Coxsackievirus Infection Research Articles

A Crucial Role of ACBD3 Required for Coxsackievirus Infection in Animal Model Developed by AAV-Mediated CRISPR Genome Editing Technique.

Genetic screens using CRISPR/Cas9 have been exploited to discover host–virus interactions. These screens have identified viral dependencies on host proteins during their life cycle and potential antiviral strategies. The acyl-CoA binding domain containing 3 (ACBD3) was identified as an essential host factor for the Coxsackievirus B3 (CVB3) infection. Other groups have also investigated the role of ACBD3 as a host factor for diverse enteroviruses in cultured cells. However, it has not been tested if ACBD3 is required in the animal model of CVB3 infection. Owing to embryonic lethality, conventional knockout mice were not available for in vivo study. As an alternative approach, we used adeno-associated virus (AAV)-mediated CRISPR genome editing to generate mice that lacked ACBD3 within the pancreas, the major target organ for CVB3. Delivery of sgRNAs using self-complementary (sc) AAV8 efficiently induced a loss-of-function mutation in the pancreas of the Cas9 knock-in mice. Loss of ACBD3 in the pancreas resulted in a 100-fold reduction in the CVB3 titer within the pancreas and a noticeable reduction in viral protein expression. These results indicate a crucial function of ACBD3 in CVB3 infection in vivo. AAV-mediated CRISPR genome editing may be applicable to many in vivo studies on the virus–host interaction and identify a novel target for antiviral therapeutics.

Coxsackievirus infection induces a non-canonical autophagy independent of the ULK and PI3K complexes

Coxsackievirus B3 (CVB3) is a single-stranded positive RNA virus that usurps cellular machinery, including the evolutionarily anti-viral autophagy pathway, for productive infections. Despite the emergence of double-membraned autophagosome-like vesicles during CVB3 infection, very little is known about the mechanism of autophagy initiation. In this study, we investigated the role of established autophagy factors in the initiation of CVB3-induced autophagy. Using siRNA-mediated gene-silencing and CRISPR-Cas9-based gene-editing in culture cells, we discovered that CVB3 bypasses the ULK1/2 and PI3K complexes to trigger autophagy. Moreover, we found that CVB3-induced LC3 lipidation occurred independent of WIPI2 and the transmembrane protein ATG9 but required components of the late-stage ubiquitin-like ATG conjugation system including ATG5 and ATG16L1. Remarkably, we showed the canonical autophagy factor ULK1 was cleaved through the catalytic activity of the viral proteinase 3C. Mutagenesis experiments identified the cleavage site of ULK1 after Q524, which separates its N-terminal kinase domain from C-terminal substrate binding domain. Finally, we uncovered PI4KIIIβ (a PI4P kinase), but not PI3P or PI5P kinases as requisites for CVB3-induced LC3 lipidation. Taken together, our studies reveal that CVB3 initiates a non-canonical form of autophagy that bypasses ULK1/2 and PI3K signaling pathways to ultimately converge on PI4KIIIβ- and ATG5–ATG12–ATG16L1 machinery.

CHRONIC PERICARDITIS SECONDARY TO COXSACKIE VIRAL INFECTION PRESENTING AS FIBROSING MEDIASTINITIS

SESSION TITLE: Fellows Chest Infections Posters SESSION TYPE: Fellow Case Report Posters PRESENTED ON: October 18-21, 2020 INTRODUCTION: Fibrosing mediastinitis (FM) is a rare fibroinflammatory disease classified as idiopathic or secondary. Most common infectious cause in the United states is histoplasma. Additional causes include malignancy, autoimmune, and sarcoidosis. CASE PRESENTATION: 45-year-old female presented with chest pain. A transthoracic echocardiogram (TTE) showed pericardial effusion with tamponade physiology and pericardial window with biopsy was done. Pathology showed fibrovascular tissue, adipose tissue with perivascular chronic inflammation. Cytology; negative for malignancy. Weakly positive Coxsackie titers were 1:16 and 1:8 for B1 & B2 respectively, negative bacterial, fungal, mycobacterial, and viral cultures. Negative Lyme antibody titers, mycoplasma IgG, cryptococcal and histoplasma antigens. Normal IgG levels. Negative rheumatological work up apart from weakly positive anti-nuclear antibody. Progressive symptoms of dyspnea despite treatment with anti-inflammatory medications. Subsequent CT showed mediastinal mass encasing the right pulmonary artery (PA) trunk (figure 1). Moderate to severe narrowing of the right PA seen on cardiac MRI. Bronchoscopy with EBUS guided TBNA revealed lymphocytes but no definite diagnosis. Endovascular biopsy of the mediastinal mass showed fibrillar sclerosing lesion with plasma cells and necrotizing granuloma. IgG staining positive for IgG4+ plasma cells but not enough to meet criteria for IgG4-related disease. Abundance of CD20+ve cells on histology. DISCUSSION: Patient did not meet criteria for IgG4-RD diagnosis. She tested negative for all secondary etiologies. Coxsackie virus infection causing chronic pericarditis leading to FM is our hypothesis. CONCLUSIONS: This represents the first report of FM caused by chronic pericarditis probably due to coxsackie viral infection. Reference #1: RadioGraphics 2019; 39:651-667 Reference #2: Ann Thorac Med. 12(3):209-212 DISCLOSURES: No relevant relationships by Sammar Alsunaid, source=Web Response no disclosure on file for Allen Burke; no disclosure on file for Joseph Friedberg; No relevant relationships by Van Holden, source=Web Response Consultant relationship with Medtronic Please note: $1001 - $5000 Added 06/11/2020 by Edward Pickering, source=Web Response, value=Consulting fee Consultant relationship with Merit Endotek Please note: $1-$1000 Added 04/06/2020 by Ashutosh Sachdeva, source=Web Response, value=Consulting fee Consultant relationship with Medtronic Please note: $1001 - $5000 Added 04/06/2020 by Ashutosh Sachdeva, source=Web Response, value=Consulting fee Speaker/Speaker's Bureau relationship with COOK Please note: $1001 - $5000 Added 04/06/2020 by Ashutosh Sachdeva, source=Web Response, value=Honoraria Consultant relationship with Medtronic Please note: $1001 - $5000 by Ashutosh Sachdeva, source=Web Response, value=Consulting fee Unrestricted education grant relationship with Olympus Please note: $1001 - $5000 by Ashutosh Sachdeva, source=Web Response, value=Grant/Research Support Consultant relationship with COOK Please note: $1001 - $5000 Added 04/27/2020 by Ashutosh Sachdeva, source=Web Response, value=Honoraria

Preliminary Anti-Coxsackie Activity of Novel 1-[4-(5,6-dimethyl(H)- 1H(2H)-benzotriazol-1(2)-yl)phenyl]-3-alkyl(aryl)ureas.

Coxsackievirus infections are associated with cases of aseptic meningitis, encephalitis, myocarditis, and some chronic disease. A series of benzo[d][1,2,3]triazol-1(2)-yl derivatives (here named benzotriazol-1(2)-yl) (4a-i, 5a-h, 6a-e, g, i, j and 7a-f, h-j) were designed, synthesized and in vitro evaluated for cytotoxicity and antiviral activity against two important human enteroviruses (HEVs) members of the Picornaviridae family [Coxsackievirus B 5 (CVB-5) and Poliovirus 1 (Sb-1)]. Compounds 4c (CC50 >100 μM; EC50 = 9 μM), 5g (CC50 >100 μM; EC50 = 8 μM), and 6a (CC50 >100 μM; EC50 = 10 μM) were found active against CVB-5. With the aim of evaluating the selectivity of action of this class of compounds, a wide spectrum of RNA (positive- and negativesense), double-stranded (dsRNA) or DNA viruses were also assayed. For none of them, significant antiviral activity was determined. These results point towards a selective activity against CVB-5, an important human pathogen that causes both acute and chronic diseases in infants, young children, and immunocompromised patients.

Single cell RNA sequence analysis reveals novel clusters unique to post-infectious myocarditis in the mouse model of Coxsackievirus infection

Abstract Myocarditis is a common occurrence in children and adolescents that can lead to dilated cardiomyopathy. Since it is difficult to identify the triggers of this complex disease, mechanistic understanding of DCM pathogenesis may create avenues to develop new treatment strategies. We have been engaged in delineating the autoimmune pathways underlying the development of DCM using various mouse models of myocarditis that resemble human disease. One such model is Coxsackievirus B3 (CVB)-induced myocarditis in A/J mice that show two distinct phases of disease, but occur in continuum. While, the acute, infectious phase (up to ~10 days post-infection) is marked by viremia and infiltrations in the hearts, infectious virions become undetected during the chronic phase (~beyond 18 days post-infection), and animals develop DCM. The latter phenotype can occur over a period of ~60 to 90 days, and hitherto, it has been a challenge to determine the underlying mechanisms. To dissect this complex nature of disease, we recently performed single cell RNA sequence analysis on heart infiltrates obtained from CVB-infected mice. Using panels of 15 to 30 marker genes for various immune and non-immune cells, we identified 18 and 26 clusters respectively in healthy and infected hearts that also include few unknown clusters unique for each group. Likewise, transcriptome analyses led us to identify a total of 14,051 genes to be differentially expressed, and of these, 206 and 345 transcripts were unique to healthy and CVB-infected groups, respectively. The data suggest that uniquely expressed genes in the CVB-infected hearts may have a role in the development of post-infectious myocarditis leading to DCM.

Single cell RNA sequence analysis reveals novel clusters unique to post-infectious myocarditis in the mouse model of Coxsackievirus infection

Abstract Myocarditis is a common occurrence in children and adolescents that can lead to dilated cardiomyopathy. Since it is difficult to identify the triggers of this complex disease, mechanistic understanding of DCM pathogenesis may create avenues to develop new treatment strategies. We have been engaged in delineating the autoimmune pathways underlying the development of DCM using various mouse models of myocarditis that resemble human disease. One such model is Coxsackievirus B3 (CVB)-induced myocarditis in A/J mice that show two distinct phases of disease, but occur in continuum. While, the acute, infectious phase (up to ~10 days post-infection) is marked by viremia and infiltrations in the hearts, infectious virions become undetected during the chronic phase (~beyond 18 days post-infection), and animals develop DCM. The latter phenotype can occur over a period of ~60 to 90 days, and hitherto, it has been a challenge to determine the underlying mechanisms. To dissect this complex nature of disease, we recently performed single cell RNA sequence analysis on heart infiltrates obtained from CVB-infected mice. Using panels of 15 to 30 marker genes for various immune and non-immune cells, we identified 18 and 26 clusters respectively in healthy and infected hearts that also include few unknown clusters unique for each group. Likewise, transcriptome analyses led us to identify a total of 14,051 genes to be differentially expressed, and of these, 206 and 345 transcripts were unique to healthy and CVB-infected groups, respectively. The data suggest that uniquely expressed genes in the CVB-infected hearts may have a role in the development of post-infectious myocarditis leading to DCM.

Identification of Immunogenic Epitopes That Permit the Detection of Antigen-Specific T Cell Responses in Multiple Serotypes of Group B Coxsackievirus Infections.

Coxsackievirus group B (CVB) contains six serotypes that can affect various organs. Some of these organ-specific diseases such as myocarditis and pancreatitis can be caused by more than one serotype. Thus, development of immunological tools common to multiple serotypes is desired. This is especially critical for analyzing antigen-specific T cell responses at a single cell level. To this end, we made efforts to identify the immunogenic epitopes of CVB3 leading us to localize three T cell epitopes within the viral protein 1 (VP1) namely, VP1 681–700, VP1 721–740 and VP1 771–790. First, we confirmed their immunogenicity in the immunization settings. Second, we sought to verify the ability of VP1 epitopes to bind major histocompatibility complex (MHC) class II (IAk) molecules. Third, we created MHC class II (IAk) dextramers and tetramers and ascertained the T cell responses to be antigen-specific. Fourth, we analyzed the T cell responses in animals infected with CVB3 and noted the magnitude of antigen-specific T cell responses occurring in the order of VP1 721–740 and VP1 681–700 followed by VP1 771–790 as verified by proliferation assay and IAk tetramer staining. All epitopes induced interferon (IFN)-γ as a major cytokine. Finally, we investigated whether the VP1 tools generated for CVB3 can also be used to verify T cell responses in infections caused by other serotypes. To this end, we established the CVB4 infection model in A/J mice and found that the CVB4 infection led to the induction of IFN-γ-producing T cell responses primarily for VP1 721–740 and VP1 681–700. Thus, the VP1-specific tools, particularly IAk tetramers can be used to monitor anti-viral T cell responses in multiple CVB serotypes.

A food-responsive switch modulates TFEB and autophagy, and determines susceptibility to coxsackievirus infection and pancreatitis

ABSTRACT Almost a billion people worldwide are chronically undernourished. Herein, using a mouse model of coxsackievirus B3 (CVB3) infection, we report that a single day of food restriction (FR) markedly increases susceptibility to attenuated enterovirus infection, replication, and disease. These “pro-viral” effects, which are rapidly-reversed by the restoration of food, are mediated by several genes whose expression is altered by FR, and which support CVB3 replication. Central to this is TFEB, a protein whose expression and activation status are rapidly increased by FR. TFEB, which regulates the transcription of >100 genes involved in macroautophagy/autophagy and lysosomal biogenesis, responds similarly to both FR and CVB3 infection and plays a pivotal role in determining host susceptibility to CVB3. We propose that, by upregulating TFEB, FR generates an intracellular environment that is more hospitable to the incoming virus, facilitating its replication. This interplay between nutritional status and enterovirus replication has implications for human health and, perhaps, for the evolution of these viruses. Abbreviations: Atg/ATG: autophagy-related; CAR: Coxsackievirus and adenovirus receptor; Cas9: CRISPR associated protein 9; Cre: recombinase that causes recombination; CRISPR: clustered regularly interspaced short palindromic repeats; Ctsb/CTSB: cathepsin B; CVB3: coxsackievirus B3; DsRedCVB3: a recombinant CVB3 that encodes the Discosoma red fluorescent protein; EL: elastase; FR: food restriction; GFP: green fluorescent protein; gRNA: guide RNA; HBSS: Hanks Buffered Salt Solution; LYNUS: lysosomal nutrient sensing machinery; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MFI: mean fluorescence intensity; MOI: multiplicity of infection; MTOR: mechanistic target of rapamycin kinase; Nluc: nanoluciferase; NlucCVB3: a recombinant CVB3 encoding nanoluciferase; pfu: plaque-forming unit(s); p.i.: post infection; rCVB: recombinant coxsackievirus B3; RPS6KB/p70S6K: ribosomal protein S6 kinase; RT: room temperature; siRNA: small interfering RNA; TFEB: transcription factor EB; tg: transgenic; TUBB: β-tubulin; UNINF: uninfected; wrt: with respect to; WT: wild type.

Imaging findings of acute idiopathic maculopathy associated with coxsackievirus infection.

Imaging findings of acute idiopathic maculopathy associated with coxsackievirus infection.

Mouse Models of Virus-Induced Type 1 Diabetes.

Virus infections have been linked to the induction of autoimmunity and disease development in human type 1 diabetes. Experimental models have been instrumental in deciphering processes leading to break of immunological tolerance and type 1 diabetes development. Animal models have also been useful for proof-of-concept studies and for preclinical testing of new therapeutic interventions. This chapter describes two robust and clinically relevant mouse models for virus-induced type 1 diabetes; acceleration of disease onset in prediabetic nonobese diabetic (NOD) mice following Coxsackievirus infection and diabetes induction by lymphocytic choriomeningitis virus (LCMV) infection of transgenic mice expressing viral neo-antigens under control of the rat insulin promoter (RIP).

Related Enteric Viruses Have Different Requirements for Host Microbiota in Mice.

Accumulating evidence suggests that intestinal bacteria promote enteric virus infection in mice. For example, previous work demonstrated that antibiotic treatment of mice prior to oral infection with poliovirus reduced viral replication and pathogenesis. Here, we examined the effect of antibiotic treatment on infection with coxsackievirus B3 (CVB3), a picornavirus closely related to poliovirus. We treated mice with a mixture of five antibiotics to deplete host microbiota and examined CVB3 replication and pathogenesis following oral inoculation. We found that, as seen with poliovirus, CVB3 shedding and pathogenesis were reduced in antibiotic-treated mice. While treatment with just two antibiotics, vancomycin and ampicillin, was sufficient to reduce CVB3 replication and pathogenesis, this treatment had no effect on poliovirus. The quantity and composition of bacterial communities were altered by treatment with the five-antibiotic cocktail and by treatment with vancomycin and ampicillin. To determine whether more-subtle changes in bacterial populations impact viral replication, we examined viral infection in mice treated with milder antibiotic regimens. Mice treated with one-tenth the standard concentration of the normal antibiotic cocktail supported replication of poliovirus but not CVB3. Importantly, a single dose of one antibiotic, streptomycin, was sufficient to reduce CVB3 shedding and pathogenesis while having no effect on poliovirus shedding and pathogenesis. Overall, replication and pathogenesis of CVB3 are more sensitive to antibiotic treatment than poliovirus, indicating that closely related viruses may differ with respect to their reliance on microbiota.IMPORTANCE Recent data indicate that intestinal bacteria promote intestinal infection of several enteric viruses. Here, we show that coxsackievirus, an enteric virus in the picornavirus family, also relies on microbiota for intestinal replication and pathogenesis. Relatively minor depletion of the microbiota was sufficient to decrease coxsackievirus infection, while poliovirus infection was unaffected. Surprisingly, a single dose of one antibiotic was sufficient to reduce coxsackievirus infection. Therefore, these data indicate that closely related viruses may differ with respect to their reliance on microbiota.

2237 Double Trouble: Coxsackie A and B Induced Hepatitis in an Adult

INTRODUCTION: Coxsackie virus which was first isolated in 1948 is a relatively common virus predominantly effecting children. There are 23 serotypes of Coxsackie A and 6 serotypes of the Coxsackie B viral strains. Clinical manifestations are generally mild in nature. We present a rare case of a 41yo female with Coxsackie A and B virus induced hepatitis. CASE DESCRIPTION/METHODS: 41yo F PMH DM, presented 36 weeks pregnant c/o 4 days of severe SOB and b/l LE edema. Home medications included metformin, insulin and pre-natals. No recent travel or use of herbal supplements. VS were T-98.3F, BP 130/92, HR 130, RR-22. PE showed LE edema and crackles on auscultation. Pt had severe features of preeclampsia and echo showed Global Cardiomyopathy (EF 21-25%). Labs showed Ph-7.21, CO2-33, HCO3-13, Cr-0.8, LDH-252, Alb-2.7, Bili-0.9/0.3, AST-40, ALT-23, ALP-184, WBC-11.8, Plt-172, INR-1.1, high urine protein. She was intubated for airway protection and taken for C-section. Over the next 3 days pt became febrile, hypotensive, tachycardic and worsening of her labs was noted, Cr-1.5, LDH-1528, Alb-2.1, Bili-0.9/0.5, AST-767, ALT-597, ALP-127, WBC-25.9, Plt-219, INR-1.3 before they started to improve. Pt was managed with aggressive diuresis, broad spectrum abx, and IV pressors for presumed septic/cardiogenic shock. Work up was positive for Coxsackie A and B virus. Pts bilirubin never increased beyond its current values. The rest of liver disease work up was unremarkable. Pt was discharged after stabilization and optimization of her medical condition. DISCUSSION: Coxsackie belongs to the Enterovirus genus, a member of the Picornaviridaefamily. It spreads via the fecal oral route, direct contact with bodily secretions or inhalation. Diagnosis is established with a positive viral culture or an increase in IgM titers. Coxsackie virus infection presents with nonspecific febrile illness, but may include pericarditis, myocarditis, hepatitis and encephalitis. Coxsackie A causes Hand foot and mouth disease, while Coxsackie B is the culprit of hepatitis. Hepatitis as a result of Coxsackie A strain is a very rare clinical phenomenon especially in the adult population. As seen in this case, the pts hepatitis and severe cardiomyopathy were likely secondary to Coxsackie virus. Treatment of Coxsackie virus infection is mainly supportive. Although commonly viral hepatitis is a results of Hepatitis A, B, C, D, E virus, HSV, EBV, and HIV, the possibility of Coxsackie virus induced hepatitis should always be considered.

Alpha-synuclein expression promotes Coxsackie virus infections in the brain and Coxsackie virus may play an important role in Lewy body formation

Alpha-synuclein expression promotes Coxsackie virus infections in the brain and Coxsackie virus may play an important role in Lewy body formation

β Cell Hypoxia-Inducible Factor-1α Is Required for the Prevention of Type 1 Diabetes

SUMMARYThe development of autoimmune disease type 1 diabetes (T1D) is determined by both genetic background and environmental factors. Environmental triggers include RNA viruses, particularly coxsackie-virus (CV), but how they induceT1D is not understood. Here, we demonstrate that deletion of the transcription factor hypoxia-inducible factor-1α (HIF-1α) from β cells increases the susceptibility of non-obese diabetic (NOD) mice to environmentally triggered T1D from coxsackieviruses and the β cell toxin streptozotocin. Similarly, knockdown of HIF-1α in human islets leads to a poorer response to coxsackievirus infection. Studies in coxsackievirus-infected islets demonstrate that lack of HIF-1α leads to impaired viral clearance, increased viral load, inflammation, pancreatitis, and loss of β cell mass. These findings show an important role for β cells and, specifically, lack of β cell HIF-1α in the development of T1D. These data suggest new strategies for the prevention of T1D.

CEACAM-1 promotes myocardial injury following coxsackievirus infection by regulating the coxsackievirus-adenovirus receptor.

Objective:To determine the effects and mechanism of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM-1, CC1)-mediated regulation of the Coxsackie and Adenovirus Receptor (CAR) after Coxsackievirus B3 (CVB3) infection.Methods:A mouse CC1 overexpression recombinant virus was constructed, followed by insertion of a pLVX-CEACAM 1-zsgreen-puro (rLV-CEACAM 1) plasmid into the recombinant retrovirus. Cardiac myocytes were assigned into different groups according to various treatments. The apoptosis rate and cell activity in each group were observed. Further, CAR expression and SYK, IL-1β, and p-SYK levels were measured.Results:The recombinant retrovirus titer was measured as 1.5 × 108 TUs/ml. The apoptosis rate of cardiac myocytes in the CC1 overexpression plus CVB3 group was significantly elevated, and the relative expression of the CAR gene was the highest in the CC1 overexpression plus CVB3 group. TNF-α and IL-1β levels increased due to CC1 overexpression and further increased after CVB3 infection. CAR protein expression also changed along with the levels of CC1, SYK, and TNF-α after infection.Conclusion:CC1 may promote CAR expression after CVB3 infection and regulate CAR protein expression by activating the CC1-SYK-TNF-α signaling axis during the infection process.

Altered exosomal miR-181d and miR-30a related to the pathogenesis of CVB3 induced myocarditis by targeting SOCS3.

MicroRNAs are a group of gene expression regulators and some of which have been confirmed to be associated with acute viral myocarditis (VM). This study aims to find new biomarkers for VM diagnosis and explore the roles of miRNAs during the pathogenesis of VM. 23 patients with acute myocarditis and 12 controls were included in this research. The expression of 10 candidate miRNAs in the serum exosome was examined by qRT-PCR. The direct targets were predicted using bioinformatics tools and then confirmed by dual luciferase assay and immunoblotting. Levels IL-6 of cell culture supernatants were determined by enzyme-linked immunosorbent assay. Six weeks old male mice were injected intraperitoneally with Coxsackievirus B3 (CVB3) and then treated by miRNA inhibitors through tail vein injection. Five miRNAs were found to have disturbed expression in the exosome and may have the potential to be used as biomarker for VM diagnosis. Meanwhile, the expression of miR-30a and -181d was also altered in the cells after CVB3 infection. We identified SOCS3 as a direct target of miR-30a and -181d. Furthermore, during CVB3 infection, up-regulated miR-30a and -181d are related to enhanced IL-6 level via modulating SOCS3 expression. miRNA inhibitors injection increased mice survival rate after CVB3 infection. miR-30a and -181d contribute to the over-activated inflammatory response to viral infection of the heart during coxsackievirus infection.

Pancreas of coxsackievirus-infected dams and their challenged pups: A complex issue

ABSTRACTEnteroviral infections are frequent, often asymptomatic in humans and during gravidity.The present study is an extension of our previous investigations where we had shown pancreatitis in challenged pups of CVB4-E2-infected dams. Present investigation describes the effect of gestational infection with this virus on the pancreas of both dams and their challenged pups. Gravid CD1 outbred mice were orally infected with CVB4-E2 virus at different gestation times. Pups were challenged orally with the same virus after 25 days of birth. Organs were collected at selected intervals postinfection (p.i.), and replicating virus and viral-RNA copies were analyzed. Additional readouts included histopathology and immunohistochemical (IHC) analysis for localization and identification of Ly6G+ cells (neutrophils), CD11b+ cells (macrophages), and viral protein in pancreatic tissue sections of the infected dams and their challenged pups. Our results show the presence of replicating virus in the pancreas of infected dams and their challenged pups, with inflammation leading to chronic necrotizing pancreatitis and atrophy of pancreatic acini of the dams and their offspring. IHC analysis of the infiltrating cells showed pronounced Ly6G+ neutrophils in dams only, whereas CD11b+ macrophages were present in tissues of both, the pups and the dams. Time of infection during gravidity as well as the p.i. intervals when mice were sacrificed influenced the pancreatic pathophysiology in both groups. We conclude that coxsackievirus infection during pregnancy is a risk factor for chronic affliction of the exocrine tissue and could affect endocrine pancreas in the mother and child.

Coxsackievirus B Tailors the Unfolded Protein Response to Favour Viral Amplification in Pancreatic β Cells

Type 1 diabetes (T1D) is an autoimmune disease characterized by islet inflammation and progressive pancreatic β cell destruction. The disease is triggered by a combination of genetic and environmental factors, but the mechanisms leading to the triggering of early innate and late adaptive immunity and consequent progressive pancreatic β cell death remain unclear. The insulin-producing β cells are active secretory cells and are thus particularly sensitive to endoplasmic reticulum (ER) stress. ER stress plays an important role in the pathologic pathway leading to autoimmunity, islet inflammation, and β cell death. We show here that group B coxsackievirus (CVB) infection, a putative causative factor for T1D, induces a partial ER stress in rat and human β cells. The activation of the PERK/ATF4/CHOP branch is blunted while the IRE1α branch leads to increased spliced XBP1 expression and c-Jun N-terminal kinase (JNK) activation. Interestingly, JNK1 activation is essential for CVB amplification in both human and rat β cells. Furthermore, a chemically induced ER stress preceding viral infection increases viral replication, in a process dependent on IRE1α activation. Our findings show that CVB tailors the unfolded protein response in β cells to support their replication, preferentially triggering the pro-viral IRE1α/XBP1s/JNK1 pathway while blocking the pro-apoptotic PERK/ATF4/CHOP pathway.

Molecular epidemiology and clinical features of hand, foot and mouth disease in northern Thailand in 2016: a prospective cohort study

BackgroundHand, foot and mouth disease (HFMD) is a major communicable disease in children ≤6 years old, particularly in several countries in the Asia-Pacific Region, including Thailand. HFMD impacts public health and the economy, especially in northern Thailand.MethodsA prospective cohort study was conducted to estimate the incidence rate and to identify the serotype and clinical features of HFMD among children in northern Thailand. A validated questionnaire and throat swab were used for data collection. Polymerase chain reaction (PCR) was used to detect human enterovirus and identify its serotypes. Participants were recruited from 14 hospitals in two provinces in northern Thailand, specifically, Chiang Rai and Pha Yao Province, between January 1, 2016, and December 31, 2016. Chi-square or Fisher’s exact test was used to detect the associations of signs and symptoms with HFMD serotype. Logistic regression was used to detect the associations of variables with a positive enterovirus at alpha = 0.05.ResultIn total, 612 children aged ≤6 years from Chiang Rai and Pha Yao Province who were diagnosed with HFMD by a throat swab were recruited for the analysis. Approximately half of the cohort was male (57.2%), 57.5% was aged < 2 years, and 57.5% lived in rural areas. The incidence rate was 279.72/100,000 person-years in Chiang Rai Province and 321.24 per 100,000 person-years in Pha Yao Province. Additionally, 42.5% of children were positive for human enterovirus; among these children, 56.1% were positive for enterovirus-A (EV-A), 17.7% were positive for coxsackievirus (CV), and 26.2% were positive for other human RNA enteroviruses. During the study period, 21 distinct outbreaks of HFMD were recognized. Four to five patients (total 92 patients) were selected from each outbreak for identifying its serotype; enterovirus-A71 (EV-A71) was detected in 34.8% of HFMD cases, coxsackievirus-A16 (CV-A16) in 26.1%, coxsackivirus-A6 (CV-A6) in 15.2%, coxsackievirus-A10 (CV-A10) in 10.9%, coxsackievirus-A4 (CV-A4) in 2.2%, coxsackievirus-B2 (CV-B2) in 2.2%, human rhinovirus in 2.2%, and unknown serotype in 6.4%. Multivariable analysis demonstrated that a history of breastfeeding for ≤6 months was associated with a higher chance of enterovirus infection than a history of breastfeeding > 6 months, and children who had mother who worked as farmers, daily wage employees, and unprofessional skilled jobs had a greater chance of enterovirus infection than those who had unemployed mothers. Coxsackievirus-infected children had a higher rate of rashes on the buttocks, knee, and elbow and fever but a lower rate of lethargy and malaise than EV-A71-infected children.ConclusionsEV-A71 is a major cause of HFMD in children < 6 years old in northern Thailand, but rash, fever, and mouth ulcers are mostly found in participants with coxsackievirus infection. Breastfeeding should be promoted during early childhood for at least 6 months to prevent HFMD particularly those mother who are working in unprofessional skill jobs.

Abstract 16756: Coxsackievirus Infection During Early Pregnancy Leads to Congenital Heart Defects

Introduction: Coxsackievirus B (CVB), the most common cause of myocarditis, targets cardiomyocytes through the Coxsackie and Adenovirus Receptor which is highly expressed in the fetal heart. Association between maternal CVB infection and fetal cardiac malformations has not been studied. Hypothesis: CVB infection during early pregnancy may play a role in the pathogenesis of congenital heart disease (CHD). Methods: Pregnant C57Bl/6J mice were infected between embryonic days (E) 5-11 with CVB3 (Nancy strain) at doses 1x10 6 , 2.5x10 6 , and 5x10 6 TCID50. Presence of the virus was confirmed by RT-PCR and infectivity assay and quantified by qPCR. Fetal heart phenotype was assessed by histology and to determine changes at the molecular level after infection, RNA was collected from fetal hearts using laser microdissection and analyzed by RNA-Seq. The results were confirmed by immunohistochemistry and qPCR. To assess the potential clinical relevance of our observations in mice, we analyzed the sera from pregnant women for presence of antibody titers against of CVB. Results: Offspring of pregnant mice infected with CVB3 at different time points developed a spectrum of CHD (38.2%, n=78/204): ventricular septal defect, double outlet right ventricle, and non-compaction of the ventricular myocardium, along with fetal death. Infection at E9 led to the highest incidence of VSD compared to infection earlier (E5, E7 or E8) or later (E11) in gestation. Transcripts found to be differentially expressed between infected fetal hearts with VSDs compared with controls were mediators of the Transforming growth factor β and Extracellular matrix signaling pathways. Consistent with the former, expression levels of BMP2 and Smad1/5/9 mRNA were elevated. Simultaneously suppressed proliferation of cardiomyocytes in these fetuses was observed. Serum analysis from 66 pregnant women who had babies with heart defects showed elevated titers of antibodies against CVB underlining the clinical significance of CVB infection during pregnancy. Conclusions: In mice, there is a critical window during pregnancy where CVB infection leads to abnormal cardiac development, likely through reduction of cardiomyocyte proliferation mediated by elevated levels of BMP2 and Smad1/5/9. Collectively, the data confirm a link between CVB infection and CHD development in mouse and human.