Angel S. Galabov

Virucidal activity of a new hand disinfectant with reduced ethanol content: comparison with other alcohol-based formulations

Summary A new formula with reduced ethanol content (55%) in combination with 10% propan-1-ol, 5.9% propan-1.2-diol, 5.7% butan-1.3-diol and 0.7% phosphoric acid exhibited a broad spectrum of virucidal activity. In quantitative suspension tests, with and without protein load, this formulation reduced the infectivity titre of seven enveloped (influenza A and B, herpes simplex 1 and 2, bovine corona, respiratory syncytial, vaccinia, hepatitis B, bovine viral diarrhoea) and four non-enveloped (hepatitis A, polio, rota, feline calici) viruses >103-fold within 30s. In comparative testing, only 95% ethanol showed similar levels of activity. In fingerpad tests, the formulation produced a log10 reduction factor of the titre of poliovirus type 1 (Sabin) of 3.04 in 30s compared with 1.32 by 60% propan-2-ol. Testing against feline calicivirus produced a log10 reduction factor of 2.38 by the test formulation; in contrast, the log10 reduction factors with 70% ethanol and 70% propan-1-ol were 0.68 and 0.70, respectively.

Rimantadine and oseltamivir demonstrate synergistic combination effect in an experimental infection with type A (H3N2) influenza virus in mice

We studied the combination effect of rimantadine hydrochloride and oseltamivir phosphate on mice infected with influenza A/Aichi/2/68 (H3N2) virus. Compounds were simultaneously administered in a 5-day-treatment course, starting 4 h before intranasal infection with 10 or 20 viral 50% mouse lethal doses. Initially, we tested combinations of oseltamivir (0.05, 0.1 and 0.2 mg/kg/day) and rimantadine (2.5, 5.0 and 7.5 mg/kg/day). Significant differences were recorded between combinationtreated groups, and groups with separately applied compounds and the placebo group, such as: protection index of oseltamivir with 5.0 or 7.5 mg/kg rimantadine varied between 34–41% and 43–87%, respectively, whereas the individual effects of oseltamivir, 5 mg/kg of rimantadine and 7.5 mg/kg of rimantadine were 0–10%, 0% and 18.7–29.6%, respectively; mean survival time in combination-treated groups was lengthened by 3.1–6.9 days, in oseltamivir groups by 0–1.9 days, and in rimantadine groups by 0.8–1.3 days at 5 mg/kg and 2.6–3.2 days at 7.5 mg/kg. The three-dimensional method of Prichard and Shipman characterized the combination effect as synergistic. Further, we studied the activity of 0.05 mg/kg/day of oseltamivir combined with 5 mg/kg of rimantadine. Lung virus titre in Madin Darby canine kidney cells, lung index and consolidation score proved the high effectiveness of the combination. When compared with the placebo group, a 2.8 log10 lower titre of 50% cell culture infectious dose (CCID50) was recorded in the combination-treated group at 48–60 h post-infection (the peak of lung virus growth). This is in contrast to the 0.1–1.0 log10 and 1.1–1.4 log10 reduction in CCID50 titre observed in the oseltamivir and rimantadine groups, respectively. These data emphasize the high anti-influenza A potential of the combination.

Oncolytic Rat Parvovirus H-1PV, a Candidate for the Treatment of Human Lymphoma: In Vitro and In Vivo Studies

The incidence of lymphomas developing in both immunocompetent and immunosuppressed patients continues to steadily increase worldwide. Current chemotherapy and immunotherapy approaches have several limitations, such as severe side toxicity and selection of resistant cell variants. Autonomous parvoviruses (PVs), in particular the rat parvovirus H-1PV, have emerged as promising anticancer agents. Although it is apathogenic in humans, H-1PV has been shown to infect and suppress various rat and human tumors in animal models. In this study, we demonstrate the capacity of H-1PV for efficiently killing, through necrosis, cell cultures originating from Burkitts lymphoma (BL), while sparing normal B lymphocytes. The cytotoxic effect was generally accompanied by a productive H-1PV infection. Remarkably, parvovirus-based monotherapy efficiently suppressed established BL at an advanced stage in a severe combined immunodeficient (SCID) mouse model of the disease. The data show for the first time that an oncolytic parvovirus deserves further consideration as a potential tool for the treatment of some non-Hodgkin B-cell lymphomas, including those resistant to apoptosis induction by rituximab.

Identification of Glycosylated Sites in Rapana Hemocyanin by Mass Spectrometry and Gene Sequence, and Their Antiviral Effect

Molluscan hemocyanins (Hcs) have recently received particular interest due to their significant immunostimulatory properties. This is mainly related to their high carbohydrate content and specific monosaccharide composition. We have now analyzed the oligosaccharides and the carbohydrate linkage sites of the Rapana venosa hemocyanin (RvH) using different approaches. We analyzed a number of glycopeptides by LC/ESI-MS/MS and identified the sugar chains and peptide sequences of 12 glycopeptides. Additionally, the potential carbohydrate linkage sites of 2 functional units, RvH-b and RvH-c, were determined by gene sequence analysis. Only RvH-c shows a potential N-glycosylation site. During this study, we discovered a highly conserved linker-intron, separating the coding exons of RVH-b and RvH-c. Following reports on antiviral properties from arthropod hemocyanin, we conducted a preliminary study of the antiviral activity of RvH and the functional units RvH-b and RvH-c. We show that the glycosylated FU RvH-c has antiviral properties against the respiratory syncytial virus (RSV), whereas native RvH and the nonglycosylated FU RvH-b have not. This is the first report of the fact that also molluscan hemocyanin functional units possess antiviral activity.

Cinnamoyl- and hydroxycinnamoyl amides of glaucine and their antioxidative and antiviral activities.

The aporphine alkaloid glaucine has been converted into 3-aminomethylglaucine and its free amino group has been linked to cinnamic, ferulic, sinapic, o-, and p-coumaric acids. The antioxidative potential of the synthesized amides was studied against DPPH(*) test. All of the tested compounds demonstrated higher radical scavenging activity than glaucine and 3-aminomethylglaucine, and lower antioxidative effect than the free hydroxycinnamic acids. The newly synthesized compounds were tested in vitro for antiviral activity against viruses belonging to different taxonomic groups.

Effect of vitamin E supplementation on lipid peroxidation in blood and lung of influenza virus infected mice.

The influenza virus infection (A/Aichi/2/68) was associated with development of oxidative stress in lung and blood of mice, accompanied by an increase in levels of lipid peroxidation products (conjugated dienes and total malondialdehyde) and a decrease in endogenous amounts of natural antioxidant vitamin E. These effects were most pronounced on the 5th day after virus inoculation, in comparison with those on the 7th. Supplementation of mice with exogenous vitamin E before virus inoculation lead to lung and blood protection against lipid peroxidation. A marked decrease in lipid peroxidation products and an increase in vitamin E content was established in blood and lung on the 5th and 7th day after virus inoculation. The stabilizing effect of vitamin E is dose-dependent in blood and dose-independent in lung, and was most pronounced on the 5th day after virus inoculation in comparison with the 7th day.

Anti-herpesvirus activities of Pseudomonas sp. S-17 rhamnolipid and its complex with alginate.

The rhamnolipid biosurfactant PS-17 and its complex with the polysaccharide alginate, both produced by the Pseudomonas sp. S-17 strain, were studied for their antiviral activity against herpes simplex virus (HSV) types 1 and 2. They significantly inhibited the herpesvirus cytopathic effect (CPE) in the Madin-Darby bovine kidney (MDBK) cell line. The investigations were carried out according to the CPE inhibition assay protocol. The suppressive effect of the compounds on HSV replication was dose-dependent and occurred at concentrations lower than the critical micelle concentration of the surfactant. The 50% inhibitory concentration (IC50) of rhamnolipid PS-17 was 14.5 μg/ml against HSV-1 and 13 μg/ml against HSV-2. The IC50 values of the complex were 435 μg/ml for HSV-1 and 482 μg/ml for HSV-2. The inhibitory effects of the substances were confirmed by measuring the infectious virus yields with the multicycle virus growth experimental design as well: ∆log CCID50 of 1.84−2.0 against the two types of herpes simplex viruses by rhamnolipid PS-17 (20 μg/ml), and a strong reduction of the HSV-2 virus yield under the effect of the alginate complex at a concentration of 450 μg/ml. The results indicate that rhamnolipid PS-17 and its alginate complex may be considered as promising substances for the development of anti-herpetic compounds.

Y-Chromosome Diversity in Modern Bulgarians: New Clues about Their Ancestry

To better define the structure and origin of the Bulgarian paternal gene pool, we have examined the Y-chromosome variation in 808 Bulgarian males. The analysis was performed by high-resolution genotyping of biallelic markers and by analyzing the STR variation within the most informative haplogroups. We found that the Y-chromosome gene pool in modern Bulgarians is primarily represented by Western Eurasian haplogroups with ∼ 40% belonging to haplogroups E-V13 and I-M423, and 20% to R-M17. Haplogroups common in the Middle East (J and G) and in South Western Asia (R-L23*) occur at frequencies of 19% and 5%, respectively. Haplogroups C, N and Q, distinctive for Altaic and Central Asian Turkic-speaking populations, occur at the negligible frequency of only 1.5%. Principal Component analyses group Bulgarians with European populations, apart from Central Asian Turkic-speaking groups and South Western Asia populations. Within the country, the genetic variation is structured in Western, Central and Eastern Bulgaria indicating that the Balkan Mountains have been permeable to human movements. The lineage analysis provided the following interesting results: (i) R-L23* is present in Eastern Bulgaria since the post glacial period; (ii) haplogroup E-V13 has a Mesolithic age in Bulgaria from where it expanded after the arrival of farming; (iii) haplogroup J-M241 probably reflects the Neolithic westward expansion of farmers from the earliest sites along the Black Sea. On the whole, in light of the most recent historical studies, which indicate a substantial proto-Bulgarian input to the contemporary Bulgarian people, our data suggest that a common paternal ancestry between the proto-Bulgarians and the Altaic and Central Asian Turkic-speaking populations either did not exist or was negligible.

Interferon γ improves the vaccination potential of oncolytic parvovirus H-1PV for the treatment of peritoneal carcinomatosis in pancreatic cancer

Oncolytic viruses with their capacity to specifically replicate in and kill tumor cells emerged as a novel class of cancer therapeutics. Rat oncolytic parvovirus (H-1PV) was used to treat different types of cancer in preclinical settings and was lately successfully combined with standard gemcitabine chemotherapy in treating pancreatic ductal adenocarcinoma (PDAC) in rats. Our previous work showed that the immune system and particularly the release of interferon-gamma (IFNγ) seem to mediate the anticancer effect of H-1PV in that model. Therefore, we reasoned that the therapeutic properties of H-1PV can be boosted with IFNγ for the treatment of late incurable stages of PDAC like peritoneal carcinomatosis. Rats bearing established orthotopic pancreatic carcinomas with peritoneal metastases were treated with a single intratumoral (i.t.) or intraperitoneal (i.p.) injection of 5x108 plaque forming units of H-1PV with or without concomitant IFNγ application. Intratumoral injection proved to be more effective than the intraperitoneal route in controlling the growth of both the primary pancreatic tumors and peritoneal carcinomatosis, accompanied by migration of virus from primary to metastatic deposits. Concomitant i.p. treatment of H-1PV with recIFNγ resulted in improved therapeutic effect yielding an extended animal survival, compared with i.p. treatment with H-1PV alone. IFNγ application enhanced the H-1PV-induced peritoneal macrophage and splenocyte responses against tumor cells while causing a significant reduction in the titers of H1-PV-neutralising antibodies in ascitic fluid. Thus, IFNγ co-application together with H-1PV might be considered as a novel therapeutic option to improve the survival of PDAC patients with peritoneal carcinomatosis.

Synthesis, antioxidative and antiviral activity of hydroxycinnamic acid amides of thiazole containing amino acid

The synthesis and the biological (antioxidant and antiviral) activities of novel hydroxycinnamic acid amides of a thiazole containing TFA.valine-4-carboxylic acid ethyl ester are reported. The amides have been synthesized from p-coumaric, ferulic and sinapic acids with the corresponding TFA.valine-thiazole-4-carboxylic acid ethyl ester using the coupling reagent N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 4-(dimethylamino) pyridine (DMAP) as a catalyst. The antioxidant properties of the newly synthesized amides have been studied for then antioxidative activity using 2,2-diphenyl-1-picrylhydrazyl (DPPH)* test. The newly synthesized compounds have been tested against the replication in vitro of influenza virus A (H3N2) and human herpes virus 1 and 2 (HSV-1 and HSV-2).