Celia E. Coto

Enterocin CRL35 inhibits late stages of HSV-1 and HSV-2 replication in vitro

The replication of herpes simplex virus (HSV) type 1 and 2 in Vero cells is inhibited in the presence of enterocin CRL35 (ECRL), a bacteriocin produced by Enterococcus faecium CRL35. Attempts to resolve the mode of action of ECRL indicate that virus adsorption and penetration are not affected. Instead, a late step of virus multiplication is hindered since the addition of 100 microg/ml of ECRL at 8h post infection still causes a 90% inhibition of virus release. The effect of ECRL on HSV antigen expression was studied by immunofluorescence using a polyclonal serum and a monoclonal antibody against glycoprotein D (gamma protein). These studies indicated that ECRL impeded the second round of infection, apparently as a consequence of the inhibition of glycoprotein D expression. The replication of syncytial mutants of HSV-1 was significantly inhibited at a ECRL concentration of 25 microg/ml. Both the percentage of fused cells and the polykaryocyte size were affected. Studies on the effect of ECRL on viral protein synthesis showed that in the presence of ECRL, HSV late gamma proteins were not synthesized. From these findings, it is concluded that inhibition of HSV spreading by ECRL is due to the prevention of mainly late glycoprotein synthesis.

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.

Treatment of arenavirus infections: from basic studies to the challenge of antiviral therapy.

Publisher Summary This chapter discusses the present state and perspectives of antiviral therapy for arenavirus infections in relation to the dramatic increase in virus emergence during the last decade. Because of the very high case-fatality rates in patients with Lassa fever, and the continuing appearance of new viruses in North and South America, including agents responsible for severe human infections, control through chemotherapy warrants special attention. Arenaviruses are enveloped, single-stranded, ambisense RNA viruses with a segmented genome consisting of two segments, designated large (L) and small (S). They are rodent-associated viruses with one exception, Tacaribe virus (TCRV), which infects bats of the genus Artibeus . They are zoonotic agents that can cause severe human diseases, known as the hemorrhagic fevers, occurring in regions of South America. The replicative cycle of arenaviruses comprises a number of steps that could be considered adequate targets for chemotherapeutic intervention. Numerous compounds have been reported to inhibit the replication of arenaviruses acting at the early stages of attachment and entry, the biosynthetic processes of replication and transcription, or the late steps of maturation, exocytosis, and budding. Numerous efforts have been devoted to obtain safe vaccines for protecting the population against hemorrhagic fever agents.

An antiviral factor from Melia azedarach L. prevents Tacaribe virus encephalitis in mice.

Treatment of neonatal mice with an antiviral factor, (AVF), obtained from the leaves ofMelia azederach L. protected them against lethal encephalitis caused by Tacaribe virus inoculation. The degree of protection obtained varied from 66% to 100% depending on the virus dose. Similarly, administration of AVF to nursing mothers protected their offspring from developing virus encephalitis. AVF does not directly inactivate Tacaribe virus; it inhibits an early step (s) in the replication process in cell cultures.

Response of cells persistently infected with arenaviruses to superinfection with homotypic and heterotypic viruses

Vero cell cultures persistently infected with the arenaviruses Junin, Pichinde, Tacaribe, and Tamiami were established and designated Vero-Jun, Vero-Pic, Vero-Tac, and Vero-Tam, respectively. Two types of carrier cultures could be easily distinguished: Vero-Jun and Vero-Tac systems were characterized by a lack of infectious virus production after a few cell transfers, whereas a more productive state with continuous release of virus was observed in Vero-Pic and Vero-Tam cultures. These differences appeared to be related to resistance of the culture to viral superinfection. In fact, Vero-Jun and Vero-Tac cultures totally excluded only the replication of the serologically more closely related arenaviruses Amapari, Junin, or Tacaribe, while the refractoriness of Vero-Pic and Vero-Tam cultures was extended to most of the virus group members. The resistance of Vero-Jun cells to superinfection by Junin or Tacaribe virus could be ascribed to the production of specific uv-resistant Junin interfering particles, which showed a specific range of interference against Junin and Tacaribe viruses. Interfering particles against homotypic and heterotypic arenaviruses were isolated from Vero-Pic cultures. However, the degree of interference developed by these Pic-interfering particles was not enough to fully explain reinfecting virus exclusion from Vero-Pic cultures. Viral susceptibility of persistent cultures is proposed as a useful tool to examine relationships of members of the arenavirus group.

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

SummaryMeliacine (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 °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.

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.

In vitro anti-junin virus activity of a peptide isolated from Melia azedarach L. leaves

Meliacine, a peptide isolated from leaves of Melia azedarach L. inhibited the multiplication of Junin virus in Vero cells treated with the compound before infection (pre-treatment) or immediately after virus adsorption. Analysis of early events following infection demonstrated that meliacine blocks virus penetration by preventing the uncoating step. The addition of meliacine at different times after infection indicated that meliacine also interferes with the release of infectious particles to the extracellular medium and inhibits the low-pH-induced fusion of infected cells. Intracellular transport of viral glycoproteins to the cell membrane was not affected by meliacine, as revealed by immunofluorescence staining. Taken together, these results suggest that meliacine affects two events of the virus replicative cycle that require membrane fusion: uncoating and budding.

Therapeutic effect of meliacine, an antiviral derived from Melia azedarach L., in mice genital herpetic infection

Since natural products are considered powerful sources of novel drug discovery, a partially purified extract (meliacine) from the leaves of Melia azedarach L., a plant used in traditional medicine in India for the treatment of several diseases, has been studied. Meliacine exhibits a potent antiviral effect against several viruses without displaying cytotoxicity. The purpose of the present study was to evaluate the therapeutic effect of intravaginal administration of meliacine in a mouse model of genital herpetic infection. BALB/c female mice were infected with MS or G strains of Herpes Simplex Virus type 2 and then treated with meliacine topically. An overall protective effect was observed. Animal survival increased, the severity of the disease was reduced, life span was extended and virus shedding in vagina fluids was diminished. In addition, meliacine reduced the amount of virus that migrated to the brain and vaginal fluids presented higher levels of IFN‐γ and TNF‐α than untreated infected mice. These results indicate that meliacine could be an alternative therapeutic compound against HSV‐2 genital infection. Copyright

Induction of a refractory state to viral infection in mammalian cells by a plant inhibitor isolated from leaves of Melia azedarach L

A partially purified plant inhibitor (Meliacin) isolated from Melia azedarach L induced in cells a refractory state to virus infection. Meliacin was active in a large variety of continuous and/or primary cell cultures. A state of maximum virus resistance was achieved after 2 h of incubation and was maintained for at least 15 h; later on it declined but it was fully regained after a second pulse of Meliacin. Interferon was not detected in the supernatant of cells treated with Meliacin and a measurable increase in ds-RNA dependent protein kinase activity was not observed in extracts of Meliacin-treated cells. The antiviral state was not transferred by either extracellular fluid or direct cell-to-cell contact. An active cell metabolism was required for Meliacin action, which was partially reversed in the presence of actinomycin D. It appears that Meliacin is not an interferon-like substance, which induces an antiviral state based on a still unexplained mechanism.