Homoseongomycin, a compound isolated from marine actinomycete bacteria K3-1, is a potent inhibitor of encephalitic alphaviruses.

Marine microorganisms have been a resource for novel therapeutic drugs for decades. In addition to anticancer drugs, the drug acyclovir, derived from a marine sponge, is FDA-approved for the treatment of human herpes simplex virus-1 infections. Most alphaviruses that are infectious to terrestrial animals and humans, such as Venezuelan and eastern equine encephalitis viruses (VEEV and EEEV), lack efficient antiviral drugs and it is imperative to develop these remedies. To push the discovery and development of anti-alphavirus compounds forward, this study aimed to isolate and screen for potential antiviral compounds from cultured marine microbes originating from the marine environment. Compounds from marine microbes were of interest as they are prolific producers of bioactive compounds across the spectrum of human diseases and infections. Homoseongomycin, an actinobacteria isolated from a marine sponge displayed impressive activity against VEEV from a total of 76 marine bioactive products. The 50% effective concentration (EC50) for homoseongomycin was 8.6 µM for suppressing VEEV TC-83 luciferase reporter virus replication. Homoseongomycin was non-toxic up to 50 µM and partially rescued cells from VEEV induced cell death. Homoseongomycin exhibited highly efficient antiviral activity with a reduction of VEEV infectious titers by 8 log10 at 50 µM. It also inhibited EEEV replication with an EC50 of 1.2 µM. Mechanism of action studies suggest that homoseongomycin affects both early and late stages of the viral life cycle. Cells treated with 25 µM of homoseongomycin had a ~90% reduction in viral entry. In comparison, later stages showed a more robust reduction in infectious titers (6 log10) and VEEV extracellular viral RNA levels (4 log10), but a lesser impact on intracellular viral RNA levels (1.5 log10). In sum, this work demonstrates that homoseongomycin is a potential anti-VEEV and anti-EEEV compound due to its low cytotoxicity and potent antiviral activity.

Review of antiviral peptides for use against zoonotic and selected non-zoonotic viruses.

Viruses remain one of the leading causes of animal and human disease. Some animal viral infections spread sporadically to human populations, posing a serious health risk. Particularly the emerging viral zoonotic diseases such as the novel, zoonotic coronavirus represent an actual challenge for the scientific and medical community. Besides human health risks, some animal viral infections, although still not zoonotic, represent important economic loses to the livestock industry. Viral infections pose a genuine concern for which there has been an increasing interest for new antiviral molecules. Among these novel compounds, antiviral peptides have been proposed as promising therapeutic options, not only for the growing body of evidence showing hopeful results but also due to the many adverse effects of chemical-based drugs. Here we review the current progress, key targets and considerations for the development of antiviral peptides (AVPs). The review summarizes the state of the art of the AVPs tested in zoonotic (coronaviruses, Rift Valley fever viruses, Eastern Equine Encephalitis Virus, Dengue and Junín virus) and also non-zoonotic farm animal viruses (avian and cattle viruses). Their molecular target, amino acid sequence and mechanism of action are summarized and reviewed. Antiviral peptides are currently on the cutting edge since they have been reported to display anti-coronavirus activity. Particularly, the review will discuss the specific mode of action of AVPs that specifically inhibit the fusion of viral and host-cell membranes for SARS-CoV-2, showing in detail some important features of the fusion inhibiting peptides that target the spike protein of these risky viruses.

Efficacy of a ML336 derivative against Venezuelan and eastern equine encephalitis viruses.

Currently, there are no licensed human vaccines or antivirals for treatment of or prevention from infection with encephalitic alphaviruses. Because epidemics are sporadic and unpredictable, and endemic disease is common but rarely diagnosed, it is difficult to identify all populations requiring vaccination; thus, an effective post-exposure treatment method is needed to interrupt ongoing outbreaks. To address this public health need, we have continued development of ML336 to deliver a molecule with prophylactic and therapeutic potential that could be relevant for use in natural epidemics or deliberate release scenario for Venezuelan equine encephalitis virus (VEEV). We report findings from in vitro assessments of four analogs of ML336, and in vivo screening of three of these new derivatives, BDGR-4, BDGR-69 and BDGR-70. The optimal dosing for maximal protection was observed at 12.5 mg/kg/day, twice daily for 8 days. BDGR-4 was tested further for prophylactic and therapeutic efficacy in mice challenged with VEEV Trinidad Donkey (TrD). Mice challenged with VEEV TrD showed 100% and 90% protection from lethal disease when treated at 24 and 48 h post-infection, respectively. We also measured 90% protection for BDGR-4 in mice challenged with Eastern equine encephalitis virus. In additional assessments of BDGR-4 in mice alone, we observed no appreciable toxicity as evaluated by clinical chemistry indicators up to a dose of 25 mg/kg/day over 4 days. In these same mice, we observed no induction of interferon. Lastly, the resistance of VEEV to BDGR-4 was evaluated by next-generation sequencing which revealed specific mutations in nsP4, the viral polymerase.

Effect of triethylamine on the recovery of selected South American alphaviruses, flaviviruses, and bunyaviruses from mosquito (Diptera: Culicidae) pools.

We evaluated the effect of triethylamine (TEA) on the recovery of infectious virus from pools of mosquitoes for two South American alphaviruses (eastern equine encephalomyelitis and Venezuelan equine encephalomyelitis subtypes IIIC and ID), one flavivirus (Ilheus) and two bunyaviruses (Mirim [Guama group] and Itaqui [group C]). Mosquitoes were inoculated intrathoracically with virus, held for 7-10 d at 26 degrees C, and handled under one of four regimens before testing for the presence of virus by plaque assay. Mosquitoes were killed by freezing at - 70 degrees C for 3 min and tested immediately for the presence of virus; killed by freezing at -70 degrees C for 3 min and then held at room temperature for 1 h before testing for the presence of virus; anesthetized with TEA and assayed immediately for the presence of virus; or anesthetized with TEA and then held at room temperature for 1 h before being assayed for the presence of virus. For each of the viruses tested, viral titers in mosquitoes anesthetized with TEA were similar to those in mosquitoes killed by freezing at-70 degrees C. Likewise, there was no significant difference in viral titers in mosquitoes anesthetized with TEA and held at room temperature for 1 h or in mosquitoes frozen at -70 degrees C and held at room temperature for 1 h before being processed for virus by isolation. Triethylamine is advantageous for the handling of mosquitoes in a field environment. The elimination of the need for a cold chain, without compromising virus recovery, increases the feasibility of conducting research projects requiring the isolation of live virus from mosquitoes in remote tropical environments.

[Ts mutants of the Eastern equine encephalomyelitis virus. Their generation, isolation and preliminary characterization]. / Ts-mutanty virusa vostochnogo éntsefalomielita loshadei. Poluchenie, vydelenie, predvaritel'naia kharakteristika.

Chick embryo fibroblast cultures of the C/O phenotype (leukemia--free) infected with eastern equine encephalomyelitis (EEE) virus were incubated in the presence of 15 micrograms/ml N-methyl-N-nitro-N-nitrosoguanidine in the culture medium. Seven (5%) temperature-sensitive mutants were isolated from cell homogenates only in those cases where cell cultures before infection had been treated with actinomycin D. The recovered ts mutants are characterized by the marked ts- phenotype and genetic stability. The method of obtaining EEE virus ts mutants under the effect of N-methyl-N-nitro-N-nitrosoguanidine in C/O phenotype (leukemia-free) chick embryo fibroblast cultures treated before virus inoculation with actinomycin D is discussed.

[Induction of alphavirus ts-mutations by N-methyl-N-nitro-N-nitrosoguanidine added to the replicating virus]. / Induktsiia ts-mutatsii al'favirusov N-metil-N-nitro-N-nitrozoguanidinom pri vozdeistvii na replitsiruiushchiisia virus.

The possibility of producing ts mutants of Sindbis and eastern equine encephalomyelitis (EEE) virus by treatment of replicating virus with N-methyl-N-nitro-N-nitrosoguanidine was studied. N-methyl-N-nitro-N-nitrosoguanidine added in a concentration of 20 micrograms/ml to chick fibroblast cultures infected with Sindbis virus for 4 hours was shown to induce ts mutations in the virus. Under similar conditions no ts mutants of EEE virus could be obtained. The content of ts mutants in the mutagenized populations of Sindbis virus was 7.9%. Eight ts mutants were isolated. Their temperature-sensitive defect was expressed to various degrees. The plaque-forming efficiency at 40 degrees C/35 degrees C ranged from 6.3 X 10(-2) to 1.7 X 10(-8), and the yield from 5.6 X 10(-2) to 2.8 X 10(-4).

[Molecular biology characteristics of an attenuated mutant of the Eastern equine encephalomyelitis virus]. / Molekuliarno-biologicheskaia kharakteristika attenuirovannogo mutanta virusa vostochnogo éntsefalomielita loshadei.

The main molecular biology parameters of an attenuated mutant DMS-20/6 of eastern equine encephalomyelitis virus derived by treatment with dimethylsulphate of the wild type virus (strain No. 627) were determined. The sedimentation coefficient of sucrose density gradient purified and concentrated virus was 280 S, the buoyant density of virions in sucrose density gradient was 1.19 g/cm3. The DMS-20/6 virion had 3 proteins with molecular weights of 56, 50, and 34 kilodaltons, and the size of virions by negative staining was 58-77 nm.

Preparation and testing of vaccines prepared from the envelopes of Venezuelan, eastern, and western equine encephalomyelitis viruses.

Envelope components were separated from Venezuelan, Eastern, and Western equine encephalomyelitis viruses after treatment of the virions with detergent. Vaccines prepared from the envelope component were capable of stimulating mice to produce humoral antibodies. Protective efficacy studies were performed using mono-, di-, and trivalent vaccine combinations. These elicited varying degrees of homologous protection, and Eastern and Venezuelan equine encephalomyelitis envelope products appeared to confer protection to mice challenged with Western equine encephalomyelitis virus.