Perturbation of Alphavirus and Flavivirus Infectivity by Components of the Bacterial Cell Wall.

The impact of the host microbiota on arbovirus infections is currently not well understood. Arboviruses are viruses transmitted through the bites of infected arthropods, predominantly mosquitoes or ticks. The first site of arbovirus inoculation is the biting site in the host skin, which is colonized by a complex microbial community that could possibly influence arbovirus infection. We demonstrated that preincubation of arboviruses with certain components of the bacterial cell wall, including lipopolysaccharides (LPS) of some Gram-negative bacteria and lipoteichoic acids or peptidoglycan of certain Gram-positive bacteria, significantly reduced arbovirus infectivity in vitro. This inhibitory effect was observed for arboviruses of different virus families, including chikungunya virus of the Alphavirus genus and Zika virus of the Flavivirus genus, showing that this is a broad phenomenon. A modest inhibitory effect was observed following incubation with a panel of heat-inactivated bacteria, including bacteria residing on the skin. No viral inhibition was observed after preincubation of cells with LPS. Furthermore, a virucidal effect of LPS on viral particles was noticed by electron microscopy. Therefore, the main inhibitory mechanism seems to be due to a direct effect on the virus particles. Together, these results suggest that bacteria are able to decrease the infectivity of alphaviruses and flaviviruses. IMPORTANCE During the past decades, the world has experienced a vast increase in epidemics of alphavirus and flavivirus infections. These viruses can cause severe diseases, such as hemorrhagic fever, encephalitis, and arthritis. Several alpha- and flaviviruses, such as chikungunya virus, Zika virus, and dengue virus, are significant global health threats because of their high disease burden, their widespread (re-)emergence, and the lack of (good) anti-arboviral strategies. Despite the clear health burden, alphavirus and flavivirus infection and disease are not fully understood. A knowledge gap in the interplay between the host and the arbovirus is the potential interaction with host skin bacteria. Therefore, we studied the effect of (skin) bacteria and bacterial cell wall components on alphavirus and flavivirus infectivity in cell culture. Our results show that certain bacterial cell wall components markedly reduced viral infectivity by interacting directly with the virus particle.

Posaconazole inhibits multiple steps of the alphavirus replication cycle.

Repurposing drugs is a promising strategy to identify therapeutic interventions against novel and re-emerging viruses. Posaconazole is an antifungal drug used to treat invasive aspergillosis and candidiasis. Recently, posaconazole and its structural analog, itraconazole were shown to inhibit replication of multiple viruses by modifying intracellular cholesterol homeostasis. Here, we show that posaconazole inhibits replication of the alphaviruses Semliki Forest virus (SFV), Sindbis virus and chikungunya virus with EC50 values ranging from 1.4 µM to 9.5 µM. Posaconazole treatment led to a significant reduction of virus entry in an assay using a temperature-sensitive SFV mutant, but time-of-addition and RNA transfection assays indicated that posaconazole also inhibits post-entry stages of the viral replication cycle. Virus replication in the presence of posaconazole was partially rescued by the addition of exogenous cholesterol. A transferrin uptake assay revealed that posaconazole considerably slowed down cellular endocytosis. A single point mutation in the SFV E2 glycoprotein, H255R, provided partial resistance to posaconazole as well as to methyl-ß-cyclodextrin, corroborating the effect of posaconazole on cholesterol and viral entry. Our results indicate that posaconazole inhibits multiple steps of the alphavirus replication cycle and broaden the spectrum of viruses that can be targeted in vitro by posaconazole, which could be further explored as a therapeutic agent against emerging viruses.

An affinity-enhanced, broadly neutralizing heavy chain-only antibody protects against SARS-CoV-2 infection in animal models.

Broadly neutralizing antibodies are an important treatment for individuals with coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Antibody-based therapeutics are also essential for pandemic preparedness against future Sarbecovirus outbreaks. Camelid-derived single domain antibodies (VHHs) exhibit potent antimicrobial activity and are being developed as SARS-CoV-2­neutralizing antibody-like therapeutics. Here, we identified VHHs that neutralize both SARS-CoV-1 and SARS-CoV-2, including now circulating variants. We observed that the VHHs bound to a highly conserved epitope in the receptor binding domain of the viral spike protein that is difficult to access for human antibodies. Structure-guided molecular modeling, combined with rapid yeast-based prototyping, resulted in an affinity enhanced VHH-human immunoglobulin G1 Fc fusion molecule with subnanomolar neutralizing activity. This VHH-Fc fusion protein, produced in and purified from cultured Chinese hamster ovary cells, controlled SARS-CoV-2 replication in prophylactic and therapeutic settings in mice expressing human angiotensin converting enzyme 2 and in hamsters infected with SARS-CoV-2. These data led to affinity-enhanced selection of the VHH, XVR011, a stable anti­COVID-19 biologic that is now being evaluated in the clinic.

The impact of obstructive sleep apnea on endothelial function during weight loss in an obese pediatric population.

BACKGROUND: Childhood obesity is an increasing problem with substantial comorbidities such as obstructive sleep apnea (OSA) and increased cardiovascular morbidity. Endothelial dysfunction is an underlying mechanism related to both obesity and OSA. RESEARCH QUESTION: To investigate the effect of weight loss on endothelial function and OSA in obese children and to determine whether a change in endothelial function can be linked to an improvement in OSA. METHODS: Obese children between 8 and 18 years of age were recruited while entering a 12-month inpatient weight loss program. Patients were followed at 3 study visits: baseline, after 10 months of weight loss, and 6 months after ending the program (18 months). Anthropometry and endothelial function (EndoPAT) were determined at all study visits. At baseline, sleep screening with a portable device (ApneaLink) was performed. This was repeated after 10 months if OSA was diagnosed at baseline. RESULTS: At baseline, 130 children were included, of which 87 had OSA (67%). Seventy-two patients attended the follow-up visit at 10 months, and 28 patients attended the follow-up visit at 18 months. The BMI z-score decreased after 10 months (from 2.7 (1.4-3.4) to 1.7 (0.5-2.7); p < 0.001) and remained stable at 18 months. Endothelial function improved significantly after weight loss, evidenced by a shorter time to peak response (TPR) and higher reactive hyperemia index (p = 0.02 and p < 0.001), and remained improved after 18 months (p < 0.001 and p = 0.007). After 10 months of weight loss, 10 patients had residual OSA. These patients had a higher TPR at 10 months (225 (75-285)s) than those without OSA (135 (45-225)s) and patients with a normalized sleep study (105 (45-285)s; p = 0.02). Linear mixed models showed that more severe OSA was associated with a worse TPR at baseline and less improvement after weight loss. CONCLUSION: Weight loss improves endothelial function in an obese pediatric population. However, even after weight loss, endothelial function improved less in the presence of OSA.

Favipiravir Does Not Inhibit Chikungunya Virus Replication in Mosquito Cells and Aedes aegypti Mosquitoes.

Favipiravir (T-705) is a broad-spectrum antiviral drug that inhibits RNA viruses after intracellular conversion into its active form, T-705 ribofuranosyl 5'-triphosphate. We previously showed that T-705 is able to significantly inhibit the replication of chikungunya virus (CHIKV), an arbovirus transmitted by Aedes mosquitoes, in mammalian cells and in mouse models. In contrast, the effect of T-705 on CHIKV infection and replication in the mosquito vector is unknown. Since the antiviral activity of T-705 has been shown to be cell line-dependent, we studied here its antiviral efficacy in Aedes-derived mosquito cells and in Aedes aegypti mosquitoes. Interestingly, T-705 was devoid of anti-CHIKV activity in mosquito cells, despite being effective against CHIKV in Vero cells. By investigating the metabolic activation profile, we showed that, unlike Vero cells, mosquito cells were not able to convert T-705 into its active form. To explore whether alternative metabolization pathways might exist in vivo, Aedes aegypti mosquitoes were infected with CHIKV and administered T-705 via an artificial blood meal. Virus titrations of whole mosquitoes showed that T-705 was not able to reduce CHIKV infection in mosquitoes. Combined, these in vitro and in vivo data indicate that T-705 lacks antiviral activity in mosquitoes due to inadequate metabolic activation in this animal species.

Itraconazole for COVID-19: preclinical studies and a proof-of-concept randomized clinical trial.

BACKGROUND: The antifungal drug itraconazole exerts in vitro activity against SARS-CoV-2 in Vero and human Caco-2 cells. Preclinical and clinical studies are required to investigate if itraconazole is effective for the treatment and/or prevention of COVID-19. METHODS: Due to the initial absence of preclinical models, the effect of itraconazole was explored in a clinical, proof-of-concept, open-label, single-center study, in which hospitalized COVID-19 patients were randomly assigned to standard of care with or without itraconazole. Primary outcome was the cumulative score of the clinical status until day 15 based on the 7-point ordinal scale of the World Health Organization. In parallel, itraconazole was evaluated in a newly established hamster model of acute SARS-CoV-2 infection and transmission, as soon as the model was validated. FINDINGS: In the hamster acute infection model, itraconazole did not reduce viral load in lungs, stools or ileum, despite adequate plasma and lung drug concentrations. In the transmission model, itraconazole failed to prevent viral transmission. The clinical trial was prematurely discontinued after evaluation of the preclinical studies and because an interim analysis showed no signal for a more favorable outcome with itraconazole: mean cumulative score of the clinical status 49 vs 47, ratio of geometric means 1.01 (95% CI 0.85 to 1.19) for itraconazole vs standard of care. INTERPRETATION: Despite in vitro activity, itraconazole was not effective in a preclinical COVID-19 hamster model. This prompted the premature termination of the proof-of-concept clinical study. FUNDING: KU Leuven, Research Foundation - Flanders (FWO), Horizon 2020, Bill and Melinda Gates Foundation.

Recent African strains of Zika virus display higher transmissibility and fetal pathogenicity than Asian strains.

The global emergence of Zika virus (ZIKV) revealed the unprecedented ability for a mosquito-borne virus to cause congenital birth defects. A puzzling aspect of ZIKV emergence is that all human outbreaks and birth defects to date have been exclusively associated with the Asian ZIKV lineage, despite a growing body of laboratory evidence pointing towards higher transmissibility and pathogenicity of the African ZIKV lineage. Whether this apparent paradox reflects the use of relatively old African ZIKV strains in most laboratory studies is unclear. Here, we experimentally compare seven low-passage ZIKV strains representing the recently circulating viral genetic diversity. We find that recent African ZIKV strains display higher transmissibility in mosquitoes and higher lethality in both adult and fetal mice than their Asian counterparts. We emphasize the high epidemic potential of African ZIKV strains and suggest that they could more easily go unnoticed by public health surveillance systems than Asian strains due to their propensity to cause fetal loss rather than birth defects.

Systematic Review of Clinical Practice Guidelines for Individuals With Amputation: Identification of Best Evidence for Rehabilitation to Develop the WHO's Package of Interventions for Rehabilitation.

OBJECTIVES: To report the results of the systematic search performed to identify interventions and related evidence for rehabilitation of individuals with amputation based on the current evidence from clinical practice guidelines (CPG). DATA SOURCES: Pubmed, Pedro, CINAHL, Embase, Google Scholar, and multiple guideline databases (date restriction, 2008-2018). STUDY SELECTION: Exclusion criteria were no CPG, not reporting on rehabilitation, published before 2008, developed for health conditions other than amputation, presence of conflict of interest (financial or nonfinancial), lack of information on the strength of the recommendation, and lack of quality assessed by the "Appraisal of Guidelines for Research and Evaluation." DATA EXTRACTION: Data extraction was done using a standardized form, which comprised information on the recommendation, the strength of recommendation and the quality of the evidence used to inform the recommendation. DATA SYNTHESIS: We included 4 guidelines, providing a total of 217 recommendations (20 on assessments, 131 on interventions, and 66 on service provision). Most recommendations concerned pain management, education, pre- and postoperative management, and residual limb care. The strength of recommendation was generally weak to intermediate. The level of evidence mostly compromised expert opinions, with only 6.9% (15 of 217) being provided by randomized controlled trials, systematic reviews, or meta-analyses. CONCLUSIONS: The field of amputation is well covered for recommended interventions, but the level of evidence is generally low and is based mostly on expert opinion. Some important domains are not covered (eg, vocation and education, sexual and/or intimate relationships, activities of daily living or leisure activities, education concerning socket/liner fitting). There is also a lack of description of the contents of training and rehabilitation programs. This should be taken into account for the development of future guidelines.

STAT2 signaling restricts viral dissemination but drives severe pneumonia in SARS-CoV-2 infected hamsters.

Emergence of SARS-CoV-2 causing COVID-19 has resulted in hundreds of thousands of deaths. In search for key targets of effective therapeutics, robust animal models mimicking COVID-19 in humans are urgently needed. Here, we show that Syrian hamsters, in contrast to mice, are highly permissive to SARS-CoV-2 and develop bronchopneumonia and strong inflammatory responses in the lungs with neutrophil infiltration and edema, further confirmed as consolidations visualized by micro-CT alike in clinical practice. Moreover, we identify an exuberant innate immune response as key player in pathogenesis, in which STAT2 signaling plays a dual role, driving severe lung injury on the one hand, yet restricting systemic virus dissemination on the other. Our results reveal the importance of STAT2-dependent interferon responses in the pathogenesis and virus control during SARS-CoV-2 infection and may help rationalizing new strategies for the treatment of COVID-19 patients.