Development and field validation of a reverse transcription loop-mediated isothermal amplification assay (RT-LAMP) for the rapid detection of chikungunya virus in patient and mosquito samples.

OBJECTIVES: We aimed to develop a reverse transcription loop-mediated isothermal amplification (RT-LAMP) platform for the rapid detection of chikungunya virus (CHIKV) in both patient and mosquito samples from Brazil. METHODS: We optimized an RT-LAMP assay and then evaluated the specificity and sensitivity using visual detection. In comparison with the RT-qPCR reference method, we validated the utility of this assay as a molecular diagnostic test in a reference laboratory for arbovirus diagnostics using 100 serum samples collected from suspected CHIKV cases. RESULTS: Our RT-LAMP assay specifically detected CHIKV without cross-reactivity against other arboviruses. The limit of detection of our RT-LAMP was estimated in -1.18 PFU (confidence interval [CI] ranging from -2.08 to 0.45), resulting in a similar analytical sensitivity when directly compared with the reference standard RT-qPCR assay. Then, we demonstrate the ability of our RT-LAMP assay to detect the virus in different human specimens (serum, urine, and saliva), and crude lysate of Aedes aegypti mosquitoes in as little as 20-30 minutes and without a separate RNA isolation step. Lastly, we showed that our RT-LAMP assay could be lyophilized and reactivated by adding water, indicating potential for room-temperature storage. Our RT-LAMP had a clinical sensitivity of 100% (95% CI, 90.97-100.00%), clinical specificity of 96.72% (95% CI, 88.65-99.60%), and overall accuracy of 98.00% (95% CI, 92.96-99.76%). DISCUSSION: Taken together, these findings indicate that the RT-LAMP assay reported here solves important practical drawbacks to the deployment of molecular diagnostics in the field and can be used to improve testing capacity, particularly in low- and middle-income countries.

Antiviral Activity of Flavonoids from Geopropolis of the Brazilian Jandaira Bee against Zika and Dengue Viruses.

Arthropod-borne viruses within the Flaviviridae family such as Zika (ZIKV) and dengue (DENV) are responsible for major outbreaks in tropical countries, and there are no specific treatments against them. Naringenin and 7-O-methyl naringenin are flavonoids that can be extracted from geopropolis, a natural material that the Brazilian Jandaira stingless bee (Melipona subnitida Ducke) produces to protect its nest. Here, these flavonoids were tested against ZIKV and DENV using Vero cells as a cellular model to perform a cytotoxicity assay and to define the effective concentrations of TCID50 as the readout method. The results demonstrated the antiviral activity of the compounds against both viruses upon the treatment of infected cells. The tested flavonoids had antiviral activity comparable with 6-methylmercaptopurine riboside (6-MMPr), used here as a positive control. In addition, to identify the possible action mechanism of the antiviral candidates, we carried out a docking analysis followed by a molecular dynamics simulation to elucidate naringenin and 7-O-methyl naringenin binding sites to each virus. Altogether, these results demonstrate that both flavonoids have potent antiviral effects against both viruses and warrant further in vivo trials.

Comparison of DNA extraction methods for COVID-19 host genetics studies.

The coronavirus disease 2019 (COVID-19) pandemic has resulted in global shortages in supplies for diagnostic tests, especially in the developing world. Risk factors for COVID-19 severity include pre-existing comorbidities, older age and male sex, but other variables are likely play a role in disease outcome. There is indeed increasing evidence that supports the role of host genetics in the predisposition to COVID-19 outcomes. The identification of genetic factors associated with the course of SARS-CoV-2 infections relies on DNA extraction methods. This study compared three DNA extraction methods (Chelex®100 resin, phenol-chloroform and the QIAamp DNA extraction kit) for COVID-19 host genetic studies using nasopharyngeal samples from patients. The methods were compared regarding number of required steps for execution, sample handling time, quality and quantity of the extracted material and application in genetic studies. The Chelex®100 method was found to be cheapest (33 and 13 times cheaper than the commercial kit and phenol-chloroform, respectively), give the highest DNA yield (306 and 69 times higher than the commercial kit and phenol-chloroform, respectively), with the least handling steps while providing adequate DNA quality for downstream applications. Together, our results show that the Chelex®100 resin is an inexpensive, safe, simple, fast, and suitable method for DNA extraction of nasopharyngeal samples from COVID-19 patients for genetics studies. This is particularly relevant in developing countries where cost and handling are critical steps in material processing.

Mouse Models of Mayaro Virus.

Mayaro virus (MAYV), the etiologic agent of Mayaro fever, leads patients to severe myalgia and arthralgia, which can have a major impact on public health in all the countries where the virus circulates. The emergence and dissemination of new viruses have led the scientific community to develop new in vivo models that can help in the fight against new diseases. So far, mice have been the most used animal model in studies with MAYV and have proved to be an adequate model for recapitulating several aspects of the disease observed in humans. Mice are widely used in in vivo research and, therefore, are well known in the scientific community, which has allowed for different strains to be investigated in the study of MAYV. In this review, we summarize the main studies with MAYV using mice as an experimental model and discuss how they can contribute to the advancement of the understanding of its pathogenesis and the development of new drugs and vaccines.

A novel N95 respirator with chitosan nanoparticles: mechanical, antiviral, microbiological and cytotoxicity evaluations.

BACKGROUND: It is known that some sectors of hospitals have high bacteria and virus loads that can remain as aerosols in the air and represent a significant health threat for patients and mainly professionals that work in the place daily. Therefore, the need for a respirator able to improve the filtration barrier of N95 masks and even inactivating airborne virus and bacteria becomes apparent. Such a fact motivated the creation of a new N95 respirator which employs chitosan nanoparticles on its intermediate layer (SN95 + CNP). RESULTS: The average chitosan nanoparticle size obtained was 165.20 ± 35.00 nm, with a polydispersity index of 0.36 ± 0.03 and a zeta potential of 47.50 ± 1.70 mV. Mechanical tests demonstrate that the SN95 + CNP respirator is more resistant and meets the safety requisites of aerosol penetration, resistance to breath and flammability, presenting higher potential to filtrate microbial and viral particles when compared to conventional SN95 respirators. Furthermore, biological in vitro tests on bacteria, fungi and mammalian cell lines (HaCat, Vero E6 and CCL-81) corroborate the hypothesis that our SN95 + CNP respirator presents strong antimicrobial activity and is safe for human use. There was a reduction of 96.83% of the alphacoronavirus virus and 99% of H1N1 virus and MHV-3 betacoronavirus after 120 min of contact compared to the conventional respirator (SN95), demonstrating that SN95 + CNP have a relevant potential as personal protection equipment. CONCLUSIONS: Due to chitosan nanotechnology, our novel N95 respirator presents improved mechanical, antimicrobial and antiviral characteristics.

Whole Yeast Vaccine Displaying ZIKV B and T Cell Epitopes Induces Cellular Immune Responses in the Murine Model.

Improving antigen presentation is crucial for the success of immunization strategies. Yeasts are classically used as biofactories to produce recombinant proteins and are efficient vehicles for antigen delivery, in addition to their adjuvant properties. Despite the absence of epidemic outbreaks, several vaccine approaches continue to be developed for Zika virus infection. The development of these prophylactic strategies is fundamental given the severity of clinical manifestations, mainly due to viral neurotropism. The present study aimed to evaluate in vivo the immune response induced by P. pastoris recombinant strains displaying epitopes of the envelope (ENV) and NS1 ZIKV proteins. Intramuscular immunization with heat-attenuated yeast enhanced the secretion of IL-6, TNF-α, and IFN-γ, in addition to the activation of CD4+ and CD8+ T cells, in BALB/c mice. P. pastoris displaying ENV epitopes induced a more robust immune response, increasing immunoglobulin production, especially IgG isotypes. Both proposed vaccines showed the potential to induce immune responses without adverse effects, confirming the safety of administering P. pastoris as a vaccine vehicle. Here, we demonstrated, for the first time, the evaluation of a vaccine against ZIKV based on a multiepitope construct using yeast as a delivery system and reinforcing the applicability of P. pastoris as a whole-cell vaccine.

Biological activity of 1,2,3-triazole-2-amino-1,4-naphthoquinone derivatives and their evaluation as therapeutic strategy for malaria control.

Malaria can be caused by several Plasmodium species and the development of an effective vaccine is challenging. Currently, the most effective tool to control the disease is the administration of specific chemotherapy; however, resistance to the frontline antimalarials is one of the major problems in malaria control and thus the development of new drugs becomes urgent. The study presented here sought to evaluate the antimalarial activities of compounds derived from 2-amino-1,4-naphthoquinones containing 1,2,3-triazole using in vivo and in vitro models. 1H-1,2,3-Triazole 2-amino-1,4-naphthoquinone derivatives were synthesized and evaluated for antimalarial activity in vitro, using P. falciparum W2 chloroquine (CQ) resistant strain and in vivo using the murine-P. berghei ANKA strain. Acute toxicity was determined as established by the OECD (2001). Cytotoxicity was evaluated against HepG2 and Vero mammalian cell lines. Transmission electron microscopy of the Plasmodium falciparum trophozoite (early and late stages) was used to evaluate the action of compounds derived at ultra-structural level. The compounds displayed low cytotoxicity CC50 > 100 µM, neither did they cause hemolysis at the tested doses and nor the signs of toxicity in the in vivo acute toxicity test. Among the five compounds tested, one showed IC50 values in submicromolar range of 0.8 µM. Compounds 7, 8 and 11 showed IC50 values < 5 µM, and selectivity index (SI) ranging from 6.8 to 343 for HepG2, and from 13.7 to 494.8 for Vero cells. Compounds 8 and 11 were partially active against P. berghei induced parasitemia in vivo. Analysis of the ultrastructural changes associated with the treatment of these two compounds, showed trophozoites with completely degraded cytoplasm, loss of membrane integrity, organelles in the decomposition stage and possible food vacuole deterioration. Our results indicated that compounds 8 and 11 may be considered hit molecules for antimalarial drug discovery platform and deserve further optimization studies.

Uncovering neglected subtypes and zoonotic transmission of Hepatitis E virus (HEV) in Brazil.

Hepatitis E virus (HEV) circulation in humans and swine has been extensively studied in South America over the last two decades. Nevertheless, only 2.1% of reported HEV strains are available as complete genome sequences. Therefore, many clinical, epidemiological, and evolutionary aspects of circulating HEV in the continent still need to be clarified. Here, we conducted a retrospective evolutionary analysis of one human case and six swine HEV strains previously reported in northeastern, southern, and southeastern Brazil. We obtained two complete and four nearly complete genomic sequences. Evolutionary analysis comparing the whole genomic and capsid gene sequences revealed high genetic variability. This included the circulation of at least one unrecognized unique South American subtype. Our results corroborate that sequencing the whole capsid gene could be used as an alternative for HEV subtype assignment in the absence of complete genomic sequences. Moreover, our results substantiate the evidence for zoonotic transmission by comparing a larger genomic fragment recovered from the sample of the autochthonous human hepatitis E case. Further studies should continuously investigate HEV genetic diversity and zoonotic transmission of HEV in South America.

Clinical and laboratory diagnosis of monkeypox (mpox): Current status and future directions.

The emergence and rapid spread of the monkeypox virus (MPXV) to non-endemic countries has brought this once obscure pathogen to the forefront of global public health. Given the range of conditions that cause similar skin lesions, and because the clinical manifestation may often be atypical in the current mpox outbreak, it can be challenging to diagnose patients based on clinical signs and symptoms. With this perspective in mind, laboratory-based diagnosis assumes a critical role for the clinical management, along with the implementation of countermeasures. Here, we review the clinical features reported in mpox patients, the available laboratory tests for mpox diagnosis, and discuss the principles, advances, advantages, and drawbacks of each assay. We also highlight the diagnostic platforms with the potential to guide ongoing clinical response, particularly those that increase diagnostic capacity in low- and middle-income countries. With the outlook of this evolving research area, we hope to provide a resource to the community and inspire more research and the development of diagnostic alternatives with applications to this and future public health crises.

Recent insights into SARS-CoV-2 omicron variant.

The SARS-CoV-2 omicron variant (B.1.1.529) was first identified in Botswana and South Africa, and its emergence has been associated with a steep increase in the number of SARS-CoV-2 infections. The omicron variant has subsequently spread very rapidly across the world, resulting in the World Health Organization classification as a variant of concern on 26 November 2021. Since its emergence, great efforts have been made by research groups around the world that have rapidly responded to fill our gaps in knowledge for this novel variant. A growing body of data has demonstrated that the omicron variant shows high transmissibility, robust binding to human angiotensin-converting enzyme 2 receptor, attenuated viral replication, and causes less severe disease in COVID-19 patients. Further, the variant has high environmental stability, high resistance against most therapeutic antibodies, and partial escape neutralisation by antibodies from convalescent patients or vaccinated individuals. With the pandemic ongoing, there is a need for the distillation of literature from primary research into an accessible format for the community. In this review, we summarise the key discoveries related to the SARS-CoV-2 omicron variant, highlighting the gaps in knowledge that guide the field's ongoing and future work.

Roles of Bothrops jararacussu toxins I and II: Antiviral findings against Zika virus.

Zika virus is the etiologic agent of Zika fever, and has been previously associated with cases of microcephaly, drawing the attention of the health authorities worldwide. However, no vaccine or antiviral are currently available. Phospholipases A2 (PLA2) isolated from snake venoms have demonstrated antiviral activity against several viruses. Here we demonstrated the anti-ZIKV activity of bothropstoxins-I and II (BthTX-I and II) isolated from Bothrops jararacussu venom. Vero E6 cells were infected with ZIKVPE243 in the presence of compounds for 72 h, when virus titers were evaluated. BthTX-I and II presented strong dose-dependent inhibition of ZIKV, with a SI of 149.1 and 1.44 × 105, respectively. These toxins mainly inhibited the early stages of the replicative cycle, such as during the entry of ZIKV into host cells, as shown by the potent virucidal effect, suggesting the action of these toxins on the virus particles. Moreover, BthTX-I and II presented significant activity towards post-entry stages of the ZIKV replicative cycle. Molecular docking analyses showed that BthTX-I and II potentially interact with DII and DIII domains from ZIKV Envelope protein. Our findings show that these PLA2s could be used as useful templates for the development of future antiviral candidate drugs against Zika fever.

Identification of Novel Zika Virus Inhibitors: A Screening using Thiosemicarbazones and Thiazoles Templates.

BACKGROUND: Zika virus (ZIKV) remains an important cause of congenital infection, fetal microcephaly, and Guillain-Barré syndrome in the population. In 2016, WHO declared a cluster of microcephaly cases and other neurological disorders reported as a global public health emergency in Brazil. There is still no specific treatment for Zika virus fever, only palliative care. Therefore, there is a need for new therapies against this disease. According to the literature, thiosemicarbazone, phthalimide and thiazole are privileged structures with several biological activities, including antiviral activity against various viruses. OBJECTIVE: Based on this, this work presents an antiviral screening using previously synthesized compounds derived from thiosemicarbazone, phthalimide, and thiazole as new hits active against ZIKV. METHODS: After synthesis and characterization, all compounds were submitted to Cytotoxicity by MTT and Antiviral activity against ZIKV assays. RESULTS: Compounds 63, 64, 65, and 73 exhibited major reductions in the ZIKV title from this evaluation. Compounds 63 (99.74%), 64 (99.77%), 65 (99.92%), and 73 (99.21%) showed a higher inhibition than the standard 6MMPr (98.74%) at the CC20 dose. These results revealed new chemical entities with anti-ZIKV activity. CONCLUSION: These derivatives are promising candidates for further assays. In addition, the current approach brings a new privileged scaffolding, which may drive future drug discovery for ZIKV.

In vitro and in vivo models for monkeypox.

The emergence and rapid spread outside of monkeypox virus (MPXV) to non-endemic areas has led to another global health emergency in the midst of the COVID-19 pandemic. The scientific community has sought to rapidly develop in vitro and in vivo models that could be applied in research with MPXV. In vitro models include two-dimensional (2D) cultures of immortalized cell lines or primary cells and three-dimensional (3D) cultures. In vitro models are considered cost-effective and can be done in highly controlled conditions; however, they do not always resemble physiological conditions. In this way, several in vivo models are being characterized to meet the growing demand for new studies related to MPXV. In this review, we summarize the main MPXV models that have already been developed and discuss how they can contribute to advance the understanding of its pathogenesis, replication, and transmission, as well as identifying antivirals to treat infected patients.

Controlled release and antiviral activity of acyclovir-loaded PLA/PEG nanofibers produced by solution blow spinning.

Herpetic dermatitis and oral recurrent herpes (ORH) are among the most common human infections. Antiviral drugs such as acyclovir (ACV) are used in the standard treatment for ORH. Despite its therapeutic efficacy, ACV is continuously and repetitively administered in high doses. In this sense, the development of controlled release drug delivery systems such as core-shell fibers have a great potential in the treatment of ORH. In this work, poly(lactic acid)/poly(ethylene glycol) (PLA/PEG) fibers were produced by solution blow spinning (SBS) for the controlled release of ACV encapsulated in the core. PLA/PEG nanofibers containing four different blend ratios (100:0, 90:10, 80:20 and 70:30 wt%) without or with 10 wt% ACV were characterized by scanning electron microscopy (SEM), thermogravimetry (TG) and differential scanning calorimetry (DSC). The ACV release profile for 21 days was accessed by UV-Vis spectroscopy. Static water contact angles of the spun fiber mats were measured by the sessile drop method to evaluate fiber wettability upon contact with skin for transdermal release. Cytotoxicity and antiviral efficacy against Herpes simplex viruses (HSV-1) were evaluated using Vero cells. ACV addition did not impact on morphology, but slightly improved thermal stability of the fibers. Addition of hydrophilic PEG in PLA/PEG blends, however, increased drug release as confirmed by contact angle measurements and release profile. The in vitro tests showed the effectiveness of the drug delivery systems developed in reducing HSV-1 viral titer, which is related to the judicious combination of polymers used in the fibrous mats, in addition to not being cytotoxic to Vero cells. These results show the great potential of PLA/PEG solution blow-spun fibers in the controlled release of ACV to develop practical devices for the treatment of cold sores, while favoring the aesthetic appearance by covering them with a soft tissue patch (fibrous mats).

Two Years into the COVID-19 Pandemic: Lessons Learned.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible and virulent human-infecting coronavirus that emerged in late December 2019 in Wuhan, China, causing a respiratory disease called coronavirus disease 2019 (COVID-19), which has massively impacted global public health and caused widespread disruption to daily life. The crisis caused by COVID-19 has mobilized scientists and public health authorities across the world to rapidly improve our knowledge about this devastating disease, shedding light on its management and control, and spawned the development of new countermeasures. Here we provide an overview of the state of the art of knowledge gained in the last 2 years about the virus and COVID-19, including its origin and natural reservoir hosts, viral etiology, epidemiology, modes of transmission, clinical manifestations, pathophysiology, diagnosis, treatment, prevention, emerging variants, and vaccines, highlighting important differences from previously known highly pathogenic coronaviruses. We also discuss selected key discoveries from each topic and underline the gaps of knowledge for future investigations.

Design to Implementation Study for Development and Patient Validation of Paper-Based Toehold Switch Diagnostics.

Access to low-burden molecular diagnostics that can be deployed into the community for testing is increasingly important and has meaningful wider implications for the well-being of societies and economic stability. Recent years have seen several new isothermal diagnostic modalities emerge to meet the need for rapid, low-cost molecular diagnostics. We have contributed to this effort through the development and patient validation of toehold switch-based diagnostics, including diagnostics for the mosquito-borne Zika and chikungunya viruses, which provided performance comparable to gold-standard reverse transcription-quantitative polymerase chain reaction (RT-qPCR) based assays. These diagnostics are inexpensive to develop and manufacture, and they have the potential to provide diagnostic capacity to low-resource environments. Here the protocol provides all the steps necessary for the development of a switch-based assay for Zika virus detection. The article takes readers through the stepwise diagnostic development process. First, genomic sequences of Zika virus serve as inputs for the computational design of candidate switches using open-source software. Next, the assembly of the sensors for empirical screening with synthetic RNA sequences and optimization of diagnostic sensitivity is shown. Once complete, validation is performed with patient samples in parallel with RT-qPCR, and a purpose-built optical reader, PLUM. This work provides a technical roadmap to researchers for the development of low-cost toehold switch-based sensors for applications in human health, agriculture, and environmental monitoring.

Antiviral activity of ouabain against a Brazilian Zika virus strain.

Zika virus (ZIKV) is an emerging arbovirus associated with neurological disorders. Currently, no specific vaccines or antivirals are available to treat the ZIKV infection. Ouabain, a cardiotonic steroid known as Na+/K+-ATPase inhibitor, has been previously described as an immunomodulatory substance by our group. Here, we evaluated for the first time the antiviral activity of this promising substance against a Brazilian ZIKV strain. Vero cells were treated with different concentrations of ouabain before and after the infection with ZIKV. The antiviral effect was evaluated by the TCID50 method and RT-qPCR. Ouabain presented a dose-dependent inhibitory effect against ZIKV, mainly when added post infection. The reduction of infectious virus was accompanied by a decrease in ZIKV RNA levels, suggesting that the mechanism of ZIKV inhibition by ouabain occurred at the replication step. In addition, our in silico data demonstrated a conformational stability and favorable binding free energy of ouabain in the biding sites of the NS5-RdRp and NS3-helicase proteins, which could be related to its mechanism of action. Taken together, these data demonstrate the antiviral activity of ouabain against a Brazilian ZIKV strain and evidence the potential of cardiotonic steroids as promising antiviral agents.

Portable sample processing for molecular assays: application to Zika virus diagnostics.

This paper introduces a digital microfluidic (DMF) platform for portable, automated, and integrated Zika viral RNA extraction and amplification. The platform features reconfigurable DMF cartridges offering a closed, humidified environment for sample processing at elevated temperatures, as well as programmable control instrumentation with a novel thermal cycling unit regulated using a proportional integral derivative (PID) feedback loop. The system operates on 12 V DC power, which can be supplied by rechargeable battery packs for remote testing. The DMF system was optimized for an RNA processing pipeline consisting of the following steps: 1) magnetic-bead based RNA extraction from lysed plasma samples, 2) RNA clean-up, and 3) integrated, isothermal amplification of Zika RNA. The DMF pipeline was coupled to a paper-based, colorimetric cell-free protein expression assay for amplified Zika RNA mediated by toehold switch-based sensors. Blinded laboratory evaluation of Zika RNA spiked in human plasma yielded a sensitivity and specificity of 100% and 75% respectively. The platform was then transported to Recife, Brazil for evaluation with infectious Zika viruses, which were detected at the 100 PFU mL-1 level from a 5 µL sample (equivalent to an RT-qPCR cycle threshold value of 32.0), demonstrating its potential as a sample processing platform for miniaturized diagnostic testing.

ZIKV B-cell epitopes for immunodiagnostic tests.

The use of serological tests is valuable to diagnose Zika virus (ZIKV) infection and carry out epidemiological surveillance. However, ZIKV serological tests may result in false positives due to cross-reactivity between antibodies against other Flavivirus, especially dengue virus that worldwide disseminated. We used three online tools to predict amino acid sequences of B-cell epitopes. We selected and synthetized two epitopes that showed appropriate features in the molecular dynamic simulation and demonstrated to be suitable for serological assays.

Field validation of the performance of paper-based tests for the detection of the Zika and chikungunya viruses in serum samples.

In low-resource settings, resilience to infectious disease outbreaks can be hindered by limited access to diagnostic tests. Here we report the results of double-blinded studies of the performance of paper-based diagnostic tests for the Zika and chikungunya viruses in a field setting in Latin America. The tests involved a cell-free expression system relying on isothermal amplification and toehold-switch reactions, a purpose-built portable reader and onboard software for computer vision-enabled image analysis. In patients suspected of infection, the accuracies and sensitivities of the tests for the Zika and chikungunya viruses were, respectively, 98.5% (95% confidence interval, 96.2-99.6%, 268 serum samples) and 98.5% (95% confidence interval, 91.7-100%, 65 serum samples) and approximately 2 aM and 5 fM (both concentrations are within clinically relevant ranges). The analytical specificities and sensitivities of the tests for cultured samples of the viruses were equivalent to those of the real-time quantitative PCR. Cell-free synthetic biology tools and companion hardware can provide de-centralized, high-capacity and low-cost diagnostics for use in low-resource settings.