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.

Antiviral effects of Melia azedarach L. leaves extracts on Sindbis virus-infected cells.

Partially purified extracts from leaves of Melia azedarach L. (MA) exert a broad range of antiviral effects on DNA and RNA viruses. The effect of MA on different stages of Sindbis virus replicative cycle in BHK cells was investigated. Under one-step growth conditions MA afforded a greater than 90% inhibition in virus yield if added to the cell cultures 2 h before or after infection, and when added 4 h after infection MA still caused a greater than 80% inhibition. Analysis of early events following Sindbis virus infection showed that MA did not affect viral adsorption to or penetration in BHK cell. In contrast, viral RNA and protein synthesis was almost totally inhibited in cells pretreated with MA 2 h before infection, while cellular macromolecular synthesis was similar in MA-treated and untreated cell cultures.

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.

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.

[Susceptibility of picornaviruses++ to an antiviral of plant origin (meliacin)]. / Susceptibilidad de picornavirus a un antiviral de origen vegetal (meliacina).

Meliacine, a peptide associated with antiviral activity isolated from the high plant Melia azedarach L (MA) inhibits the replication of different strains of foot and mouth disease virus (FMDV) and poliovirus in BHK-21 or Vero cells, respectively, infected at a multiplicity of infection (m.o.i.) of 1. A leaf extract of MA, containing meliacine was added to the culture medium after virus adsorption and maintained up to virus harvest (18 hs). FMDV O1 Campos and O69 strains showed 60% and 52% inhibition, respectively. A24 and A87 strains proved to be inhibited more than 90%, and the C3 Resende and O1 Case-ros strains were not sensitive to the extract. Regarding the three types of poliovirus, the most susceptible one was type 1 (84%). Types 2 and 3 were inhibited only 69% and 32%, respectively. Treatment of the cells with the antiviral for 24 hours before infection (pretreatment), was not effective for the two above mentioned viruses. Since the extract of MA is very active against other RNA viruses (i.e. vesicular stomatitis virus), the susceptibility of FMDV strain O1 Campos and poliovirus type 3 was tested using lower m.o.i. The results showed that MA produced an inhibition of viral replication of 99% at a m.o.i. of 0.001 in both cases. These results suggest that picornaviruses are also susceptible to MA. However, to demonstrate its antiviral effect it is necessary to slow down the rate of virus-cell killing by reducing the m.o.i.

[Subtypes of foot-and-mouth disease virus type A. Genetic markers of clones obtained from 4 strains isolated in Argentina between 1961 and 1970]. / Subtipos de virus de fiebre aftosa tipo A. Marcadores genéticos de clones obtenidos de cuatro cepas aisladas en la Argentina de 1961 a 1970.

The results obtained studying the genetic markers g, t and rtc of different clones of subtypes of foot-and-mouth disease virus type A are presented in this paper. The subtypes were isolated during outbreaks of foot and mouth disease in Argentine. No significative differences among the subtypes were observed with the t marker. For the other markers, the results seem to indicate a gradual change related with the serological variation. Because of their sensibility to guanidine hidrochloride, it is possible to conclude that the studied clones do not belong to the so called European strains.

[Multiple resistance to heavy metals and antibiotics in bacteria isolated from the Riachuelo]. / Resistencia múltiple a metales pesados y a antibióticos en bacterias aisladas del riachuelo.

Riachuelo is a heavy contaminated course of water, partially surrounding Buenos Aires city. The presence of bacteria resistant to antibiotics and heavy metals was studied. Among the isolated Gram positive colonies, 65, 59 and 48% were resistant to 60 micrograms/ml of Pb++, Zn++ and Cd++, respectively, and 20% grew in the presence of 50 micrograms/6ml gentamicin. Most of these microorganisms belonged to the order Actinomycetales. Accordingly, high percentages of resistance were detected (among the 11 Gram negative isolates (Enterobacteriaceae and Pseudomonaceae), although only one isolate was gentamicin resistant. Four Gram negative isolates also showed a broad spectrum of resistance to tetracycline, erythromyci, ampicillin, amikacin, chloramphenicol and gentamicin.

[Survival of different strains of Vibrio cholerae on fresh lettuce]. / Sobrevida de distintas cepas de Vibrio cholerae en lechuga fresca.

Temperature effect on the survival of Vibrio cholerae in fresh lettuce was studied. Remaining infectivity in leaves experimentally contaminated with different strains of Vibrio cholerae was determined at 4 degrees C and 25 degrees C. The number of colonies in TCBS agar (Vibrio cholerae) and in nutrient agar (Vibrio cholerae and secondary microorganisms) was determined at to (initial time) and at ti (different post-contamination times). The ratio ti/to was calculated in both cases. Different survival features were found among the Vibrio cholerae strains. The CDC-185 was the most sensitive one in the experimental conditions used. At 25 degrees C, Vibrio cholerae grew faster than the secondary microorganisms and perhaps in this way it would survive in nature. At 4 degrees C lettuce leaves contaminated with the CDC-185 and the Peruano strains showed an important overgrowth relative to the secondary microorganisms at the 7th day post-contamination, whereas No O1 and 425 strains were able to inhibit this multiplication. Lettuce leaves were treated with acetic acid 0,05% to determine the inactivation of Vibrio cholerae at low pH. This decontamination treatment was effective, since a reduction of 2 log was obtained in all cases.

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.

Susceptibilidad de Picornavirus a un antiviral de origen vegetal (Meliacina) / Susceptibility of picornaviruses to an antiviral of vegetal origin (meliacine)

La planta superior Melia azederach L (MA) produce un polipéptido cíclico que puede aislarse de sus hojas y que tiene un amplio espectro antiviral contra virus con RNA de distintas familias. En este trabajo se estudió su efecto sobre dos Picornavirus: el virus fiebre aftosa (VFA) y el virus polio. Para ello se probaron las cepas A24, A87, C3 Resende, O1 Campos, O1 Caseros y O69 del VFA y los tipos 1, 2 y 3 del virus polio. Se encontró que las cepas muestran una susceptibilidad diferencial a una concentración no citotóxica del antiviral de 0,3 µg/ml, siendo las cepas A24 y A87 las más susceptibles ya que resultaron inhibidas en un 90 por ciento. Para que ello ocurra el extracto de MA debe agregarse después de la adsorción viral y conservarse en el medio de cultivo hasta la cosecha de virus ya que con todas las cepas ensayadas el pretratamiento de las monocapas no resultó efectivo. Para determinar la influencia de la multiplicidad de infección (m.i.) en la diferente susceptibilidad observada se eligió la cepa O1 Campos del VFA y la tipo 3 de polio que resultaron ser poco sensibles cuando se usó una m.i. de 1, encontrándose una inhibición del 99 por ciento para ambos virus a una m.i. de 0,001. Estos resultados indican que los Picornavirus también son susceptibles a la inhibición por meliacina, que dicha inhibición varía con la cepa ensayada y que la aparente resistencia de estos virus estaría relacionada con la velocidad del ciclo de replicación, el que es superior al establecimiento del estado antiviral, cuando todas las células son infectadas simultáneamente

Resistencia múltiple a metales pesados y a antibióticos en bacterias aisladas del riachuelo / [Multiple resistance to heavy metals and antibiotics in bacteria isolated from the Riachuelo]

Riachuelo is a heavy contaminated course of water, partially surrounding Buenos Aires city. The presence of bacteria resistant to antibiotics and heavy metals was studied. Among the isolated Gram positive colonies, 65, 59 and 48

were resistant to 60 micrograms/ml of Pb++, Zn++ and Cd++, respectively, and 20

grew in the presence of 50 micrograms/6ml gentamicin. Most of these microorganisms belonged to the order Actinomycetales. Accordingly, high percentages of resistance were detected (among the 11 Gram negative isolates (Enterobacteriaceae and Pseudomonaceae), although only one isolate was gentamicin resistant. Four Gram negative isolates also showed a broad spectrum of resistance to tetracycline, erythromyci, ampicillin, amikacin, chloramphenicol and gentamicin.

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 4§C y a 25§C. 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). Hemos comprobado la existencia de variaciones en la sobrevida de distintas cepas pertenecientes al biotipo El Tor y a Vibrio cholerae No O1 en las hojas de lechuga. La cepa CDC-185 resultó ser la más sensible al almacenamiento, en las condiciones experimentales usadas. La disminución de la relación ti/to de esta cepa se debe probablemente a su incapacidad de multiplicarse. A 25§C el Vibrio cholerae ejercería un efecto competitivo dominante sobre los demás microorganismos presentes y facilitaría de esta manera su supervivencia en la naturaleza. Lo mismo sucedería a 4§C, donde puede observarse el problema estallido de la flora acompañante en las hojas de lechuga inoculadas previamente con las cepas CDC-185 y Peruano hacia el día 7 post-contaminación, mientras que las cepas No O1 y 425 lograrían contener la multiplicación de las bacterias habituales en niveles constantes, esto indicaría un comportamiento diferencial en la estabilidad de las cepas analizadas. Para comprobar el grado de inactivación del vibrio cholerae por efecto del pH, hojas de lechuga contaminadas experimentalmente fueron tratadas con ácido acético 0,05 por ciento. La decontaminación fue efectiva ya que se obtuvo una inhibición de por lo menos 2 log en todos los casos ensayados