An E460D Substitution in the NS5 Protein of Tick-Borne Encephalitis Virus Confers Resistance to the Inhibitor Galidesivir (BCX4430) and Also Attenuates the Virus for Mice.

The adenosine analogue galidesivir (BCX4430), a broad-spectrum RNA virus inhibitor, has entered a phase 1 clinical safety and pharmacokinetics study in healthy subjects and is under clinical development for treatment of Ebola and yellow fever virus infections. Moreover, galidesivir also inhibits the reproduction of tick-borne encephalitis virus (TBEV) and numerous other medically important flaviviruses. Until now, studies of this antiviral agent have not yielded resistant viruses. Here, we demonstrate that an E460D substitution in the active site of TBEV RNA-dependent RNA polymerase (RdRp) confers resistance to galidesivir in cell culture. Galidesivir-resistant TBEV exhibited no cross-resistance to structurally different antiviral nucleoside analogues, such as 7-deaza-2'-C-methyladenosine, 2'-C-methyladenosine, and 4'-azido-aracytidine. Although the E460D substitution led to only a subtle decrease in viral fitness in cell culture, galidesivir-resistant TBEV was highly attenuated in vivo, with a 100% survival rate and no clinical signs observed in infected mice. Furthermore, no virus was detected in the sera, spleen, or brain of mice inoculated with the galidesivir-resistant TBEV. Our results contribute to understanding the molecular basis of galidesivir antiviral activity, flavivirus resistance to nucleoside inhibitors, and the potential contribution of viral RdRp to flavivirus neurovirulence.IMPORTANCE Tick-borne encephalitis virus (TBEV) is a pathogen that causes severe human neuroinfections in Europe and Asia and for which there is currently no specific therapy. We have previously found that galidesivir (BCX4430), a broad-spectrum RNA virus inhibitor, which is under clinical development for treatment of Ebola and yellow fever virus infections, has a strong antiviral effect against TBEV. For any antiviral drug, it is important to generate drug-resistant mutants to understand how the drug works. Here, we produced TBEV mutants resistant to galidesivir and found that the resistance is caused by a single amino acid substitution in an active site of the viral RNA-dependent RNA polymerase, an enzyme which is crucial for replication of the viral RNA genome. Although this substitution led only to a subtle decrease in viral fitness in cell culture, galidesivir-resistant TBEV was highly attenuated in a mouse model. Our results contribute to understanding the molecular basis of galidesivir antiviral activity.

Hyaluronic Acid Surface Modified Liposomes Prepared via Orthogonal Aminoxy Coupling: Synthesis of Nontoxic Aminoxylipids Based on Symmetrically α-Branched Fatty Acids, Preparation of Liposomes by Microfluidic Mixing, and Targeting to Cancer Cells Expressing CD44.

New synthetic aminoxy lipids are designed and synthesized as building blocks for the formulation of functionalized nanoliposomes by microfluidization using a NanoAssemblr. Orthogonal binding of hyaluronic acid onto the outer surface of functionalized nanoliposomes via aminoxy coupling ( N-oxy ligation) is achieved at hemiacetal function of hyaluronic acid and the structure of hyaluronic acid-liposomes is visualized by transmission electron microscopy and cryotransmission electron microscopy. Observed structures are in a good correlation with data obtained by dynamic light scattering (size and ζ-potential). In vitro experiments on cell lines expressing CD44 receptors demonstrate selective internalization of fluorochrome-labeled hyaluronic acid-liposomes, while cells with down regulated CD44 receptor levels exhibit very low internalization of hyaluronic acid-liposomes. A method based on microfluidization mixing was developed for preparation of monodispersive unilamellar liposomes containing aminoxy lipids and orthogonal binding of hyaluronic acid onto the liposomal surface was demonstrated. These hyaluronic acid-liposomes represent a potentially new drug delivery platform for CD44-targeted anticancer drugs as well as for immunotherapeutics and vaccines.

Escape of Tick-Borne Flavivirus from 2'-C-Methylated Nucleoside Antivirals Is Mediated by a Single Conservative Mutation in NS5 That Has a Dramatic Effect on Viral Fitness.

Tick-borne encephalitis virus (TBEV) causes a severe and potentially fatal neuroinfection in humans. Despite its high medical relevance, no specific antiviral therapy is currently available. Here we demonstrate that treatment with a nucleoside analog, 7-deaza-2'-C-methyladenosine (7-deaza-2'-CMA), substantially improved disease outcomes, increased survival, and reduced signs of neuroinfection and viral titers in the brains of mice infected with a lethal dose of TBEV. To investigate the mechanism of action of 7-deaza-2'-CMA, two drug-resistant TBEV clones were generated and characterized. The two clones shared a signature amino acid substitution, S603T, in the viral NS5 RNA-dependent RNA polymerase (RdRp) domain. This mutation conferred resistance to various 2'-C-methylated nucleoside derivatives, but no cross-resistance was seen with other nucleoside analogs, such as 4'-C-azidocytidine and 2'-deoxy-2'-beta-hydroxy-4'-azidocytidine (RO-9187). All-atom molecular dynamics simulations revealed that the S603T RdRp mutant repels a water molecule that coordinates the position of a metal ion cofactor as 2'-C-methylated nucleoside analogs approach the active site. To investigate its phenotype, the S603T mutation was introduced into a recombinant TBEV strain (Oshima-IC) generated from an infectious cDNA clone and into a TBEV replicon that expresses a reporter luciferase gene (Oshima-REP-luc2A). The mutants were replication impaired, showing reduced growth and a small plaque size in mammalian cell culture and reduced levels of neuroinvasiveness and neurovirulence in rodent models. These results indicate that TBEV resistance to 2'-C-methylated nucleoside inhibitors is conferred by a single conservative mutation that causes a subtle atomic effect within the active site of the viral NS5 RdRp and is associated with strong attenuation of the virus.IMPORTANCE This study found that the nucleoside analog 7-deaza-2'-C-methyladenosine (7-deaza-2'-CMA) has high antiviral activity against tick-borne encephalitis virus (TBEV), a pathogen that causes severe human neuroinfections in large areas of Europe and Asia and for which there is currently no specific therapy. Treating mice infected with a lethal dose of TBEV with 7-deaza-2'-CMA resulted in significantly higher survival rates and reduced the severity of neurological signs of the disease. Thus, this compound shows promise for further development as an anti-TBEV drug. It is important to generate drug-resistant mutants to understand how the drug works and to develop guidelines for patient treatment. We generated TBEV mutants that were resistant not only to 7-deaza-2'-CMA but also to a broad range of other 2'-C-methylated antiviral medications. Our findings suggest that combination therapy may be used to improve treatment and reduce the emergence of drug-resistant viruses during nucleoside analog therapy for TBEV infection.

Antiviral activity of the adenosine analogue BCX4430 against West Nile virus and tick-borne flaviviruses.

There are currently no approved antiviral therapies against medically important human flaviviruses. The imino-C-nucleoside BCX4430 shows broad-spectrum antiviral activity against a wide range of RNA viruses. Here, we demonstrate that BCX4430 inhibits tick-borne species of the genus Flavivirus; however, the antiviral effect varies against individual species. Micro-molar BCX4430 levels inhibited tick-borne encephalitis virus (TBEV); while, approximately 3-8-fold higher concentrations were needed to inhibit louping ill virus and Kyasanur Forest disease virus. Moreover, the compound strongly inhibited in vitro replication of West Nile virus, a typical mosquito-transmitted flavivirus. Two chemical forms of the compound, i.e. BCX4430 and BCX4430 hydrochloride, were compared and both exerted similar inhibitory profiles in our in vitro antiviral assay systems and no or negligible cytotoxicity in porcine kidney stable and Vero cells. The obtained data indicate that, in addition to mosquito-borne flaviviruses, the compound has strong antiviral activity against members of the TBEV serocomplex.

Antiviral activities of 2,6-diaminopurine-based acyclic nucleoside phosphonates against herpesviruses: In vitro study results with pseudorabies virus (PrV, SuHV-1).

Pseudorabies virus (PrV), a causative agent of Aujeszky's disease, is deadly to most mammals with the exception of higher primates and men. This disease causes serious economic loses among farm animals, especially pigs, yet many European countries are today claimed to be Aujeszky's disease free because of the discovery of an efficient vaccination for pigs. In reality, the virus is still present in wild boar. Current vaccines are neither suitable for dogs nor are there anti-PrV drugs approved for veterinary use. Therefore, the disease still represents a high threat, particularly for expensive hunting dogs that can come into close contact with infected boars. Here we report on the anti-PrV activities of a series of synthetic diaminopurine-based acyclic nucleoside phosphonate (DAP-ANP) analogues. Initially, all synthetic DAP-ANPs under investigation are shown to exhibit minimal cytotoxicity by MTT and XTT tests (1-100µM range). Thereafter in vitro infection models are established using PrV virus SuHV-1, optimized on PK-15 and RK-13 cell lines. Out of the six DAP-ANP analogues tested, analogue VI functionalized with a cyclopropyl group on the 6-amino position of the purine ring proves the most effective antiviral DAP-ANP analogue against PrV infection, aided by sufficient hydrophobic character to enhance bioavailability to its cellular target viral DNA-polymerase. Four other DAP-ANP analogues with functional groups introduced to the C2'position are shown ineffective against PrV infection, even with favourable hydrophobic properties. Cidofovir(®), a drug approved against various herpesvirus infections, is found to exert only low activity against PrV in these same in vitro models.