Lack of abundant core virome in Culex mosquitoes from a temperate climate region despite a mosquito species-specific virome.

In arthropod-associated microbial communities, insect-specific viruses (ISVs) are prevalent yet understudied due to limited infectivity outside their natural hosts. However, ISVs might play a crucial role in regulating mosquito populations and influencing arthropod-borne virus transmission. Some studies have indicated a core virome in mosquitoes consisting of mostly ISVs. Employing single mosquito metagenomics, we comprehensively profiled the virome of native and invasive mosquito species in Belgium. This approach allowed for accurate host species determination, prevalence assessment of viruses and Wolbachia, and the identification of novel viruses. Contrary to our expectations, no abundant core virome was observed in Culex mosquitoes from Belgium. In that regard, we caution against rigidly defining mosquito core viromes and encourage nuanced interpretations of other studies. Nonetheless, our study identified 45 viruses of which 28 were novel, enriching our understanding of the mosquito virome and ISVs. We showed that the mosquito virome in this study is species-specific and less dependent on the location where mosquitoes from the same species reside. In addition, because Wolbachia has previously been observed to influence arbovirus transmission, we report the prevalence of Wolbachia in Belgian mosquitoes and the detection of several Wolbachia mobile genetic elements. The observed prevalence ranged from 83% to 92% in members from the Culex pipiens complex.IMPORTANCECulex pipiens mosquitoes are important vectors for arboviruses like West Nile virus and Usutu virus. Virome studies on individual Culex pipiens, and on individual mosquitoes in general, have been lacking. To mitigate this, we sequenced the virome of 190 individual Culex and 8 individual Aedes japonicus mosquitoes. We report the lack of a core virome in these mosquitoes from Belgium and caution the interpretation of other studies in this light. The discovery of new viruses in this study will aid our comprehension of insect-specific viruses and the mosquito virome in general in relation to mosquito physiology and mosquito population dynamics.

ß-D-N4-hydroxycytidine (NHC, EIDD-1931) inhibits chikungunya virus replication in mosquito cells and ex vivo Aedes aegypti guts, but not when ingested during blood-feeding.

Chikungunya virus (CHIKV) is a mosquito-borne virus transmitted by Aedes mosquitoes. While there are no antiviral therapies currently available to treat CHIKV infections, several licensed oral drugs have shown significant anti-CHIKV activity in cells and in mouse models. However, the efficacy in mosquitoes has not yet been assessed. Such cross-species antiviral activity could be favorable, since virus inhibition in the mosquito vector might prevent further transmission to vertebrate hosts. Here, we explored the antiviral effect of ß-d-N4-hydroxycytidine (NHC, EIDD-1931), the active metabolite of molnupiravir, on CHIKV replication in Aedes aegypti mosquitoes. Antiviral assays in mosquito cells and in ex vivo cultured mosquito guts showed that NHC had significant antiviral activity against CHIKV. Exposure to a clinically relevant concentration of NHC did not affect Ae. aegypti lifespan when delivered via a bloodmeal, but it slightly reduced the number of eggs developed in the ovaries. When mosquitoes were exposed to a blood meal containing both CHIKV and NHC, the compound did not significantly reduce virus infection and dissemination in the mosquitoes. This was confirmed by modelling and could be explained by pharmacokinetic analysis, which revealed that by 6 h post-blood-feeding, 90% of NHC had been cleared from the mosquito bodies. Our data show that NHC inhibited CHIKV replication in mosquito cells and gut tissue, but not in vivo when mosquitoes were provided with a CHIKV-infectious bloodmeal spiked with NHC. The pipeline presented in this study offers a suitable approach to identify anti-arboviral drugs that may impede replication in mosquitoes.

Design, synthesis, and lead optimization of piperazinyl-pyrimidine analogues as potent small molecules targeting the viral capping machinery of Chikungunya virus.

The worldwide re-emerge of the Chikungunya virus (CHIKV), the high morbidity associated with it, and the lack of an available vaccine or antiviral treatment make the development of a potent CHIKV-inhibitor highly desirable. Therefore, an extensive lead optimization was performed based on the previously reported CHVB compound 1b and the reported synthesis route was optimized - improving the overall yield in remarkably shorter synthesis and work-up time. Hundred analogues were designed, synthesized, and investigated for their antiviral activity, physiochemistry, and toxicological profile. An extensive structure-activity relationship study (SAR) was performed, which focused mainly on the combination of scaffold changes and revealed the key chemical features for potent anti-CHIKV inhibition. Further, a thorough ADMET investigation of the compounds was carried out: the compounds were screened for their aqueous solubility, lipophilicity, their toxicity in CaCo-2 cells, and possible hERG channel interactions. Additionally, 55 analogues were assessed for their metabolic stability in human liver microsomes (HLMs), leading to a structure-metabolism relationship study (SMR). The compounds showed an excellent safety profile, favourable physicochemical characteristics, and the required metabolic stability. A cross-resistance study confirmed the viral capping machinery (nsP1) to be the viral target of these compounds. This study identified 31b and 34 as potent, safe, and stable lead compounds for further development as selective CHIKV inhibitors. Finally, the collected insight led to a successful scaffold hop (64b) for future antiviral research studies.

Culex modestus: the overlooked mosquito vector.

Culex (Barraudius) modestus (Ficalbi 1889) are found in temperate regions across Europe, Asia, and Northern Africa. These mosquitoes thrive during the summer and prefer to breed in permanent vegetative habitats such as rice paddies and marshes. Culex modestus feed on a wide range of bird species but are highly attracted to humans, which makes them a potential 'bridge' vector for enzootic pathogens. There is compelling evidence that Culex modestus is an efficient vector for West Nile virus, potentially capable of causing epidemics in humans and other mammals. This species is also a likely vector for Usutu virus, avian malaria (Plasmodium spp.), and parasitic heartworms (Dirofilaria spp.). Culex modestus can be morphologically identified at the larval and adult stages, and a distinctive phenotype of this species is their ability to overwinter. Despite the widespread establishment of this mosquito species and their role as vectors for human pathogens, we lack sufficient knowledge on this species to implement and evaluate targeted vector control measures. Since Culex modestus can be considered a potential public health threat, there is a need for a better understanding of this mosquito species.

Belgian Culex pipiens pipiens are competent vectors for West Nile virus while Culex modestus are competent vectors for Usutu virus.

BACKGROUND: West Nile virus (WNV) and Usutu virus (USUV) are emerging arthropod-borne viruses (arboviruses) in Europe transmitted by Culex mosquitoes. In Belgium, it is currently unknown which Culex species are competent vectors for WNV or USUV and if these mosquitoes carry Wolbachia, an endosymbiotic bacterium that can block arbovirus transmission. The aims of our study were to measure the vector competence of Belgian Culex mosquitoes to WNV and USUV and determine if a naturally acquired Wolbachia infection can influence virus transmission. METHODOLOGY/PRINCIPAL FINDINGS: Female Culex mosquitoes were captured from urban and peri-urban sites in Leuven, Belgium and offered an infectious bloodmeal containing WNV lineage 2, USUV European (EU) lineage 3, or USUV African (AF) lineage 3. Blood-fed females were incubated for 14 days at 25°C after which the body, head, and saliva were collected to measure infection, dissemination, and transmission rates as well as transmission efficiency. Mosquito species were identified by qRT-PCR or Sanger sequencing, the presence of infectious virus in mosquitoes was confirmed by plaque assays, and viral genome copies were quantified by qRT-PCR. Culex pipiens pipiens were able to transmit WNV (4.3% transmission efficiency, n = 2/47) but not USUV (EU lineage: n = 0/56; AF lineage: n = 0/37). In contrast, Culex modestus were able to transmit USUV (AF lineage: 20% transmission efficiency, n = 1/5) but not WNV (n = 0/6). We found that the presence or absence of Wolbachia was species-dependent and did not associate with virus transmission. CONCLUSIONS/SIGNIFICANCE: This is the first report that Belgian Culex mosquitoes can transmit both WNV and USUV, forewarning the risk of human transmission. More research is needed to understand the potential influence of Wolbachia on arbovirus transmission in Culex modestus mosquitoes.

Ex vivo gut cultures of Aedes aegypti are efficiently infected by mosquito-borne alpha- and flaviviruses.

Aedes aegypti mosquitoes can transmit several arboviruses, including chikungunya virus (CHIKV), dengue virus (DENV), and Zika virus (ZIKV). When blood-feeding on a virus-infected human, the mosquito ingests the virus into the midgut (stomach), where it replicates and must overcome the midgut barrier to disseminate to other organs and ultimately be transmitted via the saliva. Current tools to study mosquito-borne viruses (MBVs) include 2D-cell culture systems and in vivo mosquito infection models, which offer great advantages, yet have some limitations. Here, we describe a long-term ex vivo culture of Ae. aegypti guts. Cultured guts were metabolically active for 7 d in a 96-well plate at 28°C and were permissive to ZIKV, DENV, Ross River virus, and CHIKV. Ex vivo guts from Culex pipiens mosquitoes were found to be permissive to Usutu virus. Immunofluorescence staining confirmed viral protein synthesis in CHIKV-infected guts of Ae. aegypti. Furthermore, fluorescence microscopy revealed replication and spread of a reporter DENV in specific regions of the midgut. In addition, two known antiviral molecules, ß-d-N4-hydroxycytidine and 7-deaza-2'-C-methyladenosine, were able to inhibit CHIKV and ZIKV replication, respectively, in the ex vivo model. Together, our results show that ex vivo guts can be efficiently infected with mosquito-borne alpha- and flaviviruses and employed to evaluate antiviral drugs. Furthermore, the setup can be extended to other mosquito species. Ex vivo gut cultures could thus be a new model to study MBVs, offering the advantage of reduced biosafety measures compared to infecting living mosquitoes. IMPORTANCE Mosquito-borne viruses (MBVs) are a significant global health threat since they can cause severe diseases in humans, such as hemorrhagic fever, encephalitis, and chronic arthritis. MBVs rely on the mosquito vector to infect new hosts and perpetuate virus transmission. No therapeutics are currently available. The study of arbovirus infection in the mosquito vector can greatly contribute to elucidating strategies for controlling arbovirus transmission. This work investigated the infection of guts from Aedes aegypti mosquitoes in an ex vivo platform. We found several MBVs capable of replicating in the gut tissue, including viruses of major health importance, such as dengue, chikungunya, and Zika viruses. In addition, antiviral compounds reduced arbovirus infection in the cultured gut tissue. Overall, the gut model emerges as a useful tool for diverse applications such as studying tissue-specific responses to virus infection and screening potential anti-arboviral molecules.

Tarsal exposure to atovaquone inhibits chikungunya virus transmission by Aedes aegypti mosquitoes, but not the transmission of Zika virus.

The antimalarial drug atovaquone was recently reported to inhibit the in vitro replication of different arboviruses, including chikungunya virus (CHIKV) and Zika virus (ZIKV). Furthermore, atovaquone was shown to block Plasmodium parasite transmission by Anopheles mosquitoes when the mosquitoes were exposed to low concentrations on treated surfaces (i.e. tarsal exposure). Therefore, we evaluated the anti-CHIKV and -ZIKV effects of atovaquone via tarsal exposure in Aedes aegypti mosquitoes. We first confirmed that atovaquone exerted a dose-dependent antiviral effect on CHIKV and ZIKV replication in mosquito-derived cells. The modest antiviral effect could be rescued by adding exogenous uridine. Next, we assessed the effect of tarsal exposure to atovaquone on the fitness of Ae. aegypti. Concentrations up to 100 µmol/m2 did not affect the fecundity and egg-hatching rate. No significant effect on mosquito survival was observed when mosquitoes were exposed to concentrations up to 25 µmol/m2. To evaluate the antiviral effect of atovaquone against CHIKV, we exposed female mosquitoes to 100 µmol/m2 atovaquone for 1h, after which the mosquitoes were immediately infected with CHIKV or ZIKV via bloodmeal. Atovaquone did not significantly reduce ZIKV or CHIKV infection in Ae. aegypti, but successfully blocked the transmission of CHIKV in saliva. Tarsal exposure to antiviral drugs could therefore be a potential new strategy to reduce virus transmission by mosquitoes.

Meeting report: 36th International Conference on Antiviral Research in Lyon, France - March 13-17, 2023.

The 36th International Conference on Antiviral Research (ICAR), sponsored by the International Society for Antiviral Research (ISAR), was held March 13-17, 2023, in Lyon, France, and concurrently through an interactive remote meeting platform. Here we provide a report summarizing the presentations at the 36th ICAR, including the ISAR speaker awards. We also detail special events, sessions, and additional awards conferred at the meeting. ICAR returned to in-person meetings in 2022, convening in Seattle, WA, USA. The 36th ICAR is the first in-person meeting of the society in Europe since the beginning of the COVID-19 pandemic, which restricted most events to virtual attendance to help mitigate the spread and subsequent public health impact of SARS-CoV-2. An exceptionally high number of registrants and record attendance at this year's ICAR, along with a vast array of demonstrable expertise in a variety of antiviral research-related fields, reflected a strong and growing antiviral research community committed to improving health outcomes from viral diseases, including SARS-CoV-2, and to future pandemic preparedness. This report highlights the breadth of expertise, quality of research, and notable advancements that were contributed by members of ISAR and other participants at the meeting. ICAR aims to continue to provide a platform for sharing information, fostering collaborations, and supporting trainees in the field of antiviral research. The 37th ICAR will be held in Gold Coast, Australia, May 20-24, 2024.

Monitoring of SARS-CoV-2 concentration and circulation of variants of concern in wastewater of Leuven, Belgium.

Wastewater surveillance plays an important role in the management of the coronavirus disease 2019 (COVID-19) pandemic all over the world. Using different wastewater collection points in Leuven, we wanted to investigate the use of wastewater surveillance as an early warning system for an uprise of infections and as a tool to follow the circulation of specific variants of concern (VOCs) in particular geographic areas. Wastewater samples were collected from local neighborhood sewers and from a large regional wastewater treatment plant (WWTP) in the area of Leuven, Belgium. After virus concentration, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA was quantified by real-time quantitative polymerase chain reaction (RT-qPCR) and normalized with the human fecal indicator pepper mild mottle virus (PMMoV). A combination of multiplex RT-qPCR assays was used to detect signature mutations of circulating VOCs. Fecal virus shedding of SARS-CoV-2 variants was measured in feces samples of hospitalized patients. In two residential sampling sites, a rise in wastewater SARS-CoV-2 concentration preceded peaks in positive cases. In the WWTP, viral load peaks were seen concomitant with the consecutive waves of positive cases caused by the original Wuhan SARS-CoV-2 strain and subsequent VOCs. During the Omicron BA.1 wave, the wastewater viral load increased to a lesser degree, even after normalization of SARS-CoV-2 concentration using PMMoV. This might be attributable to a lower level of fecal excretion of this variant. Circulation of SARS-CoV-2 VOCs Alpha, Delta, Omicron BA1/BA.2, and BA.4/BA.5 could be detected based on the presence of specific key mutations. The shift in variants was noticeable in the wastewater, with key mutations of two different variants being present simultaneously during the transition period. Wastewater-based surveillance is a sensitive tool to monitor SARS-CoV-2 circulation levels and VOCs in larger regions. In times of reduced test capacity, this can prove to be highly valuable. Differences in excretion levels of various SARS-CoV-2 variants should however be taken into account when using wastewater surveillance to monitor SARS-CoV-2 circulation levels in the population.

Meeting report: 34th international conference on antiviral research.

As a result of the multiple gathering and travels restrictions during the SARS-CoV-2 pandemic, the annual meeting of the International Society for Antiviral Research (ISAR), the International Conference on Antiviral Research (ICAR), could not be held in person in 2021. Nonetheless, ISAR successfully organized a remote conference, retaining the most critical aspects of all ICARs, a collegiate gathering of researchers in academia, industry, government and non-governmental institutions working to develop, identify, and evaluate effective antiviral therapy for the benefit of all human beings. This article highlights the 2021 remote meeting, which presented the advances and objectives of antiviral and vaccine discovery, research, and development. The meeting resulted in a dynamic and effective exchange of ideas and information, positively impacting the prompt progress towards new and effective prophylaxis and therapeutics.

First Report of Mutations Associated With Pyrethroid (L1014F) and Organophosphate (G119S) Resistance in Belgian Culex (Diptera: Culicidae) Mosquitoes.

The emergence of West Nile virus and Usutu virus in Europe poses a significant risk to public health. In the absence of efficient antiviral therapy or vaccine candidates, the only strategy to control these arboviruses is to target the Culex (Diptera: Culicidae) mosquito vector. However, the selection pressure caused by exposure to insecticides for vector control or agricultural pest control can lead to insecticide resistance, thereby reducing the efficacy of insecticide-based vector control interventions. In Culex mosquitoes, two of the most common amino acid substitutions associated with insecticide resistance are the kdr L1014F in voltage gated sodium channels and G119S in acetylcholinesterase. In this study, Culex pipiens biotype pipiens, Culex torrentium, and Culex modestus were sampled from 2019 to 2021 in three distinct environmental habitats (urban, peri-urban, and agricultural) in and around the city of Leuven, Belgium. Individual mosquitoes were screened for two mutations resulting in L1014F and G119S amino acid substitutions. Both mutations were observed in Cx. pipiens and Cx. modestus but not in Cx. torrentium mosquitoes across the four collection sites. Furthermore, multi-resistance or cross-resistance in Cx. pipiens could be a threat in these areas, as both mutations were observed at low frequencies. These results provide the first report of kdr L1014F and ace-1 G119S resistance mutations in Cx. pipiens and Cx. modestus mosquitoes from Belgium, highlighting the importance of mosquito surveillance to design effective arbovirus outbreak control strategies.

Bidirectional Interactions between Arboviruses and the Bacterial and Viral Microbiota in Aedes aegypti and Culex quinquefasciatus.

Mosquitoes are important vectors for many arboviruses. It is becoming increasingly clear that various symbiotic microorganisms (including bacteria and insect-specific viruses; ISVs) in mosquitoes have the potential to modulate the ability of mosquitoes to transmit arboviruses. In this study, we compared the bacteriome and virome (both eukaryotic viruses and bacteriophages) of female adult Aedes aegypti and Culex quinquefasciatus mosquitoes fed with sucrose/water, blood, or blood spiked with Zika virus (ZIKV) or West Nile virus (WNV), respectively. Furthermore, we investigated associations between the microbiota and vector competence. We show that the influence of arboviruses on the mosquito microbiome-and vice versa-is distinct for each combination of arbovirus/mosquito species. The presence of ZIKV resulted in a temporarily increased Aedes ISV diversity. However, this effect was distinct for different ISVs: some ISVs decreased following the blood meal (Aedes aegypti totivirus), whereas other ISVs increased only when the blood contained ZIKV (Guadeloupe mosquito virus). Also, the diversity of the Aedes bacteriome depended on the diet and the presence of ZIKV, with a lower diversity observed for mosquitoes receiving blood without ZIKV. In Cx. quinquefasciatus, some ISVs increased in WNV-infected mosquitoes (Guadeloupe Culex tymo-like virus). Particularly, the presence of Wenzhou sobemo-like virus 3 (WSLV3) was associated with the absence of infectious WNV in mosquito heads, suggesting that WSLV3 might affect vector competence for WNV. Distinct profiles of bacteriophages were identified in Culex mosquitoes depending on diet, despite the lack of clear changes in the bacteriome. Overall, our data demonstrate a complex three-way interaction among arboviruses, resident microbiota, and the host, which is distinct for different arbovirus-mosquito combinations. A better understanding of these interactions may lead to the identification of microbiota able to suppress the ability of arbovirus transmission to humans, and hence improved arbovirus control measures. IMPORTANCE In this study, we first utilized the single mosquito microbiome analysis, demonstrating a complex three-way interaction among arboviruses, resident microbiota, and the host, which is distinct for different arbovirus-mosquito combinations. Some of the previously described "core virus" increased in the mosquitos receiving viral blood meal, like Guadeloupe mosquito virus and Guadeloupe Culex tymo-like virus, suggesting their potential roles in ZIKV and WNV infection. Notably, Wenzhou sobemo-like virus 3 was associated with the absence of infectious WNV in heads of Culex mosquitoes, which might affect vector competence for WNV. A better understanding of these interactions will lead to the identification of microbiota able to suppress the ability of arbovirus transmission to humans, and hence improved arbovirus control measures.

HIV protease inhibitors Nelfinavir and Lopinavir/Ritonavir markedly improve lung pathology in SARS-CoV-2-infected Syrian hamsters despite lack of an antiviral effect.

Nelfinavir is an HIV protease inhibitor that has been widely prescribed as a component of highly active antiretroviral therapy, and has been reported to exert in vitro antiviral activity against SARS-CoV-2. We here assessed the effect of Nelfinavir in a SARS-CoV-2 infection model in hamsters. Despite the fact that Nelfinavir, [50 mg/kg twice daily (BID) for four consecutive days], did not reduce viral RNA load and infectious virus titres in the lung of infected animals, treatment resulted in a substantial improvement of SARS-CoV-2-induced lung pathology. This was accompanied by a dense infiltration of neutrophils in the lung interstitium which was similarly observed in non-infected hamsters. Nelfinavir resulted also in a marked increase in activated neutrophils in the blood, as observed in non-infected animals. Although Nelfinavir treatment did not alter the expression of chemoattractant receptors or adhesion molecules on human neutrophils, in vitro migration of human neutrophils to the major human neutrophil attractant CXCL8 was augmented by this protease inhibitor. Nelfinavir appears to induce an immunomodulatory effect associated with increasing neutrophil number and functionality, which may be linked to the marked improvement in SARS-CoV-2 lung pathology independent of its lack of antiviral activity. Since Nelfinavir is no longer used for the treatment of HIV, we studied the effect of two other HIV protease inhibitors, namely the combination Lopinavir/Ritonavir (Kaletra™) in this model. This combination resulted in a similar protective effect as Nelfinavir against SARS-CoV2 induced lung pathology in hamsters.

Biodistribution and environmental safety of a live-attenuated YF17D-vectored SARS-CoV-2 vaccine candidate.

New platforms are needed for the design of novel prophylactic vaccines and advanced immune therapies. Live-attenuated yellow fever vaccine YF17D serves as a vector for several licensed vaccines and platform for novel candidates. On the basis of YF17D, we developed an exceptionally potent COVID-19 vaccine candidate called YF-S0. However, use of such live RNA viruses raises safety concerns, such as adverse events linked to original YF17D (yellow fever vaccine-associated neurotropic disease [YEL-AND] and yellow fever vaccine-associated viscerotropic disease [YEL-AVD]). In this study, we investigated the biodistribution and shedding of YF-S0 in hamsters. Likewise, we introduced hamsters deficient in signal transducer and activator of transcription 2 (STAT2) signaling as a new preclinical model of YEL-AND/AVD. Compared with YF17D, YF-S0 showed improved safety with limited dissemination to brain and visceral tissues, absent or low viremia, and no shedding of infectious virus. Considering that yellow fever virus is transmitted by Aedes mosquitoes, any inadvertent exposure to the live recombinant vector via mosquito bites is to be excluded. The transmission risk of YF-S0 was hence compared with readily transmitting YF-Asibi strain and non-transmitting YF17D vaccine, with no evidence for productive infection of mosquitoes. The overall favorable safety profile of YF-S0 is expected to translate to other vaccines based on the same YF17D platform.

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.

Assessing In Vitro Resistance Development in Enterovirus A71 in the Context of Combination Antiviral Treatment.

There are currently no antivirals available to treat infection with enterovirus A71 (EV-A71) or any other enterovirus. The extensively studied capsid binders rapidly select for drug-resistant variants. We here explore whether the combination of two direct-acting enterovirus inhibitors with a different mechanism of action may delay or prevent resistance development to the capsid binders. To that end, the in vitro dynamics of resistance development to the capsid binder pirodavir was studied either alone or in combination with a viral 2C-targeting compound (SMSK_0213), a viral 3C-protease inhibitor (rupintrivir) or a viral RNA-dependent RNA polymerase inhibitor (7DMA). We demonstrate that combining pirodavir with either rupintrivir or 7DMA delays the development of resistance to pirodavir and that no resistance to the protease or polymerase inhibitor develops. The combination of pirodavir with the 2C inhibitor results in a double-resistant virus population, where only the minority carries the resistant mutation.

Comparative analysis of the molecular mechanism of resistance to vapendavir across a panel of picornavirus species.

Vapendavir is a rhino/enterovirus inhibitor that targets a hydrophobic pocket in the viral capsid preventing the virus from entering the cell. We set out to study and compare the molecular mechanisms of resistance to vapendavir among clinically relevant Picornavirus species. To this end in vitro resistance selection of drug-resistant isolates was applied in rhinovirus 2 and 14, enterovirus-D68 and Poliovirus 1 Sabin. Mutations in the drug-binding pocket in VP1 (C199R/Y in hRV14; I194F in PV1; M252L and A156T in EV-D68), typical for this class of compounds, were identified. Interestingly, we also observed mutations located outside the pocket (K167E in EV-D68 and G149C in hRV2) that contribute to the resistant phenotype. Remarkably, the G149C substitution rendered the replication of human rhinovirus 2 dependent on the presence of vapendavir. Our data suggest that the binding of vapendavir to the capsid of the G149C isolate may be required to stabilize the viral particle and to allow efficient dissemination of the virus. We observed the dependency of the G149C isolate on other compounds of this class, suggesting that this phenotype is common for capsid binders. In addition the VP1 region containing the G149C substitution has not been associated with antiviral resistance before. Our results demonstrate that the phenotype and genotype of clinically relevant vapendavir-resistant picornavirus species is more complex than generally believed.