Axel Stelzner

Apoptosis in Coxsackievirus B3-Caused Diseases: Interaction between the Capsid Protein VP2 and the Proapoptotic Protein Siva

ABSTRACT Coxsackievirus B3 (CVB3) is a common factor in human myocarditis. Apoptotic events are present in CVB3-induced disease, but it is unclear how CVB3 is involved in apoptosis and which viral proteins may induce the apoptotic pathway. In this report we demonstrate that the human and murine proapoptotic protein Siva specifically interact with the CVB3 capsid protein VP2. Furthermore, the transcription of Siva is strongly induced in tissue of CVB3-infected mice and is present in the same area which is positively stained for apoptosis, CD27, and CD70. It has been proposed that Siva is involved in the CD27/CD70-transduced apoptosis. Therefore, we suggest a molecular mechanism through which apoptotic events contributes to CVB3-caused pathogenesis.

A rapid assay for evaluation of antiviral activity against coxsackie virus B3, influenza virus A, and herpes simplex virus type 1

In order to identify new potential antiviral drugs, small amounts of extracts or compounds have to be examined for cytotoxicity and antiviral activity in primary screening using a rapid, easy, inexpensive, and highly standardised test system. In this study, high-throughput cytopathic effect (CPE) inhibitory assays were established for coxsackie virus B3 on HeLa Ohio cells, influenza virus A on Madin-Darby canine kidney cells, and herpes simplex virus type 1 (HSV-1) on green monkey kidney cells that meet these requirements. The cytotoxic and the antiviral effects were quantified using a crystal violet uptake assay allowing automated handling of large numbers of candidate agents. To ensure comparable results with plaque reduction assays, the 50 and 90% plaque inhibitory concentrations of guanidine, amantadine, and phosphonoformic acid were used to standardise the anti-coxsackie virus B3, anti-influenza virus A, and anti-HSV-1 tests, respectively. The strong correlation between the antiviral activity determined by CPE-inhibitory assays and plaque reduction assay was further proved for other antivirals. In summary, low amounts of large numbers of compounds may be tested inexpensively and standardised within 24 h (coxsackie virus B3 and influenza virus A) or 48 h (herpes simplex virus type 1) post-infection using CPE inhibitory assays.

Porcine Teschoviruses Comprise at Least Eleven Distinct Serotypes: Molecular and Evolutionary Aspects

ABSTRACT Nucleotide sequencing and phylogenetic analysis of 10 recognized prototype strains of the porcine enterovirus (PEV) cytopathic effect (CPE) group I reveals a close relationship of the viral genomes to the previously sequenced strain F65, supporting the concept of a reclassification of this virus group into a new picornavirus genus. Also, nucleotide sequences of the polyprotein-encoding genome region or the P1 region of 28 historic strains and recent field isolates were determined. The data suggest that several closely related but antigenically and molecular distinct serotypes constitute one species within the proposed genus Teschovirus. Based on sequence data and serological data, we propose a new serotype with strain Dresden as prototype. This hitherto unrecognized serotype is closely related to porcine teschovirus 1 (PTV-1, former PEV-1), but induces type-specific neutralizing antibodies. Sequencing of field isolates collected from animals presenting with neurological disorders prove that other serotypes than PTV-1 may also cause polioencephalomyelitis of swine.

Sequencing of Porcine Enterovirus Groups II and III Reveals Unique Features of Both Virus Groups

ABSTRACT The molecular classification of the porcine enterovirus (PEV) groups II and III was investigated. The sequence of the almost complete PEV-8 (group II) genome reveals that this virus has unique L and 2A gene regions. A reclassification of this group into a new picornavirus genus is suggested. PEV group III viruses are typical enteroviruses. They differ from other enteroviruses by a prolonged stem-loop D of the 5′-cloverleaf structure.

Cytokine profiles in heart, spleen, and thymus during the acute stage of experimental coxsackievirus B3-induced chronic myocarditis.

Since cytokines play an important role in the pathogenesis of virus‐induced chronic heart diseases, cytokine mRNA expression was studied in coxsackievirus B3‐infected NMRI mice during the acute phase of myocarditis until the onset of chronic cardiac disease. Virus replication, cytokine induction, inflammatory cell infiltration and myocardial damage were studied by titer determination, reverse transcription‐polymerase chain reaction (RT‐PCR), and histopathology. To investigate whether the coxsackievirus B3‐induced cytokine mRNA accumulation was only limited to the heart or generalized, spleen and thymus specimens were also included. Surprisingly, interleukin (IL)‐10 as a deactivator of T cell and macrophage functions was transcribed in the myocardium nearly in parallel with virus replication from Day 1 through Day 14. At Day 3 p.i., the mRNA of IL‐1α, tumor necrosis factor (TNF)‐α, IL‐6, and interferon (IFN)‐β accumulated. At Days 4, 7, and 14, IL‐12‐specific mRNA was produced. Furthermore, increasing amounts of IFN‐γ mRNA were found, whereas IL‐2 and IL‐4 mRNA remained undetectable. TNF‐α, IL‐1α, IL‐10, IL‐12, and IFN‐γ mRNA persisted into the late stage of myocarditis. In the spleen a closely correlated expression of virus and IL‐10‐specific mRNAs was also found, and in addition, IFN‐β, TNF‐α, and IL‐6 were detected. In striking contrast to heart and spleen tissue, the distinct expression of viral RNA in the thymus was not accompanied by an increased cytokine mRNA production. These data provide evidence for a unique coxsackievirus B3‐induced cytokine pattern in the myocardium and spleen and suggest that persistently expressed IL‐10 may play a leading role in acute and chronic myocarditis by subverting the immune response. J. Med. Virol. 61:518–526, 2000.

Nitric oxide donors inhibit the coxsackievirus B3 proteinases 2A and 3C in vitro, virus production in cells, and signs of myocarditis in virus-infected mice

The antiviral effect of nitric oxide (NO)-releasing compounds was investigated. Using bacterially expressed and purified proteinases 2A and 3C of coxsackievirus B3, in vitro assays demonstrated the inhibition of the 2A proteinase activity in the presence of S-nitroso-N-acetyl-penicillamine (SNAP), 3-morpholinosydnonimine (SIN-1), 4-phenyl-3-furoxancarbonitrile (PFC), glyceryl trinitrate (GTN), and isosorbide dinitrate (ISDN). Sodium nitroprusside (SNP), which releases NO after metabolization, had no effect. The 3C proteinase was inactivated by SNAP, GTN, and ISDN. The vasodilators GTN and ISDN, widely used in the treatment of angina pectoris, exhibited antiviral activity in CVB3-infected GMK cells. CVB3-infected NMRI outbred mice showed significantly reduced signs of myocarditis after treatment with GTN or ISDN. Inhibitors of the cellular inducible NO synthase (iNOS) such as NG-nitro-l-arginine methyl ester (L-NAME), NG-nitro-l-arginine (L-NNA), and S-methyl-isothiourea (SMT), had no deleterious effect on CVB3-infected NMRI mice, indicating that endogenous NO synthesis is unlikely to be a major defense mechanism after enterovirus infection of outbred mice.

Expression of Immunoregulatory Cytokines by Recombinant Coxsackievirus B3 Variants Confers Protection against Virus-Caused Myocarditis

ABSTRACT Clinical and laboratory investigations have demonstrated the involvement of viruses and bacteria as potential causative agents in cardiovascular disease and have specifically found coxsackievirus B3 (CVB3) to be a leading cause. Experimental data indicate that cytokines are involved in controlling CVB3 replication. Therefore, recombinant CVB3 (CVB3rec) variants expressing the T-helper-1 (TH1)-specific gamma interferon (IFN-γ) or the TH2-specific interleukin-10 (IL-10) as well as the control virus CVB3(muIL-10), which produce only biologically inactive IL-10, were established. Coding regions of murine cytokines were cloned into the 5′ end of the CVB3 wild type (CVB3wt) open reading frame and were supplied with an artificial viral 3Cpro-specific Q-G cleavage site. Correct processing releases active cytokines, and the concentration of IFN-γ and IL-10 was analyzed by enzyme-linked immunosorbent assay and bioassays. In mice, CVB3wt was detectable in pancreas and heart tissue, causing massive destruction of the exocrine pancreas as well as myocardial inflammation and heart cell lysis. Most of the CVB3wt-infected mice revealed virus-associated symptoms, and some died within 28 days postinfection. In contrast, CVB3rec variants were present only in the pancreas of infected mice, causing local inflammation with subsequent healing. Four weeks after the first infection, surviving mice were challenged with the lethal CVB3H3 variant, causing casualties in the CVB3wt- and CVB3(muIL-10)-infected groups, whereas almost none of the CVB3(IFN-γ)- and CVB3(IL-10)-infected mice died and no pathological disorders were detectable. This study demonstrates that expression of immunoregulatory cytokines during CVB3 replication simultaneously protects mice against a lethal disease and prevents virus-caused tissue destruction.

DNA vaccine-mediated immune responses in Coxsackie virus B3-infected mice

DNA immunizations with the major structural protein VP1 of Coxsackie virus B3 (CVB3) have been previously found to protect BALB/c mice from lethal challenge. Here we report that the other CVB3 capsid proteins, VP2, VP3, and VP4, were less effective at preventing CVB3-caused disease. The application of pCMV/VP1 as a vaccine caused decreased myocyte destruction, reduced viral load in the heart tissue, accelerated antibody induction, and an early cytokine expression in heart tissue. In summary, our results indicate that the induction of B cell and/or T cell memory in vaccinated mice prior to challenge is responsible for the protection observed.

Direct interferon-γ-mediated protection caused by a recombinant coxsackievirus B3

Coxsackievirus B3 (CVB3) is one of the most important causes of viral myocarditis. Cytokines are involved in the control of CVB3 replication and pathogenesis. Local expression of specific cytokines by recombinant CVB3 confers prevention of virus-caused myocarditis. Expression of IFN-γ by CVB3(IFN-γ) protected BALB/c and C57BL/6 mice when the lethal infection with the highly pathogenic CVB3H3 variant was given directly after or prior to CVB3(IFN-γ) inoculation by decreasing the viral load and spread as well as tissue destruction. This direct effect was not restricted to the homologous virus. In vitro, cocultivation of CVB3(IFN-γ)-infected cells induced a reduction of CVB3H3 replication and virus-induced cytopathogenicity.

The outcome of coxsackievirus B3-(CVB3-) induced myocarditis is influenced by the cellular immune status.

Mice develop a marked age-related susceptibility to myocardial coxsackievirus B3 (CVB3) infections. The lesions observed in mice resemble closely those seen in the human disease. Experimental murine models of CVB3-induced myocarditis have shown that both, host and viral genetic factors, can influence susceptibility to the infection as well as the persistence and progression of the disease. Recently, we have shown that CD4 T cell-deficient MHC Class II knockout mice develop a strong fibrosis with virus persistence in the heart tissue and without production of neutralizing antibodies.To examine the role of CD4+ T cells and especially the role of the T helper 1 cell response for the outcome and pathogenesis of CVB3-induced myocarditis in more detail, 2 different mouse strains with identical genetical background (H-2b) were infected with CVB3-Mü/J (Nancy strain). Immunocompetent C57BL/6 mice and mice with targeted disruption of interleukin (IL-)4 gene (IL-4−/− mice) developed a severe acute myocarditis on day 7 post infection (p. i.). The CVB3-induced inflammation was cured until the 21st day p. i. in hearts of C57BL/6 mice. IL-4−/− mice with insufficient T helper-2 cell immune response developed a severe myocardial damage between day 7 and 21 p. i. with prolonged virus persistence in the heart tissue. Therefore, we suggest that despite an obvious normal T helper-1 cell cytokine pattern, IL-4−/− mice are more susceptible to long-term heart muscle injuries after infection with CVB3.ZusammenfassungMäuse sind neben dem Menschen, der als der natürliche Wirt fungiert, für eine Infektion mit Coxsackie-Virus B3 (CVB3) empfänglich. Die myokardialen Läsionen nach Infektion mit CVB3 bei Mäusen ähneln dem humanen Krankheitsbild. Die experimentelle Reproduzierbarkeit sowie die gute Charakterisierung der Genetik und des Immunsystems erlauben das Studium der pathologischen Zusammenhänge dieser Erkrankung in diesem Tiermodell. Durch Untersuchungen in verschiedenen murinen Modellsystemen konnte gezeigt werden, dass sowohl virale als auch wirtsspezifische genetische Faktoren die Empfänglichkeit des Organismus für die Infektion, deren Verlauf sowie Ausprägung beeinflussen. Experimente mit immundefizienten MHC-Klasse-II-Knockout-Mä,usen (CD4-T-Zell-defizient) zeigten eine starke Fibrosierung des Herzens mit Viruspersistenz und ausbleibender Bildung Virus neutralisierender Antikörper.Um die Rolle der CD4-T-Zell-vermittelten Immunantwort insbesondere der CD4+-T-Helfer-1-Zellen für den Ausgang und die Pathogenese der CVB3-induzierten Myokarditis weiter aufzuklären, wurden zwei verschiedene Mäusestämme mit identischem genetischen Hintergrund (H-2b) mit der Virusvariante CVB3-Mü/J (Stamm Nancy) infiziert. Immunkompetente C57BL/6-Mäuse und Mäuse mit einem defekten Interleukin-(IL-)4-Gen (IL-4−/−-Mäuse) entwickeln eine schwre akute Myokarditis am Tag 7 post infectionem (p. i.). Die CVB3-induzierte Entzündung des Herzens heilt in immunkompetenten Mäusen bis zum Tag 21 p. i. aus. IL-4−/−-Mäuse mit einer defizienten T-Helfer-Zell-2-vermittelten Immunantwort entwickeln schwere myokardiale Schädigungen zwischen Tag 7 und 21 p. i. mit verlängerter Viruspersistenz im Herzen, obwohl ein ausgeprägtes T-Helfer-Zelltyp-1-Zytokinmuster wie in immunkompetenten Mäusen exprimiert wird. Trotz dieser scheinbaren T-Helfer-Zell-1-Immunantwort sind IL-4-defiziente Mäuse empfäglicher für eine schwere CVB3-Infektion.