Summary
Hepatitis E virus (HEV) is the causative agent of hepatitis E in humans and a member of the genus Orthohepevirus in the family Hepeviridae. Infection usually leads to acute hepatitis that can ...become fulminant, particularly among pregnant women and in patients with preexisting liver disease, or may evolve to a chronic state, especially in immunosuppressed individuals. HEV has been shown to produce a range of extra‐hepatic manifestations including aplastic anaemia, acute thyroiditis, glomerulonephritis as well as neurological disorders such as Guillain‐Barré syndrome, neuralgic amyotrophy and encephalitis. The pathogenesis of these neurological injuries remains largely unknown, and it is also uncertain whether or not HEV can directly infect neuronal cells. In this study, we investigated whether HEV is capable of completing the viral life cycle in human neuronal‐derived cell lines such as neuroepithelioma (SK‐N‐MC), desmoplastic cerebellar medulloblastoma (DAOY), glioblastoma multiforme (DBTRG), glioblastoma astrocytoma (U‐373 MG) and oligodendrocytic (M03.13) cells. Following transfection of these cells with HEV Gaussia luciferase reporter virus, all tested cell lines supported HEV RNA replication. Furthermore, extra‐ and intracellular viral capsid was detected by an HEV antigen ELISA as a marker for virus assembly and release. Permissiveness for HEV cell entry could be demonstrated for the oligodendrocytic cell line M03.13. In conclusion, these results indicate that HEV tropism is not restricted to the liver and HEV can potentially complete the full viral life cycle in neuronal‐derived tissues explaining neurologic disorders during HEV infection.
There is an urgent need for drugs, therapies and vaccines to be available to protect the human population against COVID-19. One of the first approaches taken in the COVID-19 global response was to ...consider repurposing licensed drugs. This commentary highlights an extraordinary international collaborative effort of independent researchers who have recently all come to the same conclusion—that chloroquine or hydroxchloroquine are unlikely to provide clinical benefit against COVID-19.
Epidemiological and clinical reports indicate that SARS-CoV-2 virulence hinges upon the triggering of an aberrant host immune response, more so than on direct virus-induced cellular damage. To ...elucidate the immunopathology underlying COVID-19 severity, we perform cytokine and multiplex immune profiling in COVID-19 patients. We show that hypercytokinemia in COVID-19 differs from the interferon-gamma-driven cytokine storm in macrophage activation syndrome, and is more pronounced in critical versus mild-moderate COVID-19. Systems modelling of cytokine levels paired with deep-immune profiling shows that classical monocytes drive this hyper-inflammatory phenotype and that a reduction in T-lymphocytes correlates with disease severity, with CD8+ cells being disproportionately affected. Antigen presenting machinery expression is also reduced in critical disease. Furthermore, we report that neutrophils contribute to disease severity and local tissue damage by amplification of hypercytokinemia and the formation of neutrophil extracellular traps. Together our findings suggest a myeloid-driven immunopathology, in which hyperactivated neutrophils and an ineffective adaptive immune system act as mediators of COVID-19 disease severity.
Infection with yellow fever virus (YFV), the prototypic mosquito-borne flavivirus, causes severe febrile disease with haemorrhage, multi-organ failure and a high mortality. Moreover, in recent years ...the Flavivirus genus has gained further attention due to re-emergence and increasing incidence of West Nile, dengue and Japanese encephalitis viruses. Potent and safe antivirals are urgently needed.
Starting from the crystal structure of the NS3 helicase from Kunjin virus (an Australian variant of West Nile virus), we identified a novel, unexploited protein site that might be involved in the helicase catalytic cycle and could thus in principle be targeted for enzyme inhibition. In silico docking of a library of small molecules allowed us to identify a few selected compounds with high predicted affinity for the new site. Their activity against helicases from several flaviviruses was confirmed in in vitro helicase/enzymatic assays. The effect on the in vitro replication of flaviviruses was then evaluated.
Ivermectin, a broadly used anti-helminthic drug, proved to be a highly potent inhibitor of YFV replication (EC₅₀ values in the sub-nanomolar range). Moreover, ivermectin inhibited, although less efficiently, the replication of several other flaviviruses, i.e. dengue fever, Japanese encephalitis and tick-borne encephalitis viruses. Ivermectin exerts its effect at a timepoint that coincides with the onset of intracellular viral RNA synthesis, as expected for a molecule that specifically targets the viral helicase.
The well-tolerated drug ivermectin may hold great potential for treatment of YFV infections. Furthermore, structure-based optimization may result in analogues exerting potent activity against flaviviruses other than YFV.
► Favipiravir (T-705) is an influenza drug in clinical development. ► Favipiravir inhibits (murine) norovirus-induced CPE and viral RNA synthesis. ► Favipiravir inhibits viral replication at a time ...point that coincides with the onset of viral RNA synthesis.
Human noroviruses are the primary cause of foodborne gastroenteritis. Potent and safe inhibitors are needed for the treatment/prophylaxis of norovirus infections. We demonstrate that Favipiravir T-705, a drug in advanced clinical development for the treatment of infections with the influenza virus inhibits in vitro murine norovirus replication. Time-of-drug addition studies reveal that T-705 exerts its activity at a time-point that coincides with onset of viral RNA synthesis, which is in line with the viral polymerase as the presumed target.
Potent and safe inhibitors of norovirus replication are needed for the treatment and prophylaxis of norovirus infections. We here report that the in vitro anti-norovirus activity of the protease ...inhibitor rupintrivir is extended to murine noroviruses and that rupintrivir clears human cells from their Norwalk replicon after only two passages of antiviral pressure. In addition, we demonstrate that rupintrivir inhibits the human norovirus (genogroup II GII) protease and further explain the inhibitory effect of the molecule by means of molecular modeling on the basis of the crystal structure of the Norwalk virus protease. The combination of rupintrivir with the RNA-dependent RNA polymerase inhibitors 2'-C-methylcytidine and favipiravir (T-705) resulted in a merely additive antiviral effect. The fact that rupintrivir is active against noroviruses belonging to genogroup I (Norwalk virus), genogroup V (murine norovirus), and the recombinant 3C-like protease of a GII norovirus suggests that the drug exerts cross-genotypic anti-norovirus activity and will thus most likely be effective against the clinically relevant human norovirus strains. The design of antiviral molecules targeting the norovirus protease could be a valuable approach for the treatment and/or prophylaxis of norovirus infections.
Norovirus outbreaks of acute gastroenteritis are highly prevalent, extensive and can disturb the functioning of health institutions, leading to the closure of hospital wards and causing ...life-threatening infections in long-term care facilities. There is no vaccine available; hence there is a pressing need for antivirals for the treatment (in immunodeficient patients) and prophylaxis of norovirus infections. We explored in a mouse model whether an inhibitor of norovirus replication can prevent/reduce transmission of the virus.
We reported recently that the viral polymerase inhibitor 2'-C-methylcytidine (2CMC) efficiently protects against murine norovirus (MNV)-induced diarrhoea and mortality in mice. Here, we established an MNV-transmission model, determined the 50% infectious dose and assessed the ability of an antiviral molecule to prevent or reduce transmission of (murine) norovirus when given either to the infected (seeder) mice or to the uninfected (sentinel) mice.
A robust norovirus transmission model was established using genogroup V (murine) norovirus in AG129 mice. The 50% infectious dose was determined to be ∼ 270 CCID50 (50% cell culture infectious dose). Treatment of infected mice with 2CMC reduced viral shedding and markedly reduced transmission to uninfected sentinels. Also, prophylactic treatment of sentinels with 2CMC resulted in protection against infection with MNV.
These findings constitute an important first step towards developing an efficient prophylaxis for the control of norovirus outbreaks.
Nucleoside or nucleotide analogue inhibitors of viral replication almost act as chain terminators during DNA (DNA- and retroviruses) or RNA (RNA viruses) synthesis. Following intracellular ...phosphorylation, by viral and/or cellular kinases, the 5′-triphosphate metabolites (or 2′-diphosphate metabolites in the case of acyclic nucleoside phosphonate analogues) compete with the natural substrate in the DNA or RNA polymerization reaction. Obligatory chain terminators (e.g., acy clovir) do not offer the 3′-hydroxyl function at the riboside moiety of the molecule. Nucleoside analogues that possess a hydroxyl function at a position equivalent of the 3′-hydroxyl position may act as chain terminators if this hydroxyl group is confor-mationally constrained (e.g., ganciclovir) or sterically hindered to enter into a phos-phodiester linkage with the incoming nucleotide. In case that the 3′-hydroxylgroup is correctly positioned, chain elongation may be hampered through steric hindrance from neighboring substituents (e.g., 2′-C-methyl or 4′-azido nucleoside inhibitors of HCV replication). Here, we review the molecular mechanism of action and the clinical applications of the nucleosides and nucleotides acting as chain terminators. A further discussion of clinical applications in combination therapy can be found in Chap. 12.
The chikungunya virus (CHIKV) has become a substantial global health threat due to its massive re-emergence, the considerable disease burden and the lack of vaccines or therapeutics. We discovered a ...novel class of small molecules (1,2,3triazolo4,5-dpyrimidin-7(6H)-ones) with potent in vitro activity against CHIKV isolates from different geographical regions. Drug-resistant variants were selected and these carried a P34S substitution in non-structural protein 1 (nsP1), the main enzyme involved in alphavirus RNA capping. Biochemical assays using nsP1 of the related Venezuelan equine encephalitis virus revealed that the compounds specifically inhibit the guanylylation of nsP1. This is, to the best of our knowledge, the first report demonstrating that the alphavirus capping machinery is an excellent antiviral drug target. Considering the lack of options to treat CHIKV infections, this series of compounds with their unique (alphavirus-specific) target offers promise for the development of therapy for CHIKV infections.
The clinical impact of infections with respiratory viruses belonging to the family Paramyxoviridae argues for the development of antiviral therapies with broad-spectrum activity. Favipiravir (T-705) ...has demonstrated potent antiviral activity against multiple RNA virus families and is presently in clinical evaluation for the treatment of influenza. Here we demonstrate in vitro activity of T-705 against the paramyxoviruses human metapneumovirus (HMPV), respiratory syncytial virus, human parainfluenza virus, measles virus, Newcastle disease virus, and avian metapneumovirus. In addition, we demonstrate activity against HMPV in hamsters. T-705 treatment inhibited replication of all paramyxoviruses tested in vitro, with 90% effective concentration (EC90) values of 8 to 40 μM. Treatment of HMPV-challenged hamsters with T-705 at 200 mg/kg of body weight/day resulted in 100% protection from infection of the lungs. In all treated and challenged animals, viral RNA remained detectable in the respiratory tract. The observation that T-705 treatment had a significant effect on infectious viral titers, with a limited effect on viral genome titers, is in agreement with its proposed mode of action of viral mutagenesis. However, next-generation sequencing of viral genomes isolated from treated and challenged hamsters did not reveal (hyper)mutation. Polymerase activity assays revealed a specific effect of T-705 on the activity of the HMPV polymerase. With the reported antiviral activity of T-705 against a broad range of RNA virus families, this small molecule is a promising broad-range antiviral drug candidate for limiting the viral burden of paramyxoviruses and for evaluation for treatment of infections with (re)emerging viruses, such as the henipaviruses.
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