Woodruff Strain Research Articles

Effects of double combinations of enterovirus replication inhibitors against Coxsackie B viruses

The effects of double combinations of enterovirus (EV) replication inhibitors against Coxsackieviruses B1 (neurotropic Connecticut-5 strain) and B3 (cardiotropic Woodruff and neurotropic Nancy strains) were tested in cell culture experiments. Compounds with different mechanisms of action were studied: pleconaril, guanidine.HCl, MDL-860 and oxoglaucine. A three-dimensional method was applied for determining the character of the combined effect. The study determined several synergistic double combinations: guanidine.HCL + pleconaril or MDL-860 against Coxsackievirus B1; MDL-860 + each of the other EV replication inhibitors and guanidine.HCl + pleconaril against the cardiotropic Woodruff strain of Coxsackievirus B3; MDL-860 + oxoglaucine against the neurotropic Nancy strain of Coxsackievirus B3. No increased cytotoxicity was manifested in any of the tested double combinations. Keywords: antivirals; combination activity; Coxsackieviruses.

Effect of Consecutive Alternating Administration of a Triple Combination of Anti-Enteroviral Compounds in Mice Infected with Coxsackievirus B3.

A novel approach for treatment of enterovirus infections was characterized. Application of treatment course of consecutive alternating administration (CAA) of triple combination of enterovirus replication inhibitors in experimental infections (20 MLD50) with coxsackievirus B3 (CVB3) strains in newborn mice is presented. It was established that in infection with cardiotropic Woodruff strain the combination of pleconaril, МDL-860 and oxoglaucine (PMO) subjected to the CAA scheme, a significant protective effect was observed. Monotherapeutic courses as well as simultaneously daily applied PMO were without effect. Analogous data were observed at experimental infection with the neurotriopic Nancy strain of CVB3. Following IC50 values of virus samples taken every day from target organs of infected animals during the whole period of study, a drug-resistance was established in monotherapy with compounds-partners in the PMO combination. At courses by the treatment scheme CAA of PMO development of drug-resistance was not established, but an increased susceptibility to the effect of the inhibitor-components in the combination was proven. Toxicity of PMO applied via the CAA scheme and in the monotherapeutic courses in both healthy and CVB3 infected animals was recorded. All data obtained prove the potential of the CAA treatment scheme for development of effective chemotherapy of enterovirus infections.

Inhibition of Coxsackievirus B3 cardiotropic strain Woodruff replication by silencing essential viral genes

Post-transcriptional gene silencing is an effective tool for viral replication control at the epigenetic level. In this study, we aimed to produce dsRNAs and siRNA pools targeting the genetic region for the viral protease 3C and the genetic region covering the sequences for VP1 and VP3 capsid proteins of the Coxsackievirus B3 cardiotropic Woodruff strain by the use of the polymerase complex of bacteriophage Phi6 in HEp-2 monolayer cells. The dsRNAs obtained in this study, specific for the 3C and VP1-VP3 genetic regions of the virus, were characterized by very low cytotoxicity (up to 200 nmol/L and 100 nmol/L, respectively) and high efficiency at silencing the target genes and blocking the enteroviral replication in vitro (93% and 60% reduction of the virus infected cells, respectively, at concentrations of 40 nmol/L). The generated pools of overlapping siRNAs targeting the 3C or VP1-VP3 genetic regions showed insignificant cytotoxicity at concentrations up to 200 nmol/L (< 3%) and higher efficacy than the corresponding dsRNAs. We managed to achieve 95% inhibition of the CVB3 Woodruff strain replication in vitro by using siRNAs specific for the 3C coding region of the virus at a concentration of 40 nmol/L, with no cytotoxicity.

Effectiveness of the Consecutive Alternating Administration Course of a Triple Antiviral Combination in Coxsackievirus B3 Infections in Mice.

Anti-enteroviral chemotherapeutics for clinical use are not registered so far, mainly due to the rapid development of drug-resistance. One of the possible approaches to overcome this problem is the use of combined chemotherapy. However, its application consisting of simultaneously given drugs, is not efficacious because of the development of multiple resistance. Here we present a novel approach for combined application of anti-enteroviral compounds, consisting of a consecutive alternating administration (CAA) course. CAA was tested on 2 in vivo models of Coxsackievirus B3 infection in newborn mice at inoculation dose of 20 MLD50 (50% mouse lethal dose): neurotropic (Nancy strain) and cardiotropic (Woodruff strain) infections. Compounds partnering in a triple combination were selected as enterovirus (EV) replication inhibitors with different mode of action - disoxaril (a VP1 blocker), guanidine.HCl (targeting 2C protein) and oxoglaucine (attacking 3A coding region). The application of this combination by CAA course resulted in around 40 and 60% survival rate in mice infected with Nancy and Woodruff virus, respectively, accompanied by a marked lengthening of the mean survival time (MST). The results obtained are proofs for the prospect of the treatment course by a triple combination through the CAA scheme as an approach interfering the occurrence of drug resistance at EV infections.

Amelioration of Coxsackievirus B3-Mediated Myocarditis by Inhibition of Tissue Inhibitors of Matrix Metalloproteinase-1

Coxsackievirus B3 (CVB3) is a major cause of acute myocarditis, a serious condition that is refractory to treatment. Myocardial damage results in tissue remodeling that, if too extensive, may contribute to disease. Remodeling is achieved by extracellular proteolysis mediated by the matrix metalloproteinases (MMPs), and MMP activity is counterbalanced by tissue inhibitors of MMPs (TIMPs). We show herein that TIMP-1 expression is induced in the myocardium by CVB3 infection. Surprisingly, TIMP-1 knockout mice exhibited a profound attenuation of myocarditis, with increased survival. The amelioration of disease in TIMP-1 knockout mice was not attributable to either an altered T-cell response to the virus or to reduced viral replication. These data led us to propose a novel function for TIMP-1: its highly localized up-regulation might arrest the MMP-dependent migration of inflammatory cells at sites of infection, thereby anatomically focusing the adaptive immune response. The benefits of TIMP-1 blockade in treating viral myocarditis were confirmed by administering, to wild-type animals, TIMP-1-specific siRNA or polyclonal antisera, both of which diminished CVB3-induced myocarditis. These unexpected findings indicate that increased TIMP-1 expression exacerbates, rather than ameliorates, CVB3-induced myocarditis and, thus, that TIMP-1 may represent a target for the treatment of virus-induced heart disease.

Coronary flow reserve and heart failure in experimental coxsackievirus myocarditis. A transthoracic Doppler echocardiography study

The objective of this study was to apply transthoracic Doppler echocardiography (TTDE) in mice to study coronary flow reserve (CFR), an index of coronary microvascular function, in mild and severe forms of experimental viral myocarditis. Regarding methodology, BALB/c mice were infected with cardiotropic coxsackieviruses causing either a mild (Nancy strain) or a severe (Woodruff strain) myocarditis. Left ventricular dimensions, fractional shortening, and CFR (ratio of left coronary artery flow velocity during maximal adenosine-induced vasodilatation to rest) were measured by TTDE before infection and again 1 or 2 wk after infection. As a result, the resting flow velocity did not change after infection. In contrast, CFR reduced significantly 1 wk after infection with either virus variant [from 2.5 (SD 0.3) to 1.4 (SD 0.1) in severe and from 2.4 (SD 0.4) to 2.1 (SD 0.3) in mild myocarditis], being significantly lower in the severe than mild myocarditis. CFR remained low in severe myocarditis 2 wk after infection. Fractional shortening decreased to the same levels 1 wk after infection with either virus variant [from 0.54 (SD 0.02) to 0.43 (SD 0.03) in severe and from 0.51 (SD 0.03) to 0.44 (SD 0.02) in mild myocarditis, P < 0.05]. However, 2 wk after infection, mice with severe myocarditis had enlarged left ventricles and lower fractional shortening [0.31 (SD 0.03)] than mice with mild myocarditis [0.47 (SD 0.02), P < 0.01]. In conclusion, CFR measured with TTDE is reduced in coxsackievirus myocarditis in mice. Low CFR is associated with progressive heart failure, indicating that dysfunction of coronary microcirculation is a determinant of poor outcome in viral myocarditis.

Cardiomyocyte Apoptosis after Antiviral WIN 54954 Treatment in Murine Coxsackievirus B3 Myocarditis

Objective : Cardiomyocyte apoptosis (CA) is known to occur in experimental coxsackievirus B3 (CVB3) myocarditis. However, the mechanisms of CA induction are not well known. In this study we investigate the role of direct viral induction of CA in CVB3 myocarditis. Design : A/J mice were infected with the Woodruff strain of CVB3 and treated with WIN 54954 for 5 days thereafter. WIN 54954, a compound that inhibits early events of picornavirus infection, is known to dramatically reduce mortality in murine CVB3 myocarditis without abrogating systemic or myocardial inflammation. Presence of viral RNA ( in situ hybridization), CA (TUNEL method) and histopathology were studied in transverse ventricular sections at day 7 post infection ( n = 8 treated and n = 8 non-treated). Results : The proportion of cardiomyocytes containing viral RNA was 89% lower in WIN 54954 treated mice when compared with non-treated mice (0.29 - 0.56% vs 2.76 - 1.65%, p = 0.003). Treatment also reduced the amount of CA by 52% compared with non-treated mice (0.20 - 0.06% vs 0.42 - 0.06%, p < 0.001). The reduction of CA by WIN treatment did not result in any increase of necrosis, in fact treatment reduced the area of necrotic lesions by 77% (2.51 - 1.64% vs 11.10 - 8.76%, p = 0.028). Conclusion : Taking the results of the reduced CA, necrosis and viral RNA with no effect on inflammation into account, our findings suggest the importance of direct viral effect in cardiomyocyte damage by both apoptosis and necrosis in CVB3 myocarditis.

Expression and distribution of the receptors for coxsackievirus B3 during fetal development of the Balb/c mouse and of their brain cells in culture

This study was designed mainly to determine the relationships between the expression and distribution of the cellular receptor proteins for coxsackievirus B3 (CVB3) and susceptibility of mouse brain cells during fetal development of Balb/c mice. Immunoblot analysis of fetal extracts demonstrated that the CVB3 receptor proteins were first expressed at day 14 of the fetal stage, and that maximal expression of the cellular receptor occurred at near term or newborn stage. Results also suggested that newborn mouse brain tissue expressed much larger quantities of viral receptor proteins, compared to other tissues. In vitro studies showed that both mouse neurons and astrocytes could be infected by two CVB3 strains, pantropic CVB3 Nancy strain (CVB3N) and myocardiotropic CVB3 Woodruff strain (CVB3W). CVB3N, however, replicated and grew to high titer in primary astrocyte cultures and in primary neuron cultures, whereas, primary astrocyte cultures were relatively resistant to CVB3W. Virus binding assays revealed that CVB3N bound faster and in greater amounts to mouse brain cells than CVBW. These two virus strains, however, were found to share the same receptor specificity by virus competition assays. The number of virus binding sites for CVB3 on newborn mouse brain cells was approximately 1.8 × 10 4 per cell. The data suggested that preferential expression of the cellular receptors on newborn mouse brain cells may be related to their high susceptibilities to CVB3 infection.