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Zika virus (ZIKV) has re-emerged in recent decades, leading to outbreaks of Zika fever in Africa, Asia, and Central and South America. Despite its drastic re-emergence and clinical impact, no vaccines or antiviral compounds are available to prevent or control ZIKV infection. This study evaluated the potential antiviral activity of quercetin hydrate against ZIKV infection and demonstrated that this substance inhibits virus particle production in A549 and Vero cells under different treatment conditions. In vitro antiviral activity was long-lasting (still observed 72 h post-infection), suggesting that quercetin hydrate affects multiple rounds of ZIKV replication. Molecular docking indicates that quercetin hydrate can efficiently interact with the specific allosteric binding site cavity of the NS2B-NS3 proteases and NS1-dimer. These results identify quercetin as a potential compound to combat ZIKV infection in vitro.

Ultraviolet (UV) light can inactivate SARS-CoV-2. However, the practicality of UV light is limited by the carcinogenic potential of mercury vapor-based UV lamps. Recent advances in the development of krypton chlorine (KrCl) excimer lamps hold promise, as these emit a shorter peak wavelength (222 nm), which is highly absorbed by the skin's stratum corneum and can filter out higher wavelengths. In this sense, UV 222 nm irradiation for the inactivation of virus particles in the air and surfaces is a potentially safer option as a germicidal technology. However, these same physical properties make it harder to reach microbes present in complex solutions, such as saliva, a critical source of SARS-CoV-2 transmission. We provide the first evaluation for using a commercial filtered KrCl excimer light source to inactivate SARS-CoV-2 in saliva spread on a surface. A conventional germicidal lamp (UV 254 nm) was also evaluated under the same condition. Using plaque-forming units (PFU) and Median Tissue Culture Infectious Dose (TCID

Made available in DSpace on 2020-12-11T03:29:36Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-11-27 Royal Society -Newton Advanced Fellowship Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Wellcome Trust L'Oreal-UNESCO-ABC Para Mulheres na Ciencia L'Oreal-UNESCO International Rising Talents Program Zika virus (ZIKV) is a mosquito-transmitted Flavivirus, originally identified in Uganda in 1947 and recently associated with a large outbreak in South America. Despite extensive efforts there are currently no approved antiviral compounds for treatment of ZIKV infection. Here we describe the antiviral activity of diarylamines derived from anthranilic acid (FAMs) against ZIKV. A synthetic FAM (E3) demonstrated anti-ZIKV potential by reducing viral replication up to 86%. We analyzed the possible mechanisms of action of FAM E3 by evaluating the intercalation of this compound into the viral dsRNA and its interaction with the RNA polymerase of bacteriophage SP6. However, FAM E3 did not act by these mechanisms. In silico results predicted that FAM E3 might bind to the ZIKV NS3 helicase suggesting that this protein could be one possible target of this compound. To test this, the thermal stability and the ATPase activity of the ZIKV NS3 helicase domain (NS3(Hel)) were investigated in vitro and we demonstrated that FAM E3 could indeed bind to and stabilize NS3(Hel). Univ Fed Uberlandia, ICBIM, Inst Biomed Sci, Lab Virol, Uberlandia, MG, Brazil Sao Paulo State Univ, IBILCE, Sao Jose Do Rio Preto, SP, Brazil Sao Paulo State Univ, IBILCE, Lab Antibiot & Chemotherapeut, Sao Jose Do Rio Preto, SP, Brazil Univ Fed Goias, Fac Farm, LabMol Lab Mol Modeling & Drug Design, BR-74605170 Goiania, Go, Brazil Univ Sao Paulo, Inst Phys Sao Carlos, Sao Carlos, SP, Brazil Rutgers State Univ, New Jersey Med Sch, Dept Pharmacol Physiol & Neurosci, Newark, NJ 07103 USA Repare Therapeut, 7210 Rue Frederick Banting,Suite 100, Montreal, PQ H4S 2A1, Canada Collaborat Pharmaceut Inc, 840 Main Campus Dr,Lab 3510, Raleigh, NC 27606 USA Univ Fed Uberlandia, Lab Nanobiotechnol, Uberlandia, MG, Brazil Univ Fed Uberlandia, Integrat Physiol & Salivary Nanobiotechnol Grp, Uberlandia, MG, Brazil Univ Tartu, Inst Technol, Nooruse 1, EE-50411 Tartu, Estonia Univ Leeds, Sch Mol & Cellular Biol, Fac Biol Sci, Leeds LS2 9JT, W Yorkshire, England Univ Leeds, Astbury Ctr Struct Mol Biol, Leeds LS2 9JT, W Yorkshire, England Sao Paulo State Univ, IBILCE, Sao Jose Do Rio Preto, SP, Brazil Sao Paulo State Univ, IBILCE, Lab Antibiot & Chemotherapeut, Sao Jose Do Rio Preto, SP, Brazil Royal Society -Newton Advanced Fellowship: NA 150195 CNPq: 445021/2014-4 CNPq: 300508/2017-4 CNPq: 201710267000063 FAPEMIG: APQ-00587-14 FAPEMIG: SICONV 793988/2013 CAPES: 001 CNPq: CNPq-465669/2014-0 CNPq: 440610/2016-8 CAPES: CNPq-465669/2014-0 CAPES: 440610/2016-8 FAPEMIG: CNPq-465669/2014-04 FAPEMIG: 40610/2016-8 Wellcome Trust: 096670

Hepatitis C virus (HCV) is responsible for more than 180 million infections worldwide, and about 80 % of infections are reported in Low and Middle-income countries (LMICs). Therapy is based on the administration of interferon (INF), ribavirin (RBV) or more recently Direct-Acting Antivirals (DAAs). However, amino acid substitutions associated with resistance (RAS) have been extensively described and can contribute to treatment failure, and diagnosis of RAS requires considerable infrastructure, not always locally available. Dried serum spots (DSS) sampling is an alternative specimen collection method, which embeds drops of serum onto filter paper to be transported by posting to a centralized laboratory. Here, we assessed feasibility of genotypic analysis of HCV from DSS in a cohort of 80 patients from São Paulo state Brazil. HCV RNA was detected on DSS specimens in 83 % of samples of HCV infected patients. HCV genotypes 1a, 1b, 2a, 2c and 3a were determined using the sequence of the palm domain of NS5B region, and RAS C316N/Y, Q309R and V321I were identified in HCV 1b samples. Concerning therapy outcome, 75 % of the patients who used INF +RBV as a previous protocol of treatment did not respond to DAAs, and 25 % were end-of-treatment responders. It suggests that therapy with INF plus RBV may contribute for non-response to a second therapeutic protocol with DAAs. One patient that presented RAS (V321I) was classified as non-responder, and combination of RAS C316N and Q309R does not necessarily imply in resistance to treatment in this cohort of patients. Data presented herein highlights the relevance of studying circulating variants for a better understanding of HCV variability and resistance to the therapy. Furthermore, the feasibility of carrying out genotyping and RAS phenotyping analysis by using DSS card for the potential of informing future treatment interventions could be relevant to overcome the limitations of processing samples in several location worldwide, especially in LMICs.

Made available in DSpace on 2021-06-25T11:03:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-12-01 Chikungunya virus (CHIKV) is the etiologic agent of Chikungunya fever, a globally spreading mosquito-borne disease. There is no approved antiviral or vaccine against CHIKV, highlighting an urgent need for novel therapies. In this context, snake venom proteins have demonstrated antiviral activity against several viruses, including arboviruses which are relevant to public health. In particular, the phospholipase A2CB (PLA2CB), a protein isolated from the venom of Crotalus durissus terrificus was previously shown to possess anti-inflammatory, antiparasitic, antibacterial and antiviral activities. In this study, we investigated the multiple effects of PLA2CB on the CHIKV replicative cycle in BHK-21 cells using CHIKV-nanoluc, a marker virus carrying nanoluciferase reporter. The results demonstrated that PLA2CB possess a strong anti-CHIKV activity with a selectivity index of 128. We identified that PLA2CB treatment protected cells against CHIKV infection, strongly impairing virus entry by reducing adsorption and post-attachment stages. Moreover, PLA2CB presented a modest yet significant activity towards post-entry stages of CHIKV replicative cycle. Molecular docking calculations indicated that PLA2CB may interact with CHIKV glycoproteins, mainly with E1 through hydrophobic interactions. In addition, infrared spectroscopy measurements indicated interactions of PLA2CB and CHIKV glycoproteins, corroborating with data from in silico analyses. Collectively, this data demonstrated the multiple antiviral effects of PLA2CB on the CHIKV replicative cycle, and suggest that PLA2CB interacts with CHIKV glycoproteins and that this interaction blocks binding of CHIKV virions to the host cells. Institute of Biomedical Science (ICBIM) Federal University of Uberlândia (UFU), Avenida Amazonas, 4C- Room 216, Umuarama Institute of Biosciences Humanities and Exact Sciences (Ibilce) São Paulo State University (Unesp), Campus São José do Rio Preto Department of Clinical Toxicological and Bromatological Analyses School of Pharmaceutical Sciences of Ribeirao Preto University of São Paulo (USP) Institute of Biotechnology Federal University of Uberlândia (UFU) Institute of Technology University of Tartu Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology University of Leeds Institute of Biosciences Humanities and Exact Sciences (Ibilce) São Paulo State University (Unesp), Campus São José do Rio Preto

Made available in DSpace on 2020-12-10T17:35:32Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-07-01 Royal Society - Newton Advanced Fellowship Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Wellcome Trust Chikungunya virus (CHIKV) is a mosquito-transmitted virus of special concern as it causes Chikungunya fever, characterized by an acute febrile illness, rash, and arthralgia that can progress to chronic and debilitating arthritic symptoms. The effects of climate change on the geographic distribution of the mosquito vector has the potential to expose more of the globe to this virus. No antiviral agents or vaccines are currently available against CHIKV infection and the development of novel therapies that may lead to a future treatment is therefore necessary. In this context, the ADP-ribose binding site of the CHIKV nsP3 macro domain has been reported as a potential target for the development of antivirals. Mutations in the ADP-ribose binding site demonstrated decreased viral replication in cell culture and reduced virulence. In this study, 48,750 small molecules were screened in silico for their ability to bind to the ADP-ribose binding site of the CHIKV nsP3 macro domain. From this in silico analysis, 12 molecules were selected for in vitro analysis using a CHIKV subgenomic replicon in Huh-7 cells. Cell viability and CHIKV replication were evaluated and molecules C5 and C13 demonstrated 53 and 66% inhibition of CHIKV replication, respectively. By using a CHIKV-Dual luciferase replicon contain two reporter genes, we also demonstrated that the treatment with either compounds are probably interfering in the early replication rather than after RNA replication has occurred. Sao Paulo State Univ, IBILCE, Sao Jose Do Rio Preto, SP, Brazil Univ Fed Uberlandia, Inst Biomed Sci, Lab Virol, ICBIM, Uberlandia, MG, Brazil Univ Leeds, Fac Engn & Phys Sci, Sch Chem, Leeds LS2 9JT, W Yorkshire, England Univ Leeds, Astbury Ctr Struct Mol Biol, Leeds LS2 9JT, W Yorkshire, England Univ Leeds, Fac Biol Sci, Sch Mol & Cellular Biol, Leeds LS2 9JT, W Yorkshire, England Univ Tartu, Inst Technol, Nooruse 1, EE-50411 Tartu, Estonia Sao Paulo State Univ, IBILCE, Sao Jose Do Rio Preto, SP, Brazil Royal Society - Newton Advanced Fellowship: NA 150195 CNPq: 445021/2014-4 FAPEMIG: APQ-00587-14 FAPEMIG: SICONV 793988/2013 CAPES: 001 Wellcome Trust: 096670

Made available in DSpace on 2020-12-10T17:38:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-08-01 Nottingham Molecular Pathology Node (United Kingdom MRC/EPSRC) National Institute for Health Research (NIHR) Nottingham Biomedical Research Centre Background: Effective drug regimens for the treatment of hepatitis B virus (HBV) infections are essential to achieve the World Health Organisation commitment to eliminate viral hepatitis by 2030. Lamivudine (3TC) is widely used in countries with high levels of chronic HBV, however resistance has been shown to occur in up to 50 % of individuals receiving continuous monotherapy for 4 years. Telbivudine (LdT) is now more commonly used in place of lamivudine but is ineffective against 3TC-resistant HBV. Genotyping and identification of resistance-associated substitutions (RAS) is not practical in many locations. Objectives: A novel assay was designed to enable HBV genotyping and characterisation of resistance mutations directly from serum samples stored on filter paper, using Sanger and MinION sequencing. Study design: The assay was applied to a cohort of 30 samples stored on filter paper for several years with HBV viral loads ranging from 8.2 x 10(8) to 635 IU/mL. A set of 6 high-titre samples were used in a proof-of-principle study using the MinION sequencer. Results: The assay allowed determination of HBV genotype and elucidation of RAS down to 600 IU/mL using a 550bp amplicon. Sequencing of a 1.2 kb amplicon using a MinION sequencer gave results consistent with Sanger sequencing and allowed the identification of minor populations of variants. Conclusions: We present two approaches for reliable HBV sequencing and RAS identification using methods suitable for resource-limited environments. This is the first demonstration of extraction-free DNA sequencing direct from DSS using MinION and these workflows are adaptable to the investigation of other DNA viruses. Univ Nottingham, Nottingham Digest Dis Ctr, Sch Med, Nottingham, England Nottingham Univ Hosp NHS Trust, NIHR Nottingham Biomed Res Ctr, Nottingham, England Univ Nottingham, Nottingham, England Univ Nottingham, MRC EPSRC Nottingham Mol Pathol Node, Nottingham, England Brazilian Minist Hlth, Inst Adolfo Lutz, Sao Paulo, SP, Brazil Univ Nottingham, Sch Life Sci, Nottingham, England Univ Fed Uberlandia, Inst Biomed Sci, Uberlandia, MG, Brazil Univ Sulaimani, Fac Sci, Biol Dept, Sulaymaniyah, Iraq Hawler Med Univ, Med Res Ctr, Erbil, Iraq Cent Publ Hlth Lab, Erbil, Iraq Sao Paulo State Univ, IBILCE, Sao Jose Do Rio Preto, SP, Brazil Sao Paulo State Univ, IBILCE, Sao Jose Do Rio Preto, SP, Brazil Nottingham Molecular Pathology Node (United Kingdom MRC/EPSRC): MR/N005953/1

In order to achieve the commitment made by the World Health Organisation to eliminate viral hepatitis by 2030, it is essential that clinicians can obtain basic sequencing data for hepatitis B virus (HBV) infected patients. While accurate diagnosis of HBV is achievable in most clinical settings, genotyping and identification of resistance-associated substitutions (RAS) present a practical challenge in regions with limited healthcare and biotechnology infrastructure. Here we outline two workflows for generating clinically relevant HBV sequence data directly from dried serum spot (DSS) cards without DNA extraction using either Sanger, or the portable MinION sequencing platforms. Data obtained from the two platforms were highly consistent and allowed determination of HBV genotype and RAS. This is the first demonstration of MinION sequencing from DSS, illustrating the broad utility of this sequencing technology. We demonstrated the clinical application of this technology using sera sampled on DSS cards obtained from both Iraq and Brazil. The sample stability provided by DSS cards, combined with the rapid PCR and sequencing protocols will enable regional/national centres to provide information relevant to patient management. By providing viable workflows for both the Sanger and MinION sequencing platforms, which vary greatly in the infrastructure and expertise required, we demonstrate that MinION sequencing is a viable method for HBV genotyping in resource-limited settings. These workflows could also be applied to sequencing of other blood borne DNA viruses and bacterial pathogens.

Made available in DSpace on 2018-12-11T17:33:26Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-07-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) Hepatitis C virus (HCV) affects about 170 million people worldwide. The current treatment has a high cost and variable response rates according to the virus genotype. Acridones, a group of compounds extracted from natural sources, showed potential antiviral actions against HCV. Thus, this study aimed to evaluate the effect of a panel of 14 synthetic acridones on the HCV life cycle. The compounds were screened using an Huh7.5 cell line stably harbouring the HCV genotype 2a subgenomic replicon SGR-Feo-JFH-1. Cells were incubated in the presence or absence of compounds for 72 h and cell viability and replication levels were assessed by MTT and luciferase assays, respectively. At a concentration of 5 μM the acridone Fac4 exhibited a >90%inhibition of HCV replication with no effect on cell viability. The effects of Fac4 on virus replication, entry and release steps were evaluated in Huh7.5 cells infected with the JFH-1 isolate of HCV (HCVcc). Fac4 inhibited JFH-1 replication to approximately 70 %, while no effect was observed on virus entry. The antiviral activity of Fac4 was also observed on viral release, with almost 80% of inhibition. No inhibitory effect was observed against genotype 3 replication. Fac4 was able to intercalate into dsRNA, however did not inhibit NS5B polymerase activity or translation driven by the HCV IRES. Although its mode of action is partly understood, Fac4 presents significant inhibition of HCV replication and can therefore be considered as a candidate for the development of a future anti-HCV treatment. Institute of Bioscience Language and Exact Science IBILCE UNESP –São Paulo State University Institute of Biomedical Science ICBIM UFU –Federal University of Uberlândia School of Molecular and Cellular Biology Faculty of Biological Sciences Astbury Centre for Structural Molecular Biology University of Leeds Institute of Chemistry São Paulo State University Institute of Bioscience Language and Exact Science IBILCE UNESP –São Paulo State University Institute of Chemistry São Paulo State University CNPq: 165802/2015-4 FAPESP: 2012/01403-9 FAPESP: 2014/22198-0 CNPq: 445021/2014-4 FAPEMIG: APQ-00587-14 CNPq: SICONV 793988/2013