Ultrastructural analysis of monkeypox virus replication in Vero cells.

In early May 2022, the first worldwide monkeypox virus (MPXV) outbreak was reported, with different clinical aspects from previously studied human monkeypox infections. Despite monkeypox medical importance, much of its biological aspects remain to be further investigated. In the present work, we evaluated ultrastructural aspects of MPXV asynchronous infections in Vero cells by transmission electron microscopy (TEM). The viral strain was isolated from a male patient infected during the 2022 outbreak. TEM analysis showed: (i) adhered intracellular mature virus particles before entry of the host cell; (ii) a reorganization of the rough endoplasmic reticulum cisternae into the so-called "mini-nuclei" structure associated with genome replication; and (iii) noticeably different sites within the viral factory presenting granular or fibrillar aspects. We also observed viral crescents, different MPXV particle morphotypes, and cellular alterations induced by infection, such as changes in the cytoskeleton structure and multimembrane vesicles abundance. Taken together, to the best of our knowledge, these results revealed for the first-time ultrastructural aspects of different steps of the MPXV cycle.

Low occurrence of Hepatitis A virus in water samples from an urban area of Southern Brazil.

Hepatitis A virus (HAV), a member of Picornaviridae family, is the main causative agent of acute viral hepatitis in the world, mainly in developing countries. HAV may be present in contaminated water and food and its presence is often associated to a lesser extent with socioeconomic factors and environmental quality. The main goals in the present study were to standardize a cell culture combined to a polymerase chain reaction protocol for the detection and quantification of viral viability and analyze whether the virus could be found in water samples collected in four urban streams of Sinos River watershed. Virus recovery was assayed from known virus concentrations measured in experimentally contaminated raw and ultrapure water (MilliQ®). Recovery rates ranged from 270% in raw water to 15,000% in ultrapure water. In a second step, a qPCR coupled to a previous passage in cells, demonstrated more analytical sensitivity when compared to samples assayed without a previous passage in cell cultures. HAV genome was detected in only 1 of 84 samples analyzed, pointing to a very low occurrence of HAV in water samples in the studied region. These findings are remarkable, since no more than 5% of the domestic sewage in this area is treated pointing to a low occurrence of HAV in the population living nearby during the study period.

Recovery rate of multiple enteric viruses artificially seeded in water and concentrated by adsorption-elution with negatively charged membranes: interaction and interference between different virus species.

Viral concentration method by adsorption-elution with negative membranes has been widely employed for concentrating viruses from environmental samples. In order to provide an adequate assessment of its recovery efficiency, this study was conducted to assess viral recovery rates for viral species commonly found in water (HAdV-5, EV, RV, BAdV and CAV-2), quantifying viral genomes at the end of the five different steps of the process. Recovery rates were analyzed for several viruses combined in a single water sample and for each virus assayed separately. Ultrapure water samples were artificially contaminated and analyzed by real-time quantitative polymerase chain reaction (qPCR). High recovery rates were found after the final stage when assessed individually (89 to 125%) and combined in the same sample (23 to > 164%). HAdV-5 exhibited >100% recovery when assayed with human viruses and other AdVs, whereas BAdV and CAV-2 were not detected. These data suggest that recovery efficiency could be related to viral structural characteristics, their electric charges and other interactions, so that they are retained with greater or lesser efficiency when coupled. This protocol could be applied to environmental samples, since high recovery rates were observed and infectious viruses were detected at the end of the concentration process.