Virucidal effect of lipids on visna virus, a lentivirus related to HIV.

Natural lipids and fatty alcohols show virucidal activities against enveloped viruses. A virucidal profile of these compounds against visna virus (VV), a lentivirus related to HIV, or against other viruses of the genus Lentivirus has not been established before and could help elucidate how lipids inactivate enveloped viruses and assist in the development of virucidal drugs. The activity profile for VV may not exactly reflect the profile for HIV or for the lentivirus subgroup in general, but the results for VV are in agreement with earlier studies, which have shown that lipids become generally more virucidal at low pH.

Deaza- and deoxyadenosine derivatives: synthesis and inhibition of animal viruses as human infection models.

N6-Cycloalkyl-2',3'-dideoxyadenosine derivatives and (2-chloro)-N6-cycloheptyl-3-deazaadenosine have been synthesized and tested, along with other (deaza)purine (deoxy)nucleosides from our chemical library, as inhibitors of virus replication against Bovine Herpes Virus 1 (BHV-1) and sheep Maedi/Visna Virus (MVV). Most compounds demonstrated good antireplicative activity against MVV, showing also low cell toxicity.

Inhibition of nitric oxide enhances ovine lentivirus replication in monocyte-derived macrophages.

Ovine lentivirus (OvLV) also known as maedi-visna virus, infects and replicates primarily in macrophages. This investigation examined the role of nitric oxide in the replication of OvLV in cultured macrophages. Peripheral blood mononuclear cells were collected from OvLV-free sheep and cultured in Teflon coated flasks at a high concentration of lamb serum. The cells were subsequently infected with OvLV strain 85/34. OvLV replication was assessed under different experimental treatments by comparison of reverse transcriptase (RT) activity in culture supernatant. Cultures that were treated with exogenous nitric oxide via S-nitroso-acetylpenicillamine did not have altered levels of RT activity compared to cultures treated with the inactive control compound, acetylpenicillamine. However, blockage of nitric oxide production by treatment with aminoguanidine, a competitive inhibitor of inducible nitric oxide synthase (iNOS), led to a significant rise in RT activity. This rise in RT activity was partially reversed in aminoguanidine treated cultures by L-arginine, the normal substrate for iNOS. Finally, the number of viral antigen producing cells was also quantified after aminoguanidine treatment and found to be significantly higher than untreated cultures. Collectively, these results indicate that nitric oxide is a negative regulator of OvLV replication in macrophages.

Granulocyte macrophage colony stimulating factor is elevated in alveolar macrophages from sheep naturally infected with maedi-visna virus and stimulates maedi-visna virus replication in macrophages in vitro.

Infection by maedi-visna virus, a lentivirus of sheep, leads to chronic inflammatory reactions of various tissues. In this report we have analysed the role of specific cytokines in the disease process. A significant increase in expression of interleukin-6, interleukin-10, granulocyte macrophage-colony stimulating factor (GM-CSF) and transforming growth factor-beta1 mRNA was observed in alveolar macrophages isolated from the lungs of naturally infected animals when compared with lungs of seronegative controls. Levels of GM-CSF mRNA expression in alveolar macrophages correlated with the presence of lung lesions, but there was no correlation of interleukin-10, interleukin-6, tumour necrosis factor-alpha and transforming growth factor-beta1 mRNA levels in alveolar macrophages from animals with pulmonary lesions. In vitro investigation showed that GM-CSF in the range 0.1-10 ng/ml induced a significant increase in viral p25 production after 7 days in acutely infected blood monocyte-derived macrophages. The production of p25 peaked between 7 and 14 days exposure to 10 ng/ml of GM-CSF. Quantitative polymerase chain reaction showed that the level of viral DNA in monocyte-derived macrophages was dose-dependent following GM-CSF treatment in the range 0.1-100 ng/ml after 7 days. Viral mRNA expression was also enhanced. These findings indicate a role for GM-CSF in the pathogenesis of lymphoid interstitial pneumonia in infected animals.

Adenine and deazaadenine nucleoside and deoxynucleoside analogues: inhibition of viral replication of sheep MVV (in vitro model for HIV) and bovine BHV-1.

A series of N(6)-cycloalkyl-2',3'-dideoxyadenosine derivatives has been prepared by coupling of 2,6-dichloropurine to protected 2,3-dideoxyribose, followed by reaction with appropriate cycloalkylamines. Synthesized compounds, along with other purine nucleoside analogues previously synthesized in our laboratory, have been tested for their antiviral activity against Bovine herpesvirus 1 (BHV-1) and sheep Maedi/Visna Virus (MVV), the latter being an in vitro and in vivo model of Human Immunodeficiency Virus (HIV). All compounds showed good antireplicative activity against MVV, with the N(6)-cycloheptyl-2',3'-dideoxyadenosine (5b) being the most active [effective concentration (EC(50)) causing 50% reduction of cytopatic effects (CPE)=27 nM]. All compounds showed also a from low to very low cell toxicity, resulting in a cytotoxic dose 50 (CD(50))/EC(50) ratio in some cases higher than 1000.

Visna virus-induced activation of MAPK is required for virus replication and correlates with virus-induced neuropathology.

It is well accepted that viruses require access to specific intracellular environments in order to proliferate or, minimally, to secure future proliferative potential as latent reservoirs. Hence, identification of essential virus-cell interactions should both refine current models of virus replication and proffer alternative targets for therapeutic intervention. In the present study, we examined the activation states of mitogen-activated protein kinases (MAPKs), ERK-1/2, in primary cells susceptible to visna virus and report that virus infection induces and sustains activation of the ERK/MAPK pathway. Treatment of infected cells with PD98059, a specific inhibitor of the ERK/MAPK pathway, abolishes visna virus replication, as evidenced by extremely low levels of Gag protein expression and reverse transcriptase activity in culture supernatants. In addition, although visna virus-induced activation of MAPK is detectable within 15 min, early events of viral replication (i.e., reverse transcription, integration, and transcription) are largely unaffected by PD98059. Interestingly, further examination demonstrated that treatment with PD98059 results in decreased cytoplasmic expression of gag and env, but not rev, mRNA, highly suggestive of an ERK/MAPK-dependent defect in Rev function. In vivo analysis of ERK-1/2 activation in brains derived from visna virus-infected sheep demonstrates a strong correlation between ERK/MAPK activation and virus-associated encephalitis. Moreover, double-labeling experiments revealed that activation of MAPK occurs not only in cells classically infected by visna virus (i.e., macrophages and microglia), but also in astrocytes, cells not considered to be major targets of visna virus replication, suggesting that activation of the ERK/MAPK pathway may contribute to the virus-induced processes leading to neurodegenerative pathology.

Inactivation of visna virus and other enveloped viruses by free fatty acids and monoglycerides.

Human milk was found to become antiviral during storage at 4 degrees C because of the release of fatty acids by lipolysis. The stored milk caused more than a 10,000-fold inactivation of visna virus during incubation at 37 degrees C for 30 minutes. Medium-chain saturated and long-chain unsaturated fatty acids inactivated visna virus and other enveloped viruses causing more than a 3000-fold to 10,000-fold reduction in virus titer. 1-Monoglycerides and ethers of medium-chain fatty acids were more antiviral than the corresponding free fatty acids. Antiviral fatty acids were found to affect the viral envelope, causing leakage and, at higher concentrations, a complete disintegration of the envelope and the viral particles. Lipids commonly found in natural products could possibly be used as antiviral agents against enveloped viruses.

Inhibition of visna virus replication by 2',3'-dideoxynucleosides and acyclic nucleoside phosphonate analogs.

A series of acyclic nucleoside phosphonate (ANP) and 2',3'-dideoxynucleoside (ddN) derivatives were evaluated for their inhibitory effects on visna virus replication and maedi/visna virus-induced syncytium formation in sheep choroid plexus cells. Most ANP derivatives inhibited virus replication and syncytium formation within a concentration range of 0.2 to 1.8 microM. Among the most active ANP derivatives ranked (R)-9-(2-phosphonomethoxypropyl)adenine, (R)-9-(2-phosphonomethoxypropyl)-2,6-diaminopurine, and (S)-9-(3-fluoro-2-phosphonomethoxypropyl)adenine. Of the ddN derivatives, 2',3'-dideoxycytidine (ddCyd) proved to be the most inhibitory to visna virus-induced syncytium formation (50% effective concentration, 0.02 microM). The purine ddN analogs (i.e., 2',3'-dideoxyinosine, 2',3'-dideoxyadenosine, 2',3'-dideoxyguanosine, and 2,6-diaminopurine-2',3'-dideoxyribosine) were 10- to 30-fold less effective, and the thymidine derivatives 2',3'-didehydro-2',3'-dideoxythymidine (D4T) and 3'-azido-2',3'-dideoxythymidine (AZT) were more than 500-fold less inhibitory to visna virus than ddCyd. The 5'-triphosphate forms of AZT and D4T were 100- to 600-fold more inhibitory to visna virus particle-derived reverse transcriptase than was the 5'-triphosphate of ddCyd. The apparent discrepancy between the inhibitory effects of these ddN derivatives on virus replication and viral reverse transcriptase activity most likely reflects differences in the metabolic conversion of ddCyd versus D4T and AZT in sheep choroid plexus cells.

Antiviral activities of 2'-deoxyribofuranosyl and arabinofuranosyl analogs of sangivamycin against retro- and DNA viruses.

Eight sugar-modified pyrrolopyrimidine nucleoside analogs related to the antibiotic sangivamycin were evaluated in cell culture against herpes simplex types 1 (HSV-1) and 2 (HSV-2), cytomegalovirus (CMV), adenovirus, and visna virus. Five of the compounds were highly active against most of the viruses with 50% inhibition (ED50) values of 1-10 microM. The selectivity of the agents was low, with inhibition of uninfected cell proliferation occurring within 5-fold that of the virus ED50 for most of the viruses. The compounds did not possess RNA virus-inhibitory activity when evaluated against certain myxo-, paramyxo-, picorna-, reo-, rhabdo-, and togaviruses. Two of the nucleosides were tested further in a cell line persistently infected with Friend leukemia virus where they were inhibitory to both virus yield and cell proliferation at 4-5 microM. Several of the sangivamycin analogs were tested in animal models using a twice-a-day treatment regimen. They proved to be inactive against HSV-1, murine CMV and/or Friend leukemia virus infections in mice.

Visna virus as an in vitro model for human immunodeficiency virus and inhibition by ribavirin, phosphonoformate, and 2',3'-dideoxynucleosides.

Inhibition of visna virus replication in vitro by several compounds previously reported to inhibit replication of human immunodeficiency virus (HIV) was examined. Ribavirin concentrations as high as 1 mM reduced virus production by less than 50% relative to controls. The concentration of phosphonoformate reducing virus replication by 50% was 80 microM. 2',3'-Dideoxynucleosides were potent inhibitors of visna virus replication. The 50% inhibitory concentrations for dideoxyguanosine, dideoxyadenosine, and dideoxycytidine were 0.1, 0.2, and 0.3 microM, respectively. In contrast, weak inhibition was produced by 100 microM dideoxythymidine. These results are consistent with the reported susceptibility of HIV replication to inhibition by these compounds in vitro. The interaction of visna virus reverse transcriptase with several inhibitors was also examined. Reverse transcriptase was inhibited by phosphonoformate, ribavirin 5'-triphosphate, ddATP, ddCTP, ddGTP, and ddTTP. The last four compounds inhibited incorporation of homologous 2'-deoxynucleoside 5'-triphosphates into polynucleotides by a competitive mechanism. In view of the biological similarities between visna virus and HIV and the similar in vitro susceptibility of visna virus replication to known inhibitors of HIV, visna virus may provide a good model for studying the inhibition of HIV replication in vitro. Because visna virus is not pathogenic to humans, this model may facilitate the identification of compounds for further investigation into the treatment of HIV-induced disease.