Antiviral agents : discovery to resistance

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Bright and early : inhibiting human cytomegalovirus by targeting major immediate-early gene expression or protein function

Funding: UK Medical Research Council, grant number MR/P022146/1 (M.M.N.).The human cytomegalovirus (HCMV), one of eight human herpesviruses, establishes lifelong latent infections in most people worldwide. Primary or reactivated HCMV infections cause severe disease in immunosuppressed patients and congenital defects in children. There is no vaccine for HCMV, and the currently approved antivirals come with major limitations. Most approved HCMV antivirals target late molecular processes in the viral replication cycle including DNA replication and packaging. “Bright and early” events in HCMV infection have not been exploited for systemic prevention or treatment of disease. Initiation of HCMV replication depends on transcription from the viral major immediate-early (IE) gene. Alternative transcripts produced from this gene give rise to the IE1 and IE2 families of viral proteins, which localize to the host cell nucleus.The IE1 and IE2 proteins are believed to control all subsequent early and late events in HCMV replication, including reactivation from latency, in part by antagonizing intrinsic and innate immune responses. Here we provide an update on the regulation of major IE gene expression and the functions of IE1 and IE2 proteins. We will relate this insight to experimental approaches that target IE gene expression or protein function via molecular gene silencing and editing or small chemical inhibitors.Publisher PDFPeer reviewe

Identification of novel inhibitors of the type I interferon induction pathway using cell-based high-throughput screening

Production of type I interferon (IFN) is an essential component of the innate immune response against invading pathogens. However, its production must be tightly regulated to avoid harmful effects. Compounds that modulate the IFN response are potentially valuable for a variety of applications due to IFNs beneficial and detrimental roles. We developed and executed a cell-based high-throughput screen (HTS) targeting components that participate in and/or regulate the IRF3 and NF-κB branches of the IFN-induction pathway. The assay detects activation of the IFN-induction pathway via an eGFP reporter gene under the control of the IFN-β promoter and was optimized, miniaturized and demonstrated suitable for HTS as robust Z’ factor scores of >0.6 were consistently achieved. A diversity screening set of 15,667 small molecules was assayed and two novel hit compounds validated that specifically inhibit the IFN- induction pathway. We demonstrate that one of these compounds acts at, or upstream of IRF3 phosphorylation. A second cell-based assay to detect activation of the IFN- signaling (Jak-Stat) pathway via an eGFP reporter gene under the control of an ISRE containing MxA promoter also performed well (robust Z’ factor = >0.7), and may therefore be similarly used to identify small molecules that modulate the IFN-signaling pathway.Publisher PDFPeer reviewe

Structure-activity relationships of the Human Immunodeficiency Virus type 1 maturation inhibitor PF-46396

This work was funded by the University of St Andrews and Society for Applied Microbiology New Lecturer Research Grant awarded to CSA, Wellcome Trust grant (093228) awarded to TKS.HIV-1 maturation inhibitors are a novel class of antiretroviral compounds, which consist of two structurally distinct chemical classes; betulinic acid derivatives and the pyridone-based compound PF-46396. It is currently believed that both classes act by a similar mode of action to generate aberrant non-infectious particles via inhibition of CA-SP1 cleavage during Gag proteolytic processing. In this study we utilized a series of novel analogues, with decreasing similarity to PF-46396, to determine the chemical groups within PF-46396 that contribute to antiviral activity, Gag binding and the relationship between these essential properties. A spectrum of antiviral activity (active, intermediate, inactive) was observed across the analogue series with respect to CA-SP1 cleavage and HIV-1 (NL4-3) replication kinetics in Jurkat T cells. We demonstrate that selected inactive analogues are incorporated into WT immature particles and that one inactive analogue is capable of interfering with PF-46396 inhibition of CA-SP1 cleavage. Mutations that confer PF-46396 resistance can impose a defective phenotype on HIV-1 that can be rescued in a compound-dependent manner. Some inactive analogues retained the capacity to rescue PF-46396-dependent mutants (SP1-A3V, SP1-A3T, CA-P157S), implying that they can also interact with mutant Gag. The structure-activity relationships observed in this study demonstrate that (i) the tert-butyl group is essential for antiviral activity, but not an absolute requirement for Gag binding, (ii) the trifluromethyl group is optimal but not essential for antiviral activity and (iii) the 2-aminoindan group is important for antiviral activity and Gag binding but not essential as its replacement is tolerated.Publisher PDFPeer reviewe

Use of the internet for sexual health among sexually experienced persons aged 16 to 44 years: evidence from a nationally representative survey of the British population

BACKGROUND: Those who go online regarding their sexual health are potential users of new Internet-based sexual health interventions. Understanding the size and characteristics of this population is important in informing intervention design and delivery. OBJECTIVE: We aimed to estimate the prevalence in Britain of recent use of the Internet for key sexual health reasons (for chlamydia testing, human immunodeficiency virus [HIV] testing, sexually transmitted infection [STI] treatment, condoms/contraceptives, and help/advice with one's sex life) and to identify associated sociodemographic and behavioral factors. METHODS: Complex survey analysis of data from 8926 sexually experienced persons aged 16-44 years in a 2010-2012 probability survey of Britain's resident population. Prevalence of recent (past year) use of Internet sources for key sexual health reasons was estimated. Factors associated with use of information/support websites were identified using logistic regression to calculate age-adjusted odds ratios (AORs). RESULTS: Recent Internet use for chlamydia/HIV testing or STI treatment (combined) was very low (men: 0.31%; women: 0.16%), whereas 2.35% of men and 0.51% of women reported obtaining condoms/contraceptives online. Additionally, 4.49% of men and 4.57% of women reported recent use of information/support websites for advice/help with their sex lives. Prevalence declined with age (men 16-24 years: 7.7%; 35-44 years: 1.84%, P<.001; women 16-24 years: 7.8%; 35-44 years: 1.84%, P<.001). Use of information/support websites was strongly associated with men's higher socioeconomic status (managerial/professional vs semiroutine/routine: AOR 1.93, 95% CI 1.27-2.93, P<.001). Despite no overall association with area-level deprivation, those in densely populated urban areas were more likely to report use of information/support websites than those living in rural areas (men: AOR 3.38, 95% CI 1.68-6.77, P<.001; women: AOR 2.51, 95% CI 1.34-4.70, P<.001). No statistically significant association was observed with number of sex partners reported after age adjustment, but use was more common among men reporting same-sex partners (last 5 years: AOR 2.44, 95% CI 1.27-4.70), women reporting sex with multiple partners without condoms (last year: AOR 1.90, 95% CI 1.11-3.26), and, among both sexes, reporting seeking sex online (last year, men: AOR 1.80, 95% CI 1.16-2.79; women: AOR 3.00, 95% CI 1.76-5.13). No association was observed with reporting STI diagnosis/es (last 5 years) or (after age adjustment) recent use of any STI service or non-Internet sexual health seeking. CONCLUSIONS: A minority in Britain used the Internet for the sexual health reasons examined. Use of information/support websites was reported by those at greater STI risk, including younger people, indicating that demand for online STI services, and Internet-based sexual health interventions in general, may increase over time in this and subsequent cohorts. However, the impact on health inequalities needs addressing during design and evaluation of online sexual health interventions so that they maximize public health benefit

Antiviral drug discovery : preparing for the next pandemic

Acknowledgements The authors also gratefully acknowledge financial support from the South African Medical Research Council (MRC) with funds received from the South African National Department of Health and the UK Government's Newton Fund (R. A. D., RL. M. G., K. C.), the UK Engineering and Physical Sciences Research Council EQATA (R. J. M. G.), the UK Global Challenge Research Fund (R. J. M. G., R. A. D.), the University of Cape Town (K. C.) and the South African Research Chairs Initiative of the Department of Science and Innovation, administered through the South African National Research Foundation (NRF) to K. C. (UID: 64767) and R. A. D. (UID: 87583). C. S. A. acknowledges financial support for SARS-CoV-2/Covid-19 research from UKMRC (CVG-1725-2020) and UKRI-DHSC (MR/Vo28464/1). The authors acknowledge Bronwyn Tweedie of the Rhodes University Print Services Unit who provided the graphics for Fig. 1 and thank Gordon Cragg for his insightful comments and encouragement during the preparation of this manuscript.Peer reviewedPublisher PD

Far-UVC (222 nm) efficiently inactivated an airborne pathogen in a room-sized chamber

Funding: We acknowledge the financial assistance of the United Kingdom’s Department for Health and Social Care (2020/092).Many infectious diseases, including COVID-19, are transmitted by airborne pathogens. There is a need for effective environmental control measures which, ideally, are not reliant on human behaviour. One potential solution is Krypton Chloride (KrCl) excimer lamps (often referred to as Far-UVC), which can efficiently inactivate pathogens, such as coronaviruses and influenza, in air. Research demonstrates that when KrCl lamps are filtered to remove longer-wavelength ultraviolet emissions they do not induce acute reactions in the skin or eyes, nor delayed effects such as skin cancer. While there is laboratory evidence for Far-UVC efficacy, there is limited evidence in full-sized rooms. For the first time, we show that Far-UVC deployed in a room-sized chamber effectively inactivates aerosolised Staphylococcus aureus. At a room ventilation rate of 3 air-changes-per-hour (ACH), with 5 filtered-sources the steady-state pathogen load was reduced by 98.4% providing an additional 184 equivalent air changes (eACH). This reduction was achieved using Far-UVC irradiances consistent with current American Conference of Governmental Industrial Hygienists threshold limit values for skin for a continuous 8-h exposure. Our data indicate that Far-UVC is likely to be more effective against common airborne viruses, including SARS-CoV-2, than bacteria and should thus be an effective and “hands-off” technology to reduce airborne disease transmission. The findings provide room-scale data to support the design and development of effective Far-UVC systems.Publisher PDFPeer reviewe

The role of ion dissolution in metal and metal oxide surface inactivation of SARS CoV-2

Funding: This work was funded by UKRI-NIHR (MRC MR/V028464/1) COVID-19 Rapid Response Initiative.Anti-viral surface coatings are under development to prevent viral fomite transmission from high-traffic touch surfaces in public spaces. Copper’s anti-viral properties have been widely documented, but the anti-viral mechanism of copper surfaces is not fully understood. We screened a series of metal and metal oxide surfaces for anti-viral activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease (COVID-19). Copper and copper oxide surfaces exhibited superior anti-SARS-CoV-2 activity; however, the level of anti-viral activity was dependent on the composition of the carrier solution used to deliver virus inoculum. We demonstrate that copper ions released into solution from test surfaces can mediate virus inactivation, indicating a copper ion dissolution-dependent anti-viral mechanism. The level of anti-viral activity is, however, not dependent on the amount of copper ions released into solution per se. Instead, our findings suggest that degree of virus inactivation is dependent on copper ion complexation with other biomolecules (e.g., proteins/metabolites) in the virus carrier solution that compete with viral components. Although using tissue culture-derived virus inoculum is experimentally convenient to evaluate the anti-viral activity of copper-derived test surfaces, we propose that the high organic content of tissue culture medium reduces the availability of “uncomplexed” copper ions to interact with the virus, negatively affecting virus inactivation and hence surface anti-viral performance. We propose that laboratory anti-viral surface testing should include virus delivered in a physiologically relevant carrier solution (saliva or nasal secretions when testing respiratory viruses) to accurately predict real-life surface anti-viral performance when deployed in public spaces.PostprintPeer reviewe

Polymorphisms in Gag spacer peptide 1 confer varying levels of resistance to the HIV- 1maturation inhibitor bevirimat

Background: The maturation inhibitor bevirimat (BVM) potently inhibits human immunodeficiency virus type 1 (HIV-1) replication by blocking capsid-spacer peptide 1 (CA-SP1) cleavage. Recent clinical trials demonstrated that a significant proportion of HIV-1-infected patients do not respond to BVM. A patient’s failure to respond correlated with baseline polymorphisms at SP1 residues 6-8. Results: In this study, we demonstrate that varying levels of BVM resistance are associated with point mutations at these residues. BVM susceptibility was maintained by SP1-Q6A, -Q6H and -T8A mutations. However, an SP1-V7A mutation conferred high-level BVM resistance and SP1-V7M and T8Δ mutations conferred intermediate levels of BVM resistance. Conclusions: Future exploitation of the CA-SP1 cleavage site as an antiretroviral drug target will need to overcome the baseline variability in the SP1 region of Gag.Publisher PDFPeer reviewe