Mpox outbreak in Rio de Janeiro, Brazil: A translational approach.

Mpox is a zoonotic disease historically reported in Africa. Since 2003, limited outbreaks have occurred outside Africa. In 2022, the global spread of cases with sustained interhuman transmission and unusual disease features raised public health concerns. We explore the mpox outbreak in Rio de Janeiro (RJ) state, Brazil, in an observational study of mpox-suspected cases from June to December 2022. Data collection relied on a public healthcare notification form. Diagnosis was determined by MPXV-PCR. In 46 confirmed cases, anti-OPXV IgG was determined by ELISA, and seven MPXV genomes were sequenced. A total of 3095 cases were included, 816 (26.3%) with positive MPXV-PCR results. Most positive cases were men in their 30 s and MSM. A total of 285 (34.9%) MPXV-PCR+ patients live with HIV. Eight were coinfected with varicella-zoster virus. Anogenital lesions and adenomegaly were associated with the diagnosis of mpox. Females and individuals under 18 represented 9.4% and 5.4% of all confirmed cases, respectively, showing higher PCR cycle threshold (Ct) values and fewer anogenital lesions compared to adult men. Anti-OPXV IgG was detected in 29/46 (63.0%) patients. All analyzed sequences belonged to clade IIb. In RJ state, mpox presented a diverse clinical picture, represented mainly by mild cases with low complication rates and prominent genital involvement. The incidence in females and children was higher than usually reported. The observation of a bimodal distribution of Ct values, with few positive results, may suggest the need to review the diagnostic criteria in these groups.

Cost-effective 3D lung tissue spheroid as a model for SARS-CoV-2 infection and drug screening.

BACKGROUND: The SARS-CoV-2 pandemic has spurred an unparalleled scientific endeavor to elucidate the virus' structure, infection mechanisms, and pathogenesis. Two-dimensional culture systems have been instrumental in shedding light on numerous aspects of COVID-19. However, these in vitro systems lack the physiological complexity to comprehend the infection process and explore treatment options. Three-dimensional (3D) models have been proposed to fill the gap between 2D cultures and in vivo studies. Specifically, spheroids, composed of lung cell types, have been suggested for studying SARS-CoV-2 infection and serving as a drug screening platform. METHODS: 3D lung spheroids were prepared by coculturing human alveolar or bronchial epithelial cells with human lung stromal cells. The morphology, size, and ultrastructure of spheroids before and after SARS-CoV-2 infection were analyzed using optical and electron microscopy. Immunohistochemistry was used to detect spike protein and, thus, the virus presence in the spheroids. Multiplex analysis elucidated the cytokine release after virus infection. RESULTS: The spheroids were stable and kept their size and morphology after SARS-CoV-2 infection despite the presence of multivesicular bodies, endoplasmic reticulum rearrangement, tubular compartment-enclosed vesicles, and the accumulation of viral particles. The spheroid responded to the infection releasing IL-6 and IL-8 cytokines. CONCLUSION: This study demonstrates that coculture spheroids of epithelial and stromal cells can serve as a cost-effective infection model for the SARS-CoV-2 virus. We suggest using this 3D spheroid as a drug screening platform to explore new treatments related to the cytokines released during virus infection, especially for long COVID treatment.

SARS-CoV-2 egress from Vero cells: a morphological approach.

Despite being extensively studied because of the current coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) interactions with mammalian cells are still poorly understood. Furthermore, little is known about this coronavirus cycle within the host cells, particularly the steps that lead to viral egress. This study aimed to shed light on the morphological features of SARS-CoV-2 egress by utilizing transmission and high-resolution scanning electron microscopy, along with serial electron tomography, to describe the route of nascent virions towards the extracellular medium. Electron microscopy revealed that the clusters of viruses in the paracellular space did not seem to result from collective virus release. Instead, virus accumulation was observed on incurved areas of the cell surface, with egress primarily occurring through individual vesicles. Additionally, our findings showed that the emission of long membrane projections, which could facilitate virus surfing in Vero cells infected with SARS-CoV-2, was also observed in non-infected cultures, suggesting that these are constitutive events in this cell lineage.

Endoplasmic Stress Affects the Coinfection of Leishmania Amazonensis and the Phlebovirus (Bunyaviridae) Icoaraci.

Viral coinfections can modulate the severity of parasitic diseases, such as human cutaneous leishmaniasis. Leishmania parasites infect thousands of people worldwide and cause from single cutaneous self-healing lesions to massive mucosal destructive lesions. The transmission to vertebrates requires the bite of Phlebotomine sandflies, which can also transmit Phlebovirus. We have demonstrated that Leishmania infection requires and triggers the Endoplasmic stress (ER stress) response in infected macrophages. In the present paper, we tested the hypothesis that ER stress is increased and required for the aggravation of Leishmania infection due to coinfection with Phlebovirus. We demonstrated that Phlebovirus Icoaraci induces the ER stress program in macrophages mediated by the branches IRE/XBP1 and PERK/ATF4. The coinfection with L. amazonensis potentiates and sustains the ER stress, and the inhibition of IRE1α or PERK results in poor viral replication and decreased parasite load in macrophages. Importantly, we observed an increase in viral replication during the coinfection with Leishmania. Our results demonstrated the role of ER stress branches IRE1/XBP1 and PERK/ATF4 in the synergic effect on the Leishmania increased load during Phlebovirus coinfection and suggests that Leishmania infection can also increase the replication of Phlebovirus in macrophages.

Culex Flavivirus Isolation from Naturally Infected Mosquitoes Trapped at Rio de Janeiro City, Brazil.

Culex Flavivirus (CxFV) is a classical insect-specific virus, which has aroused interest after the first indication that it can produce in nature superinfection exclusion of viruses of medical interest such as West Nile. Despite the detection of CxFV in different regions, CxFV ecology and the influence of co-circulation of arboviruses remains poorly understood. Therefore, our primary goals are to observe the occurrence of CxFV infection in mosquitoes trapped in an urban area of Rio de Janeiro, Brazil, characterize the virus circulating, and provide isolates. A prospective study was carried out for eight months on the campus of the Federal University of Rio de Janeiro, trapping adult mosquitoes. The CxFV minimum infection rates were determined in this period, and the virus isolation process is fully described. Samples from this study were grouped into genotype 2, along with CxFV sequences from Latin America and Africa.

Route of Zika virus infection in Aedes aegypti by transmission electron microscopy.

BACKGROUND: Zika fever has been a global health security threat, especially in the tropical and subtropical regions where most of the cases occur. The disease is caused by Zika virus (ZIKV), which belongs to the family Flaviviridae, genus Flavivirus. The virus is transmitted by Aedes mosquitoes, mostly by Aedes aegypti, during its blood meal. In this study we present a descriptive analysis, by transmission electron microscopy (TEM), of ZIKV infection in A. aegypti elected tissues at the 3rd day of infection. ZIKV vertical transmission experiments by oral infection were conducted to explore an offspring of natural infection. RESULTS: Gut and ovary tissues harbored a higher number of viral particles. The ZIKV genome was also detected, by RT-qPCR technique, in the organism of orally infected female mosquitoes and in their eggs laid. CONCLUSIONS: The data obtained suggest that the ovary is an organ susceptible to be infected with ZIKV and that virus can be transmitted from mother to a fraction of the progeny.

Chemical composition and anti-Mayaro virus activity of Schinus terebinthifolius fruits.

Brazilian traditional medicine has explored the antiviral properties of many plant extracts, including those from the Brazilian pepper tree, Schinus terebinthifolius. In the present study, we investigated the chemical composition and anti-mayaro virus (MAYV) activity of S. terebinthifolius fruit. Extensive virucidal activity (more than 95%) was detected for the ethyl acetate extract and the isolated biflavonoids. From the ethyl acetate extract of Schinus terebinthifolius fruits, two bioflavonoids were isolated ((2S, 2″S)-2,3,2″,3″-tetrahydroamentoflavone and agathisflavone), which showed strong virucidal activity against Mayaro virus. Furthermore, several other compounds like terpenes and phenolics were identified by hyphenated techniques (GC-MS, LC-MS and HPLC-UV), as well as by mass spectrometry. Immunofluorescence assay confirmed antiviral activity and transmission electron microscopy revealed damage in viral particles treated with biflavonoids. The data suggest the direct action of the extract and the biflavonoids on the virus particles. The biflavonoids tetrahydroamentoflavone and agathisflavone had strong virucidal activity and reduced MAYV infection. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13337-021-00698-z.

Virucidal and antiviral activities of pomegranate (Punica granatum) extract against the mosquito-borne Mayaro virus.

BACKGROUND: The arthropod-borne Mayaro virus (MAYV) causes "Mayaro fever," a disease of medical significance, primarily affecting individuals in permanent contact with forested areas in tropical South America. Recently, MAYV has attracted attention due to its likely urbanization. There are currently no licensed drugs against most mosquito-transmitted viruses. Punica granatum (pomegranate) fruits cultivated in Brazil have been subjected to phytochemical investigation for the identification and isolation of antiviral compounds. In the present study, we explored the antiviral activity of pomegranate extracts in Vero cells infected with Mayaro virus. METHODS: The ethanol extract and punicalagin of pomegranate were extracted solely from the shell and purified by chromatographic fractionation, and were chemically identified using spectroscopic techniques. The cytotoxicity of the purified compounds was measured by the dye uptake assay, while their antiviral activity was evaluated by a virus yield inhibition assay. RESULTS: Pomegranate ethanol extract (CC50 = 588.9, IC50 = 12.3) and a fraction containing punicalagin as major compound (CC50 = 441.5, IC50 = 28.2) were shown to have antiviral activity (SI 49 and 16, respectively) against Mayaro virus, an alphavirus. Immunofluorescence analysis showed the virucidal effect of pomegranate extract, and transmission electron microscopy (TEM) revealed damage in viral particles treated with this extract. CONCLUSIONS: The P. granatum extract is a promising source of antiviral compounds against the alphavirus MAYV and represents an excellent candidate for future studies with other enveloped RNA viruses.

Intracellular host cell membrane remodelling induced by SARS-CoV-2 infection in vitro.

BACKGROUND INFORMATION: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection induces an alteration in the endomembrane system of the mammalian cells. In this study, we used transmission electron microscopy and electron tomography to investigate the main structural alterations in the cytoplasm of Vero cells infected with a SARS-CoV-2 isolate from São Paulo state (Brazil). RESULTS: Different membranous structures derived from the zippered endoplasmic reticulum were observed along with virus assembly through membrane budding. Also, we demonstrated the occurrence of annulate lamellae in the cytoplasm of infected cells and the presence of virus particles in the perinuclear space. CONCLUSIONS AND SIGNIFICANCE: This study contributes to a better understanding of the cell biology of SARS-CoV-2 and the mechanisms of the interaction of the virus with the host cell that promote morphological changes, recruitment of organelles and cell components, in a context of a virus-induced membrane remodelling.

Ultrastructural analysis of SARS-CoV-2 interactions with the host cell via high resolution scanning electron microscopy.

SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Here, we investigated the interaction of this new coronavirus with Vero cells using high resolution scanning electron microscopy. Surface morphology, the interior of infected cells and the distribution of viral particles in both environments were observed 2 and 48 h after infection. We showed areas of viral processing, details of vacuole contents, and viral interactions with the cell surface. Intercellular connections were also approached, and viral particles were adhered to these extensions suggesting direct cell-to-cell transmission of SARS-CoV-2.

Microscopy analysis of Zika virus morphogenesis in mammalian cells.

Zika virus (ZIKV) is an arbovirus that recently emerged in the Americas as an important pathogen mainly because of its expanded pathogenesis, and elevated tropism for neuronal cells, transposition across the placental barrier, and replication in reproductive tract cells. Thus, transmission modes are eventually independent of an invertebrate vector, which is an atypical behavior for the flavivirus genus and indicates the need to study the replication of this virus in different cell types. Although ZIKV became a target for public health programs, the interaction of this flavivirus with the infected cell is still poorly understood. Herein, we analyzed the main stages of virus morphogenesis in mammalian cells, from establishment of the viroplasm-like zone to viral release from infected cells, using super-resolution fluorescence microscopy and electron microscopy. In addition, we compared this with other host cell types and other members of the Flaviviridae family that present a similar dynamic.

Prostaglandin A1 inhibits the replication of bovine viral diarrhea virus

ABSTRACT Bovine viral diarrhea virus can cause acute disease in livestock, leading to economic losses. We show that Prostaglandin A1 inhibits bovine viral diarrhea virus replication in Madin-Darby bovine kidney cells (94% inhibition using 5 µg/mL). Light and electron microscopy of infected cells shows that Prostaglandin A1 also prevents virus-induced vacuolization, but at higher concentrations (10 µg/mL).

Prostaglandin A1 triggers Mayaro virus inhibition and heat shock protein 70 expression in an epithelial cell model.

INTRODUCTION: The Mayaro virus (MAYV), which is an arbovirus closely related to the Chikungunya virus, causes a dengue-like acute illness that is endemic to Central and South America. We investigated the anti-MAYV activity of prostaglandin A1 (PGA1), a hormone which exhibits antiviral activity against both ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) viruses. Further, we examined the effects of inducting the stress protein HSP70 following PGA1 treatment. METHODS: Hep-2 cells infected with MAYV were treated with PGA1 (0.1-6µg/ml) 12h before infection and for different periods post-infection. Inhibition of viral replication inhibition was analyzed via viral titer determination, whereas the effect of PGA1 on viral morphogenesis was examined via transmission electron microscopy (TEM). Autoradiography (with 35S methionine labeling) and western blotting were used to assess the effect of PGA1 treatment on viral and cellular protein synthesis, and on HSP70 induction, respectively. RESULTS: PGA1 strongly reduced viral replication in Hep-2 cells, particularly when added during the early stages of viral replication. Although PGA1 treatment inhibited viral replication by 95% at 24 hours post-infection (hpi), viral structural protein synthesis was inhibited only by 15%. TEM analysis suggested that PGA1 inhibited replication before viral morphogenesis. Western blot and densitometry analyses showed that PGA1 treatment increased HSP70 protein levels, although this was not detectable via autoradiography. CONCLUSIONS: PGA1 inhibits MAYV replication in Hep-2 cells at early stages of viral replication, prior to production of viral structural proteins, possibly via HSP70 induction.

Prostaglandin A1 triggers Mayaro virus inhibition and heat shock protein 70 expression in an epithelial cell model

Abstract INTRODUCTION: The Mayaro virus (MAYV), which is an arbovirus closely related to the Chikungunya virus, causes a dengue-like acute illness that is endemic to Central and South America. We investigated the anti-MAYV activity of prostaglandin A1 (PGA1), a hormone which exhibits antiviral activity against both ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) viruses. Further, we examined the effects of inducting the stress protein HSP70 following PGA1 treatment. METHODS: Hep-2 cells infected with MAYV were treated with PGA1 (0.1-6μg/ml) 12h before infection and for different periods post-infection. Inhibition of viral replication inhibition was analyzed via viral titer determination, whereas the effect of PGA1 on viral morphogenesis was examined via transmission electron microscopy (TEM). Autoradiography (with 35S methionine labeling) and western blotting were used to assess the effect of PGA1 treatment on viral and cellular protein synthesis, and on HSP70 induction, respectively. RESULTS: PGA1 strongly reduced viral replication in Hep-2 cells, particularly when added during the early stages of viral replication. Although PGA1 treatment inhibited viral replication by 95% at 24 hours post-infection (hpi), viral structural protein synthesis was inhibited only by 15%. TEM analysis suggested that PGA1 inhibited replication before viral morphogenesis. Western blot and densitometry analyses showed that PGA1 treatment increased HSP70 protein levels, although this was not detectable via autoradiography. CONCLUSIONS: PGA1 inhibits MAYV replication in Hep-2 cells at early stages of viral replication, prior to production of viral structural proteins, possibly via HSP70 induction.

A Window to Toxoplasma gondii Egress.

The Toxoplasma gondii cellular cycle has been widely studied in many lifecycle stages; however, the egress event still is poorly understood even though different types of molecules were shown to be involved. Assuming that there is no purpose or intentionality in biological phenomena, there is no such question as "Why does the parasite leaves the host cell", but "Under what conditions and how?". In this review we aimed to summarize current knowledge concerning T. gondii egress physiology (signalling pathways), structures, and route.

Prostaglandin A1 inhibits the replication of bovine viral diarrhea virus.

Bovine viral diarrhea virus can cause acute disease in livestock, leading to economic losses. We show that Prostaglandin A1 inhibits bovine viral diarrhea virus replication in Madin-Darby bovine kidney cells (94% inhibition using 5µg/mL). Light and electron microscopy of infected cells shows that Prostaglandin A1 also prevents virus-induced vacuolization, but at higher concentrations (10µg/mL).

A structural analysis of the natural egress of Toxoplasma gondii.

Previous studies have analysed the process of Toxoplasma gondii egress with the aid of inducers, such as calcium ionophores. Although calcium transients have been successful in triggering T. gondii egress, the structural panorama of "natural" and artificial events should match. The present study approaches the natural egress of this parasite using super-resolution and electron microscopy and reveals lytic and non-lytic events of individual egress; this corroborates the use of calcium ionophore as a reliable tool to trigger parasite egress. Altogether, our data suggest that different signalling routes can converge to similar structural aspects in natural and induced egress.

Thieno[2,3-b]pyridine derivatives: a new class of antiviral drugs against Mayaro virus.

Mayaro virus (MAYV) is an arthropod-borne virus and a member of the family Togaviridae, genus Alphavirus. Its infection leads to an acute illness accompanied by long-lasting arthralgia. To date, there are no antiviral drugs or vaccines against infection with MAYV and resources for the prevention or treatment of other alphaviruses are very limited. MAYV has served as a model to study the antiviral potential of several substances on alphavirus replication. In this work we evaluated the antiviral effect of seven new derivatives of thieno[2,3-b]pyridine against MAYV replication in a mammalian cell line. All derivatives were able to reduce viral production effectively at concentrations that were non-toxic for Vero cells. Molecular modeling assays predicted low toxicity risk and good oral bioavailability of the substances in humans. One of the molecules, selected for further study, demonstrated a strong anti-MAYV effect at early stages of replication, as it protected pre-treated cells and also during the late stages, affecting virus morphogenesis. This study is the first to demonstrate the antiviral effect of thienopyridine derivatives on MAYV replication in vitro, suggesting the potential application of these substances as antiviral molecules against alphaviruses. Additional in vivo research will be needed to expand the putative therapeutic applications.

Monitoring of dynamin during the Toxoplasma gondii cell cycle.

The obligate intracellular protozoan parasite Toxoplasma gondii actively invades virtually all warm-blooded nucleated cells. This process results in a non-fusogenic vacuole, inside which the parasites replicate continuously until egress signaling is triggered. In this work, we investigated the role of the large GTPase dynamin in the interaction of T. gondii with the host cell by using laser and electron microscopy during three key stages: invasion, development and egress. The detection of dynamin during invasion indicates the occurrence of endocytosis, while T. gondii egress appeared to be independent of dynamin participation. However, the presence of dynamin during T. gondii development suggests that this molecule plays undescribed roles in the tachyzoite's cell cycle.

On the debate about teleology in biology: the notion of "teleological obstacle" / O debate sobre teleologia na biologia: a noção de "obstáculo teleológico"

Among the epistemological obstacles described by Gaston Bachelard, we contend that unitary and pragmatic knowledge is correlated to the teleological categories of Ernst Mayr and is the basis for prevailing debate on the notion of "function" in biology. Given the proximity of the aspects highlighted by these authors, we propose to associate the role of teleological thinking in biology and the notion of unitary and pragmatic knowledge as an obstacle to scientific knowledge. Thus, teleological thinking persists acting as an epistemological obstacle in biology, according to Bachelardian terminology. Our investigation led us to formulate the "teleological obstacle," which we consider important for the future of biology and possibly other sciences.

Dentre os obstáculos epistemológicos descritos por Gaston Bachelard, propomos que o conhecimento unitário e pragmático se relaciona com as categorias de teleologia propostas por Ernst Mayr e fundamenta as discussões atuais sobre a noção de "função" em biologia. Dada a proximidade dos aspectos salientados por ambos, propomos relacionar o papel do pensamento teleológico na biologia e a noção do conhecimento unitário e pragmático como obstáculo ao conhecimento científico. O pensamento teleológico, portanto, ainda atua como obstáculo epistemológico na biologia, segundo a terminologia bachelardiana. Nossas investigações nos levaram à formulação do "obstáculo teleológico", que entendemos ser importante para o desenvolvimento da biologia e possivelmente para outras ciências.