| Congresso Brasileiro de Microbiologia 2023 | Resumo: 109-2 | ||||
Resumo:Part of the Poxviridae family, Monkeypox virus (MPV) has a linear double stranded DNA genome and typical brick-shape morphology similar to Vaccinia and Variola virus, existing in two distinct infectious forms; Mature Virion (MV) and Enveloped Virion (EV). The major viral population produced during the infection is composed by MVs, virus with only one membrane that remain intracellular until released exclusively after cell lysis. Alternatively, MVs can be transported out of the viral factories to the Golgi apparatus for wrapping with two additional lipid bilayers generating the EVs. Both particles count with distinct mechanisms to invade host cells, and the antisera against MVs fail to neutralize EVs. During infection process, EVs binds to the host cells losing its outer lipoprotein membrane allowing the fuse of membranes. Unlike most of DNA viruses, the replication process occurs at the cytoplasm inside a replication factory. Two genetically distinct clades of MPV have been identified, the Congo Basin (Central African) clade with higher mortality reaching case fatality rate of 10%, and West African clade that causes a milder disease leading to fatal outcomes in less than 1% of cases, the latter being responsible for the recent outbreak in Brazil. Cases in humans have been reported since 1970, and the recent Monkeypox (Mpox) outbreak in 2022 involving multiple countries in different continents where the disease was not endemic. This led the World Health Organization (WHO) to declare a global emergency escalating the global Mpox outbreak as a Public Health Emergency of International Concern (PHEIC). Previous reports, including Brazilian ones, described that, due the similarity of this virus to others from the same family, the serum of patients vaccinated against smallpox can inactivate MPV. The scarce literature and few information about this virus motivated us to study and characterize a Brazilian MPV isolate from the 2022 outbreak. All the experiments were performed with biosafety level 3 practices unless the virus was boiled or fixed with formaldehyde. The first step was to establish a functional protocol to yield high purity viral particles, which was successfully defined by collecting a T1 3 day-inoculum from Vero Cells ATCC-CCL 81, scraping and centrifugating at 15.500 x g to collect cells and extracellular virus. The sample was then sonicated to lyse the cells and release the intracellular MVs on the supernatant. The lysate was positioned on the top of a 36% sucrose cushion and centrifuged at 30.000 x g to pelletize the virus and eliminate most of cellular contaminants. Using a 1.20, 1.25, and 1.30 g/cm³ CsCl discontinuous gradient centrifuged at 160.000 x g the MVs and EVs were fractionated and independently collected. An aliquot saved after every step was used to compare the purity level by transmission electron microscopy negatively stained with 2.5% uranyl acetate. MV and EV protein profile were analyzed by electrophoresis on a reducing SDS-PAGE and Western Blot assay was performed using the serum of a patient vaccinated against variola for sensibilization with primary antibodies (Abs). Anti-human IgG conjugated with peroxidase was used as secondary Ab, showing the recognition of distinct protein from MT and EV that will be identified by proteomic revealing which proteins are the target of Abs from vaccinated patients. For the next steps, MV and EV will also be characterized by proteomic and lipidomic. Palavras-chave: Monkeypox, Vaccine, Virus, Transmission Electron Microscopy, Human Antibody Agência de fomento:Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) | |||||