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.

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.

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.

[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.

Índices larvais de Aedes aegypti e incidência de dengue: um estudo ecológico no Estado do Rio de Janeiro, Brasil / Aedes aegypti larval indices and dengue incidence: an ecological study in the state of Rio de Janeiro, Brazil / Índices larvales de Aedes aegypti e incidencia de dengue: un estudio ecológico en el estado de Río de Janeiro, Brasil

Resumo: 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.

Abstract: 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.

Resumen: 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.

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.

Inhibition by Marine Algae of Chikungunya Virus Isolated From Patients in a Recent Disease Outbreak in Rio de Janeiro.

Chikungunya virus (CHIKV) infection is one of the most challenging re-emergent diseases caused by a virus, and with no specific antiviral treatment it has now become a major public health concern. In this investigation, 25 blood samples were collected from patients with characteristic CHIKV symptoms and submitted to a virus isolation protocol, which detected 3 CHIKV isolates. These samples were evaluated by sequencing for the characterization of the strains and any homology to viruses circulating in Brazil during a recent outbreak. These viruses were used for the development of antiviral assays. Subsequently, the inhibitory effects of seaweed extracts on CHIKV replication were studied. The marine species of algae tested were Bryothamnion triquetrum, Caulerpa racemosa, Laurencia dendroidea, Osmundaria obtusiloba, Ulva fasciata, and Kappaphycus alvarezii, all of which are found in different countries including Brazil. The results revealed high levels of CHIKV inhibition, including extracts of O. obtusiloba with inhibition values of 1.25 µg/mL and a selectivity index of 420. Viral inhibition was dependent on the time of addition of extract of O. obtusiloba to the infected cells, with the optimal inhibition occurring up to 16 h after infection. Neuron evaluations with O. obtusiloba were performed and demonstrated low toxicity, and in infected neurons we observed high inhibitory activity in a dose-dependent manner. These results indicate that the algal extracts may be promising novel candidates for the development of therapeutic agents against CHIKV infections.

Prostaglandin A1 triggers Mayaro virus inhibition and heat shock protein 70 expression in an epithelial cell model.

INTRODUCTION: The Mayaro virus (MAYV), which is an arbovirus closely related to the Chikungunya virus, causes a dengue-like acute illness that is endemic to Central and South America. We investigated the anti-MAYV activity of prostaglandin A1 (PGA1), a hormone which exhibits antiviral activity against both ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) viruses. Further, we examined the effects of inducting the stress protein HSP70 following PGA1 treatment. METHODS: Hep-2 cells infected with MAYV were treated with PGA1 (0.1-6µg/ml) 12h before infection and for different periods post-infection. Inhibition of viral replication inhibition was analyzed via viral titer determination, whereas the effect of PGA1 on viral morphogenesis was examined via transmission electron microscopy (TEM). Autoradiography (with 35S methionine labeling) and western blotting were used to assess the effect of PGA1 treatment on viral and cellular protein synthesis, and on HSP70 induction, respectively. RESULTS: PGA1 strongly reduced viral replication in Hep-2 cells, particularly when added during the early stages of viral replication. Although PGA1 treatment inhibited viral replication by 95% at 24 hours post-infection (hpi), viral structural protein synthesis was inhibited only by 15%. TEM analysis suggested that PGA1 inhibited replication before viral morphogenesis. Western blot and densitometry analyses showed that PGA1 treatment increased HSP70 protein levels, although this was not detectable via autoradiography. CONCLUSIONS: PGA1 inhibits MAYV replication in Hep-2 cells at early stages of viral replication, prior to production of viral structural proteins, possibly via HSP70 induction.

Prostaglandin A1 triggers Mayaro virus inhibition and heat shock protein 70 expression in an epithelial cell model

Abstract INTRODUCTION: The Mayaro virus (MAYV), which is an arbovirus closely related to the Chikungunya virus, causes a dengue-like acute illness that is endemic to Central and South America. We investigated the anti-MAYV activity of prostaglandin A1 (PGA1), a hormone which exhibits antiviral activity against both ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) viruses. Further, we examined the effects of inducting the stress protein HSP70 following PGA1 treatment. METHODS: Hep-2 cells infected with MAYV were treated with PGA1 (0.1-6μg/ml) 12h before infection and for different periods post-infection. Inhibition of viral replication inhibition was analyzed via viral titer determination, whereas the effect of PGA1 on viral morphogenesis was examined via transmission electron microscopy (TEM). Autoradiography (with 35S methionine labeling) and western blotting were used to assess the effect of PGA1 treatment on viral and cellular protein synthesis, and on HSP70 induction, respectively. RESULTS: PGA1 strongly reduced viral replication in Hep-2 cells, particularly when added during the early stages of viral replication. Although PGA1 treatment inhibited viral replication by 95% at 24 hours post-infection (hpi), viral structural protein synthesis was inhibited only by 15%. TEM analysis suggested that PGA1 inhibited replication before viral morphogenesis. Western blot and densitometry analyses showed that PGA1 treatment increased HSP70 protein levels, although this was not detectable via autoradiography. CONCLUSIONS: PGA1 inhibits MAYV replication in Hep-2 cells at early stages of viral replication, prior to production of viral structural proteins, possibly via HSP70 induction.

History, epidemiology and diagnostics of dengue in the American and Brazilian contexts: a review.

Dengue virus (DENV), an arbovirus transmitted by mosquitoes, has become a major threat to American human life, reaching approximately 23 million cases from 1980 to 2017. Brazil is among the countries most affected by this terrible viral disease, with 13.6 million cases. DENV has four different serotypes, DENV1-4, which show a broad clinical spectrum. Dengue creates a staggering epidemiological and economic burden for endemic countries. Without a specific therapy and with a commercial vaccine that presents some problems relative to its full effectiveness, initiatives to improve vector control strategies, early disease diagnostics and the development of vaccines and antiviral drugs are priorities. In this study, we present the probable origins of dengue in America and the trajectories of its spread. Overall, dengue diagnostics are costly, making the monitoring of dengue epidemiology more difficult and affecting physicians' therapeutic decisions regarding dengue patients, especially in developing countries. This review also highlights some recent and important findings regarding dengue in Brazil and the Americas. We also summarize the existing DENV polymerase chain reaction (PCR) diagnostic tests to provide an improved reference since these tests are useful and accurate at discriminating DENV from other flaviviruses that co-circulate in the Americas. Additionally, these DENV PCR assays ensure virus serotyping, enabling epidemiologic monitoring.

Detection and characterization of a novel rhabdovirus in Aedes cantans mosquitoes and evidence for a mosquito-associated new genus in the family Rhabdoviridae.

Thanks to recent advances in random amplification technologies, metagenomic surveillance expanded the number of novel, often unclassified viruses within the family Rhabdoviridae. Using a vector-enabled metagenomic (VEM) tool, we identified a novel rhabdovirus in Aedes cantans mosquitoes collected from Germany provisionally named Ohlsdorf virus (OHSDV). The OHSDV genome encodes the canonical rhabdovirus structural proteins (N, P, M, G and L) with alternative ORF in the P gene. Sequence analysis indicated that OHSDV exhibits a similar genome organization and characteristics compared to other mosquito-associated rhabdoviruses (Riverside virus, Tongilchon virus and North Creek virus). Complete L protein based phylogeny revealed that all four viruses share a common ancestor and form a deeply rooted and divergent monophyletic group within the dimarhabdovirus supergroup and define a new genus, tentatively named Ohlsdorfvirus. Although the Ohlsdorfvirus clade is basal within the dimarhabdovirus supergroup phylogeny that includes genera of arthropod-borne rhabdoviruses, it remains unknown if viruses in the proposed new genus are vector-borne pathogens. The observed spatiotemporal distribution in mosquitoes suggests that members of the proposed genus Ohlsdorfvirus are geographically restricted/separated. These findings increase the current knowledge of the genetic diversity, classification and evolution of this virus family. Further studies are needed to determine the host range, transmission route and the evolutionary relationships of these mosquito-associated viruses with those infecting vertebrates.

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.

Charge neutralization as the major factor for the assembly of nucleocapsid-like particles from C-terminal truncated hepatitis C virus core protein.

BACKGROUND: Hepatitis C virus (HCV) core protein, in addition to its structural role to form the nucleocapsid assembly, plays a critical role in HCV pathogenesis by interfering in several cellular processes, including microRNA and mRNA homeostasis. The C-terminal truncated HCV core protein (C124) is intrinsically unstructured in solution and is able to interact with unspecific nucleic acids, in the micromolar range, and to assemble into nucleocapsid-like particles (NLPs) in vitro. The specificity and propensity of C124 to the assembly and its implications on HCV pathogenesis are not well understood. METHODS: Spectroscopic techniques, transmission electron microscopy and calorimetry were used to better understand the propensity of C124 to fold or to multimerize into NLPs when subjected to different conditions or in the presence of unspecific nucleic acids of equivalent size to cellular microRNAs. RESULTS: The structural analysis indicated that C124 has low propensity to self-folding. On the other hand, for the first time, we show that C124, in the absence of nucleic acids, multimerizes into empty NLPs when subjected to a pH close to its isoelectric point (pH ≈ 12), indicating that assembly is mainly driven by charge neutralization. Isothermal calorimetry data showed that the assembly of NLPs promoted by nucleic acids is enthalpy driven. Additionally, data obtained from fluorescence correlation spectroscopy show that C124, in nanomolar range, was able to interact and to sequester a large number of short unspecific nucleic acids into NLPs. DISCUSSION: Together, our data showed that the charge neutralization is the major factor for the nucleocapsid-like particles assembly from C-terminal truncated HCV core protein. This finding suggests that HCV core protein may physically interact with unspecific cellular polyanions, which may correspond to microRNAs and mRNAs in a host cell infected by HCV, triggering their confinement into infectious particles.

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.

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.