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.

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.

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.

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.

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.

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.

Antiviral Drug Discovery and Development for Mayaro Fever - What do we have so far?

Tropical infectious diseases cause millions of deaths every year in developing countries, with about half of the world population living at risk. Mayaro virus (MAYV) is an emerging arbovirus that causes Mayaro fever, which is characterized by fever, headache, diarrhea, arthralgia, and rash. These symptoms can be clinically indistinguishable from other arboviruses, such as Dengue, Zika, and Chikungunya, which makes the diagnosis and treatment of the disease more difficult. Though, the Mayaro virus is a potential candidate to cause large-scale epidemics on the scale of ZIKV and CHIKV. Despite this, there is no licensed vaccine or antiviral for the treatment of Mayaro fever and most arboviruses, so the design and development of candidates for antiviral drugs are urgently needed. In this context, this mini-review aims to provide an overview of studies of anti-MAYV derivatives and highlight the importance of the discovery and development of promising drug candidates for Mayaro fever.

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.

High Prevalence of a Newly Discovered Wutai Mosquito Phasivirus in Mosquitoes from Rio de Janeiro, Brazil.

Many RNA viruses have recently emerged, threatening humans and causing harm to animals and plants. Bunyaviruses represent one of the largest groups of RNA viruses and are able to infect a wide range of hosts (invertebrates, vertebrates, and plants). Recently, new insect-specific viruses have been isolated from mosquitoes and phlebotomine sandflies worldwide. Little is known regarding the impact of these viruses on the vector life cycles and the stages of oviposition, breeding, blood feeding, and the mosquito's lifespan. This study describes, for the first time in South America, the detection and characterization of a recently discovered bunyavirus corresponding to the Wutai mosquito phasivirus, confirming its high prevalence in the Culex spp. and Aedes spp. mosquitoes collected in the urban environment of Rio de Janeiro city, Brazil. The knowledge of the mosquito's insect-specific virus infection can improve virus evolution studies and may contribute to the understanding of intrinsic factors that influence vector competence to transmit pathogenic viruses.

Purification of yellow fever virus produced in Vero cells for inactivated vaccine manufacture.

Yellow fever (YF) is a high-lethality viral disease, endemic in tropical regions of South America and Africa, with a population of over 900 million people under risk. A highly effective attenuated vaccine, produced in embryonated eggs, has been used for about 80 years. However, egg-based production limits manufacturing capacity, and vaccine shortage led to the emergency use of a fractional dose (1/5) by the WHO in an outbreak in Africa in 2016 and by Brazilian authorities during an outbreak in 2018. In addition, rare but fatal adverse events of this vaccine have been reported since 2001. These two aspects make clear the need for the development of a new vaccine. In an effort to develop an inactivated YF vaccine, Bio-Manguinhos/FIOCRUZ started developing a new vaccine based on the production of the attenuated 17DD virus in serum-free conditions in Vero cells propagated in bioreactors, followed by chromatography-based purification and ß-propiolactone inactivation. Virus purification was studied in this work. Capture was performed using an anion-exchange membrane adsorber (Sartobind® Q), resulting in a virus recovery of 80.2 ±â€¯4.8% and a residual DNA level of 1.3 ±â€¯1.6 ng/dose, thus in accordance with the recommendations of the WHO (<10 ng/dose). However, the level of host cell proteins (HCP) was still high for a human vaccine, so a second chromatography step was developed based on a multimodal resin (Capto™ Core 700). This step resulted in a virus recovery of 65.7 ±â€¯4.8% and decreased HCP levels to 345 ±â€¯25 ppm. The overall virus recovery in these chromatography steps was 52.7%. SDS-PAGE of the purified sample showed a band with molecular mass of 56 kDa, thus consistent with the virus envelope protein (E) and corresponding to 96.7% of identified proteins. A Western blot stained with an antibody against the E protein showed a single band, confirming the identity of the sample.

Purification and Proteomic Analysis of Alphavirus Particles from Sindbis Virus Grown in Mammalian and Insect Cells.

Current mass spectrometry (MS) methods and new instrumentation now allow for more accurate identification of proteins in low abundance than previous protein fractionation and identification methods. It was of interest if this method could serve to define the virus proteome of a membrane-containing virus. To evaluate the efficacy of mass spec to determine the proteome of medically important viruses, Sindbis virus (SINV), the prototypical alphavirus was chosen for evaluation. This model system was chosen specifically because the alphaviruses contain members which are human pathogens, this virus is well defined biochemically and structurally, and grows to high titers in both vertebrate and non-vertebrate host cells. The SINV proteome was investigated using this method to determine if host proteins are specifically packaged into infectious virions. It was also of interest if the SINV proteome, when grown in multiple host cells representing vertebrate and mosquito hosts, incorporated specific host proteins from all hosts. Observation of recurrent or distinctive proteins in the virus proteome aided in the determination of proteins incorporated into the virion as opposed to those bound to the particle exterior. Mass spectrometry analysis identified the total protein content of purified virions within limits of detection. The most significant finding was that in addition to the host proteins, SINV non-structural protein 2 (nsP2) was detected within virions grown in all host cells examined. This analysis identified host factors not previously associated with alphavirus entry, replication, or egress, identifying at least one host factor integrally involved in alphavirus replication. Key to the success of this analysis is the method of virus purification which must deliver measurably infectious virus free of high levels of contaminants. For SINV and other members of the alphavirus family, this is accomplished by isopycnic centrifugation through potassium tartrate, followed by a high salt wash.

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.

Role of the vacuolar ATPase in the Alphavirus replication cycle.

We have shown that Alphaviruses can enter cells by direct penetration at the plasma membrane (R. Vancini, G. Wang, D. Ferreira, R. Hernandez, and D. Brown, J Virol, 87:4352-4359, 2013). Direct penetration removes the requirement for receptor-mediated endocytosis exposure to low pH and membrane fusion in the process of RNA entry. Endosomal pH as well as the pH of the cell cytoplasm is maintained by the activity of the vacuolar ATPase (V-ATPase). Bafilomycin is a specific inhibitor of V-ATPase. To characterize the roll of the V-ATPase in viral replication we generated a Bafilomycin A1(BAF) resistant mutant of Sindbis virus (BRSV). BRSV produced mature virus and virus RNA in greater amounts than parent virus in BAF-treated cells. Sequence analysis revealed mutations in the E2 glycoprotein, T15I/Y18H, were responsible for the phenotype. These results show that a functional V-ATPase is required for efficient virus RNA synthesis and virus maturation in Alphavirus infection.

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.