Comparative analysis of SARS-CoV-2 variants Alpha (B.1.1.7), Gamma (P.1), Zeta (P.2) and Delta (B.1.617.2) in Vero-E6 cells: ultrastructural characterization of cytopathology and replication kinetics.

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.

Comparative analysis of SARS-CoV-2 variants Alpha (B.1.1.7), Gamma (P.1), Zeta (P.2) and Delta (B.1.617.2) in Vero-E6 cells: ultrastructural characterization of cytopathology and replication kinetics

Abstract 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.

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.

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.

SARS-CoV-2: Ultrastructural Characterization of Morphogenesis in an In Vitro System.

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.

Transmission Electron Microscopy Studies of Dengue Viruses.

Despite the advancement of molecular biology techniques, morphological studies using transmission electron microscopy (TEM) are still being of great importance to elucidate some aspects of viral structures, morphogenesis, and pathogenesis. In relation to dengue viruses (DENV), several studies report the use of TEM to obtain a clearer definition of viral morphology, the events involved in its morphogenesis, aspects of pathogenesis, and cell tropism. In this chapter, the main technical protocols and their respective reagents for studies of DENV infection by TEM are described, both in cell culture and in biological tissue samples.

Immunocompetent Mice Infected by Two Lineages of Dengue Virus Type 2: Observations on the Pathology of the Lung, Heart and Skeletal Muscle.

Dengue virus (DENV) infection by one of the four serotypes (DENV-1 to 4) may result in a wide spectrum of clinical manifestations, with unpredictable evolution and organ involvement. Due to its association with severe epidemics and clinical manifestations, DENV-2 has been substantially investigated. In fact, the first emergence of a new lineage of the DENV-2 Asian/American genotype in Brazil (Lineage II) in 2008 was associated with severe cases and increased mortality related to organ involvement. A major challenge for dengue pathogenesis studies has been a suitable animal model, but the use of immune-competent mice, although sometimes controversial, has proven to be useful, as histological observations in infected animals reveal tissue alterations consistent to those observed in dengue human cases. Here, we aimed to investigate the outcomes caused by two distinct lineages of the DENV-2 Asian/American genotype in the lung, heart and skeletal muscle tissues of infected BALB/c mice. Tissues were submitted to histopathology, immunohistochemistry, histomorphometry and transmission electron microscopy (TEM) analysis. The viral genome was detected in heart and skeletal muscle samples. The viral antigen was detected in cardiomyocytes and endothelial cells of heart tissue. Heart and lung tissue samples presented morphological alterations comparable to those seen in dengue human cases. Creatine kinase serum levels were higher in mice infected with both lineages of DENV-2. Additionally, statistically significant differences, concerning alveolar septa thickening and heart weight, were observed between BALB/c mice infected with both DENV-2 lineages, which was demonstrated to be an appropriate experimental model for dengue pathogenesis studies on lung, heart and skeletal muscle tissues.

Brazilian Dengue Virus Type 2-Associated Renal Involvement in a Murine Model: Outcomes after Infection by Two Lineages of the Asian/American Genotype.

Dengue virus type 2 (DENV-2) is, traditionally, the most studied serotype due to its association with explosive outbreaks and severe cases. In Brazil, almost 20 years after the first introduction in the 1990s, a new lineage (Lineage II) of the DENV-2 Asian/American genotype emerged and caused an epidemic with severe cases and hospitalizations. Severe dengue includes multiple organ failure, and renal involvement can be potentially related to increased mortality. In order to better understand the role of DENV infection in renal injury, here we aimed to investigate the outcomes of infection with two distinct lineages of DENV-2 Asian/American genotype in the kidney of a murine model. BALB/c mice were infected with Lineages I and II and tissues were submitted to histopathology, immunohistochemistry, histomorphometry and ultrastructural analysis. Blood urea nitrogen (BUN) was detected in blood sample accessed by cardiac puncture. A tendency in kidney weight increase was observed in mice infected with both lineages, but urea levels, on average, were increased only in mice infected with Lineage II. The DENV antigen was detected in the tissue of mice infected with Lineage II and morphological changes were similar to those observed in human dengue cases. Furthermore, the parameters such as organ weight, urea levels and morphometric analysis, showed significant differences between the two lineages in the infected BALB/c, which was demonstrated to be a suitable experimental model for dengue pathophysiology studies in kidneys.

Comparative analysis of liver involvement caused by two DENV-2 lineages using an immunocompetent murine model.

Dengue (DEN) is the most prevalent arbovirus among humans, and four billion people live at risk of infection. The clinical manifestations of DEN are variable, and the disease may present subclinically or asymptomatically. A quarter of patients develop classical dengue (CD) or severe dengue (SD), which is potentially lethal and involves vascular permeability changes, severe hemorrhage and organ damage. The involvement of the liver is a fairly common feature in DEN, and alterations range from asymptomatic elevation of transaminases to acute liver failure. Since its introduction in Brazil in 1990, two strains of Dengue virus (DENV) serotype 2 (DENV-2) have been detected: Lineage I, which is responsible for an outbreak in 1991, and Lineage II, which caused an epidemic greater than the previous one and had a different epidemiological profile. To date, studies on different strains of the same serotype/genotype and their association with disease severity are scarce. In addition, one of the greatest challenges regarding the study of DEN pathogenesis and the development of drug and vaccine therapies is the absence of an animal model that reproduces the disease as it occurs in humans. The main goals of this study were to assess BALB/c mouse susceptibility experimentally infected by two distinct DENV-2 strains and characterize possible differences in the clinical signs and alterations induced in the liver resulting from those infections. Mice infected by the two DENV-2 lineages gained less weight than uninfected mice; however, their livers were slightly heavier. Increased AST and AST levels were observed in infected mice, and the number of platelets increased in the first 72 h of infection and subsequently decreased. Mice infected with both lineages presented leukocytosis but at different times of infection. The histopathological changes induced by both lineages were similar and comparable to the changes observed in DEN fatal cases. The viral genome was detected in two liver samples. The results demonstrate the susceptibility of BALB/c mice to both DENV-2 lineages and suggest that the changes induced by those strains are similar, although for some parameters, they are manifested at different times of infection.

Dengue, Yellow Fever, Zika and Chikungunya epidemic arboviruses in Brazil: ultrastructural aspects.

BACKGROUND: The impact of arbovirus cocirculation in Brazil is unknown. Dengue virus (DENV) reinfection may result in more intense viraemia or immunopathology, leading to more severe disease. The Zika virus (ZIKV) epidemic in the Americas provided pathogenicity evidence that had not been previously observed in flavivirus infections. In contrast to other flaviviruses, electron microscopy studies have shown that ZIKV may replicate in viroplasm-like structures. Flaviviruses produce an ensemble of structurally different virions, collectively contributing to tissue tropism and virus dissemination. OBJECTIVES AND METHODS: In this work, the Aedes albopictus mosquito cell lineage (C6/36 cells) and kidney epithelial cells from African green monkeys (Vero cells) were infected with samples of the main circulating arboviruses in Brazil [DENV-1, DENV-2, DENV-3, DENV-4, ZIKV, Yellow Fever virus (YFV) and Chikungunya virus (CHIKV)], and ultrastructural studies by transmission electron microscopy were performed. FINDINGS: We observed that ZIKV, the DENV serotypes, YFV and CHIKV particles are spherical. ZIKV, DENV-1, -2, -3 and -4 presented diameters of 40-50 nm, and CHIKV presented approximate diameters of 50-60 nm. Viroplasm-like structures was observed in ZIKV replication cycle. MAIN CONCLUSIONS: The morphogenesis of these arboviruses is similar to what has been presented in previous studies. However, we understand that further studies are needed to investigate the relationship between viroplasm-like structures and ZIKV replication dynamics.

Morphology and morphogenesis of SARS-CoV-2 in Vero-E6 cells.

BACKGROUND: The coronaviruses (CoVs) called the attention of the world for causing outbreaks of severe acute respiratory syndrome (SARS-CoV), in Asia in 2002-03, and respiratory disease in the Middle East (MERS-CoV), in 2012. In December 2019, yet again a new coronavirus (SARS-CoV-2) first identified in Wuhan, China, was associated with a severe respiratory infection, known today as COVID-19. This new virus quickly spread throughout China and 30 additional countries. As result, the World Health Organization (WHO) elevated the status of the COVID-19 outbreak from emergency of international concern to pandemic on March 11, 2020. The impact of COVID-19 on public health and economy fueled a worldwide race to approve therapeutic and prophylactic agents, but so far, there are no specific antiviral drugs or vaccines available. In current scenario, the development of in vitro systems for viral mass production and for testing antiviral and vaccine candidates proves to be an urgent matter. OBJECTIVE: The objective of this paper is study the biology of SARS-CoV-2 in Vero-E6 cells at the ultrastructural level. METHODS: In this study, we documented, by transmission electron microscopy and real-time reverse transcription polymerase chain reaction (RT-PCR), the infection of Vero-E6 cells with SARS-CoV-2 samples isolated from Brazilian patients. FINDINGS: The infected cells presented cytopathic effects and SARS-CoV-2 particles were observed attached to the cell surface and inside cytoplasmic vesicles. The entry of the virus into cells occurred through the endocytic pathway or by fusion of the viral envelope with the cell membrane. Assembled nucleocapsids were verified inside rough endoplasmic reticulum cisterns (RER). Viral maturation seemed to occur by budding of viral particles from the RER into smooth membrane vesicles. MAIN CONCLUSIONS: Therefore, the susceptibility of Vero-E6 cells to SARS-CoV-2 infection and the viral pathway inside the cells were demonstrated by ultrastructural analysis.

Foraging preferences of the native stingless bee Melipona seminigra pernigra (Apidae: Meliponini) in campo rupestre on canga of Serra dos Carajás, southeastern Amazonia / Preferências de forrageamento da abelha nativa sem ferrão Melipona seminigra pernigra (Apidae: Meliponini) em campo rupestre na canga da Serra dos Carajás, sudeste da Amazônia

Abstract: Honey pollen samples of Melipona seminigra pernigraMoure & Kerr 1950 sampled between 2017 and 2019 from experimental apiaries installed in campo rupestre on canga (CRC) vegetation of the Serra dos Carajás aimed to evaluated seasonal floral availability of undisturbed and mining-influenced areas. Around one hundred pollen types were identified mainly belonging to Fabaceae, Myrtaceae and Euphorbiaceae (31, 6 and 5 species, respectively). Mining area presented the highest pollen richness, almost twice those identified in the undisturbed areas. 80% of the pollen types are rare with concentrations ≤ 2,000 pollen grains/10 g, while the remaining were the most abundant, frequent and the primary bee sources. These latter correspond mostly to native plants species such as Tapirira guianensis Aubl., Protium spp., Aparisthmium cordatum (A.Juss.) Baill., Mimosa acutistipula var. ferrea Barneby, Periandra mediterranea (Vell.) Taub., Miconia spp., Pleroma carajasense K.Rocha, Myrcia splendens (Sw.) DC., Serjania spp. and Solanum crinitum Lam. All pollen types were identified during both seasons, but higher concentration values are related to the dry period (June-September). The statistical analysis of the pollen data indicated that there was no significant difference between undisturbed and mining-influenced areas, since primary bee sources of this study are widespread used in revegetation of mined areas.

Resumo: O conteúdo polínico de amostras de mel coletadas nos anos de 2017 e 2019 de apiários experimentais de Melipona seminigra pernigraMoure & Kerr 1950, instalado dentro de uma vegetação de campo rupestre em um afloramento de canga na Serra dos Carajás, sudeste da Amazônia, foi analisado para entender a variabilidade local dos recursos florais em áreas naturais e perturbadas. Aproximadamente 100% dos tipos polínicos foram identificados e pertencem principalmente às famílias Fabaceae, Myrtaceae e Euphorbiaceae (31, 6 e 5 espécies, respectivamente). Áreas de mineração apresentaram a maior riqueza de pólen, quase o dobro daquelas identificadas em áreas perturbadas. 80% dos tipos de pólen são raros com concentrações ≤ 2.000 grãos de pólen/10g, enquanto que os restantes foram os mais abundantes, frequentes e fontes primárias para as abelhas. Este últimos correspondem principalmente a plantas nativas como Tapirira guianensis Aubl., Protium spp., Aparisthmium cordatum (A.Juss.) Baill., Mimosa acutistipula var. ferrea Barneby, Periandra mediterrânea (Vell.) Taub., Miconia spp., Pleroma carajasense K.Rocha, Myrcia splendens (Sw.) DC., Serjania spp. e Solanum crinitum Lam. Todos os tipos polínicos foram identificados durante ambas as estações, mas altas concentrações estão relacionadas ao período seco (junho-setembro). A análise estatística indicou que não houve diferença significativa nos dados de pólen de mel entre áreas naturais e áreas anteriormente degradadas, uma vez que as fontes primárias das abelhas deste estudo são amplamente utilizadas na revegetação de áreas mineradas.

Different aspects of platelet evaluation in dengue: Measurement of circulating mediators, ability to interact with the virus, the degree of activation and quantification of intraplatelet protein content.

Platelets play a role in hemostasis, coagulation, angiogenesis, inflammation and immune response is one of the most affected cells in dengue. Here we describe some aspects of platelets by observing their specific circulating mediators, the ability to interact with the virus and morphological consequences of this interaction, activation markers and intraplatelet protein contents in dengue. We conducted this study using dengue-patients as well as healthy donors. Immunoenzymatic assay, flow cytometry, transmission electron microscopy and intraplatelet proteins expression assays were carried out. Briefly, we found an increase in sCD62L, NO or TBX2 ratio in platelet count, mostly in patients with the worse clinical outcome. After in vitro DENV infection or during natural infection, platelets underwent morphological alteration with increased expression of platelet activation markers, particularly in natural infections. Analysis of intraplatelet protein contents revealed different angiogenic and inflammatory profiles, maintaining or not extracellular matrix integrity between DF and DFWS patients. Thus, platelets are frequently affected by dengue, either by altering their own functionality, as "carrier" of the virus, or as an antiviral and mediator-secreting effector cell. Thus, strategies aimed at recovering platelet amounts in dengue seem to be essential for a better clinical outcome of the patients.

First detection of dengue virus in the saliva of immunocompetent murine model.

The lack of an experimental animal model for the study of dengue pathogenesis is a limiting factor for the development of vaccines and drugs. In previous studies, our group demonstrated the susceptibility of BALB/c mice to infection by dengue virus (DENV) 1 and 2, and the virus was successfully isolated in several organs. In this study, BALB/c mice were experimentally infected intravenously with DENV-4, and samples of their saliva were collected. Viral RNA extracted from the saliva samples was subjected to qRT-PCR, with a detection limit of 0.002 PFU/mL. The presence of DENV-4 viral RNA was detected in the saliva of two mice, presenting viral titers of 109 RNA/mL. The detection of DENV RNA via saliva sampling is not a common practice in dengue diagnosis, due to the lower detection rates in human patients. However, the results observed in this study seem to indicate that, as in humans, detection rates of DENV RNA in mouse saliva are also low, correlating the infection in both cases. This study reports the first DENV detection in the saliva of BALB/c immunocompetent mice experimentally infected with non-neuroadapted DENV-4.

Detection of Latino virus (Arenaviridae: Mammarenavirus) naturally infecting Calomys callidus.

Mammarenavirus species are associated with a specific rodent host species, although an increasing number of virus has been associated to more than one host, suggesting that co-evolution is less robust than initially thought. There are few eco-epidemiological studies of South America mammarenaviruses in non-endemic areas of Arenavirus Hemorrhagic Fever, affecting specially our current knowledge about animal reservoirs and virus range and host-virus relations. In Brazil, seven arenavirus species were described in seven different rodent species. Here in we describe a new rodent reservoir species in Brazil related to the previously described Latino mammarenavirus (LATV) MARU strain. Samples of 148 rodents from Mato Grosso state, Brazil were analyzed. Amplification of the glycoprotein precursor gene (GPC) was observed in six Calomys callidus rodents. According to phylogenetic inferences, is observed a well-supported monophyletic clade of LATV from C. callidus and other Clade C mammarenavirus. In addition, the phylogenetic relations of both genes showed a close relation between LATV MARU and Capão Seco strains, two distinct lineages. Additionally, the results obtained in this study point out to a change of scenario and in previously stabilized patterns in the dynamics of South American mammarenaviruses, showing that with more studies in AHF non-endemic or silent areas, more potential hosts for this virus will be discovered.

Structural investigation of C6/36 and Vero cell cultures infected with a Brazilian Zika virus.

Zika virus (ZIKV) is a member of the flavivirus genus, and its genome is approximately 10.8 kilobases of positive-strand RNA enclosed in a capsid and surrounded by a membrane. Studies on the replication dynamics of ZIKV are scarce, which limits the development of antiviral agents and vaccines directed against ZIKV. In this study, Aedes albopictus mosquito lineage cells (C6/36 cells) and African green monkey kidney epithelial cells (Vero cells) were inoculated with a ZIKV sample isolated from a Brazilian patient, and the infection was characterized by immunofluorescence staining, phase contrast light microscopy, transmission electron microscopy and real-time RT-PCR. The infection was observed in both cell lineages, and ZIKV particles were observed inside lysosomes, the rough endoplasmic reticulum and viroplasm-like structures. The susceptibility of C6/36 and Vero cells to ZIKV infection was demonstrated. Moreover, this study showed that part of the replicative cycle may occur within viroplasm-like structures, which has not been previously demonstrated in other flaviviruses.

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.