Background: After the isolation of Acanthamoeba polyphaga mimivirus (APMV), the study and search for new giant viruses has been intensified. Most giant viruses are associated with free-living amoebae of the genusAcanthamoeba; however other giant viruses have been isolated inVermamoeba vermiformis, such as Faustovirus, Kaumoebavirus and Orpheovirus. These studies have considerably expanded our knowledge about the diversity, structure, genomics, and evolution of giant viruses. Until now, there has been only one Orpheovirus isolate, and many aspects of its life cycle remain to be elucidated.Methods: In this study, we performed an in-depth characterization of the replication cycle and particles of Orpheovirus by transmission and scanning electron microscopy, optical microscopy and IF assays.Results: We observed, through optical and IF microscopy, morphological changes inV. vermiformiscells during Orpheovirus infection, as well as increased motility at 12 h post infection (h.p.i.). The viral factory formation and viral particle morphogenesis were analysed by transmission electron microscopy, revealing mitochondria and membrane recruitment into and around the electron-lucent viral factories. Membrane traffic inhibitor (Brefeldin A) negatively impacted particle morphogenesis. The first structure observed during particle morphogenesis was crescent-shaped bodies, which extend and are filled by the internal content until the formation of multi-layered mature particles. We also observed the formation of defective particles with different shapes and sizes. Virological assays revealed that viruses are released from the host by exocytosis at 12 h.p.i., which is associated with an increase of particle counts in the supernatant.Conclusions: The results presented here contribute to a better understanding of the biology, structures and important steps in the replication cycle of Orpheovirus.

Background: Viruses are the most numerous entities on Earth and have also been central to many episodes in the history of humankind. As the study of viruses progresses further and further, there are several limitations in transferring this knowledge to undergraduate and high school students. This deficiency is due to the difficulty in designing hands-on lessons that allow students to better absorb content, given limited financial resources and facilities, as well as the difficulty of exploiting viral particles, due to their small dimensions. The development of tools for teaching virology is important to encourage educators to expand on the covered topics and connect them to recent findings. Discoveries, such as giant DNA viruses, have provided an opportunity to explore aspects of viral particles in ways never seen before. Coupling these novel findings with techniques already explored by classical virology, including visualization of cytopathic effects on permissive cells, may represent a new way for teaching virology. This work aimed to develop a slide microscope kit that explores giant virus particles and some aspects of animal virus interaction with cell lines, with the goal of providing an innovative approach to virology teaching. Methods: Slides were produced by staining, with crystal violet, purified giant viruses and BSC-40 and Vero cells infected with viruses of the genera Orthopoxvirus, Flavivirus, and Alphavirus. Slides with amoebae infected with different species of giant viruses and stained with hemacolor reagents were also produced. Results: Staining of the giant viruses allowed better visualization of the viral particles, and this technique highlights the diversity in morphology and sizes among them. Hemacolor staining enabled visualization of viral factories in amoebae, and the staining of infected BSC-40 and Vero cell monolayers with crystal violet highlights plaque-forming units. Conclusions: This kit was used in practical virology classes for the Biological Sciences course (UFMG, Brazil), and it will soon be made available at a low-cost for elementary school teachers in institutions that have microscopes. We hope this tool will foster an inspiring learning environment.

Numerous studies have shown that viruses are present in a variety of environments on Earth, acting as drivers of biogeochemical cycles and powerful selective forces. Among them, giant viruses of amoebae have garnered attention from the scientific community due to their large particles and extensive genomes. Here, we describe the discovery of one of the largest tailed viruses in the known virosphere (averaging 1350 nm), named Naiavirus. This virus, isolated from a swamp biome in Brazil, has particles with a never-before-seen morphology and composition, and represents the first giant amoeba virus with an external envelope covering the capsid and extending over a flexible tail region. The Naiavirus genome, with nearly 1 million base pairs, reveals a unique set of genes, and does not resemble any other virus previously isolated so far.

Chikungunya virus (CHIKV) is an arbovirus that causes acute and chronic disease by strong stimulation of the immune system. It activates innate and adaptive immune cells, including monocytes, natural killer (NK) cells, and lymphocytes, as well as soluble mediators such as IL-17A, IFN-γ, IL-13, and IL-7. Despite the significant morbidity associated with CHIKV, no specific antiviral therapy is currently available. In this context, natural compounds with immunomodulatory potential, such as cannabidiol (CBD), represent promising candidates. Here, we investigated whether CBD modulates immune responses following CHIKV stimulation. Peripheral blood mononuclear cells (PBMCs) were stimulated with inactivated CHIKV and treated with CBD. Immune cell subsets were analyzed by flow cytometry, and soluble factors were profiled by Luminex assay. CBD treatment fine-tuned NK and monocyte subsets in vitro and attenuated key inflammatory pathways, particularly the IL-17A-IFN-γ axis, in response to CHIKV stimulation. These findings identify CBD as a potential immunomodulatory candidate for innovative therapeutic strategies against CHIKV and other alphaviruses.

The constant emergence of SARS-CoV-2 resistance drives the search for new antivirals. We screened the SARS-CoV-2 cysteine proteases, the main protease (M^pro) and papain-like protease (PL^pro), with 141 peptidyl and peptidomimetic inhibitors designed to target a trypanosome cysteine protease. Five compounds (1a-5a) inhibited M^pro (IC₅₀ of 0.1601-16.42 µM), whereas none inhibited PL^pro. Compounds 1a-4a inhibited human cathepsin L (hCatL; 0.184-10.74 µM), which is important for viral entry into human cells. Compounds 1a and 5a, and its synthesised (R,S) enantiomer, 5b, which share a benzyl carbamate moiety, inhibited the M^pro of SARS-CoV/MERS-CoV (0.0732-0.8295 µM). The three compounds were biochemically characterised as covalent reversible inhibitors. Compounds 5a and 5b, which contain vinyl ketone warheads, were specific for M^pro, and this behaviour was supported by covalent and noncovalent computational simulations. This study highlights the importance of revisiting legacy assets to identify starting points for new antiviral drugs.

The present study sought to search for the immunodominance related to the N-terminal, Central and C-terminal regions of HTLV-1 Tax using novel, cutting-edge peptide microarray analysis. In addition, in silico predictions were performed to verify the presence of nine amino acid peptides present along Tax restricted to the human leukocyte antigen (HLA)-A2.02*01 haplotype, as well as to verify the ability to induce pro-inflammatory and regulatory cytokines, such as IFN-γ and IL-4, respectively. Our results indicated abundant dose-dependent reactivity for HLA-A*02:01 in all regions (N-terminal, Central and C-terminal), but with specific hotspots. Furthermore, the results of fold-change over the Tax^11–19 reactivity obtained at lower concentrations of HLA-A*02:01 reveal that peptides from the three regions contain sequences that react 100 times more than Tax^11–19. On the other hand, Tax^11–19 has similar or superior HLA-A*02:01 reactivity at higher concentrations of this haplotype. The in silico analysis showed a higher frequency of IFN-γ-inducing peptides in the N-terminal portion, while the C-terminal portion showed a higher frequency of IL-4 inducers. Taken together, these results shed light on the search for new Tax immunodominant epitopes, in addition to the canonic Tax^11–19, for the rational design of immunomodulatory strategies for HTLV-1 chronic diseases.