The combination of pleconaril, rupintrivir, and remdesivir efficiently inhibits enterovirus infections in vitro, delaying the development of drug-resistant virus variants.

Enteroviruses are a significant global health concern, causing a spectrum of diseases from the common cold to more severe conditions like hand-foot-and-mouth disease, meningitis, myocarditis, pancreatitis, and poliomyelitis. Current treatment options for these infections are limited, underscoring the urgent need for effective therapeutic strategies. To find better treatment option we analyzed toxicity and efficacy of 12 known broad-spectrum anti-enterovirals both individually and in combinations against different enteroviruses in vitro. We identified several novel, synergistic two-drug and three-drug combinations that demonstrated significant inhibition of enterovirus infections in vitro. Specifically, the triple-drug combination of pleconaril, rupintrivir, and remdesivir exhibited remarkable efficacy against echovirus (EV) 1, EV6, EV11, and coxsackievirus (CV) B5, in human lung epithelial A549 cells. This combination surpassed the effectiveness of single-agent or dual-drug treatments, as evidenced by its ability to protect A549 cells from EV1-induced cytotoxicity across seven passages. Additionally, this triple-drug cocktail showed potent antiviral activity against EV-A71 in human intestinal organoids. Thus, our findings highlight the therapeutic potential of the pleconaril-rupintrivir-remdesivir combination as a broad-spectrum treatment option against a range of enterovirus infections. The study also paves the way towards development of strategic antiviral drug combinations with virus family coverage and high-resistance barriers.

Effect of castalagin against HSV-1 infection in newborn mice.

We tested in vivo anti-herpetic effect of castalagin, an ellagitannin compound, extracted from pedunculate oak (Quercus robur). Previous investigations found that castalagin possesses a strong inhibitory effect in vitro against HSV-1/2 equal to acyclovir (ACV). It is also effective against ACV-resistant mutants and shows a synergistic effect with ACV. We study castalagin's activity towards HSV-1 infection in newborn mice. Acute toxicity determination in mice showed LD50 value of 295 mg/kg. Prolonged toxicity was also constructed. Castalagin manifested a marked activity against HSV-1 (LD90/0.02 ml) administered in 7-day course at 0.02 ml s.c. doses of 7.5 or 10 mg/kg (PI 57-58%). ACV course demonstrated a marked activity at 20 mg/kg. The selectivity ratio LD50/ED50 (295/7.5) could be accepted as ≥ 33.

Consecutive alternating administration as an effective anti-coxsackievirus B3 in vivo treatment scheme.

Although chemotherapy is generally indicated for treatment of enterovirus infections, antivirals are currently not used in clinical practice. The use of monotherapy is the main reason for this unfavourable state. This is related to the fact that enterovirus progeny consist of innumerable quasispecies, allowing the virus to develop drug resistance quickly. Here, we present a consecutive alternating administration (CAA) treatment scheme for combining enterovirus inhibitors. Applying the CAA approach with a combination of pleconaril (capsid binder), guanidine HCl (viral 2C inhibitor), and oxoglaucine (PI4KB inhibitor) (PGO) was found to be effective in the treatment of newborn mice infected with a massive inoculum (20 MLD50) of the coxsackievirus B3 cardiotropic Woodruff or neurotropic Nancy strain. In addition to preventing drug resistance, the CAA approach resulted in the parallel development of increased susceptibility to the compounds in the PGO combination. These observations demonstrate the therapeutic potential of the CAA approach for treatment of enterovirus infections.

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.

Anti-enteroviral activity of new MDL-860 analogues: Synthesis, in vitro/in vivo studies and QSAR analysis.

A series of 60 nitrobenzonitrile analogues of the anti-viral agent MDL-860 were synthesized (50 of which are new) and evaluated for their activity against three types of enteroviruses (coxsackievirus B1, coxsackievirus B3 and poliovirus 1). Among them, six diaryl ethers (20e, 27e, 28e, 29e, 33e and 35e) demonstrated high in vitro activity (SI > 50) towards at least one of the tested viruses and very low cytotoxicity against human cells. Compound 27e possesses the broadest spectrum of activity towards all tested viruses in the same way as MDL-860 does. The most active derivatives (27e, 29e and 35e) against coxsackievirus B1 were tested in vivo in newborn mice experimentally infected with 20 MLD50 of coxsackievirus B1. Compound 29e showed promising in vivo activity (protection index 26% and 4 days lengthening of mean survival time). QSAR analysis of the substituent effects on the in vitro cytotoxicity (CC50) and anti-viral activity of the nitrobenzonitrile derivatives was carried out and adequate QSAR models for the anti-viral activity of the compounds against poliovirus 1 and coxsackievirus B1 were constructed.

Anti-Herpes Simplex Virus Type 1 Activity of Specially Selected Groups of Tannins.

Anti-herpes simplex virus (HSV-1) activity of 9 ellagitannins, including 6 natural compounds (castalin, vescalin, acutissimin A, epiacutissimins A and B, mongolicain) and 3 vescalagin synthetic derivatives (VgSBuSH, VgSOctSH, VgOMe), and 13 gallotannin-type compounds [Gal-01A, Gal-01B, Gal-02A, Gal-02B, Gal-03M, Gal-04A, Gal-04B, Gal-05M, Gal-07, Gal-08, Gal-09, Gal-11M (tannic acid), as well as Gal-12 (gallic acid), Gal-13 and Gal-14 (ellagic acid)] were examined in MDBK monolayer cell culture. Their antiviral activity was determined by the cytopathic effect (CPE) inhibition test and their cytotoxicity was evaluated through the neutral red uptake assay. In general, the series of ellagitannins showed a significantly stronger activity against HSV-1 replication than that of the gallotannins. Six of the tested ellagitannins manifested a well-pronounced activity: epiacutissimin B (selectivity index, SI>60.6), epiacutissimin A (SI>55.5), acutissimin A (SI>34.8), mongolicain (SI>32.5), VgSBuSH (SI>24.6) and VgOMe (SI>22.0). Four gallotannin-type compounds inhibited the replication of HSV-1 at a lower but still significant extent: Gal-04B (SI>35.7), Gal-04A (SI>28.5), Gal-11M (tannic acid) (SI>25) and Gal-05M (SI=15.6).

Tailoring acyclovir prodrugs with enhanced antiviral activity: rational design, synthesis, human plasma stability and in vitro evaluation.

Bile acid prodrugs have served as a viable strategy for refining the pharmaceutical profile of parent drugs through utilizing bile acid transporters. A series of three ester prodrugs of the antiherpetic drug acyclovir (ACV) with the bile acids cholic, chenodeoxycholic and deoxycholic were synthesized and evaluated along with valacyclovir for their in vitro antiviral activity against herpes simplex viruses type 1 and type 2 (HSV-1, HSV-2). The in vitro antiviral activity of the three bile acid prodrugs was also evaluated against Epstein-Barr virus (EBV). Plasma stability assays, utilizing ultra-high performance liquid chromatography coupled with tandem mass spectrometry, in vitro cytotoxicity and inhibitory experiments were conducted in order to establish the biological profile of ACV prodrugs. The antiviral assays demonstrated that ACV-cholate had slightly better antiviral activity than ACV against HSV-1, while it presented an eight-fold higher activity with respect to ACV against HSV-2. ACV-chenodeoxycholate presented a six-fold higher antiviral activity against HSV-2 with respect to ACV. Concerning EBV, the highest antiviral effect was demonstrated by ACV-chenodeoxycholate. Human plasma stability assays revealed that ACV-deoxycholate was more stable than the other two prodrugs. These results suggest that decorating the core structure of ACV with bile acids could deliver prodrugs with amplified antiviral activity.

Synthesis and anti-enterovirus activity of new analogues of MDL-860.

A series of twelve novel compounds, analogues of antiviral agent MDL-860 were synthesized and their antiviral activity was evaluated in vitro against enteroviruses poliovirus 1 (PV1), Coxsackieviruses B1 (CVB1) and Coxsackieviruses B3 (CVB3). Compounds 14, 24 and 25 manifested strong antiviral effects against CVB1 and PV1 (SI values of 405 and 118 for CVB1 and PV1 respectively). In contrast to the wide anti-enteroviral activity of MDL-860, these three compounds were inactive against CVB3. Compounds 14, 24 and 25 along with MDL-860 were tested in vivo in mice infected with CVB1. Marked protective effects of compounds 14 and 24 were established, PI values of 50% and 33.3%, respectively. In addition, almost all of the tested compounds manifested very low toxicity.

Cycluridine: A novel antiviral effective against flaviviruses.

This review describes the contemporary state of research for antivirals effective against flaviviruses, especially focusing on inhibitors of the pestivirus causative agent of bovine viral diarrhoea virus. We highlight cycluridine, an originally synthesized Mannich's base [a tetrahydro-2(1H)-pyrimidinones derivative], as a highly effective antiviral possessing a strong inhibitory effect on bovine viral diarrhoea virus replication. Cycluridine was active against replication of a wide variety of bovine viral diarrhoea virus strains in cell cultures. The drug-sensitive period in the bovine viral diarrhoea virus replication cycle included the latent period and the exponential phase; a 90-min delay in the peak of viral RNA synthesis was observed. Cycluridine administered orally manifested a pronounced protective effect in calves with natural mucosal disease/viral diarrhoea and calves experimentally infected with bovine viral diarrhoea virus. Its magnitude of activity and selectivity places cycluridine in the lead among all known substances with anti- bovine viral diarrhoea virus activity. Additionally, cycluridine applied subcutaneously showed anti-tick-born encephalitis virus activity, manifesting a marked protective effect in mice infected with tick-born encephalitis virus. Cycluridine could be a prospective antiviral in veterinary and medical practice for the treatment of bovine viral diarrhoea virus and other flavivirus infections.

Allosteric Regulation of Phosphatidylinositol 4-Kinase III Beta by an Antipicornavirus Compound MDL-860.

MDL-860 is a broad-spectrum antipicornavirus compound discovered in 1982 and one of the few promising candidates effective in in vivo virus infection. Despite the effectiveness, the target and the mechanism of action of MDL-860 remain unknown. Here, we have characterized antipoliovirus activity of MDL-860 and identified host phosphatidylinositol-4 kinase III beta (PI4KB) as the target. MDL-860 treatment caused covalent modification and irreversible inactivation of PI4KB. A cysteine residue at amino acid 646 of PI4KB, which locates at the bottom of a surface pocket apart from the active site, was identified as the target site of MDL-860. This work reveals the mechanism of action of this class of PI4KB inhibitors and offers insights into novel allosteric regulation of PI4KB activity.

Inhibitory Effect of Abitylguanide on Adenovirus Replication.

N'N'-anhydro-bis(ß-hydroxy-ethyl)biguanide.HCl (abitylguanide) demonstrated a marked inhibitory effect on replication in cell cultures of a broad spectrum of human adenoviruses both standard laboratory strains and strains isolated from epidemic keratoconjunctivitis patients. The strongest inhibitory activity was found in viruses belonging to subgroup C (Rosen's subgroup III). The compound susceptible period of human adenovirus 5 replication in primary cell cultures of human embryo kidney cells included the total replication cycle and was especially pronounced during the exponential phase of the virus growth curve. Electron microscopy established that the compound decreased the percentage of cells in which mature or empty virions with the characteristic nuclear localization were observed; a complete absence of paracrystals was registered and the number of cells with virus particles arranged in crystals in the nucleoplasm was strongly decreased. Abitylguanide can be considered as a ligand of adenovirus capsid protein(s).

The Oncolytic Virotherapy Era in Cancer Management: Prospects of Applying H-1 Parvovirus to Treat Blood and Solid Cancers.

Non-Hodgkin lymphoma (NHL) and leukemia are among the most common cancers worldwide. While the treatment of NHL/leukemia of B-cell origin has much progressed with the introduction of targeted therapies, few treatment standards have been established for T-NHL/leukemia. As presentation in both B- and T-NHL/leukemia patients is often aggressive and as prognosis for relapsed disease is especially dismal, this cancer entity poses major challenges and requires innovative therapeutic approaches. In clinical trials, oncolytic viruses (OVs) have been used against refractory multiple myeloma (MM). In preclinical settings, a number of OVs have demonstrated a remarkable ability to suppress various types of hematological cancers. Most studies dealing with this approach have used MM or B- or myeloid-cell-derived malignancies as models. Only a few describe susceptibility of T-cell lymphoma/leukemia to OV infection and killing. The rat H-1 parvovirus (H-1PV) is an OV with considerable promise as a novel therapeutic agent against both solid tumors (pancreatic cancer and glioblastoma) and hematological malignancies. The present perspective article builds on previous reports of H-1PV-driven regression of Burkitt's lymphoma xenografts and on unpublished observations demonstrating effective killing by H-1PV of cells from CHOP-resistant diffuse large B-cell lymphoma, cutaneous T-cell lymphoma, and T-cell acute lymphoblastic leukemia. On the basis of these studies, H-1PV is proposed for use as an adjuvant to (chemo)therapeutic regimens. Furthermore, in the light of a recently completed first parvovirus clinical trial in glioblastoma patients, the advantages of H-1PV for systemic application are discussed.

Virus inactivation under the photodynamic effect of phthalocyanine zinc(II) complexes.

Various metal phthalocyanines have been studied for their capacity for photodynamic effects on viruses. Two newly synthesized water-soluble phthalocyanine Zn(II) complexes with different charges, cationic methylpyridyloxy-substituted Zn(II)- phthalocyanine (ZnPcMe) and anionic sulfophenoxy-substituted Zn(II)-phthalocyanine (ZnPcS), were used for photoinactivation of two DNA-containing enveloped viruses (herpes simplex virus type 1 and vaccinia virus), two RNA-containing enveloped viruses (bovine viral diarrhea virus and Newcastle disease virus) and two nude viruses (the enterovirus Coxsackie B1, a RNA-containing virus, and human adenovirus 5, a DNA virus). These two differently charged phthalocyanine complexes showed an identical marked virucidal effect against herpes simplex virus type 1, which was one and the same at an irradiation lasting 5 or 20 min (Δlog=3.0 and 4.0, respectively). Towards vaccinia virus this effect was lower, Δlog=1.8 under the effect of ZnPcMe and 2.0 for ZnPcS. Bovine viral diarrhea virus manifested a moderate sensitivity to ZnPcMe (Δlog=1.8) and a pronounced one to ZnPcS at 5- and 20-min irradiation (Δlog=5.8 and 5.3, respectively). The complexes were unable to inactivate Newcastle disease virus, Coxsackievirus B1 and human adenovirus type 5.

Characterization of genomic changes in the cervical pre-cancerous lesions and tumors induced by different types of human papillomaviruses.

Cervical carcinoma is the second most common malignancy among women in both incidence and mortality. Although much is known about the etiology and treatment of cervical cancer, the role of genetic alterations in the multistep pathway of cervical tumorigenesis is largely unknown. The aim of this study was to characterize the genomic changes in the cervical pre-cancerous lesions and tumors, induced by different types of human papillomaviruses. In this research was used the BlueGnome CytoChip oligo 2 × 105 K microarray for whole-genome oligo-array CGH. Microarray CGH analysis of 40 specimens was carried out-12 specimens from patients with early-stage squamous cell carcinomas; 19 specimens from patients with mild to moderate dysplasia and 9 with severe dysplasia. First we performed microarray CGH analysis of five DNA pools which contained the DNA from homogeneous groups of patients. The results revealed presence of micro chromosomal aberrations in chromosome region 14q11.2. According to the genome database these aberrations represent polymorphisms. Microarray analysis of DNA from 9 separate carcinoma lesions revealed a total of 26 aberrations in 14 chromosomes of nine patients. Our results showed the advantages of high-resolution chips in the clinical diagnosis of patients with cancerous and precancerous lesions caused by viral infection with HPV, but also highlight the need for extensive population studies revealing the molecular nature and clinical significance of different CNVs and the creation of detailed maps of variations in the Bulgarian population. This would facilitate extremely precise interpretation of specific genomic imbalances in the clinical aspect.

Effect of consecutive alternating administration (CAA) of a triple anti-enteroviral combination on Coxsackievirus B1 neuroinfection in mice.

Currently, clinically effective antivirals for use in the treatment of enteroviral (EV) infections do not exist. The main reason is the development of drug resistance, the principle obstacle in the development of EV infection chemotherapy, based til now on monotherapy. The most important achievement of our previous studies was the development of a novel scheme for in vivo application of a triple combination of EV inhibitors with different modes of action against Coxsackievirus B (CVB) infections in mice. It consists of consecutive alternating administration (CAA) of the substances in the combination. Here, we tested the effect of the triple combination pleconaril, guanidine-HCl, and oxoglaucine (PGO) via CAA in newborn mice infected with a neurotropic strain of CVB1 (20 LD50 per mouse). This combination manifested a considerable protective effect with pleconaril doses of 25-200mg/kg: it decreased mortality rate (protection index, PI, between 31.3% and 67.7%) and increased mean survival time (MST) by 4-6days. Pleconaril monotherapy demonstrated activity similar to that of PGO via CAA, as measured by PI values, but MST values were slightly lower. However, it also greatly suppressed growth of infected suckling mice, especially at 200mg/kg. This toxic effect was avoided with CAA of PGO at pleconaril doses of 25-100mg/kg. Pleconaril monotherapy administered every 3days was ineffective. The PGO with CAA treatment course decreased infectious virus content, whereas pleconaril monotherapy did not. Analysis of drug-sensitivity in brain samples from CVB1 infected mice, based on IC50 (50% inhibitory concentration) values from cell culture experiments, showed that the CAA course counteracted the development of drug resistance to pleconaril and oxoglaucine in the triple PGO combination and increased drug sensitivity. In contrast, pleconaril and oxoglaucine monotherapies resulted in drug resistance. This data clearly proves the effectiveness of the proposed novel approach-the CAA treatment course-for combined application of EV replication inhibitors.

Phosphatidylinositol 4-kinase III beta is the target of oxoglaucine and pachypodol (Ro 09-0179) for their anti-poliovirus activities, and is located at upstream of the target step of brefeldin A.

In recent years, phosphatidylinositol 4-kinase III beta (PI4KB) has emerged as a conserved target of anti-picornavirus compounds. In the present study, PI4KB was identified as the direct target of the plant-derived anti-picornavirus compounds, oxoglaucine and pachypodol (also known as Ro 09-0179). PI4KB was also identified as the target via which pachypodol interferes with brefeldin A (BFA)-induced Golgi disassembly in non-infected cells. Oxysterol-binding protein (OSBP) inhibitor also has interfering activity against BFA. It seems that this interference is not essential for the anti-poliovirus (PV) activities of BFA and PI4KB/OSBP inhibitors. BFA inhibited early to late phase PV replication (0 to 6 hr postinfection) as well as PI4KB inhibitor, but with some delay compared to guanidine hydrochloride treatment. In contrast with PI4KB/OSBP inhibitors, BFA inhibited viral nascent RNA synthesis, suggesting that BFA targets some step of viral RNA synthesis located downstream of the PI4KB/OSBP pathway in PV replication. Our results suggest that PI4KB is a major target of anti-picornavirus compounds identified in vitro for their anti-picornavirus activities and for some uncharacterized biological phenomena caused by these compounds, and that BFA and PI4KB/OSBP inhibitors synergistically repress PV replication by targeting distinct steps in viral RNA replication.

Combination activity of neuraminidase inhibitor oseltamivir and α-tocopherol in influenza virus A (H3N2) infection in mice.

BACKGROUND: Influenza is a highly contagious viral infection of the respiratory system. To attack two processes involved in flu pathogenesis-viral replication in the infected body and oxidative damages, we studied the combination effect of neuraminidase inhibitor oseltamivir and antioxidant α-tocopherol in experimental model of influenza. METHODS: After inoculation of albino mice with 10 MLD50 (50% mouse lethal dose) of influenza virus A/Aichi/2/68 (H3N2), oseltamivir was applied orally at three doses, 2.5 mg/kg, 1.25 mg/kg, and 0.625 mg/kg, for five days post infection. α-Tocopherol (120 mg/kg, in sunflower oil) was administered intraperitoneally. Three schemes of α-tocopherol five-day course were tested: onset five or two days before infection, or on the virus inoculation day. RESULTS: Strongly dose-dependent augmented antiviral effect of the combination α-tocopherol and 0.625 mg/kg oseltamivir was demonstrated when α-tocopherol was administered simultaneously with oseltamivir: a pronounced decrease in mortality rate (a 78% protection), and a lengthening of mean survival time by 3.2-4 days. Lung parameters showed a substantial decrease in infectious virus content (Δ logs = 3.8/4.1) and a marked diminishment of lung index and pathology. Combination α-tocopherol with 1.25 mg/kg oseltamivir manifested a marked protective effect, but the effect on lung parameters was less. The combination effect of α-tocopherol with 2.5 mg/kg oseltamivir did not surpass the monotherapeutic effect of oseltamivir. When α-tocopherol was applied in courses starting five or two days before infection, its combination with oseltamivir was ineffective. CONCLUSIONS: Evidently, α-tocopherol could be considered as prospective component of influenza therapy in combination with oseltamivir.

Antiviral Combination Approach as a Perspective to Combat Enterovirus Infections.

Human enteroviruses distributed worldwide are causative agents of a broad spectrum of diseases with extremely high morbidity, including a series of severe illnesses of the central nervous system, heart, endocrine pancreas, skeleton muscles, etc., as well as the common cold contributing to the development of chronic respiratory diseases, including the chronic obstructive pulmonary disease. The above mentioned diseases along with the significantly high morbidity and mortality in children, as well as in the high-risk populations (immunodeficiencies, neonates) definitely formulate the chemotherapy as the main tool for the control of enterovirus infections. At present, clinically effective antivirals for use in the treatment of enteroviral infection do not exist, in spite of the large amount of work carried out in this field. The main reason for this is the development of drug resistance. We studied the process of development of resistance to the strongest inhibitors of enteroviruses, WIN compounds (VP1 protein hydrophobic pocket blockers), especially in the models in vivo, Coxsackievirus B (CV-B) infections in mice. We introduced the tracing of a panel of phenotypic markers (MIC50 value, plaque shape and size, stability at 50℃, pathogenicity in mice) for characterization of the drug-mutants (resistant and dependent) as a very important stage in the study of enterovirus inhibitors. Moreover, as a result of VP1 RNA sequence analysis performed on the model of disoxaril mutants of CVB1, we determined the molecular basis of the drug-resistance. The monotherapy courses were the only approach used till now. For the first time in the research for anti-enterovirus antivirals our team introduced the testing of combination effect of the selective inhibitors of enterovirus replication with different mode of action. This study resulted in the selection of a number of very effective in vitro double combinations with synergistic effect and a broad spectrum of sensitive enteroviruses. The most prospective attainment in our examinations in this field was the development of a novel scheme for the combined application of anti-enteroviral substances in coxsackievirus B1 neuroinfection in newborn mice. It consisted of a consecutive, alternating and non simultaneous administration of the substances in the combination. The triple combination - disoxaril- guanidine. HCl-oxoglaucine (DGO) showed a high effectiveness expressed in the marked reduction of the mortality rate in infected mice as compared both to the placebo group, and to the partner compounds used alone every day, and to the same combination applied simultaneously every day. The studies of the drug sensitivity of viral brain isolates from mice treated with DGO combination showed not only preserved, but even increased sensitivity to the drugs included in the combination. Obviously, the consecutive alternating administration of anti-enteroviral substances hinders the occurrence of drug-resistance in the course of the experimental enteroviral infections in mice.