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. 2023 Aug 28:118:e230090.
doi: 10.1590/0074-02760230090. eCollection 2023.

Monkeypox (Mpox) virus isolation and ultrastructural characterisation from a Brazilian human sample case

Milene Dias Miranda  1 , Gabriela Cardoso Caldas  1   2 , Vivian Neuza Ferreira  1 , Ortrud Monika Barth  1 , Aline de Paula Dias da Silva  1   3 , Mayara Secco Torres Silva  4 , Beatriz Grinsztejn  4 , Valdiléa Gonçalves Veloso  4 , Thiago Moreno Souza  3 , Edson Elias da Silva  5 , Debora Ferreira Barreto-Vieira  1
Affiliations

Monkeypox (Mpox) virus isolation and ultrastructural characterisation from a Brazilian human sample case

Milene Dias Miranda et al. Mem Inst Oswaldo Cruz. .

Abstract

Background: According to the last 2023 Monkeypox (Mpox) Outbreak Global Map from the Centres for Disease Control and Prevention (CDC), more than 100 countries with no Mpox infection report cases. Brazil stands out in this group and is the second country with the highest number of cases in the last outbreak.

Objective: To contribute to knowledge of the virus infection effects in a cellular model, which is important for diagnosis infections not yet included in a provider´s differential diagnosis and for developing viral inhibition strategies.

Methods: We describe a virus isolation protocol for a human clinical sample from a patient from Brazil, the viral growth in a cell model through plaque forming units (PFU) assay, reverse transcriptase polymerase chain reaction (RT-PCR) and transmission electron microscopy (TEM).

Findings: We follow the viral isolation in Vero cell culture from a Mpox positive clinically diagnosed sample and show the infection effects on cellular structures using a TEM.

Main conclusions: Understanding the impact of viral growth on cellular structures and its replication kinetics may offer better strategies for the development of new drugs with antiviral properties.

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Conflict of interest statement

All authors have read and agreed to the published version of the manuscript. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Fig. 1:
Fig. 1:. Monkeypox (Mpox) cytophatic effect (CPE) at three- and six-days post-infection. Images of Vero cells without virus inoculation (A) and inoculated with a 1:10 diluted positive sample were obtained at three days post-infection at 100x (B), and six days post-infection at 100x (C); and 200x (D). Images were obtained through an inverted microscope, EVOS XL Core Imaging System (AMEX1200, Thermo Fisher Scientific).
Fig. 2:
Fig. 2:. Monkeypox virus (MPXV) particles inside Vero cells six days post-infection. MPXV infected cell (A) with MPXV particles (white arrow) in cytosol (B-C), microscope images show pyknotic nuclei (*) (C), nuclei presenting an altered chromatin profile (N) and mitochondria alteration (M) (A, B, D), and numerous vesicles (V) (C-D). Images were obtained using a Hitachi HT 7800 (Hitachi, Tokyo, Japan) transmission electron microscope (TEM).
Fig. 3:
Fig. 3:. Vero cells six days post-infection (A-D) with Monkeypox virus (MPXV) particles (arrow) inside the cytoplasm (C). Virus particles presenting 391 rectangular shapes with lateral bodies and a central core. Virus particles measuring between 250 and 300 nanometers were observed. 392 nucleus (N). images were obtained using a Hitachi HT 7800 (Hitachi, Tokyo, Japan) transmission electron microscope (TEM).
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