The pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has impacted public health and the world economy and fueled a worldwide race to approve therapeutic and prophylactic agents, but so far there are no specific antiviral drugs. Understanding the biology of the virus is the first step in structuring strategies to combat it, and in this context several studies have been conducted with the aim of understanding the replication mechanism of SARS-CoV-2 in vitro systems. In this work, studies using transmission and scanning electron microscopy and 3D electron microscopy modeling were performed with the goal of characterizing the morphogenesis of SARS-CoV-2 in Vero-E6 cells. Several ultrastructural changes were observed—such as syncytia formation, cytoplasmic membrane projections, lipid droplets accumulation, proliferation of double-membrane vesicles derived from the rough endoplasmic reticulum, and alteration of mitochondria. The entry of the virus into cells occurred through endocytosis. Viral particles were observed attached to the cell membrane and in various cellular compartments, and extrusion of viral progeny took place by exocytosis. These findings allow us to infer that Vero-E6 cells are highly susceptible to SARS-CoV-2 infection as described in the literature and their replication cycle is similar to that described with SARS-CoV and MERS-CoV in vitro models.

This study compares the effects of virus-cell interactions among SARS-CoV-2 variants of concern (VOCs) isolated in Brazil in 2021, hypothesizing a correlation between cellular alterations and mortality and between viral load and transmissibility. For this purpose, reference isolates of Alpha, Gamma, Zeta, and Delta variants were inoculated into monolayers of Vero-E6 cells. Viral RNA was quantified in cell supernatants by RT‒PCR, and infected cells were analyzed by Transmission Electron Microscopy (TEM) for qualitative and quantitative evaluation of cellular changes 24, 48, and 72 hours postinfection (hpi). Ultrastructural analyses showed that all variants of SARS-CoV-2 altered the structure and function of mitochondria, nucleus, and rough endoplasmic reticulum of cells. Monolayers infected with the Delta variant showed the highest number of modified cells and the greatest statistically significant differences compared to those of other variants. Viral particles were observed in the cytosol and the cell membrane in 100 % of the cells at 48 hpi. Alpha showed the highest mean particle diameter (79 nm), and Gamma and Delta were the smallest (75 nm). Alpha and Gamma had the highest particle frequency per field at 48 hpi, while the same was observed for Zeta and Delta at 72 hpi and 24 hpi, respectively. The cycle threshold of viral RNA varied among the target protein, VOC, and time of infection. The findings presented here demonstrate that all four VOCs evaluated caused ultrastructural changes in Vero-E6 cells, which were more prominent when infection occured with the Delta variant.

The Oropouche virus (OROV) is a segmented negative-sense RNA arbovirus member of the Peribunyaviridae family, associated with recurring epidemics of Oropouche fever in Central and South America. Since its identification in 1955, OROV has been responsible for outbreaks in both rural and urban areas, with transmission involving sylvatic and urban cycles. This study focuses on the characterization of an OROV isolate from a human clinical sample collected in the state of Rio de Janeiro, a non-endemic region in Brazil, highlighting ultrastructural and morphological aspects of the viral replicative cycle in Vero cells. OROV was isolated in Vero cell monolayers which, following viral inoculation, exhibited marked cytopathic effects (CPEs), mainly represented by changes in cell morphology, including membrane protrusions and vacuolization, as well as cell death. Studies by transmission electron microscopy (TEM) revealed significant ultrastructural changes, such as apoptosis, intense remodeling of membrane-bound organelles and signs of rough endoplasmic reticulum and mitochondrial stress. Additionally, the formation of specialized cytoplasmic vacuoles and intra- and extracellular vesicles emphasized trafficking and intercellular communication as essential mechanisms in OROV infection. RT-qPCR studies confirmed the production of viral progeny in high titers, corroborating the efficiency of this experimental model. These findings contribute to a better understanding of the cytopathogenic mechanisms of OROV infection and the contribution of cellular alterations in OROV morphogenesis.

An unprecedented global outbreak caused by the monkeypox virus (MPXV) prompted the World Health Organization to declare a public health emergency of international concern on July 23, 2022. Therapeutics and vaccines for MPXV are not widely available, necessitating further studies, particularly in drug repurposing area. To this end, the standardization of in vitro infection systems is essential. The most robust in vitro studies on poxviruses concern the Vaccinia virus, and there are significant gaps in understanding the replicative cycle of MPXV. Herein, we conducted ultrastructural studies using transmission and scanning electron microscopies and 3D reconstruction to describe and elucidate the step‐by‐step morphogenesis of MPXV. Vero cells, derived from the kidney lineage of Cercopithecus aethiops monkeys, were infected with a strain isolated from an oropharyngeal swab of a patient with suspected Mpox, collected during an observational cohort study conducted between June 12 and August 19, 2022, in Rio de Janeiro, Brazil. Infected Vero cells exhibited several morphological alterations, including cell lysis plaque formation, nuclei with altered chromatin profiles, thickening of the rough endoplasmic reticulum (RER), presence of myelin figures, disorganization of mitochondrial cristae, and the formation of a granular and fibrous matrix (viral factory) surrounded by mitochondria and RER cisternae in a perinuclear space. Viral entry into cells occurred via endocytosis MPXV particles were observed adhering to cytoskeletal filaments, and viral progeny extrusion occurred through exocytosis. This article presents novel data on the morphogenesis of MPXV that have not been previously documented in the literature.

Ever since its brief introduction in the Brazilian territory in 1981, dengue virus serotype 4 (DENV-4) remained absent from the national epidemiological scenario for almost 25 years. The emergence of DENV-4 in 2010 resulted in epidemics in most Brazilian states. DENV-4, however, remains one of the least studied among the four DENV serotypes. Despite being known as a mild serotype, DENV-4 is associated with severe cases and deaths and deserves to be investigated; however, the lack of suitable experimental animal models is a limiting factor for pathogenesis studies. Here, we aimed to investigate the susceptibility and potential tropism of DENV-4 for liver, lung and heart of an immunocompetent mice model, and to evaluate and investigate the resulting morphological and ultrastructural alterations upon viral infection. BALB/c mice were inoculated intravenously with non-neuroadapted doses of DENV-4 isolated from a human case. The histopathological analysis of liver revealed typical alterations of DENV, such as microsteatosis, edema and vascular congestion, while in lung, widespread areas of hemorrhage and interstitial pneumonia were observed. While milder alterations were present in heart, characterized by limited hemorrhage and discrete presence of inflammatory infiltrate, the disorganization of the structure of the intercalated disc is of particular interest. DENV-4 RNA was detected in liver, lung, heart and serum of BALB/c mice through qRT-PCR, while the NS3 viral protein was observed in all of the aforementioned organs through immunohistochemistry. These findings indicate the susceptibility of the model to the serotype and further reinforce the usefulness of BALB/c mice in studying the many alterations caused by DENV.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has revealed a complex interplay between respiratory and neurological manifestations. This study utilized K18-hACE2 transgenic mice to investigate the morphological, ultrastructural, and transcriptomic changes induced by SARS-CoV-2 infection in both lungs and brain tissues. Histopathological analysis at seven days post-infection revealed significant pulmonary damage characterized by interstitial pneumonia, alveolar septal thickening, with a marked inflammatory infiltrate predominantly consisting of neutrophils and lymphocytes, and an abnormal profile of type II pneumocytes. Concurrently, in the brain, we observed vasculitis, gliosis, and edema, indicating an inflammatory response and vascular compromise that can disturb the blood–brain barrier. In addition, gene expression in lung tissue presented increased CCL2, IL10, and GDDA45D in infected mice and the downregulation of proinflammatory genes. However, in brain tissue, the increased expression of CCL2, CASP1, IL6, IFNB1, and GDDA45G inflammatory genes was observed in infected K18-hACE2 mice.