Tracking the turnover of SARS-CoV-2 VOCs Gamma to Delta in a Brazilian state (Minas Gerais) with a high-vaccination status.

The emergence and global dissemination of Severe Acute Respiratory Syndrome virus 2 (SARS-CoV-2) variants of concern (VOCs) have been described as the main factor driving the Coronavirus Disease 2019 pandemic. In Brazil, the Gamma variant dominated the epidemiological scenario during the first period of 2021. Many Brazilian regions detected the Delta variant after its first description and documented its spread. To monitor the introduction and spread of VOC Delta, we performed Polymerase Chain Reaction (PCR) genotyping and genome sequencing in ten regional sentinel units from June to October 2021 in the State of Minas Gerais (MG). We documented the introduction and spread of Delta, comprising 70 per cent of the cases 8 weeks later. Comparing the viral loads of the Gamma and Delta dominance periods, we provide additional evidence that the latter is more transmissible. The spread and dominance of Delta did not culminate in the increase in cases and deaths, suggesting that the vaccination may have restrained the epidemic growth. Analysis of 224 novel Delta genomes revealed that Rio de Janeiro state was the primary source for disseminating this variant in the state of MG. We present the establishment of Delta, providing evidence of its enhanced transmissibility and showing that this variant shift did not aggravate the epidemiological scenario in a high immunity setting.

Delta Variant of SARS-CoV-2 Replacement in Brazil: A National Epidemiologic Surveillance Program.

Coronavirus disease 2019 (COVID-19) pandemic has caused immeasurable impacts on the health and socioeconomic system. The real-time identification and characterization of new Variants of Concern (VOCs) are critical to comprehend its emergence and spread worldwide. In this sense, we carried out a national epidemiological surveillance program in Brazil from April to October 2021. Genotyping by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and sequencing were performed to monitor the dynamics and dissemination of VOCs in samples from 15 federative units. Delta VOC was first detected on June 2021 and took sixteen weeks to replace Gamma. To assess the transmissibility potential of Gamma and Delta VOCs, we studied the dynamics of RT-qPCR cycle threshold (Ct) score in the dominance period of each variant. The data suggest that Delta VOC has a higher transmission rate than Gamma VOC. We also compared relevant symptom patterns in individuals infected with both VOCs. The Delta-infected subjects were less likely to have low oxygen saturation or fatigue, altered results on chest computed tomography, and a propensity for altered X-rays. Altogether, we described the replacement of Gamma by Delta, Delta enhanced transmissibility, and differences in symptom presentation.

Spatial and temporal fluctuations in COVID-19 fatality rates in Brazilian hospitals.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Gamma variant of concern has spread rapidly across Brazil since late 2020, causing substantial infection and death waves. Here we used individual-level patient records after hospitalization with suspected or confirmed coronavirus disease 2019 (COVID-19) between 20 January 2020 and 26 July 2021 to document temporary, sweeping shocks in hospital fatality rates that followed the spread of Gamma across 14 state capitals, during which typically more than half of hospitalized patients aged 70 years and older died. We show that such extensive shocks in COVID-19 in-hospital fatality rates also existed before the detection of Gamma. Using a Bayesian fatality rate model, we found that the geographic and temporal fluctuations in Brazil's COVID-19 in-hospital fatality rates were primarily associated with geographic inequities and shortages in healthcare capacity. We estimate that approximately half of the COVID-19 deaths in hospitals in the 14 cities could have been avoided without pre-pandemic geographic inequities and without pandemic healthcare pressure. Our results suggest that investments in healthcare resources, healthcare optimization and pandemic preparedness are critical to minimize population-wide mortality and morbidity caused by highly transmissible and deadly pathogens such as SARS-CoV-2, especially in low- and middle-income countries.

Identification of Glycycometus malaysiensis (for the first time in Brazil), Blomia tropicalis and Dermatophagoides pteronyssinus through multiplex PCR.

Blomia tropicalis and Dermatophagoides pteronyssinus play an important role in triggering allergy. Glycycometus malaysiensis causes IgE reaction in sensitive people, but is rarely reported in domestic dust, because it is morphologically similar to B. tropicalis making the identification of these species difficult. The identification of mites is mostly based on morphology, a time-consuming and ambiguous approach. Herein, we describe a multiplex polymerase chain reaction (mPCR) assay based on ribosomal DNA capable to identify mixed cultures of B. tropicalis, D. pteronyssinus and G. malaysiensis, and/or to identify these species from environmental dust. For this, the internal transcribed spacer 2 (ITS2) regions, flanked by partial sequences of the 5.8S and 28S genes, were PCR-amplified, cloned and sequenced. The sequences obtained were aligned with co-specific sequences available in the GenBank database for primer design and phylogenetic studies. Three pairs of primers were chosen to compose the mPCR assay, which was used to verify the frequency of different mites in house dust samples (n = 20) from homes of Salvador, Brazil. Blomia tropicalis was the most frequent, found in 95% of the samples, followed by G. malaysiensis (70%) and D. pteronyssinus (60%). Besides reporting for the first time the occurrence of G. malaysiensis in Brazil, our results confirm the good resolution of the ITS2 region for mite identification. Furthermore, the mPCR assay proved to be a fast and reliable tool for identifying these mites in mixed cultures and could be applied in future epidemiological studies, and for quality control of mite extract production for general use.

Global Characterization of Fungal Mitogenomes: New Insights on Genomic Diversity and Dynamism of Coding Genes and Accessory Elements.

Fungi comprise a great diversity of species with distinct ecological functions and lifestyles. Similar to other eukaryotes, fungi rely on interactions with prokaryotes and one of the most important symbiotic events was the acquisition of mitochondria. Mitochondria are organelles found in eukaryotic cells whose main function is to generate energy through aerobic respiration. Mitogenomes (mtDNAs) are double-stranded circular or linear DNA from mitochondria that may contain core genes and accessory elements that can be replicated, transcribed, and independently translated from the nuclear genome. Despite their importance, investigative studies on the diversity of fungal mitogenomes are scarce. Herein, we have evaluated 788 curated fungal mitogenomes available at NCBI database to assess discrepancies and similarities among them and to better understand the mechanisms involved in fungal mtDNAs variability. From a total of 12 fungal phyla, four do not have any representative with available mitogenomes, which highlights the underrepresentation of some groups in the current available data. We selected representative and non-redundant mitogenomes based on the threshold of 90% similarity, eliminating 81 mtDNAs. Comparative analyses revealed considerable size variability of mtDNAs with a difference of up to 260 kb in length. Furthermore, variation in mitogenome length and genomic composition are generally related to the number and length of accessory elements (introns, HEGs, and uORFs). We identified an overall average of 8.0 (0-39) introns, 8.0 (0-100) HEGs, and 8.2 (0-102) uORFs per genome, with high variation among phyla. Even though the length of the core protein-coding genes is considerably conserved, approximately 36.3% of the mitogenomes evaluated have at least one of the 14 core coding genes absent. Also, our results revealed that there is not even a single gene shared among all mitogenomes. Other unusual genes in mitogenomes were also detected in many mitogenomes, such as dpo and rpo, and displayed diverse evolutionary histories. Altogether, the results presented in this study suggest that fungal mitogenomes are diverse, contain accessory elements and are absent of a conserved gene that can be used for the taxonomic classification of the Kingdom Fungi.

Report 46: Factors driving extensive spatial and temporal fluctuations in COVID-19 fatality rates in Brazilian hospitals.

The SARS-CoV-2 Gamma variant spread rapidly across Brazil, causing substantial infection and death waves. We use individual-level patient records following hospitalisation with suspected or confirmed COVID-19 to document the extensive shocks in hospital fatality rates that followed Gamma's spread across 14 state capitals, and in which more than half of hospitalised patients died over sustained time periods. We show that extensive fluctuations in COVID-19 in-hospital fatality rates also existed prior to Gamma's detection, and were largely transient after Gamma's detection, subsiding with hospital demand. Using a Bayesian fatality rate model, we find that the geographic and temporal fluctuations in Brazil's COVID-19 in-hospital fatality rates are primarily associated with geographic inequities and shortages in healthcare capacity. We project that approximately half of Brazil's COVID-19 deaths in hospitals could have been avoided without pre-pandemic geographic inequities and without pandemic healthcare pressure. Our results suggest that investments in healthcare resources, healthcare optimization, and pandemic preparedness are critical to minimize population wide mortality and morbidity caused by highly transmissible and deadly pathogens such as SARS-CoV-2, especially in low- and middle-income countries. NOTE: The following manuscript has appeared as 'Report 46 - Factors driving extensive spatial and temporal fluctuations in COVID-19 fatality rates in Brazilian hospitals' at https://spiral.imperial.ac.uk:8443/handle/10044/1/91875 . ONE SENTENCE SUMMARY: COVID-19 in-hospital fatality rates fluctuate dramatically in Brazil, and these fluctuations are primarily associated with geographic inequities and shortages in healthcare capacity.

Genome-wide identification of miRNAs and target regulatory network in the invasive ectoparasitic mite Varroa destructor.

Varroa destructor is an ectoparasite mite that attacks bees leading to colony disorders worldwide. microRNAs (miRNAs) are key molecules used by eukaryotes to post-transcriptional control of gene expression. Nevertheless, still lack information aboutV. destructor miRNAs and its regulatory networks. Here, we used an integrative strategy to characterize the miRNAs in the V. destructor mite. We identified 310 precursors that give rise to 500 mature miRNAs, which 257 are likely mite-specific elements. miRNAs showed canonical length ranging between 18 and 25 nucleotides and 5' uracil preference. Top 10 elements concentrated over 80% of total miRNA expression, with bantam alone representing ~50%. We also detected non-templated bases in precursor-derived small RNAs, indicative of miRNA post-transcriptional regulatory mechanisms. Finally, we note that conserved miRNAs control similar processes in different organisms, suggesting a conservative role. Altogether, our findings contribute to the better understanding of the mite biology that can assist future studies on varroosis control.

Comparative mitogenomics of Agaricomycetes: Diversity, abundance, impact and coding potential of putative open-reading frames.

The mitochondrion is an organelle found in eukaryote organisms, and it is vital for different cellular pathways. The mitochondrion has its own DNA molecule and, because its genetic content is relatively conserved, despite the variation of size and structure, mitogenome sequences have been widely used as a promising molecular biomarker for taxonomy and evolution in fungi. In this study, the mitogenomes of two fungal species of Agaricomycetes class, Phellinotus piptadeniae and Trametes villosa, were assembled and annotated for the first time. We used these newly sequenced mitogenomes for comparative analyses with other 55 mitogenomes of Agaricomycetes available in public databases. Mitochondrial DNA (mtDNA) size and content are highly variable and non-coding and intronic regions, homing endonucleases (HEGs), and unidentified ORFs (uORFs) significantly contribute to the total size of the mitogenome. Furthermore, accessory genes (most of them as HEGs) are shared between distantly related species, most likely as a consequence of horizontal gene transfer events. Conversely, uORFs are only shared between taxonomically related species, most probably as a result of vertical evolutionary inheritance. Additionally, codon usage varies among mitogenomes and the GC content of mitochondrial features may be used to distinguish coding from non-coding sequences. Our results also indicated that transposition events of mitochondrial genes to the nuclear genome are not common. Despite the variation of size and content of the mitogenomes, mitochondrial genes seemed to be reliable molecular markers in our time-divergence analysis, even though the nucleotide substitution rates of mitochondrial and nuclear genomes of fungi are quite different. We also showed that many events of mitochondrial gene shuffling probably happened amongst the Agaricomycetes during evolution, which created differences in the gene order among species, even those of the same genus. Altogether, our study revealed new information regarding evolutionary dynamics in Agaricomycetes.

Exploring the Relationship Among Divergence Time and Coding and Non-coding Elements in the Shaping of Fungal Mitochondrial Genomes.

The order Hypocreales (Ascomycota) is composed of ubiquitous and ecologically diverse fungi such as saprobes, biotrophs, and pathogens. Despite their phylogenetic relationship, these species exhibit high variability in biomolecules production, lifestyle, and fitness. The mitochondria play an important role in the fungal biology, providing energy to the cells and regulating diverse processes, such as immune response. In spite of its importance, the mechanisms that shape fungal mitogenomes are still poorly understood. Herein, we investigated the variability and evolution of mitogenomes and its relationship with the divergence time using the order Hypocreales as a study model. We sequenced and annotated for the first time Trichoderma harzianum mitochondrial genome (mtDNA), which was compared to other 34 mtDNAs species that were publicly available. Comparative analysis revealed a substantial structural and size variation on non-coding mtDNA regions, despite the conservation of copy number, length, and structure of protein-coding elements. Interestingly, we observed a highly significant correlation between mitogenome length, and the number and size of non-coding sequences in mitochondrial genome. Among the non-coding elements, group I and II introns and homing endonucleases genes (HEGs) were the main contributors to discrepancies in mitogenomes structure and length. Several intronic sequences displayed sequence similarity among species, and some of them are conserved even at gene position, and were present in the majority of mitogenomes, indicating its origin in a common ancestor. On the other hand, we also identified species-specific introns that advocate for the origin by different mechanisms. Investigation of mitochondrial gene transfer to the nuclear genome revealed that nuclear copies of the nad5 are the most frequent while atp8, atp9, and cox3 could not be identified in any of the nuclear genomes analyzed. Moreover, we also estimated the divergence time of each species and investigated its relationship with coding and non-coding elements as well as with the length of mitogenomes. Altogether, our results demonstrated that introns and HEGs are key elements on mitogenome shaping and its presence on fast-evolving mtDNAs could be mostly explained by its divergence time, although the intron sharing profile suggests the involvement of other mechanisms on the mitochondrial genome evolution, such as horizontal transference.

Virome analyses of Hevea brasiliensis using small RNA deep sequencing and PCR techniques reveal the presence of a potential new virus.

BACKGROUND: Hevea brasiliensis is an important commercial crop due to the high quality of the latex it produces; however, little is known about viral infections in this plant. The only virus described to infect H. brasiliensis until now is a Carlavirus, which was described more than 30 years ago. Virus-derived small interfering RNA (vsiRNAs) are the product of the plant's antiviral defense triggered by dsRNA viral intermediates generated, during the replication cycle. These vsiRNAs are complementar to viral genomes and have been widely used to identify and characterize viruses in plants. METHODS: In the present study, we investigated the virome of leaf and sapwood samples from native H. brasiliensis trees collected in two geographic areas in the Brazilian Amazon. Small RNA (sRNA) deep sequencing and bioinformatic tools were used to assembly, identify and characterize viral contigs. Subsequently, PCR amplification techniques were performed to experimentally verify the presence of the viral sequences. Finally, the phylogenetic relationship of the putative new virus with related viral genomes was analyzed. RESULTS: Our strategy allowed the identification of 32 contigs with high similarity to viral reference genomes, from which 23 exhibited homology to viruses of the Tymoviridae family. The reads showed a predominant size distribution at 21 nt derived from both strands, which was consistent with the vsiRNAs profile. The presence and genome position of the viral contigs were experimentally confirmed using droplet digital PCR amplifications. A 1913 aa long fragment was obtained and used to infer the phylogenetic relationship of the putative new virus, which indicated that it is taxonomically related to the Grapevine fleck virus, genus Maculavirus. The putative new virus was named Hevea brasiliensis virus (HBrV) in reference to its host. CONCLUSION: The methodological strategy applied here proved to be efficient in detecting and confirming the presence of new viral sequences on a 'very difficult to manage' sample. This is the second time that viral sequences, that could be ascribed as a putative novel virus, associated to the rubber tree has been identified.

A multiscale study of fungal endophyte communities of the foliar endosphere of native rubber trees in Eastern Amazon.

Hevea brasiliensis is a native hyperdiverse tree species in the Amazon basin with great economic importance since it produces the highest quality natural rubber. H. brasiliensis, in its natural habitat, may harbor fungal endophytes that help defend against phytopathogenic fungi. In this work, we investigated the fungal endophytic communities in two pristine areas in Eastern Amazon (Anavilhanas National Park - ANP and Caxiuanã National Forest - CNF) at different spatial scales: regional, local, individual (tree), and intra-individual (leaflet). Using a culture-based approach, 210 fungal endophytes were isolated from 240 sampling units and assigned to 46 distinct MOTUs based on sequencing of the nrITS DNA. The community compositions of the endophytomes are different at both regional and local scales, dominated by very few taxa and highly skewed toward rare taxa, with many endophytes infrequently isolated across hosts in sampled space. Colletotrichum sp. 1, a probably latent pathogen, was the most abundant endophytic putative species and was obtained from all individual host trees in both study areas. Although the second most abundant putative species differed between the two collection sites, Clonostachys sp. 1 and Trichoderma sp. 1, they are phylogenetically related (Hypocreales) mycoparasites. Thus, they probably exhibit the same ecological function in the foliar endosphere of rubber tree as antagonists of its fungal pathogens.