Early Evidence for Zika Virus Circulation among Aedes aegypti Mosquitoes, Rio de Janeiro, Brazil.

During 2014-2016, we conducted mosquito-based Zika virus surveillance in Rio de Janeiro, Brazil. Results suggest that Zika virus was probably introduced into the area during May-November 2013 via multiple in-country sources. Furthermore, our results strengthen the hypothesis that Zika virus in the Americas originated in Brazil during October 2012-May 2013.

Thieno[2,3-b]pyridine derivatives: a new class of antiviral drugs against Mayaro virus.

Mayaro virus (MAYV) is an arthropod-borne virus and a member of the family Togaviridae, genus Alphavirus. Its infection leads to an acute illness accompanied by long-lasting arthralgia. To date, there are no antiviral drugs or vaccines against infection with MAYV and resources for the prevention or treatment of other alphaviruses are very limited. MAYV has served as a model to study the antiviral potential of several substances on alphavirus replication. In this work we evaluated the antiviral effect of seven new derivatives of thieno[2,3-b]pyridine against MAYV replication in a mammalian cell line. All derivatives were able to reduce viral production effectively at concentrations that were non-toxic for Vero cells. Molecular modeling assays predicted low toxicity risk and good oral bioavailability of the substances in humans. One of the molecules, selected for further study, demonstrated a strong anti-MAYV effect at early stages of replication, as it protected pre-treated cells and also during the late stages, affecting virus morphogenesis. This study is the first to demonstrate the antiviral effect of thienopyridine derivatives on MAYV replication in vitro, suggesting the potential application of these substances as antiviral molecules against alphaviruses. Additional in vivo research will be needed to expand the putative therapeutic applications.

Cytokine kinetics of Zika virus-infected patients from acute to reconvalescent phase.

Zika virus is an emerging mosquito-borne flavivirus currently causing large epidemics in the Pacific Ocean region and Brazil. Clinically, Zika fever resembles dengue fever, but is less severe. Whereas the clinical syndrome and laboratory diagnostic procedures have been described, little attention was paid to the immunology of the disease and its possible use for clinical follow-up of patients. Here, we investigate the role of cytokines in the pathogenesis of Zika fever in travelers returning from Asia, the Pacific, and Brazil. Polyfunctional T cell activation (Th1, Th2, Th9, and Th17 response) was seen during the acute phase characterized by respective cytokine level increases, followed by a decrease in the reconvalescent phase.

Extracellular peptidases from Deinococcus radiodurans.

The extremophile Deinococcus radiodurans wild type R1 produces peptidases (metallo- and serine-) in TGY medium and in the media supplemented with human hair (HMY) and chicken feathers (FMY). Enzymatic screening on agar plates revealed peptidase activity. In TGY medium metallopeptidases were detected corresponding to a molecular mass range of 300-85 kDa (gelatinases); 280-130 (caseinases) and a 300 and a 170 kDa (keratinases); and a gelatinolytic serine peptidase (75 kDa). In HMY medium after 144 h, D. radiodurans produced keratinase (290 U/ml), gelatinase (619 U/ml) and sulfite (26 µg/ml). TGY medium produced higher proteolytic activity: 950 U/ml of gelatinolytic (24 h); 470 U/ml of keratinolytic (24 h) and 110 U/ml of caseinolytic (72 h). In the FMY medium, we found gelatinolytic (317 U/ml), keratinolytic (43 U/ml) and caseinolytic (85 U/ml) activities. The sulfite had a maximum release at 48 h (8.1 µg/ml). Enzymography analysis revealed that the keratinases degraded keratin after 24 h of reaction. The addition of sodium sulfite (1.0 %) improved the keratin degradation. Environmental Scanning Electron microscopy revealed alterations such as damage and holes in the hair fiber cuticle after D. radiodurans growth. This work presents for the first time D. radiodurans as a new keratinolytic microorganism.

Marine extremophiles: a source of hydrolases for biotechnological applications.

The marine environment covers almost three quarters of the planet and is where evolution took its first steps. Extremophile microorganisms are found in several extreme marine environments, such as hydrothermal vents, hot springs, salty lakes and deep-sea floors. The ability of these microorganisms to support extremes of temperature, salinity and pressure demonstrates their great potential for biotechnological processes. Hydrolases including amylases, cellulases, peptidases and lipases from hyperthermophiles, psychrophiles, halophiles and piezophiles have been investigated for these reasons. Extremozymes are adapted to work in harsh physical-chemical conditions and their use in various industrial applications such as the biofuel, pharmaceutical, fine chemicals and food industries has increased. The understanding of the specific factors that confer the ability to withstand extreme habitats on such enzymes has become a priority for their biotechnological use. The most studied marine extremophiles are prokaryotes and in this review, we present the most studied archaea and bacteria extremophiles and their hydrolases, and discuss their use for industrial applications.

Alphavirus genome delivery occurs directly at the plasma membrane in a time- and temperature-dependent process.

It is widely held that arboviruses such as the alphavirus Sindbis virus gain entry into cells by a process of receptor-mediated endocytosis followed by membrane fusion in the acid environment of the endosome. We have used an approach of direct observation of Sindbis virus entry into cells by electron microscopy and immunolabeling of virus proteins with antibodies conjugated to gold beads. We found that upon attaching to the cell surface, intact RNA-containing viruses became empty shells that could be identified only by antibody labeling. We found that the rate at which full particles were converted to empty particles increased with time and temperature. We found that this entry event takes place at temperatures that inhibit both endosome formation and membrane fusion. We conclude that entry of alphaviruses is by direct penetration of cell plasma membranes through a pore structure formed by virus and, possibly, host proteins.

APOL1-mediated monovalent cation transport contributes to APOL1-mediated podocytopathy in kidney disease.

Two coding variants of apolipoprotein L1 (APOL1), called G1 and G2, explain much of the excess risk of kidney disease in African Americans. While various cytotoxic phenotypes have been reported in experimental models, the proximal mechanism by which G1 and G2 cause kidney disease is poorly understood. Here, we leveraged 3 experimental models and a recently reported small molecule blocker of APOL1 protein, VX-147, to identify the upstream mechanism of G1-induced cytotoxicity. In HEK293 cells, we demonstrated that G1-mediated Na+ import/K+ efflux triggered activation of GPCR/IP3-mediated calcium release from the ER, impaired mitochondrial ATP production, and impaired translation, which were all reversed by VX-147. In human urine-derived podocyte-like epithelial cells (HUPECs), we demonstrated that G1 caused cytotoxicity that was again reversible by VX-147. Finally, in podocytes isolated from APOL1 G1 transgenic mice, we showed that IFN-γ-mediated induction of G1 caused K+ efflux, activation of GPCR/IP3 signaling, and inhibition of translation, podocyte injury, and proteinuria, all reversed by VX-147. Together, these results establish APOL1-mediated Na+/K+ transport as the proximal driver of APOL1-mediated kidney disease.

Espirito Santo virus: a new birnavirus that replicates in insect cells.

Espirito Santo virus (ESV) is a newly discovered virus recovered as contamination in a sample of a virulent strain of dengue-2 virus (strain 44/2), which was recovered from a patient in the state of Espirito Santo, Brazil, and amplified in insect cells. ESV was found to be dependent upon coinfection with a virulent strain of dengue-2 virus and to replicate in C6/36 insect cells but not in mammalian Vero cells. A sequence of the genome has been produced by de novo assembly and was not found to match to any known viral sequence. An incomplete match to the nucleotide sequence of the RNA-dependent RNA polymerase from Drosophila X virus (DXV), another birnavirus, could be detected. Mass spectrometry analysis of ESV proteins found no matches in the protein data banks. However, peptides recovered by mass spectrometry corresponded to the de novo-assembled sequence by BLAST analysis. The composition and three-dimensional structure of ESV are presented, and its sequence is compared to those of other members of the birnavirus family. Although the virus was found to belong to the family Birnaviridae, biochemical and sequence information for ESV differed from that of DXV, the representative species of the genus Entomobirnavirus. Thus, significant differences underscore the uniqueness of this infectious agent, and its relationship to the coinfecting virus is discussed.

Prophage induction in lysogenic Aggregatibacter actinomycetemcomitans cells co-cultured with human gingival fibroblasts, and its effect on leukotoxin release.

Lysogeny is common among strains of the periodontal pathogen Aggregatibacter actinomycetemcomitans. Since lysogenic induction is known to result in the increased synthesis and release of bacterial toxins from lysogens, it would be important to elucidate the conditions under which induction of these bacteria may occur. Co-cultures of A. actinomycetemcomitans strains (either lysogenic or non-lysogenic) and human cells (either gingival fibroblasts or pharyngeal epithelial cells) were prepared. Following incubation, bacteriophage titers of up to 6.2 × 10(7) pfu/ml were detected in the cell-free, spent culture media from the co-cultures of the lysogenic A. actinomycetemcomitans strains and the fibroblasts. Little (maximum of 2 × 10(0) pfu/ml) or no titers of phage could be detected in the mono-cultures of the lysogenic A. actinomycetemcomitans strains alone. In contrast, no phage were detectable in the cell-free spent culture media of the lysogens cocultured with the epithelial cells. Futhermore, co-culture of the A. actinomycetemcomitans lysogens with the fibroblasts resulted in enhanced release of the A. actinomycetemcomitans leukotoxin into the culture medium, in comparison with the spent culture media from mono-cultures of the lysogens alone. These results are consistent with the concept that interaction with fibroblasts may mediate prophage induction in lysogenic strains of A. actinomycetemcomitans, and that leukotoxin release is greatly augmented following induction of the lysogens.

Tizoxanide Antiviral Activity on Dengue Virus Replication.

Dengue virus is an important circulating arbovirus in Brazil responsible for high morbidity and mortality worldwide, representing a huge economic and social burden, in addition to affecting public health. In this study, the biological activity, toxicity, and antiviral activity against dengue virus type 2 (DENV-2) of tizoxanide (TIZ) was evaluated in Vero cell culture. TIZ has a broad spectrum of action in inhibiting different pathogens, including bacteria, protozoa, and viruses. Cells were infected for 1 h with DENV-2 and then treated for 24 h with different concentrations of the drug. The quantification of viral production indicated the antiviral activity of TIZ. The protein profiles in infected Vero cells treated and not treated with TIZ were analyzed using the label-free quantitative proteomic approach. TIZ was able to inhibit virus replication mainly intracellularly after DENV-2 penetration and before the complete replication of the viral genome. Additionally, the study of the protein profile of infected not-treated and infected-treated Vero cells showed that TIZ interferes with cellular processes such as intracellular trafficking and vesicle-mediated transport and post-translational modifications when added after infection. Our results also point to the activation of immune response genes that would eventually lead to a decrease of DENV-2 production. TIZ is a promising therapeutic molecule for the treatment of DENV-2 infections.

Envelope lipid-packing as a critical factor for the biological activity and stability of alphavirus particles isolated from mammalian and mosquito cells.

Alphaviruses are enveloped arboviruses. The viral envelope is derived from the host cell and is positioned between two icosahedral protein shells (T = 4). Because the viral envelope contains glycoproteins involved in cell recognition and entry, the integrity of the envelope is critical for the success of the early events of infection. Differing levels of cholesterol in different hosts leads to the production of alphaviruses with distinct levels of this sterol loaded in the envelope. Using Mayaro virus, a New World alphavirus, we investigated the role of cholesterol on the envelope of alphavirus particles assembled in either mammalian or mosquito cells. Our results show that although quite different in their cholesterol content, Mayaro virus particles obtained from both cells share a similar high level of lateral organization in their envelopes. This organization, as well as viral stability and infectivity, is severely compromised when cholesterol is depleted from the envelope of virus particles isolated from mammalian cells, but virus particles isolated from mosquito cells are relatively unaffected by cholesterol depletion. We suggest that it is not cholesterol itself, but rather the organization of the viral envelope, that is critical for the biological activity of alphaviruses.

Antibacterial activity and cytotoxicity analysis of halistanol trisulphate from marine sponge Petromica citrina.

OBJECTIVES: An aqueous extract and fraction from the marine sponge Petromica citrina have antibacterial activity. We performed a chemical and biological characterization of the antibiotic substance from P. citrina and investigated its mode of action on Staphylococcus aureus cells. METHODS: The inhibitory activity of the aqueous extract of P. citrina was determined against 14 bacteria belonging to type strains and clinical antibiotic-resistant strains. The aqueous extract was fractionated under bioassay guidance and the bioactive substance was identified by its (1)H-NMR, (13)C-NMR and mass spectra. The MIC and the MBC of this substance were determined. This substance was also subjected to cytotoxic bioassays. The mode of action on S. aureus cells was investigated by light and transmission electron microscopy analysis. RESULTS: P. citrina showed a large spectrum of activity against type strains and resistant-bacteria such as S. aureus, Staphylococcus epidermidis, Enterococcus faecalis, Mycobacterium fortuitum and Neisseria gonorrhoeae. The aqueous extract was fractionated and halistanol trisulphate (24ε,25-dimethylcholestane-2ß,3α,6α-triol trisodium sulphate) was isolated for the first time from P. citrina. Halistanol trisulphate had a bactericidal effect on exponentially growing S. aureus cells at the MIC (512 mg/L). Cytotoxicity biossays showed moderate toxicity against cancer cell line L929 (fibrosarcoma). This substance apparently acts by damaging the cell membrane, with subsequent cell lysis. CONCLUSIONS: Halistanol trisulphate is a broad-spectrum antibiotic isolated from P. citrina with a mode of action involving disruption of the cytoplasmic membrane. It is a new candidate for research on antibacterial substances.

Entomological Surveillance of Aedes Mosquitoes: Comparison of Different Collection Methods in an Endemic Area in RIO de Janeiro, Brazil.

Using collection methods for Aedes adults as surveillance tools provides reliable indices and arbovirus detection possibilities. This study compared the effectiveness of different methods for collecting Ae. aegypti and Ae. albopictus and detecting arboviruses circulating in field-caught female specimens. Collection sites were defined in urban, peri-urban, and rural landscapes in two Brazilian cities. Collections were performed using Adultraps (ADT), BG-Sentinel (BGS), CDC-like traps (CDC), and indoor (ASP-I) and outdoor (ASP-O) aspiration during the rainy and dry seasons of 2015 and 2016. Generalized linear mixed models were used to model the effectiveness of each collection method. A total of 434 Ae. aegypti and 393 Ae. albopictus were collected. In total, 64 Ae. aegypti and sixteen Ae. albopictus female pools were tested for DENV, CHIKV, ZIKV, or YFV; none were positive. Positivity and density were linear at low densities (<1 specimen); thereafter, the relationship became non-linear. For Ae. aegypti, ADT and CDC were less effective, and ASP-I and ASP-O were as effective as BGS. For Ae. albopictus, all collection methods were less effective than BGS. This study highlights the need for an integrated surveillance method as an effective tool for monitoring Aedes vectors.

Genomic surveillance of SARS-CoV-2 Spike gene by sanger sequencing.

The SARS-CoV-2 responsible for the ongoing COVID pandemic reveals particular evolutionary dynamics and an extensive polymorphism, mainly in Spike gene. Monitoring the S gene mutations is crucial for successful controlling measures and detecting variants that can evade vaccine immunity. Even after the costs reduction resulting from the pandemic, the new generation sequencing methodologies remain unavailable to a large number of scientific groups. Therefore, to support the urgent surveillance of SARS-CoV-2 S gene, this work describes a new feasible protocol for complete nucleotide sequencing of the S gene using the Sanger technique. Such a methodology could be easily adopted by any laboratory with experience in sequencing, adding to effective surveillance of SARS-CoV-2 spreading and evolution.

[Aedes aegypti larval indices and dengue incidence: an ecological study in the state of Rio de Janeiro, Brazil]. / Índices larvais de Aedes aegypti e incidência de dengue: um estudo ecológico no Estado do Rio de Janeiro, Brasil.

The simultaneous circulation of dengue, Zika, and chikungunya poses major challenges for Brazil. Due to climate changes and other associated factors, more than two billion people in the world may be exposed to these arbovirus infections, according to the World Health Organization. The principal strategy for Aedes aegypti control programs is based on the Infestation Index Rapid Survey for Ae. aegypti (LIRAa), a sample survey in which the Building Infestation Index (BII) is used to prioritize areas for intervention. This study analyzed the performance of LIRAa in terms of its sensitivity for predicting dengue epidemics in municipalities in the state of Rio de Janeiro in epidemic years. Incidence rates per municipality for the years 2011, 2012, 2013, 2015, and 2016, plus the BII in October of the previous years. Scatterplots were created, aimed at an exploratory analysis and graphic visualizations of the relationship between the above-mentioned variables, as well as analyses of the Spearman correlation between the BII and the Breteau Index for each year, aimed at estimating the quality of the LIRAa. Comparative analysis of the values for the BII and the respective incidence rates in the period only indicated significant correlation between these variables in 2011/2012 (rs = 0.479; p < 0.01). There was also a correlation between BII and Breteau Index. It is urgent to rethink the parameters established by the LIRAa methodology and invest in alternative methodologies in entomological and epidemiological surveillance that reliably measure transmission risk in the territory and thus design more effective strategies to control these arbovirus infections.

A circulação simultânea da dengue, Zika e chikungunya impõe desafios importantes para o Brasil, que em decorrência das mudanças climáticas e outros fatores associados, estas arboviroses podem expor mais de 2 bilhões de pessoas no mundo, segundo a Organização Mundial da Saúde. A principal estratégia dos programas de controle do Aedes aegypti baseia-se no Levantamento de Índice Rápido para o Ae. aegypti (LIRAa), um inquérito amostral no qual o Índice de Infestação Predial (IIP) obtido é utilizado para priorizar áreas de intervenção. Neste estudo, analisou-se o desempenho do LIRAa quanto à sua sensibilidade na previsão de epidemias de dengue em municípios do Estado do Rio de Janeiro, em anos considerados epidêmicos. Foram obtidas as taxas de incidência (TI) por município nos anos de 2011, 2012, 2013, 2015 e 2016, e os IIP de outubro dos anos anteriores. Foram elaborados diagramas de dispersão, visando à análise exploratória e à visualização gráfica da relação entre as referidas variáveis, assim como análises de correlação de Spearman entre o IIP e o Índice de Breteau (IB) de cada ano, a fim de estimar a qualidade do LIRAa. A análise comparativa dos valores dos IIP e as respectivas TI no período indicou correlação significativa entre estas variáveis apenas em 2011/2012 (rs = 0,479; p < 0,01). Adicionalmente, foi observada correlação entre os IIP e IB. É urgente repensar os parâmetros estabelecidos pela metodologia LIRAa e investir em metodologias alternativas de vigilância entomoepidemiológica, que mensurem de forma confiável o risco de transmissão no território e assim delinear estratégias mais efetivas para o controle dessas arboviroses.

La circulación simultánea del dengue, Zika y chikungunya impone desafíos importantes para Brasil, que, a consecuencia del cambio climático y otros factores asociados, pueden estar expuestas a estas arbovirosis más de 2 billones de personas en el mundo, según la Organización Mundial de la Salud. La principal estrategia de los programas de control del Aedes aegypti se basa en el Levantamento de Índice Rápido para el Ae. aegypti (LIRAa), una encuesta de muestreo en la que el Índice de Infestación de Edificios (IIP) obtenido es utilizado para priorizar áreas de intervención. En este estudio se analizó el desempeño del LIRAa, en cuanto a su sensibilidad en la previsión de epidemias de dengue en municipios del estado de Río de Janeiro, durante años considerados epidémicos. Se obtuvieron tasas de incidencia (TI) por municipio de los años de 2011, 2012, 2013, 2015 y 2016, y los IIP de octubre de años anteriores. Se elaboraron diagramas de dispersión, visando el análisis exploratorio y visualización gráfica de la relación entre las referidas variables, así como análisis de correlación de Spearman entre el IIP y el Índice de Breteau (IB) de cada año, con el objetivo estimar la calidad del LIRAa. El análisis comparativo de los valores de los IIP y las respectivas TI en el período indicó correlación significativa entre esas variables solamente en 2011/2012 (rs = 0,479; p < 0,01). Asimismo, se observó correlación entre los IIP e IB. Es urgente repensar los parámetros establecidos por la metodología LIRAa, e invertir en metodologías alternativas de vigilancia entomoepidemiológica, que midan de forma confiable el riesgo de transmisión en el territorio y así delinear estrategias más efectivas para el control de esas arbovirosis.

Inactivation of avian influenza viruses by hydrostatic pressure as a potential vaccine development approach.

Vaccines are a recommended strategy for controlling influenza A infections in humans and animals. Here, we describe the effects of hydrostatic pressure on the structure, morphology and functional characteristics of avian influenza A H3N8 virus. The effect of hydrostatic pressure for 3 h on H3N8 virus revealed that the particles were resistant to this condition, and the virus displayed only a discrete conformational change. We found that pressure of 3 kbar applied for 6 h was able to inhibit haemagglutination and infectivity while virus replication was no longer observed, suggesting that full virus inactivation occurred at this point. However, the neuraminidase activity was not affected at this approach suggesting the maintenance of neutralizing antibody epitopes in this key antigen. Our data bring important information for the area of structural virology of enveloped particles and support the idea of applying pressure-induced inactivation as a tool for vaccine production.

Virucidal and antiviral activities of pomegranate (Punica granatum) extract against the mosquito-borne Mayaro virus.

BACKGROUND: The arthropod-borne Mayaro virus (MAYV) causes "Mayaro fever," a disease of medical significance, primarily affecting individuals in permanent contact with forested areas in tropical South America. Recently, MAYV has attracted attention due to its likely urbanization. There are currently no licensed drugs against most mosquito-transmitted viruses. Punica granatum (pomegranate) fruits cultivated in Brazil have been subjected to phytochemical investigation for the identification and isolation of antiviral compounds. In the present study, we explored the antiviral activity of pomegranate extracts in Vero cells infected with Mayaro virus. METHODS: The ethanol extract and punicalagin of pomegranate were extracted solely from the shell and purified by chromatographic fractionation, and were chemically identified using spectroscopic techniques. The cytotoxicity of the purified compounds was measured by the dye uptake assay, while their antiviral activity was evaluated by a virus yield inhibition assay. RESULTS: Pomegranate ethanol extract (CC50 = 588.9, IC50 = 12.3) and a fraction containing punicalagin as major compound (CC50 = 441.5, IC50 = 28.2) were shown to have antiviral activity (SI 49 and 16, respectively) against Mayaro virus, an alphavirus. Immunofluorescence analysis showed the virucidal effect of pomegranate extract, and transmission electron microscopy (TEM) revealed damage in viral particles treated with this extract. CONCLUSIONS: The P. granatum extract is a promising source of antiviral compounds against the alphavirus MAYV and represents an excellent candidate for future studies with other enveloped RNA viruses.

Sulphate-reducing bacterial community structure from produced water of the Periquito and Galo de Campina onshore oilfields in Brazil.

Sulphate-reducing bacteria (SRB) cause fouling, souring, corrosion and produce H2S during oil and gas production. Produced water obtained from Periquito (PQO) and Galo de Campina (GC) onshore oilfields in Brazil was investigated for SRB. Produced water with Postgate B, Postgate C and Baars media was incubated anaerobically for 20 days. DNA was extracted, 16S rDNA PCR amplified and fragments were sequenced using Illumina TruSeq. 4.2 million sequence reads were analysed and deposited at NCBI SAR accession number SRP149784. No significant differences in microbial community composition could be attributed to the different media but significant differences in the SRB were observed between the two oil fields. The dominant bacterial orders detected from both oilfields were Desulfovibrionales, Pseudomonadales and Enterobacteriales. The genus Pseudomonas was found predominantly in the GC oilfield and Pleomorphominas and Shewanella were features of the PQO oilfield. 11% and 7.6% of the sequences at GC and PQO were not classified at the genus level but could be partially identified at the order level. Relative abundances changed for Desulfovibrio from 29.8% at PQO to 16.1% at GC. Clostridium varied from 2.8% at PQO and 2.4% at GC. These data provide the first description of SRB from onshore produced water in Brazil and reinforce the importance of Desulfovibrionales, Pseudomonadales, and Enterobacteriales in produced water globally. Identifying potentially harmful microbes is an important first step in developing microbial solutions that prevent their proliferation.

Chemical composition and anti-Mayaro virus activity of Schinus terebinthifolius fruits.

Brazilian traditional medicine has explored the antiviral properties of many plant extracts, including those from the Brazilian pepper tree, Schinus terebinthifolius. In the present study, we investigated the chemical composition and anti-mayaro virus (MAYV) activity of S. terebinthifolius fruit. Extensive virucidal activity (more than 95%) was detected for the ethyl acetate extract and the isolated biflavonoids. From the ethyl acetate extract of Schinus terebinthifolius fruits, two bioflavonoids were isolated ((2S, 2″S)-2,3,2″,3″-tetrahydroamentoflavone and agathisflavone), which showed strong virucidal activity against Mayaro virus. Furthermore, several other compounds like terpenes and phenolics were identified by hyphenated techniques (GC-MS, LC-MS and HPLC-UV), as well as by mass spectrometry. Immunofluorescence assay confirmed antiviral activity and transmission electron microscopy revealed damage in viral particles treated with biflavonoids. The data suggest the direct action of the extract and the biflavonoids on the virus particles. The biflavonoids tetrahydroamentoflavone and agathisflavone had strong virucidal activity and reduced MAYV infection. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13337-021-00698-z.

In vitro Studies on The Inhibition of Replication of Zika and Chikungunya Viruses by Dolastane Isolated from Seaweed Canistrocarpus cervicornis.

The lack of vaccines and antiviral treatment, along with the increasing number of cases of Zika virus (ZIKV) and Chikungunya virus (CHIKV) infections, emphasize the need for searching for new therapeutic strategies. In this context, the marine brown seaweed Canistrocarpus cervicornis has been proved to hold great antiviral potential. Hence, the aim of this work was to evaluate the anti-ZIKV and anti-CHIKV activity of a marine dolastane isolated from brown seaweed C. cervicornis and its crude extract. Vero cells were used in antiviral assays, submitted to ZIKV and CHIKV, and treated with different concentrations of C. cervicornis extract or dolastane. The crude extract of C. cervicornis showed inhibitory activities for both ZIKV and CHIKV, with EC50 values of 3.3 µg/mL and 3.1 µg/mL, respectively. However, the isolated dolastane showed a more significant and promising inhibitory effect (EC50 = 0.95 µM for ZIKV and 1.3 µM for CHIKV) when compared to both the crude extract and ribavirin, which was used as control. Also, the dolastane showed a very potent virucidal activity against CHIKV and was able to inhibit around 90% of the virus infectivity at 10 µM. For the ZIKV, the effects were somewhat lower, although interesting, at approximately 64% in this same concentration. Further, we observed that both the extract and the dolastane were able to inhibit the replication of ZIKV and CHIKV at different times of addition post-infection, remaining efficient even if added after 8 hours post-infection, but declining soon after. A synergistic effect using sub-doses of the extract and isolates was associated with ribavirin, inhibiting above 80% replication even at the lowest concentrations. Therefore, this work has unveiled the anti-ZIKV and CHIKV potential of C. cervicornis crude extract and an isolated dolastane, which, in turn, can be used as a preventive or therapeutic strategy in the future.