The COVID-19 pandemic caused by SARS-CoV-2 has affected millions of people since its beginning in 2019. The propagation of new lineages and the discovery of key mechanisms adopted by the virus to overlap the immune system are central topics for the entire public health policies, research and disease management. Since the second semester of 2020, the mutation E484K has been progressively found in the Brazilian territory, composing different lineages over time. It brought multiple concerns related to the risk of reinfection and the effectiveness of new preventive and treatment strategies due to the possibility of escaping from neutralizing antibodies. To better characterize the current scenario we performed genomic and phylogenetic analyses of the E484K mutated genomes sequenced from Brazilian samples in 2020. From October 2020, more than 40% of the sequenced genomes present the E484K mutation, which was identified in three different lineages (P.1, P.2 and B.1.1.33 - posteriorly renamed as N.9) in four Brazilian regions. We also evaluated the presence of E484K associated mutations and identified selective pressures acting on the spike protein, leading us to some insights about adaptive and purifying selection driving the virus evolution.

The COVID-19 pandemic caused by SARS-CoV-2 has affected millions of people since its beginning in 2019. The propagation of new lineages and the discovery of key mechanisms adopted by the virus to overlap the immune system are central topics for the entire public health policies, research and disease management. Since the second semester 2020, the mutation E484K has been progressively found in the Brazilian territory, composing different lineages over time. It brought multiple concerns related to the risk of reinfection and the effectiveness of new preventive and treatment strategies due to the possibility of escaping from neutralizing antibodies. To better characterize the current scenario we performed genomic and phylogenetic analyses of the E484K mutated genomes sequenced from Brazilian samples in 2020. From October, 2020, 43.9% of the sequenced genomes present the E484K mutation, which was identified in three different lineages (P1, P2 and B.1.1.33) in four Brazilian regions. We also evaluated the presence of E484K associated mutations and identified selective pressures acting on the spike protein, leading us to some insights about adaptive and purifying selection driving the virus evolution.

Background Brazil is the third country most affected by Coronavirus disease-2019 (COVID-19), but viral evolution in municipality resolution is still poorly understood in Brazil and it is crucial to understand the epidemiology of viral spread. We aimed to track molecular evolution and spread of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Esteio (Southern Brazil) using phylogenetics and phylodynamics inferences from 21 new genomes in global and regional context. Importantly, the case fatality rate (CFR) in Esteio (3.26%) is slightly higher compared to the Rio Grande do Sul (RS) state (2.56%) and the entire Brazil (2.74%). Results We provided a comprehensive view of mutations from a representative sampling from May to October 2020, highlighting two frequent mutations in spike glycoprotein (D614G and V1176F), an emergent mutation (E484K) in spike Receptor Binding Domain (RBD) characteristic of the B.1.351 and P.1 lineages, and the adjacent replacement of 2 amino acids in Nucleocapsid phosphoprotein (R203K and G204R). E484K was found in two genomes from mid-October, which is the earliest description of this mutation in Southern Brazil. Lineages containing this substitution must be subject of intense surveillance due to its association with immune evasion. We also found two epidemiologically-related clusters, including one from patients of the same neighborhood. Phylogenetics and phylodynamics analysis demonstrates multiple introductions of the Brazilian most prevalent lineages (B.1.1.33 and B.1.1.248) and the establishment of Brazilian lineages ignited from the Southeast to other Brazilian regions. Conclusions Our data show the value of correlating clinical, epidemiological and genomic information for the understanding of viral evolution and its spatial distribution over time. This is of paramount importance to better inform policy making strategies to fight COVID-19.

Evaluating the quality of a de novo annotation of a complex fungal genome based on RNA-seq data remains a challenge. In this study, we sequentially optimized a Cufflinks-CodingQuary-based bioinformatics pipeline fed with RNA-seq data using the manually annotated model pathogenic yeasts Cryptococcus neoformans and Cryptococcus deneoformans as test cases. Our results show that the quality of the annotation is sensitive to the quantity of RNA-seq data used and that the best quality is obtained with 5–10 million reads per RNA-seq replicate. We also showed that the number of introns predicted is an excellent a priori indicator of the quality of the final de novo annotation. We then used this pipeline to annotate the genome of the RNAi-deficient species Cryptococcus deuterogattii strain R265 using RNA-seq data. Dynamic transcriptome analysis revealed that intron retention is more prominent in C. deuterogattii than in the other RNAi-proficient species C. neoformans and C. deneoformans. In contrast, we observed that antisense transcription was not higher in C. deuterogattii than in the two other Cryptococcus species. Comparative gene content analysis identified 21 clusters enriched in transcription factors and transporters that have been lost. Interestingly, analysis of the subtelomeric regions in these three annotated species identified a similar gene enrichment, reminiscent of the structure of primary metabolic clusters. Our data suggest that there is active exchange between subtelomeric regions, and that other chromosomal regions might participate in adaptive diversification of Cryptococcus metabolite assimilation potential.

Brazil is the third country most affected by Covid-19 pandemic. In spite of this, viral evolution in municipality resolution is poorly understood in Brazil and it is crucial to understand the epidemiology of viral spread. We identified four main circulating lineages in Esteio (Southern Brazil) and their relationship with global, national and regional lineages using phylogenetics and phylodynamics inferences from 21 SARS-CoV-2 genome sequences. We provided a comprehensive view of viral mutations from a time- and age-representative sampling from May to October 2020, in Esteio (RS, Brazil), highlighting two frequent mutations in Spike glycoprotein (D614G and V1176F), an emergent mutation (E484K) in Spike Receptor Binding Domain (RBD) characteristic of the South African lineage B.1.351, and the adjacent replacement of 2 amino acids in Nucleocapsid phosphoprotein (R203K and G204R). A significant viral diversity was evidenced with the identification of 80 different SNPs. The E484K replacement was found in two genomes (9.5%) from samples obtained in mid-October, which is to our best knowledge the earliest description of E484K harboring SARS-CoV-2 in South Brazil. This mutation identified in a small municipality from the RS state demonstrates that it was probably widely distributed in the Brazilian territory, but went unnoticed so far by the lack of genomic surveillance in Brazil. The introduction of E484K mutants shows temporal correlation with later increases in new cases in our state. Importantly, since it has been associated with immune evasion and enhanced interaction with hACE-2, lineages containing this substitution must be the subject of intense surveillance. Our date demonstrates multiple introductions of the most prevalent lineages (B.1.1.33 and B.1.1.248) and the major role of community transmission in viral spreading and the establishment of Brazilian lineages. This represents an important contribution to the epidemiology of SARS-CoV-2.

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Cryptococcus gattii is a human and animal pathogen that infects healthy hosts and caused the Pacific Northwest outbreak of cryptococcosis. The inhalation of infectious propagules can lead to internalization of cryptococcal cells by alveolar macrophages, a niche in which C. gattii cells can survive and proliferate. Although the nutrient composition of macrophages is relatively unknown, the high induction of amino acid transporter genes inside the phagosome indicates a preference for amino acid uptake instead of synthesis. However, the presence of countable errors in the R265 genome annotation indicates significant inhibition of transcriptomic analysis in this hypervirulent strain. Thus, we analyzed RNA-Seq data from in vivo and in vitro cultures of C. gattii R265 to perform the reannotation of the genome. In addition, based on in vivo transcriptomic data, we identified highly expressed genes and pathways of amino acid metabolism that would enable C. gattii to survive and proliferate in vivo. Importantly, we identified high expression in three APC amino acid transporters as well as the GABA permease. The use of amino acids as carbon and nitrogen sources, releasing ammonium and generating carbohydrate metabolism intermediaries, also explains the high expression of components of several degradative pathways, since glucose starvation is an important host defense mechanism.