Coxsackievirus Type B3 Research Articles

Novel N-benzenesulfonyl sophocarpinol derivatives as coxsackie B virus inhibitors.

Novel N-benzenesulfonyl sophocarpinic acid/ester and sophocarpinol derivatives were synthesized and evaluated for their antienteroviral activities against coxsackievirus type B3 (CVB3) from sophocarpine (1), a natural medicine isolated from Chinese herb. Structure-activity relationship (SAR) analysis revealed that the double bond and its geometrical configuration and position at the C-11 attachment did not greatly affect the potency. Among these derivatives, sophocarpinol 24d exerted the promising activities against not only CVB3 but also CVB1, CVB2, CVB5, and CVB6 with IC50 ranging from 0.62 to 3.63 μM (SI from 46 to 275), indicating a broad-spectrum antienteroviral characteristic. The SAR results provided the powerful information for further strategic optimization and development of a novel scaffold of broad-spectrum antiviral candidates against enteroviruses.

Antimicrobial and anti-biofilm activity of thiourea derivatives incorporating a 2-aminothiazole scaffold.

A series of new thiourea derivatives of 1,3-thiazole have been synthesized. All obtained compounds were tested in vitro against a number of microorganisms, including Gram-positive cocci, Gram-negative rods and Candida albicans. Compounds were also tested for their in vitro tuberculostatic activity against the Mycobacterium tuberculosis H37Rv strain, as well as two 'wild' strains isolated from tuberculosis patients. Compounds 3 and 9 showed significant inhibition against Gram-positive cocci (standard strains and hospital strain). The range of MIC values is 2-32 µg/mL. Products 3 and 9 effectively inhibited the biofilm formation of both methicillin-resistant and standard strains of S. epidermidis. The halogen atom, especially at the 3rd position of the phenyl group, is significantly important for this antimicrobial activity. Moreover, all obtained compounds resulted in cytotoxicity and antiviral activity on a large set of DNA and RNA viruses, including Human Immunodeficiency Virus type 1 (HIV-1) and other several important human pathogens. Compound 4 showed activity against HIV-1 and Coxsackievirus type B5. Seven compounds resulted in cytotoxicity against MT-4 cells (CC50<10 µM).

PO-0526 Neonatal Enterovirus Infection As A Cause Of Cardiogenic Shock

Neonatal enterovirus infection can show a septic disease with different organ manifestation and acute heart failure. 2 neonates admitted at the age of 10 and 18 days in cardiogenic shock with respiratory failure, massive lactic acidosis, significantly elevated proBNP and troponin values. Exclusion of congenital heart lesions. No detection of a bacterial infection. No sign of a congenital metabolic disorder. Radiologically significant cardiomegaly and pulmonary oedema. Echocardiography pronounced left ventricular dysfunction and suprasystemic pressures in right ventricle. In one patient, invasive exclusion of coronary pathology. Intensive care treatment with catecholamines and phosphodiesterase inhibitors and mechanical ventilation. Detection of human Coxsackie Virus type B3 in the blood, stool and tracheal secretions of both children. Stabilisation of both children after therapy but in echocardiography persistent left ventricular dysfunction. Neonatal infection with enteroviruses may present as a decompensated congenital heart defect or neonatal bacterial sepsis clinically. After exclusion of these causes an enterovirus infection is to take into consideration. The prognosis is depending on recovery of cardiac function and other organ functions. In extreme cases, ECMO therapy has to be considered. The duration of the cardiac therapy (beta- blockers, diuretics, ACE inhibitors, etc.) is unclear.

Abstract 18: Coxsackievirus B3 Escapes Infected Cardiac Stem Cells via Ejected Autophagosomes

Many viruses have been shown to upregulate host autophagic processes during the course of infection. Originally this process was thought to be a response mechanism of the host cell in order to engulf viral particles and destroy them to prevent viral spread and host cell death. However, certain viruses have been shown to strategically hijack the autophagic pathway to evade host immunity and increase viral replication. Coxsackievirus B (CVB) is a non-enveloped picornavirus that most commonly causes a self-limited febrile illness in young children, but in rare severe cases can progress to myocarditis, pancreatitis, or meningo-encephalitis. It has also been associated with late-onset idiopathic dilated cardiomyopathy. CVB type 3 (CVB3) has previously been documented to upregulate host autophagic machinery upon infection, and we report here that the virus can infect cardiac stem cells and other muscle progenitors and allow itself to be engulfed in autophagosomes which later get shed from the surface of the host cell. This is a novel method of viral dissemination especially for a “naked” virus which would typically escape the infected host by triggering cytolysis. However, cytolytic viruses subsequently become exposed to neutralizing antibodies. The fact that CVB3 can hide itself in host membranes presents several fascinating possibilities. Not only could the ejected autophagosome mask the virus from adaptive immunity, but it may also bypass receptor-mediated endocytosis and infect cell-types that might lack CVB3 receptors. This mode of viral escape represents a unique evolutionary adaptation which obscures the line dividing enveloped and non-enveloped viruses.

The antiviral effect of jiadifenoic acids C against coxsackievirus B3

Coxsackievirus B type 3 (CVB3) is one of the major causative pathogens associated with viral meningitis and myocarditis, which are widespread in the human population and especially prevalent in neonates and children. These infections can result in dilated cardiomyopathy (DCM) and other severe clinical complications. There are no vaccines or drugs approved for the prevention or therapy of CVB3-induced diseases. During screening for anti-CVB3 candidates in our previous studies, we found that jiadifenoic acids C exhibited strong antiviral activities against CVB3 as well as other strains of Coxsackie B viruses (CVBs). The present studies were carried out to evaluate the antiviral activities of jiadifenoic acids C. Results showed that jiadifenoic acids C could reduce CVB3 RNA and proteins synthesis in a dose-dependent manner. Jiadifenoic acids C also had a similar antiviral effect on the pleconaril-resistant variant of CVB3. We further examined the impact of jiadifenoic acids C on the synthesis of viral structural and non-structural proteins, finding that jiadifenoic acids C could reduce VP1 and 3D protein production. A time-course study with Vero cells showed that jiadifenoic acids C displayed significant antiviral activities at 0–6h after CVB3 inoculation, indicating that jiadifenoic acids C functioned at an early step of CVB3 replication. However, jiadifenoic acids C had no prophylactic effect against CVB3. Taken together, we show that jiadifenoic acids C exhibit strong antiviral activities against all strains of CVB, including the pleconaril-resistant variant. Our study could provide a significant lead for anti-CVB3 drug development.

A new transcriptional role for matrix metalloproteinase-12 in antiviral immunity.

Interferon-α (IFN-α) is essential for antiviral immunity, but in the absence of matrix metalloproteinase-12 (MMP-12) or IκBα (encoded by NFKBIA) we show that IFN-α is retained in the cytosol of virus-infected cells and is not secreted. Our findings suggest that activated IκBα mediates the export of IFN-α from virus-infected cells and that the inability of cells in Mmp12(-/-) but not wild-type mice to express IκBα and thus export IFN-α makes coxsackievirus type B3 infection lethal and renders respiratory syncytial virus more pathogenic. We show here that after macrophage secretion, MMP-12 is transported into virus-infected cells. In HeLa cells MMP-12 is also translocated to the nucleus, where it binds to the NFKBIA promoter, driving transcription. We also identified dual-regulated substrates that are repressed both by MMP-12 binding to the substrate's gene exons and by MMP-12-mediated cleavage of the substrate protein itself. Whereas intracellular MMP-12 mediates NFKBIA transcription, leading to IFN-α secretion and host protection, extracellular MMP-12 cleaves off the IFN-α receptor 2 binding site of systemic IFN-α, preventing an unchecked immune response. Consistent with an unexpected role for MMP-12 in clearing systemic IFN-α, treatment of coxsackievirus type B3-infected wild-type mice with a membrane-impermeable MMP-12 inhibitor elevates systemic IFN-α levels and reduces viral replication in pancreas while sparing intracellular MMP-12. These findings suggest that inhibiting extracellular MMP-12 could be a new avenue for the development of antiviral treatments.

Production of a Dominant-Negative Fragment Due to G3BP1 Cleavage Contributes to the Disruption of Mitochondria-Associated Protective Stress Granules during CVB3 Infection

Stress granules (SGs) are dynamic cytosolic aggregates containing messenger ribonucleoproteins and target poly-adenylated (A)-mRNA. A key component of SGs is Ras-GAP SH3 domain binding protein-1 (G3BP1), which in part mediates protein-protein and protein-RNA interactions. SGs are modulated during infection by several viruses, however, the function and significance of this process remains poorly understood. In this study, we investigated the interplay between SGs and Coxsackievirus type B3 (CVB3), a member of the Picornaviridae family. Our studies demonstrated that SGs were formed early during CVB3 infection; however, G3BP1-positive SGs were actively disassembled at 5 hrs post-infection, while poly(A)-positive RNA granules persisted. Furthermore, we confirmed G3BP1 cleavage by 3Cpro at Q325. We also demonstrated that overexpression of G3BP1-SGs negatively impacted viral replication at the RNA, protein, and viral progeny levels. Using electron microscopy techniques, we showed that G3BP1-positive SGs localized near mitochondrial surfaces. Finally, we provided evidence that the C-terminal cleavage product of G3BP1 inhibited SG formation and promoted CVB3 replication. Taken together, we conclude that CVB3 infection selectively targets G3BP1-SGs by cleaving G3BP1 to produce a dominant-negative fragment that further inhibits G3BP1-SG formation and facilitates viral replication.

Human pathogenic viruses are retained in and released by Candida albicans biofilm in vitro

Candida albicans is the most prevalent human fungal pathogen associated with biofilm formation on indwelling medical devices. Under this form, Candida represents an infectious reservoir difficult to eradicate and possibly responsible for systemic, often lethal infections. Currently, no information is available on the occurrence and persistence of pathogenic viruses within C. albicans biofilm. Therefore, the aim of this study was to investigate whether Herpes Simplex Virus type 1 (HSV-1) and Coxsackievirus type B5 (CVB5) can be encompassed in Candida biofilm, retain their infectivity and then be released. Thus, cell-free virus inocula or HSV-1-infected cells were added to 24h-old fungal biofilm in tissue culture plates; 48h later, the biofilm was detached by washing and energetic scratching and the presence of virus in the rescued material was end-point titrated on VERO cells. Planktonic Candida cultures and samples containing only medium were run in parallel as controls. We found that both HSV-1 and CVB5 free virus particles, as well as HSV-1 infected cells remain embedded in the biofilm retaining their infectivity. As a second step, the influence of biofilm on virus sensitivity to sodium hypochlorite and to specific neutralizing antibodies was investigated. The results showed that virus encompassment in fungal biofilm reduces virus sensitivity to chemical inactivation but does not affect antibody neutralization. Overall, these data provide the first in vitro evidence that viruses can be encompassed within Candida biofilm and then be released. Thus, it may be speculated that Candida biofilm can be a reservoir of viruses too, posing a further health risk.

Synthesis and Biological Evaluation of N‐Substituted Sophocarpinic Acid Derivatives as Coxsackievirus B3 Inhibitors

A series of novel N-substituted sophocarpinic acid derivatives was designed, synthesized, and evaluated for their anti-enteroviral activities against coxsackievirus type B3 (CVB3) and coxsackievirus type B6 (CVB6) in Vero cells. Structure-activity relationship analysis revealed that the introduction of a benzenesulfonyl moiety on the 12-nitrogen atom in (E)-β,γ-sophocarpinic acid might significantly enhance anti-CVB3 activity. Among the derivatives, (E)-12-N-(m-cyanobenzenesulfonyl)-β,γ-sophocarpinic acid (11 m), possessing a meta-cyanobenzenesulfonyl group, exhibited potent activity against CVB3 with a selectivity index (SI) of 107. Furthermore, compound 11 m also showed a good oral pharmacokinetic profile, with an AUC value of 7.29 μM h⁻¹ in rats, and good safety through the oral route in mice, with an LD₅₀ value of >1000 mg kg⁻¹; these values suggest a druggable characteristic. Therefore, compound 11 m was selected for further investigation as a promising CVB3 inhibitor. We consider (E)-β,γ-N-(benzenesulfonyl)sophocarpinic acids to be a novel class of anti-CVB3 agents.

Efficient synthesis and anti-enteroviral activity of 9-arylpurines

To further explore the anti-enteroviral activity of 9-aryl-6-chloropurines, three different series of compounds with a dialkylamino, (alkyl)amido, or oxazolidinone substituent at the aryl ring have been synthesized, in most cases with the aid of microwave-assisted synthesis. The resulting compounds efficiently inhibit Coxsackie virus type B3 (CVB3) replication with EC50 values varying from 3 to 15 μM, and with no significant toxicity in Vero cells. The most potent compounds also selectively inhibit the replication of other enteroviruses including Coxsackie virus B4 and Echo virus 11. The cross-resistance studies performed with different 9-aryl-6-chloropurines indicate that they all belong to the same pharmacological family and differ from other CVB3 drugs such as enviroxime.

Surveillance of Poliovirus Circulation Among Refugees in Italy, 2008–2011: Table 1

The aim of this study was to evaluate the presence of wild poliovirus or sabin-like poliovirus in 152 stool samples from migrants in the Accommodation Center in Italy and liquid waste from the sewage systems. Two surveys were performed in 2008 and 2011. All stool samples were negative for enterovirus and poliovirus. One of the liquid samples analyzed was positive for Coxsackievirus type B5.

MiR-342-5p suppresses coxsackievirus B3 biosynthesis by targeting the 2C-coding region

Coxsackievirus B type 3 (CVB3) is one of the major pathogens associated with human heart disease. miRNAs are a class of short, noncoding RNA that can post-transcriptionally modulate gene expression. By comparing the CVB3 genome and miR-342-5p sequences, we found there were potential miR-342-5p targets in the CVB3 genome. To verify the effect of miR-342-5p on CVB3 biosynthesis, HeLa cells were infected with a Renilla luciferase (RLuc)-expressing CVB3 variant (RLuc-CVB3). We observed that miR-342-5p could significantly inhibit the expression of RLuc in infected cells. In HeLa cells infected with an enhanced green fluorescence protein (EGFP)-expressing CVB3 variant (EGFP-CVB3), EGFP expression was also significantly inhibited by miR-342-5p. The inhibitory effect of miR-342-5p on EGFP expression in EGFP-CVB3-infected cells could be reversed by transfection with anti-miR-342-5p oligonucleotide (AMO-miR-342-5p). Moreover, RNA and protein biosynthesis in wild-type CVB3 was significantly inhibited by miR-342-5p. By mutating the putative targets of miR-342-5p in the 2C-coding region, a sequence, nt4989–nt5015, was identified as the miR-342-5p target. The conserved nt4989–nt5015 sequences of CVB type 1–5 suggest miR-342-5p may exert its inhibitory effect in other types of coxsackievirus besides CVB3. Western blotting indicated that miR-342-5p could indeed suppress protein expression in CVB type 1 and 5. There was a moderate abundance of miR-342-5p in the gut, heart, and brain of Balb/c mice, suggesting that miR-342-5p may interact with CVB3 in vivo. Taken together, these results indicate that miR-342-5p can inhibit CVB3 biosynthesis by targeting its 2C-coding region and therefore may be a potential therapeutic agent in the treatment of CVB3 infection.

Echoviruses are a major cause of aseptic meningitis in infants and young children in Kuwait

BackgroundThe etiologic agents of aseptic meningitis (AM) often include human enteroviruses. The role of enteroviruses causing AM in young children was investigated during a 3-year period in Kuwait.ResultsEnteroviral RNA was detected in cerebrospinal fluid (CSF) by reverse transcription-PCR and specific genotypes of enteroviruses were identified by direct DNA sequencing of VP4-VP2 region. Enteroviral RNA was detected in 92 of 387 (24%) suspected AM cases and the results were confirmed by hybridization of amplicons with an internal, enterovirus-specific probe. The CSF samples from 75 of 281 (27%) children < 2 years old but only from 3 of 38 (8%) 4-12 year-old children were positive for enteroviral RNA (p = 0.011). Majority of infections in children < 2 years old (49 of 75, 65%) were due to three echoviruses; echovirus type 9 (E9), E11 and E30. Only three other enteroviruses, namely coxsackievirus type B4, coxsackievirus type B5 and enterovirus 71 were detected among AM cases in Kuwait.ConclusionsOur data show that three types of echoviruses (E9, E11 and E30) are associated with the majority of AM cases in Kuwait. To the best of our knowledge, this is the first report to characterize different enterovirus genotypes associated with AM in the Arabian Gulf region.

ERK MAP kinase-activated Arf6 trafficking directs coxsackievirus type B3 into an unproductive compartment during virus host-cell entry

Clathrin- and caveolae-mediated endocytosis have been implicated in the productive entry of many viruses into host cells. ADP-ribosylation factor 6 (Arf6)-dependent endocytosis is another endocytosis pathway that traffics from the cell surface and it is the only Arf that traffics at the plasma membrane. However, little is known about Arf6-dependent trafficking during virus entry. This study showed that coxsackievirus type B3 (CVB3) associated with decay-accelerating factor in non-polarized HeLa cells can be redirected into non-productive compartments by Arf6-dependent internalization, thus restricting infection. Overexpression of wild-type (WT) and constitutively active (CA) Arf6 in HeLa cells resulted in a 2.3- and 3.6-fold decrease in infection, respectively. A dominant-negative inhibitor of Arf6 recovered restriction of infection by WT-Arf6 and CA-Arf6. RNA interference of endogenous Arf6 resulted in a 3.3-fold increase in CVB3 titre in HeLa cells. It was shown that coxsackie-adenovirus receptor (CAR) ligation by virus or CAR-specific antibody could activate extracellular signal-regulated kinase (ERK) of the mitogen-activated protein kinase family and lead to Arf6-mediated viral restriction. In the absence of ERK activation, CVB3 internalization into early endosomes was inhibited and subsequent infection was reduced, but Arf6-mediated restriction was also abolished. In conclusion, receptor-mediated signalling enhances CVB3 entry whilst also activating non-productive pathways of virus entry; thus, virus infection is an equilibrium of productive and non-productive pathways of entry.

Chronic Hereditary Pancreatitis in a Girl With a Serine Protease Inhibitor Kazal Type I (SPINK-1) Gene Mutation and a Coxsackie Type B5 Infection

To the Editors: A 3-year-old girl presented with epigastric pain, vomiting, diarrhea, and anorexia. Blood results showed a serum lipase value of 10.784 U/L (normal 0–200 U/L), amylase 150 U/L (normal 0–85 U/L), alkaline phosphatase 218 U/L, ALT 23 U/L, AST 32 U/L, LD 695 U/L, and a total bilirubin of 3 μmol/L. Serologic tests showed a high antibody titer to Coxsackie virus type B5 (1:2048). IgM antibody for mumps, varicella zoster, rubella, measles, para-influenza, and cytomegalovirus were all undetectable. After a second episode of acute pancreatitis, a mutation in the serine protease inhibitor kazal type I (SPINK-1) was identified. Endoscopic ultrasonography (EUS) and retrograde cholangiopancreaticography (ERCP) showed a short distal stenosis in the pancreatic head. A 5 cm 7 Fr endoprosthesis was placed in the pancreatic duct. Exocrine pancreatic function remained undisturbed (elastase-1 levels >0.500). The child's mother also had a SPINK-1 mutation. SPINK-1 mutations are also found in 2% of patients without chronic pancreatitis.1,2 Therefore, the mutation by itself does not cause pancreatitis. Other factors (like in this case a Coxsackie type B5 infection3) must be present to cause pancreatitis. SPINK-1 mutation might predispose to chronic pancreatitis. Under normal conditions, SPINK-1 inhibits the transformation of trypsinogen in trypsin in the pancreas, by which auto-digestion is prevented. A SPINK-1 gene mutation leads to an increased trypsin activity in the pancreas, causing auto-digestion.2 In the case of a Coxsackie type B5 induced acute pancreatitis, screening for a SPINK-1 mutation is indicated if episodes of acute pancreatitis recur. Endoscopic ultrasonography is a useful diagnostic technique to visualize pancreatic duct abnormalities in children with chronic pancreatitis.4,5 Michael Groeneweg, MD, PhD Dept of Paediatrics, Sophia Children's Hospital Erasmus Medical Center Rotterdam Rotterdam, the Netherlands Dept of Paediatrics Maasstad Hospital Rotterdam, the Netherlands Jan W. Poley, MD, PhD Dept of Gastroenterology and Hepatology Erasmus Medical Center Rotterdam Rotterdam, the Netherlands Marlies Dansen, MD Dept of Paediatrics, Sophia Children's Hospital Erasmus Medical Center Rotterdam Rotterdam, the Netherlands Jan G. Brinkman, MD Dept of Paediatrics Maasstad Hospital Rotterdam, the Netherlands Johanna C. Escher, MD, PhD Edward E. S. Nieuwenhuis, MD, PhD Dept of Paediatrics, Sophia Children's Hospital Erasmus Medical Center Rotterdam Rotterdam, the Netherlands

Molecular detection and characterization of human enteroviruses in Korean surface water

In this study, the genetic epidemiology of enteroviruses (EVs) in Korean surface water was evaluated by conducting phylogenetic analyses of the nucleotide sequences of the 5' non-coding region (5' NCR), which was determined by RT-PCR analysis of total culturable virus assay-positive samples. The results showed that the nucleotide sequences of the EVs could be classified into 4 genetic clusters, and that the predominant presence of Korea EVs were very similar to echoviruses type 30. Interestingly, two nucleotide sequences were very similar to those of coxsackievirus type B1 isolated from aseptic meningitis patients in Seoul, Korea, implying the possibility of a common source for the viruses circulated in water systems and humans. In addition, 3 nucleotide sequences clustered strongly with the nucleotide sequences from China or Japan, and one fell into the same cluster as echovirus type 11 from Taiwan, which suggests that EVs in Asia may have evolved in a region-specific manner. Taken together, the results of this study revealed that EVs from Korea surface waters could be genetically classified as coxsackieviruses or echoviruses, and that they evolved in Asia in a region-specific manner.

Serologic Prevalence of Coxsackievirus Group B in Greece

Coxsackieviruses are human enteroviruses, which have been associated with myocarditis/pericarditis and sudden death. In one investigation (Spanakis N, Manolis EN, Tsakris A, Tsiodras S, Panagiotopoulos T, Saroglou G, and Legakis NJ: J Clin Pathol 2005;58:357-360), a cluster of cases of fatal myocarditis in Greece was linked to coxsackievirus B3. The information from this investigation prompted us to study serologically the prevalence of coxsackieviruses B throughout Greece. Sera were obtained from 506 healthy blood donors from various transfusion centers, covering the entire country. All sera were tested for the presence of IgG and IgM antibodies, using ELISAs with various antigenic specificities: (1) heat-denatured coxsackievirus type B1 and B5 virions, (2) a synthetic peptide from the N terminus of the VP1 protein of coxsackievirus B3, and (3) a synthetic peptide from the N terminus of the VP1 protein of coxsackievirus B4. Sera positive for IgG antibodies against coxsackieviruses B1/B5, B3, and B4 were detected in 6.7 to 21.6% of the individuals tested in the various regions of Greece. Statistical analysis revealed that the highest prevalence of IgG antibodies against coxsackieviruses B1/B5 was found in blood donors from Crete (p = 0.025), whereas the highest prevalence against coxsackievirus B4 was detected in blood donors from Athens (p = 0.01). IgM antibodies against coxsackievirus B were detected at low percentage, less than 5%, with no significant viral preference for particular geographic regions. The preference of anti-coxsackievirus IgG antibodies for particular geographic regions could be potentially related to the previously reported clustering of cases of insulin-dependent diabetes mellitus and myocarditis in Athens and Crete, respectively.

Detection of Enteroviruses and Mammalian Reoviruses in Korean Environmental Waters

Using the standard total culturable virus assay-most probable number (TCVA-MPN) method, we evaluated a total of 348 samples, including surface water, finished water, and tap water samples, collected from randomly selected water treatment plants in Korea from August 2001 through July 2005 according to the Information Collection Rule. All the TCVA-positive samples were also subjected to integrated cell culture-PCR (ICC-PCR) methods for the detection of enteroviruses, hepatitis A virus, adenoviruses, and reoviruses. The most probable number of infectious units per 100 liters for the environmental water samples ranged from 0.5 to 47.3. Nine of the 13 TCVA-positive samples (69.2%) were found by ICC-PCR to be positive for human enteroviruses, which were confirmed to be coxsackievirus type B3, coxsackievirus type B4, coxsackievirus type B6, echovirus type 30, and vaccine strain poliovirus type 3 by direct sequencing. Eleven of the 13 TCVA-positive samples (84.6%) were found by ICC-PCR assay to be positive for reoviruses. The serotype of all the reoviruses was the same as reovirus type 1 by direct sequencing. Both enteroviruses and reoviruses were concurrently detected in seven TCVA-positive samples (53.8%).

Cardiomyocytes upregulate matrix metalloproteinases following coxsackievirus infection

Introduction: Coxsackievirus type B3 (CVB3) is the most common causative agent of infectious myocarditis. To enhance survival of the host cell and to eliminate the infectious agents, virus-infected cardiomyocytes induce expression of a variety of proteins, including pro-inflammatory molecules. Matrix metalloproteinases (MMPs) are endopeptidases that can collectively breakdown all components of the extracellular matrix and are crucial for matrix remodeling of wounds. They have also been shown to facilitate immune cell influx into wounds and to aid in pro-inflammatory molecule processing. Hypothesis: We hypothesize that virus infection of mouse heart cells stimulates production of MMPs in order to facilitate inflammatory cell migration and remodeling of acute wounds. Methods: Adolescent A/J mice were either infected intraperitoneally with 105 pfu of CVB3 or sham-infected with saline and sacrificed on days 3, 9, and 30 post-infection (pi). Ventricular tissues were formalin-fixed for MMP-2 immunohistochemistry (IHC) and histological staining. Apices were flash-frozen and homogenized for MMP-12 Western blot and gelatin zymography. Transmission electron microscopy (TEM) of ventricular tissue was utilized to detect orientation and distribution of collagens. HL-1 cells, a murine cardiomyocyte cell line, were infected with CVB3 at an MOI of 50. The conditioned media from these cells were collected at 4, 6, 10, and 12 hours pi, and subjected to gelatin zymography and MMP-12 Western blot analyses. Results: The gelatinase MMP-2 was shown to be increased within cardiomyocytes as well as extracellularly by immunohistochemistry. MMP-2 immunopositivity, which colocalized to damaged necrotic tissue, increased as early as 3 days pi and was further upregulated at days 9 and 30 days pi. Zymography further confirmed that the activities of the gelatinases, MMP-2 and MMP-9, were upregulated at the later timepoints. MMP-12 protein expression decreased at day 9 but significantly increased at day 30. TEM analysis showed no disruption of the matrix at day 3 but massive collagen deposition and reorganization of the matrix were observed at day 9. CVB3-infected HL-1 cells showed upregulation of both gelatinases beginning from 4 hours pi and was maintained until 12 hours pi. Interestingly, MMP-12 expression was significantly increased at 4 hours and remained increased throughout the infection. Conclusions: MMPs-2, -9, and -12 are upregulated in cardiomyocytes during viral myocarditis, leading to disruption of the collagen matrix.

Metallothionein is induced and trace element balance changed in target organs of a common viral infection.

In experimental studies on the common human coxsackievirus B type 3 (CB3) infection, administered cadmium (Cd) is known to accumulate in the liver and kidneys. CB3 adapted to Balb/c mice was used to study whether infection affects the Cd-binding protein, metallothionein (MT) and if this alters the normal physiological trace element balance in the liver, kidney, spleen and brain. On day 3 of infection, degradation of liver proteins (44%, P<0.01) occurred, whereas in the spleen, protein increased (63%, P<0.05). The infection increased MT five-fold (P<0.01) in liver and kidneys, and in spleen by 34% (P<0.05). A redistribution of Cd and copper (Cu) from the liver to the kidney was associated with this increase in MT, resulting in an increased (P<0.01) kidney/liver ratio for both elements. The infection increased the zinc (Zn) concentration more in the kidney than in the liver, but the kidney/liver ratio was not significantly affected. Results show that MT is increased in several organs during the early phase of infection and is associated with redistribution of both essential and non-essential trace elements. This may be a normal response in common infections that could adversely influence the pathogenesis when the host is concomitantly exposed to potentially toxic trace elements, even at levels in the physiological range.