Exposure to ambient airborne particulate matter (PM) has been associated with almost 9 million premature deaths per year worldwide. Ports are essential infrastructure for trade and transport, with over 80% of the world’s international trade volume travelling by sea, yet there is little understanding of the potential health effects of PM from ships. Here, we studied the elemental and toxicological characteristics of size-fractionated PM collected at different areas of a busy European mixed-use port. We found that unregulated ultrafine PM, a PM size fraction <100 nm in aerodynamic diameter, originating from a cruise ship source, was highly enriched compared to ultrafine PM from other sites in tracers of heavy fuel oil combustion (V and Ni), with Co identified as a novel tracer. In cell models of airway and alveolar epithelium, this cruise ship-associated ultrafine PM was markedly more pro-inflammatory than PM from other sites, while simultaneously reducing expression of a range of genes associated with the innate antiviral response. Investigation of the effects of the three metals found to be enriched in our cruise ship ultrafine PM showed that these effects could be reproduced by vanadium exposure, which reduced interferon signalling and increased permissiveness to viral infection, facilitating replication of the respiratory viruses rhinovirus-16 and SARS-CoV-2. Our findings suggest that, despite a series of regulations introduced over the last 20 years, cruise ship-associated emissions of PM from heavy fuel oil combustion may exert disproportionate effects on health compared to PM from other sources. In particular, the effects manifest themselves through induction of a response with characteristics of a poor viral infection prognosis. More broadly, current regulations, which neglect PM source- and composition-related variations, and ultrafine PM in general, may need strengthening to better mitigate health effects resulting from exposure to PM.

Clade Ib mpox is a newly emerged strain of the mpox virus (MPXV). The antiviral efficacy of 12 different therapeutic drugs was evaluated, in vitro using a live-virus, foci reduction assay, against MPXV clade Ib. We report that antiviral activity is retained against clade Ib with inhibitory concentrations (required to reduce the viral foci count by 50% (IC50)) of 0.025 ± 0.018 and 43.8 ± 15.2 μM for tecovirimat and cidofovir, respectively. These values are not significantly different from those observed for clade IIb, when measured in the same foci reduction assay (IC50 values of 0.010 ± 0.02 and 15.7 ± 14.3 μM for tecovirimat and cidofovir, respectively). Activity was also demonstrated for other antivirals, with the IC50 of the active metabolites of molnupiravir (EIDD-1931; 4.47 ± 1.72 μM) and remdesivir (GS-441524; 11.8 ± 6.43 μM) and with other licensed antivirals such as ribavirin (34.4 ± 10.3 μM) and baloxavir marboxil (22.6 ± 10.5 μM). In contrast, no inhibitory activity was observed with acyclovir, L-valacyclovir hydrochloride or favipiravir (IC50 >100 μM). Interestingly, the anti-parasitic drugs nitazoxanide, mefloquine hydrochloride and chloroquine diphosphate, showed inhibitory activity against the clade Ib virus, with IC50 values of 14.5 ± 3.41, 5.37 ± 1.37 and 24.7 ± 2.38 μM, respectively. This study shows that several therapeutics, including several licensed antivirals, may offer alternative treatment options for mpox clade Ib. ### Competing Interest Statement The authors have declared no competing interest.

Severe coronavirus disease 2019 (COVID-19) patients who require hospitalization are at high risk of invasive pulmonary mucormycosis. Amphotericin B (AmB), which is the first-line therapy for invasive pulmonary mucormycosis, has been shown to promote or inhibit replication of a spectrum of viruses. In this study, we first predicted that AmB and nystatin had strong interactions with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins using in silico screening, indicative of drugs with potential therapeutic activity against this virus. Subsequently, we investigated the impact of AmB, nystatin, natamycin, fluconazole, and caspofungin on SARS-CoV-2 infection and replication in vitro. Results showed that AmB and nystatin actually increased SARS-CoV-2 replication in Vero E6, Calu-3, and Huh7 cells. At optimal concentrations, AmB and nystatin increase SARS-CoV-2 replication by up to 100- and 10-fold in Vero E6 and Calu-3 cells, respectively. The other antifungals tested had no impact on SARS-CoV-2 infection in vitro. Drug kinetic studies indicate that AmB enhances SARS-CoV-2 infection by promoting viral entry into cells. Additionally, knockdown of genes encoding for interferon-induced transmembrane (IFITM) proteins 1, 2, and 3 suggests AmB enhances SARS-CoV-2 cell entry by overcoming the antiviral effect of the IFITM3 protein. This study further elucidates the role of IFITM3 in viral entry and highlights the potential dangers of treating COVID-19 patients, with invasive pulmonary mucormycosis, using AmB.IMPORTANCEAmB and nystatin are common treatments for fungal infections but were predicted to strongly interact with SARS-CoV-2 proteins, indicating their potential modulation or inhibition against the virus. However, our tests revealed that these antifungals, in fact, enhance SARS-CoV-2 infection by facilitating viral entry into cells. The magnitude of enhancement could be up to 10- or 100-fold, depending on cell lines used. These findings indicate that AmB and nystatin have the potential to enhance disease when given to patients infected with SARS-CoV-2 and therefore should not be used for treatment of fungal infections in active COVID-19 cases.

The COVID-19 pandemic saw unprecedented resources and funds driven into research for the development, and subsequent rapid distribution, of vaccines, diagnostics and directly acting antivirals (DAAs). DAAs have undeniably prevented progression and life-threatening conditions in patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, there are concerns of antimicrobial resistance (AMR), antiviral resistance specifically, for DAAs. To preserve activity of DAAs for COVID-19 therapy, as well as detect possible mutations conferring resistance, antimicrobial stewardship and surveillance were rapidly implemented in England. This paper expands on the ubiquitous ongoing public health activities carried out in England, including epidemiologic, virologic and genomic surveillance, to support the stewardship of DAAs and assess the deployment, safety, effectiveness and resistance potential of these novel and repurposed therapeutics.

The development of new therapies against SARS-CoV-2 is required to extend the toolkit of intervention strategies to combat the global pandemic. In this study, hyperimmune plasma from sheep immunised with whole spike SARS-CoV-2 recombinant protein has been used to generate candidate products. In addition to purified IgG, we have refined candidate therapies by removing non-specific IgG via affinity binding along with fragmentation to eliminate the Fc region to create F(ab′) 2 fragments. These preparations were evaluated for in vitro activity and demonstrated to be strongly neutralising against a range of SARS-CoV-2 strains, including Omicron B2.2. In addition, their protection against disease manifestations and viral loads were assessed using a hamster SARS-CoV-2 infection model. Results demonstrated protective effects of both IgG and F(ab′) 2 , with the latter requiring sequential dosing to maintain in vivo activity due to rapid clearance from the circulation.

Ebola virus infection can have dramatic effects on the human body which manifest in Ebola virus disease. The outcome of infection is either survival or death and in the former group with the potential of longer-term health consequences and persistent infection.

The 2',5'- oligoadenylate synthetase (OAS) – ribonuclease L (RNAseL) - phosphodiesterase 12 (PDE12) pathway is an essential interferon-induced effector mechanism against RNA virus infection. Inhibition of PDE12 leads to selective amplification of RNAseL activity in infected cells. We aimed to investigate PDE12 as a potential pan-RNA virus antiviral drug target and develop PDE12 inhibitors that elicit antiviral activity against a range of viruses. A library of 18 000 small molecules was screened for PDE12 inhibitor activity using a fluorescent probe specific for PDE12. The lead compounds (CO-17 or CO-63) were tested in cell-based antiviral assays using encephalomyocarditis virus (EMCV), hepatitis C virus (HCV), dengue virus (DENV), West Nile virus (WNV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in vitro. Cross reactivity of PDE12 inhibitors with other PDEs and in vivo toxicity were measured. In EMCV assays, CO-17 potentiated the effect of IFNα by 3 log10. The compounds were selective for PDE12 when tested against a panel of other PDEs and non-toxic at up to 42 mg kg−1 in rats in vivo. Thus, we have identified PDE12 inhibitors (CO-17 and CO-63), and established the principle that inhibitors of PDE12 have antiviral properties. Early studies suggest these PDE12 inhibitors are well tolerated at the therapeutic range, and reduce viral load in studies of DENV, HCV, WNV and SARS-CoV-2 in human cells and WNV in a mouse model.

Background The mutational landscape of SARS-CoV-2 varies at the dominant viral genome sequence and minor genomic variant population. During the COVID-19 pandemic, an early substitution in the genome was the D614G change in the spike protein, associated with an increase in transmissibility. Genomes with D614G are accompanied by a P323L substitution in the viral polymerase (NSP12). However, P323L is not thought to be under strong selective pressure. Results Investigation of P323L/D614G substitutions in the population shows rapid emergence during the containment phase and early surge phase during the first wave. These substitutions emerge from minor genomic variants which become dominant viral genome sequence. This is investigated in vivo and in vitro using SARS-CoV-2 with P323 and D614 in the dominant genome sequence and L323 and G614 in the minor variant population. During infection, there is rapid selection of L323 into the dominant viral genome sequence but not G614. Reverse genetics is used to create two viruses (either P323 or L323) with the same genetic background. L323 shows greater abundance of viral RNA and proteins and a smaller plaque morphology than P323. Conclusions These data suggest that P323L is an important contribution in the emergence of variants with transmission advantages. Sequence analysis of viral populations suggests it may be possible to predict the emergence of a new variant based on tracking the frequency of minor variant genomes. The ability to predict an emerging variant of SARS-CoV-2 in the global landscape may aid in the evaluation of medical countermeasures and non-pharmaceutical interventions.

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.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a complex strategy for the transcription of viral subgenomic mRNAs (sgmRNAs), which are targets for nucleic acid diagnostics. Each of these sgmRNAs has a unique 5' sequence, the leader-transcriptional regulatory sequence gene junction (leader-TRS junction), that can be identified using sequencing. High-resolution sequencing has been used to investigate the biology of SARS-CoV-2 and the host response in cell culture and animal models and from clinical samples. LeTRS, a bioinformatics tool, was developed to identify leader-TRS junctions and can be used as a proxy to quantify sgmRNAs for understanding virus biology. LeTRS is readily adaptable for other coronaviruses such as Middle East respiratory syndrome coronavirus or a future newly discovered coronavirus. LeTRS was tested on published data sets and novel clinical samples from patients and longitudinal samples from animal models with coronavirus disease 2019. LeTRS identified known leader-TRS junctions and identified putative novel sgmRNAs that were common across different mammalian species. This may be indicative of an evolutionary mechanism where plasticity in transcription generates novel open reading frames, which can then subject to selection pressure. The data indicated multiphasic abundance of sgmRNAs in two different animal models. This recapitulates the relative sgmRNA abundance observed in cells at early points in infection but not at late points. This pattern is reflected in some human nasopharyngeal samples and therefore has implications for transmission models and nucleic acid-based diagnostics. LeTRS provides a quantitative measure of sgmRNA abundance from sequencing data. This can be used to assess the biology of SARS-CoV-2 (or other coronaviruses) in clinical and nonclinical samples, especially to evaluate different variants and medical countermeasures that may influence viral RNA synthesis.

Background: Administration of potent anti-receptor-binding domain (RBD) monoclonal antibodies has been shown to curtail viral shedding and reduce hospitalization in patients with SARS-CoV-2 infection. However, the structure-function analysis of potent human anti-RBD monoclonal antibodies and its links to the formulation of antibody cocktails remains largely elusive. Methods: Previously, we isolated a panel of neutralizing anti-RBD monoclonal antibodies from convalescent patients and showed their neutralization efficacy in vitro. Here, we elucidate the mechanism of action of antibodies and dissect antibodies at the epitope level, which leads to a formation of a potent antibody cocktail. Results: We found that representative antibodies which target non-overlapping epitopes are effective against wild type virus and recently emerging variants of concern, whilst being encoded by antibody genes with few somatic mutations. Neutralization is associated with the inhibition of binding of viral RBD to ACE2 and possibly of the subsequent fusion process. Structural analysis of representative antibodies, by cryo-electron microscopy and crystallography, reveals that they have some unique aspects that are of potential value while sharing some features in common with previously reported neutralizing monoclonal antibodies. For instance, one has a common VH 3-53 public variable region yet is unusually resilient to mutation at residue 501 of the RBD. We evaluate the in vivo efficacy of an antibody cocktail consisting of two potent non-competing anti-RBD antibodies in a Syrian hamster model. We demonstrate that the cocktail prevents weight loss, reduces lung viral load and attenuates pulmonary inflammation in hamsters in both prophylactic and therapeutic settings. Although neutralization of one of these antibodies is abrogated by the mutations of variant B.1.351, it is also possible to produce a bi-valent cocktail of antibodies both of which are resilient to variants B.1.1.7, B.1.351 and B.1.617.2. Conclusions: These findings support the up-to-date and rational design of an anti-RBD antibody cocktail as a therapeutic candidate against COVID-19.

There is need for effective and affordable vaccines against SARS-CoV-2 to tackle the ongoing pandemic. In this study, we describe a protein nanoparticle vaccine against SARS-CoV-2. The vaccine is based on the display of coronavirus spike glycoprotein receptor-binding domain (RBD) on a synthetic virus-like particle (VLP) platform, SpyCatcher003-mi3, using SpyTag/SpyCatcher technology. Low doses of RBD-SpyVLP in a prime-boost regimen induce a strong neutralising antibody response in mice and pigs that is superior to convalescent human sera. We evaluate antibody quality using ACE2 blocking and neutralisation of cell infection by pseudovirus or wild-type SARS-CoV-2. Using competition assays with a monoclonal antibody panel, we show that RBD-SpyVLP induces a polyclonal antibody response that recognises key epitopes on the RBD, reducing the likelihood of selecting neutralisation-escape mutants. Moreover, RBD-SpyVLP is thermostable and can be lyophilised without losing immunogenicity, to facilitate global distribution and reduce cold-chain dependence. The data suggests that RBD-SpyVLP provides strong potential to address clinical and logistic challenges of the COVID-19 pandemic.

Background and PurposeCurrently, there are no licensed vaccines and limited antivirals for the treatment of COVID‐19. Heparin (delivered systemically) is currently used to treat anticoagulant anomalies in COVID‐19 patients. Additionally, in the United Kingdom, Brazil and Australia, nebulised unfractionated heparin (UFH) is being trialled in COVID‐19 patients as a potential treatment. A systematic comparison of the potential antiviral effect of various heparin preparations on live wild type SARS‐CoV‐2, in vitro, is needed.Experimental ApproachSeven different heparin preparations including UFH and low MW heparins (LMWH) of porcine or bovine origin were screened for antiviral activity against live SARS‐CoV‐2 (Australia/VIC01/2020) using a plaque inhibition assay with Vero E6 cells. Interaction of heparin with spike protein RBD was studied using differential scanning fluorimetry and the inhibition of RBD binding to human ACE2 protein using elisa assays was examined.Key ResultsAll the UFH preparations had potent antiviral effects, with IC50 values ranging between 25 and 41 μg·ml−1, whereas LMWHs were less inhibitory by ~150‐fold (IC50 range 3.4–7.8 mg·ml−1). Mechanistically, we observed that heparin binds and destabilizes the RBD protein and furthermore, we show heparin directly inhibits the binding of RBD to the human ACE2 protein receptor.Conclusion and ImplicationsThis comparison of clinically relevant heparins shows that UFH has significantly stronger SARS‐CoV‐2 antiviral activity compared to LMWHs. UFH acts to directly inhibit binding of spike protein to the human ACE2 protein receptor. Overall, the data strongly support further clinical investigation of UFH as a potential treatment for patients with COVID‐19.

A novel coronavirus, SARS-CoV-2, has been identified as the causative agent of the current COVID-19 pandemic. Animal models, and in particular non-human primates, are essential to understand the pathogenesis of emerging diseases and to assess the safety and efficacy of novel vaccines and therapeutics. Here, we show that SARS-CoV-2 replicates in the upper and lower respiratory tract and causes pulmonary lesions in both rhesus and cynomolgus macaques. Immune responses against SARS-CoV-2 are also similar in both species and equivalent to those reported in milder infections and convalescent human patients. This finding is reiterated by our transcriptional analysis of respiratory samples revealing the global response to infection. We describe a new method for lung histopathology scoring that will provide a metric to enable clearer decision making for this key endpoint. In contrast to prior publications, in which rhesus are accepted to be the preferred study species, we provide convincing evidence that both macaque species authentically represent mild to moderate forms of COVID-19 observed in the majority of the human population and both species should be used to evaluate the safety and efficacy of interventions against SARS-CoV-2. Importantly, accessing cynomolgus macaques will greatly alleviate the pressures on current rhesus stocks.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Recent reemergence of Ebola in Guinea that caused over 28,000 cases between 2013 and 2016 has been linked to the original virus from that region. It appears the virus has remained in the region for at least 5 years and is likely to have been maintained in humans.

There is a vital need for authentic COVID-19 animal models to enable the pre-clinical evaluation of candidate vaccines and therapeutics. Here we report a dose titration study of SARS-CoV-2 in the ferret model. After a high (5 × 10 6 pfu) and medium (5 × 10 4 pfu) dose of virus is delivered, intranasally, viral RNA shedding in the upper respiratory tract (URT) is observed in 6/6 animals, however, only 1/6 ferrets show similar signs after low dose (5 × 10 2 pfu) challenge. Following sequential culls pathological signs of mild multifocal bronchopneumonia in approximately 5–15% of the lung is seen on day 3, in high and medium dosed groups. Ferrets re-challenged, after virus shedding ceased, are fully protected from acute lung pathology. The endpoints of URT viral RNA replication & distinct lung pathology are observed most consistently in the high dose group. This ferret model of SARS-CoV-2 infection presents a mild clinical disease.

Introduction SARS-CoV-2 has a complex strategy for the transcription of viral subgenomic mRNAs (sgmRNAs), which are targets for nucleic acid diagnostics. Each of these sgRNAs has a unique 5’ sequence, the leader-transcriptional regulatory sequence gene junction (leader-TRS-junction), that can be identified using sequencing. Results High resolution sequencing has been used to investigate the biology of SARS-CoV-2 and the host response in cell culture models and from clinical samples. LeTRS, a bioinformatics tool, was developed to identify leader-TRS-junctions and be used as a proxy to quantify sgmRNAs for understanding virus biology. This was tested on published datasets and clinical samples from patients and longitudinal samples from animal models with COVID-19. Discussion LeTRS identified known leader-TRS-junctions and identified novel species that were common across different species. The data indicated multi-phasic abundance of sgmRNAs in two different animal models, with spikes in sgmRNA abundance reflected in human samples, and therefore has implications for transmission models and nucleic acid-based diagnostics.