Antiherpes simplex virus type 2 activity of the antimicrobial peptide subtilosin.

AIMS: In this study, we evaluated the antiviral activity of subtilosin, a cyclical peptide isolated from Bacillus amyloliquefaciens, against herpes simplex virus type 2 (HSV-2) in cell cultures and we investigated subtilosin mode of action. METHODS AND RESULTS: We determined, using a virus yield inhibition assay, that noncytotoxic concentrations of subtilosin inhibit HSV-2 replication in Vero cell cultures. Subtilosin strongly inhibited extracellular and total virus production even when it was added at 8 h postinfection indicating that not only virus release but also viral particle formation is impeded by the antiviral peptide. Although viral glycoprotein gD level of expression is not affected by the bacteriocin, an altered pattern of gD intracellular localization was detected by immunofluorescence assay in subtilosin-treated culture. On the other hand, at high concentrations, subtilosin displays virucidal action. CONCLUSIONS: Subtilosin displays antiviral and virucidal actions against HSV-2. The target of subtilosin inhibitory effect would be late stages of the viral replicative cycle such as viral glycoprotein intracellular transport. SIGNIFICANCE AND IMPACT OF THE STUDY: Given its antimicrobial activity and its safety for human tissues, subtilosin could represent a valuable alternative to be considered in the development of new microbicide formulations.

Boza, a natural source of probiotic lactic acid bacteria.

AIMS: To evaluate the probiotic properties of strains isolated from boza, a traditional beverage produced from cereals. METHODS AND RESULTS: The strains survived low pH conditions (pH 3.0), grew well at pH 9.0 and were not inhibited by the presence of 0.3% (w/v) oxbile. Cytotoxicity levels of the bacteriocins, expressed as CC(50), ranged from 38 to 3776 microg ml(-1). Bacteriocin bacST284BZ revealed high activity (EC(50) = 735 microg ml(-1)) against herpes simplex virus type 1. Growth of Mycobacterium tuberculosis was 69% repressed after 5 days in the presence of bacST194BZ. Various levels of auto-cell aggregation and co-aggregation with Listeria innocua LMG 13568 were observed. Adhesion of the probiotic strains to HT-29 cells ranged from 18 to 22%. CONCLUSIONS: Boza is a rich source of probiotic lactic acid bacteria. All strains survived conditions simulating the gastrointestinal tract and produced bacteriocins active against a number of pathogens. Adherence to HT-29 and Caco-2 cells was within the range reported for Lactobacillus rhamnosus GG, a well-known probiotic. In addition, the high hydrophobicity readings recorded define the strains as good probiotics. SIGNIFICANCE AND IMPACT OF THE STUDY: Boza contains a number of different probiotic lactic acid bacteria and could be marketed as a functional food product.

Performance and use of a ribonucleotide reductase herpes simplex virus type-specific serological assay.

In response to the increasingly evident need for herpes simplex virus (HSV) serotype-specific serologic assays that rely on proteins other than glycoprotein-G (gG), we developed a rapid serologic assay that is based on type-specific epitopes within the large subunit of HSV ribonucleotide reductase (R1). The assay (Au-2 enzyme-linked immunosorbent assay [ELISA]) uses an HSV type 2 (HSV-2) R1 peptide antigen. It provides a reliable method for detecting serotype-specific antibody to a protein other than gG-2. The Au-2 ELISA has high sensitivity and specificity as determined by direct comparison to Western blotting, a widely accepted "gold standard," and to ELISA with an HSV-1 R1 peptide (Au-1). The use of the Au-2 ELISA in conjunction with the gG-2-based assays will improve the sensitivity and specificity of serologic diagnosis and patient management.

A growth and latency compromised herpes simplex virus type 2 mutant (ICP10DeltaPK) has prophylactic and therapeutic protective activity in guinea pigs.

A growth compromised herpes simplex virus type 2 (HSV-2) mutant which is deleted in the PK domain of the large subunit of ribonucleotide reductase (ICP10DeltaPK) protects from fatal HSV-2 challenge in the mouse model (Aurelian L, Kokuba H, Smith CC. Vaccine potential of a Herpes Simplex Virus type 2 mutant deleted in the PK domain of the large subunit of ribonucleotide reductase (ICP10). Vaccine 1999;17:1951-1963). Here we report the results of our studies with ICP10DeltaPK in the guinea pig model of recurrent HSV-2 disease. ICP10DeltaPK was also compromised for growth and disease causation in this model. It was not isolated from latently infected ganglia by explant co-cultivation. The proportions of latently infected ganglia were significantly lower for ICP10DeltaPK than HSV-2 [3/25 (12%) and 7/10 (70%), respectively]. Similar results were obtained for the levels of viral DNA (8 x 10(3) and 2 x 10(5) molecules/ganglion for ICP10DeltaPK and HSV-2, respectively]. ICP10DeltaPK immunization caused a significant (P< or = 0.001) decrease in the proportion of animals with primary [1/14 (6%) and 16/16 (100%) for ICP10DeltaPK and PBS, respectively) and recurrent [1/14 (6%) and 11/14 (79%) for ICP10DeltaPK and PBS, respectively) HSV-2 skin lesions. It also protected from genital HSV-2 disease [1/10 and 10/10 for ICP10DeltaPK and PBS, respectively] and decreased the severity of the lesions in both models. Quantitative PCR (Q-PCR) with primers that distinguish between HSV-2 and ICP10DeltaPK indicated that immunization reduced the proportion of ganglia positive for HSV-2 DNA [8/25 (32%) and 7/10 (70%) for ICP10DeltaPK and PBS, respectively) and its levels [3 x 10(3) and 2 x 10(5) molecules/ganglion for ICP10DeltaPK and PBS, respectively]. The proportion of HSV-2 infected animals with recurrent disease was also significantly (P < or = 0.001) decreased by immunization with ICP10DeltaPK [1/15 (7%) and 11/14 (79%) with recurrent disease for ICP10DeltaPK and PBS, respectively], suggesting that ICP10DeltaPK has prophylactic and therapeutic activity in the guinea pig.

The use of time step simulations and difference equations (TSSADEQ) in modeling heparin pharmacokinetics.

A model in which the clearance of heparin requires the binding of heparin to a finite and regenerated pool of binding was constructed using time step simulations and difference equations (TSSADEQ). A simulation of a heparin i.v. bolus demonstrated a dose-dependent, triphasic pharmacokinetic curve with (1) an initial log-linear phase representing first-order association of heparin with binding sites, (2) an intermediate plateau phase representing constant regeneration of heparin binding sites, and (3) a terminal log-linear phase occurring when the quantity of regenerated sites exceeded the remaining heparin. Sensitivity analysis based on the literature produced estimates of the k at 1.39 to 2.77 h-1, the pool of binding sites of 50 units/kg, and the regeneration rate of 15 to 20 unit/kg/h--virtually identical to the empirically derived guidelines for the bolus size and infusion rate for unfractionated heparin. A pilot study of bolus dosing of unfractionated heparin in normal volunteers confirmed the model.

Antiviral effect of brassinosteroids against herpes virus and arenaviruses.

A natural brassinosteroid and a series of synthetic derivatives were found to be good inhibitors of herpes simplex virus type 1 (HSV-1) and arenavirus replication in cell culture. The synthetic compounds tested were analogues of the 24(S) ethylbrassinone. Compounds (22 R,23 R,24S)-2alpha, 3alpha,5alpha,22,23-pentahydroxy-stigmastan-6-one and (22R,23R,24S)-3beta-bromo-5alpha,22,23-trihydroxy stigmastan-6-one were cytotoxic at concentrations of 20-40 microM. (22S,23S,24S)-2alpha,3alpha,22,23-tetrahydroxy-5alpha, stigmastan-6-one, (22R,23R,24S)-3beta-acetoxy-22,23-dihydroxy-5alpha-choles tan-6-one, (22S,23S,24S)-3beta-bromo-22,23-dihydroxy-5alpha-cholestan-6 -one and (22S,23S,24S)-3beta-bromo-5alpha,22,23-trihydroxy-stigmastan -6-one were the most active of the series against HSV-1, with selectivity index (SI) values (CC50/EC50) ranging from 10.6 to 16.5. The majority of the compounds were potent inhibitors of arenaviruses, (22S,23S,24S)-3beta-bromo-5alpha,22,23-trihydroxy-stigmastan -6-one being the most active, with SI values of 307.8 and 692.5 for Tacaribe and Junin viruses, respectively. The antiviral activity of brassinosteroid derivatives was not because of direct inactivation; time-of-addition experiments suggested that a late step in HSV-1 multiplication was affected, whereas arenaviruses remained susceptible to the compounds throughout the replicative cycle.

Antiviral activity of enterocin CRL35 against herpesviruses.

Enterocin CRL35 is an antibacterial polypeptide of 3.5 x 10(3) Da produced by Enterococcus faecium CRL35. A series of experiments are described that show the enterocin also had antiviral activity against thymidine-kinase positive (tk+) and deficient (tk-) strains of herpes simplex (HSV) type 1 and 2 in Vero and BHK-21 cells. This activity was observed at 100 microg/ml, 15-fold lower than the cytotoxic concentration. In both cell lines there was a 2 log inhibition of infectivity. The compound inhibited viral multiplication in a dose-dependent manner and had no virucidal effect. Enterocin CRL35 also inhibited the virion-associated host shutoff in infected Vero cells showing that intracellular viral multiplication was affected.

Inhibition of foot and mouth disease virus (FMDV) uncoating by a plant-derived peptide isolated from Melia azedarach L leaves.

Meliacine (MA), a peptide isolated from leaves of the high plant Melia azedarach L inhibited the multiplication of foot and mouth disease virus (FMDV) in BHK-21 cells. In this report, we establish that the MA-inhibitable process takes place within the first hour of the viral reproductive cycle. MA had no virucidal effect and did not affect adsorption and penetration of the virus in cells. In experiments with neutral red-labeled virus, it was found that MA significantly suppressed the development of photoresistance of the virus in infected cells. In untreated cultures nearly all virus which adsorbed to cells was uncoated within 1 h at 37 degrees C, whereas in treated cultures, even after 3 h only 3% of the virus was uncoated. Labeling of BHK-21 cells with acridine orange showed that MA affects the pH of intracellular acidic vesicles. Therefore, it is concluded that MA prevents the process of uncoating of FMDV in BHK-21 cells by inhibiting vacuolar acidification.

Sobrevida de distintas cepas de Vibrio cholerae en lechuga fresca / Survival of different Vibrio cholerae strains in fresh lettuce

A fin de estudiar el efecto de la temperatura en la sobrevida de Vibrio cholerae en lechuga fresca se determinó el número de unidades formadoras de colonias remanentes en esta verdura contaminada experimentalmente con las siguientes cepas de este microorganismo: Peruano, CDC-185, 425 y No O1. Los estudios de estabilidad se realizaron a 4ºC y a 25ºC. Asimismo, a fin de establecer una relación entre Vibrio cholerae y flora acompañante, se determinó el número de colonias en agar TCBS y en agar nutritivo inmediatamente luego de contaminar (to) y a distintos tiempos post-contaminación (ti). He