Tonsils are major sites of persistence of SARS-CoV-2 in children.

In the present study, we show that SARS-CoV-2 can infect palatine tonsils, adenoids, and secretions in children without symptoms of COVID-19, with no history of recent upper airway infection. We studied 48 children undergoing tonsillectomy due to snoring/OSA or recurrent tonsillitis between October 2020 and September 2021. Nasal cytobrushes, nasal washes, and tonsillar tissue fragments obtained at surgery were tested by RT-qPCR, immunohistochemistry (IHC), flow cytometry, and neutralization assay. We detected the presence of SARS-CoV-2 in at least one specimen tested in 27% of patients. IHC revealed the presence of the viral nucleoprotein in epithelial surface and in lymphoid cells in both extrafollicular and follicular regions, in adenoids and palatine tonsils. Also, IHC for the SARS-CoV-2 non-structural protein NSP-16 indicated the presence of viral replication in 53.8% of the SARS-CoV-2-infected tissues. Flow cytometry showed that CD20+ B lymphocytes were the most infected phenotypes, followed by CD4+ lymphocytes and CD123 dendritic cells, CD8+ T lymphocytes, and CD14+ macrophages. Additionally, IF indicated that infected tonsillar tissues had increased expression of ACE2 and TMPRSS2. NGS sequencing demonstrated the presence of different SARS-CoV-2 variants in tonsils from different tissues. SARS-CoV-2 antigen detection was not restricted to tonsils but was also detected in nasal cells from the olfactory region. Palatine tonsils and adenoids are sites of prolonged RNA presence by SARS-CoV-2 in children, even without COVID-19 symptoms. IMPORTANCE This study shows that SRS-CoV-2 of different lineages can infect tonsils and adenoids in one quarter of children undergoing tonsillectomy. These findings bring advancement to the area of SARS-CoV-2 pathogenesis, by showing that tonsils may be sites of prolonged infection, even without evidence of recent COVID-19 symptoms. SARS-CoV-2 infection of B and T lymphocytes, macrophages, and dendritic cells may interfere with the mounting of immune responses in these secondary lymphoid organs. Moreover, the shedding of SARS-CoV-2 RNA in respiratory secretions from silently infected children raises concern about possible diagnostic confusion in the presence of symptoms of acute respiratory infections caused by other etiologies.

Search for a platelet-activating factor receptor in the Trypanosoma cruzi proteome: a potential target for Chagas disease chemotherapy.

Chagas disease (CD) causes the highest burden of parasitic diseases in the Western Hemisphere and is therefore a priority for drug research and development. Platelet-activating factor (PAF) causes the CD parasite Trypanosoma cruzi to differentiate, which suggests that the parasite may express PAF receptors. Here, we explored the T. cruzi proteome for PAF receptor-like proteins. From a total of 23,000 protein sequences, we identified 29 hypothetical proteins that are predicted to have seven transmembrane domains (TMDs), which is the main characteristic of the G protein-coupled receptors (GPCRs), including the PAF receptor. The TMDs of these sequences were independently aligned with domains from 25 animal PAF receptors and the sequences were analysed for conserved residues. The conservation score mean values for the TMDs of the hypothetical proteins ranged from 31.7-44.1%, which suggests that if the putative T. cruzi PAF receptor is among the sequences identified, the TMDs are not highly conserved. These results suggest that T. cruzi contains several GPCR-like proteins and that one of these GPCRs may be a PAF receptor. Future studies may further validate the PAF receptor as a target for CD chemotherapy.

Coding-Complete Genome Sequence of a Yellow Fever Virus Isolated from a Baby Howler Monkey (Alouatta caraya) from São Paulo State, Brazil, in 2016.

We report a coding-complete sequence of a yellow fever virus, strain JabSPM02, containing the 3' untranslated region and all coding regions. The virus was recovered from an infected howler monkey from a rural area in São Paulo State, Brazil. Our findings show that it belongs to the South America 1E genotype.

Evidence for current circulation of an ancient West Nile virus strain (NY99) in Brazil.

INTRODUCTION: In Brazil, West Nile virus (WNV) was first detected, in 2018, in horses with neurological disease. AIM: We report the first case of WNV infection in a horse from Ceará state and the complete genome sequence of an isolate from Espírito Santo state. Both infections occurred in 2019. METHODS: WNV was isolated from the tissues of a horse with neurological signs in Espírito Santo and sequenced by MiSeq. RESULTS: Phylogenetic analysis revealed that the isolate belongs to lineage 1a, clustering with the NY99 strain, a strain that has not circulated in the USA since 2005. CONCLUSIONS: Our findings reinforce the hypothesis that WNV has been silently circulating in Brazil for many years.

Evidence for current circulation of an ancient West Nile virus strain (NY99) in Brazil

Abstract INTRODUCTION: In Brazil, West Nile virus (WNV) was first detected, in 2018, in horses with neurological disease. AIM: We report the first case of WNV infection in a horse from Ceará state and the complete genome sequence of an isolate from Espírito Santo state. Both infections occurred in 2019. METHODS: WNV was isolated from the tissues of a horse with neurological signs in Espírito Santo and sequenced by MiSeq. RESULTS: Phylogenetic analysis revealed that the isolate belongs to lineage 1a, clustering with the NY99 strain, a strain that has not circulated in the USA since 2005. CONCLUSIONS: Our findings reinforce the hypothesis that WNV has been silently circulating in Brazil for many years.

CodonShuffle: a tool for generating and analyzing synonymously mutated sequences.

Because synonymous mutations do not change the amino acid sequence of a protein, they are generally considered to be selectively neutral. Empiric data suggest, however, that a significant fraction of viral mutational fitness effects may be attributable to synonymous mutation. Bias in synonymous codon usage in viruses may result from selection for translational efficiency, mutational bias, base pairing requirements in RNA structures, or even selection against specific dinucleotides by innate immune effectors. Experimental analyses of codon usage and genome evolution have been facilitated by advances in synthetic biology, which now make it feasible to generate viral genomes that contain large numbers of synonymous mutations. The generally pleiotropic effects of synonymous mutation on viral fitness have, at times, made it difficult to define the mechanistic basis for the observed attenuation of these heavily mutated viruses. We have addressed this problem by developing a bioinformatic tool for the generation and analysis of viral sequences with large-scale synonymous mutation. A variety of permutation strategies are applied to shuffle codons within an open reading frame. After measuring the dinucleotide frequency, codon usage, codon pair bias, and free energy of RNA folding for each permuted genome, we used z-score normalization and a least squares regression model to quantify their overall distance from the starting sequence. Using this approach, the user can easily identify a large number of synonymously mutated sequences with varying similarity to a wild-type genome across a range of nucleic-acid-based determinants of viral fitness. We believe that this tool will be useful in designing genomes for subsequent experimental studies of the fitness impacts of synonymous mutation.

Transport genes of Chromobacterium violaceum: an overview.

The complete genome sequence of the free-living bacterium Chromobacterium violaceum has been determined by a consortium of laboratories in Brazil. Almost 500 open reading frames (ORFs) coding for transport-related membrane proteins were identified in C. violaceum, which represents 11% of all genes found. The main class of transporter proteins is the primary active transporters (212 ORFs), followed by electrochemical potential-driven transporters (154 ORFs) and channels/pores (62 ORFs). Other classes (61 ORFs) include group translocators, transport electron carriers, accessory factors, and incompletely characterized systems. Therefore, all major categories of transport-related membrane proteins currently recognized in the Transport Protein Database (http://tcdb.ucsd.edu/tcdb) are present in C. violaceum. The complex apparatus of transporters of C. violaceum is certainly an important factor that makes this bacterium a dominant microorganism in a variety of ecosystems in tropical and subtropical regions. From a biotechnological point of view, the most important finding is the transporters of heavy metals, which could lead to the exploitation of C. violaceum for bioremediation.

Search for a platelet-activating factor receptor in the Trypanosoma cruzi proteome: a potential target for Chagas disease chemotherapy

Chagas disease (CD) causes the highest burden of parasitic diseases in the Western Hemisphere and is therefore a priority for drug research and development. Platelet-activating factor (PAF) causes the CD parasite Trypanosoma cruzi to differentiate, which suggests that the parasite may express PAF receptors. Here, we explored the T. cruzi proteome for PAF receptor-like proteins. From a total of 23,000 protein sequences, we identified 29 hypothetical proteins that are predicted to have seven transmembrane domains (TMDs), which is the main characteristic of the G protein-coupled receptors (GPCRs), including the PAF receptor. The TMDs of these sequences were independently aligned with domains from 25 animal PAF receptors and the sequences were analysed for conserved residues. The conservation score mean values for the TMDs of the hypothetical proteins ranged from 31.7-44.1 percent, which suggests that if the putative T. cruzi PAF receptor is among the sequences identified, the TMDs are not highly conserved. These results suggest that T. cruzi contains several GPCR-like proteins and that one of these GPCRs may be a PAF receptor. Future studies may further validate the PAF receptor as a target for CD chemotherapy.