A potential antiviral against COVID-19 obtained from Byrsonima coccolobifolia leaves extract.

In this study, we specifically focused on the crude methanolic leaf extract of Byrsonima coccolobifolia, investigating its antifungal potential against human pathogenic fungi and its antiviral activity against COVID-19. Through the use of high-performance liquid chromatography coupled with electrospray ionization ion trap tandem mass spectrometry, direct infusion electrospray ionization ion trap tandem mass spectrometry, and chromatographic dereplication procedures, we identified galloyl quinic acid derivatives, catechin derivatives, proanthocyanidins, and flavonoid glycosides. The broth dilution assay revealed that the methanolic leaf extract of B. coccolobifolia exhibits antifungal activity against Cryptococcus neoformans (IC50 = 4 µg/mL). Additionally, docking studies were conducted to elucidate the interactions between the identified compounds and the central residues at the binding site of biological targets associated with COVID-19. Furthermore, the extract demonstrated an in vitro half-maximum effective concentration (EC50 = 7 µg/mL) and exhibited significant selectivity (>90%) toward SARS-CoV-2.

Transcriptome profiling and Calreticulin expression in Zika virus - infected Aedes aegypti

Zika virus (ZIKV) may cause febrile illness and neurological damage, such as microcephaly in fetuses. ZIKV is transmitted to humans by Aedes aegypti, a nearly cosmopolitan mosquito. Understanding the virus-vector molecular interactions has been promising to enhance the knowledge towards disease mitigation. Since ZIKV infection alters gene physiology of mosquitoes, we examined the expression profile of ZIKV-infected Ae. aegypti by several approaches to identify genes altered by viral infection. Transcriptomics were performed by comparing between ZIKV-infected and uninfected Ae. aegypti females, which revealed some differentially expressed genes. Most of these genes appear to be involved with immune response as evidenced by an interactome analysis, and a prominent finding was a calreticulin-like (CRT) gene, which was upregulated during the infection. Expression of CRT was also experimentally quantified by qPCR, however, it revealed no significant differences between infected and uninfected females. Instead, expression levels were highly variable among individuals and negatively correlated to viral load. We also tested the possibility of this gene to be silenced, but the double-stranded RNA did not reduce CRT expression, and actually increased the inter-individuals' expressional variability. Present results differed from our original hypothesis of upregulation by infection. They also diverged between them (comparing qPCR to Transcriptomics) and from the literature which reported augmented CRT levels in Aedes species during viral infection. Present case probably underlies a more complex virus-host interaction system than we expected. Regulation of this gene seems not to be a linear correlation between expression and viremy. As infection takes place, a complex homeostatic mechanism may act to prevent expression and other cellular tasks from drifting. It is also possible that CRT expression is simply randomly disturbed by viral infection. Taken together, results show that CRT expression profile during ZIKV infection is complex and requires different investigative approaches to be understood. Studies focused on the biochemical function of CRT protein and on its role in the native mosquito metabolic network could unravel how it is actually influenced by ZIKV. Current work contributes more by getting incidental findings and by posing new hypotheses than by answering the original questions.

Transcriptome profiling and Calreticulin expression in Zika virus-infected Aedes aegypti

Zika virus (ZIKV) may cause febrile illness and neurological damage, such as microcephaly in fetuses. ZIKV is transmitted to humans by Aedes aegypti, a nearly cosmopolitan mosquito. Understanding the virus-vector molecular interactions has been promising to enhance the knowledge towards disease mitigation. Since ZIKV infection alters gene physiology of mosquitoes, we examined the expression profile of ZIKV-infected Ae. aegypti by several approaches to identify genes altered by viral infection. Transcriptomics were performed by comparing between ZIKV-infected and uninfected Ae. aegypti females, which revealed some differentially expressed genes. Most of these genes appear to be involved with immune response as evidenced by an interactome analysis, and a prominent finding was a calreticulin-like (CRT) gene, which was upregulated during the infection. Expression of CRT was also experimentally quantified by qPCR, however, it revealed no significant differences between infected and uninfected females. Instead, expression levels were highly variable among individuals and negatively correlated to viral load. We also tested the possibility of this gene to be silenced, but the double-stranded RNA did not reduce CRT expression, and actually increased the inter-individuals' expressional variability. Present results differed from our original hypothesis of upregulation by infection. They also diverged between them (comparing qPCR to Transcriptomics) and from the literature which reported augmented CRT levels in Aedes species during viral infection. Present case probably underlies a more complex virus-host interaction system than we expected. Regulation of this gene seems not to be a linear correlation between expression and viremy. As infection takes place, a complex homeostatic mechanism may act to prevent expression and other cellular tasks from drifting. It is also possible that CRT expression is simply randomly disturbed by viral infection. Taken together, results show that CRT expression profile during ZIKV infection is complex and requires different investigative approaches to be understood. Studies focused on the biochemical function of CRT protein and on its role in the native mosquito metabolic network could unravel how it is actually influenced by ZIKV. Current work contributes more by getting incidental findings and by posing new hypotheses than by answering the original questions.

A Metagenomic Approach Identified a Novel Phasi Charoen-Like Virus Coinfecting a Chikungunya Virus-Infected Aedes aegypti Mosquito in Brazil.

Insect-specific viruses do not replicate in vertebrates. Here, we report the genome sequence of a novel strain of a Phasi Charoen-like virus (PCLV) that was isolated from a wild Aedes aegypti mosquito collected in Aracajú, Sergipe State, Brazil. The coding-complete genome of the PCLV is described in this report.

Transgenic Aedes aegypti Mosquitoes Transfer Genes into a Natural Population.

In an attempt to control the mosquito-borne diseases yellow fever, dengue, chikungunya, and Zika fevers, a strain of transgenically modified Aedes aegypti mosquitoes containing a dominant lethal gene has been developed by a commercial company, Oxitec Ltd. If lethality is complete, releasing this strain should only reduce population size and not affect the genetics of the target populations. Approximately 450 thousand males of this strain were released each week for 27 months in Jacobina, Bahia, Brazil. We genotyped the release strain and the target Jacobina population before releases began for >21,000 single nucleotide polymorphisms (SNPs). Genetic sampling from the target population six, 12, and 27-30 months after releases commenced provides clear evidence that portions of the transgenic strain genome have been incorporated into the target population. Evidently, rare viable hybrid offspring between the release strain and the Jacobina population are sufficiently robust to be able to reproduce in nature. The release strain was developed using a strain originally from Cuba, then outcrossed to a Mexican population. Thus, Jacobina Ae. aegypti are now a mix of three populations. It is unclear how this may affect disease transmission or affect other efforts to control these dangerous vectors. These results highlight the importance of having in place a genetic monitoring program during such releases to detect un-anticipated outcomes.

Oviplate: A Convenient and Space-Saving Method to Perform Individual Oviposition Assays in Aedes aegypti.

Aedes aegypti is the principal vector of the urban arboviruses and the blood ingestion is important to produce the eggs in this species. To analyze the egg production in Ae. aegypti, researchers frequently use small cages or Drosophila vials to collect eggs from gravid females. Although it is affordable, the setup is time- and space-consuming, mainly when many mosquitoes need to be individually analyzed. This study presents an easy, cheap, and space-saving method to perform individual oviposition assays in Ae. aegypti using cell culture plates. This new method to access fecundity rate was named "oviplate". The oviplates are setup with 12- or 24-well plates, distilled water and filter paper and they are 78 to 88% cheaper than the traditional Drosophila vial assay, respectively. Furthermore, to allocate 72 vitellogenic females in an insectary using Drosophila vial is necessary 4100 cm³ against 1400 cm³ and 700 cm³ when using 12- and 24-well plates, respectively. No statistical differences were found between the number of eggs laid in Drosophila vials and the oviplates, validating the method. The oviplate method is an affordable, and time- and space-efficient device, and it is simpler to perform individual fecundity analyses in Ae. aegypti.

First report of naturally infected Aedes aegypti with chikungunya virus genotype ECSA in the Americas.

BACKGROUND: The worldwide expansion of new emergent arboviruses such as Chikungunya and Zika reinforces the importance in understanding the role of mosquito species in spreading these pathogens in affected regions. This knowledge is essential for developing effective programs based on species specificity to avoid the establishment of endemic transmission cycles sustained by the identified local vectors. Although the first autochthonous transmission of Chikungunya virus was described in 2014 in the north of Brazil, the main outbreaks were reported in 2015 and 2016 in the northeast of Brazil. METHODOLOGY/PRINCIPAL FINDINGS: During 5 days of February 2016, we collected mosquitoes in homes of 6 neighborhoods of Aracaju city, the capital of Sergipe state. Four mosquito species were identified but Culex quinquefasciatus and Aedes aegypti were the most abundant. Field-caught mosquitoes were tested for Chikungunya (CHIKV), Zika (ZIKV) and Dengue viruses (DENV) by qRT-PCR and one CHIKV-infected Ae. aegypti female was detected. The complete sequence of CHIKV genome was obtained from this sample and phylogenetic analysis revealed that this isolate belongs to the East-Central-South-African (ECSA) genotype. CONCLUSIONS: Our study describes the first identification of a naturally CHIKV-infected Ae. aegypti in Brazil and the first report of a CHIKV from ECSA genotype identified in this species in the Americas. These findings support the notion of Ae. aegypti being a vector involved in CHIKV outbreaks in northeast of Brazil.

Correction: Laboratory strains of Aedes aegypti are competent to Brazilian Zika virus.

[This corrects the article DOI: 10.1371/journal.pone.0171951.].

Laboratory strains of Aedes aegypti are competent to Brazilian Zika virus.

The Zika virus outbreaks are unprecedented human threat in relation to congenital malformations and neurological/autoimmune complications. Since this virus has high potential to spread in regions presenting the vectors, improvement in mosquito control is a top priority. Thus, Aedes aegypti laboratory strains will be fundamental to support studies in different research fields implicated on Zika-mosquito interactions which are the basis for the development of innovative control methods. In this sense, our aim was to determine the main infection aspects of a Brazilian Zika strain in reference Aedes aegypti laboratory mosquitoes. We orally exposed Rockefeller, Higgs and Rexville mosquitoes to the Brazilian ZIKV (ZIKVBR) and qRT-PCR was applied to determine the infection, dissemination and detection rates of ZIKV in the collected saliva as well as viral levels in mosquito tissues. The three strains sustain the virus development but Higgs showed significantly lower viral loads in bodies at 14 days post-infection (dpi) and the lowest prevalences in bodies and heads. The Rockefeller strain was the most susceptible at 7 dpi but similar dissemination rates were observed at 14 dpi. Although variations exist, the ZIKVBR RNA shows detectable levels in saliva of the three strains at 14 dpi but is only detected in Rockefeller at 7 dpi. Moreover, saliva samples from the three strains were confirmed to be infectious when intrathoracically injected into mosquitoes. The ZIKVBR kinetics was monitored in Rockefeller mosquitoes and virus could be identified in the heads at 4 dpi but was more consistently detected late in infection. Our study presents the first evaluation on how Brazilian Zika virus behaves in reference Aedes aegypti strains and shed light on how the infection evolves over time. Vector competence and hallmarks of the ZIKVBR development were revealed in laboratory mosquitoes, providing additional information to accelerate studies focused on ZIKV-mosquito interactions.

Antiplasmodial activity study of angiotensin II via Ala scan analogs.

Angiotensin II (AII) as well as analog peptides shows antimalarial activity against Plasmodium gallinaceum and Plasmodium falciparum, but the exact mechanism of action is still unknown. This work presents the solid-phase synthesis and characterization of eight peptides corresponding to the alanine scanning series of AII plus the amide-capped derivative and the evaluation of the antiplasmodial activity of these peptides against mature P. gallinaceum sporozoites. The Ala screening data indicates that the replacement of either the Ile(5) or the His(6) residues causes minor effects on the in vitro antiplasmodial activity compared with AII, i.e. AII (88%), [Ala(6) ]-AII (79%), and [Ala(5) ]-AII (75%). Analogs [Ala(3) ]-AII, [Ala(1) ]-AII, and AII-NH2 showed antiplasmodial activity around 65%, whereas the activity of the [Ala(8) ]-AII, [Ala(7) ]-AII, [Ala(4) ]-AII, and [Ala(2) ]-AII analogs is lower than 45%. Circular dichroism data suggest that AII and the most active analogs adopt a ß-fold conformation in different solutions. All AII analogs, except [Ala(4) ]-AII and [Ala(8) ]-AII, show contractile responses and interact with the AT1 receptor, [Ala(5) ]-AII and [Ala(6) ]-AII. In conclusion, this approach is helpful to understand the contribution of each amino acid residue to the bioactivity of AII, opening new perspectives toward the design of new sporozoiticidal compounds.

Glytube: a conical tube and parafilm M-based method as a simplified device to artificially blood-feed the dengue vector mosquito, Aedes aegypti.

Aedes aegypti, the main vector of dengue virus, requires a blood meal to produce eggs. Although live animals are still the main blood source for laboratory colonies, many artificial feeders are available. These feeders are also the best method for experimental oral infection of Ae. aegypti with Dengue viruses. However, most of them are expensive or laborious to construct. Based on principle of Rutledge-type feeder, a conventional conical tube, glycerol and Parafilm-M were used to develop a simple in-house feeder device. The blood feeding efficiency of this apparatus was compared to a live blood source, mice, and no significant differences (p = 0.1189) were observed between artificial-fed (51.3% of engorgement) and mice-fed groups (40.6%). Thus, an easy to assemble and cost-effective artificial feeder, designated "Glytube" was developed in this report. This simple and efficient feeding device can be built with common laboratory materials for research on Ae. aegypti.