Secondary dengue infection in immunocompetent murine model leads to heart tissue damage.

Dengue, considered the most important arthropod-borne viral disease affecting humans, is transmitted by the bite of mosquitoes of the genus Aedes and caused by one of the four distinct serotypes of dengue virus (DENV-1, -2, -3 and -4). Infection with one of the four serotypes provides lifelong homotypic immunity. However, immunity against the heterologous serotypes is transient. As a consequence, secondary infection may lead to severer manifestations due to cross-reactivity of antibodies and T-cells. Over 500,000 people are hospitalized every year and around 2,5 million, living in endemic areas, are at risk of infection. Given the background, the development of vaccines and anti-DENV drugs is of the utmost importance, as is the characterization of an animal model for testing them. The purpose of this study was to investigate ultrastructural alterations caused by DENV secondary infection in BALB/c mice heart. To achieve our goal, six BALB/c mice were infected with DENV-1 and, 4 months later, reinfected with DENV-2. Uninfected mice were used as negative controls. Heart samples were collected and processed for ultrastructural and histopathological analysis. Our results showed edema, endothelium activation characterized by the presence of transport vesicles, free platelets in interstitium, mitochondria presenting rarefied matrix and degenerated cristae, and disorganization of muscle fibers. These results point not only to BALB/c mice susceptibility to DENV infection, but also to the fact that, although it is not an often reported occurrence, dengue can lead to heart damage. Keywords: dengue; experimental model; reinfection; BALB/c mice.

Fungi infection in honeybee hives in regions affected by Brazilian sac brood / Contaminação fúngica em colmeias de abelhas de regiões afetadas pela cria ensacada brasileira

The Brazilian Sac Brood is a disease that affects apiaries of Africanized bee hives in Brazil, thereby making them susceptible to high losses. This study investigated the pathogenicity of Africanized bee hives by the entomopathogenic fungi in a Brazilian Sac Brood endemic region. The degree of fungal contamination, presence of mycotoxins in beehive elements, and vulnerability of healthy beehives in environments subjected and not subjected to the disease were investigated. From the contaminating fungal load, species that are mycotoxin producers and pathogenic causing mortality in the bees have been isolated. The analysis of bee pollen and bee bread samples did not show the presence of the toxic pollen of Stryphnodendron (Fabaceae), which has been indicated as the causative agent of mortality in pre-pupal stage larvae. However, bee bread showed the highest correlation between substrate and fungal contamination...

A cria ensacada brasileira é uma doença que afeta apiários de colmeias de abelhas africanizadas no Brasil, tornando-os suscetíveis a perdas elevadas. Este estudo investigou a patogenicidade de fungos entomopatogênicos em colmeias de abelhas africanizadas de uma região endêmica de cria ensacada brasileira. O grau de contaminação fúngica, a presença de micotoxinas em elementos colmeia e a vulnerabilidade das colmeias saudáveis em ambientes sujeitos e não sujeitos à doença foram investigados. A partir da carga fúngica contaminante, espécies produtoras de micotoxinas e patogênicas, que provocam a mortalidade de abelhas, foram isoladas. A análise do pólen e do pão de abelha não demonstrou a presença do pólen tóxico de Stryphnodendron (Fabaceae), que tem sido apontado como agente causador da mortalidade de larvas em fase de pré-pupa. No entanto, o pão de abelha foi o substrato mais correlacionado com a contaminação fúngica...

Human and animal infections by vaccinia-like viruses in the state of Rio de Janeiro: a novel expanding zoonosis.

Since 1999, vesicular infections caused by Orthopoxvirus in humans and animals, mainly in dairy cattle, have been identified in 20 municipalities in the Rio de Janeiro state of Brazil. This paper describes studies conducted in counties of the northwestern, middle-Paraíba Valley and southern regions of the Rio de Janeiro state where 77 human, 346 bovine and 78 rodent samples were collected over the past ten years. Laboratory investigations using virus isolation, electron microscopy, molecular biology (PCR) and serological analysis confirmed Orthopoxvirus infections in 77.9% of human, 49.2% of dairy cattle and 17.9% of rodent samples. The characterisation of the Cantagalo/IOC strain reconfirmed that this virus was a vaccinia-like virus. In other regions of the Rio de Janeiro state, vesicular/pustular infections in animals and humans are suspected but these have not yet been confirmed. A continuous surveillance system has been established to monitor these regions in addition to several other states of the Brazilian Federation.

Identification and characterization of novel senescence-associated genes from barley (Hordeum vulgare) primary leaves.

Leaf senescence is the final developmental stage of a leaf. The progression of barley primary leaf senescence was followed by measuring the senescence-specific decrease in chlorophyll content and photosystem II efficiency. In order to isolate novel factors involved in leaf senescence, a differential display approach with mRNA populations from young and senescing primary barley leaves was applied. In this approach, 90 senescence up-regulated cDNAs were identified. Nine of these clones were, after sequence analyses, further characterized. The senescence-associated expression was confirmed by Northern analyses or quantitative RealTime-PCR. In addition, involvement of the phytohormones ethylene and abscisic acid in regulation of these nine novel senescence-induced cDNA fragments was investigated. Two cDNA clones showed homologies to genes with a putative regulatory function. Two clones possessed high homologies to barley retroelements, and five clones may be involved in degradation or transport processes. One of these genes was further analysed. It encodes an ADP ribosylation factor 1-like protein (HvARF1) and includes sequence motifs representing a myristoylation site and four typical and well conserved ARF-like protein domains. The localization of the protein was investigated by confocal laser scanning microscopy of onion epidermal cells after particle bombardment with chimeric HvARF1-GFP constructs. Possible physiological roles of these nine novel SAGs during barley leaf senescence are discussed.

Pathologic study of a fatal case of dengue-3 virus infection in Rio de Janeiro, Brazil.

Dengue hemorrhagic fever (DHF) is a severe febrile disease, characterized by abnormalities in hemostasis and increased vascular permeability, which in some cases results in hypovolemic shock syndrome and in dengue shock syndrome. The clinical features of DHF include plasma leakage, bleeding tendency and liver involvement. We studied the histopathological features of a fatal case of dengue-3 virus infection. The patient, a 63-year old male, presented with an acute onset of severe headache, myalgia and maculopapular rash. Tissue fragments (liver, spleen, lung, heart, kidney and lymph nodes) were collected for light microscopy studies and stained by standard methods. Histopathology revealed severe tissue damage, caused by intense hemorrhage, interstitial edema and inflammation. Some tissue sections were also processed with the immunoperoxidase reaction, which revealed the dengue viral antigen. Dengue-3 virus was isolated and identified with electron microscopy in a C6/36 cell culture inoculated with the patient's serum. Viral particles were detected in the infected cell culture.

Pathologic study of a fatal case of dengue-3 virus infection in Rio de Janeiro, Brazil

Dengue hemorrhagic fever (DHF) is a severe febrile disease, characterized by abnormalities in hemostasis and increased vascular permeability, which in some cases results in hypovolemic shock syndrome and in dengue shock syndrome. The clinical features of DHF include plasma leakage, bleeding tendency and liver involvement. We studied the histopathological features of a fatal case of dengue-3 virus infection. The patient, a 63-year old male, presented with an acute onset of severe headache, myalgia and maculopapular rash. Tissue fragments (liver, spleen, lung, heart, kidney and lymph nodes) were collected for light microscopy studies and stained by standard methods. Histopathology revealed severe tissue damage, caused by intense hemorrhage, interstitial edema and inflammation. Some tissue sections were also processed with the immunoperoxidase reaction, which revealed the dengue viral antigen. Dengue-3 virus was isolated and identified with electron microscopy in a C6/36 cell culture inoculated with the patient's serum. Viral particles were detected in the infected cell culture.

Liver injury and viremia in mice infected with dengue-2 virus.

The goal of this study was to test the feasibility of BALB/c mice as an experimental model in the study of dengue disease. BALB/c mice were intraperitoneal infected with DENV-2 obtained from a human patient. Histopathological analysis of infected animals revealed liver injury with viral antigens detection. In initial stages, the most prominent lesions were vacuolization and diffuse steatosis in hepatocytes. Serum levels of ALT and AST increased progressively, reaching the highest values 7 days p.i. and decreasing at the 14th day. Since levels of circulating virus were very low, viremia was analyzed in C6/36 cells. Virus presence was detected by ultrastructural analysis, confirmed by RT-PCR assays. Period of viremia was analyzed by flow cytometry with cells incubated with mouse-infected sera collected in different days, revealing peak virus levels at the 7th day p.i. All such data correlate to the development of the disease described in humans.

Histopathological aspects of Dengue-2 virus infected mice tissues and complementary virus isolation.

The difficulty in studying dengue virus (DENV) infection in humans and in developing a virus vaccine is the absence of a suitable animal model which develops the full spectra of the Dengue haemorrhagic fever (DHF) and Dengue shock syndrome (DSS). Despite the fact that viruses have been found in various animal tissues, we isolated DENV from tissues of adult BALB/c mice, inoculated with DENV serotype 2 (DENV-2) obtained from human serum. Viruses were ultrastructurally identified and immunolocalized by immunofluorescence techniques in C6/36 mosquito cell cultures, inoculated with tissues (liver, lung, kidney and cerebellum) macerate supernatant from mice, 48 h post-infection (p.i.). These organs, collected at the same stage of infection, were examined histologically. The histopathological analysis revealed focal alterations in all tissues examined. Liver contained focal ballooned hepatocytes, but without modifying the average diameter of the majority of hepatocytes. Sinusoidal lumen was significantly diminished at this stage but portal and centrolobular veins became congested. Lungs exhibited hemorrhagic foci in the alveolar space, vascular congestion and focal alveolitis. Cerebellar tissue showed rare foci of neuronal compactation (Purkinje cells) and perivascular oedema. In kidneys it was observed an increase in glomerular volume with augmented endocapillary and mesangial cellularity, with reactivity to anti-IgM in all glomeruli of infected mice. In conclusion, DENV-2 was found in all tissues examined early in the evolution of infection. Presence of viruses in tissues has mainly led to hemodynamic alterations with generalized vascular congestion and increased permeability, and mast cell recruitment in lungs. The latter could participate in the vascular modifications in tissues.

Construction of yellow fever-influenza A chimeric virus particles.

In order to obtain a better understanding of the functional mechanisms involved in the fusogenesis of enveloped viruses, the influenza A (X31) and the yellow fever (17DD) virus particles were used to construct a chimeric structure based on their distinct pH requirements for fusion, and the distinct malleability of their nucleocapsids. The malleable nucleocapsid of the influenza A virus particle is characterized by a pleomorphic configuration when observed by electron microscopy. A heat inactivated preparation of X31 virus was used as a lectin to interact with the sialic acid domains present in the 17DD virus envelope. The E spikes of 17DD virus were induced to promote fusion of both envelopes, creating a double genome enveloped structure, the chimeric yellow fever-influenza A virus particle. These chimeric viral particles, originally denominated 'partículas virais quiméricas' (PVQ), were characterized by their infectious capacity for different biological systems. Cell inoculation with PVQ resulted in viral products that showed similar characteristics to those obtained after 17DD virus infections. Our findings open new opportunities towards the understanding of both virus particles and aspects of cellular physiologic quality control. The yellow fever-influenza A chimeric particles, by means of their hybrid composition, should be a valuable tool in the study of cell biology and the function of viral components.

Detection of hepatitis B virus antigens in paraffin-embedded liver specimens from the Amazon region, Brazil.

Hepatic viscerotomy of paraffin-preserved old specimens, collected in the period from 1934 to 1967, were analyzed by immunohistochemical assays to detect hepatitis B, hepatitis D, dengue and yellow fever virus antigens. The material belongs to the Yellow Fever Collection, Department of Pathology, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil and the cases were diagnosed at that time according to clinical aspects and histopathological findings reporting viral hepatitis, yellow fever, focal necrosis and hepatic atrophy. From the 79 specimens, 69 were collected at the Labrea Region and the other 10 in difFerent other localities in the Amazon Region. The five micra thick histological slices were analyzed for the presence of hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg) by immunoperoxidase technique. An immunofluorescence assay was applied to the detection of hepatitis D, yellow fever and dengue virus antigens. Nine (11.4%) histological samples were HBsAg reactive and 5 (6.3%) were HBcAg reactive. The oldest reactive sample was from 1934. Viral antigens related to the other pathologies were not detected in this study. Our results confirm that the methodology described may be used to elucidate the aetiology of hepatitis diseases even after a long time of conservation of the specimens.

Detection of hepatitis B virus antigens in paraffin-embedded liver specimens from the Amazon Region, Brazil

Hepatic viscerotomy of paraffin-preserved old specimens, collected in the period from 1934 to 1967, were analyzed by immunohistochemical assays to detect hepatitis B, hepatitis D, dengue and yellow fever virus antigens. The material belongs to the Yellow Fever Collection, Department of Pathology, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil and the cases were diagnosed at that time according to clinical aspects and histopathological findings reporting viral hepatitis, yellow fever, focal necrosis and hepatic atrophy. From the 79 specimens, 69 were collected at the Labrea Region and the other 10 in different other localities in the Amazon Region. The five micra thick histological slices were analyzed for the presence of hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg) by immunoperoxidase technique. An immunofluorescence assay was applied to the detection of hepatitis D, yellow fever and dengue virus antigens. Nine (11.4 percent) histological samples were HBsAg reactive and 5 (6.3 percent) were HBcAg reactive. The oldest reactive sample was from 1934. Viral antigens related to the other pathologies were not detected in this study. Our results confirm that the methodology described may be used to elucidate the aetiology of hepatitis diseases even after a long time of conservation of the specimens

Detection of poxvirus in cattle associated with human cases in the State of Rio de Janeiro: preliminary report.

This preliminary report describes human and cow cases of poxvirus that recently occurred in the State of Rio de Janeiro. The electron microscopic findings were consistent with parapoxviral and orthopoxviral infection. Orthopoxvirus strains were isolated from human and cow cases. Detailed viral characterization by means of genetical techniques is under investigation. Based on these informations, poxviral diseases should be also considered an emerging viral zoonosis that can affect human beings.

Ultrastructural aspects of the dengue virus (flavivirus) particle morphogenesis.

The pathway of dengue virus infection in both mosquito and Vero cells in culture has been described. However, a number of stages associated with dengue virus morphogenesis remain unclear. For this reason further study involving electron microscopic in situ hybridisation of viral RNA and immunolocalisation of envelope proteins was carried out. The data obtained support the hypothesis that both viral RNA and viral proteins assemble when anchored to the viral-induced smooth membrane structures which occur within the lumen of the rER. Following the formation of the nucleocapsid, virus particles acquire their envelopes inside the lumen of the rER and associated structures. Some virus particles only are transferred to the Golgi system for maturation and are delivered from the cell by exocytosis. Nevertheless, the majority of virus particles do not pass the Golgi system but instead remain enclosed in rER-derived vesicles, even after cell and syncytial lysis. The virus replication pathway is a cell line independent process.

Intracellular localisation of dengue-2 RNA in mosquito cell culture using electron microscopic in situ hybridisation.

Non-isotopic in situ hybridisation was used at the electron microscope level to determine the localisation of viral RNA in dengue-2 infected mosquito cells at 14, 24, 48 and 72 h post-infection. In situ hybridisation was carried out on sections of dengue-2 infected mosquito cells using a digoxigenin-labelled DNA probe to the envelope protein gene sequence of the virus. Viral RNA was consistently localised over the rough endoplasmic reticulum and the virus-induced smooth membrane structures which form within the endoplasmic reticulum. During the later stages of infection electron-dense areas were observed to develop in close proximity to the smooth membrane structures. Electron microscopic in situ hybridisation showed that these denser areas contained both viral RNA and virus particles. Our results show that in dengue-2 infected mosquito cells the smooth membrane structures are an important site for the concentration of dengue viral RNA and its possible subsequent encapsidation into virus particles.

Ultrastructural aspects of spermatogenesis in Metamicrocotyla macracantha (Alexander, 1954) Koratha, 1955 (Monogenea; Microcotylidae).

Ultrastructural aspects of spermatogenesis, spermiogenesis and of the mature spermatozoon of a microcotylid monogenean Metamicrocotyla macracantha parasite from Mugil liza, are described. The irregularly-shaped spermatogonia divides by successive mitoses, forming the primary spermatocytes, identified by the presence of synaptonemal complexes in their nuclei. The spermatids formed by meiotic cell divisions of the secondary spermatocytes, differentiate into a mature spermatozoon. Cross sections of the head and the middle region of mature spermatozoa show the nucleus with strong condensed chromatin, the mitochondria with short cristae, peripheral microtubules and two axonemes with a 9 + 1 pattern, confirming the characteristics of this genus.

Scanning electron microscopic study of Prosorhynchoides arcuatus (Linton, 1990) (Bucephalidae: Digenea).

Prosorhynchoides arcuatus (Linton, 1900) from the intestine of Pomatomus saltator (L.) from the Atlantic coast of the State of Rio de Janeiro is studied by scanning electron microscopy, with detailed description of tegumental spines. Comments on the synonymy of this species with Bucephalopsis callicotyle Kohn, 1962 are made. The tegument of adult P. arcuatus presents scale like and serrated spines and uniciliated sensory papillae, distributed over the body surface and is compared with other digenetic trematodes.

Ultrastructural study on experimental infection of rotavirus in a murine heterologous model.

Viral replication, histopathological and ultrastructural changes were observed for a period of nine days in the small intestine of suckling mice infected with a simian rotavirus (SA11). Samples taken from duodenum, jejunum and ileum were prepared for light microscopy, transmission and scanning electron microscopy analysis. Histopathologic effect could be detected within 8 hr post-infection, when only a few altered cells were observed. Damage was extensive after 16 hr post-infection, showing swollen enterocytes and reduced and irregularly oriented microvilli at intestinal villi tips. Virus particles were detected at 16 and 48 hr post-infection, budding from the viroplasm into the rough endoplasmic reticulum cisternae in ileum enterocytes. Clear evidence of viral replication, observed by electron microscopy was not described before in heterologous murine models. Regeneration of the intestinal villi began at the third day post-infection. Despite some differences observed in clinical symptoms and microscopic analysis of homologous and heterologous rotavirus infections, we concluded that mechanisms of heterologous rotavirus infection in mice follow similar patterns to those observed in the homologous models.

Ultrastructural aspects of virus replication in one fatal case and several other isolates from a dengue type 2 outbreak in Rio de Janeiro.

Dengue virus replication in mosquito cell cultures was observed by electron microscopy in one fatal and 40 classical isolates from a dengue type 2 outbreak in Rio de Janeiro and compared with the prototype New Guinea C strain. All the Brazilian isolates presented, beside the classical structured dengue virus particles, fuzzy coated virus-like particles, never observed in the referencial New Guinea C virus strain. More numerous DEN-2 virus particles, fuzzy coated virus-like particles, defective virus particles and smooth membrane structures inside the rough endoplasmic reticulum characterized the unique fatal isolate examined.

[Morphological study of ovum and nymphs of Dipetalogaster maximus (Uhler, 1894) (Hemiptera, Reduviidae, Triatominae)]. / Estudo morfológico de ovos e ninfas de Dipetalogaster maximus (Uhler, 1894) (Hemiptera, Reduviidae, Triatominae).

Intending to provide new parameters to be used in Triatomines' Taxonomy, vectors of Chaga's Disease and to amplify a large range of knowledge about this species, a study of the structure of the egg and external morphology of the five nymphal instars was done. The eggs show under optical microscope (M.O.), the chorium's surface of the body and the operculum formed by poligonal areas, clearer under scanning electronic microscope (MEV). The apex of the third rostrum segment in all 5 instars nymphs, show 1 + 1 invaginated and elongated structures and the apical portion of the second tarsal segment presents a group of more or less numerous, very long, delicated and golden hairs.