Expression of recombinant dengue virus type 1 non-structural protein 1 in mammalian cells and preliminary assessment of its suitability to detect human IgG antibodies elicited by viral infection.

In recent years dengue has become a rapidly growing public health problem worldwide, however, the availability of accurate and affordable diagnostic immunoassays is limited, partly due to the difficulty of producing large quantities of purified antigen. Non-structural protein 1 (NS1) has shown to be a good candidate for inclusion in diagnostic assays and for serosurveys, particularly in endemic countries as a prerequisite for vaccination. In this work the NS1 antigen derived from dengue virus type-1 (DENV1) was expressed in HEK293-T cells and purified by affinity chromatography. The recombinant protein was recovered properly folded as dimers, highly purified and with good yield (1.5 mg/L). It was applied as a serological probe in an indirect ELISA developed in this work to detect human IgG antibodies. Preliminary comparative performance values of 81.1% sensitivity and 83.0% specificity of the developed and preliminary validated iELISA, relative to a commercial kit were obtained, suggesting that the purified recombinant DENV1 NS1 antigen is suitable to detect IgG antibodies, indicative of past DENV infection.

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.

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.

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.

Microscopy analysis of Zika virus morphogenesis in mammalian cells.

Zika virus (ZIKV) is an arbovirus that recently emerged in the Americas as an important pathogen mainly because of its expanded pathogenesis, and elevated tropism for neuronal cells, transposition across the placental barrier, and replication in reproductive tract cells. Thus, transmission modes are eventually independent of an invertebrate vector, which is an atypical behavior for the flavivirus genus and indicates the need to study the replication of this virus in different cell types. Although ZIKV became a target for public health programs, the interaction of this flavivirus with the infected cell is still poorly understood. Herein, we analyzed the main stages of virus morphogenesis in mammalian cells, from establishment of the viroplasm-like zone to viral release from infected cells, using super-resolution fluorescence microscopy and electron microscopy. In addition, we compared this with other host cell types and other members of the Flaviviridae family that present a similar dynamic.

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 inhibits the replication of bovine viral diarrhea virus

ABSTRACT Bovine viral diarrhea virus can cause acute disease in livestock, leading to economic losses. We show that Prostaglandin A1 inhibits bovine viral diarrhea virus replication in Madin-Darby bovine kidney cells (94% inhibition using 5 µg/mL). Light and electron microscopy of infected cells shows that Prostaglandin A1 also prevents virus-induced vacuolization, but at higher concentrations (10 µg/mL).

Prostaglandin A1 inhibits the replication of bovine viral diarrhea virus.

Bovine viral diarrhea virus can cause acute disease in livestock, leading to economic losses. We show that Prostaglandin A1 inhibits bovine viral diarrhea virus replication in Madin-Darby bovine kidney cells (94% inhibition using 5µg/mL). Light and electron microscopy of infected cells shows that Prostaglandin A1 also prevents virus-induced vacuolization, but at higher concentrations (10µg/mL).

Challenges for molecular and serological ZIKV infection confirmation.

BACKGROUND: Zika Virus (ZIKV), member of Flaviviridae family and Flavivirus genus, has recently emerged as international public health emergency after its association with neonatal microcephaly cases. Clinical diagnosis hindrance involves symptom similarities produced by other arbovirus infections, therefore laboratory confirmation is of paramount importance. DISCUSSION: The most reliable test available is based on ZIKV RNA detection from body fluid samples. However, short viremia window periods and asymptomatic infections diminish the success rate for RT-PCR positivity. Beyond molecular detection, all serology tests in areas where other Flavivirus circulates proved to be a difficult task due to the broad range of cross-reactivity, especially with dengue pre-exposed individuals. CONCLUSION: Altogether, lack of serological diagnostic tools brings limitations to any retrospective evaluation. Those studies are central in the context of congenital infection that could occur asymptomatically and mask prevalence and risk rates.