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Background: The Golden Syrian hamster (Mesocricetus auratus), Ferrets (Mustela putorius furo), and macaques have been described as useful laboratory animals naturally susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection., Objectives: To study the mechanism of lung injury, we describe the histopathological features of SARS-CoV-2 infection in Golden Syrian hamsters inoculated intranasally with the A.2 Brazilian strain., Methods: Hamsters were intranasally inoculated with the A.2 variant and euthanised at 3-, 5-, 10- and 15-days post-inoculation. The physical examination and body weight were recorded daily. Neutralising antibodies and viral RNA load of the respiratory tract were assessed during necropsies., Findings: The coronavirus disease 2019 (COVID-19) model presented body weight loss, high levels of respiratory viral RNA load, severe segmentary pneumonitis, and bronchial fistula besides lymphatic trapping and infiltration, like the human SARS-COV-2 pathogenesis. The presence of subepithelial lymphoeosinophilic infiltrate was highlighted in our results; it contributed to the detachment of SARS-CoV-2 nucleocapsid-positive epithelial cells resulting in the infectious cell plugs., Main Conclusions: The SARS-CoV-2 caused segmentary pneumonia and vascular damage. In our comprehension, the infectious cell plugs, as being aspirated from the upper respiratory tract into the terminal bronchial lumen, work as a "Trojan horse", thus contributing to the dissemination of the SARS-CoV-2 infection into specific regions of the deep lung parenchyma.

Background: The SARS-CoV-2 pandemic has spurred an unparalleled scientific endeavor to elucidate the virus' structure, infection mechanisms, and pathogenesis. Two-dimensional culture systems have been instrumental in shedding light on numerous aspects of COVID-19. However, these in vitro systems lack the physiological complexity to comprehend the infection process and explore treatment options. Three-dimensional (3D) models have been proposed to fill the gap between 2D cultures and in vivo studies. Specifically, spheroids, composed of lung cell types, have been suggested for studying SARS-CoV-2 infection and serving as a drug screening platform., Methods: 3D lung spheroids were prepared by coculturing human alveolar or bronchial epithelial cells with human lung stromal cells. The morphology, size, and ultrastructure of spheroids before and after SARS-CoV-2 infection were analyzed using optical and electron microscopy. Immunohistochemistry was used to detect spike protein and, thus, the virus presence in the spheroids. Multiplex analysis elucidated the cytokine release after virus infection., Results: The spheroids were stable and kept their size and morphology after SARS-CoV-2 infection despite the presence of multivesicular bodies, endoplasmic reticulum rearrangement, tubular compartment-enclosed vesicles, and the accumulation of viral particles. The spheroid responded to the infection releasing IL-6 and IL-8 cytokines., Conclusion: This study demonstrates that coculture spheroids of epithelial and stromal cells can serve as a cost-effective infection model for the SARS-CoV-2 virus. We suggest using this 3D spheroid as a drug screening platform to explore new treatments related to the cytokines released during virus infection, especially for long COVID treatment., (© 2024 International Center for Artificial Organ and Transplantation (ICAOT) and Wiley Periodicals LLC.)

The LABEXTRACT plant extract bank, featuring diverse members of the Myrtaceae family from Brazilian hot spot regions, provides a promising avenue for bioprospection. Given the pivotal roles of the Spike protein and 3CL pro and PL pro proteases in SARS-CoV-2 infection, this study delves into the correlations between the Myrtaceae species from the Atlantic Forest and these targets, as well as an antiviral activity through both in vitro and in silico analyses. The results uncovered notable inhibitory effects, with Eugenia prasina and E. mosenii standing out, while E. mosenii proved to be multitarget, presenting inhibition values above 72% in the three targets analyzed. All extracts inhibited viral replication in Calu-3 cells (EC 50 was lower than 8.3 µg·mL -1 ). Chemometric analyses, through LC-MS/MS, encompassing prediction models and molecular networking, identified potential active compounds, such as myrtucommulones, described in the literature for their antiviral activity. Docking analyses showed that one undescribed myrtucommulone (m/z 841 [M - H] - ) had a higher fitness score when interacting with the targets of this study, including ACE2, Spike, PL pro and 3CL pro of SARS-CoV-2. Also, the study concludes that Myrtaceae extracts, particularly from E. mosenii and E. prasina , exhibit promising inhibitory effects against crucial stages in SARS-CoV-2 infection. Compounds like myrtucommulones emerge as potential anti-SARS-CoV-2 agents, warranting further exploration.

Tuberculosis (TB) disease, caused by Mycobacterium tuberculosis (Mtb) is the leading cause of death among people with human immunodeficiency virus (HIV) infection. No dual-target drug is currently being used to simultaneously treat both infections. This work aimed to obtain new multitarget HIV-TB agents, with the goal of optimizing treatments and preventing this coinfection. These compounds incorporate the structural features of azaaurones as anti-Mtb and zidovudine (AZT) as the antiretroviral moiety. The azaaurone scaffold displayed submicromolar activities against Mtb, and AZT is a potent antiretroviral drug. Six derivatives were synthetically generated, and five were evaluated against both infective agents. Evaluations of anti-HIV activity were carried out in HIV-1-infected MT-4 cells and on endogenous HIV-1 reverse transcriptase (RT) activity. The H37Rv strain was used for anti-Mtb assessments. Most compounds displayed potent antitubercular and moderate anti-HIV activity. (E)-12 exhibited a promising multitarget profile with an MIC 90 of 2.82 µM and an IC 50 of 1.98 µM in HIV-1-infected T lymphocyte cells, with an 84% inhibition of RT activity. Therefore, (E)-12 could be the first promising compound from a family of multitarget agents used to treat HIV-TB coinfection. In addition, the compound could offer a prototype for the development of new strategies in scientific research to treat this global health issue., (© 2023 Deutsche Pharmazeutische Gesellschaft.)

Despite being extensively studied because of the current coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) interactions with mammalian cells are still poorly understood. Furthermore, little is known about this coronavirus cycle within the host cells, particularly the steps that lead to viral egress. This study aimed to shed light on the morphological features of SARS-CoV-2 egress by utilizing transmission and high-resolution scanning electron microscopy, along with serial electron tomography, to describe the route of nascent virions towards the extracellular medium. Electron microscopy revealed that the clusters of viruses in the paracellular space did not seem to result from collective virus release. Instead, virus accumulation was observed on incurved areas of the cell surface, with egress primarily occurring through individual vesicles. Additionally, our findings showed that the emission of long membrane projections, which could facilitate virus surfing in Vero cells infected with SARS-CoV-2, was also observed in non-infected cultures, suggesting that these are constitutive events in this cell lineage., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Severe acute respiratory syndrome-related coronavirus (SARS-CoV-2) transmission occurs even among fully vaccinated individuals; thus, prompt identification of infected patients is central to control viral circulation. Antigen rapid diagnostic tests (Ag-RDTs) are highly specific, but sensitivity is variable. Discordant RT-qPCR vs. Ag-RDT results are reported, raising the question of whether negative Ag-RDT in positive RT-qPCR samples could imply the absence of infectious viruses. To study the relationship between negative Ag-RDT results with virological, molecular, and serological parameters, we selected a cross-sectional and a follow-up dataset and analyzed virus culture, subgenomic RNA quantification, and sequencing to determine infectious viruses and mutations. We demonstrated that RT-qPCR positive while SARS-CoV-2 Ag-RDT negative discordant results correlate with the absence of infectious virus in nasopharyngeal samples. A decrease in sgRNA detection together with an expected increase in detectable anti-S and anti-N IgGs was also verified in these samples. The data clearly demonstrate that a negative Ag-RDT sample is less likely to harbor infectious SARS-CoV-2 and, consequently, has a lower transmissible potential., Competing Interests: LG, MR, MB, RV, GC, and JH were employed by Abbott Laboratories Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Corrêa, Faffe, Galliez, Gonçalves, Maia, da Silva, Moreira, Mariani, Campos, Leitão, de Souza, Cunha, Nascimento, Ribeiro, da Cruz, Policarpo, Gonzales, Rodgers, Berg, Vijesurier, Cloherty, Hackett Jr., Ferreira Jr., Castiñeiras, Tanuri and da Costa.)

Community testing is a crucial tool for the early identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and transmission control. The emergence of the highly mutated Omicron variant (B.1.1.529) raised concerns about its primary site of replication, impacting sample collection and its detectability by rapid antigen tests. We tested the performance of the Panbio antigen rapid diagnostic test (Ag-RDT) using nasal and oral specimens for COVID-19 diagnosis in 192 symptomatic individuals, with quantitative reverse transcription-PCR (RT-qPCR) of nasopharyngeal samples as a control. Variant of concern (VOC) investigation was performed with the 4Plex SARS-CoV-2 screening kit. The SARS-CoV-2 positivity rate was 66.2%, with 99% of the positive samples showing an amplification profile consistent with that of the Omicron variant. Nasal Ag-RDT showed higher sensitivity (89%) than oral (12.6%) Ag-RDT. Our data showed good performance of the Ag-RDT in a pandemic scenario dominated by the Omicron VOC. Furthermore, our data also demonstrated that the Panbio COVID-19 antigen rapid diagnostic test does not provide good sensitivity with oral swabs for Omicron Ag-RDT detection. IMPORTANCE This study showed that the antigen rapid test for COVID19 worked fine using nasal swabs when it was utilized in patients infected with the Omicron variant, showing a concordance with PCR in 93% of patients tested. The nasal swab yielded more reliable results than the oral swab when an antigen rapid diagnosis test (the Panbio COVID-19 antigen rapid diagnostic test) was used in patients infected with the Omicron variant.

RT-qPCR is the gold standard technique available for SARS-CoV-2 detection. However, the long test run time and costs associated with this type of molecular testing are a challenge in a pandemic scenario. Due to high testing demand, especially for monitoring highly vaccinated populations facing the emergence of new SARS-CoV-2 variants, strategies that allow the increase in testing capacity and cost savings are needed. We evaluated a RT-qPCR pooling strategy either as a simplex and multiplex assay, as well as performed in-silico statistical modeling analysis validated with specimen samples obtained from a mass testing program of Industry Federation of the State of Rio de Janeiro (Brazil). Although the sensitivity reduction in samples pooled with 32 individuals in a simplex assay was observed, the high-test sensitivity was maintained even when 16 and 8 samples were pooled. This data was validated with the results obtained in our mass testing program with a cost saving of 51.5% already considering the expenditures with pool sampling that were analyzed individually. We also demonstrated that the pooling approach using 4 or 8 samples tested with a triplex combination in RT-qPCR is feasible to be applied without sensitivity loss, mainly combining Nucleocapsid (N) and Envelope (E) gene targets. Our data shows that the combination of pooling in a RT-qPCR multiplex assay could strongly contribute to mass testing programs with high-cost savings and low-reagent consumption while maintaining test sensitivity. In addition, the test capacity is predicted to be considerably increased which is fundamental for the control of the virus spread in the actual pandemic scenario., (© 2022. The Author(s).)

Coronaviruses are the etiologic agents of several diseases. Coronaviruses of critical medical importance are characterized by highly inflammatory pathophysiology, involving severe pulmonary impairment and infection of multiple cell types within the body. Here, we discuss the interplay between coronaviruses and autophagy regarding virus life cycle, cell resistance, and inflammation, highlighting distinct mechanisms by which autophagy restrains inflammatory responses, especially those involved in coronavirus pathogenesis. We also address different autophagy modulators available and the rationale for drug repurposing as an attractive adjunctive therapy. We focused on pharmaceuticals being tested in clinical trials with distinct mechanisms but with autophagy as a common target. These autophagy modulators act in cell resistance to virus infection and immunomodulation, providing a double-strike to prevent or treat severe disease development and death from coronaviruses diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Silva, Ribeiro, da Silva, da Costa and Travassos.)

Background: During routine Coronavirus disease 2019 (COVID-19) diagnosis, an unusually high viral load was detected by reverse transcription real-time polymerase chain reaction (RT-qPCR) in a nasopharyngeal swab sample collected from a patient with respiratory and neurological symptoms who rapidly succumbed to the disease. Therefore we sought to characterise the infection., Objectives: We aimed to determine and characterise the etiological agent responsible for the poor outcome., Methods: Classical virological methods, such as plaque assay and plaque reduction neutralisation test combined with amplicon-based sequencing, as well as a viral metagenomic approach, were performed to characterise the etiological agents of the infection., Findings: Plaque assay revealed two distinct plaque phenotypes, suggesting either the presence of two severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains or a productive coinfection of two different species of virus. Amplicon-based sequencing did not support the presence of any SARS-CoV-2 genetic variants that would explain the high viral load and suggested the presence of a single SARS-CoV-2 strain. Nonetheless, the viral metagenomic analysis revealed that Coronaviridae and Herpesviridae were the predominant virus families within the sample. This finding was confirmed by a plaque reduction neutralisation test and PCR., Main Conclusions: We characterised a productive coinfection of SARS-CoV-2 and Herpes simplex virus 1 (HSV-1) in a patient with severe symptoms that succumbed to the disease. Although we cannot establish the causal relationship between the coinfection and the severity of the clinical case, this work serves as a warning for future studies focused on the interplay between SARS-CoV-2 and HSV-1 coinfection and COVID-19 severity.

Current guidelines for patient isolation in COVID-19 cases recommend a symptom-based approach, averting the use of control real-time reverse transcription PCR (rRT-PCR) testing. However, we hypothesized that patients with persistently positive results by RT-PCR for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could be potentially infectious for a prolonged time, even if immunocompetent and asymptomatic, which would demand a longer social isolation period than presently recommended. To test this hypothesis, 72 samples from 51 mildly symptomatic immunocompetent patients with long-lasting positive rRT-PCR results for SARS-CoV-2 were tested for their infectiousness in cell culture. The serological response of samples from those patients and virus genomic integrity were also analyzed. Infectious viruses were successfully isolated from 34.38% (22/64) of nasopharynx samples obtained 14 days or longer after symptom onset. Indeed, we observed successful virus isolation up to 128 days. Complete SARS-COV-2 genome integrity was demonstrated, suggesting the presence of replication-competent viruses. No correlation was found between the isolation of infectious viruses and rRT-PCR cycle threshold values or the humoral immune response. These findings call attention to the need to review current isolation guidelines, particularly in scenarios involving high-risk individuals. IMPORTANCE In this study, we evaluated mildly symptomatic immunocompetent patients with long-lasting positive rRT-PCR results for SARS-CoV-2. Infectious viruses were successfully isolated in cell cultures from nasopharynx samples obtained 14 days or longer after symptom onset. Indeed, we observed successful virus isolation for up to 128 days. Moreover, SARS-CoV-2 genome integrity was demonstrated by sequencing, suggesting the presence of replication-competent viruses. These data point out the risk of continuous SARS-CoV-2 transmission from patients with prolonged detection of SARS-CoV-2 in the upper respiratory tract, which has important implications for current precaution guidelines, particularly in settings where vulnerable individuals may be exposed (e.g., nursing homes and hospitals).

As a part of the efforts to quickly develop pharmaceutical treatments for COVID-19 through repurposing existing drugs, some researchers around the world have combined the recently released crystal structure of SARS-CoV-2 M pro in complex with a covalently bonded inhibitor with virtual screening procedures employing molecular docking approaches. In this context, protease inhibitors (PIs) clinically available and currently used to treat infectious diseases, particularly viral ones, are relevant sources of promising drug candidates to inhibit the SARS-CoV-2 M pro , a key viral enzyme involved in crucial events during its life cycle. In the present perspective, we summarized the published studies showing the promising use of HIV and HCV PIs as potential repurposing drugs against the SARS-CoV-2 M pro ., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The integral membrane protein ATG9A plays a key role in autophagy. It displays a broad intracellular distribution and is present in numerous compartments, including the plasma membrane (PM). The reasons for the distribution of ATG9A to the PM and its role at the PM are not understood. Here, we show that ATG9A organizes, in concert with IQGAP1, components of the ESCRT system and uncover cooperation between ATG9A, IQGAP1 and ESCRTs in protection from PM damage. ESCRTs and ATG9A phenocopied each other in protection against PM injury. ATG9A knockouts sensitized the PM to permeabilization by a broad spectrum of microbial and endogenous agents, including gasdermin, MLKL and the MLKL-like action of coronavirus ORF3a. Thus, ATG9A engages IQGAP1 and the ESCRT system to maintain PM integrity., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Here, we investigated the interaction of this new coronavirus with Vero cells using high resolution scanning electron microscopy. Surface morphology, the interior of infected cells and the distribution of viral particles in both environments were observed 2 and 48 h after infection. We showed areas of viral processing, details of vacuole contents, and viral interactions with the cell surface. Intercellular connections were also approached, and viral particles were adhered to these extensions suggesting direct cell-to-cell transmission of SARS-CoV-2.

The ProTide approach has emerged as a powerful tool to improve the intracellular delivery of nucleotide analogs with antiviral and anticancer activity. Here, we characterized the anti-ZIKV (ZIKV, Zika virus) activity of two ProTides of 2'-C-β-methylguanosine. ProTide UMN-1001 is a 2'-C-β-methylguanosine tryptamine phosphoramidate monoester, and ProTide UMN-1002 is a 2-(methylthio)-ethyl-2'-C-β-methylguanosine tryptamine phosphoramidate diester. UMN-1002 undergoes stepwise intracellular activation to the corresponding nucleotide monophosphate followed by P-N bond cleavage by intracellular histidine triad nucleotide binding protein 1 (Hint1). UMN-1001 is activated by Hint1 but is less cell-permeable than UMN-1002 . UMN-1001 and UMN-1002 were found to be more potent than 2'-C-β-methylguanosine against ZIKV in human-derived microvascular endothelial and neuroblastoma cells and in reducing ZIKV RNA replication. Studies with a newborn mouse model of ZIKV infection demonstrated that, while treatment with 2'-C-β-methylguanosine and UMN-1001 was lethal, treatment with UMN-1002 was nontoxic and significantly reduced ZIKV infection. Our data suggests that anchimeric activated ProTides of 2'-C-β-methyl nucleosides should be further investigated for their potential as anti-ZIKV therapeutics.

Chikungunya virus (CHIKV) is an arthropod-borne virus (arbovirus) of epidemic concern, transmitted by Aedes ssp. mosquitoes, and is the etiologic agent of a febrile and incapacitating arthritogenic illness responsible for millions of human cases worldwide. After major outbreaks starting in 2004, CHIKV spread to subtropical areas and western hemisphere coming from sub-Saharan Africa, South East Asia, and the Indian subcontinent. Even though CHIKV disease is self-limiting and non-lethal, more than 30% of the infected individuals will develop chronic disease with persistent severe joint pain, tenosynovitis, and incapacitating polyarthralgia that can last for months to years, negatively impacting an individual's quality of life and socioeconomic productivity. The lack of specific drugs or licensed vaccines to treat or prevent CHIKV disease associated with the global presence of the mosquito vector in tropical and temperate areas, representing a possibility for CHIKV to continually spread to different territories, make this virus an agent of public health burden. In South America, where Dengue virus is endemic and Zika virus was recently introduced, the impact of the expansion of CHIKV infections, and co-infection with other arboviruses, still needs to be estimated. In Brazil, the recent spread of the East/Central/South Africa (ECSA) and Asian genotypes of CHIKV was accompanied by a high morbidity rate and acute cases of abnormal disease presentation and severe neuropathies, which is an atypical outcome for this infection. In this review, we will discuss what is currently known about CHIKV epidemics, clinical manifestations of the human disease, the basic concepts and recent findings in the mechanisms underlying virus-host interaction, and CHIKV-induced chronic disease for both in vitro and in vivo models of infection. We aim to stimulate scientific debate on how the characterization of replication, host-cell interactions, and the pathogenic potential of the new epidemic viral strains can contribute as potential developments in the virology field and shed light on strategies for disease control., (Copyright © 2020 Cunha, Costa, Correa, de Souza, Calil, da Silva, Costa, Fonseca and da Costa.)

This work describes the synthesis and biological evaluation of an anchimerically activated proTide of 2'- C -β-methylguanosine as an inhibitor of dengue virus 2 (DENV-2). The proTide incorporates a chemically cleavable 2-(methylthio)ethyl moiety and a HINT1 hydrolyzable tryptamine phosphoramidate. Inhibition of DENV-2 replication by proTide 6 was 5-fold greater than the parent nucleoside while displaying no apparent cytotoxicity. Furthermore, we demonstrate with a HINT1 inhibitor that the anti DENV-2 activity of the proTide correlates with the activity of HINT1. Taken together, these results demonstrate that a phosphoramidate based pronucleotide that undergoes an initial nonenzymatic activation step based on anchimeric assistance followed by P-N bond cleavage by HINT1 can be prepared.

Background: The occurrence of outbreaks of human rabies transmitted by Desmodus rotundus in Brazil in 2004 and 2005 reinforced the need for further research into this zoonosis. Studies of knowledge and practices related to the disease will help to define strategies for the avoidance of new cases, through the identification of gaps that may affect the preventive practices., Methodology/principal Findings: A semi-structured questionnaire was applied to 681 residents of twelve communities of northeastern Pará state involved in the 2004 and 2005 outbreaks mentioned above. The objective was to evaluate the local knowledge and practices related to the disease. We found a highly significant difference (p<0.0001) in the knowledge of rabies among education levels, indicating that education is a primary determinant of knowledge on this disease. More than half of the respondents (63%) recognized the seriousness of the zoonosis, and 50% were aware of the importance of bats for its transmission, although few individuals (11%) were familiar with the symptoms, and only 40% knew methods of prevention. Even so, 70% of pet owners maintained their animals vaccinated, and 52% of the respondents bitten by bats had received post-exposure vaccination. Most of the respondents (57%) reported being familiarized with rabies through informal discussions, and only a few (23%) mentioned public health agents as the source of their information., Conclusion/significance: We identified many gaps in the knowledge and practices of the respondents regarding rabies. This may be the result of the reduced participation of public health agents in the transfer of details about the disease. The lack of knowledge may be a direct determinant in the occurrence of new outbreaks. Given these findings, there is a clear need for specific educational initiatives involving the local population and the public health entities, with the primary aim of contributing to the prevention of rabies.

Aim: The aims of the current study were to compare the levels of HIV-1 in the subgingival biofilm (SHVL) between detectable and undetectable plasmatic HIV-1 viral load (PHVL) in HIV-infected patients as well as to determine the association of SHVL with PHVL and clinical periodontal parameters., Material and Methods: Forty-one HIV-infected individuals were divided into two groups: detectable (21) and undetectable (20) PHVL. Subgingival biofilm samples were obtained for detection and quantification of HIV-1 by real-time RT-PCR. To estimate the effect of co-variables on the outcome undetectable SHVL, the Generalized Estimation Equation (GEE) was employed., Results: Detectable SHVL was observed only in the detectable PHVL group and the detection of the HIV-1 was observed in 40% of these individuals. In the bivariate analysis between co-variables from the individual level and the outcome SHVL, significant difference was observed only for the CD4+ T lymphocytes levels (p = 0.017). The multiple logistic model demonstrated that only CD4+ T lymphocytes levels had a significant effect on the outcome undetectable SHVL [OR 8.85 (CI 3.6-9.2), p = 0.002]., Conclusion: HIV-1 can be detected and quantified in the subgingival biofilm of HIV-infected individuals, but these findings are not associated with PHVL and periodontal clinical parameters., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Nef is an accessory protein of human immunodeficiency viruses that promotes viral replication and progression to AIDS through interference with various host trafficking and signaling pathways. A key function of Nef is the down-regulation of the coreceptor CD4 from the surface of the host cells. Nef-induced CD4 down-regulation involves at least two independent steps as follows: acceleration of CD4 endocytosis by a clathrin/AP-2-dependent pathway and targeting of internalized CD4 to multivesicular bodies (MVBs) for eventual degradation in lysosomes. In a previous work, we found that CD4 targeting to the MVB pathway was independent of CD4 ubiquitination. Here, we report that this targeting depends on a direct interaction of Nef with Alix/AIP1, a protein associated with the endosomal sorting complexes required for transport (ESCRT) machinery that assists with cargo recruitment and intraluminal vesicle formation in MVBs. We show that Nef interacts with both the Bro1 and V domains of Alix. Depletion of Alix or overexpression of the Alix V domain impairs lysosomal degradation of CD4 induced by Nef. In contrast, the V domain overexpression does not prevent cell surface removal of CD4 by Nef or protein targeting to the canonical ubiquitination-dependent MVB pathway. We also show that the Nef-Alix interaction occurs in late endosomes that are enriched in internalized CD4. Together, our results indicate that Alix functions as an adaptor for the ESCRT-dependent, ubiquitin-independent targeting of CD4 to the MVB pathway induced by Nef., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

There is controversy concerning the effect of pilocarpine in the reversal of radio-induced xerostomia; however, the tests are usually performed at the end of radiotherapy. The present study evaluated the radioprotective effects of pilocarpine when ingested during radiation treatment. Eleven patients (recently diagnosed with head and neck cancer who were not undergoing radiotherapy) were divided into two groups: the control group (saline solution intake n=6) and the pilocarpine-treated (5 mg pilocarpine three times daily, n=5) group, in a prospective and double-blinded study. For five weeks, oral conditions, unstimulated salivary flow and stimulated saliva flow were collected weekly, with the first collection occurring prior to radiation therapy. As early as the second week, the control group exhibited oral complications and greater reduction in salivary flow rate. At the end of the study, the pilocarpine-treated group presented mean values of salivary flow greater than those of the control group. Pilocarpine intake applied simultaneously with radiotherapy demonstrated encouraging results with regard to lowering salivary flow reduction and incidence of xerostomia, as well as of oral complications.

The 30 different species of mRNAs synthesized during the HIV-1 replication cycle are all capped and polyadenilated. Internal ribosome entry sites have been recognized in the 5' untranslated region of some mRNA species of HIV-1, which would contribute to an alternative mechanism of initiation of mRNA translation. However, the Cap-dependent translation is assumed to be the main mechanism driving the initiation of HIV-1 protein synthesis. In this work, we describe a cell system in which lower to higher levels of transient expression of the poliovirus 2A protease strongly inhibited cellular Cap-dependent translation with no toxic effect to the cells during a 72-hour time frame. In this system, the synthesis of HIV-1 proteins was inhibited in a temporal dose-dependent way. Higher levels of 2A protease expression severely inhibited HIV-1 protein synthesis during the first 24 hours of infection consequently inhibiting viral production and infectivity. Intermediate to lower levels of 2A Protease expression caused the inhibition of viral protein synthesis only during the first 48 hours of viral replication. After this period both protein synthesis and viral release were recovered to the control levels. However, the infectivity of viral progeny was still partially inhibited. These results indicate that two mechanisms of mRNA translation initiation contribute to the synthesis of HIV-1 proteins; during the first 24-48 hours of viral replication HIV-1 protein synthesis is strongly dependent on Cap-initiation, while at later time points IRES-driven translation initiation is sufficient to produce high amounts of viral particles.

Dengue virus infection is a serious public health problem in endemic areas of the world where 2.5 billion people live. Clinical manifestations of the Dengue infection range from a mild fever to fatal cases of hemorrhagic fever. Although being the most rapidly spreading mosquito-borne viral infection in the world, until now no strategies are available for effective prevention or control of Dengue infection. In this scenario, the development of compounds that specifically inhibit viral replication with minimal effects to the human hosts will have a substantial effect in minimizing the symptoms of the disease and help to prevent viral transmission in the affected population. The aim of this study was to screen compounds with potential activity against dengue virus from a library of synthetic naphthoquinones. Several 1,2- and 1,4-pyran naphthoquinones were synthesized by a three-component reaction of lawsone, aldehyde (formaldehyde or arylaldehydes) and different dienophiles adequately substituted. These compounds were tested for the ability to inhibit the ATPase activity of the viral NS3 enzyme in in vitro assays and the replication of dengue virus in cultured cells. We have identified two 1,4-pyran naphthoquinones, which inhibited dengue virus replication in mammal cells by 99.0% and three others that reduced the dengue virus ATPase activity of NS3 by two-fold in in vitro assays.

Aseptic meningitis is one of the most common neurological disorders caused by enteroviruses. Among them, Echovirus 30 (E30) is described as the main etiological agent of many outbreaks and sporadic cases. This study investigated the genomic variability of E30 isolated from the cerebrospinal fluid (CSF) of aseptic meningitis cases that occurred from 1998 to 2008 in Brazil. Over a 10-year period (1998-2008), 302 non-polio enteroviruses were isolated, of which 177 were identified as E30 (58.6%). Phylogenetic analysis of the complete VP1 gene (876 nt) of 48 E30 isolates was performed and compared with additional Brazilian and foreign strains. E30 VP1 sequences segregated into three distinct major groups and seven subgroups, which were linked to the isolation year. In general, sequence divergence among E30 strains ranged from 0.2% to 13.8%. A common direct ancestor for this set of E30 strains was not defined. Brazilian isolates from Group I were related genetically to a 1997 USA isolate and both may have a common origin. Group III representatives showed close relationship to the 2007 Argentinean isolates. The present results complement existing data on the molecular characterization and genetic variability of E30 and may contribute to the understanding of the epidemiology of aseptic meningitis in the region., (Copyright © 2011 Wiley Periodicals, Inc.)

Objective: The aim of the present study was to analyse the characteristics of salivary production and its composition in individuals with burning mouth syndrome (BMS)., Study Design: Salivary flow rate, concentrations of potassium, iron, chloride, thiocyanate, magnesium, calcium, phosphorus, glucose, total protein and urea, as well as the expression profile of salivary proteins were analysed by SDS-PAGE., Results: The mean salivary flow rate among control patients was lower than that of BMS patients. Chloride, phosphorus and potassium levels were elevated in patients with BMS (p = 0.041, 0.001 and 0.034, respectively). Total salivary protein concentration was reduced in individuals with BMS (p = 0.223). Analysis of the expression of salivary proteins by Coomassie blue SDS-PAGE revealed a lower expression of low molecular weight proteins in individuals with BMS compared to healthy controls., Conclusions: These results indicate that the identification and characterisation of low molecular weight salivary proteins in BMS may be important in understanding BMS pathogenesis, thus contributing to its diagnosis and treatment.

The present paper aimed at evaluating the validity of the Portuguese version of the Research Diagnostic Criteria for Temporomandibular Disorders (RDC/TMD) Axis II Questionnaire. The sample was comprised of 155 patients with signs and symptoms of Temporomandibular Disorders (TMD), evaluated at the Orofacial Pain Control Center, School of Dentistry, University of Pernambuco, Brazil, between July 2003 and February 2004. Data collection was performed with the following tools: the RDC/TMD Axis I (clinical evaluation and TMD classification), and Axis II (psychosocial evaluation), as well as specific questionnaires for evaluation of Oral Health Related Quality of Life, namely, Oral Impacts on Daily Performances and the Oral Health Impact Profile-14, considered to be gold standard criteria. Validity evaluation consisted of internal consistency evaluation by the Cronbach alfa reliability test, reliability and reproducibility estimated by the Kappa test and the Spearman's correlation, and concurring validation through Spearman's correlation. The Portuguese version of the RDC/TMD Axis II questionnaire was considered consistent (Cronbach alfa = 0.72), reproducible (Kappa values from 0.73 to 0.91, p < 0.01), and valid (p < 0.01). It was concluded that this version showed valid and reproducible results for the Brazilian population, thus paving the way for including Brazil in transcultural epidemiological studies on TMD.

It has been recently demonstrated that HIV-1 reverse transcriptase is the target of two diterpenes, (6 R)-6-hydroxydichotoma-3,14-diene-1,17-dial (compound 1) and (6 R)-6-acetoxydichotoma-3,14-diene-1,17-dial (compound 2), that inhibit HIV-1 replication in vitro. In this work, the effects of both diterpenes on the kinetic properties of the recombinant HIV-1 reverse transcriptase (RT) enzyme were evaluated. RNA-dependent DNA-polymerase (RDDP) activity assays demonstrated that both diterpenes behave as non-competitive inhibitors with respect to dTTP and uncompetitive inhibitors with respect to poly(rA).oligo(dT) template primers. The K(i) values obtained for compounds 1 and 2 were 10 and 35 microM, respectively. Neither of these diterpenes affected the DNA-dependent DNA-polymerase (DDDP) activity of the HIV-1 RT. The RDDP activities of AMV-RT and MMLV-RT enzymes were also inhibited by compounds 1 and 2. In contrast to the HIV-1 enzyme, the DDDP activities of AMV-RT and MMLV-RT enzymes were significantly reduced by compound 1. Taken together, our results demonstrate that compound 1 is a more effective inhibitor of the viral reverse transcriptases from HIV-1, AMV and MMLV than compound 2. The kinetic behavior analyses of the HIV-1 RT demonstrate that both diterpenes have similar mechanisms of inhibition of RDDP activity.

Brefeldin A (BFA), a fungal metabolite that blocks transport of newly synthesized proteins from the endoplasmic reticulum, was found to inhibit Mayaro virus replication. At the concentration of 0.05 microgram/ml, the yield of the virus was inhibited by 94% in Aedes albopictus cells and by 99.5% in Vero cells. Treatment of A. albopictus cells with BFA did not inhibit the virus protein synthesis. However, this compound drastically reduced viral protein synthesis in Vero cells. The inhibitory effect progressively declined when BFA was added at late times post infection (p.i.). The effect of BFA on protein glycosylation is discussed.

A method is described for the efficient substitution, deletion or insertion of any desired DNA sequence into any viral infectious clones without the limitation of naturally occurring restriction sites. The technique employs the polymerase chain reaction combined with the resistance of 2'-deoxynucleotides 5'-O-(1-thiotriphosphate) dNTPs [S] bonds (phosphorothiate bonds) to the 5'-3' double strand specific T7 gene 6 exonuclease (T7 Exo) digestion. Primers used to amplify the DNA target regions being manipulated present three phosphorothioate bonds from the fifteenth base at the 5' end. The enzyme activity was shown to be completely inhibited by the presence of more than one phosphorothioate residue at the 5' end of the DNA molecules. When the amplification products are submitted to the exonuclease digestion the hydrolytic T7 Exo activity generates a short single strand DNA tail which contains the nucleotide integrity of the 3' strand. Since the ends of two independently amplified products overlap they can regenerate a stable recombinant structure when further combined in the same reaction tube in the presence of T4 DNA ligase. This new method can be used for manipulating an HIV-1 full-length clone belonging to subtype D replacing the env (gp120) gene for an F subtype sequence.