Coxsackievirus B3 HFMD animal models in Syrian hamster and rhesus monkey.

Coxsackievirus B3 (CVB3) is the pathogen causing hand, foot and mouth disease (HFMD), which manifests across a spectrum of clinical severity from mild to severe. However, CVB3-infected mouse models mainly demonstrate viral myocarditis and pancreatitis, failing to replicate human HFMD symptoms. Although several enteroviruses have been evaluated in Syrian hamsters and rhesus monkeys, there is no comprehensive data on CVB3. In this study, we have first tested the susceptibility of Syrian hamsters to CVB3 infection via different routes. The results showed that Syrian hamsters were successfully infected with CVB3 by intraperitoneal injection or nasal drip, leading to nasopharyngeal colonization, acute severe pathological injury, and typical HFMD symptoms. Notably, the nasal drip group exhibited a longer viral excretion cycle and more severe pathological damage. In the subsequent study, rhesus monkeys infected with CVB3 through nasal drips also presented signs of HFMD symptoms, viral excretion, serum antibody conversion, viral nucleic acids and antigens, and the specific organ damages, particularly in the heart. Surprisingly, there were no significant differences in myocardial enzyme levels, and the clinical symptoms resembled those often associated with common, mild infections. In summary, the study successfully developed severe Syrian hamsters and mild rhesus monkey models for CVB3-induced HFMD. These models could serve as a basis for understanding the disease pathogenesis, conducting pre-trial prevention and evaluation, and implementing post-exposure intervention.

ISG15 blocks cardiac glycolysis and ensures sufficient mitochondrial energy production during Coxsackievirus B3 infection.

AIMS: Virus infection triggers inflammation and, may impose nutrient shortage to the heart. Supported by type I interferon (IFN) signalling, cardiomyocytes counteract infection by various effector processes, with the IFN-stimulated gene of 15 kDa (ISG15) system being intensively regulated and protein modification with ISG15 protecting mice Coxsackievirus B3 (CVB3) infection. The underlying molecular aspects how the ISG15 system affects the functional properties of respective protein substrates in the heart are unknown. METHODS AND RESULTS: Based on the protective properties due to protein ISGylation, we set out a study investigating CVB3-infected mice in depth and found cardiac atrophy with lower cardiac output in ISG15-/- mice. By mass spectrometry, we identified the protein targets of the ISG15 conjugation machinery in heart tissue and explored how ISGylation affects their function. The cardiac ISGylome showed a strong enrichment of ISGylation substrates within glycolytic metabolic processes. Two control enzymes of the glycolytic pathway, hexokinase 2 (HK2) and phosphofructokinase muscle form (PFK1), were identified as bona fide ISGylation targets during infection. In an integrative approach complemented with enzymatic functional testing and structural modelling, we demonstrate that protein ISGylation obstructs the activity of HK2 and PFK1. Seahorse-based investigation of glycolysis in cardiomyocytes revealed that, by conjugating proteins, the ISG15 system prevents the infection-/IFN-induced up-regulation of glycolysis. We complemented our analysis with proteomics-based advanced computational modelling of cardiac energy metabolism. Our calculations revealed an ISG15-dependent preservation of the metabolic capacity in cardiac tissue during CVB3 infection. Functional profiling of mitochondrial respiration in cardiomyocytes and mouse heart tissue by Seahorse technology showed an enhanced oxidative activity in cells with a competent ISG15 system. CONCLUSION: Our study demonstrates that ISG15 controls critical nodes in cardiac metabolism. ISG15 reduces the glucose demand, supports higher ATP production capacity in the heart, despite nutrient shortage in infection, and counteracts cardiac atrophy and dysfunction.

MicroRNA-22-3p displaces critical host factors from the 5' UTR and inhibits the translation of Coxsackievirus B3 RNA.

Coxsackievirus B3 (CVB3) is known to cause acute myocarditis and pancreatitis in humans. We investigated the microRNAs (miRNAs) that can potentially govern the viral life cycle by binding to the untranslated regions (UTRs) of CVB3 RNA. MicroRNA-22-3p was short-listed, as its potential binding site overlapped with the region crucial for recruiting internal ribosome entry site trans-acting factors (ITAFs) and ribosomes. We demonstrate that miR-22-3p binds CVB3 5' UTR, hinders recruitment of key ITAFs on viral mRNA, disrupts the spatial structure required for ribosome recruitment, and ultimately blocks translation. Likewise, cells lacking miR-22-3p exhibited heightened CVB3 infection compared to wild type, confirming its role in controlling infection. Interestingly, miR-22-3p level was found to be increased at 4 hours post-infection, potentially due to the accumulation of viral 2A protease in the early phase of infection. 2Apro enhances the miR-22-3p level to dislodge the ITAFs from the SD-like sequence, rendering the viral RNA accessible for binding of replication factors to switch to replication. Furthermore, one of the cellular targets of miR-22-3p, protocadherin-1 (PCDH1), was significantly downregulated during CVB3 infection. Partial silencing of PCDH1 reduced viral replication, demonstrating its proviral role. Interestingly, upon CVB3 infection in mice, miR-22-3p level was found to be downregulated only in the small intestine, the primary target organ, indicating its possible role in influencing tissue tropism. It appears miR-22-3p plays a dual role during infection by binding viral RNA to aid its life cycle as a viral strategy and by targeting a proviral protein to restrict viral replication as a host response.IMPORTANCECVB3 infection is associated with the development of end-stage heart diseases. Lack of effective anti-viral treatments and vaccines for CVB3 necessitates comprehensive understanding of the molecular players during CVB3 infection. miRNAs have emerged as promising targets for anti-viral strategies. Here, we demonstrate that miR-22-3p binds to 5' UTR and inhibits viral RNA translation at the later stage of infection to promote viral RNA replication. Conversely, as host response, it targets PCDH1, a proviral factor, to discourage viral propagation. miR-22-3p also influences CVB3 tissue tropism. Deciphering the multifaced role of miR-22-3p during CVB3 infection unravels the necessary molecular insights, which can be exploited for novel intervening strategies to curb infection and restrict viral pathogenesis.

Pathological Characteristics of Echovirus 30 Infection in a Mouse Model.

Echovirus 30 (E30), a member of species B enterovirus, is associated with outbreaks of aseptic meningitis and has become a global health emergency. However, the pathogenesis of E30 remains poorly understood due to the lack of appropriate animal models. In this study, we established a mouse infection model to explore the pathogenicity of E30. The 2-day-old IFNAR-/- mice infected with E30 strain WZ16 showed lethargy and paralysis, and some died. Obvious pathological changes were observed in the skeletal muscle, brain tissue, and other tissues, with the highest viral load in the skeletal muscles. Transcriptome analysis of brain and skeletal muscle tissues from infected mice showed that significant differentially expressed genes were enriched in complement response and neuropathy-related pathways. Using immunofluorescence assay, we found that the viral double-stranded RNA (dsRNA) was detected in the mouse brain region and could infect human glioma (U251) cells. These results indicated that E30 affects the nervous system, and they provide a theoretical basis for understanding its pathogenesis. IMPORTANCE Echovirus 30 (E30) infection causes a wide spectrum of diseases with mild symptoms, such as hand, foot, and mouth disease (HFMD), acute flaccid paralysis, and aseptic meningitis and other diseases, especially one of the most common pathogens causing aseptic meningitis outbreaks. We established a novel mouse model of E30 infection by inoculating neonatal mice with clinical isolates of E30 and observed the pathological changes induced by E30. Using the E30 infection model, we found complement responses and neuropathy-related genes in the mice tissues at the transcriptome level. Moreover, we found that the viral dsRNA localized in the mouse brain and could replicate in human glioma cell line U251 rather than in the neuroblastoma cell line, SK-N-SH.

Simulating coxsackievirus B3 infection with an accessible computational model of its complete kinetics.

We describe how to use a publicly available computational model for coxsackievirus B3 (CVB3) infection that we recast as a graphical user interface (GUI). The GUI-based implementation enables non-computationalists to incorporate systems-biology modeling into their research and teaching. The model simulates the full life cycle of CVB3, including the host antiviral response, and includes 44 alterable parameters. The model simplifies some viral life cycle processes to improve interpretability and utility when performing in silico experiments. For complete details on the use and execution of this protocol, please refer to Lopacinski et al. (2021).

Crocetin Exerts a Cardio-protective Effect on Mice with Coxsackievirus B3-induced Acute Viral Myocarditis.

Previous research has proven that coxsackievirus B3 (CVB3) is broadly considered virus used in the experimental model of animals, which causes myocarditis in humans. To investigate whether there exists a cardio-protective effect of crocetin in an experimental murine model of acute viral myocarditis (AVM). Male BALB/c mice were randomly assigned to three groups: control, myocarditis treated with placebo and myocarditis treated with crocetin (n = 40 animals per group). Myocarditis was established by intraperitoneal injection with CVB3. Twenty-four hours after infection, crocetin was intraperitoneally administered for 14 consecutive days. Twenty mice were randomly selected from each group to monitor a 14-day survival rate. On day 7 and day 14, eight surviving mice from each group were sacrificed and their hearts and blood were obtained to perform serological and histological examinations. Expression of ROCKs, interleukin-17 (IL-17), interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNFα), RORγt, and Foxp3 was quantified by RT-PCR. Plasma levels of TNFα, IL-1ß and IL-17 were measured by ELISA. In addition, protein levels of IL-17 and ROCK2 in cardiac tissues were analyzed by Western blot. Crocetin treatment significantly increased survival, attenuated myocardial necrotic lesions, reduced CVB3 replication and expression of ROCK2 and IL-17 in the infected hearts. ROCK pathway inhibition was cardio-protective in viral myocarditis with increased survival, decreased viral replication, and inflammatory response. These findings suggest that crocetin is a potential therapeutic agent for patients with viral myocarditis.

Coxsackievirus B3 Exploits the Ubiquitin-Proteasome System to Facilitate Viral Replication.

Infection by RNA viruses causes extensive cellular reorganization, including hijacking of membranes to create membranous structures termed replication organelles, which support viral RNA synthesis and virion assembly. In this study, we show that infection with coxsackievirus B3 entails a profound impairment of the protein homeostasis at virus-utilized membranes, reflected by an accumulation of ubiquitinylated proteins, including K48-linked polyubiquitin conjugates, known to direct proteins to proteasomal degradation. The enrichment of membrane-bound ubiquitin conjugates is attributed to the presence of the non-structural viral proteins 2B and 3A, which are known to perturb membrane integrity and can cause an extensive rearrangement of cellular membranes. The locally increased abundance of ubiquitinylated proteins occurs without an increase of oxidatively damaged proteins. During the exponential phase of replication, the oxidative damage of membrane proteins is even diminished, an effect we attribute to the recruitment of glutathione, which is known to be required for the formation of infectious virus particles. Furthermore, we show that the proteasome contributes to the processing of viral precursor proteins. Taken together, we demonstrate how an infection with coxsackievirus B3 affects the cellular protein and redox homeostasis locally at the site of viral replication and virus assembly.

Sex-Specific Effects of Plastic Caging in Murine Viral Myocarditis.

BACKGROUND: Myocarditis is an inflammatory heart disease caused by viral infections that can lead to heart failure, and occurs more often in men than women. Since animal studies have shown that myocarditis is influenced by sex hormones, we hypothesized that endocrine disruptors, which interfere with natural hormones, may play a role in the progression of the disease. The human population is exposed to the endocrine disruptor bisphenol A (BPA) from plastics, such as water bottles and plastic food containers. METHODS: Male and female adult BALB/c mice were housed in plastic versus glass caging, or exposed to BPA in drinking water versus control water. Myocarditis was induced with coxsackievirus B3 on day 0, and the endpoints were assessed on day 10 post infection. RESULTS: We found that male BALB/c mice that were exposed to plastic caging had increased myocarditis due to complement activation and elevated numbers of macrophages and neutrophils, whereas females had elevated mast cell activation and fibrosis. CONCLUSIONS: These findings show that housing mice in traditional plastic caging increases viral myocarditis in males and females, but using sex-specific immune mechanisms.

CVB3-Mediated Mitophagy Plays an Important Role in Viral Replication via Abrogation of Interferon Pathways.

Coxsackievirus B3 (CVB3) is a common enterovirus that causes systemic inflammatory diseases, such as myocarditis, meningitis, and encephalitis. CVB3 has been demonstrated to subvert host cellular responses via autophagy to support viral replication in neural stem cells. Mitophagy, a specialized form of autophagy, contributes to mitochondrial quality control via degrading damaged mitochondria. Here, we show that CVB3 infection induces mitophagy in human neural progenitor cells, HeLa and H9C2 cardiomyocytes. In particular, CVB3 infection triggers mitochondrial fragmentation, loss of mitochondrial membrane potential, and Parkin/LC3 translocation to the mitochondria. Rapamycin or carbonyl cyanide m-chlorophenyl hydrazone (CCCP) treatment led to increased CVB3 RNA copy number in a dose-dependent manner, suggesting enhanced viral replication via autophagy/mitophagy activation, whereas knockdown of PTEN-induced putative kinase protein 1(PINK1) led to impaired mitophagy and subsequent reduction in viral replication. Furthermore, CCCP treatment inhibits the interaction between mitochondrial antiviral signaling protein (MAVS) and TANK-binding kinase 1(TBK1), thus contributing to the abrogation of type I and III interferon (IFN) production, suggesting that mitophagy is essential for the inhibition of interferon signaling. Our findings suggest that CVB3-mediated mitophagy suppresses IFN pathways by promoting fragmentation and subsequent sequestration of mitochondria by autophagosomes.

Coxsackievirus B4 sewage-isolate induces pancreatitis after oral infection of mice.

Numerous serotypes which belong to the genus Enterovirus (EV) show variability in their virulence and clinical manifestations. They are also known to undergo changes caused by mutations and recombination during their circulation in the environment and the population. Various EV serotypes are prevalent in groundwater, wastewater and surface waters. Our previous studies showed that oral infection induces pancreatitis depending on specific conditions, such as gravidity, in an outbred murine model. Our aim in the present study was to further explore the pancreatic histopathology in an outbred mouse model following oral infection with clinical isolates from a patient who had aseptic meningitis and an isolate from a treated-sewage sample recovered from the residential area of the patient. The isolates were identified as coxsackievirus B4 (CVB4) in tissue culture. The CVB4 sewage-isolate induced pancreatitis after oral infection. In contrast, pancreatitis was absent following infection with the clinical isolates. Comparison of polyprotein sequences showed that the treated-sewage strains differed from the patient's isolates by 9 and 11 amino acids. We conclude that the isolates of clinical and environmental origin differed in their pathogenic properties and showed genetic variation.

Retrospective Characterization of the 2006-2007 Swine Vesicular Disease Epidemic in Northern Italy by Whole Genome Sequence Analysis.

Advances in the epidemiological tracing of pathogen transmission have been largely driven by the increasing characterisation of whole-genome sequence data obtained at a finer resolution from infectious disease outbreaks. Dynamic models that integrate genomic and epidemiological data further enhance inference on the evolutionary history and transmission dynamics of epidemic outbreaks by reconstructing the network of 'who-infected-whom'. Swine Vesicular Disease (SVD) was present in Italy from 1966 until 2015, and since the mid-1990s, it has mainly been circulating within Italy's central-southern regions with sporadic incursions to the north of the country. However, a recrudescence of SVD in northern Italy was recorded between November 2006 and October 2007, leading to a large-scale epidemic that significantly affected the intensive pig industry of the Lombardy region. In this study, by using whole-genome sequence data in combination with epidemiological information on disease occurrences, we report a retrospective epidemiological investigation of the 2006-2007 SVD epidemic, providing new insights into the transmission dynamics and evolutionary mode of the two phases that characterised the epidemic event. Our analyses support evidence of undetected premises likely missed in the chain of observed infections, of which the role as the link between the two phases is reinforced by the tempo of SVD virus evolution. These silent transmissions, likely resulting from the gradual loss of a clear SVD clinical manifestation linked to sub-clinical infections, may pose a risk of failure in the early detection of new cases. This study emphasises the power of joint inference schemes based on genomic and epidemiological data integration to inform the transmission dynamics of disease epidemics, ultimately aimed at better disease control.

Exploration of Analgesia with Tramadol in the Coxsackievirus B3 Myocarditis Mouse Model.

Infection of mice with Coxsackievirus B3 (CVB3) triggers inflammation of the heart and this mouse model is commonly used to investigate underlying mechanisms and therapeutic aspects for viral myocarditis. Virus-triggered cytotoxicity and the activity of infiltrating immune cells contribute to cardiac tissue injury. In addition to cardiac manifestation, CVB3 causes cell death and inflammation in the pancreas. The resulting pancreatitis represents a severe burden and under such experimental conditions, analgesics may be supportive to improve the animals' well-being. Notably, several known mechanisms exist by which analgesics can interfere with the immune system and thereby compromise the feasibility of the model. We set up a study aiming to improve animal welfare while ensuring model integrity and investigated how tramadol, an opioid, affects virus-induced pathogenicity and immune response in the heart. Tramadol was administered seven days prior to a CVB3 infection in C57BL/6 mice and treatment was continued until the day of analysis. Tramadol had no effect on the virus titer or viral pathogenicity in the heart tissue and the inflammatory response, a hallmark of myocardial injury, was maintained. Our results show that tramadol exerts no disruptive effects on the CVB3 myocarditis mouse model and, therefore, the demonstrated protocol should be considered as a general analgesic strategy for CVB3 infection.

High Heterogeneity of Echoviruses in Brazilian Children with Acute Gastroenteritis.

Echoviruses (E) are a diverse group of viruses responsible for various pathological conditions in humans including aseptic meningitis, myocarditis, and acute flaccid paralysis. The detection and identification of echovirus genotypes in clinical samples is challenging due to its high genetic diversity. Here, we report the complete genome sequences of nine echoviruses, obtained by next-generation sequencing of 238 fecal samples from individuals with gastroenteritis in regions of Brazil. Detected viruses were classified into six genotypes: Three E1 sequences (BRA/TO-028, BRA/TO-069 and BRA/TO-236), one E3 (BRA/TO-018), one E11 (BRA/TO-086), one E20 (BRA/TO-016), two E29 (BRA/TO-030 and BRA/TO-193), and one E30 sequence (BRA/TO-032). Phylogenetic analysis indicated that the echoviruses E1 and E29 circulating in Brazil are divergent from strains circulating worldwide. The genotype diversity identified in our study may under-represent the total echovirus diversity in Brazil because of the small sample size and the restricted geographical distribution covered by the survey.

CVB3 VP1 interacts with MAT1 to inhibit cell proliferation by interfering with Cdk-activating kinase complex activity in CVB3-induced acute pancreatitis.

Coxsackievirus B3 (CVB3) belongs to the genus Enterovirus of the family Picornaviridae and can cause acute acinar pancreatitis in adults. However, the molecular mechanisms of pathogenesis underlying CVB3-induced acute pancreatitis have remained unclear. In this study, we discovered that CVB3 capsid protein VP1 inhibited pancreatic cell proliferation and exerted strong cytopathic effects on HPAC cells. Through yeast two-hybrid, co-immunoprecipitation, and confocal microscopy, we show that Menage a trois 1 (MAT1), a subunit of the Cdk-Activating Kinase (CAK) complex involved in cell proliferation and transcription, is a novel interaction protein with CVB3 VP1. Moreover, CVB3 VP1 inhibited MAT1 accumulation and localization, thus interfering with its interaction with CDK7. Furthermore, CVB3 VP1 could suppress CAK complex enzymic phosphorylation activity towards RNA Pol II and CDK4/6, direct substrates of CAK. VP1 also suppresses phosphorylation of retinoblastoma protein (pRb), an indirect CAK substrate, especially at phospho-pRb Ser780 and phospho-pRb Ser807/811 residues, which are associated with cell proliferation. Finally, we present evidence using deletion mutants that the C-terminal domain (VP1-D8, 768-859aa) is the minimal VP1 region required for its interaction with MAT1, and furthermore, VP1-D8 alone was sufficient to arrest cells in G1/S phase as observed during CVB3 infection. Taken together, we demonstrate that CVB3 VP1 can inhibit CAK complex assembly and activity through direct interaction with MAT1, to block MAT1-mediated CAK-CDK4/6-Rb signaling, and ultimately suppress cell proliferation in pancreatic cells. These findings substantially extend our basic understanding of CVB3-mediated pancreatitis, providing strong candidates for strategic therapeutic targeting.

Mitigated viral myocarditis in A/J mice by the immunoproteasome inhibitor ONX 0914 depends on inhibition of systemic inflammatory responses in CoxsackievirusB3 infection.

A preclinical model of troponin I-induced myocarditis (AM) revealed a prominent role of the immunoproteasome (ip), the main immune cell-resident proteasome isoform, in heart-directed autoimmunity. Viral infection of the heart is a known trigger of cardiac autoimmunity, with the ip enhancing systemic inflammatory responses after infection with a cardiotropic coxsackievirusB3 (CV). Here, we used ip-deficient A/J-LMP7-/- mice to investigate the role of ip-mediated effects on adaptive immunity in CV-triggered myocarditis and found no alteration of the inflammatory heart tissue damage or cardiac function in comparison to wild-type controls. Aiming to define the impact of the systemic inflammatory storm under the control of ip proteolysis during CV infection, we targeted the ip in A/J mice with the inhibitor ONX 0914 after the first cycle of infection, when systemic inflammation has set in, well before cardiac inflammation. During established acute myocarditis, the ONX 0914 treatment group had the same reduction in cardiac output as the controls, with inflammatory responses in heart tissue being unaffected by the compound. Based on these findings and with regard to the known anti-inflammatory role of ONX 0914 in CV infection, we conclude that the efficacy of ip inhibitors for CV-triggered myocarditis in A/J mice relies on their immunomodulatory effects on the systemic inflammatory reaction.

MicroRNA-30a-5p silencing polarizes macrophages toward M2 phenotype to alleviate cardiac injury following viral myocarditis by targeting SOCS1.

Viral myocarditis (VMC) is a life-threatening disease characterized by severe cardiac inflammation generally caused by coxsackievirus B3 (CVB3) infection. Several microRNAs (miRNAs or miRs) are known to play crucial roles in the pathogenesis of VMC. The study aimed to decipher the role of miR-30a-5p in the underlying mechanisms of VMC pathogenesis. We first quantified miR-30a-5p expression in a CVB3-induced mouse VMC model. The physiological characteristics of mouse cardiac tissues were then detected by hematoxylin and eosin (HE) and Picrosirius red staining. We established the correlation between miR-30a-5p and SOCS1, using dual-luciferase gene assay and Pearson's correlation coefficient. The expression of inflammatory factors (IFN-γ, IL-6, IL-10, and IL-13), M1 polarization markers [TNF-α, inducible nitric oxide synthase (iNOS)], M2 polarization markers (Arg-1, IL-10), and myocardial hypertrophy markers [atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP)] was detected by RT-qPCR and Western blot analysis. miR-30a-5p was found to be highly expressed in VMC mice. Silencing of miR-30a-5p improved the cardiac function index and reduced heart weight-to-body weight ratio, myocardial tissue pathological changes and fibrosis degree, serological indexes, as well as proinflammatory factor levels, while enhancing anti-inflammatory factor levels in VMC mice. Furthermore, silencing of miR-30a-5p inhibited M1 polarization of macrophages while promoting M2 polarization in vivo and in vitro. SOCS1 was a target gene of miR-30a-5p, and the aforementioned cardioprotective effects of miR-30a-5p silencing were reversed upon silencing of SOCS1. Overall, this study shows that silencing of miR-30a-5p may promote M2 polarization of macrophages and improve cardiac injury following VMC via SOCS1 upregulation, constituting a potential therapeutic target for VMC treatment.NEW & NOTEWORTHY We found in this study that microRNA (miR)-30a-5p inhibition might improve cardiac injury following viral myocarditis (VMC) by accelerating M2 polarization of macrophages via SOCS1 upregulation. Furthermore, the anti-inflammatory mechanisms of miR-30a-5p inhibition may contribute to the development of new therapeutic strategies for VMC.

Coxsackievirus B3 Infection Early in Pregnancy Induces Congenital Heart Defects Through Suppression of Fetal Cardiomyocyte Proliferation.

Background Coxsackievirus B (CVB) is the most common cause of viral myocarditis. It targets cardiomyocytes through coxsackie and adenovirus receptor, which is highly expressed in the fetal heart. We hypothesized CVB3 can precipitate congenital heart defects when fetal infection occurs during critical window of gestation. Methods and Results We infected C57Bl/6 pregnant mice with CVB3 during time points in early gestation (embryonic day [E] 5, E7, E9, and E11). We used different viral titers to examine possible dose-response relationship and assessed viral loads in various fetal organs. Provided viral exposure occurred between E7 and E9, we observed characteristic features of ventricular septal defect (33.6%), abnormal myocardial architecture resembling noncompaction (23.5%), and double-outlet right ventricle (4.4%) among 209 viable fetuses examined. We observed a direct relationship between viral titers and severity of congenital heart defects, with apparent predominance among female fetuses. Infected dams remained healthy; we did not observe any maternal heart or placental injury suggestive of direct viral effects on developing heart as likely cause of congenital heart defects. We examined signaling pathways in CVB3-exposed hearts using RNA sequencing, Kyoto Encyclopedia of Genes and Genomes enrichment analysis, and immunohistochemistry. Signaling proteins of the Hippo, tight junction, transforming growth factor-ß1, and extracellular matrix proteins were the most highly enriched in CVB3-infected fetuses with ventricular septal defects. Moreover, cardiomyocyte proliferation was 50% lower in fetuses with ventricular septal defects compared with uninfected controls. Conclusions We conclude prenatal CVB3 infection induces congenital heart defects. Alterations in myocardial proliferate capacity and consequent changes in cardiac architecture and trabeculation appear to account for most of observed phenotypes.

Human FcRn expression and Type I Interferon signaling control Echovirus 11 pathogenesis in mice.

Neonatal echovirus infections are characterized by severe hepatitis and neurological complications that can be fatal. Here, we show that expression of the human homologue of the neonatal Fc receptor (hFcRn), the primary receptor for echoviruses, and ablation of type I interferon (IFN) signaling are key host determinants involved in echovirus pathogenesis. We show that expression of hFcRn alone is insufficient to confer susceptibility to echovirus infections in mice. However, expression of hFcRn in mice deficient in type I interferon (IFN) signaling, hFcRn-IFNAR-/-, recapitulate the echovirus pathogenesis observed in humans. Luminex-based multianalyte profiling from E11 infected hFcRn-IFNAR-/- mice revealed a robust systemic immune response to infection, including the induction of type I IFNs. Furthermore, similar to the severe hepatitis observed in humans, E11 infection in hFcRn-IFNAR-/- mice caused profound liver damage. Our findings define the host factors involved in echovirus pathogenesis and establish in vivo models that recapitulate echovirus disease in humans.

Coxsackievirus B3 Infection of Human iPSC Lines and Derived Primary Germ-Layer Cells Regarding Receptor Expression.

The association of members of the enterovirus family with pregnancy complications up to miscarriages is under discussion. Here, infection of two different human induced pluripotent stem cell (iPSC) lines and iPSC-derived primary germ-layer cells with coxsackievirus B3 (CVB3) was characterized as an in vitro cell culture model for very early human development. Transcriptomic analysis of iPSC lines infected with recombinant CVB3 expressing enhanced green fluorescent protein (EGFP) revealed a reduction in the expression of pluripotency genes besides an enhancement of genes involved in RNA metabolism. The initial distribution of CVB3-EGFP-positive cells within iPSC colonies correlated with the distribution of its receptor coxsackie- and adenovirus receptor (CAR). Application of anti-CAR blocking antibodies supported the requirement of CAR, but not of the co-receptor decay-accelerating factor (DAF) for infection of iPSC lines. Among iPSC-derived germ-layer cells, mesodermal cells were especially vulnerable to CVB3-EGFP infection. Our data implicate further consideration of members of the enterovirus family in the screening program of human pregnancies. Furthermore, iPSCs with their differentiation capacity into cell populations of relevant viral target organs could offer a reliable screening approach for therapeutic intervention and for assessment of organ-specific enterovirus virulence.

Clinical characteristics of echovirus 11 and coxsackievirus B5 infections in Taiwanese children requiring hospitalization.

BACKGROUND: Severe illness can occur in young children infected with certain types of enteroviruses including echovirus 11 (Echo11) and coxsackievirus B5 (CoxB5). The manifestations and outcomes of Echo11 and CoxB5 diseases across all ages of children remained not comprehensively characterized in Taiwan. METHODS: Culture-confirmed Echo11 (60 patients) or CoxB5 (65 patients) infections were identified in a hospital from 2010 to 2018. The demographics, clinical presentations, laboratory data and outcomes were abstracted and compared between the two viruses infections. RESULTS: Echo11 and CoxB5 was respectively identified in 7 (77.8%) and 2 (22.2%) of 9 calendar years. The median age of all patients was 15 months (range, 1 day-14.5 years). For infants ≤3 months old, Echo11 (23 cases) was associated with higher incidence of aseptic meningitis (35% versus 0%, P = 0.003), and a lower rate of upper respiratory tract infections (URI) (22% versus 65%, P = 0.004) compared to CoxB5 (20 cases) infections. For patients >3 months old, URI was the cardinal diagnosis (60%) for both viruses. Aseptic meningitis was also more commonly identified in elder children with Echo11 infections (27% versus 11%), though with marginal significance (P = 0.07). Acute liver failure was identified in four young infants with Echo11 infections including one neonate dying of severe sepsis and myocarditis. All patients with CoxB5 infections recovered uneventfully. CONCLUSION: Aseptic meningitis, sepsis-like illness and acute liver failure were more commonly identified in children with Echo11 than those with CoxB5 infections, suggesting greater neurological tropism and virulence toward Echo11.