Although enveloped viruses canonically mediate particle entry through virus-cell fusion, certain viruses can spread by cell-cell fusion, brought about by receptor engagement and triggering of ...membrane-bound, viral-encoded fusion proteins on the surface of cells. The formation of pathogenic syncytia or multinucleated cells is seen
, but their contribution to viral pathogenesis is poorly understood. For the negative-strand paramyxoviruses respiratory syncytial virus (RSV) and Nipah virus (NiV), cell-cell spread is highly efficient because their oligomeric fusion protein complexes are active at neutral pH. The recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has also been reported to induce syncytia formation in infected cells, with the spike protein initiating cell-cell fusion. Whilst it is well established that fusion protein-specific antibodies can block particle attachment and/or entry into the cell (canonical virus neutralization), their capacity to inhibit cell-cell fusion and the consequences of this neutralization for the control of infection are not well characterized, in part because of the lack of specific tools to assay and quantify this activity. Using an adapted bimolecular fluorescence complementation assay, based on a split GFP
luciferase reporter, we have established a micro-fusion inhibition test (mFIT) that allows the identification and quantification of these neutralizing antibodies. This assay has been optimized for high-throughput use and its applicability has been demonstrated by screening monoclonal antibody (mAb)-mediated inhibition of RSV and NiV fusion and, separately, the development of fusion-inhibitory antibodies following NiV vaccine immunization in pigs. In light of the recent emergence of coronavirus disease 2019 (COVID-19), a similar assay was developed for SARS-CoV-2 and used to screen mAbs and convalescent patient plasma for fusion-inhibitory antibodies. Using mFITs to assess antibody responses following natural infection or vaccination is favourable, as this assay can be performed entirely at low biocontainment, without the need for live virus. In addition, the repertoire of antibodies that inhibit cell-cell fusion may be different to those that inhibit particle entry, shedding light on the mechanisms underpinning antibody-mediated neutralization of viral spread.
The emergence of SARS-CoV-2 variants alters the efficacy of existing immunity towards the viral spike protein, whether acquired from infection or vaccination. Mutations that impact N-glycosylation of ...spike may be particularly important in influencing antigenicity, but their consequences are difficult to predict. Here, we compare the glycosylation profiles and antigenicity of recombinant viral spike of ancestral Wu-1 and the Gamma strain, which has two additional N-glycosylation sites due to amino acid substitutions in the N-terminal domain (NTD). We found that a mutation at residue 20 from threonine to asparagine within the NTD caused the loss of NTD-specific antibody COVA2-17 binding. Glycan site-occupancy analyses revealed that the mutation resulted in N-glycosylation switching to the new sequon at N20 from the native N17 site. Site-specific glycosylation profiles demonstrated distinct glycoform differences between Wu-1, Gamma, and selected NTD variant spike proteins, but these did not affect antibody binding. Finally, we evaluated the specificity of spike proteins against convalescent COVID-19 sera and found reduced cross-reactivity against some mutants, but not Gamma spike compared to Wuhan spike. Our results illustrate the impact of viral divergence on spike glycosylation and SARS-CoV-2 antibody binding profiles.
The COVID-19 pandemic has revealed gaps in our understanding of safe, effective and efficient means of disinfecting high use public spaces. Whilst this creates an opportunity for development and ...application of innovative approaches such as unmanned aerial vehicle (UAV) based disinfection, unregulated outdoor disinfection using chlorine has led to environmental and public health risks. This study has quantified the efficiency, safety and efficacy of UAV-based spraying of aqueous ozone. Optimised UAV flight characteristics of 4.7 km/h at 1.7 m elevation spraying 2.4 L/min were able to provide >97% and >92% coverage of a 1 m and 2 m wide swath respectively. During spraying operations using 1 mg/L aqueous ozone, atmospheric concentrations of ozone remained within background levels (<0.04 ppm). Highly efficient inactivation of two different isolates of SARS-CoV-2 virus was achieved at aqueous ozone concentrations of 0.75 mg/L after an incubation period of only 5 min, with 0.375 mg/L achieving 82–91.5% inactivation in this time. Exposure of diamondback moth larvae and parasitic wasps to 1 mg/L aqueous ozone did not significantly affect their survivorship. These results indicate for the first time that aqueous ozone may provide the required balance between human and environmental safety and viral inactivation efficacy for targeted application in high risk outdoor settings.
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•Aqueous ozone achieves highly efficient inactivation of SARS-CoV-2.•Spraying aqueous ozone from unmanned aerial vehicles (UAV) has potential for broad scale disinfection.•UAV-based spraying can achieve >97% coverage with optimised flight characteristics.•Aqueous ozone represents lower environmental and human health risks than other disinfectants.
The ongoing SARS-CoV-2 pandemic continues to pose an enormous health challenge globally. The ongoing emergence of variants of concern has resulted in decreased vaccine efficacy necessitating booster ...immunizations. This was particularly highlighted by the recent emergence of the Omicron variant, which contains over 30 mutations in the spike protein and quickly became the dominant viral strain in global circulation. We previously demonstrated that delivery of a SARS-CoV-2 subunit vaccine via a high-density microarray patch (HD-MAP) induced potent immunity resulting in robust protection from SARS-CoV-2 challenge in mice. Here we show that serum from HD-MAP immunized animals maintained potent neutralisation against all variants tested, including Delta and Omicron. These findings highlight the advantages of HD-MAP vaccine delivery in inducing high levels of neutralising antibodies and demonstrates its potential at providing protection from emerging viral variants.
Stachyonic acid A, arising from the first in‐depth phytochemical investigation of the herb Basilicum polystachyon, was found to display potent inhibitory activity against dengue virus, with limited ...cytotoxicity. Andrographolide, a known dengue virus inhibitor and closely related labdane‐type diterpene, is structurally more complex but displayed poor antiviral activity in the PRNT assay, and increased cytotoxicity in comparison. Furthermore, a Diels–Alder reaction with PTAD identified the active pharmacophore of stachyonic acid to be the conjugated diene.
Stachyonic acid A, isolated from the herb Basilicum polystachyon, was found to display potent inhibitory activity against dengue virus, and out‐perform the structurally more complex, albeit related labdane, andrographolide. In addition, the active pharmacophore was identified as the conjugated diene opening up a new avenue for anti‐DENV discovery.
Background
Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is unlikely to be a major transfusion‐transmitted pathogen; however, convalescent plasma is a treatment option used in some ...regions. The risk of transfusion‐transmitted infections can be minimized by implementing Pathogen Inactivation (PI), such as THERAFLEX MB‐plasma and THERAFLEX UV‐Platelets systems. Here we examined the capability of these PI systems to inactivate SARS‐CoV‐2.
Study Design and Methods
SARS‐CoV‐2 spiked plasma units were treated using the THERAFLEX MB‐Plasma system in the presence of methylene blue (~0.8 μmol/L; visible light doses: 20, 40, 60, and 120 standard J/cm2). SARS‐CoV‐2 spiked platelet concentrates (PCs) were treated using the THERAFLEX UV‐platelets system (UVC doses: 0.05, 0.10, 0.15, and 0.20 standard J/cm2). Samples were taken prior to the first and after each illumination dose, and viral infectivity was assessed using an immunoplaque assay.
Results
Treatment of spiked plasma with the THERAFLEX MB‐Plasma system resulted in an average ≥5.03 log10 reduction in SARS‐CoV‐2 infectivity at one third (40 J/cm2) of the standard visible light dose. For the platelet concentrates (PCs), treatment with the THERAFLEX UV‐Platelets system resulted in an average ≥5.18 log10 reduction in SARS‐CoV‐2 infectivity at the standard UVC dose (0.2 J/cm2).
Conclusions
SARS‐CoV‐2 infectivity was reduced in plasma and platelets following treatment with the THERAFLEX MB‐Plasma and THERAFLEX UV‐Platelets systems, to the limit of detection, respectively. These PI technologies could therefore be an effective option to reduce the risk of transfusion‐transmitted emerging pathogens.
Pirfenidone, an oral anti‐fibrotic and anti‐inflammatory medication used for the treatment of idiopathic pulmonary fibrosis (IPF), has been proposed to treat post‐COVID pneumonia pulmonary fibrosis ...(PF). However, pirfenidone displays side effects which include hepatotoxicity and anorexia. Cubane and cyclooctatetraene (COT) derivatives of pirfenidone were prepared as bioisostere/biomotif replacements of the phenyl ring to explore potential changes in activity. The key intermediate, aminocubane, enabled the synthesis of both the cubane and cyclooctatetraene (COT) derived pirfenidone analogues in 6 steps. The COT derivative was observed to display similar activity and cytotoxicity with pirfenidone in an anti‐virus and ACE2 inhibition assay, although limited effect was observed from pirfenidone and the analogues described herein.
Cubane and cyclooctatetraene (COT) derived pirfenidone analogues were synthesized from aminocubane. Cubane derived pirfenidone illustrated relatively high cytotoxicity while the COT analogue was observed to behave similarly to pirfenidone.
Heparan sulfate (HS) is a cell surface polysaccharide recently identified as a coreceptor with the ACE2 protein for the S1 spike protein on SARS-CoV-2 virus, providing a tractable new therapeutic ...target. Clinically used heparins demonstrate an inhibitory activity but have an anticoagulant activity and are supply-limited, necessitating alternative solutions. Here, we show that synthetic HS mimetic pixatimod (PG545), a cancer drug candidate, binds and destabilizes the SARS-CoV-2 spike protein receptor binding domain and directly inhibits its binding to ACE2, consistent with molecular modeling identification of multiple molecular contacts and overlapping pixatimod and ACE2 binding sites. Assays with multiple clinical isolates of SARS-CoV-2 virus show that pixatimod potently inhibits the infection of monkey Vero E6 cells and physiologically relevant human bronchial epithelial cells at safe therapeutic concentrations. Pixatimod also retained broad potency against variants of concern (VOC) including B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta), and B.1.1.529 (Omicron). Furthermore, in a K18-hACE2 mouse model, pixatimod significantly reduced SARS-CoV-2 viral titers in the upper respiratory tract and virus-induced weight loss. This demonstration of potent anti-SARS-CoV-2 activity tolerant to emerging mutations establishes proof-of-concept for targeting the HS–Spike protein–ACE2 axis with synthetic HS mimetics and provides a strong rationale for clinical investigation of pixatimod as a potential multimodal therapeutic for COVID-19.
Mosquito-borne viruses are a major worldwide health problem associated with high morbidity and mortality rates and significant impacts on national healthcare budgets. The development of antiviral ...drugs for both the treatment and prophylaxis of these diseases is thus of considerable importance. To address the need for therapeutics with antiviral activity, a library of heparan sulfate mimetic polymers was screened against dengue virus (DENV), Yellow fever virus (YFV), Zika virus (ZIKV), and Ross River virus (RRV). The polymers were prepared by RAFT polymerization of various acidic monomers with a target MW of 20 kDa (average M n ∼ 27 kDa by GPC). Among the polymers, poly(SS), a homopolymer of sodium styrenesulfonate, was identified as a broad spectrum antiviral with activity against all the tested viruses and particularly potent inhibition of YFV (IC50 = 310 pM). Our results further uncovered that poly(SS) exhibited a robust inhibition of ZIKV infection in both mosquito and human cell lines, which points out the potential functions of poly(SS) in preventing mosquito-borne viruses associated diseases by blocking viral transmission in their mosquito vectors and mitigating viral infection in patients.
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