Antiviral activity of Spirulina maxima against herpes simplex virus type 2

Spirulina has been used in a variety of practical applications in biotechnology and medical sciences. This paper presents the antiviral activity found in a hot water extract (HWE) of a commercial preparation of Spirulina maxima, studied by a microplate inhibition assay, using several viruses. The HWE inhibited the infection for: herpes simplex virus type 2 (HSV-2), pseudorabies virus (PRV), human cytomegalovirus (HCMV), and HSV-1, and the 50% effective inhibition doses (ED(50)) were 0.069, 0.103, 0.142, and 0.333 mg/ml for each virus, respectively. For adenovirus the inhibition was less than 20%, and no inhibition was found for measles virus, subacute sclerosing panencephalitis virus (SSPE), vesicular stomatitis virus (VSV), poliovirus 1 and rotavirus SA-11, at concentrations of 2 mg/ml of the HWE. The highest antiviral activity was for HSV-2, with a selectivity index of 128. The antiviral activity was not due to a virucidal effect. Herpesvirus infection was inhibited at the initial events (adsorption and penetration) of the viral cycle. To initiate the isolation and identification of the compound that exhibits the antiviral activity of S. maxima, some extracts made by using several solvents with different polarity were evaluated by microplate inhibition assay using HSV-2. The highest antiviral activity was detected in the methanol-water 3:1, which suggests that the antiviral activity is probably due to highly polar compounds.     

Anti-HSV activity of lactoferricin analogues is only partly related to their affinity for heparan sulfate

Earlier studies have shown that the heparan sulfate (HS) on the cell surface acts as a receptor for herpes simplex virus (HSV). We have recently shown that bovine lactoferricin (LfcinB), a small part of the milk protein lactoferrin, inhibits HSV-1 and HSV-2 infection, probably by blocking the entry of the virus. The human homologue (18-42), which shares 36% sequence similarity with LfcinB (17-41), displayed much lower antiviral activity. In the present study, a set of cyclic and linear human and bovine Lfcin derivatives were constructed to investigate the relation between their affinity to HS and chondroitin sulfate (CS) and their antiviral activity against HSV-1 and HSV-2. The lactoferrin (LF) proteins and several of the Lfcin derivatives exhibited similar affinity for HS, but the LF proteins possess a much higher antiviral activity than the smaller peptides. Our structure-activity relationship studies on the Lfcin derivates confirmed that affinity for HS, that was correlated to the net positive charge, is an important factor, but does not well predict the antiviral activity. Structural parameters such as hydrophobicity, molecular size, spatial distribution of charged and lipophilic amino acids, and the cyclic structure of Lfcin also seem to be important factors to govern antiviral activity against HSV.     

Inhibition of herpes simplex virus type 1 and adenovirus type 5 by heterocyclic Schiff bases of aminohydroxyguanidine tosylate

Eleven heterocyclic Schiff bases of aminohydroxyguanidine tosylate (SB-AHGs), compounds I-XI, were tested for antiviral activity against herpes simplex virus type 1 (HSV-1) and adenovirus type 5 (Ad 5) via plaque reduction and virus yield reduction assays. This work was undertaken to test the hypothesis that low molecular weight SB-AHGs (MW < 235 for the free SB) make better antiviral agents than high MW SB-AHGs (MW > 300). The plaque reduction assay method demonstrated that three compounds, I, VII and IX, had moderate activity against HSV-1, with 50% inhibitory concentration (IC50) values of 38.0, 23.5 and 52.1 microM, respectively. Against Ad 5, compounds I, VIII and XI exhibited moderate activity, with IC50 values of 52.7, 19.3 and 5.1 microM, respectively. Among the compounds screened, compound I (1-[(3'-hydroxy-6'-methyl-2'-pyridyl)methylene]amino-3-hydroxyguanidi ne tosylate) was the most promising antiviral candidate, with selectivity indices (SI) of 10.2 (HSV-1) and 7.6 (Ad 5), respectively. Virus yield reduction assays indicated that compound I had less antiviral potency against HSV-1 than against Ad 5. The antiviral effects of compound I at a high input virus multiplicity of infection (MOI > 5) indicated that compound I had effective anti-adenoviral activity at 24 h post infection. This work demonstrated that some of SB-AHGs only have moderate antiviral activities against Ad 5 and HSV-1 viruses. In general, low MW SB-AHGs have low cytotoxicities to the host cells.     

Oligomeric proanthocyanidins from Rumex acetosa L. inhibit the attachment of herpes simplex virus type-1

The polyphenole-enriched acetone-water extract R2 from the aerial parts of Rumex acetosa L. containing high amounts of oligomeric and polymeric proanthocyanidins and flavonoids was tested for antiviral activity. R2 exhibited strong antiviral activity against herpes simplex virus type-1 (HSV-1) while the replication of adenovirus 3 was not affected. By plaque reduction test and MTT assay on Vero cells, the HSV-1-specific inhibitory concentration (IC(50)) and cytotoxic concentration (CC(50)) were determined. R2 exibited an IC(50) of 0.8 μg/mL and a selectivity index (SI) (ratio of IC(50) to CC(50)) of approximately 100 when added to the virus inoculum for 1h at 37°C prior to infection. The antiviral activity was due to the presence of flavan-3-ols and oligomeric proanthocyanidins in the extract. Structure-activity analyses indicated that flavan-3-ols and proanthocyanidins with galloylation at position O-3 are highly potent compounds (SI>40), while ungalloylated compounds did not exhibit antiviral effects (SI<1). R2 and a major proanthocyanidin from R2, epicatechin-3-O-gallate-(4β→8)-epicatechin-3-O-gallate abolished virus entry into the host cell by blocking attachment to the cell surface. When added after attachment at a concentration of ≥ 12.5 μg/mL, R2 inhibited also penetration of HSV-1 into the host cell. R2 and epicatechin-3-O-gallate-(4β→8)-epicatechin-3-O-gallate were shown to directly interact with viral particles leading to the oligomerisation of envelope proteins as demonstrated for the essential viral glycoprotein gD. Using raft cultures with three-dimensional organotypic human skin equivalents it was shown that treatment of cultures with R2 after infection with HSV-1 resulted in a reduced viral spread.     

Involvement of bovine lactoferrin moieties in the inhibition of herpes simplex virus type 1 infection

Bovine lactoferrin (BLf) is an iron binding protein folded in two lobes, N- and C-lobes. In this study we have reported the inhibitory activity towards herpes simplex virus type 1 (HSV-1) in vitro infection of BLf tryptic digested N- and C-lobes in comparison with the whole protein. The N-lobe and C-lobe exerted an anti-herpesvirus activity 50- and 10-fold lower than native BLf, respectively. In order to assess the phase of viral replication affected, lactoferrin-derived lobes were added to the cells at different non cytotoxic concentrations, during the whole cycle of viral infection or during viral attachment step, demonstrating that both lobes interfered with the early phases of infection. Among the BLf tryptic digested fragments, two negatively-charged small peptides deriving from N-lobe, previously shown effective towards HSV-1, have been further studied. We assessed that the net negative charge of these peptides was not responsible for the antiviral activity since their activity was not modified when the aspartic and glutamic acidic residues of these peptides were replaced with asparagine and glutamine, respectively. The experiments here reported confirm a pivotal role of N-lobe in inhibiting viral infection. However, the residual inhibiting activity of C-lobe and the similar efficacy shown by negatively or positively charged peptides strongly support the idea that the antiviral activity of bovine lactoferrin cannot be fully explained simply on the basis of competition between the protein and viral recognition sites for binding to glycosaminoglycans.     

Antiviral activity of Plantago major extracts and related compounds in vitro

Plantago major L., a popular traditional Chinese medicine, has long been used for treating various diseases varying from cold to viral hepatitis. The aim of present study was to examine the antiviral activity of aqueous extract and pure compounds of P. major. Studies were conducted on a series of viruses, namely herpesviruses (HSV-1, HSV-2) and adenoviruses (ADV-3, ADV-8, ADV-11). The antiviral activity of EC50 was defined as the concentration achieved 50% cyto-protection against virus infection and the selectivity index (SI) was determined by the ratio of CC50 (concentration of 50% cellular cytotoxicity) to EC50. Results showed that aqueous extract of P. major possessed only a slight anti-herpes virus activity. In contrast, certain pure compounds belonging to the five different classes of chemicals found in extracts of this plant exhibited potent antiviral activity. Among them, caffeic acid exhibited the strongest activity against HSV-1 (EC50=15.3 microg/ml, SI=671), HSV-2 (EC50=87.3 microg/ml, SI=118) and ADV-3 (EC50=14.2 microg/ml, SI=727), whereas chlorogenic acid possessed the strongest anti-ADV-11 (EC50=13.3 microg/ml, SI=301) activity. The present study concludes that pure compounds of P. major, which possess antiviral activities are mainly derived from the phenolic compounds, especially caffeic acid. Its mode of action against HSV-2 and ADV-3 was found to be at multiplication stages (postinfection of HSV-1: 0-12 h; ADV-3: 0-2 h), and with SI values greater than 400, suggesting the potential use of this compound for treatment of the infection by these two viruses.     

Opioid receptor ligands derived from food proteins

During the last two decades a variety of food protein fragments has been demonstrated to elicit biological effects in various in vitro or in vivo test systems. A considerable part of these bioactive peptides are opioid receptor ligands, which may be regarded as exogenous supplements to the endogenous opioidergic systems of the human organism. Most of these food-derived opioid receptor ligands are fragments of the milk proteins alpha-, beta- or kappa-casein, alpha-lactalbumin, beta-lactoglobulin or lactotransferrin; however, also wheat gluten, rice albumin, bovine serum albumin or hemoglobin, i.e. possible constituents of meat, and even a protein from spinach could be demonstrated to contain fragments behaving like opioid receptor ligands. Practically all of these compounds display opioid agonist activity; only very few of them behave like opioid antagonists. Bioactive food protein derivatives have been termed " food hormones", which implies that these compounds display their bioactivities when released from food constituents, i.e. from their precursor molecules due to the action of gastrointestinal enzymes. The critical point in case of food protein-derived opioid receptor ligands is that only a minority of their bioactive effects demonstrated as yet has been observed upon oral or intragastric administration of these peptides or their precursor proteins and that most of these studies have been performed in animals. Thus, in terms of "evidence-based dietary supplementation" more studies are needed to prove effects of food protein-derived opioid receptor ligands or their precursors after oral administration in humans and, moreover, to prove a benefit for the consumer's organism.     

Antiviral effect of octyl gallate against DNA and RNA viruses

The effects of gallic acid (3,4,5-trihydroxybenzoic acid) and its alkyl esters on virus growth and virion infectivity were examined. All the compounds tested showed an inhibitory effect on the growth of herpes simplex virus type 1 (HSV-1) in HEp-2 or Vero cells. The antiviral activity of gallic acid alkyl esters was enhanced by increasing the number of carbon in the alkyl moieties of the compounds, reaching maximum at a carbon number of 12 (lauryl gallate), but both cytocidal activity and cytopathic effect of the compounds were also significantly increased simultaneously. Among these compounds, octyl gallate showed a marked antiviral effect with a relatively moderate cytotoxity. In addition, octyl gallate suppressed the multiplication of RNA viruses, such as vesicular stomatitis virus and poliovirus. Quantitative characterization of the HSV-1 infection in the presence of octyl gallate revealed that: (1) this reagent can directly inactivate HSV-1 (virucidal activity), (2) it suppresses both the intracellar multiplication and the release of the virus, (3) it selectively accelerates death of the virus-infected cells and (4) the addition of the reagent even at 6-h post infection completely abolishes the formation of progeny virus in the infected cells.     

Enhancement of non-specific resistance to viral infection by muramyldipeptide and its analogs

Antiviral activity of muramyldipeptide (MDP) and its lipophilic derivatives, B30-MDP and MDP-Lys(L18), was investigated in mice infected with vaccinia virus (VV) and herpes simplex virus type 2 (HSV-2). Mice administered these compounds subcutaneously or orally were protected against VV in tail lesion tests and against HSV-2 in skin lesion tests, respectively. Since in vitro antiviral activity was not demonstrated with these compounds in cultured mammalian cells infected with either VV or HSV-2, host-mediated defense mechanisms may play a role in the activity of the compounds. As for serum interferon (IFN) induction, MDP and its analogs showed no activity in mice, suggesting that IFN does not participate in the antiviral mechanisms against VV and HSV-2. An extrinsic antiviral activity was demonstrated when peritoneal macrophages from the mice administered these compounds were cocultivated with VV-infected 3T3 cells. The results indicate that macrophage activation by MDP and its analogs plays a role in the defense mechanisms against viral infection. This activity was not virus-specific. We also demonstrate that the introduction of lipophilic residue(s) into MDP enhances the antiviral activity of mice against VV and HSV-2.     

Antiadenovirus activity of milk proteins: lactoferrin prevents viral infection.

Different milk proteins were analysed for their inhibitory effect on adenovirus infection in vitro. Proteins investigated were mucin, alpha-lactalbumin, beta-lactoglobulin, bovine lactoferrin, and human lactoferrin. Results obtained demonstrated that mucin, alpha-lactalbumin, and beta-lactoglobulin did not prevent the viral cytopathic effect, whereas lactoferrin was able to inhibit adenovirus replication in a dose-dependent manner. Further experiments were carried out in which lactoferrin was added to the cells during different phases of viral infection. Results obtained showed that lactoferrin was able to prevent viral replication when added both before, or during the viral adsorption step, or when present during the entire replicative cycle of adenovirus, demonstrating that its action takes place on an early phase of viral replication.     

Antiviral activity of hop constituents against a series of DNA and RNA viruses

We investigated whether crude hop extracts and purified hop components representing every major chemical class of hop compound have antiviral activity. These hop constituents were tested for antiviral activity against bovine viral diarrhea virus (BVDV) as a surrogate model of hepatitis C virus (HCV), human immunodeficiency virus (HIV), influenza A virus (FLU-A), influenza B virus (FLU-B), rhinovirus (Rhino), respiratory syncytial virus (RSV), yellow fever virus (YFV), cytomegalovirus (CMV), hepatitis B virus (HBV), and herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2). The extracts all failed to prevent the replication of HIV, FLU-A, FLU-B, RSV and YFV. A xanthohumol-enriched hop extract displayed a weak to moderate antiviral activity against BVDV (therapeutic index (TI)=6.0), HSV-2 (TI=>5.3), Rhino (TI=4.0) and HSV-1 (TI=>1.9) with IC(50) values in the low microg/ml range. Pure iso-alpha-acids demonstrated low to moderate antiviral activity against both BVDV (TI=9.1) and CMV (TI=4.2) with IC(50) values in the low microg/ml range. No antiviral activity was detected using beta-acids or a hop oil extract. Ultra-pure preparations (>99% pure) were used to show that xanthohumol accounted for the antiviral activity observed in the xanthohumol-enriched hop extract against BVDV, HSV-1 and HSV-2. Xanthohumol was found to be a more potent antiviral agent against these viruses than the isomer iso-xanthohumol. With Rhino, the opposite trend was observed with iso-xanthohumol showing superior antiviral activity to that observed with xanthohumol. Xanthohumol also showed antiviral activity against CMV, suggesting that it might have a generalized anti-herpesvirus antiviral activity. Again, superior antiviral activity was observed with the xanthohumol isomer against CMV. In summary, iso-alpha-acids and xanthohumol were shown to have a low-to-moderate antiviral activity against several viruses. These hop constituents might serve as interesting lead compounds from which more active anti-HCV, anti-Rhino and anti-herpesvirus antiviral agents could be synthesized.     

Virucidal activity of polysaccharide extracts from four algal species against herpes simplex virus

Herpes simplex virus types 1 and 2 (HSV-1, HSV-2) infections are common, but can cause serious infections in neonates and the immunocompromised. Drugs currently used to treat cutaneous or genital HSV infections are effective in limiting disease, but the emergence of drug resistant viruses in immunocompromised individuals can be problematic. While the prophylactic oral treatment with antiviral drugs can reduce virus shedding and transmission, there is a need for topical microbicides that have the potential to limit sexual transmission of the virus. Previous reports demonstrated the antiviral activity of complex sulfated polysaccharides extracted from various species of marine algae and suggested that they interfered with the attachment of virions to host cells. Here, we evaluated the antiviral activity of extracts from Undaria pinnatifida, Splachnidium rugosum, Gigartina atropurpurea, and Plocamium cartilagineum against HSV-1 and HSV-2. These extracts exhibited good activity when added during the first hour of viral infection, but were ineffective if added later. Plaque reduction assays, when the extracts were added prior to viral inoculation, yielded EC₅₀ values that ranged from 2.5–3.6 μg/ml for HSV-1 and 0.7–6.6 μg/ml for HSV-2. None of the extracts exhibited significant toxicity in a neutral red uptake assay (IC₅₀ >100 μg/ml). Subsequent assays showed that the compounds had potent virucidal activity and were active at very low concentrations. We conclude that these extracts are nontoxic and effective virucidal agents that warrant further investigation to examine their potential role in the prevention of HSV infections of humans.     

Characterization of the anti-HIV effects of native lactoferrin and other milk proteins and protein-derived peptides

In a search for natural proteins with anti-HIV activity, we screened a large set of purified proteins from bovine milk and peptide fragments thereof. Because several charged proteins and peptides are known to inhibit the process of virus entry, we selected proteins with an unusual charge composition or hydrophobicity profile. In contrast with some chemically modified (strongly negative) milk proteins, unmodified alpha(s2)-, beta- and kappa-casein, as well as several negatively and positively charged fragments thereof, did not show significant inhibition of virus replication. In fact, HIV-1 replication was elevated in the presence of beta-casein or amphiphilic fragments thereof. Bovine lactoferrin (bLF), a milk protein of 80 kDa, showed considerable inhibitory activity against HIV-1 with an IC50 of 0.4 microM. Modest inhibition was obtained with lactoferricin, a highly positively charged loop domain of bLF, indicating that other domains within the native bLF protein may also be required for inhibition. bLF blocked HIV-1 variants that use either the CXCR4 or the CCR5 coreceptor. In order to obtain further insight into the mechanism of action of this antiviral protein, we selected a bLF-resistant HIV-1 variant. The bLF-resistance phenotype is mediated by the viral envelope protein, which contains two interesting mutations that have previously been associated with an altered virus-host interaction and a modified receptor-coreceptor interaction. These results demonstrate that bLF targets the HIV-1 entry process.     

Phenoxazine derivatives inactivate human cytomegalovirus, herpes simplex virus-1, and herpes simplex virus-2 in vitro.

We examined whether phenoxazine derivatives, 2-amino-4,4alpha-dihydro-4alpha-7-dimethyl-3H-phenoxazine-3-one (Phx-1), 3-amino-1,4alpha-dihydro-4alpha-8-dimethyl-2H-phenoxazine-2-one (Phx-2), and 2-amino-phenoxazine-3-one (Phx-3) may have antiviral activity against herpes family viruses: human cytomegalovirus (HCMV), herpes simplex virus type 1 (HSV-1), and herpes simplex virus type 2 (HSV-2). The antiviral activity was evaluated by the selectivity index (SI), which is the ratio of 50% cytotoxic concentration (CC(50)) and 50% antiviral concentration (IC(50)). Among these phenoxazines, Phx-2 exerted strong antiviral activity to HCMV with the SI of 200, while Phx-1 and Phx-3 exerted no marked anti-HCMV activity. Phx-2 also showed moderate inhibition of HSV-1 and HSV-2, with the SI of 6.7 and 17, respectively. In the time-of-addition experiments, inhibitory effect of Phx-2 against HCMV was active even when applied to cells at 100 h after HCMV infection, while ganciclovir (GCV) showed potent inhibition when applied to cells before 42-h post-infection, but its inhibitory effects disappeared thereafter. Attachment and penetration of HCMV was not affected by the presence of Phx-2. When HCMV was pretreated with Phx-2, concentration-dependent virucidal action was observed, suggesting that Phx-2 inactivates HCMV directly. From these data, it was found that Phx-2 might have a different anti-HCMV target from GCV.     

In vitro antiviral activity of dehydroepiandrosterone, 17 synthetic analogs and ERK modulators against herpes simplex virus type 1

In the present study the in vitro antiviral activity of dehydroepiandrosterone (DHEA) and 17 synthetic derivatives against herpes simplex type 1 (HSV-1) was determined. DHEA, epiandrosterone (EA), two synthetic DHEA analogs and three synthetic EA analogs showed a selective inhibitory effect on HSV in vitro multiplication. DHEA and E2, a synthetic derivative of EA, were not found to be virucidal to cell-free HSV-1 and did not impair virus adsorption or penetration. We determined that treatment with both compounds decreased viral protein synthesis. Moreover, inhibitory effect of DHEA and E2 on extracellular viral titer was stronger than the inhibition found on total viral infectivity, suggesting that the antiherpetic activity of these compounds may also be in part due to an inhibition in virus formation and release. Since DHEA is a known Raf/MEK/ERK signaling pathway activator, we studied the role of this pathway on HSV-1 infection. ERK1/2 phosphorylation was stimulated in HSV-1 infected cultures. UO126, a Raf/MEK/ERK signaling pathway inhibitor, impaired viral multiplication, while anisomycin, an activator of this pathway, enhanced it. Treatment with DHEA 6 h before infection enhanced HSV-1 multiplication. On the contrary, pre-treatment with E2, which does not modulate Raf/MEK/ERK signaling pathway, did not produce an increase of viral replication. Taking together these results, the antiviral activity of DHEA seems to occur via a mechanism independent of its ability to modulate ERK phosphorylation.     

Antiviral activity of cyclosaligenyl prodrugs of the nucleoside analogue bromovinyldeoxyuridine against herpes viruses

A series of 42 lipophilic bromovinyldeoxyuridine monophosphates (BVDUMPs) are presented as potential prodrugs of the antiviral agent (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU). The 5'-cycloSal-masking group technique has been applied to this cyclic nucleoside analogue to achieve delivery of the monophosphate of BVDU inside the target cells. The new substances have been tested for their antiviral activity against herpes simplex virus types 1 and 2 (HSV-1 and -2), thymidine kinase-deficient (TK(-)) HSV-1, varicella-zoster virus (VZV), human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV). The XTT-based tetrazolium reduction assay EZ4U (for HSV), the plaque inhibition test (for VZV and HCMV) and a DNA hybridisation assay (for EBV) were used to assess antiviral activity. The results indicate that cycloSal-BVDUMP triesters proved to be potent and selective inhibitors of HSV-1 comparable with aciclovir. VZV replication was inhibited by very low concentrations, and two substances had a slightly better anti-VZV activity than the parent compound BVDU. No antiviral effect could be demonstrated against TK(-)-HSV-1, HSV-2 and HCMV, most likely owing to the lack of phosphorylation to BVDU diphosphate. Most remarkably, several cycloSal-BVDUMP triesters yielded promising anti-EBV activity whereas the parent compound BVDU was entirely inactive.     

Antibacterial and antiviral effects of milk proteins and derivatives thereof

Milk forms a rich source of biologically interesting components. In particular, its protein fraction is known to encompass many kinds of biological functions. In this review we focus on antibacterial and antiviral properties of milk proteins and milk protein derivatives. The latter include chemically modified proteins and enzymatically induced peptides. If such peptides are released by enzymes present within the digestive tract (e.g. trypsin or pepsin), it is likely that they play a role in the health defense system. This is especially the case when the active fragments can survive the intestinal conditions long enough to arrive at the right place to exert their beneficial function. In the first part of this paper attention is paid to the antibacterial proteins lactoferrin, lactoperoxidase, and lysozyme. Furthermore, antibacterial peptides originating from caseins and whey proteins are described. The second part reports on studies of antiviral effects of milk proteins and derivatives thereof. Special focus is directed to the antiviral action towards the human immunodeficiency virus (HIV) and the human cytomegalovirus (HCMV). Unmodified milk proteins are generally not active against these viruses. An exception is lactoferrin, which shows significant antiviral activity against both HIV and HCMV. Several other milk proteins tested showed strong antiviral effects only after chemical modification, i.e. by making them polyanionic (for anti-HIV activity) or polycationic (for anti-HCMV activity). In a number of cases, conclusions are drawn concerning possible relationships between antibacterial/antiviral activity and molecular structure of the components described.     

Novel thiosemicarbazones derived from formyl- and acyldiazines: synthesis, effects on cell proliferation, and synergism with antiviral agents.

The synthesis of a series of novel thiosemicarbazones (TSC's) derived from various alkyl diazinyl (3-pyridazinyl, 4-pyrimidinyl, 2-pyrazinyl) ketones and 3-pyridazinecarbaldehyde and their evaluation against herpes simplex virus (HSV) and human immunodeficiency virus (HIV) as well as the determination of their cytotoxicity are described. In addition, the effects of combination of such TSC's with the well-known antiviral drugs acyclovir (ACV) and 3'-azido-3'-deoxythymidine (AZT) were studied. Under our experimental conditions, i.e. determination of virus-induced cytopathic effect upon infection of HUT78 cells with HSV-1 and upon infection of MT4 cells with HIV-1, no antiviral activity could be detected with any of the TSC's. However, pronounced effects on proliferation of these rapidly growing T4 lymphocyte cell lines were observed. Clear structure-activity relationships with regard to these cytotoxic effects could be established: compared to pyridine, pyrazine, or pyrimidine-derived TSC's most of the 3-pyridazinyl congeners investigated are less cytotoxic; introduction of a methyl group into C-6 of the pyridazine system or prolongation of the acyl moiety in these compounds has essentially no influence; all compounds bearing an N,N-dimethylamino or a cycloamino substituent are much more toxic than those with an NH2 or NHR substituent; the nature of R in the latter type of compounds has only moderate influence. It has been reported that combination of TSC's with the antiviral agent acyclovir (ACV) results in potentiation of this well-known drug. We evaluated the potential of our series of novel TSC's in combination with ACV for inhibition of HSV-1-induced cytopathic effect in HUT78 cells and in combination with 3'-azido-3'-deoxythymidine (AZT) for inhibition of HIV-1-induced cytopathic effect in MT4 cells. Only four compounds out of this series, all characterized by an unsubstituted NH2 group, exhibited moderate synergism with the above mentioned antiviral drugs. Our results do not support the previously expressed opinion that TSC's are selective antiviral agents. In our test systems no evidence for inhibition of virus-induced cytopathic effect was obtained. The TSC derivatives exhibited a broad range of cytotoxic effects, some at concentrations considerably below those reported to have antiviral efficacy. Several of our novel diazine-derived compounds proved advantageous over the previously described pyridine analogues with regard to cytotoxicity. Moderate synergism could be detected for relatively noncytotoxic TSC's with the antiviral drugs ACV (antiherpes) and AZT (anti-HIV).     

Inhibition of HSV cell-to-cell spread by lactoferrin and lactoferricin

The milk protein lactoferrin (Lf) has multiple functions, including immune stimulation and antiviral activity towards herpes simplex virus 1 and 2 (HSV-1 and HSV-2); antiviral activity has also been reported for the N-terminal pepsin-derived fragment lactoferricin (Lfcin). The anti-HSV mode of action of Lf and Lfcin is assumed to involve, in part, their interaction with the cell surface glycosaminoglycan heparan sulfate, thereby blocking of viral entry. In this study we investigated the ability of human and bovine Lf and Lfcin to inhibit viral cell-to-cell spread as well as the involvement of cell surface glycosaminoglycans during viral cell-to-cell spread. Lf and Lfcin from both human and bovine origin, inhibited cell-to-cell spread of both HSV-1 and HSV-2. Inhibition of cell-to-cell spread by bovine Lfcin involved cell surface chondroitin sulfate. Based on transmission electron microscopy studies, human Lfcin, like bovine Lfcin, was randomly distributed intracellularly, thus differences in their antiviral activity could not be explained by differences in their distribution. In contrast, the cellular localization of iron-saturated (holo)-Lf appeared to differ from that of apo-Lf, indicating that holo- and apo-Lf may exhibit different antiviral mechanisms.     

十味板蓝根颗粒剂抗病毒作用的实验研究

目的:探讨十味板蓝根颗粒剂体外抗柯萨奇病毒B3株(CVB3)、单纯疱疹病毒II型(HSV II)的作用.方法:采用组织细胞培养法,观察药物毒性和细胞病变效应,评价十味板蓝根颗粒剂的细胞毒性及抗病毒效果.结果:十味板蓝根颗粒剂对Hep-2和Hela细胞毒性均较低(TD0为1 mg·mL-1),浓度在125μg·mL-1以上时,可以明显抑制HSV-II病毒对Hela细胞感染;浓度达250 μg·mL-1以上时,明显抑制CVB3病毒对Hep-2细胞的致病变作用,对Hep-2和Hela细胞有保护作用,并且随着药物浓度的增加,CPE特征逐渐减弱,病毒抑制率(细胞存活率)明显升高.结论:十味板蓝根颗粒剂在体外能明显抑制CVB3和HSV-II病毒的致细胞病变作用.