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

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.

Primary infection of BALB/c mice with a dengue virus type 4 strain leads to kidney injury

BACKGROUND Dengue is a disease caused by dengue virus (DENV-1 through -4). Among the four serotypes, DENV-4 remains the least studied. Acute kidney injury is a potential complication of dengue generally associated with severe dengue infection. OBJECTIVES The goal of this study was to investigate the alterations caused by experimental dengue infection in the kidney of adult BALB/c mice. METHODS In this study, BALB/c mice were infected through the intravenous route with a DENV-4 strain, isolated from a human patient. The kidneys of the mice were procured and subject to histopathological and ultrastructural analysis. FINDINGS The presence of the viral antigen was confirmed through immunohistochemistry. Analysis of tissue sections revealed the presence of inflammatory cell infiltrate throughout the parenchyma. Glomerular enlargement was a common find. Necrosis of tubular cells and haemorrhage were also observed. Analysis of the kidney on a transmission electron microscope allowed a closer look into the necrotic tubular cells, which presented nuclei with condensed chromatin, and loss of cytoplasm. MAIN CONCLUSIONS Even though the kidney is probably not a primary target of dengue infection in mice, the inoculation of the virus in the blood appears to damage the renal tissue through local inflammation.

Ultrastructure of Zika virus particles in cell cultures

Zika virus (ZIKV) has infected thousands of Brazilian people and spread to other American countries since 2015. The introduction of ZIKV brought a strong impact to public health in Brazil. It is of utmost importance to identify a susceptible cell line that will enable the isolation and identification of the virus from patient samples, viral mass production, and testing of drug and vaccine candidates. Besides real-time reverse transcriptase polymerase chain reaction diagnosis for detecting the viral genome, virus isolation in cell lines was useful in order to study the structure of the viral particle and its behaviour inside cells. Analysis of ZIKV infected cell lines was achieved using transmission electron microscopy (TEM). Blood was obtained from a Brazilian patient during the first days after presenting with signs of the disease, and ZIKV from the patient’s blood was isolated in the C6/36 mosquito cell line. Afterwards, Vero cells were inoculated with the viral suspension, fixed six days after inoculation, embedded in polymers, and ultra-thin cut. Like dengue viruses, this flavivirus showed numerous virus particles present inside cellular vesicles thereby confirming the susceptibility of the Vero cell line to ZIKV replication. TEM is a unique technique available to make the virus visible.

Low impact to fixed cell processing aiming transmission electron microscopy

In cell culture, cell structures suffer strong impact due to centrifugation during processing for electron microscope observation. In order to minimise this effect, a new protocol was successfully developed. Using conventional reagents and equipments, it took over one week, but cell compression was reduced to none or the lowest deformation possible.