Enhancement of varicella-zoster virus replication in cultured human embryonic lung cells treated with the pesticide carbaryl.

In studies designed to determine the factors responsible for control of herpesvirus replicaton in an infected cell, we examined the interaction of varicella-zoster (VZ) virus-infected human embryonic lung cells with the pesticide carbaryl. The replication of the cell-associated VZ virus was enhanced 2- to 13-fold as compared to control cultures in Sevin 4 Oil-treated cultures and in cultures treated with the pesticide's active ingredient, carbaryl. The replication of VZ virus in cultures teated with the base oil plus inert ingredients found in the pesticide formulation was not enhanced. Possible differences in cytotoxicity induced by Seven 4 Oil, pure carbaryl, or the base oil preparation were ruled out since treated and control cultures were shown to have similar numbers of viable cells when measured by trypan blue exclusion tests or by the ability of treated cells to form foci. A dose response study showed a decrease in viral enhancement in cells treated with decreasing carbaryl concentrations.     

Enhancement of goldfish virus-2 in vitro replication by the pesticides carbaryl and toxaphene

Goldfish virus-2 replication was enhanced in vitro by pretreatment of CAR cells with subcytotoxic concentrations of carbaryl and toxaphene. This phenomenon was time and temperature dependent. Shortening of pretreatment with carbaryl eliminated enhancement, which was observed for toxaphene only with substantially increased concentrations. Decreasing the temperature of pretreatment (4 degrees C) abrogated any enhancement by carbaryl and resulted in enhancement by toxaphene only at increased concentrations. Increased absorption of input virus was ruled out as a mechanism for enhancement, as was stimulation of cell division in the presence of pesticides over that of control cultures. Pretreatment of virus rather than cells did not result in enhancement.     

Enhancement of goldfish virus-2 in vitro replication by the pesticides carbaryl and toxaphene.

Goldfish virus-2 replication was enhanced in vitro by pretreatment of CAR cells with subcytotoxic concentrations of carbaryl and toxaphene. This phenomenon was time and temperature dependent. Shortening of pretreatment with carbaryl eliminated enhancement, which was observed for toxaphene only with substantially increased concentrations. Decreasing the temperature of pretreatment (4 degrees C) abrogated any enhancement by carbaryl and resulted in enhancement by toxaphene only at increased concentrations. Increased absorption of input virus was ruled out as a mechanism for enhancement, as was stimulation of cell division in the presence of pesticides over that of control cultures. Pretreatment of virus rather than cells did not result in enhancement.     

Characterization of varicella-zoster virus enhancement by the pesticide carbaryl.

Further characterization of the viral enhancement system of varicella-zoster virus by the pesticide carbaryl (1-naphthyl-N-methylcarbamate) is presented. It was necessary to expose cells to the enhancing chemical during the period of virus replication to detect enhancement. The optimum time for the pretreatment is 20 to 24 h. Maximum enhancement of virus expression occurs 48 to 72 h post-inoculation. Treated cells cannot pass on to daughter cells the ability to produce increased amounts of virus. alpha-Naphthol, a metabolite of carbaryl, is also capable of enhancing virus replication; Guthion, another pesticide tested, did not enhance varicella-zoster virus. The stable cell line HEP-2 can be used in place of human embryonic lung cells to detect enhancement. Differences in enhancement levels were not due to cell lot, cell passage, or a change in the stability of the cell membrane to sonic disruption.     

Characterization of varicella-zoster virus enhancement by the pesticide carbaryl

Further characterization of the viral enhancement system of varicella-zoster virus by the pesticide carbaryl (1-naphthyl-N-methylcarbamate) is presented. It was necessary to expose cells to the enhancing chemical during the period of virus replication to detect enhancement. The optimum time for the pretreatment is 20 to 24 h. Maximum enhancement of virus expression occurs 48 to 72 h post-inoculation. Treated cells cannot pass on to daughter cells the ability to produce increased amounts of virus. alpha-Naphthol, a metabolite of carbaryl, is also capable of enhancing virus replication; Guthion, another pesticide tested, did not enhance varicella-zoster virus. The stable cell line HEP-2 can be used in place of human embryonic lung cells to detect enhancement. Differences in enhancement levels were not due to cell lot, cell passage, or a change in the stability of the cell membrane to sonic disruption.     

Suppression of interferon synthesis by the pesticide carbaryl as a mechanism for enhancement of goldfish virus-2 replication

Interferon production was demonstrated by the goldfish-derived CAR cell line in response to infection by goldfish virus-2. Supernatants of infected cultures provided antiviral protection to CAR cells and another cell line derived from goldfish, ABIII. The protective factor retained activity after ultracentrifugation, dialysis, freezing and thawing, acid treatment (pH 2), or heating to 56 degrees C but was sensitive to trypsin. Supernatants of infected cultures did not affect adsorption of virus. Previous studies have shown that replication of goldfish virus type 2 is enhanced by pretreatment of cultures with subcytotoxic concentrations of carbaryl. In the present study, pesticide-treated cultures were found to synthesize reduced levels of interferon.     

Suppression of interferon synthesis by the pesticide carbaryl as a mechanism for enhancement of goldfish virus-2 replication.

Interferon production was demonstrated by the goldfish-derived CAR cell line in response to infection by goldfish virus-2. Supernatants of infected cultures provided antiviral protection to CAR cells and another cell line derived from goldfish, ABIII. The protective factor retained activity after ultracentrifugation, dialysis, freezing and thawing, acid treatment (pH 2), or heating to 56 degrees C but was sensitive to trypsin. Supernatants of infected cultures did not affect adsorption of virus. Previous studies have shown that replication of goldfish virus type 2 is enhanced by pretreatment of cultures with subcytotoxic concentrations of carbaryl. In the present study, pesticide-treated cultures were found to synthesize reduced levels of interferon.     

Spindle disturbances in mammalian cells. II. Induction of viable aneuploid/polyploid cells and multiple chromatid exchanges after treatment of V79 Chinese hamster cells with carbaryl. Modifying effects of glutathione and S9

Treatment of V79 cells with carbaryl increased the frequency of cells with elevated chromosome numbers (greater than 22). This effect of carbaryl was inhibited by simultaneous addition of glutathione or S9. Although selective forces seemed to act against cells with increased chromosome numbers, such cells were still significantly more frequent in treated compared with control cultures 74 h after treatment. In addition, a high adaptive value for these remaining aneuploid/polyploid cells was indicated because they were able to go through 2 successive rounds of replication immediately before fixation to the same extent as cells with chromosome numbers considered normal (21/22) for this cell line. Multiple chromatid exchanges were also observed after carbaryl treatment. The lack of single exchange events indicates that the effect may be systemic. However, additional experiments are required before this hypothesis can be confirmed or discarded. Considering the results obtained and the possible importance of gene dosage for tumor promotion, it is suggested that carbaryl should be tested for tumor promotion in vivo.     

Enhancement of Goldfish Virus Type 2 Replication by 1- Naphthol, the Major Hydrolysis Product of the Pesticide Carbaryl

The major hydrolysis product of the pesticide carbaryl, 1-naphthol, enhanced the replication of goldfish virus type 2 in piscine cell culture at concentrations below any detectable cytotoxicity. Enhancement indices of 2.3, 3.7, and 7.1 were observed at 1, 5, and 10 ppm (μl/liter), respectively. Replication at 0.5 ppm was equivalent to that of controls.     

Variation in human herpesvirus susceptibility to enhancement by the pesticide carbaryl

The ability of various human herpesviruses to be enhanced by the pretreatment of human embryonic lung cells with the pesticide carbaryl (1-naphthyl-N-methyl-carbamate) differs according to the virus tested. Different strains of varicella-zoster virus produced different patterns of susceptibility to enhancement. Laboratory-adapted strains were less sensitive to enhancement than were wild-type strains recently isolated from clinical specimens. The related human herpes simplex viruses types 1 and 2 and cytomegalovirus were negative for susceptibility to enhancement when either laboratory-adapted or wild-type strains were tested. No difference in the pattern of susceptibility was detected whether virus yields were determined by cell-associated or cell-free virus assay or when the input multiplicity was varied 10-fold.     

Variation in human herpesvirus susceptibility to enhancement by the pesticide carbaryl.

The ability of various human herpesviruses to be enhanced by the pretreatment of human embryonic lung cells with the pesticide carbaryl (1-naphthyl-N-methyl-carbamate) differs according to the virus tested. Different strains of varicella-zoster virus produced different patterns of susceptibility to enhancement. Laboratory-adapted strains were less sensitive to enhancement than were wild-type strains recently isolated from clinical specimens. The related human herpes simplex viruses types 1 and 2 and cytomegalovirus were negative for susceptibility to enhancement when either laboratory-adapted or wild-type strains were tested. No difference in the pattern of susceptibility was detected whether virus yields were determined by cell-associated or cell-free virus assay or when the input multiplicity was varied 10-fold.     

Cytomegalovirus Replication in Cells Pretreated with 5-Iodo-2′-Deoxyuridine

The replication of human cytomegalovirus (CMV) in cells pretreated with 5-iodo-2′-deoxyuridine (IUdR) was studied. Pretreatment of cells with IUdR enhanced several parameters of virus replication. Virus grown in drug-treated cells exhibited a shorter eclipse period and the cells produced more infectious virus sooner than did untreated cells. There was an approximate fivefold increase in virus yield per cell in the drug-treated samples when compared to control cultures. The time required for plaque development was shortened by 6 days in drug-treated cultures. Pretreatment of cells with IUdR also increased plaquing efficiency of the virus by approximately 10-fold. The enhancement of virus replication by IUdR was further demonstrated by varying the multiplicity of infection. In a 7-day period there was a 100-fold increase in sensitivity of the cultures for virus detection when the cells had been previously exposed to IUdR. The data presented indicate the possibility that IUdR interferes with the production of a cellular product inhibitory for CMV replication.     

Effect of cyclophosphamide in vitro and on vaccinia virus replication in tissue culture.

The effect of cyclophosphamide on the growth of Vero, BSC-1, and HeLa cells in monolayer cultures was studied. By using hemocytometer counts and tritiated thymidine uptake as indicators of growth, it was found that cyclophosphamide significantly interfered with the metabolism of Vero and BSC-1 cells when sustained in Leibovitz medium. Vero cells and HeLa cells grown in Eagle medium were not affected by exposure to cyclophosphamide. Vaccinia virus replication in Vero cell monolayer cultures incubated with cyclophosphamide was markedly augmented, and this enhanced growth was reflected by virus quantitation techniques and metabolic studies using tritiated thymidine uptake. No difference in the distribution of infectious particles was found when cyclophosphamide-treated and control infected cultures were compared. Pathways other than through hepatic enzymes appear available to activate cyclophosphamide in vitro. These effects are dependent on both the cell type and the medium in which the cells are grown. Cyclophosphamide can facilitate vaccinia virus replication in vitro through metabolic interactions at the cellular level. The precise mechanisms underlying this effect require further study.     

18beta-glycyrrhetinic acid inhibits rotavirus replication in culture

ABSTRACT: BACKGROUND: Glycyrrhizin (GA) and primary metabolite 18beta-glycyrrhetinic acid (GRA) are pharmacologically active components of the medicinal licorice root, and both have been shown to have antiviral and immunomodulatory properties. Although these properties are well established, the mechanisms of action are not completely understood. In this study, GA and GRA were tested for the ability to inhibit rotavirus replication in cell culture, toward a long term goal of discovering natural compounds that may complement existing vaccines. METHODS: Epithelial cells were treated with GA or GRA various times pre- or post-infection and virus yields were measured by immunofluorescent focus assay. Levels of viral proteins VP2, VP6, and NSP2 in GRA treated cells were measured by immunoblot to determine if there was an effect of GRA treatment on the accumulation of viral protein. RESULTS: GRA treatment reduced rotavirus yields by 99% when added to infected cultures post- virus adsorption, whereas virus yields in GA treated cultures were similar to mock treated controls. Time of addition experiments indicated that GRA-mediated replication inhibition likely occurs at a step or steps subsequent to virus entry. The amounts of VP2, VP6 and NSP2 were substantially reduced when GRA was added to cultures up to two hours post-entry. CONCLUSIONS: GRA, but not GA, has significant antiviral activity against rotavirus replication in vitro, and studies to determine whether GRA attenuates rotavirus replication in vivo are underway.     

Epstein-barr virus latently infected cells are selectively deleted in simulated-microgravity cultures

Summary Rotating-wall vessels (RWVs) allow for the cultivation of cells in simulated microgravity. Previously, we showed that the cultivation of lymphoblastoid cells in simulated microgravity results in the suppression of Epstein—Barr virus (EBV) reactivation. To determine if the suppression generated by simulated microgravity could be reversed by changing to static culture conditions, cells were cultured in an RWV for 5 d, and then switched to static conditions. Following the switch to static conditions, viral reactivation remained suppressed (significantly lower) relative to static control cultures over a 4-d period. Additionally, experiments were conducted to determine if chemical treatment could induce viral reactivation in cells from simulated-microgravity cultures. Cells were cultured in static flask cultures and in simulated microgravity in RWVs for 4–7 d. The cells were then transferred to 50-cm³ tubes, and treated with 3 mM n-butyrate for 48 h, or 18 ng/ml of phorbol ester, viz., 12-0-tetradecanoylphorbol-13 acetate (TPA) for either 2 or 48 h, under static conditions. Although EBV was inducible, the cells from simulated-microgravity cultures treated withn-butyrate displayed significantly lower levels of viral-antigen expression compared with the treated cells from static cultures. Also, incubation with TPA for 2–3 h, but not for 48 h, reactivated EBV in cells from RWV cultures. In contrast, EBV was inducible in cells from static cultures treated for either 2–3 or 48 h with TPA. TPA reactivation of EBV following a 2–3-h period of treatment indicates that the protein kinase C signal-transduction pathway is not impaired in lymphoblastoid cells cultured in simulated microgravity. However, the exposure of B-lymphoblastoid cells from simulated-microgravity cultures to TPA for more than 3–4 h triggered a lytic event (apoptosis or necrosis), which prevented replication of the virus. Thus, EBV-infected cells in simulated microgravity were negatively selected in the absence of any cytotoxic cells.     

T lymphocyte cytotoxicity with natural varicella-zoster virus infection and after immunization with live attenuated varicella vaccine

Varicella-zoster virus (VZV) specific cytotoxicity was investigated during acute primary VZV infection, in naturally immune subjects and after vaccination with the live attenuated varicella vaccine by using T cell cultures (TCC) generated by stimulating PBMC with VZV Ag and autologous VZV-superinfected lymphoblastoid cell lines as targets. Lysis of VZV-infected lymphoblastoid cell lines was observed by TCC from acutely infected subjects, naturally immune subjects, and recipients of the varicella vaccine. VZV glycoprotein I induced cytotoxic T cells but killing was less efficient than killing by TCC stimulated with VZV Ag. The TCC were primarily CD4+ (mean 86.6%) T lymphocytes with 15.2% of the cells coexpressing Leu-19. TCC were predominantly restricted by HLA class II as demonstrated by lack of any blocking using class I mAb and blocking of 15 to 71% by L243, a mAb to class II. Unrestricted killing as measured by killing of K562 cells occurred in all TCC but was minimally greater than that observed against uninfected autologous targets. Phenotypes of PBMC during acute infection had an initial increase in CD4+ cells and an overall decrease in the percentage of circulating Leu-11+ (CD16). No enhanced K562 killing was demonstrated in PBMC from subjects with acute infection compared to subjects without infection. CD4+ CTL may function as an important primary host response in acute varicella. Immunization with live attenuated varicella vaccine induced VZV-specific, memory CTL responses comparable to those of naturally immune subjects. The demonstration of their persistence long after primary VZV infection may indicate a role for CTL in restriction of viral replication during episodes of VZV reactivation from latency.     

The inhibitory effect of essential oils on herpes simplex virus type-1 replication in vitro

The antiviral effect of 12 essential oils on herpes simplex virus type-1 (HSV-1) replication was examined in vitro. The replication ability of HSV-1 was suppressed by incubation of HSV-1 with 1% essential oils at 4 C for 24 hr. Especially, lemongrass completely inhibited the viral replication even at a concentration of 0.1%, and its antiviral activity was dependent on the concentrations of the essential oil. When Vero cells were treated with the essential oil before or after viral adsorption, no antiviral activity was found, which suggests that the antiviral activity of essential oils including lemongrass may be due to the direct interaction with virions.