-linked immunosorbent assay, luciferase assay, flow cytometry, over-expression and knockdown techniques were used to understand the molecular mechanisms of TRIM18 in regulating type I interferon (IFN) production after virus infection in this study.ResultsWe find that knockdown or deletion of TRIM18 in human or mouse macrophages enhances production of type I IFN in response to double strand (ds) RNA and dsDNA or RNA and DNA virus infection. Importantly, deletion of TRIM18 protects mice from viral myocarditis, viral pneumonia, and herpes simplex encephalitis due to enhanced type I IFN production in vivo. Mechanistically, we show that TRIM18 recruits protein phosphatase 1A (PPM1A) to dephosphorylate TANK binding kinase 1 (TBK1), which inactivates TBK1 to block TBK1 from interacting with its upstream adaptors, mitochondrial antiviral signaling (MAVS) and stimulator of interferon genes (STING), thereby dampening antiviral signaling during viral infections. Moreover, TRIM18 stabilizes PPM1A by inducing K63-linked ubiquitination of PPM1A.ConclusionsOur results indicate that TRIM18 serves as a negative regulator of viral myocarditis, lung inflammation and brain damage by downregulating innate immune activation induced by both RNA and DNA viruses. Our data reveal that TRIM18 is a critical regulator of innate immunity in viral induced diseases, thereby identifying a potential therapeutic target for treatment.

Jessel Josephs

<h3>ABSTRACT</h3> ISG15 is an interferon-stimulated, ubiquitin-like protein that can conjugate to substrate proteins (ISGylation) to counteract microbial infection, but the underlying mechanisms remain elusive. Here, we used a viral-like particle...

Virus-induced inhibition of cardiac pacemaker channel HCN4 triggers bradycardia in human-induced stem cell system

The enterovirus Coxsackievirus B3 (CVB3) is known to be a major source for the development of cardiac dysfunctions like viral myocarditis (VMC) and dilatative cardiomyopathy (DCM), but also results in bradycardia and fatal cardiac arrest. Besides clinical reports on bradycardia and sudden cardiac death, very little is known about the influence of CVB3 on the activity of human cardiac pacemaker cells. Here, we address this issue using the first human induced pluripotent stem cell (hiPSC)-derived pacemaker-like cells, in which the expression of a transgenic non-infectious variant of CVB3 can be controlled dose- and time-dependently. We found that CVB3 drastically changed hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) distribution and function in hiPSC-derived pacemaker-like tissue. In addition, using HCN4 cell expression systems, we found that HCN4 currents were decreased with altered voltage dependency of activation when CVB3 was expressed. Increased autophagosome formation and autophagosomal HCN4 insertion was observed in hiPSC-derived pacemaker-like cells under CVB3 expression as well. Individual effects of single, non-structural CVB3 proteins were analyzed and demonstrated that CVB3 proteins 2C and 3A had the most robust effect on HCN4 activity. Treatment of cells with the Rab7 inhibitor CID 106770 or the CVB3-3A inhibitor GW5074 led to the recovery of the cytoplasmatic HCN4 accumulation into a healthy appearing phenotype, indicating that malfunctioning Rab7-directed autophagosome transport is involved in the disturbed, cytoplasmatic HCN4 accumulation in CVB3-expressing human pacemaker-like cells. Summarizing, the enterovirus CVB3 inhibits human cardiac pacemaker function by reducing the pacemaker channel plasma membrane density, an effect that can be corrected by pharmacological intervention of endocytic vesicle trafficking.