Analysis of the Genomic Features and Evolutionary History of Pithovirus-Like Isolates Reveals Two Major Divergent Groups of Viruses.

New representatives of the phylum Nucleocytoviricota have been rapidly described in the last decade. Despite this, not all viruses of this phylum are allocated to recognized taxonomic families, as is the case for orpheovirus, pithovirus, and cedratvirus, which form the proposed family Pithoviridae. In this study, we performed comprehensive comparative genomic analyses of 8 pithovirus-like isolates, aiming to understand their common traits and evolutionary history. Structural and functional genome annotation was performed de novo for all the viruses, which served as a reference for pangenome construction. The synteny analysis showed substantial differences in genome organization between these viruses, with very few and short syntenic blocks shared between orpheovirus and its relatives. It was possible to observe an open pangenome with a significant increase in the slope when orpheovirus was added, alongside a decrease in the core genome. Network analysis placed orpheovirus as a distant and major hub with a large fraction of unique clusters of orthologs, indicating a distant relationship between this virus and its relatives, with only a few shared genes. Additionally, phylogenetic analyses of strict core genes shared with other viruses of the phylum reinforced the divergence of orpheovirus from pithoviruses and cedratviruses. Altogether, our results indicate that although pithovirus-like isolates share common features, this group of ovoid-shaped giant viruses presents substantial differences in gene contents, genomic architectures, and the phylogenetic history of several core genes. Our data indicate that orpheovirus is an evolutionarily divergent viral entity, suggesting its allocation to a different viral family, Orpheoviridae. IMPORTANCE Giant viruses that infect amoebae form a monophyletic group named the phylum Nucleocytoviricota. Despite being genomically and morphologically very diverse, the taxonomic categories of some clades that form this phylum are not yet well established. With advances in isolation techniques, the speed at which new giant viruses are described has increased, escalating the need to establish criteria to define the emerging viral taxa. In this work, we performed a comparative genomic analysis of representatives of the putative family Pithoviridae. Based on the dissimilarity of orpheovirus from the other viruses of this putative family, we propose that orpheovirus be considered a member of an independent family, Orpheoviridae, and suggest criteria to demarcate families consisting of ovoid-shaped giant viruses.

High SARS-CoV-2 tropism and activation of immune cells in the testes of non-vaccinated deceased COVID-19 patients.

BACKGROUND: Cellular entry of SARS-CoV-2 has been shown to rely on angiotensin-converting enzyme 2 (ACE2) receptors, whose expression in the testis is among the highest in the body. Additionally, the risk of mortality seems higher among male COVID-19 patients, and though much has been published since the first cases of COVID-19, there remain unanswered questions regarding SARS-CoV-2 impact on testes and potential consequences for reproductive health. We investigated testicular alterations in non-vaccinated deceased COVID-19-patients, the precise location of the virus, its replicative activity, and the immune, vascular, and molecular fluctuations involved in the pathogenesis. RESULTS: We found that SARS-CoV-2 testicular tropism is higher than previously thought and that reliable viral detection in the testis requires sensitive nanosensors or RT-qPCR using a specific methodology. Through an in vitro experiment exposing VERO cells to testicular macerates, we observed viral content in all samples, and the subgenomic RNA's presence reinforced the replicative activity of SARS-CoV-2 in testes of the severe COVID-19 patients. The cellular structures and viral particles, observed by transmission electron microscopy, indicated that macrophages and spermatogonial cells are the main SARS-CoV-2 lodging sites, where new virions form inside the endoplasmic reticulum Golgi intermediate complex. Moreover, we showed infiltrative infected monocytes migrating into the testicular parenchyma. SARS-CoV-2 maintains its replicative and infective abilities long after the patient's infection. Further, we demonstrated high levels of angiotensin II and activated immune cells in the testes of deceased patients. The infected testes show thickening of the tunica propria, germ cell apoptosis, Sertoli cell barrier loss, evident hemorrhage, angiogenesis, Leydig cell inhibition, inflammation, and fibrosis. CONCLUSIONS: Our findings indicate that high angiotensin II levels and activation of mast cells and macrophages may be critical for testicular pathogenesis. Importantly, our findings suggest that patients who become critically ill may exhibit severe alterations and harbor the active virus in the testes.

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.

Dengue virus 3 genotype I shows natural changes in heparan sulphate binding sites, cell interactions, and neurovirulence in a mouse model.

Dengue virus (DENV) is the most prevalent pathogen of the Flaviviridae family. Due to the considerable increase in DENV incidence and spread, symptoms such as CNS involvement have increased. Heparan sulphate (HS) was the first molecule identified as an adhesion factor for DENV in mammalian cells. Viral phenotypes with different HS interactions are associated with various clinical symptoms, including neurological alterations. Here, using in silico analyses, in vitro studies, and the in vivo mouse model, we characterized two natural circulating DENV3 genotype I (GI) lineage 1 (L1) in Brazil-DENV3 MG-20 (from Minas Gerais) and DENV3 PV_BR (from Rondônia) that present divergent neurovirulent profiles and sensitivity to sulphated molecules. We identified substitutions at the viral envelope (E) in positions 62 and 123 as likely responsible for the differences in neurovirulence. The E62K and E123Q substitutions in DENV3 MG-20 and DENV3 PV_BR, respectively, greatly influenced in silico electrostatic density and heparin docking results. In vivo, mice inoculated with DENV3 MG-20 died, but not those infected with DENV3 PV_BR. The clinical symptoms, such as paralysis of the lower limbs and meningoencephalitis, and histopathology, also differed between the inoculated groups. In vitro heparin and heparinases assays further demonstrated the biological impact of these substitutions. Other characteristics that have been previously associated with alterations in cell tropism and neurovirulence, such as changes in the size of lysis plaques and differences in cytopathic effects in glioblastoma cells, were also observed.

The morphogenesis of different giant viruses as additional evidence for a common origin of Nucleocytoviricota.

Large and giant DNA viruses are a monophyletic group constituting the recently established phylum Nucleocytoviricota. The virus particle morphogenesis of these viruses exhibit striking similarities. Viral factories are established in the host cells where new virions are assembled by recruiting host membranes, forming an inner lipid layer. An outer protein layer starts as a lamellar structure, commonly referred to as viral crescents, coded by the major capsid protein gene. Also, these viruses have a conserved ATPase-coding gene related to genome encapsidation. Similar properties are described for tectiviruses, putative small ancestors of giant viruses. Here we review the morphogenesis of giant viruses and discuss how the process similarities constitute additional evidence to the common origin of Nucleocytoviricota.

Microbial risk assessment in recreational freshwaters from southern Brazil.

In this study, total coliforms (TC), Escherichia coli, enterovirus (EV), rotavirus (RV), and human mastadenovirus species C and F (HAdV-C and HAdV-F) were evaluated in water samples from Belo Stream. For HAdV-C and F, the infectivity was assessed by integrated cell culture quantitative real-time polymerase chain reaction (ICC-qPCR). Samples were collected monthly (May/2015 to April/2016) at four sites. Viral analyses were performed for both ultracentrifuge-concentrated and unconcentrated samples. For site P4 (used for recreational purposes), QMRA was applied to estimate health risks associated with exposure to E. coli and HAdV-C and F. TC and E. coli were present throughout the collection period. EV and RV were not detected. HAdV-C were present in 8.51% (1.89E + 06 to 2.28E + 07 GC (Genomic Copies)/L) and 21.27% (2.36E + 05 to 1.29E + 07 GC/L) for unconcentrated and concentrated samples, respectively. For HAdV-F were 12.76% (2.77E + 07 to 3.31E + 08 GC/L) and 48.93% (1.10E + 05 to 4.50E + 08 GC/L) for unconcentrated and concentrated samples, respectively. For unconcentrated samples, infectivity for HAdV-C was detected in 37.20% (1st ICC-qPCR) and 25.58% (2nd ICC-qPCR). For HAdV-F, infectivity was detected in 6.97% (1st ICC-qPCR) and 6.97% (2nd ICC-qPCR). For concentrated samples, HAdV-C infectious was observed in 17.02% (1st ICC-qPCR) and in 8.51% (2nd ICC-qPCR). For HAdV-F, were present in 8.51% for both 1st and 2nd ICC-qPCR. Statistical analyzes showed significant difference between the collection sites when analyzed the molecular data of HAdV-F, data of TC and E. coli. Correlation tests showed direct correlation between HAdV-F with E. coli and TC. E. coli concentrations translated to the lowest estimates of infection risks (8.58E-05 to 2.17E-03). HAdV-F concentrations were associated with the highest infection risks at 9.99E-01 and for group C, 1.29E-01 to 9.99E-01. These results show that commonly used bacterial indicators for water quality may not infer health risks associated with viruses in recreational freshwaters.

Acute stressor-selective effects on homocysteine metabolism and oxidative stress parameters in female rats.

Homocysteine levels are affected by diet factors such as vitamin deficiencies, non-diet factors such as genetic disorders, and stress exposure. Hyperhomocysteinemia has been implicated in several disorders, including cardiovascular disease, depression, schizophrenia, Alzheimer's and Parkinson's disease. Since sex differences play a role both in stress responses and in susceptibility to various diseases, the objective of this study was to evaluate possible alterations in homocysteine metabolism including cysteine, folate, and vitamin B(6), and oxidative stress markers in female rats exposed to different types of acute stress. Female rats were randomly distributed into eight groups according to stress manipulation (restraint, swimming, cold and control) and estrous cycle (diestrus and estrus). In general no significant differences were seen between rats in estrus and diestrus. Restraint stress was the only type of stress that altered homocysteine concentrations (+33% relative to controls). An increase in levels of thiobarbituric acid reactive substances (TBARS) and a decrease in total glutathione (GSHt) concentration were also observed in animals subjected to restraint and swimming stress, suggesting the possibility of oxidative damage. Thus, both the homocysteine results and the oxidative stress data indicated that restraint stress was the most powerful stress manipulation in female rats, as previously observed in male rats. These findings indicate that hormonal and gonadal differences do not interfere with stress responses related to homocysteine metabolism and suggest that putative gender-related differences in homocysteine responses are probably not involved in the differential prevalence of some diseases in human males and females.