Broad-spectrum non-toxic antiviral nanoparticles with a virucidal inhibition mechanism

Viral infections kill millions yearly. Available antiviral drugs are virus-specific and active against a limited panel of human pathogens. There are broad-spectrum substances that prevent the first step of virus–cell interaction by mimicking heparan sulfate proteoglycans (HSPG), the highly conserved target of viral attachment ligands (VALs). The reversible binding mechanism prevents their use as a drug, because, upon dilution, the inhibition is lost. Known VALs are made of closely packed repeating units, but the aforementioned substances are able to bind only a few of them. We designed antiviral nanoparticles with long and flexible linkers mimicking HSPG, allowing for effective viral association with a binding that we simulate to be strong and multivalent to the VAL repeating units, generating forces (∼190 pN) that eventually lead to irreversible viral deformation. Virucidal assays, electron microscopy images, and molecular dynamics simulations support the proposed mechanism. These particles show no cytotoxicity, and in vitro nanomolar irreversible activity against herpes simplex virus (HSV), human papilloma virus, respiratory syncytial virus (RSV), dengue and lenti virus. They are active ex vivo in human cervicovaginal histocultures infected by HSV-2 and in vivo in mice infected with RSV.

Intercapsomeric disulfide bonds in papillomavirus assembly and disassembly

Peter Martin Beard

In order to analyze bonding contacts that stabilize the virion or promote capsid assembly, bovine papillomavirus (BPV) virions were subjected to buffer conditions known to disrupt polyomavirus virions. At physiologic ionic strength, incubation with dithiothreitol (DTT), EGTA, or DTT plus EGTA did not disrupt BPV virions as determined by electron microscopy. However, incubation of virions with DTT rendered the BPV L1 protein susceptible to trypsin cleavage at its carboxy terminus and rendered the genome susceptible to digestion with DNase I. When DTT-treated BPV virions were analyzed by analytical ultracentrifugation, they sedimented at 230S compared with 273S for untreated virions, suggesting a capsid shell expansion. Incubation with EGTA had no effect on trypsin or DNase I sensitivity and only a small effect upon the virion S value. A single cysteine residue conserved among BPV and human papillomavirus (HPV) L1 proteins resides within the trypsin-sensitive carboxy terminus of L1, which is required for capsid assembly. A recombinant HPV type 11 L1 protein, which was purified after expression in Escherichia coli and which has a Cys-to-Gly change at this position (Cys424), formed pentamers; however, unlike the wild-type protein, these mutant pentamers could no longer assemble in vitro into capsid-like structures. These results indicate an important role for interpentamer disulfide bonds in papillomavirus capsid assembly and disassembly and suggest a mechanism of virus uncoating in the reducing environment of the cytoplasm.

1998

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

Vibrio coralliilyticus infection triggers a behavioural response and perturbs nutritional exchange and tissue integrity in a symbiotic coral

Emma Mary Gibbin, Anders Meibom

Under homoeostatic conditions, the relationship between the coralPocillopora damicornisandVibrio coralliilyticusiscommensal. An increase in temperature, or in the abundance ofV. coralliilyticus, can turn this association pathogenic,causing tissue lysis, expulsion of the corals’symbiotic algae (genusSymbiodinium), and eventually coral death. Using acombination of microfluidics,fluorescence microscopy, stable isotopes, electron microscopy and NanoSIMS isotopicimaging, we provide insights into the onset and progression ofV.coralliilyticusinfection in the daytime and at night, atthe tissue and (sub-)cellular level. The objective of our study was to connect the macro-scale behavioural response ofthe coral to the micro-scale nutritional interactions that occur between the host and its symbiont. In the daytime, polypsenhanced their mucus production, and actively spewed pathogens.Vibrioinfection primarily resulted in the formationof tissue lesions in the coenosarc. NanoSIMS analysis revealed infection reduced13C-assimilation inSymbiodinium, butincreased13C-assimilation in the host. In the night incubations, no mucus spewing was observed, and a mucusfilm wasformed on the coral surface.Vibrioinoculation and infection at night showed reduced13C-turnover inSymbiodinium, but didnot impact host13C-turnover. Our results show that both the nutritional interactions that occur between the two symbioticpartners and the behavioural response of the host organism play key roles in determining the progression and severity ofhost-pathogen interactions. More generally, our approach provides a new means of studying interactions (ranging frombehavioural to metabolic scales) between partners involved in complex holobiont systems, under both homoeostatic andpathogenic conditions.

2018

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

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

2016