Brazil experienced a large dengue virus (DENV) epidemic in 2019, highlighting a continuous struggle with effective control and public health preparedness. Using Oxford Nanopore sequencing, we led field and classroom initiatives for the monitoring of DENV in Brazil, generating 227 novel genome sequences of DENV1-2 from 85 municipalities (2015–2019). This equated to an over 50% increase in the number of DENV genomes from Brazil available in public databases. Using both phylogenetic and epidemiological models we retrospectively reconstructed the recent transmission history of DENV1-2. Phylogenetic analysis revealed complex patterns of transmission, with both lineage co-circulation and replacement. We identified two lineages within the DENV2 BR-4 clade, for which we estimated the effective reproduction number and pattern of seasonality. Overall, the surveillance outputs and training initiative described here serve as a proof-of-concept for the utility of real-time portable sequencing for research and local capacity building in the genomic surveillance of emerging viruses.

The recent emergence of a previously unknown coronavirus (SARS-CoV-2), first confirmed in the city of Wuhan in China in December 2019, has caused serious public health and economic issues due to its rapid dissemination worldwide. Although 61,888 confirmed cases had been reported in Brazil by 28 April 2020, little was known about the SARS-CoV-2 epidemic in the country. To better understand the recent epidemic in the second most populous state in southeast Brazil (Minas Gerais, MG), we looked at existing epidemiological data from 3 states and sequenced 40 complete genomes from MG cases using Nanopore. We found evidence of multiple independent introductions from outside MG, both from genome analyses and the overly dispersed distribution of reported cases and deaths. Epidemiological estimates of the reproductive number using different data sources and theoretical assumptions all suggest a reduction in transmission potential since the first reported case, but potential for sustained transmission in the near future. The estimated date of introduction in Brazil was consistent with epidemiological data from the first case of a returning-traveler from Lombardia, Italy. These findings highlight the unique reality of MGs epidemic and reinforce the need for real-time and continued genomic surveillance strategies as a way of understanding and therefore preparing against the epidemic spread of emerging viral pathogens.

A chimeric protein was constructed expressing three epitopes of LiD1, a dermonecrotic toxin from the venom of Loxosceles intermedia spider. This species is responsible for a large number of accidents involving spiders in Brazil. We demonstrated that the chimeric protein (rCpLi) generated is atoxic and that antibodies previously developed in rabbits against synthetic epitopes reactive with rCpLi in ELISA and immunoblot assays. The antibody response in rabbits against the rCpLi was evaluated by ELISA and we have detected an antibody response in all immunized animals. Overlapping peptides covering the amino acid sequence of the rCpLi were synthesized on a cellulose membrane, and their recognition by rabbit anti-rCpLi serum assessed. Three different antigenic regions were identified. The percentage of inhibition of the dermonecrotic, hemorrhagic and edematogenic activities caused by the recombinant protein LiD1r in naïve rabbits was assessed by pre-incubation with anti-rCpLi antibodies. Anti-rCpLi induced good dermonecrotic and hemorrhagic protection. The levels of protection were similar to the antiboides anti-LiD1r. In summary, we have developed a polyepitope recombinant chimeric protein capable of inducing multiple responses of neutralizing antibodies in a rabbit model. This engineered protein may be a promising candidate for therapeutic serum development or vaccination.

The main goal of this work was to develop a strategy to identify B-cell epitopes on four different three finger toxins (3FTX) and one phospholipase A2 (PLA2) from Micrurus corallinus snake venom. 3FTx and PLA2 are highly abundant components in Elapidic venoms and are the major responsibles for the toxicity observed in envenomation by coral snakes. Overlapping peptides from the sequence of each toxin were prepared by SPOT method and three different anti-elapidic sera were used to map the epitopes. After immunogenicity analysis of the spot-reactive peptides by EPITOPIA, a computational method, nine sequences from the five toxins were chemically synthesized and antigenically and immunogenically characterized. All the peptides were used together as immunogens in rabbits, delivered with Freund's adjuvant for a first cycle of immunization and Montanide in the second. A good antibody response against individual synthetic peptides and M. corallinus venom was achieved. Anti-peptide IgGs were also cross-reactive against Micrurus frontalis and Micrurus lemniscatus crude venoms. In addition, anti-peptide IgGs inhibits the lethal and phospholipasic activities of M. corallinus crude venom. Our results provide a rational basis to the identification of neutralizing epitopes on coral snake toxins and show that their corresponding synthetic peptides could improve the generation of immuno-therapeutics. The use of synthetic peptide for immunization is a reasonable approach, since it enables poly-specificity, low risk of toxic effects and large scale production.

Loxoscelism is a necrotic-hemolytic syndrome caused by bites of brown spiders belonging to the genus Loxosceles. Many approaches for the treatment of Loxosceles poisoning have already been proposed, among which administration of specific antivenom is thought to be the more specific. We have evaluated the use of peptides as immunogen to raise in rabbits an antibody response that could protect animals from a challenge by the Loxtox isoform LiD1, one of the main toxic component of Loxosceles intermedia venom. Six antigenic regions of LiD1 were mapped by using the SPOT method. The corresponding peptides were further chemically synthesized, mixed, and used as immunogens in rabbits. Control animal received recombinant LiD1 alone or together with peptides. We found that the rabbit antibody response to peptides was cross-reactive with LiD1, although only one peptide from the mix of six was immunogenic. The dermonecrotic, hemorrhagic and oedema forming activities induced by LiD1 in naïve rabbits were inhibited by 82%, 35% and 35% respectively, by preincubation of LiD1 with anti-peptide antibodies prepared from immunized rabbits. Animals that were immunized with peptides or LiD1r, were found to be protected from the dermonecrotic, hemorrhagic and oedema forming activities induced by a challenge with LiD1. The protection conferred by peptides was, however, lower than that provided by the peptide protein combination or by the full-length protein. These results encourage us in the utilization of synthetic peptides for therapeutic serum development or vaccination approaches.