A long-term prospecting study on giant viruses in terrestrial and marine Brazilian biomes.

The discovery of mimivirus in 2003 prompted the search for novel giant viruses worldwide. Despite increasing interest, the diversity and distribution of giant viruses is barely known. Here, we present data from a 2012-2022 study aimed at prospecting for amoebal viruses in water, soil, mud, and sewage samples across Brazilian biomes, using Acanthamoeba castellanii for isolation. A total of 881 aliquots from 187 samples covering terrestrial and marine Brazilian biomes were processed. Electron microscopy and PCR were used to identify the obtained isolates. Sixty-seven amoebal viruses were isolated, including mimiviruses, marseilleviruses, pandoraviruses, cedratviruses, and yaraviruses. Viruses were isolated from all tested sample types and almost all biomes. In comparison to other similar studies, our work isolated a substantial number of Marseillevirus and cedratvirus representatives. Taken together, our results used a combination of isolation techniques with microscopy, PCR, and sequencing and put highlight on richness of giant virus present in different terrestrial and marine Brazilian biomes.

The genomic and phylogenetic analysis of Marseillevirus cajuinensis raises questions about the evolution of Marseilleviridae lineages and their taxonomical organization.

Marseilleviruses (MsV) are a group of viruses that compose the Marseilleviridae family within the Nucleocytoviricota phylum. They have been found in different samples, mainly in freshwater. MsV are classically organized into five phylogenetic lineages (A/B/C/D/E), but the current taxonomy does not fully represent all the diversity of the MsV lineages. Here, we describe a novel strain isolated from a Brazilian saltwater sample named Marseillevirus cajuinensis. Based on genomics and phylogenetic analyses, M. cajuinensis exhibits a 380,653-bp genome that encodes 515 open reading frames. Additionally, M. cajuinensis encodes a transfer RNA, a feature that is rarely described for Marseilleviridae. Phylogeny suggests that M. cajuinensis forms a divergent branch within the MsV lineage A. Furthermore, our analysis suggests that the common ancestor for the five classical lineages of MsV diversified into three major groups. The organization of MsV into three main groups is reinforced by a comprehensive analysis of clusters of orthologous groups, sequence identities, and evolutionary distances considering several MsV isolates. Taken together, our results highlight the importance of discovering new viruses to expand the knowledge about known viruses that belong to the same lineages or families. This work proposes a new perspective on the Marseilleviridae lineages organization that could be helpful to a future update in the taxonomy of the Marseilleviridae family. IMPORTANCE: Marseilleviridae is a family of viruses whose members were mostly isolated from freshwater samples. In this work, we describe the first Marseillevirus isolated from saltwater samples, which we called Marseillevirus cajuinensis. Most of M. cajuinensis genomic features are comparable to other Marseilleviridae members, such as its high number of unknown proteins. On the other hand, M. cajuinensis encodes a transfer RNA, which is a gene category involved in protein translation that is rarely described in this viral family. Additionally, our phylogenetic analyses suggested the existence of, at least, three major Marseilleviridae groups. These observations provide a new perspective on Marseilleviridae lineages organization, which will be valuable in future updates to the taxonomy of the family since the current official classification does not capture all the Marseilleviridae known diversity.

Immunogenicity and safety of inactivated SARS-CoV-2 vaccine (CoronaVac) using two-dose primary protocol in children and adolescents (Immunita-002, Brazil): A phase IV six-month follow up.

Introduction: Vaccines are essential for the prevention and control of several diseases, indeed, monitoring the immune response generated by vaccines is crucial. The immune response generated by vaccination against SARS-CoV-2 in children and adolescents is not well defined regarding to the intensity and medium to long-term duration of a protective immune response, which may point out the need of booster doses and might support the decisions in public health. Objective: The study aims to evaluate the immunogenicity and safety of inactivated SARS-CoV-2 vaccine (CoronaVac) in a two-dose primary protocol in children and adolescent aging from 3 to 17 years old in Brazil. Methods: Participants were invited to participate in the research at two public healthcare centers located in Serrana (São Paulo) and Belo Horizonte (Minas Gerais), Brazil. Participants underwent medical interviews to gather their medical history, including COVID-19 history and medical records. Physical exams were conducted, including weight, blood pressure, temperature, and pulse rate measurements. Blood samples were obtained from the participants before vaccination, 1 month after the first dose, and 1, 3, and 6 months after the second dose and were followed by a virtual platform for monitoring post-vaccination reactions and symptoms of COVID-19. SARS-CoV-2 genome from Swab samples of COVID-19 positive individuals were sequenced by NGS. Total antibodies were measured by ELISA and neutralizing antibodies to B.1 lineage and Omicron variant (BA.1) quantified by PRNT and VNT. The cellular immune response was evaluated by flow cytometry by the quantification of systemic soluble immune mediators. Results: The follow-up of 640 participants showed that the CoronaVac vaccine (Sinovac/Butantan Institute) was able to significantly induce the production of total IgG antibodies to SARS-CoV-2 and the production of neutralizing antibodies to B.1 lineage and Omicron variant. In addition, a robust cellular immune response was observed with wide release of pro-inflammatory and regulatory mediators in the early post-immunization moments. Adverse events recorded so far have been mild and transient except for seven serious adverse events reported on VigiMed. Conclusions: The results indicate a robust and sustained immune response induced by the CoronaVac vaccine in children and adolescents up to six months, providing evidences to support the safety and immunogenicity of this effective immunizer.

New anti-SARS-CoV-2 aminoadamantane compounds as antiviral candidates for the treatment of COVID-19.

Here, the antiviral activity of aminoadamantane derivatives were evaluated against SARS-CoV-2. The compounds exhibited low cytotoxicity to Vero, HEK293 and CALU-3 cells up to a concentration of 1,000 µM. The inhibitory concentration (IC50) of aminoadamantane was 39.71 µM in Vero CCL-81 cells and the derivatives showed significantly lower IC50 values, especially for compounds 3F4 (0.32 µM), 3F5 (0.44 µM) and 3E10 (1.28 µM). Additionally, derivatives 3F5 and 3E10 statistically reduced the fluorescence intensity of SARS-CoV-2 protein S from Vero cells at 10 µM. Transmission microscopy confirmed the antiviral activity of the compounds, which reduced cytopathic effects induced by the virus, such as vacuolization, cytoplasmic projections, and the presence of myelin figures derived from cellular activation in the face of infection. Additionally, it was possible to observe a reduction of viral particles adhered to the cell membrane and inside several viral factories, especially after treatment with 3F4. Moreover, although docking analysis showed favorable interactions in the catalytic site of Cathepsin L, the enzymatic activity of this enzyme was not inhibited significantly in vitro. The new derivatives displayed lower predicted toxicities than aminoadamantane, which was observed for either rat or mouse models. Lastly, in vivo antiviral assays of aminoadamantane derivatives in BALB/cJ mice after challenge with the mouse-adapted strain of SARS-CoV-2, corroborated the robust antiviral activity of 3F4 derivative, which was higher than aminoadamantane and its other derivatives. Therefore, aminoadamantane derivatives show potential broad-spectrum antiviral activity, which may contribute to COVID-19 treatment in the face of emerging and re-emerging SARS-CoV-2 variants of concern.

Ecological drivers of sustained enzootic yellow fever virus transmission in Brazil, 2017-2021.

Beginning December 2016, sylvatic yellow fever (YF) outbreaks spread into southeastern Brazil, and Minas Gerais state experienced two sylvatic YF waves (2017 and 2018). Following these massive YF waves, we screened 187 free-living non-human primate (NHPs) carcasses collected throughout the state between January 2019 and June 2021 for YF virus (YFV) using RTqPCR. One sample belonging to a Callithrix, collected in June 2020, was positive for YFV. The viral strain belonged to the same lineage associated with 2017-2018 outbreaks, showing the continued enzootic circulation of YFV in the state. Next, using data from 781 NHPs carcasses collected in 2017-18, we used generalized additive mixed models (GAMMs) to identify the spatiotemporal and host-level drivers of YFV infection and intensity (an estimation of genomic viral load in the liver of infected NHP). Our GAMMs explained 65% and 68% of variation in virus infection and intensity, respectively, and uncovered strong temporal and spatial patterns for YFV infection and intensity. NHP infection was higher in the eastern part of Minas Gerais state, where 2017-2018 outbreaks affecting humans and NHPs were concentrated. The odds of YFV infection were significantly lower in NHPs from urban areas than from urban-rural or rural areas, while infection intensity was significantly lower in NHPs from urban areas or the urban-rural interface relative to rural areas. Both YFV infection and intensity were higher during the warm/rainy season compared to the cold/dry season. The higher YFV intensity in NHPs in warm/rainy periods could be a result of higher exposure to vectors and/or higher virus titers in vectors during this time resulting in the delivery of a higher virus dose and higher viral replication levels within NHPs. Further studies are needed to better test this hypothesis and further compare the dynamics of YFV enzootic cycles between different seasons.

Diversity of HLA-A2-Restricted and Immunodominant Epitope Repertoire of Human T-Lymphotropic Virus Type 1 (HTLV-1) Tax Protein: Novel Insights among N-Terminal, Central and C-Terminal Regions.

The present study sought to search for the immunodominance related to the N-terminal, Central and C-terminal regions of HTLV-1 Tax using novel, cutting-edge peptide microarray analysis. In addition, in silico predictions were performed to verify the presence of nine amino acid peptides present along Tax restricted to the human leukocyte antigen (HLA)-A2.02*01 haplotype, as well as to verify the ability to induce pro-inflammatory and regulatory cytokines, such as IFN-γ and IL-4, respectively. Our results indicated abundant dose-dependent reactivity for HLA-A*02:01 in all regions (N-terminal, Central and C-terminal), but with specific hotspots. Furthermore, the results of fold-change over the Tax11-19 reactivity obtained at lower concentrations of HLA-A*02:01 reveal that peptides from the three regions contain sequences that react 100 times more than Tax11-19. On the other hand, Tax11-19 has similar or superior HLA-A*02:01 reactivity at higher concentrations of this haplotype. The in silico analysis showed a higher frequency of IFN-γ-inducing peptides in the N-terminal portion, while the C-terminal portion showed a higher frequency of IL-4 inducers. Taken together, these results shed light on the search for new Tax immunodominant epitopes, in addition to the canonic Tax11-19, for the rational design of immunomodulatory strategies for HTLV-1 chronic diseases.

A study of the MAYV replication cycle: Correlation between the kinetics of viral multiplication and viral morphogenesis.

Mayaro virus (MAYV) is mainly found in Central and South America and causes a febrile illness followed by debilitating arthritis and arthralgia similar to chikungunya virus (CHIKV). Infection leads to long-term sequelae with a direct impact on the patient's productive capacity, resulting in economic losses. Mayaro fever is a neglected disease due to the limited epidemiological data. In Brazil, it is considered a potential public health risk with the number of cases increasing every year. Most of our knowledge about MAYV biology is inferred from data obtained from other alphaviruses as well as more recent studies on MAYV. Here, we analyzed the kinetics of viral replication through standard growth curves, quantification of intracellular and extracellular particles, and RNA quantification. We compared transmission electron microscopy data during different stages of infection. This approach allowed us to establish a chronological order of events during MAYV replication and its respective timepoints including cell entry through clathrin-mediated endocytosis occurring at 15-30 min, genome replication at 2-3 h, morphogenesis at 4 hpi, and release at 4-6 hpi. We also present evidence of uncharacterized events such as ribosome reorganization as well as clusters of early viral precursors and release through exocytosis in giant forms. Our work sheds new and specific light on the MAYV replication cycle and may contribute to future studies on the field.

In-Depth Characterization of the Chikungunya Virus Replication Cycle.

The chikungunya virus has spread globally with a remarkably high attack rate. Infection causes arthralgic sequelae that can last for years. Nevertheless, there are no specific drugs or vaccines to contain the virus. Understanding the biology of the virus, such as its replication cycle, is a powerful tool to identify new drugs and comprehend virus-host interactions. Even though the chikungunya virus has been known for a long time (it was first described in 1952), many aspects of the replication cycle remain unclear. Furthermore, part of the cycle is based on observations of other alphaviruses. In this study, we used electron and scanning microscopy, as well as biological assays, to analyze and investigate the stages of the chikungunya virus replication cycle. Based on our data, we found infection cellular activities other than those usually described for the chikungunya virus replication cycle, i.e., we show particles enveloping intracellularly without budding in a membrane-delimited morphogenesis area, and we also observed virion release by membrane protrusions. Our work provides novel details regarding the biology of chikungunya virus and fills gaps in our knowledge of its replication cycle. These findings may contribute to a better understanding of virus-host interactions and support the development of antivirals. IMPORTANCE The understanding of virus biology is essential to containing virus dissemination, and exploring the virus replication cycle is a powerful tool to do this. There are many points in the biology of the chikungunya virus that need to be clarified, especially regarding its replication cycle. Our incomplete understanding of chikungunya virus infection stages is based on studies with other alphaviruses. We systematized the chikungunya virus replication cycle using microscopic imaging in the order of infection stages, as follows: entry, replication, protein synthesis, assembly/morphogenesis, and release. The imaging evidence shows novel points in the replication cycle of enveloping without budding, as well as particle release by cell membrane protrusion.

Case report: hepatitis in a child infected with SARS-CoV-2 presenting toll-like receptor 7 Gln11Leu single nucleotide polymorphism.

BACKGROUND: Covid-19 has the respiratory tract as the main target of infection, and patients present mainly dyspnea, pneumonia, dry cough, and fever. Nevertheless, organs outside the respiratory tract had been reported in recent studies, including the gastrointestinal tract and liver. The host innate immune system recognizes pathogen-associated molecular patterns (PAMPs) through their pattern recognition receptor (PRRs). Toll-like receptor 7 (TLR-7) is a pattern recognition receptor recognizing ssRNA (SARS-CoV-2 is an ssRNA). Polymorphisms are characterized by two or more alternative forms of a distinct phenotype in the same population. Polymorphisms in tlrs genes can negatively influence the immune response to infectious diseases. There are several references in the literature to non-synonymous single nucleotide (rs) polymorphisms related to several genes. Some of them are important for the innate immunity, as rs 179008 (tlr-7), rs3775291 (tlr3), rs8177374 (tir domain-containing adaptor protein, tirap), rs1024611 (monocyte chemoattractant protein-1, mcp-1) and rs61942233 (2'-5'-oligoadenylate synthase-3, oas-3). CASE PRESENTATION: We identified a 5-year-old-male child with gastrointestinal symptoms and fever presenting acholic stool and jaundice, who was positive for SARS-CoV-2 IgM, IgA, and IgG and presenting the Gln11Leu rs 179008 in tlr-7. The child presented high levels of aspartate aminotransferase, alanine aminotransferase, bilirubin, C-reactive protein, D-dimer, gamma-glutamyl transferase, alkaline phosphatase, and was negative for serological tests for hepatitis A, B, C, E, HIV 1 and 2, herpes virus, cytomegalovirus, Epstein-Barr virus, and negative for RTqPCR for Influenza A and B, RSV and SARS-CoV-2. We also investigated other SNPs in the tlr-3 (rs3775291), tirap (rs8177374), mcp-1 (rs1024611), and oas-3 (rs61942233) genes, and no mutation was detected. After an interview with the child's caregivers, any possible accidental ingestion of drugs or hepatotoxic substances was ruled out. CONCLUSION: To our knowledge, this is the first report of a SARS-CoV-2 caused hepatitis in a male child that has the tlr-7 Gln11Leu rs 179008, which could impair an efficient initial immune response. The knowledge of the patient's immune deficiency could improve the treatment to correct this deficiency with specific medications.

Dengue virus 3 genotype I shows natural changes in heparan sulphate binding sites, cell interactions, and neurovirulence in a mouse model.

Dengue virus (DENV) is the most prevalent pathogen of the Flaviviridae family. Due to the considerable increase in DENV incidence and spread, symptoms such as CNS involvement have increased. Heparan sulphate (HS) was the first molecule identified as an adhesion factor for DENV in mammalian cells. Viral phenotypes with different HS interactions are associated with various clinical symptoms, including neurological alterations. Here, using in silico analyses, in vitro studies, and the in vivo mouse model, we characterized two natural circulating DENV3 genotype I (GI) lineage 1 (L1) in Brazil-DENV3 MG-20 (from Minas Gerais) and DENV3 PV_BR (from Rondônia) that present divergent neurovirulent profiles and sensitivity to sulphated molecules. We identified substitutions at the viral envelope (E) in positions 62 and 123 as likely responsible for the differences in neurovirulence. The E62K and E123Q substitutions in DENV3 MG-20 and DENV3 PV_BR, respectively, greatly influenced in silico electrostatic density and heparin docking results. In vivo, mice inoculated with DENV3 MG-20 died, but not those infected with DENV3 PV_BR. The clinical symptoms, such as paralysis of the lower limbs and meningoencephalitis, and histopathology, also differed between the inoculated groups. In vitro heparin and heparinases assays further demonstrated the biological impact of these substitutions. Other characteristics that have been previously associated with alterations in cell tropism and neurovirulence, such as changes in the size of lysis plaques and differences in cytopathic effects in glioblastoma cells, were also observed.

Evaluation of kinase inhibitors as potential therapeutics for flavivirus infections.

The recent introduction of Zika virus (ZIKV), the recurrence of dengue virus (DENV), and the lethality of yellow fever virus (YFV) have had a significant impact on Brazilian society and public health. Here, we targeted two cellular kinases implicated in cell proliferation and cancer that are also important for viral replication: mitogen-activated protein kinase kinase (MEK) and Src. We used two MEK inhibitors - trametinib and selumetinib - and two Src inhibitors - saracatinib and bosutinib - to inhibit ZIKV, DENV, and YFV replication in cell culture. The cytotoxicity of the four inhibitors was determined by the observation of abnormal morphology and quantification of adherent cells by crystal violet staining. The antiviral activity of these drugs was assessed based on the reduction of plaque-forming units in cell culture as evidence of the inhibition of the replication of the selected flaviviruses. All four inhibitors showed antiviral activity, but among them, trametinib was the safest and most efficacious against all of the viruses, inhibiting the replication of ZIKV and YFV by 1000-fold, and DENV2/3 by nearly 100-fold. This pan-antiviral effect shows that trametinib could be repurposed for the treatment of flaviviral infections.

Detection of SARS-CoV-2 RNA on public surfaces in a densely populated urban area of Brazil: A potential tool for monitoring the circulation of infected patients.

The world is experiencing the worst global health crisis in recent decades since December/2019 due to a new pandemic coronavirus. The COVID-19 disease, caused by SARS-CoV-2, has resulted in more than 30 million cases and 950 thousand deaths worldwide as of September 21, 2020. Determining the extent of the virus on public surfaces is critical for understanding the potential risk of infection in these areas. In this study, we investigated the presence of SARS-CoV-2 RNA on public surfaces in a densely populated urban area in Brazil. Forty-nine of 933 samples tested positive (5.25%) for SARS-CoV-2 RNA, including samples collected from distinct material surfaces, including metal and concrete, and distinct places, mainly around hospital care units and public squares. Our data indicated the contamination of public surfaces by SARS-CoV-2, suggesting the circulation of infected patients and the risk of infection for the population. Constant monitoring of the virus in urban areas is required as a strategy to fight the pandemic and prevent further infections.

Análise do reparo ósseo da superfície de implante revestida com raloxifeno através das análises topográfica, biomecânica, histológica, histométrica e imunoistoquímica / Analysis of bone repair of the implant surface coated with raloxifene through topographic, biomechanical, histological, histometric and immunohistochemical analyzes

Introdução: A fim de melhorar a osseointegração, pesquisas tem buscado tratamentos para superfícies de implantes. Objetivo: Avaliar resposta óssea na interface osso/implante, em modelo padronizado em tíbia de rato, de superfícies de implantes de titânio comercialmente puro (Ti-cp) tratadas com raloxifeno (RLX). Materiais e Métodos: Foram utilizados 144 implantes de Ti-cp, divididos em dois grupos (n=36) de acordo com o tratamento de superfície: 1-Controle (CRT); e 2-RLX. Após tratamentos, cada animal recebeu um implante em cada tíbia. Os animais foram eutanaziados após 7, 15, 30 e 40 dias para análises histológica, histométrica e imunoistoquimica. Dados quantitativos foram submetidos à ANOVA dois fatores e pós-teste de Tukey (α=0,05). Resultados: A análise histológica aos 7 dias do grupo RLX apresentou maior quantidade de tecido conjuntivo comparada ao grupo CRT. Nos demais períodos, a modelação óssea foi semelhante. A análise histométrica, em relação à área óssea neoformada (AON), apresentou diferença significante entre RLX e CRT após 7 (p=0,005) e 40 (p=0,04) dias. Em relação à análise de extensão linear de contato entre tecido ósseo e superfície do implante (ELCOI), houve diferença significante entre RLX e CRT após 7 e 15 dias (p=0,003). Na análise imunoistoquímica aos 40 dias, RLX apresentou menos matriz óssea mineralizada e AON em relação ao CRT. Conclusão: Uso do RLX para modificação de superfície de implante demonstrou características de AON semelhantes ao CRT(AU)

Background: To improve osseointegration, researches have sought implant surface treatments. Purpose: To evaluate bone response at bone/implant interface, in standardized rat tibia model, of commercially pure titanium (cp-Ti) implant treated with raloxifene (RLX). Materials and Method: 144 cp-Ti implants were divided into two groups (n=36) according to the surface treatment: 1-Control (CRT); and 2-RLX. After treatments, each animal received one implant in each tibia. The animals were euthanized after 7, 15, 30 and 40 days for histological, histometric and immunohistochemical analysis. Quantitative data were submitted to two-way ANOVA and Tukey's test (α=0.05). Results: Histological analysis at 7 days in RLX showed greater amount of connective tissue compared to CRT. In other timepoints, bone remodeling was similar. Histometric analysis, regarding the newly formed bone area (NBA), showed significant difference between RLX and CRT after 7 (p=0.005) and 40 (p=0.04) days. Regarding the analysis of linear extension of contact between bone tissue and implant surface (LECBI), there was significant difference between RLX and CRT after 7 and 15 days (p=0.003). In the immunohistochemical analysis at 40 days, RLX showed less mineralized bone matrix and NBA compared to CRT. Conclusion: Use of RLX to modify implant surface demonstrated NBA characteristics similar to CRT(AU)

Bone formation around two titanium implant surfaces placed in bone defects with and without a bone substitute material: A histological, histomorphometric, and micro-computed tomography evaluation.

OBJECTIVE: To evaluate the histological and microtomographic response of peri-implant bone tissue around titanium implants with different surface treatments, placed in bone defects filled or not filled with bone substitute materials. MATERIALS AND METHODS: Thirty rabbits were divided into two groups according to the implant surface treatment. A bone defect was created in both tibias of all the rabbits, followed by the placement of one implant in each of these defects. On the left tibia, the defect was filled with a blood clot (BC), and on the right tibia, the defect was filled with biphasic hydroxyapatite/ß-tricalcium-phosphate (HA/TCP); thus, there were four groups in total: BC-N: bone defect filled with a BC and porous surface titanium implant (control group); BC-A: bone defect filled with a BC and porous-hydrophilic surface titanium implant; HA/TCP-N: bone defect filled with a bone substitute material and porous surface titanium implant; HA/TCP-A: bone defect filled with a bone substitute material and porous-hydrophilic surface titanium implant. The animals were submitted for euthanasia at three distinct periods: 15, 30, and 60 days after implant installation. The samples were evaluated histologically and histometrically, to assess the quantity and quality of cells and the remaining bone substitute material in the grafted areas. The bone quantity was assessed by micro-computed tomography (CT). RESULTS: For both surface types, the presence of a bone substitute material led to higher values in all evaluated micro-CT parameters, except in the bone surface/volume ratio parameter. No significant statistical difference was found for new bone formation between the four groups (P < .05; CI 95%). At all periods, the HA/TCP-A group had a higher percentage of new bone formation. CONCLUSION: These results suggest that a porous hydrophilic surface in the presence of bone substitute material can accelerate peri-implant bone tissue formation.

Virus goes viral: an educational kit for virology classes.

BACKGROUND: Viruses are the most numerous entities on Earth and have also been central to many episodes in the history of humankind. As the study of viruses progresses further and further, there are several limitations in transferring this knowledge to undergraduate and high school students. This deficiency is due to the difficulty in designing hands-on lessons that allow students to better absorb content, given limited financial resources and facilities, as well as the difficulty of exploiting viral particles, due to their small dimensions. The development of tools for teaching virology is important to encourage educators to expand on the covered topics and connect them to recent findings. Discoveries, such as giant DNA viruses, have provided an opportunity to explore aspects of viral particles in ways never seen before. Coupling these novel findings with techniques already explored by classical virology, including visualization of cytopathic effects on permissive cells, may represent a new way for teaching virology. This work aimed to develop a slide microscope kit that explores giant virus particles and some aspects of animal virus interaction with cell lines, with the goal of providing an innovative approach to virology teaching. METHODS: Slides were produced by staining, with crystal violet, purified giant viruses and BSC-40 and Vero cells infected with viruses of the genera Orthopoxvirus, Flavivirus, and Alphavirus. Slides with amoebae infected with different species of giant viruses and stained with hemacolor reagents were also produced. RESULTS: Staining of the giant viruses allowed better visualization of the viral particles, and this technique highlights the diversity in morphology and sizes among them. Hemacolor staining enabled visualization of viral factories in amoebae, and the staining of infected BSC-40 and Vero cell monolayers with crystal violet highlights plaque-forming units. CONCLUSIONS: This kit was used in practical virology classes for the Biological Sciences course (UFMG, Brazil), and it will soon be made available at a low-cost for elementary school teachers in institutions that have microscopes. We hope this tool will foster an inspiring learning environment.

In-depth analysis of the replication cycle of Orpheovirus.

BACKGROUND: After the isolation of Acanthamoeba polyphaga mimivirus (APMV), the study and search for new giant viruses has been intensified. Most giant viruses are associated with free-living amoebae of the genus Acanthamoeba; however other giant viruses have been isolated in Vermamoeba vermiformis, such as Faustovirus, Kaumoebavirus and Orpheovirus. These studies have considerably expanded our knowledge about the diversity, structure, genomics, and evolution of giant viruses. Until now, there has been only one Orpheovirus isolate, and many aspects of its life cycle remain to be elucidated. METHODS: In this study, we performed an in-depth characterization of the replication cycle and particles of Orpheovirus by transmission and scanning electron microscopy, optical microscopy and IF assays. RESULTS: We observed, through optical and IF microscopy, morphological changes in V. vermiformis cells during Orpheovirus infection, as well as increased motility at 12 h post infection (h.p.i.). The viral factory formation and viral particle morphogenesis were analysed by transmission electron microscopy, revealing mitochondria and membrane recruitment into and around the electron-lucent viral factories. Membrane traffic inhibitor (Brefeldin A) negatively impacted particle morphogenesis. The first structure observed during particle morphogenesis was crescent-shaped bodies, which extend and are filled by the internal content until the formation of multi-layered mature particles. We also observed the formation of defective particles with different shapes and sizes. Virological assays revealed that viruses are released from the host by exocytosis at 12 h.p.i., which is associated with an increase of particle counts in the supernatant. CONCLUSIONS: The results presented here contribute to a better understanding of the biology, structures and important steps in the replication cycle of Orpheovirus.

Cervicofacial Necrotizing Fasciitis and Drugs.

Necrotizing fasciitis (NF) is a devastating disease that typically affects immunocompromised patients, chronically debilitated patients or drug users, but can also affect healthy patients. Necrotizing fasciitis can rapidly produce septic shock and requires immediate surgical management of the necrotic tissue. It is a bacterial infection that progresses rapidly and has a high mortality generally caused by aerobic and anaerobic bacteria. The patient was immunocompromised and drug user. During treatment, a combination of broad-spectrum antibiotic therapy with Ciprofloxacin and Metronidazole, besides the use of activated charcoal dressing composed of carbonized fabric and impregnated with 0.15% silver nitrate enveloped by layer of fabric without activated carbon, chemical-mechanical debridement with hydrogen peroxide, 0.9% saline, and povidone iodine. According to the patient presented, for the treatment of NF there is a need for broad-spectrum antibiotic therapy associated with surgical debridement, use of activated charcoal for antiseptic compression and general intensive care.

Influência do número de roscas e da densidade dos blocos de poliuretano na estabilidade primária de implantes do sistema intraoss / The influence of implant thread number and polyurethane block density on primary stability values of the Intraoss implant system

Objetivo: avaliar e comparar a estabilidade primária dos implantes cilíndricos de 3,75 mm x 11 mm Titaoss e Titaoss dupla rosca, do Sistema Intraoss, instalados em blocos de poliuretano com baixa densidade. Material e métodos: foram realizadas 32 cavidades em dois blocos de poliuretano, simulando as densidades de ossos tipo III e IV. As estabilidades dos implantes foram mensuradas por meio do torque de inserção e frequência de ressonância. Resultados: os implantes Titaoss instalados no osso tipo III apresentaram valores de torque de inserção de 43,1 ± 14,87 Ncm, enquanto os valores dos implantes Titaoss dupla rosca foram 46,9 ± 5,3 Ncm. No osso tipo IV, os implantes Titaoss apresentaram valores de 30 ± 0,0 Ncm e os dupla rosca de 29,4 ± 1,77 Ncm. Não houve diferença significante na estabilidade primária entre os grupos dos implantes (p > 0,05) instalados no mesmo tipo de osso. Os implantes Titaoss instalados no osso tipo III apresentaram valores de ISQ de 57,3 ± 4,69, ao passo que os valores para os implantes Titaoss dupla rosca foram 54,9 ± 3,98. No osso tipo IV, os implantes Titaoss apresentaram valores de 48,4 ± 4,07 ISQ e os dupla rosca de 50,8 ± 3,98 ISQ. As estabilidades primárias dos implantes foram maiores no osso tipo III quando comparado ao IV (p < 0,05). Conclusão: os dois desenhos de implantes permitem a obtenção da estabilidade primária para a técnica da carga mediata. No entanto, para submeter esses tipos de implante à carga imediata, indica-se a técnica da subfresagem.

Objectives: this in vitro study aimed to evaluate and compare the primary stability of the cylindrical implants (3.75 mm x 11 mm) Titaoss and Titaoss double thread from Titaoss System placed in cavities made of low-density polyurethane blocks. Material and methods: 32 cavities were made in two types of polyurethane blocks simulating the type III and IV bone densities. The implant stability values were measured using the final insertion torque and resonance frequency analysis. Results: the Titaoss implants placed in type III bone achieved 43.1 ± 14.87 Ncm, while the double-thread values were 46.9 ± 5.3 Ncm. For bone type IV, the Titaoss implants reached 30Ncm and the double thread 29.4± 1.77 Ncm. No statistically significant differences on implant stability were seen for both groups (p > 0.05) for the same bone type. the Titaoss implants in bone type III had 57.3 ± 4.69 and the double thread 54.9 ± 3.98 ISQ values. In type IV bone, Titaoss reached 48.4 ± 4.07 and the double thread 50.8 ± 3.98 ISQ units. The primary stability values were greater at type III than type IV bone (p < 0.05). Conclusion: both implant designs allow for primary stability. However, the use of an underpreparation technique is recommended in cases of immediate loading protocols.

Respiratory Obstruction Due to Richards Type 4 Fracture.

Facial fractures can be potentially fatal in polytraumatized patients due to retropositioning tissues to upper airway obstruction. In the first aids, this situation can be reversed using a tracheostomy procedure to break out the airways. However, fractures leading to airway obstruction are rare and according to Richards Classification can be classified into 5 types. The purpose of this article is to report a clinical patient with a bilateral condylar and mandibular symphysis fractures that resulted in airway obstruction and severe mandibular rear projection.

Assessment of bone repair in critical-size defect in the calvarium of rats after the implantation of tricalcium phosphate beta (ß-TCP).

OBJECTIVES: Evaluating the osteoconductive property of tricalcium phosphate beta (ß-TCP) in comparison to that of inorganic bovine bone for repair in a critical-size defect in the rat calvarium. MATERIALS AND METHODS: Critical-size defects of 7mm were made with a trephine in the calvaria of 48 Wistar rats. The animals were divided into four groups, and the defects in each group were filled with tricalcium phosphate beta (ß-TCP), inorganic bovine bone (Bio-Oss), autogenous bone, or left empty. The animals were euthanized at two different time points (30 and 60days post-operation). All defects were recovered with a absorbable membrane of bovine cortical bone. Histological, histometric, and immunohistochemical (osteocalcin) assessments were carried out at 30 and 60days post-operation. RESULTS: At 30days post-operation, all groups showed areas of bone formation, predominantly when autogenous grafts were used. However, there were no statistically significant differences between the treatment groups (p>0.05). After 60days, there were similarities in the bone formation patterns between the ß-TCP (26.32±) and Bio-Oss (17.35±) groups (p=0.549). In terms of the immunohistochemical assessment of osteocalcin, the clot group showed light to moderate staining at 30 and 60days. The autogenous group showed moderate staining at 30days and moderate to intense staining after 60days. The Bio-Oss group showed light to moderate staining after 30days and intense staining at 60days. The ß-TCP group showed moderate staining at 30 and 60days post-operation. CONCLUSION: ß-TCP is a good osteoconductive material with similar effects to those of inorganic bovine bone graft and is suitable for utilization in the repair of bone defects.