Identification of Novel Non-Nucleoside Inhibitors of Zika Virus NS5 Protein Targeting MTase Activity.

Zika virus (ZIKV) is a positive-sense single-stranded virus member of the Flaviviridae family. Among other arboviruses, ZIKV can cause neurological disorders such as Guillain Barré syndrome, and it can have congenital neurological manifestations and affect fertility. ZIKV nonstructural protein 5 (NS5) is essential for viral replication and limiting host immune detection. Herein, we performed virtual screening to identify novel small-molecule inhibitors of the ZIKV NS5 methyltransferase (MTase) domain. Compounds were tested against the MTases of both ZIKV and DENV, demonstrating good inhibitory activities against ZIKV MTase. Extensive molecular dynamic studies conducted on the series led us to identify other derivatives with improved activity against the MTase and limiting ZIKV infection with an increased selectivity index. Preliminary pharmacokinetic parameters have been determined, revealing excellent stability over time. Preliminary in vivo toxicity studies demonstrated that the hit compound 17 is well tolerated after acute administration. Our results provide the basis for further optimization studies on novel non-nucleoside MTase inhibitors.

SARS-CoV-2 inhibitory activity of a short peptide derived from internal fusion peptide of S2 subunit of spike glycoprotein.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has posed a great concern in human population. To fight coronavirus emergence, we have dissected the conserved amino acid region of the internal fusion peptide in the S2 subunit of Spike glycoprotein of SARS-CoV-2 to design new inhibitory peptides. Among the 11 overlapping peptides (9-23-mer), PN19, a 19-mer peptide, exhibited a powerful inhibitory activity against different SARS-CoV-2 clinical isolate variants in absence of cytotoxicity. The PN19 inhibitory activity was found to be dependent on conservation of the central Phe and C-terminal Tyr residues in the peptide sequence. Circular dichroism spectra of the active peptide exhibited an alpha-helix propensity, confirmed by secondary structure prediction analysis. The PN19 inhibitory activity, exerted in the first step of virus infection, was reduced after peptide adsorption treatment with virus-cell substrate during fusion interaction. Additionally, PN19 inhibitory activity was reduced by adding S2 membrane-proximal region derived peptides. PN19 showed binding ability to the S2 membrane proximal region derived peptides, confirmed by molecular modelling, playing a role in the mechanism of action. Collectively, these results confirm that the internal fusion peptide region is a good candidate on which develop peptidomimetic anti SARS-CoV-2 antivirals.

Antiviral Activity against SARS-CoV-2 of Conformationally Constrained Helical Peptides Derived from Angiotensin-Converting Enzyme 2.

Despite the availability of vaccines, COVID-19 continues to be aggressive, especially in immunocompromised individuals. Therefore, the development of a specific therapeutic agent with antiviral activity against SARS-CoV-2 is necessary. The infection pathway starts when the receptor binding domain of the viral spike protein interacts with the angiotensin converting enzyme 2 (ACE2), which acts as a host receptor for the RBD expressed on the host cell surface. In this scenario, ACE2 analogs binding to the RBD and preventing the cell entry can be promising antiviral agents. Most of the ACE2 residues involved in the interaction belong to the α1 helix, more specifically to the minimal fragment ACE2(24-42). In order to increase the stability of the secondary structure and thus antiviral activity, we designed different triazole-stapled analogs, changing the position and the number of bridges. The peptide called P3, which has the triazole-containing bridge in the positions 36-40, showed promising antiviral activity at micromolar concentrations assessed by plaque reduction assay. On the other hand, the double-stapled peptide P4 lost the activity, showing that excessive rigidity disfavors the interaction with the RBD.

Teledentistry and COVID-19 molecular surveillance strategy for a safe clinical environment in Paediatric Dentistry / Teleodontología y estrategia de vigilancia molecular para COVID-19 proporcionando un entorno clínico seguro en Odontología Pediátrica / Teleodontologia e estratégia de vigilância molecular para a COVID-19 proporcionando um ambiente clínico seguro na Odontopediatria

The aim of this manuscript is to describe a hybrid care model to paediatricpatients on a virtual basis for dental treatment before conducting an in-person surveillance by using combined nasal/oral swabbing (NOS).This longitudinal study used a convenience sample of paediatric patients and members of the dental team from an undergraduate paediatric dentistry clinic at the University of São Paulo School of Dentistry during the COVID-19 pandemic. Firstly, parents werecontacted and teledentistry was used for screening children who need dental treatment. Appointments were scheduled once a week for two months, in which a pre-COVID-19 screening was performed. Dental team and children's parents completed a questionnaire addressing COVID-19-related symptoms. Members of the dental team and children were tested for COVID-19 before entering the dental clinic, by NOS and RT-PCR screening.Ninety-three individuals were enrolled and all of them completed the electronic questionnaire on symptoms and had NOS collected weekly, totalising 241 pairs of swabs. No participant reported COVID-19 symptomsbefore entering the clinic for treatment. Only one child tested positive in the third week of sampling. The hybrid care model associated with molecular testing for asymptomatics provided a safe clinical environment regarding the transmission of SARS-CoV-2 (AU).

El propósito de este manuscrito es describir un modelo híbrido de atención odontológica, a través de una plataforma virtual que precede a la realización de vigilancia molecular presencial mediante hisopado nasal/oral combinado, en pacientes pediátricos. Eneste estudio longitudinal se utilizó una muestra de conveniencia de pacientes pediátricos y miembros del equipo odontológico en la clínica de pregrado de la Facultad de Odontología de la Universidade de São Paulo durante la pandemia de COVID-19. En primerlugar, se contactó con los padres y se utilizó la consulta virtual para seleccionar a los niños que requerían tratamiento odontológico. Se programaron citas una vez a la semana durante dos meses, en las que se realizó el cribado previo a COVID-19. El equipo dental y los padres de los niños rellenaron un cuestionario en el que se abordaban los síntomas relacionados con la COVID-19. A continuación, los miembros del equipo dental y los niños fueron sometidos a pruebas de detección de COVID-19 antes de entrar en la clínica para atendimiento mediante cribado con hisopo nasal/oral y RT-PCR. Se inscribieron 93 personas, todas las cuales cumplimentaron el cuestionario electrónico sobre síntomas y se les recogieron muestras semanalmente, con un total de 241 pares dehisopos. Ningún participante declaró síntomas de COVID-19 antes de entrar en la clínica para recibir tratamiento. Sólo un niño dio positivo en la tercera semana de toma de muestras. El modelo de tratamiento híbrido unido a las pruebas moleculares para asintomáticos proporcionó un entorno clínico seguro con respecto a la transmisión del SARS-CoV-2 (AU).

O objetivo deste manuscrito é descrever um modelo de atendimento odontológico híbrido, por meio de uma plataforma virtual anterior a realização de uma vigilância molecular presencial usando esfregaço combinado nasal/oral, em pacientes pediátricos. Este estudo longitudinal utilizou uma amostra de conveniência de pacientes pediátricos e membros da equipe odontológica na clínica de graduação da Faculdade de Odontologia da Universidade de São Paulo durante a pandemia COVID-19. Primeiro, os pais foram contactados e a consulta virtual foi utilizada para o rastreio de crianças que necessitavam de tratamento odontológico. Foram agendadas consultas uma vez por semana durante dois meses, nas quais foi realizado um rastreio pré-COVID-19. A equipe odontológica e os pais das crianças preencheram um questionário que abordava os sintomas relacionados com a COVID-19. Em sequência, os membros da equipe odontológica e as crianças foram testados para o COVID-19 antes de entrarem na clínica para atendimento, através do rastreio com esfregaço nasal/oral e RT-PCR. Noventa e três indivíduos foram inscritos e todos eles preencheram o questionário eletrônico sobre os sintomas e tiveram amostras coletadas semanalmente, totalizando 241 pares de cotonetes. Nenhum participante comunicou sintomas de COVID-19 antes de entrar na clínica para tratamento. Apenas uma criança testou positivo na terceira semana de amostragem. O modelo de tratamento híbrido associado a testes moleculares para assintomática proporcionou um ambiente clínico seguro no que diz respeito à transmissão da SARS-CoV-2 (AU).

Severe acute respiratory syndrome by influenza and factors associated with death in older adults: a population study.

INTRODUCTION: Influenza is characterized by an acute viral infection, which can lead to severe conditions and death, especially in vulnerable populations, such as older adults. Therefore, we sought to analyze cases of severe acute respiratory syndrome (SARS) due to influenza in older adults registered in Brazil and investigate the factors related to death due to this disease. METHODOLOGY: This is a cross-sectional, population-based study that used secondary data from the Influenza Epidemiological Surveillance Information System (IESIS-Influenza). Older adults aged 60 years and above with laboratory diagnosis of influenza were included. RESULTS: A total of 3,547 older adults with SARS due to influenza were included, out of which 1,185 cases with death as the outcome were identified. Among older adults with death as the outcome, 87.4% were not vaccinated against influenza. The main risk factors for death were invasive ventilatory support use, intensive care unit admission, brown skin color and dyspnea (p < 0.001). CONCLUSIONS: This study described the profile of older adults with SARS due to influenza in Brazil. Factors associated with death in this population were identified. Moreover, the need to encourage compliance with vaccination among older adults is evident in order to prevent severe cases and unfavorable outcomes related to influenza.

Antiviral Activity of Oligonucleotides Targeting the SARS-CoV-2 Genomic RNA Stem-Loop Sequences within the 3'-End of the ORF1b.

Increased evidence shows vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exhibited no long-term efficacy and limited worldwide availability, while existing antivirals and treatment options have only limited efficacy. In this study, the main objective was the development of antiviral strategies using nucleic acid-based molecules. To this purpose, partially overlapped 6-19-mer phosphorothioate deoxyoligonucleotides (S-ONs) designed on the SARS-CoV-2 genomic RNA stem-loop packaging sequences within the 3' end of the ORF1b were synthetized using the direct and complementary sequence. Among the S-ONs tested, several oligonucleotides exhibited a fifty percent inhibitory concentration antiviral activity ranging from 0.27 to 34 µM, in the absence of cytotoxicity. The S-ON with a scrambled sequence used in the same conditions was not active. Moreover, selected 10-mer S-ONs were tested using different infectious doses and against different SARS-CoV-2 variants, showing comparable antiviral activity that was abrogated when the central sequence was mutated. Experiments to evaluate the intracellular functional target localization of the S-ON inhibitory activity were also performed. Collectively the data indicate that the SARS-CoV-2 packaging region in the 3' end of the ORF1b may be a promising target candidate for further investigation to develop innovative nucleic-acid-based antiviral therapy.

Factors associated with death due to severe acute respiratory syndrome caused by influenza: Brazilian population study.

INTRODUCTION: Influenza infection is characterized by acute viral infection of high transmissibility. Worsening of the case can lead to the need for hospitalization, severe acute respiratory syndrome (SARS) and even death. METHOD: This is a cross-sectional population-based study that used secondary database from the Brazilian Influenza Epidemiological Surveillance Information System. Only cases of adults with diagnosis of influenza by RT-PCR and case evolution recorded were included. RESULTS: We identified 2273 adults with SARS by influenza, 343 of which had death as an outcome. The main risk factors for death were lack of hospitalization, not having cough and age, both with p < 0.001. In addition, without asthma, having black skin color, not receiving flu vaccine, having brown skin color and not having a sore throat (p ≤ 0.005) were risk factors too. CONCLUSION: Factors associated with death due to SARS caused by influenza in Brazil, risk factors and protective factors to death were identified. It was evident that those who did not receive the flu vaccine presented twice the risk of unfavorable outcome, reinforcing the need to stimulate adherence to vaccination adhering and propose changes in public policies to make influenza vaccines available to the entire population, in order to prevent severe cases and unfavorable outcomes.

Plasma Torquetenovirus (TTV) microRNAs and severity of COVID-19.

BACKGROUND: Torquetenovirus (TTV), a widespread anellovirus recognized as the main component of the healthy human virome, displays viremia that is highly susceptible to variations in immune competence. TTV possesses microRNA (miRNA)-coding sequences that might be involved in viral immune evasion. Among TTV-encoded miRNAs, miRNA t1a, t3b, and tth8 have been found in biological fluids. Here, the presence of TTV DNA and TTV miRNAs in the plasma of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected subjects was investigated to monitor the possible association with coronavirus disease 2019 (COVID-19) severity. METHODS: Detection of TTV DNA and miRNA t1a, t3b, and tth8 was investigated in plasma samples of 56 SARS-CoV-2-infected subjects with a spectrum of different COVID-19 outcomes. TTV DNA and TTV miRNAs were assessed with a universal single step real-time TaqMan PCR assay and miRNA quantitative RT-PCR miRNA assay, respectively. RESULTS: The TTV DNA prevalence was 59%, whereas at least one TTV miRNA was found in 94% of the patients tested. miRNA tth8 was detected in 91% of subjects, followed by miRNAs t3b (64%) and miRNAt1a (30%). Remarkably, although TTV DNA was unrelated to COVID-19 severity, miRNA tth8 was significantly associated with the degree of disease (adjusted incidence rate ratio (IRR) 2.04, 95% CI 1.14-3.63, for the subjects in the high severity group compared to those in the low severity group). CONCLUSIONS: Our findings encourage further investigation to understand the potential role of TTV miRNAs in the different outcomes of COVID-19 at early and late stages.

Use of saliva and RT-PCR screening for SARS-CoV-2 variants of concern: Surveillance and monitoring.

Genomic surveillance has been applied since the beginning of the COVID-19 pandemic to track the spread of the virus, leading to the characterization of multiple SARS-CoV-2 variants, including variants of concern (VOC). Although sequencing is the standard method, a rapid molecular test for screening and surveillance of VOC is considered for detection. Furthermore, using alternative saliva as specimen collection facilitates the implementation of a less invasive, self-collected sample. In this study, we applied a combinatory strategy of saliva collection and reverse transcription polymerase chain reaction (RT-PCR) for SARS-CoV-2 VOC detection. Saliva samples from patients attending a tertiary hospital with suspected COVID-19 were collected and SARS-CoV-2 RNA was detected using SARS-CoV-2 RT-qPCR reagent kit (PerkinElmer). Positive saliva samples were screened for SARS-CoV-2 VOC with previously described RT-PCR for Alpha, Beta, and Gamma variants. Saliva samples were positive in 171 (53%) of 324 tested. A total of 108 (74%) from positive samples were also positive for VOC by RT-PCR screening. Those samples were found between January and August 2021. This approach allowed us to successfully use an alternative and complementary tool to genomic surveillance to monitor the circulation of SARS-CoV-2 VOC in the studied population.

MALDI-TOF mass spectrometry of saliva samples as a prognostic tool for COVID-19.

BACKGROUND: The SARS-CoV-2 infections are still imposing a great public health challenge despite the recent developments in vaccines and therapy. Searching for diagnostic and prognostic methods that are fast, low-cost and accurate are essential for disease control and patient recovery. The MALDI-TOF mass spectrometry technique is rapid, low cost and accurate when compared to other MS methods, thus its use is already reported in the literature for various applications, including microorganism identification, diagnosis and prognosis of diseases. METHODS: Here we developed a prognostic method for COVID-19 using the proteomic profile of saliva samples submitted to MALDI-TOF and machine learning algorithms to train models for COVID-19 severity assessment. RESULTS: We achieved an accuracy of 88.5%, specificity of 85% and sensitivity of 91.5% for classification between mild/moderate and severe conditions. When we tested the model performance in an independent dataset, we achieved an accuracy, sensitivity and specificity of 67.18, 52.17 and 75.60% respectively. CONCLUSION: Saliva is already reported to have high inter-sample variation; however, our results demonstrates that this approach has the potential to be a prognostic method for COVID-19. Additionally, the technology used is already available in several clinics, facilitating the implementation of the method. Further investigation using a larger dataset is necessary to consolidate the technique.

Lack of direct association between oral mucosal lesions and SARS-CoV- 2 in a cohort of patients hospitalised with COVID-19.

Background: COVID-19 is a disease affecting various human organs and systems, in which the virus seeks to interact with angiotensin-converting enzyme 2 receptors. These receptors are present in the oral cavity, but the direct relationship between such an interaction and possible oral manifestations of COVID-19 is still unclear. Aim: The present study evaluated oral manifestations in a cohort of COVID-19 patients during the period of hospitalisation. Methods: In total, 154 patients presenting moderate-to-severe forms of COVID-19 had their oral mucosa examined twice a week until the final outcome, either discharge or death. The oral alterations observed in the patients were grouped into Group 1 (pre-existing conditions and opportunistic oral lesions) and Group 2 (oral mucosal changes related to hospitalization). Results: Oral lesions found in the patients of Group 1 are not suggestive of SARS-CoV-2 infection as they are mainly caused by opportunistic infections. On the other hand, oral alterations found in the patients of Group 2 were statistically (P < 0.001) related to intubation and longer period of hospitalisation. Conclusion: It is unlikely that ulcerative lesions in the oral cavity are a direct manifestation of SARS-CoV-2 or a marker of COVID-19 progression.

Screening of the essential oil effects on human H1N1 influenza virus infection: an in vitro study in MDCK cells.

Nineteen essential oils (EOs) obtained from different plants have been evaluated for their potential in vitro anti-H1N1 influenza virus efficacy. Both multivariate analyses and bivariate correlation were performed to better understand how the composition influences the activity. The results evidenced that for the laboratory distilled EOs both rosemary hybrids (S. x lavandulaceus and S. x mendizabalii) showed a good antiviral activity with low cytotoxic effect. Concerning the commercial ones: Eucalyptus globulus and Juniperus communis EOs exhibited virtuous effects on influenza virus. These results were confirmed by the multivariate analyses and only eucalyptol showed a positive correlation with cell viability. On the contrary, o-cymene and terpinolene correlated to the inhibitory effect. Rosemary hybrids, E. globulus and J. communis could be considered as promising candidate to develop new alternative anti-H1N1 natural agent.

Quantification of torque teno virus (TTV) DNA in saliva and plasma samples in patients at short time before and after kidney transplantation.

BACKGROUND: Several reports have proposed that the viral load of torque teno virus (TTV) in plasma is a biomarker of immune function in solid organ transplantation (SOT) and in allogeneic hematopoietic stem cell transplantation. Additionally, for the latter one, TTV-DNA quantification in saliva has also been suggested. AIM: to investigate the correlation between the TTV viral load and immune function in paired saliva and plasma samples in patients on kidney transplantation. MATERIALS AND METHODS: TTV-DNA viral load was quantified in paired samples of saliva and plasma from 71 patients before and a short-time after renal-transplantation by real-time PCR. RESULTS: The data obtained from 213 paired samples showed a slight consistency in the comparison between saliva and plasma, with prevalence of TTV-DNA being 58%, 52% and 60% in saliva samples and 60%, 73% and 90% in plasma samples before and at 15-20 and 45-60 days after transplantation, respectively. Additionally, a high TTV viral load was observed in plasma at 15-20 and 45-60 days after transplantation compared to that observed in saliva at the same time. CONCLUSIONS: Overall, monitoring TTV-DNA in saliva samples could be an additional fast non-invasive option to assess the immune functionality in SOT populations.

West Nile Virus Seroprevalence in the Italian Tuscany Region from 2016 to 2019.

Although in humans West Nile virus is mainly the cause of mild or sub-clinical infections, in some cases a neuroinvasive disease may occur predominantly in the elderly. In Italy, several cases of West Nile virus infection are reported every year. Tuscany was the first Italian region where the virus was identified; however, to date only two cases of infection have been reported in humans. This study aimed at evaluating the prevalence of antibodies against West Nile virus in the area of Siena Province to estimate the recent circulation of the virus. Human serum samples collected in Siena between 2016 and 2019 were tested for the presence of antibodies against West Nile virus by ELISA. ELISA positive samples were further evaluated using immunofluorescence, micro neutralization, and plaque reduction neutralization assays. In total, 1.9% (95% CI 1.2-3.1) and 1.4% (95% CI 0.8-2.4) of samples collected in 2016-2017 were positive by ELISA and immunofluorescence assay, respectively. Neutralizing antibodies were found in 0.7% (95% CI 0.3-1.5) of samples. Additionally, 0.9% (95% CI 0.4-1.7) and 0.65% (95% CI 0.3-1.45) of samples collected in 2018-2019 were positive by ELISA and immunofluorescence assay, respectively. The prevalence of neutralizing antibodies was 0.5% (95% CI 0.2-1.3). Although no human cases of West Nile infection were reported in the area between 2016 and 2019 and virus prevalence in the area of Siena Province was as low as less than 1%, the active asymptomatic circulation confirms the potential concern of this emergent virus for human health.

Volatiles and Antifungal-Antibacterial-Antiviral Activity of South African Salvia spp. Essential Oils Cultivated in Uniform Conditions.

Spontaneous emissions of S. dentata Aiton and S. scabra Thunb., as well as the essential oil (EO) composition of the cited species, together with S. aurea L., were investigated. The chemical profile of the first two species is reported here for the first time. Moreover, in vitro tests were performed to evaluate the antifungal activity of these EOs on Trichophyton mentagrophytes, Microsporum canis, Aspergillus flavus, Aspergillus niger, and Fusarium solani. Secondly, the EO antibacterial activity against Escherichia coli, Staphylococcus aureus, and Staphylococcus pseudointermedius was examined, and their antiviral efficacy against the H1N1 influenza virus was assessed. Leaf volatile organic compounds (VOCs), as well as the EOs obtained from the arial part of Salvia scabra, were characterized by a high percentage of sesquiterpene hydrocarbons (97.8% and 76.6%, respectively), mostly represented by an equal amount of germacrene D (32.8% and 32.7%, respectively). Both leaf and flower spontaneous emissions of S. dentata, as well as the EO composition, showed a prevalence of monoterpenes divided into a more or less equal amount of hydrocarbon and oxygenated compounds. Interestingly, its EO had a non-negligible percentage of oxygenated sesquiterpenes (29.5%). S. aurea EO, on the contrary, was rich in sesquiterpenes, both hydrocarbons and oxygenated compounds (41.5% and 33.5%, respectively). S. dentata EO showed good efficacy (Minimal Inhibitory Concentration (MIC): 0.5%) against M. canis. The tested EOs were not active against E. coli and S. aureus, whereas a low inhibition of S. dentata EO was observed on S. pseudointermedius (MIC = 10%). Once again, S. dentata EO showed a very good H1N1 inhibition; contrariwise, S. aurea EO was completely inactive against this virus. The low quantity of S. scabra EO made it impossible to test its biological activity. S. dentata EO exhibited interesting new perspectives for medicinal and industrial uses.

Detection of polyomavirus microRNA-5p expression in saliva shortly after kidney transplantation.

Background: MicroRNAs (miRNAs) of polyomavirus (PyV) are present in several biological fluids and are suggested to be relevant viral factors for monitoring its persistence. Aim: To evaluate the effect of an immunosuppressive regimen on the status of PyV-miRNA-5p in the oral cavity. Materials and Methods: The JCPyV, BKPyV, MCPyV miRNA-5p were investigated in paired saliva and plasma samples obtained from 23 patients before and shortly after renal-transplantation by using real-time RT-PCR. Results: Overall, within a short-time after transplantation, patients exhibited decreased numbers of leukocyte and lymphocyte as well as low levels of creatinine. During the clinical management of the patients, a significant amount of saliva samples were positive for JCPyV and BKPyV miRNA-5p (range: 26%-91%) compared to paired plasma samples (range: 9%-35%). Among the two polyomaviruses showing positive expression of miRNA-5p, BKPyV presented the highest positivity in saliva (91%) and MCPyV-miRNA-5p was constantly negative in both saliva and plasma samples. Compared to the time before transplantation, a significant reduction in the expression of JCPyV-miRNA-5p was observed in saliva samples obtained after transplantation. Conclusions: Altogether, these data suggest that additional investigations of polyomavirus miRNA-5p in saliva should be performed shortly after renal-transplantation to evaluate the potential role in early viral reactivation.

Torque teno virus microRNA detection in cerebrospinal fluids of patients with neurological pathologies.

BACKGROUND: Torque teno virus (TTV) is a widespread anellovirus that establishes persistent infections in humans and represents the most abundant component of the human virome. TTV encodes microRNAs (miRNA) which are found both in viremic and not viremic subjects being potentially ideal tools for the virus to evade the immune system response and to maintain chronic infection in the host. OBJECTIVE: To investigate TTV-DNA loads and TTV-miRNAs expression in cerebrospinal fluids (CSF) from subjects under analysis for the assessment of neurological diseases. STUDY DESIGN: Detection of TTV-DNA and TTV-miRNAs (e. g. miRNA t1a, t3b, and tth8) were carried out from CSF samples of 93 subjects with neurological diseases by using universal real-time PCR, real-time RT-PCR, and next-generation sequencing (NGS) analyses. RESULTS: TTV-DNA was detected in 11 of 93 (12 %) CSFs with a mean TTV load of 155 copies/mL. Conversely, 29 CSF samples (31 %) were positive for at least one TTV-miRNA, while 15 (16 %) CSFs contained all the TTV-miRNAs examined. Overall, TTV-miRNA tth8 was detected in 62 % of samples, followed by TTV miRNA t3b (56 %), and t1a (29 %). Interestingly, TTV-miRNAs were found in CSF samples that were negative for the presence of TTV-DNA. Next-generation sequencing analysis carried out from 4 TTV-DNA negative CSF samples detected reads mapped in TTV-miRNA sequences region. CONCLUSIONS: These results shed novel light on the relationship between TTV and the central nervous system and make compelling furthered studies for investigating the potential role of TTV-miRNAs in neurological disorders.

Unique Domain for a Unique Target: Selective Inhibitors of Host Cell DDX3X to Fight Emerging Viruses.

Emerging viruses like dengue, West Nile, chikungunya, and Zika can cause widespread viral epidemics. Developing novel drugs or vaccines against specific targets for each virus is a difficult task. As obligate parasites, all viruses exploit common cellular pathways, providing the possibility to develop broad-spectrum antiviral agents targeting host factors. The human DEAD-box RNA helicase DDX3X is an essential cofactor for viral replication but dispensable for cell viability. Herein, we exploited the presence of a unique structural motif of DDX3X not shared by other cellular enzymes to develop a theoretical model to aid in the design of a novel class of highly selective inhibitors acting against such specific targets, thus limiting off-targeting effects. High-throughput virtual screening led us to identify hit compound 5, endowed with promising antienzymatic activity. To improve its aqueous solubility, 5 and its two enantiomers were synthesized and converted into their corresponding acetate salts (compounds 11, 12, and 13). In vitro mutagenesis and biochemical and cellular assays further confirmed that the developed molecules were selective for DDX3X and were able to suppress replication of West Nile and dengue viruses in infected cells in the micromolar range while showing no toxicity for uninfected cells. These results provide proof of principle for a novel strategy in developing highly selective and broad-spectrum antiviral molecules active against emerging and dangerous viral pathogens. This study paves the way for the development of larger focused libraries targeting such domain to expand SAR studies and fully characterize their mode of interaction.