Design and characterization of BSA-mycophenolic acid nanocomplexes: Antiviral activity exploration.

The interactions between bovine serum albumin (BSA) and mycophenolic acid (MPA) were investigated in silico through molecular docking and in vitro, using fluorescence spectroscopy. Dynamic light scattering and scanning electron microscopy were used to figure out the structure of MPA-Complex (MPA-C). The binding affinity between MPA and BSA was determined, yielding a Kd value of (12.0 ± 0.7) µM, and establishing a distance of 17 Å between the BSA and MPA molecules. The presence of MPA prompted protein aggregation, leading to the formation of MPA-C. The cytotoxicity of MPA-C and its ability to fight Junín virus (JUNV) were tested in A549 and Vero cell lines. It was found that treating infected cells with MPA-C decreased the JUNV yield and was more effective than free MPA in both cell line models for prolonged time treatments. Our results represent the first report of the antiviral activity of this type of BSA-MPA complex against JUNV, as assessed in cell culture model systems. MPA-C shows promise as a candidate for drug formulation against human pathogenic arenaviruses.

Experimental Aspects Suggesting a "Fluxus" of Information in the Virions of Herpes Simplex Virus Populations.

Our perspective on nature has changed throughout history and at the same time has affected directly or indirectly our perception of biological processes. In that sense, the "fluxus" of information in a viral population arises a result of a much more complex process than the encoding of a protein by a gene, but as the consequence of the interaction between all the components of the genome and its products: DNA, RNA, and proteins and its modulation by the environment. Even modest "agents of life" like viruses display an intricate way to express their information. This conclusion can be withdrawn from the huge quantity of data furnished by new and potent technologies available now to analyze viral populations. Based on this premise, evolutive processes for viruses are now interpreted as a simultaneous and coordinated phenomenon that leads to global (i.e., not gradual or 'random') remodeling of the population. Our system of study involves the modulation of herpes simplex virus populations through the selective pressure exerted by carrageenans, natural compounds that interfere with virion attachment to cells. On this line, we demonstrated that the passaging of virus in the presence of carrageenans leads to the appearance of progeny virus phenotipically different from the parental seed, particularly, the emergence of syncytial (syn) variants. This event precedes the emergence of mutations in the population which can be readily detected five passages after from the moment of the appearance of syn virus. This observation can be explained taking into consideration that the onset of phenotypic changes may be triggered by "environmental-sensitive" glycoproteins. These "environmental-sensitive" glycoproteins may act by themselves or may transmit the stimulus to "adapter" proteins, particularly, proteins of the tegument, which eventually modulate the expression of genomic products in the "virocell." The modulation of the RNA network is a common strategy of the virocell to respond to environmental changes. This "fast" adaptive mechanism is followed eventually by the appearance of mutations in the viral genome. In this paper, we interpret these findings from a philosophical and scientific point of view interconnecting epigenetic action, exerted by carragenans from early RNA network-DNA interaction to late DNA mutation. The complexity of HSV virion structure is an adequate platform to envision new studies on this topic that may be complemented in a near future through the analysis of the genetic dynamics of HSV populations.

In vitro and in vivo immunomodulatory activity of sulfated polysaccharides from red seaweed Nemalion helminthoides.

Water-soluble sulfated polysaccharides from the red seaweed Nemalion helminthoides: two xylomannan fractions (N3 and N4) and a mannan fraction (N6) were investigated to determine their in vitro and in vivo immunomodulatory activities. N3 and N4 induced in vitro proliferation of macrophages of the murine cell line RAW 264.7 and significantly stimulated the production of nitric oxide (NO) and cytokines (IL-6 and TNF-α) in the same cells, whereas this response was not observed with the mannan N6. The cytokine production was also stimulated by sulfated xylomannans in vivo in BALB/c mice inoculated intravenously with these polysaccharides. Remarkably, when mice were treated with N3 and N4 for 1 h before being infected with Herpes simplex virus type 2, they remained asymptomatic with no signs of disease. The in vitro and in vivo results suggest that sulfated xylomannans could be strong immunomodulators.

Chemical modifications of algal mannans and xylomannans: effects on antiviral activity.

The structures of two sulfated xylomannans extracted from the red alga Nemalion helminthoides were determined. These two fractions plus a sulfated mannan, isolated from the same alga and whose structure was previously reported, were subjected to chemical modification. The mannan was oversulfated with SO(3)-pyridine in dimethyl sulfoxide at 60 °C during two and three hours and the xylomannans were subjected to Smith degradation in order to eliminate xylose side-chains. Structural analysis of all derivatives was carried out by methylation analysis and (13)C NMR spectroscopy. Antiviral activity against herpes simplex virus type 1 and 2, and dengue virus type 2 of native and modified mannans and xylomannans was estimated. Anticoagulant effect of the active fractions was also determined.

Emergence of herpes simplex virus-1 syncytial variants with altered virulence for mice after selection with a natural carrageenan.

BACKGROUND: Antiviral therapy against herpes simplex virus based on sulfated polysaccharides, like carrageenans, represents a new alternative for genital herpes infections treatment and arises the concern about the appearance of resistant viral populations. METHODS: We characterized the F strain of herpes simplex virus-1 passaged in the presence of a natural carrageenan isolated from the red seaweed Gigartina skottbergii in view of the virulence for mice of isolated viral clones. RESULTS: Viral clones (syn14-1 and syn17-2) showed a syncytial phenotype and a mild resistance to carrageenan, heparin, acyclovir, and brivudine. Both clones were avirulent for BALB/c mice when inoculated intravaginally, whereas F strain produced high mortality. Attenuation correlated with low levels of TNF-[alpha], interleukin-6, and IFN-[gamma] in vaginal lavages although virus titers were similar to those obtained for F strain. On the contrary, they showed a marked virulence when inoculated intranasally leading to a generalized spreading of virus. CONCLUSIONS: Results confirm the hypothesis that selection of herpes simplex virus-1 with a carrageenan in vitro leads to the emergence of variants with a differential virulence when compared to the original virus. This finding should be addressed when an antiviral therapy against genital herpes infection employing a natural carrageenan is under consideration.

Sulfated mannans from the red seaweed Nemalion helminthoides of the South Atlantic.

Nemalion helminthoides, collected in the Argentine South Atlantic coast, was extracted with hot water and the crude product fractionated using cetrimide. The complexed material was subjected to fractional solubilization in solutions of increasing sodium chloride concentration and seven fractions were separated and analyzed. Structural analysis of the main fractions, those soluble in 3.0 and 4.0 M NaCl (yields 21.0% and 13.8%, respectively) and those insoluble in 4.0 M NaCl (yield 20.0%), indicated that this seaweed biosynthesizes (1-->3)-linked alpha-D-mannans that are sulfated at positions 4 and 6. Three mannan fractions comprising considerable amounts of xylose were also isolated in very low total yield (2.0%). The fractions that were soluble in 3.0 and 4.0 M NaCl showed low antiherpetic activity whereas this activity was considerable for the fraction solubilized in 2.0 M NaCl (yield 0.5%) which contained single stubs of beta-D-xylose. A xylan, soluble in cetrimide solution, containing (1-->3, 1-->4)-linked beta-D-xylose residues, was also isolated in minor amount.

Polysaccharides from Gracilaria corticata: sulfation, chemical characterization and anti-HSV activities.

In this study, we have analyzed water-extracted polysaccharides of Gracilaria corticata. The water extract (WE), a galactan-containing sub-fraction (F3) and their hyper sulfated derivatives (WES1, WES2, F3S1 and F3S2) had anti-HSV activity with inhibitory concentration 50% (IC50) from 1.1 to 27.4 microg/ml. Sub-fraction F3, which has a molecular mass of 30 kDa, consists of a backbone of beta-(1-->3) and alpha-(1-->4)-linked-galactopyranosyl residues. This linear galactan contained Gal2Xyl1, Gal2AnGal2, Gal4 and Me-Gal3AnGal2 as oligomeric building subunits. Sulfate group was located at C-4 of (1-->3)-linked galactopyranosyl residues of the native galactan, and appeared to be very important for the anti-herpetic activity.

Anti-herpetic activity of a sulfated xylomannan from Scinaia hatei.

Many viruses display affinity for cell surface heparan sulfate proteoglycans with biological relevance in virus entry. This raises the possibility of the application of sulfated polysaccharides in antiviral therapy. In this study we have analyzed polysaccharide fractions isolated from Scinaia hatei. The crude water extract (ShWE) as well as one fraction (F1) obtained by size exclusion chromatography had potent anti-HSV activity. Their inhibitory concentration 50% (IC50) values ranging from 0.5 to 4.6 microg/ml were much lower than the cytotoxic concentration 50% (CC50) values (1000 microg/ml). These fractions had very low anticoagulant activity. Furthermore, they had a weak inactivating effect on virions in a virucidal assay at concentrations in the range of 60-100 microg/ml. Chemical, chromatographic and spectroscopic methods showed that the major polysaccharide, which had 0.4 sulfate group per monomer unit and an apparent molecular mass of 160 kDa, contained a backbone of alpha-(1-->3)-linked D-mannopyranosyl residues substituted at C-6, C-4 and C-2 with single stub of beta-d-xylopyranosyl residues. Sulfate groups, when present, are located at C-4 of alpha-(1-->3)-linked D-mannopyranosyl units, and appeared to be very important for the anti-herpetic activity of this polymer.

ACIDFORM inactivates herpes simplex virus and prevents genital herpes in a mouse model: optimal candidate for microbicide combinations.

The acidic vaginal milieu is presumed to inactivate pathogens but is neutralized by semen. This notion fostered the development of acid-buffering products, such as ACIDFORM (developed by Program for Topical Prevention of Conception and Disease, Rush University, and licensed by Instead), as microbicides. However, the extent and mechanism of protective activity provided by buffering gels is not known. Exposure of herpes simplex virus (HSV) to pH 4.5 or lower irreversibly inactivated HSV and reduced HSV yields by at least 90%; exposure to pH 5.0 had little or no effect. Pretreatment of HSV-2 with pH 3.5-4.5 triggered proteolysis, disrupting the HSV particle and resulting in a reduction in binding and invasion. ACIDFORM protected 21 (81%) of 26 mice from genital herpes, compared with 3 (12%) of 25 mice who received a placebo gel. ACIDFORM retained significant activity if mice were challenged with HSV delivered in seminal fluid. These findings suggest that ACIDFORM offers considerable protection against HSV and may be an optimal candidate for developing combination microbicides.

Cervicovaginal secretions contribute to innate resistance to herpes simplex virus infection.

Defining and preserving the innate antiviral activity found in cervicovaginal secretions is critical. Cervicovaginal lavage (CVL) samples were obtained from 20 healthy women and evaluated for anti-herpes simplex virus (HSV) activity. CVL samples reduced HSV-2 yields by 23-fold (median), and the anti-HSV activity of CVL samples correlated with the concentration of human neutrophil peptides (HNP)-1-3. Both CVL samples and HNP-1-3 interacted with virus and prevented entry after binding. Substantially less protective activity was observed in CVL samples obtained from 20 human immunodeficiency virus--infected subjects, but the addition of CVL samples from healthy subjects enhanced the antiviral activity. The significance of the innate activity was further demonstrated by showing that CVL samples prevented murine genital herpes. Fourteen of 15 mice were protected from genital herpes if they were challenged with HSV-2 pretreated with CVL samples from healthy subjects. In contrast, all 15 mice challenged with untreated HSV-2 died. These findings are evidence that cervicovaginal secretions contribute to innate resistance to HSV-2 and identify defensins as contributors to this activity.

In vitro anti-herpetic activity of sulfated polysaccharide fractions from Caulerpa racemosa.

A sulfated polysaccharide fraction was isolated from the hot water extract of the green alga Caulerpa racemosa and designated HWE. This polymer, which contained galactose, glucose, arabinose and xylose as the major component sugars, had [alpha](D)(30) + 46.2 degrees in water and contained 9% sulfate hemiester groups. Sugar linkage analysis indicates that HWE was branched and mainly contained 1,3- and 1,3,6-linked galactose, 1,3,4-linked arabinose, 1,4-linked glucose and terminal- and 1,4-linked xylose residues. Sulfation was deduced from infrared spectroscopy and methylation analysis to occur on O-6 of galactose and O-3 of arabinose. The native polysaccharide could be fractionated by size exclusion chromatography into two overlapping fractions and the major fraction has a hydrodynamic volume similar to that of 70 kDa dextran. HWE was a selective inhibitor of reference strains and TK(-) acyclovir-resistant strains of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in Vero cells, with antiviral effective concentration 50% (EC(50)) values in the range of 2.2-4.2 microg/ml and lacking cytotoxic effects. Furthermore, HWE did not exhibit anticoagulant properties at concentrations near the EC(50).

Isolation, chemical investigation and antiviral activity of polysaccharides from Gracilaria corticata (Gracilariaceae, Rhodophyta).

Polysaccharides were sequentially extracted from the agarophyte Gracilaria corticata. Chemical analysis combined with infrared spectroscopy showed that the cold water extracted material consists mainly of a high molecular weight sulfated galactan. Most of the sulfate groups are alkali labile and are located at C-4 of the 1,3-linked D-galactose units and C-6 of the 1,4-linked L-galactose residues. The autoclaved extracts contain agar type polysaccharide having a high pyruvate content and a variable degree of methylation, but were contaminated with floridean starch. 1H-NMR studies indicate that methoxyl groups, when present, occur at C-6 of the 1,3-linked D-galactose units and C-2 of the 1,4-linked L-galactose residues of agar polymer. Bioassays showed that a high molecular weight galactan sulfate, exhibited selective antiviral activity against herpes simplex virus types 1 and 2, likely due to an inhibition of the initial virus attachment to the host cell.