In Silico Drug Repurposing Uncovered the Antiviral Potential of the Antiparasitic Drug Oxibendazole Against the Chikungunya Virus.

Chikungunya virus (CHIKV) has been reported in over 120 countries and is the causative agent of Chikungunya fever. The debilitating nature of this disease, which can persist months to years after acute infection, drastically impacts the quality of life of patients. Yet, specific antivirals are lacking for the treatment of this disease, which makes the search for new drugs necessary. In this context, the nsP2 protease emerges as an attractive therapeutic target, and drug repurposing strategies have proven to be valuable. Therefore, we combined in silico and in vitro methods to identify known drugs as potential CHIKV nsP2 protease inhibitors with antiviral properties within DrugBank. Herein, we developed a hybrid virtual screening pipeline comprising pharmacophore- and target-based screening, drug-like, and pharmaceutical filtering steps. Six virtual hits were obtained, and two of them, capecitabine (CPB) and oxibendazole (OBZ), were evaluated against CHIKV replication in Vero cells. CPB did not present antiviral activity, whereas OBZ inhibited the replication of two different strains of CHIKV, namely, 181-25 (Asian genotype) and BRA/RJ/18 (clinical isolate from ECSA genotype). OBZ showed potent antiviral activity against the CHIKV BRA/RJ/18 (EC50 = 11.4 µM) with a high selectivity index (>44). Analogs of OBZ (albendazole, fenbendazole, and mebendazole) were also evaluated, but none exhibited anti-CHIKV activity, and further, their stereoelectronic features were analyzed. Additionally, we observed that OBZ acts mainly at post-entry steps. Hence, our results support further in vivo studies to investigate the antiviral potential of OBZ, which offers a new alternative to fight CHIKV infections.

WSV2023 - The second meeting of the world society for virology: One health - One world - One virology.

The Second International Conference of the World Society for Virology (WSV), hosted by Riga Stradins University, was held in Riga, Latvia, on June 15-17th, 2023. It prominently highlighted the recent advancements in different disciplines of virology. The conference had fourteen keynote speakers covering diverse topics, including emerging virus pseudotypes, Zika virus vaccine development, herpesvirus capsid mobility, parvovirus invasion strategies, influenza in animals and birds, West Nile virus and Marburg virus ecology, as well as the latest update in animal vaccines. Discussions further explored SARS-CoV-2 RNA replicons as vaccine candidates, SARS-CoV-2 in humans and animals, and the significance of plant viruses in the 'One Health' paradigm. The presence of the presidents from three virology societies, namely the American, Indian, and Korean Societies for Virology, highlighted the event's significance. Additionally, past president of the American Society for Virology (ASV), formally declared the partnership between ASV and WSV during the conference.

Antiviral evaluation of 1,4-disubstituted-1,2,3-triazole derivatives against Chikungunya virus.

Aim: This work aimed to investigate the antiviral activity of two 1,4-disubstituted-1,2,3-triazole derivatives (1 and 2) against Chikungunya virus (CHIKV) replication. Materials & methods: Cytotoxicity was analyzed using colorimetric assays and the antiviral potential was evaluated using plaque assays and computational tools. Results: Compound 2 showed antiviral activity against CHIKV 181-25 in BHK-21 and Vero cells. Also, this compound presented a higher activity against CHIKV BRA/RJ/18 in Vero cells, like compound 1. Compound 2 exhibited virucidal activity and inhibited virus entry while compound 1 inhibited virus release. Molecular docking suggested that these derivatives inhibit nsP1 protein while compound 1 may also target capsid protein. Conclusion: Both compounds exhibit promising antiviral activity against CHIKV by blocking different steps of virus replication.

A Prototype-Pathogen Approach for the Development of Flavivirus Countermeasures.

Flaviviruses are a genus within the Flaviviridae family of positive-strand RNA viruses and are transmitted principally through mosquito and tick vectors. These viruses are responsible for hundreds of millions of human infections worldwide per year that result in a range of illnesses from self-limiting febrile syndromes to severe neurotropic and viscerotropic diseases and, in some cases, death. A vaccine against the prototype flavivirus, yellow fever virus, has been deployed for 85 years and is highly effective. While vaccines against some medically important flaviviruses are available, others have proven challenging to develop. The emergence and spread of flaviviruses, including dengue virus and Zika virus, demonstrate their pandemic potential. This review highlights the gaps in knowledge that need to be addressed to allow for the rapid development of vaccines against emerging flaviviruses in the future.

Structure of Vibrio Phage XM1, a Simple Contractile DNA Injection Machine.

Antibiotic resistance poses a growing risk to public health, requiring new tools to combat pathogenic bacteria. Contractile injection systems, including bacteriophage tails, pyocins, and bacterial type VI secretion systems, can efficiently penetrate cell envelopes and become potential antibacterial agents. Bacteriophage XM1 is a dsDNA virus belonging to the Myoviridae family and infecting Vibrio bacteria. The XM1 virion, made of 18 different proteins, consists of an icosahedral head and a contractile tail, terminated with a baseplate. Here, we report cryo-EM reconstructions of all components of the XM1 virion and describe the atomic structures of 14 XM1 proteins. The XM1 baseplate is composed of a central hub surrounded by six wedge modules to which twelve spikes are attached. The XM1 tail contains a fewer number of smaller proteins compared to other reported phage baseplates, depicting the minimum requirements for building an effective cell-envelope-penetrating machine. We describe the tail sheath structure in the pre-infection and post-infection states and its conformational changes during infection. In addition, we report, for the first time, the in situ structure of the phage neck region to near-atomic resolution. Based on these structures, we propose mechanisms of virus assembly and infection.

Efficacy of an isoxazole-3-carboxamide analog of pleconaril in mouse models of Enterovirus-D68 and Coxsackie B5.

Enteroviruses (EV) cause a number of life-threatening infectious diseases. EV-D68 is known to cause respiratory illness in children that can lead to acute flaccid myelitis. Coxsackievirus B5 (CVB5) is commonly associated with hand-foot-mouth disease. There is no antiviral treatment available for either. We have developed an isoxazole-3-carboxamide analog of pleconaril (11526092) which displayed potent inhibition of EV-D68 (IC50 58 nM) as well as other enteroviruses including the pleconaril-resistant Coxsackievirus B3-Woodruff (IC50 6-20 nM) and CVB5 (EC50 1 nM). Cryo-electron microscopy structures of EV-D68 in complex with 11526092 and pleconaril demonstrate destabilization of the EV-D68 MO strain VP1 loop, and a strain-dependent effect. A mouse respiratory model of EV-D68 infection, showed 3-log decreased viremia, favorable cytokine response, as well as statistically significant 1-log reduction in lung titer reduction at day 5 after treatment with 11526092. An acute flaccid myelitis neurological infection model did not show efficacy. 11526092 was tested in a mouse model of CVB5 infection and showed a 4-log TCID50 reduction in the pancreas. In summary, 11526092 represents a potent in vitro inhibitor of EV with in vivo efficacy in EV-D68 and CVB5 animal models suggesting it is worthy of further evaluation as a potential broad-spectrum antiviral therapeutic against EV.

Structural constraints link differences in neutralization potency of human anti-Eastern equine encephalitis virus monoclonal antibodies.

Selection and development of monoclonal antibody (mAb) therapeutics against pathogenic viruses depends on certain functional characteristics. Neutralization potency, or the half-maximal inhibitory concentration (IC50) values, is an important characteristic of candidate therapeutic antibodies. Structural insights into the bases of neutralization potency differences between antiviral neutralizing mAbs are lacking. In this report, we present cryo-electron microscopy (EM) reconstructions of three anti-Eastern equine encephalitis virus (EEEV) neutralizing human mAbs targeting overlapping epitopes on the E2 protein, with greater than 20-fold differences in their respective IC50 values. From our structural and biophysical analyses, we identify several constraints that contribute to the observed differences in the neutralization potencies. Cryo-EM reconstructions of EEEV in complex with these Fab fragments reveal structural constraints that dictate intravirion or intervirion cross-linking of glycoprotein spikes by their IgG counterparts as a mechanism of neutralization. Additionally, we describe critical features for the recognition of EEEV by these mAbs including the epitope-paratope interaction surface, occupancy, and kinetic differences in on-rate for binding to the E2 protein. Each constraint contributes to the extent of EEEV inhibition for blockade of virus entry, fusion, and/or egress. These findings provide structural and biophysical insights into the differences in mechanism and neutralization potencies of these antibodies, which help inform rational design principles for candidate vaccines and therapeutic antibodies for all icosahedral viruses.

prM-reactive antibodies reveal a role for partially mature virions in dengue virus pathogenesis.

Cleavage of the flavivirus premembrane (prM) structural protein during maturation can be inefficient. The contribution of partially mature flavivirus virions that retain uncleaved prM to pathogenesis during primary infection is unknown. To investigate this question, we characterized the functional properties of newly-generated dengue virus (DENV) prM-reactive monoclonal antibodies (mAbs) in vitro and using a mouse model of DENV disease. Anti-prM mAbs neutralized DENV infection in a virion maturation state-dependent manner. Alanine scanning mutagenesis and cryoelectron microscopy of anti-prM mAbs in complex with immature DENV defined two modes of attachment to a single antigenic site. In vivo, passive transfer of intact anti-prM mAbs resulted in an antibody-dependent enhancement of disease. However, protection against DENV-induced lethality was observed when the transferred mAbs were genetically modified to inhibit their ability to interact with Fcγ receptors. These data establish that in addition to mature forms of the virus, partially mature infectious prM+ virions can also contribute to pathogenesis during primary DENV infections.

Positive-strand RNA viruses-a Keystone Symposia report.

Positive-strand RNA viruses have been the cause of several recent outbreaks and epidemics, including the Zika virus epidemic in 2015, the SARS outbreak in 2003, and the ongoing SARS-CoV-2 pandemic. On June 18-22, 2022, researchers focusing on positive-strand RNA viruses met for the Keystone Symposium "Positive-Strand RNA Viruses" to share the latest research in molecular and cell biology, virology, immunology, vaccinology, and antiviral drug development. This report presents concise summaries of the scientific discussions at the symposium.

Exploring the Expression and Function of cTyro3, a Candidate Zika Virus Receptor, in the Embryonic Chicken Brain and Inner Ear.

The transmembrane protein Axl was proposed as an entry receptor for Zika virus (ZIKV) infection in vitro, but conflicting results from in vivo studies have made it difficult to establish Axl as a physiologically relevant ZIKV receptor. Both the functional redundancy of receptors and the experimental model used can lead to variable results. Therefore, it can be informative to explore alternative animal models to analyze ZIKV receptor candidates as an aid in discovering antivirals. This study used chicken embryos to examine the role of chicken Tyro3 (cTyro3), the equivalent of human Axl. Results show that endogenous cTyro3 mRNA expression overlaps with previously described hot spots of ZIKV infectivity in the brain and inner ear. We asked if ectopic expression or knockdown of cTyro3 influenced ZIKV infection in embryos. Tol2 vectors or replication-competent avian retroviruses were used in ovo to introduce full-length or truncated (presumed dominant-negative) cTyro3, respectively, into the neural tube on embryonic day two (E2). ZIKV was delivered to the brain 24 h later. cTyro3 manipulations did not alter ZIKV infection or cell death in the E5/E6 brain. Moreover, delivery of truncated cTyro3 variants to the E3 otocyst had no effect on inner ear formation on E6 or E10.

Surface decorated reporter-tagged chikungunya virus-like particles for clinical diagnostics and identification of virus entry inhibitors.

The ever-evolving and versatile VLP technology is becoming an increasingly popular area of science. This study presents surface decorated reporter-tagged VLPs of CHIKV, an enveloped RNA virus of the genus alphavirus and its applications. Western blot, IFA and live-cell imaging confirm the expression of reporter-tagged CHIK-VLPs from transfected HEK293Ts. CryoEM micrographs reveal particle diameter as ∼67nm and 56-70 nm, respectively, for NLuc CHIK-VLPs and mCherry CHIK-VLPs. Our study demonstrates that by exploiting NLuc CHIK-VLPs as a detector probe, robust ratiometric luminescence signal in CHIKV-positive sera compared to healthy controls can be achieved swiftly. Moreover, the potential activity of the Suramin drug as a CHIKV entry inhibitor has been validated through the reporter-tagged CHIK-VLPs. The results reported in this study open new avenues in the eVLPs domain and offer potential for large-scale screening of clinical samples and antiviral agents targeting entry of CHIKV and other alphaviruses.

A Zika virus-specific IgM elicited in pregnancy exhibits ultrapotent neutralization.

Congenital Zika virus (ZIKV) infection results in neurodevelopmental deficits in up to 14% of infants born to ZIKV-infected mothers. Neutralizing antibodies are a critical component of protective immunity. Here, we demonstrate that plasma IgM contributes to ZIKV immunity in pregnancy, mediating neutralization up to 3 months post-symptoms. From a ZIKV-infected pregnant woman, we isolated a pentameric ZIKV-specific IgM (DH1017.IgM) that exhibited ultrapotent ZIKV neutralization dependent on the IgM isotype. DH1017.IgM targets an envelope dimer epitope within domain II. The epitope arrangement on the virion is compatible with concurrent engagement of all ten antigen-binding sites of DH1017.IgM, a solution not available to IgG. DH1017.IgM protected mice against viremia upon lethal ZIKV challenge more efficiently than when expressed as an IgG. Our findings identify a role for antibodies of the IgM isotype in protection against ZIKV and posit DH1017.IgM as a safe and effective candidate immunotherapeutic, particularly during pregnancy.

Requirement of a functional ion channel for Sindbis virus glycoprotein transport, CPV-II formation, and efficient virus budding.

Many viruses encode ion channel proteins that oligomerize to form hydrophilic pores in membranes of virus-infected cells and the viral membrane in some enveloped viruses. Alphavirus 6K, human immunodeficiency virus type 1 Vpu (HIV-Vpu), influenza A virus M2 (IAV-M2), and hepatitis C virus P7 (HCV-P7) are transmembrane ion channel proteins that play essential roles in virus assembly, budding, and entry. While the oligomeric structures and mechanisms of ion channel activity are well-established for M2 and P7, these remain unknown for 6K. Here we investigated the functional role of the ion channel activity of 6K in alphavirus assembly by utilizing a series of Sindbis virus (SINV) ion channel chimeras expressing the ion channel helix from Vpu or M2 or substituting the entire 6K protein with full-length P7, in cis. We demonstrate that the Vpu helix efficiently complements 6K, whereas M2 and P7 are less efficient. Our results indicate that while SINV is primarily insensitive to the M2 ion channel inhibitor amantadine, the Vpu inhibitor 5-N, N-Hexamethylene amiloride (HMA), significantly reduces SINV release, suggesting that the ion channel activity of 6K similar to Vpu, promotes virus budding. Using live-cell imaging of SINV with a miniSOG-tagged 6K and mCherry-tagged E2, we further demonstrate that 6K and E2 colocalize with the Golgi apparatus in the secretory pathway. To contextualize the localization of 6K in the Golgi, we analyzed cells infected with SINV and SINV-ion channel chimeras using transmission electron microscopy. Our results provide evidence for the first time for the functional role of 6K in type II cytopathic vacuoles (CPV-II) formation. We demonstrate that in the absence of 6K, CPV-II, which originates from the Golgi apparatus, is not detected in infected cells, with a concomitant reduction in the glycoprotein transport to the plasma membrane. Substituting a functional ion channel, M2 or Vpu localizing to Golgi, restores CPV-II production, whereas P7, retained in the ER, is inadequate to induce CPV-II formation. Altogether our results indicate that ion channel activity of 6K is required for the formation of CPV-II from the Golgi apparatus, promoting glycoprotein spike transport to the plasma membrane and efficient virus budding.

The Japanese Encephalitis Antigenic Complex Viruses: From Structure to Immunity.

In the last three decades, several flaviviruses of concern that belong to different antigenic groups have expanded geographically. This has resulted in the presence of often more than one virus from a single antigenic group in some areas, while in Europe, Africa and Australia, additionally, multiple viruses belonging to the Japanese encephalitis (JE) serogroup co-circulate. Morphological heterogeneity of flaviviruses dictates antibody recognition and affects virus neutralization, which influences infection control. The latter is further impacted by sequential infections involving diverse flaviviruses co-circulating within a region and their cross-reactivity. The ensuing complex molecular virus-host interplay leads to either cross-protection or disease enhancement; however, the molecular determinants and mechanisms driving these outcomes are unclear. In this review, we provide an overview of the epidemiology of four JE serocomplex viruses, parameters affecting flaviviral heterogeneity and antibody recognition, host immune responses and the current knowledge of the cross-reactivity involving JE serocomplex flaviviruses that leads to differential clinical outcomes, which may inform future preventative and therapeutic interventions.

Contribution of the Golgi apparatus in morphogenesis of a virus-induced cytopathic vacuolar system.

The Golgi apparatus (GA) in mammalian cells is pericentrosomally anchored and exhibits a stacked architecture. During infections by members of the alphavirus genus, the host cell GA is thought to give rise to distinct mobile pleomorphic vacuoles known as CPV-II (cytopathic vesicle-II) via unknown morphological steps. To dissect this, we adopted a phased electron tomography approach to image multiple overlapping volumes of a cell infected with Venezuelan equine encephalitis virus (VEEV) and complemented it with localization of a peroxidase-tagged Golgi marker. Analysis of the tomograms revealed a pattern of progressive cisternal bending into double-lamellar vesicles as a central process underpinning the biogenesis and the morphological complexity of this vacuolar system. Here, we propose a model for the conversion of the GA to CPV-II that reveals a unique pathway of intracellular virus envelopment. Our results have implications for alphavirus-induced displacement of Golgi cisternae to the plasma membrane to aid viral egress operating late in the infection cycle.

Cryo-EM structures of alphavirus conformational intermediates in low pH-triggered prefusion states.

Alphaviruses can cause severe human arthritis and encephalitis. During virus infection, structural changes of viral glycoproteins in the acidified endosome trigger virus-host membrane fusion for delivery of the capsid core and RNA genome into the cytosol to initiate virus translation and replication. However, mechanisms by which E1 and E2 glycoproteins rearrange in this process remain unknown. Here, we investigate prefusion cryoelectron microscopy (cryo-EM) structures of eastern equine encephalitis virus (EEEV) under acidic conditions. With models fitted into the low-pH cryo-EM maps, we suggest that E2 dissociates from E1, accompanied by a rotation (∼60°) of the E2-B domain (E2-B) to expose E1 fusion loops. Cryo-EM reconstructions of EEEV bound to a protective antibody at acidic and neutral pH suggest that stabilization of E2-B prevents dissociation of E2 from E1. These findings reveal conformational changes of the glycoprotein spikes in the acidified host endosome. Stabilization of E2-B may provide a strategy for antiviral agent development.

Construction of Genomic and Sub-Genomic Dengue Infectious Replicons.

Dengue replicons are powerful tools used in studying virus biology as well as in high-throughput screening of drug candidates. Replicon constructs are developed as genomic (consists of all the viral protein genes) or sub-genomic (consists of only nonstructural protein genes) and are used to study various aspects of the virus life cycle such as genome replication and virus assembly. In addition, a replicon usually includes a reporter gene used in monitoring virus replication. In this chapter, we provide methods to develop both genomic and sub-genomic dengue replicons with a luciferase reporter and describe different assays to utilize these systems.

Embryonic and Neonatal Mouse Cochleae Are Susceptible to Zika Virus Infection.

Congenital Zika Syndrome (CZS) is caused by vertical transmission of Zika virus (ZIKV) to the gestating human fetus. A subset of CZS microcephalic infants present with reduced otoacoustic emissions; this test screens for hearing loss originating in the cochlea. This observation leads to the question of whether mammalian cochlear tissues are susceptible to infection by ZIKV during development. To address this question using a mouse model, the sensory cochlea was explanted at proliferative, newly post-mitotic or maturing stages. ZIKV was added for the first 24 h and organs cultured for up to 6 days to allow for cell differentiation. Results showed that ZIKV can robustly infect proliferating sensory progenitors, as well as post-mitotic hair cells and supporting cells. Virus neutralization using ZIKV-117 antibody blocked cochlear infection. AXL is a cell surface molecule known to enhance the attachment of flavivirus to host cells. While Axl mRNA is widely expressed in embryonic cochlear tissues susceptible to ZIKV infection, it is selectively downregulated in the post-mitotic sensory organ by E15.5, even though these cells remain infectible. These findings may offer insights into which target cells could potentially contribute to hearing loss resulting from fetal exposure to ZIKV in humans.

Levels of Circulating NS1 Impact West Nile Virus Spread to the Brain.

Dengue virus (DENV) and West Nile virus (WNV) are arthropod-transmitted flaviviruses that cause systemic vascular leakage and encephalitis syndromes, respectively, in humans. However, the viral factors contributing to these specific clinical disorders are not completely understood. Flavivirus nonstructural protein 1 (NS1) is required for replication, expressed on the cell surface, and secreted as a soluble glycoprotein, reaching high levels in the blood of infected individuals. Extracellular DENV NS1 and WNV NS1 interact with host proteins and cells, have immune evasion functions, and promote endothelial dysfunction in a tissue-specific manner. To characterize how differences in DENV NS1 and WNV NS1 might function in pathogenesis, we generated WNV NS1 variants with substitutions corresponding to residues found in DENV NS1. We discovered that the substitution NS1-P101K led to reduced WNV infectivity in the brain and attenuated lethality in infected mice, although the virus replicated efficiently in cell culture and peripheral organs and bound at wild-type levels to brain endothelial cells and complement components. The P101K substitution resulted in reduced NS1 antigenemia in mice, and this was associated with reduced WNV spread to the brain. Because exogenous administration of NS1 protein rescued WNV brain infectivity in mice, we conclude that circulating WNV NS1 facilitates viral dissemination into the central nervous system and impacts disease outcomes. IMPORTANCE Flavivirus NS1 serves as an essential scaffolding molecule during virus replication but also is expressed on the cell surface and is secreted as a soluble glycoprotein that circulates in the blood of infected individuals. Although extracellular forms of NS1 are implicated in immune modulation and in promoting endothelial dysfunction at blood-tissue barriers, it has been challenging to study specific effects of NS1 on pathogenesis without disrupting its key role in virus replication. Here, we assessed WNV NS1 variants that do not affect virus replication and evaluated their effects on pathogenesis in mice. Our characterization of WNV NS1-P101K suggests that the levels of NS1 in the circulation facilitate WNV dissemination to the brain and affect disease outcomes. Our findings facilitate understanding of the role of NS1 during flavivirus infection and support antiviral strategies for targeting circulating forms of NS1.