Favipiravir treatment prolongs the survival in a lethal mouse model intracerebrally inoculated with Jamestown Canyon virus.

BACKGROUND: Jamestown Canyon virus (JCV) is a mosquito-borne orthobunyavirus that causes acute febrile illness, meningitis, and meningoencephalitis, primarily in North American adults. Currently, there are no available vaccines or specific treatments against JCV infections. METHODOLOGY/PRINCIPAL FINDINGS: The antiviral efficacy of favipiravir (FPV) against JCV infection was evaluated in vitro and in vivo in comparison with that of ribavirin (RBV) and 2'-fluoro-2'-deoxycytidine (2'-FdC). The in vitro inhibitory effect of these drugs on JCV replication was evaluated in Vero and Neuro-2a (N2A) cells. The efficacy of FPV in the treatment of JCV infection in vivo was evaluated in C57BL/6J mice inoculated intracerebrally with JCV, as per the survival, viral titers in the brain, and viral RNA load in the blood. The 90% inhibitory concentrations (IC90) of FPV, RBV, and 2'-FdC were 41.0, 61.8, and 13.6 µM in Vero cells and 20.7, 25.8, and 8.8 µM in N2A cells, respectively. All mice infected with 1.0×104 TCID50 died or were sacrificed within 10 days post-infection (dpi) without treatment. However, mice treated with FPV for 5 days [initiated either 2 days prior to infection (-2 dpi-2 dpi) or on the day of infection (0 dpi-4 dpi)] survived significantly longer than control mice, administered with PBS (p = 0.025 and 0.011, respectively). Moreover, at 1 and 3 dpi, the virus titers in the brain were significantly lower in FPV-treated mice (0 dpi-4 dpi) versus PBS-treated mice (p = 0.002 for both 1 and 3 dpi). CONCLUSIONS/SIGNIFICANCE: Although the intracerebral inoculation route is thought to be a challenging way to evaluate drug efficacy, FPV inhibits the in vitro replication of JCV and prolongs the survival of mice intracerebrally inoculated with JCV. These results will enable the development of a specific antiviral treatment against JCV infections and establishment of an effective animal model.

The variability of the large genomic segment of Tahyna orthobunyavirus and an all-atom exploration of its anti-viral drug resistance.

Tahyna virus (TAHV), a member of the Bunyaviridae family (California complex), is an important but neglected human mosquito-borne pathogen. The virus genome is composed of three segments, i.e., small (S), medium (M), and large (L). Previous studies on genetic variability of viruses within the California complex were focused on S and M segments, but the L segment remains relatively unstudied. To assess the genetic variation and the relation to virus phenotype we analyzed the L segment sequences of biologically diverse TAHV strains isolated in the Czech Republic and Slovakia. Phylogenetic analysis covering all available sequences of the L segment of TAHV clearly revealed two distinguished lineages, tentatively named as "European" and "Asian". The L segment strains within the European lineage are highly conserved (identity 99.3%), whilst Asian strains are more genetically diverse (identity 97%). Based on sequence comparison with other bunyaviruses, several non-synonymous nucleotide substitutions unique for TAHV in the L segment were identified. We also identified specific residue substitutions in the endonuclease domain of TAHV compared with the La Crosse virus. Since the endonuclease domain of the La Crosse virus has been resolved, we employed an all energy landscape algorithm to analyze the ligand migration of a viral polymerase inhibitor. This allowed us to demonstrate, at the atomic level, that this viral polymerase inhibitor randomly explored the specific residue substitutions in the endonuclease domain of the TAHV L segment.

[In vitro activity of Russian ribavirin on the models of Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, and Tahyna and Dhori viruses].

The high activity of ribavirin made by effective biotechnology in Russia was established in in vitro experiments using the models Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, and Tahyna and Dhori viruses, which suggests that it is promising in using the drug in the treatment of infection with these viruses.

Adsorptive endocytosis of California encephalitis virus into mosquito and mammalian cells: a role for G1.

The G1 glycoprotein of California encephalitis (CE) virus plays a critical role in the infection of mosquito and mammalian cells. We found that CE virus enters baby hamster kidney (BHK-21) and Aedes albopictus (C6/36) cells by the endocytic pathway. Ammonium chloride, a lysosomotropic amine that prevents release of virus from endosomes, inhibited infection of both cell types when added within 10 min after viral adsorption. In addition, infected cells formed polykaryons when the extracellular pH was lowered to 6.3; optimal fusion occurred at pH 5.8 and 6.0 (C6/36 and BHK-21 cells, respectively). Two neutralizing G1 MAba, 6D5.5 and 7D4.5, inhibited low pH-induced syncytia formation without affecting viral attachment, suggesting a role for G1 in viral entry. Since viral fusion proteins have been demonstrated to undergo conformational changes at low pH, acid-induced changes in G1 and G2 were assessed. While both G1 and G2 demonstrated low pH-induced alterations in detergent binding, only G1 displayed an altered protease cleavage pattern at the fusion pH. These results indicate that the G1 protein of CE virus undergoes conformational changes necessary for low pH-mediated entry into both mosquito and mammalian cells.

Mechanism of La Crosse virus inhibition by ribavirin.

The effect of ribavirin on the growth and replication of La Crosse virus was examined. The data suggest that low concentrations of ribavirin have a marked effect on the initial steps of La Crosse virus transcription. The therapeutic potential of ribavirin in the treatment of human California encephalitis serotype infections is discussed in light of these findings.

[Antiviral activity of ribamidyl in experimental infections with California encephalitis group viruses]. / Protivovirusnaia aktivnost' ribamidila pri éksperimental'nykh infektsiiakh virusami kompleksa kaliforniiskogo éntsefalita.

A preparation of ribamydil, an analogue of natural nucleosides, synthesized at the Latvian SSR Institute of Organic Chemistry showed a sufficiently high activity against bunyaviruses of California encephalitis complex both in vitro and in vivo. Various modifications of the enzyme immunoassay may be used for control of the effectiveness of treatment with this drug. Some advantages of the subcutaneous route over the intramuscular one were found. Ribamydil may be useful for treatment of infections of California encephalitis complex.

Inhibitors of protein synthesis inhibit both La Crosse virus S-mRNA and S genome syntheses in vivo.

The effect of drugs such as puromycin and cycloheximide, which inhibit protein synthesis, on the accumulation of La Crosse virus S genome RNAs in vivo has been examined. We have found that if these drugs are added to the cultures before infection, minuscule amounts of S-mRNA can be detected late in infection. Genome replication, on the other hand, cannot be detected at any time. When these drugs are added later in infection when RNA synthesis is well established, S-mRNA accumulation decreases in a dose-dependent manner proportional to the effect of these drugs on protein synthesis. This decrease cannot be accounted for by increased turnover of the mRNA in the presence of the drug. S genome replication, curiously, was found to be hypersensitive to the effects of these drugs. Our results confirm those of Abraham and Pattnaik (1983) that ongoing protein synthesis is required for the accumulation of complete bunyavirus S-mRNA.

Inhibition of La Crosse virus replication by monensin, monovalent ionophore.

The effect of monensin, a monovalent ionophore, on La Crosse virus particle formation and polypeptide synthesis was examined. Monensin inhibited the release of virus particles (both infectious and non-infectious) from infected BHK-21 cells. Monensin had no detectable effect on the synthesis of polypeptides G1, G2, and N.

La crosse virus production and export have a colchicine-sensitive step.

In the present investigation, the effect of colchicine on La Crosse virus production and export was tested. Colchicine-treated, La Crosse virus-infected cells: (1) had decreased mean virus titers compared with those of control cells; (2) had a ratio of released to cell-associated virus of 1-1.9 whereas control cells had a ratio of 13. A colchicine-sensitive step, possibly involving microtubules, may be involved in virus production and/or release from the cell.

Sensitivities of neurotropic arboviruses to human interferon.

The sensitivities of selected neurotropic arboviruses (St. Louis encephalitis, Western equine encephaltis, and La Crosse viruses) to human interferon and polyriboinosinic-polyribocytidylic acid (poly I-poly C) were determined in primary human fetal glial cell cultures. The sensitivities were measured by an end point (inhibition of virus yield). All of the viruses were sensitive to human interferon and poly I-poly C; St. Louis encephalitis and Western equine encephalitis viruses were inhibited at concentrations of interferon actually measured in the brains of persons who died from these infections. For St. Louis encephalitis and Western equine encephalitis viruses, viral replication and induction of interferon varied among cell cultures derived from individual fetuses. The present experiments may provide an approach toward understanding the problem of different host susceptibilities to neurotropic arbovirus infections.