Discovery of potent broad spectrum antivirals derived from marine actinobacteria.

Natural products provide a vast array of chemical structures to explore in the discovery of new medicines. Although secondary metabolites produced by microbes have been developed to treat a variety of diseases, including bacterial and fungal infections, to date there has been limited investigation of natural products with antiviral activity. In this report, we used a phenotypic cell-based replicon assay coupled with an iterative biochemical fractionation process to identify, purify, and characterize antiviral compounds produced by marine microbes. We isolated a compound from Streptomyces kaviengensis, a novel actinomycetes isolated from marine sediments obtained off the coast of New Ireland, Papua New Guinea, which we identified as antimycin A1a. This compound displays potent activity against western equine encephalitis virus in cultured cells with half-maximal inhibitory concentrations of less than 4 nM and a selectivity index of greater than 550. Our efforts also revealed that several antimycin A analogues display antiviral activity, and mechanism of action studies confirmed that these Streptomyces-derived secondary metabolites function by inhibiting the cellular mitochondrial electron transport chain, thereby suppressing de novo pyrimidine synthesis. Furthermore, we found that antimycin A functions as a broad spectrum agent with activity against a wide range of RNA viruses in cultured cells, including members of the Togaviridae, Flaviviridae, Bunyaviridae, Picornaviridae, and Paramyxoviridae families. Finally, we demonstrate that antimycin A reduces central nervous system viral titers, improves clinical disease severity, and enhances survival in mice given a lethal challenge with western equine encephalitis virus. Our results provide conclusive validation for using natural product resources derived from marine microbes as source material for antiviral drug discovery, and they indicate that host mitochondrial electron transport is a viable target for the continued development of broadly active antiviral compounds.

Light-independent inactivation of dengue-2 virus by carboxyfullerene C3 isomer.

Carboxyfullerene (C60) is known as a photosensitizer for virus inactivation. Its regioisomer with C3 symmetry, named the C3 isomer, could also inactivate the dengue-2 virus without light when the dose of C3 isomer was increased to 40 microM, indicating the possible involvement of a light-independent mechanism. Further analysis showed that the C3 isomer blocked viral replication at the attachment and penetration stages, suggesting that a direct interaction between the C3 isomer and the virion is required for inactivation. The C3 isomer with a bipolar structure showed better lipid interaction and dengue-2 virus suppression than D3, another isomer that contains evenly distributed hydrophilic side chains. Moreover, the C3 isomer selectively inactivated enveloped viruses (viz., dengue-2 virus and Japanese encephalitis virus) instead of nonenveloped viruses (viz., enterovirus 71 and coxsackievirus B3). Collectively, these findings support the hypothesis that C3 isomer suppression of enveloped viruses is effected through its hydrophobic interaction with the viral lipid envelope. Our report, which demonstrates the light-dependent and -independent mechanisms of C60 on viral inactivation, will aid in the development of novel anti-viral agents for use against enveloped viruses.

Hyaluronidase enhances cell fusion and synthesis of viral DNA during infection with caprine arthritis encephalitis virus.

Caprine arthritis encephalitis virus (CAEV) is a lentivirus which infects goats and causes chronic progressive arthritis after a prolonged incubation period. CAEV replicates productively in cultures of goat synovial membrane cells and causes cytopathic effects characterized by multinucleated giant cell formation. The enzyme hyaluronidase was found to accelerate this virus induced fusion of GSM cells. Hyaluronidase treatment also resulted in synthesis of increased levels of unintegrated viral DNA early after infection. However, there was no significant increase in viral RNA in the infected cells or in the amount of virus produced. These studies suggest that hyaluronidase facilitates the interaction of CAEV with the target cells. Further it suggests that only a few copies of viral DNA are required to achieve maximal levels of virus replication. Additional copies of viral DNA appear to be redundant not contributing to viral specific transcription or increased production of virus.

A simple method for the inactivation of St. Louis encephalitis virus preparations for immunofluorescent microscopy.

A study was made of different treatments for the inactivation of St. Louis encephalitis virus in smears prepared for immunofluorescence microscopy. Treatment of infected cells with 0.3% betapropiolactone at 56 degrees C for 40 min resulted in an inactivated virus suitable for immunofluorescence studies.

An attenuated variant of Eastern encephalitis virus: biological properties and protection induced in mice.

Wild type Eastern equine encephalitis virus (E) was compared with a mutant (Em) derived from it. The latter was tested as an attenuated vaccine in mice. They differed in the following properties: Em formed smaller plaques on chick embryo (CE) cell monolayers and, unlike E, did not plaque on mouse embryo (ME) monolayers. Futher, Em had a longer latent period and attained a lower peak titer than E after infection of CE cells, was more senssitive than E to chick interferon, and was less virulent for mice (SC and IP routes) and hamsters (IP route) than E. Both viruses were similar in several other properties tested. The mutant was found to induce a gradient in the specificity of protection in mice against challenge by selected viruses after a single subcutaneous injection of living virus. The protection was best against autologous (Em) challenge, was next best against challenge by the virulent parent (E) virus, but was not demonstrable against cross challenge by Venezuelan encephalitis (V) virus. Conventional hemagglutination-inhibiting (HI), complement-fixing (F), and neutralizing (N) antibodies could not be detected in Em-immunized mice even when fresh monkey or guinea pig serum was included in Ntests to provide complement and/or accessory factor(s). However, N antibodies were detected in protected mice by an indirect antiglobulin test. Passive protection by serum or ascites fluids (a.f.) was characterized by a lower but otherwise similar protection gradient like that found after active immunization with virus as described above. Interferon was not detected in the a.f. used for passive protection, nor was heterologous interference evident in Em immunized mice challenged 18 days later with vaccinia or vesicular stomatitis virus. Immunized mice that survived autologous (Em) challenge showed broadened protection against a second challenge by parent E virus, and cross protection against V virus. This typical protection was associated with the presence of HI and conventional N antibodies, except for V which showed no detectable neutralizing antibodies by either a standard or antiglobulin technique.

Lead aggravates viral disease and represses the antiviral activity of interferon inducers.

Lead acetate was administered continuously in the drinking water to CD-1 male mice beginning at 4 weeks of age. An LD(10-20) of the lytic viruses or 300 plaque-forming units of RLV was inoculated intrapertioneally at 6 weeks of age. Lead increased the response of the mice to all classes of viruses against which it was tested: an RNA picornavirus-encephalomyocarditis (EMCV), a DNA herpesvirus-pseudoribies, an RNA leukemia-virus-Rauscher leukemia (RLV), an RNA arbovirus B-St. Louis encephalitis, and an RNA arbovirus A-western encephalitis. Most studies were performed between lead and EMCV. Increases in EMCV mortality in lead treated mice over controls ranged from 2x at a lead level of 0.004M to 7x (100% mortality) at a 0.1M lead level. Splenomegaly with spleens 800 to 1100 mg in weight containing high titers of RLV occurred in lead (0.03M)-treated mice 3 and 6 weeks after RLV inoculation; spleens or RLV controls were normal in weight (200 mg) and were free of virus. Lead did not reduce the protective effect of mouse interferon (IF) against the lethal action of EMCV, but it did repress the EMCV antiviral effect of poly I/poly C (PIC) and of Newcastle disease virus (NDV) against EMCV mortality. These data indicate several new facts concerning adverse effects lead may have on an animal: (1) lead aggravates viral disease, most likely in part, through reduced IF synthesis; (2) lead represses the anti-EMCV protective effects of both PIC and of NDV, which, in other reports, were shown to induce IF in radioresistant macrophages (PIC) or in radiosensitive lymphocytes (NDV); (3) lead may then be said to repress IF induction in two kinds of cells; (4) however, lead does not inhibit IF action.

Elimination of Mycoplasma contaminants from virus stocks by treatment with nonionic detergents.

Five nonionic detergents (Tweens 20, 40, 60, and 80, and Triton WR-1339) were tested for their ability to inactivate four Mycoplasma species which are common contaminants of animal cell cultures. Tween 20 was found to be the most effective, in that a concentration of 2.5 mg/ml completely inactivated cultures of M. hominis, M. hyorhinis, and Acholeplasma laidlawii within 1 hr and a culture of M. orale within 3 hr. The other detergents exhibited various degree of activity against the different mycoplasmas, with Triton WR-1339 being the least effective. The virucidal activity of the detergents was determined for six viruses. All four Tween compounds were highly virucidal for herpes simplex virus. Tween 20 also exhibited virucidal effects against vesicular stomatitis virus, California encephalitis virus, and Newcastle disease virus, and Tween 80 was found to be active against California encephalitis and Newcastle disease viruses. Detergent treatment procedures were effective in two instances in eliminating mycoplasma contaminants from virus preparations while the preparations retained most of the viral infectivity. The limitations of this technique for routine use are discussed.