The coronavirus disease 2019 (COVID-19) pandemic, driven by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has highlighted the urgent need for effective therapeutics, particularly as limitations of existing antiviral treatments persist. Establishing the proper strategy to set target concentrations for human dose projection is essential for any novel drug development project. In this study, we investigated the translatability of various preclinical models, specifically in vitro A549-hACE2 cells and air-liquid interface human nasal epithelial cultures, and in vivo Syrian golden hamsters and K18-hACE2 transgenic mice, using 3-chymotrypsin-like protease (3CLpro) inhibitors as model direct-acting antivirals. We assessed the minimal efficacious concentrations in these models of both ensitrelvir (ETV) and nirmatrelvir (NTV) co-dosed with ritonavir (Paxlovid) and compared this to clinical human data. Notably, in vitro models and the K18-hACE2 mice proved to be the most predictive for human efficacious exposures. Interestingly, lower efficacious exposures were needed in the more severe K18-hACE2 mouse model than in the Syrian golden hamster model for mild infection. Overall, we recommend applying a factor of 4-fold to the in vitro 90% effective concentration (EC90) for a conservative estimate of human efficacious exposure and recommend validation using K18-hACE2 mice. These insights are critical for guiding the development of effective SARS-CoV-2 therapeutics and optimizing clinical trial design.

The membrane (M) protein of betacoronaviruses is well conserved and has a key role in viral assembly 1,2 . Here we describe the identification of JNJ-9676, a small-molecule inhibitor targeting the coronavirus M protein. JNJ-9676 demonstrates in vitro nanomolar antiviral activity against SARS-CoV-2, SARS-CoV and sarbecovirus strains from bat and pangolin zoonotic origin. Using cryogenic electron microscopy (cryo-EM), we determined a binding pocket of JNJ-9676 formed by the transmembrane domains of the M protein dimer. Compound binding stabilized the M protein dimer in an altered conformational state between its long and short forms, preventing the release of infectious virus. In a pre-exposure Syrian golden hamster model, JNJ-9676 (25 mg per kg twice per day) showed excellent efficacy, illustrated by a significant reduction in viral load and infectious virus in the lung by 3.5 and 4 log 10 -transformed RNA copies and 50% tissue culture infective dose (TCID 50 ) per mg lung, respectively. Histopathology scores at this dose were reduced to the baseline. In a post-exposure hamster model, JNJ-9676 was efficacious at 75 mg per kg twice per day even when added at 48 h after infection, when peak viral loads were observed. The M protein is an attractive antiviral target to block coronavirus replication, and JNJ-9676 represents an interesting chemical series towards identifying clinical candidates addressing the current and future coronavirus pandemics.

Mosquito-borne flaviviruses have emerged as global health threats due to their rapid spread and high disease burden. The initial site of virus replication is the mosquito biting site in the host skin, yet the role of host skin bacteria remains unclear. Here, we observed accelerated progression of Zika virus (ZIKV)-induced disease in mice with depleted skin bacteria by topical antibiotic treatment prior to ZIKV infection. Increased viral loads in the blood, draining lymph node, spleen and brain of antibiotic-treated mice suggested faster ZIKV dissemination. Flow cytometry revealed increased frequencies of T cells in the lymph nodes, along with enhanced activation of T cells in the brain upon antibiotic treatment. The antibiotic-induced phenotype was rescued by restoring the skin bacteria prior to infection. Our findings demonstrate an increased susceptibility to ZIKV infection in mice with depleted skin microbiomes, suggesting a potential role of skin bacteria in modulating the infection outcome. ### Competing Interest Statement The authors have declared no competing interest.

Abstract The membrane (M) protein of betacoronaviruses is well-conserved and plays a key role in viral assembly. Here, we describe the discovery of JNJ-9676, a novel small molecule targeting the coronaviral (CoV) M protein. JNJ-9676 demonstrates in vitro nanomolar antiviral activity against SARS-CoV2, SARS-CoV, and sarbecovirus strains from bat and pangolin zoonotic origin. Using cryogenic electron microscopy, we determined a novel binding pocket of JNJ-9676 in the M protein's transmembrane domain. Compound binding stabilized the M protein in an altered conformational state between its long- and short-forms, preventing the release of infectious virus. In a pre-exposure Syrian golden hamster model, JNJ-9676 (25 mg/kg BID) showed excellent efficacy illustrated by a significant reduction in viral load and infectious virus in the lung by 3.5 log10 and 4 log10, respectively. Histopathology scores at this dose were reduced to baseline. In a post-exposure hamster model, JNJ-9676 was efficacious at 75mg/kg BID even when added at 48 h post-infection, when peak viral loads were observed. The M protein is an attractive novel antiviral target to block coronavirus replication with JNJ-9676 representing an interesting chemical series towards identifying clinical candidates addressing the current and future CoV pandemics.

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-β-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.

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 pre-incubation 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 pre-incubation 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 directly interacting with the virus particle. ### Competing Interest Statement The authors have declared no competing interest.

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.

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

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.

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.

We have identified camelid single-domain antibodies (VHHs) that cross-neutralize SARS-CoV-1 and −2, such as VHH72, which binds to a unique highly conserved epitope in the viral receptor-binding domain (RBD) that is difficult to access for human antibodies. Here, we establish a protein engineering path for how a stable, long-acting drug candidate can be generated out of such a VHH building block. When fused to human IgG1-Fc, the prototype VHH72 molecule prophylactically protects hamsters from SARS-CoV-2. In addition, we demonstrate that both systemic and intranasal application protects hACE-2-transgenic mice from SARS-CoV-2 induced lethal disease progression. To boost potency of the lead, we used structure-guided molecular modeling combined with rapid yeast-based Fc-fusion prototyping, resulting in the affinity-matured VHH72_S56A-Fc, with subnanomolar SARS-CoV-1 and −2 neutralizing potency. Upon humanization, VHH72_S56A was fused to a human IgG1 Fc with optimized manufacturing homogeneity and silenced effector functions for enhanced safety, and its stability as well as lack of off-target binding was extensively characterized. Therapeutic systemic administration of a low dose of VHH72_S56A-Fc antibodies strongly restricted replication of both original and D614G mutant variants of SARS-CoV-2 virus in hamsters, and minimized the development of lung damage. This work led to the selection of XVR011 for clinical development, a highly stable anti-COVID-19 biologic with excellent manufacturability. Additionally, we show that XVR011 is unaffected in its neutralizing capacity of currently rapidly spreading SARS-CoV-2 variants, and demonstrate its unique, wide scope of binding across the Sarbecovirus clades.

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.

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-2–neutralizing 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 anti–COVID-19 biologic that is now being evaluated in the clinic.

Summary The global emergence of Zika virus (ZIKV) in the last decade revealed the unprecedented ability for a mosquito-borne virus to cause congenital birth defects such as microcephaly. 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 compared the transmissibility and pathogenicity of seven low-passage ZIKV strains representing the recently circulating viral genetic diversity. We found that recent African ZIKV strains largely outperformed their Asian counterparts in mosquito transmission kinetics experiments, which translated into a markedly higher epidemic potential in outbreak computer simulations. In addition, African ZIKV strains were significantly more lethal than Asian ZIKV strains in immunocompromised adult mice. Finally, prenatal infection of immunocompetent mouse embryos with an African ZIKV strain resulted in embryonic death whereas it caused microcephaly with Asian ZIKV strains. Together, our results demonstrate the high epidemic potential and pathogenicity of recent ZIKV strains from Africa. Importantly, they also imply that the African ZIKV lineage could more easily go unnoticed by public health surveillance systems than the Asian ZIKV lineage due to its propensity to cause fetal loss rather than birth defects.

Alphaviruses are (mainly) arthropod-borne viruses that belong to the family of the Togaviridae. Based on the disease they cause, alphaviruses are divided into an arthritogenic and an encephalitic group. Arthritogenic alphaviruses such as the chikungunya virus (CHIKV), the Ross River virus (RRV) and the Mayaro virus (MAYV) have become a serious public health concern in recent years. Epidemics are associated with high morbidity and the infections cause in many patients debilitating joint pain that can persist for months to years. The recent (2013–2014) introduction of CHIKV in the Americas resulted in millions of infected persons. Massive outbreaks of CHIKV and other arthritogenic alphaviruses are likely to occur in the future. Despite the worldwide (re-)emergence of these viruses, there are no antivirals or vaccines available for the treatment or prevention of infections with alphaviruses. It is therefore of utmost importance to develop antiviral strategies against these viruses. We here review the possible molecular targets in the replication cycle of these viruses for the development of antivirals. In addition, we provide an overview of the currently available in vitro systems and mouse infection models that can be used to assess the potential antiviral effect against these viruses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapidly spread around the globe after its emergence in Wuhan in December 2019. With no specific therapeutic and prophylactic options available, the virus has infected millions of people of which more than half a million succumbed to the viral disease, COVID-19. The urgent need for an effective treatment together with a lack of small animal infection models has led to clinical trials using repurposed drugs without preclinical evidence of their in vivo efficacy. We established an infection model in Syrian hamsters to evaluate the efficacy of small molecules on both infection and transmission. Treatment of SARS-CoV-2-infected hamsters with a low dose of favipiravir or hydroxychloroquine with(out) azithromycin resulted in, respectively, a mild or no reduction in virus levels. However, high doses of favipiravir significantly reduced infectious virus titers in the lungs and markedly improved lung histopathology. Moreover, a high dose of favipiravir decreased virus transmission by direct contact, whereas hydroxychloroquine failed as prophylaxis. Pharmacokinetic modeling of hydroxychloroquine suggested that the total lung exposure to the drug did not cause the failure. Our data on hydroxychloroquine (together with previous reports in macaques and ferrets) thus provide no scientific basis for the use of this drug in COVID-19 patients. In contrast, the results with favipiravir demonstrate that an antiviral drug at nontoxic doses exhibits a marked protective effect against SARS-CoV-2 in a small animal model. Clinical studies are required to assess whether a similar antiviral effect is achievable in humans without toxic effects.

Alphaviruses are (mainly) arthropod-borne viruses that belong to the family of the Togaviridae. Based on the disease they cause, alphaviruses are divided into an arthritogenic and an encephalitic group. Arthritogenic alphaviruses such as the chikungunya virus (CHIKV), the Ross River virus (RRV) and the Mayaro virus (MAYV) have become a serious public health concern in recent years. Epidemics are associated with high morbidity and the infections cause in many patients debilitating joint pain that can persist for months to years. The recent (2013–2014) introduction of CHIKV in the Americas resulted in millions of infected persons. Massive outbreaks of CHIKV and other arthritogenic alphaviruses are likely to occur in the future. Despite the worldwide (re-)emergence of these viruses, there are no antivirals or vaccines available for the treatment or prevention of infections with alphaviruses. It is therefore of utmost importance to develop antiviral strategies against these viruses. We here review the possible molecular targets in the replication cycle of these viruses for the development of antivirals. In addition, we provide an overview of the currently available in vitro systems and mouse infection models that can be used to assess the potential antiviral effect against these viruses.

Background: Drug repurposing is an attractive strategy to rapidly develop affordable therapy against COVID-19. The antifungal drug itraconazole exerts in vitro activity against SARS-CoV-2 comparable to that of hydroxychloroquine. 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 patients with COVID-19 were randomly assigned to receive standard of care with or without itraconazole. The 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. Other outcomes included time to sustained clinical improvement, duration of supplemental oxygen and evolution of nasopharyngeal viral load. 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 interim analysis that showed no trends 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), median time to clinical improvement 10 vs 9 days, hazard ratio 0.94 (95% CI 0.56 to 1.60) for itraconazole vs standard of care.  Interpretation: Despite in vitro activity, itraconazole was not effective in a preclinical COVID-19 hamster model. A proof-of-concept clinical study was ended prematurely because of futility.  Trial Registration: (EudraCT 2020-001243-15) Funding: Covid-19-Fund KU Leuven, Research Foundation - Flanders (FWO), Horizon 2020, Bill and Melinda Gates Foundation Declaration of Interests: Initial dug screening and discovery of the antiviral effect of itraconazole was done in collaboration with Johnson & Johnson and described in a separate manuscript. Scientists from Johnson & Johnson also performed drug measurements on hamster samples and provided guidance on the dosing regimens for the preclinical studies. The company had no role in the design, execution, analysis, publication or funding of the clinical trial. Author Conflict of Interests: None to declare. Ethics Approval Statement: The institutional Ethical Committee approved all animal experiments (license P065-2020). The study was conducted in accordance with the International Conference on Harmonization Guidelines for Good Clinical Practice and the Declaration of Helsinki. The protocol was approved by the institutional Ethics Committee and by the Belgian Federal Agency for Medicines and Health Products (EudraCT 2020-001243-15). The trial was part of the DAWn clinical studies.

Arthropod-borne viruses (arboviruses) are important human pathogens for which there are no specific antiviral medicines. The abundance of genetically distinct arbovirus species, coupled with the unpredictable nature of their outbreaks, has made the development of virus-specific treatments challenging. Instead, we have defined and targeted a key aspect of the host innate immune response to virus at the arthropod bite that is common to all arbovirus infections, potentially circumventing the need for virus-specific therapies. Using mouse models and human skin explants, we identify innate immune responses by dermal macrophages in the skin as a key determinant of disease severity. Post-exposure treatment of the inoculation site by a topical TLR7 agonist suppressed both the local and subsequent systemic course of infection with a variety of arboviruses from the Alphavirus, Flavivirus, and Orthobunyavirus genera. Clinical outcome was improved in mice after infection with a model alphavirus. In the absence of treatment, antiviral interferon expression to virus in the skin was restricted to dermal dendritic cells. In contrast, stimulating the more populous skin-resident macrophages with a TLR7 agonist elicited protective responses in key cellular targets of virus that otherwise proficiently replicated virus. By defining and targeting a key aspect of the innate immune response to virus at the mosquito bite site, we have identified a putative new strategy for limiting disease after infection with a variety of genetically distinct arboviruses.