Interferon Lambda Delays the Emergence of Influenza Virus Resistance to Oseltamivir

Influenza viruses are a leading cause of morbidity and mortality worldwide. These air-borne pathogens are able to cross the species barrier, leading to regular seasonal epidemics and sporadic pandemics. Influenza viruses also possess a high genetic variability, which allows for the acquisition of resistance mutations to antivirals. Combination therapies with two or more drugs targeting different mechanisms of viral replication have been considered an advantageous option to not only enhance the effectiveness of the individual treatments, but also reduce the likelihood of resistance emergence. Using an in vitro infection model, we assessed the barrier to viral resistance of a combination therapy with the neuraminidase inhibitor oseltamivir and human interferon lambda against the pandemic H1N1 A/Netherlands/602/2009 (H1N1pdm09) virus. We serially passaged the virus in a cell line derived from human bronchial epithelial cells in the presence or absence of increasing concentrations of oseltamivir alone or oseltamivir plus interferon lambda. While the treatment with oseltamivir alone quickly induced the emergence of antiviral resistance through a single mutation in the neuraminidase gene, the co-administration of interferon lambda delayed the emergence of drug-resistant influenza virus variants. Our results suggest a possible clinical application of interferon lambda in combination with oseltamivir to treat influenza.

Novel broad-spectrum virucidal antivirals

Matteo Gasbarri

The current COVID-19 pandemic has showed the threats that viruses can cause to the whole world. Over 200 million infections and over 4.5 million deaths and enormous socio-economic impact were the output of the emergence of a single new pathogen: SARS-CoV-2. The development of vac-cines is the paramount solution to overcome the pandemic. However, even with extraordinary and unprecedented worldwide efforts, it took almost one year to have a first validated vaccine and it will take longer to have a global vaccination. In addition, vaccines are preventive drugs, that need to be administered before the infection to stimulate the immune-system of the patient. In the meanwhile, in case of an infection, we were harmless in the fight against the virus.Antivirals are curative drugs, that inhibit one step of the viral replication. Many approaches have been investigated: antibodies, peptides, peptomimics, small molecules. Most of them are specif-ic to a single family of virus, making the development slow and expensive. Entry inhibitors are a class of antivirals that binds to the virus, preventing the attachment to the cell membrane. Such antivirals usually have a broad-spectrum of action, since they mimic cell receptors, such as heparan sulfate pro-teoglycans (HSPGs), exploited by different viruses. Many compounds have been developed throughout the years; however, the reversible nature of the virus inhibition mechanism has prevented their trans-lation to the clinic. Our group has recently shown that the simple addition of a long hydrophobic linker makes the inhibition irreversible, proving this hypothesis with two compounds: gold-nanoparticles and beta-cyclodextrins. The goal of this thesis is to develop novel antivirals able to inhibit a broad-spectrum of viruses with an irreversible mechanism of action and to investigate the key features responsible of such properties. In this thesis, we further expand the broad-spectrum activity of gold-nanoparticles and beta-cyclodextrins showing that they are capable of inhibiting viruses beyond HSPG-dependent ones. In addition, we show their ability of reversibly inactivating SARS-CoV-2. Moreover, we have investigated the role of the hydrophobic linker's length in the virucidal inhibition in case of cyclodextrins, proving the need of such moiety to have an irreversible viral inactivation. Envisioning a translation of such drug, we have performed a preliminar pharmacokinetic study performed via intranasal administration.A novel class of compounds has been also proposed: sulfated/sulfonated dendritic polyglyc-erol. A broad investigation of the molecular design rules needed to achieve nanomolar irreversible viral inhibition is discussed. We also show that the most active compound proves to be active against HSV-2 and RSV, thus with a broad-spectrum activity. In addition, we demonstrate that the virucidal activity is leading to the release of viral DNA upon interaction between the virus and our dendritic polyglycerol. A different modification of dendritic polyglycerol, bearing carboxylic acid, showed activi-ty against SARS-CoV-2; such inhibition results to be irreversible. This compound has been further test-ed in a hamster model, where initial studies confirm its feasibility as antiviral. In addition to these studies on development of virucidal antivirals, different technological so-lutions to prevent viral spreading are proposed. In particular, we focused on an antiviral filter and antiviral surfaces.

EPFL2021

Multi-resistance of human enterovirus to UV-C disinfection and mutagenic nucleoside analogs

Virginie Bachmann, Anna Carratala Ripolles, Tamar Kohn, Qingxia Zhong

Disinfection is an important strategy to control the environmental transmission of human viruses. Specifically, UV254 is increasingly used to disinfect from clinical items or surfaces to drinking water. RNA virus populations have high mutation rates and large population numbers that allow them to rapidly adapt to stressors. It is therefore likely that variants with enhanced resistance to disinfection may eventually emerge in a virus population. The genetic basis and mechanisms that underpin the emergence of virus resistance to disinfection remain, however, unknown. The goal of this study was to investigate the emergence of echoviruses resistant to UV254 disinfection in vitro, and to characterize the genotypic and phenotypic changes in the resistant populations as compared to the wild-type. Resistant virus populations emerged in mutation-accumulation laboratory experiments. Each experiment consisted in exposing echovirus suspensions to a UV254 fluence such that a 4.5 log10 reduction in infective viruses was achieved, and then regrowing the survivors by infecting BGM cells at a very low multiplicity of infection (MOI>0.001). Unexposed control experiments were conducted in parallel by diluting the virus suspensions instead of exposing them to UV254 prior to regrowth. We demonstrated that echovirus populations became increasingly resistant to UV254. Interestingly, a similar extent of resistance was observed in the unexposed control experiments. Fixed mutations were repeatedly identified by next generation sequencing in the 2C and 3D protein region, both involved in the genome replication. The wild-type and resistant echovirus populations exhibited an equivalent replicative fitness under standard culturing conditions. However, the UV254-resistant mutants were more resistant to ribavirin and guanidine hydrochloride. Finally, the exposed populations were able to maintain the production of infective progeny despite the effect of such chemical mutagens. Combined, these findings suggest that multi-resistance of echovirus to UV254 disinfection and treatment with mutagenic nucleotide analogs may arise in virus populations that, subjected to repeated dramatic bottlenecks, evolve towards an increased replication fidelity. The outcomes of this work show that enteric human pathogens may exhibit multi-resistance to disinfection and clinical antiviral therapies. Ultimately, the results from this work may impact future strategies to ensure adequate control of virus environmental transmission.

2016

Use of consensus sequences for the design of high density resequencing microarrays: the influenza virus paradigm

Stewart Cole, Iain Old

BACKGROUND: A resequencing microarray called PathogenID v2.0 has been developed and used to explore various strategies of sequence selection for its design. The part dedicated to influenza viruses was based on consensus sequences specific for one gene generated from global alignments of a large number of influenza virus sequences available in databanks. RESULTS: For each HA (H1, H2, H3, H5, H7 and H9) and NA (N1, N2 and N7) molecular type chosen to be tested, 1 to 3 consensus sequences were computed and tiled on the microarray. A total of 12 influenza virus samples from different host origins (humans, pigs, horses and birds) and isolated over a period of about 50 years were used in this study. Influenza viruses were correctly identified, and in most cases with the accurate information of the time of their emergence. CONCLUSIONS: PathogenID v2.0 microarray demonstrated its ability to type and subtype influenza viruses, often to the level of viral variants, with a minimum number of tiled sequences. This validated the strategy of using consensus sequences, which do not exist in nature, for our microarray design. The versatility, rapidity and high discriminatory power of the PathogenID v2.0 microarray could prove critical to detect and identify viral genome reassortment events resulting in a novel virus with epidemic or pandemic potential and therefore assist health authorities to make efficient decisions about patient treatment and outbreak management.