Rio Negro virophage: Sequencing of the near complete genome and transmission electron microscopy of viral factories and particles.

Rio Negro virophage (RNV) was co-isolated with a strain of mimivirus named sambavirus, from Brazilian Amazon. We report the near complete genome sequence of RNV, the first virophage isolated in Brazil. We also present new microscopical data demonstrating that RNV particles have similar dimensions to that described to sputnik virophages.

Rio Negro virophage: Sequencing of the near complete genome and transmission electron microscopy of viral factories and particles

ABSTRACT Rio Negro virophage (RNV) was co-isolated with a strain of mimivirus named sambavirus, from Brazilian Amazon. We report the near complete genome sequence of RNV, the first virophage isolated in Brazil. We also present new microscopical data demonstrating that RNV particles have similar dimensions to that described to sputnik virophages.

Haemorrhagic smolt syndrome (HSS) in Norway: pathology and associated virus-like particles.

Atlantic salmon Salmo salar pre-smolt, smolt and post-smolt, with clinical signs of haemorrhagic smolt syndrome (HSS) have been found in several locations along the Norwegian coast (Rogaland to Troms). Affected fish had pale gills and bleeding at the fin bases, but seemed to be in good physical condition with no obvious weight loss. The internal organs and body cavity showed distinct bleedings. Petechiae were found on the gastrointestinal tract, swim bladder and peritoneum, visceral adipose tissue, heart and somatic musculature. The liver was bright yellow and sometimes mottled with petechiae and ecchymoses. Acitic fluid was found in the visceral cavity and fluid was also present in the pericardial cavity. Histological examination revealed haemorrhage in most organs. The glomeruli were degenerated and the renal tubules were filled with erythrocytes. The aims of this study were to describe the pathology and discover, if possible, the aetiology of the HSS. Tissues were collected for light and transmission electron microscopy (TEM), immunofluorescence (IFAT), reverse transcription (RT)-PCR diagnostics (screening for infectious salmon anaemia virus [ISAV], viral haemorrhagic septicaemia virus [VHSV], salmon pancreas disease virus [SPDV], sleeping disease virus [SDV] and infectious haematopoetic necrosis virus [IHNV]), and tissue homogenates (heart, liver, kidney and spleen) were sterile-filtered and inoculated into cell cultures. Homogenates made from several tissues were also injected intraperitoneally into salmon and rainbow trout Oncorhynchus mykiss. The diagnostic tests revealed no consistent findings of any pathogens, with the exception of TEM which showed 2 types of virus-like particles: Type I was 50 to 60 nm in diameter and Type II about 50 nm in diameter. These virus-like particles were found in salmon from all farms affected by HSS and screened by TEM. Several different cells, blood vessel endothelial cells, endocardial cells, heart myofibres, and leukocytes were associated with the 2 virus-like particles. The Type I particle seems to be an infectious pancreatic necrosis (IPN)-like virus, while (based on the number of target cells, particle morphology, budding and uptake into target cells) Type II particle could be a togavirus.

[The arboviruses of genus alphavirus family togaviridae were isolated from mosquitoes captured from Yantai].

OBJECTIVE: To isolate arbovirus from mosquitoes caught from Yantai. METHODS: The isolated viruses were tested for their physico- chemical properties, examined by electron microscopy and specific immuno-reactivity. RESULTS: Fifteen strains of virus were isolated from mosquitoes in 1994 from Yantai. Three of them were further assayed. The results showed that the viruses could multiply on C6/36 cell and produce typical cytopathogenic effect. The viruses couldn't cause regular sickness and death of suckling mice by intracerebral inoculation. The viruses were sensitive to ether, but resistant to 5, -Idu. One sample was examined with electron microscope, and spherical virus particles were observed. The diameter of the virus particles is about 55 +/- 2. 3nm. It did not react with the JBE virus and Bunyaviridae group specific immuno-ascitic fluids but cross-reacted with the group A Togaviridae specific immuno-ascitic fluid. CONCLUSIONS: The results showed that the viruses isolated from Yantai belong to alphavirus of togaviridae.

Mayaro virus proteins.

Mayaro virus was grown in BHK-21 cells and purified by centrifugation in a potassium-tartrate gradient (5-50%). The electron microscopy analyses of the purified virus showed an homogeneous population of enveloped particles with 69 +/- 2.3 nm in diameter. Three structural virus proteins were identified and designated p1, p2 and p3. Their average molecular weight were p1, 54 KDa; p2, 50 KDa and p3, 34 KDa. In Mayaro virus infected Aedes albopictus cells and in BHK-21 infected cells we detected six viral proteins, in which three of them are the structural virus proteins and the other three were products from processing of precursors of viral proteins, whose molecular weights are 62 KDa, 64 KDa and 110 KDa. The 34 KDa protein was the first viral protein synthesized at 5 hours post-infection in both cell lines studied.

Toga virus-like particles in acute liver failure attributed to sporadic non-A, non-B hepatitis and recurrence after liver transplantation.

Toga virus-like particles (typically 60-70 nm: enveloped with small surface spikes) were detected in the native hepatectomy specimens in 7 of 18 patients grafted for acute liver failure attributed to sporadic non-A, non-B hepatitis and in 2 patients grafted for fulminant hepatitis attributed to anti-epileptic drug hepatotoxicity. These particles were not detected in the hepatectomies from 12 other patients grafted for other causes of acute liver failure, 12 for various chronic liver diseases, and 2 histologically normal livers. Acute hepatic failure, characterized histologically by severe haemorrhagic necrosis, developed 7 days after grafting in 5 patients, all in the non-A, non-B group with toga virus-like particles in native liver. Similar virus-like particles were detected in all grafts and were in greater abundance than in the native livers. The agent may be novel because pre- and post-grafting sera were negative for antibodies against representative panels of arboviruses and in first and second generation antibody tests for hepatitis C virus.

A highly virulent togavirus-like agent associated with the fulminating disease of guinea fowl.

During a 1986 natural lethal outbreak of fulminating disease in guinea poult flocks in southwestern France, enveloped virus particles were consistently observed in the gut contents of infected birds. For the present study, a protocol was developed for the purification of these particles. Sucrose-banded virus obtained from birds infected experimentally with virus from the outbreak was found to have a buoyant density of 1.18 g/ml. The purified virus showed hemagglutinating activity, was shown by electron microscopy to have a togavirus-like morphology, and also was shown to be transmissible and pathogenic through oral ingestion. In addition, other enveloped particles have been occasionally detected in gut contents of both infected and uninfected birds; the improbability of the viral nature of these interfering particles is discussed.

Different pathogenicity of encephalitic togaviruses in organotypic cultures of spinal cord slices.

The pathogenicity of two encephalitic Togaviruses, Sindbis virus (SV), an alphavirus, and West Nile virus (WNV), a flavivirus, was studied in organotypic cultures of fetal mouse spinal cord slices grown in roller tubes. After about 3 weeks in vitro, during which time the cultures became abundantly myelinated, they were infected either by 5 X 10(5) PFU SV or by 5 X 10(6) PFU WNV per culture. The viruses caused different patterns of cytopathogenicity: SV induced severe cytotoxicity in all glia cells and neurons with concomitant demyelination within 48 hr. In contrast, WNV, even 4 days after infection, caused only mild cytopathic effects mainly to neurons and astrocytes and a slight degree of damage to the myelin sheath. A most remarkable finding was the entrapment of WNV particles in the interperiod lines of the myelin sheaths. Treatment of cultures with mouse alpha and beta interferon prior to their infection with either virus protected the cultures from any viral damage. Long-term exposure of non-infected control organotypic cultures of fetal spinal cord slices to mouse interferons had no significant effect on neuronal and glial differentiation, and myelin formation.

Toga-like virus as a cause of fulminant hepatitis attributed to sporadic non-A, non-B.

Virus-like particles (60-70 nm) with spiked surfaces budding into cell vacuoles and rod-shaped inclusions were detected in nuclei of hepatocytes from a British patient transplanted for sporadic non-A, non-B fulminant hepatitis (NANB-FHF), probably contracted in Kenya. Identical particles were seen in two successive grafts (days 2 and 10) at regrafting for recurrent FHF. Ultrastructural features resembled those of the RNA-containing arbovirus, Rift Valley fever virus, but serological markers against a representative panel for arboviruses (Togaviruses) and transmission in mice proved negative. The particles shared features with the different arboviruses seen in the hepatectomy specimen of a second patient with NANB-FHF, and in both patients an insect vector was implicated in the clinical history. The particles were identical in size to those of a third patient with NANB-FHF, who had remained in the United Kingdom. These findings, together with the recent report of isolation of an RNA-containing virus resembling the Togaviridae, in parenteral NANB, suggest that several exotic virus-like agents resembling the arboviruses may be involved in the aetiology of NANB, including in the sporadic forms of FHF in the United Kingdom.

Non-A, non-B hepatitis: visualization of virus-like particles from chimpanzee and human sera.

Virus-like particles (VLP), morphologically similar to togavirus particles, were detected in the sera of chimpanzees and humans with chronic non-A, non-B (NANB) hepatitis. Particles resembling the virus were also found in the cytoplasm of hepatocytes obtained from NANB hepatitis infected chimpanzees. Furthermore, two chimpanzees inoculated with materials containing the VLP developed NANB hepatitis. These results strongly suggest that the VLP may be closely associated with the agent of NANB hepatitis.

Virus and virus-like particles in the faeces of cats with and without diarrhoea.

Negative staining electron microscopy was used to identify viruses in 166 normal and 62 diarrhoeal faecal samples from 208 cats admitted to an animal shelter during a 16-month period (March 1984 to June 1985). On the basis of size and shape 7 distinct viral types were detected: 24 nm parvovirus-like particles, 30 nm astrovirus, 30 nm picornavirus-like particles, reovirus, rotavirus, coronavirus and a 75 nm "togavirus-like" particle. The incidence of these particles in the 208 cats was 11%, 7%, 6%, 0.4%, 5%, 1% and 1% respectively. Virus isolation studies using 40 of the faecal samples succeeded in isolating reovirus 1 in 2 cases. Immune electron microscope studies demonstrated the presence of antibody in a human serum to cat astrovirus, but failed to clarify the identity of the parvovirus-like particles and picornavirus-like particles, other than showing that some of the parvovirus-like particles were not related to feline panleukopenia virus. It was found that parvovirus-like particles, astrovirus, picornavirus-like particles, reovirus and rotavirus could be excreted by cats with normal faeces as well as cats with diarrhoeal faeces. Parvovirus-like particles, astrovirus, picornavirus-like particles and rotavirus could be excreted in high concentration in normal faeces. There was no simple relationship between age and diarrhoea in the population of cats studied. Age was not a critical factor in the excretion of parvovirus-like particles, astrovirus, picornavirus-like particles and rotavirus. The incidence of diarrhoea was not clearly associated with the seasons.

Togaviridae.

The family Togaviridae comprises four genera: Alphavirus (with 26 species), Rubivirus (one species), Pestivirus (three species), and Arterivirus (one species). The main characteristics of the member viruses are: (i) the virus particles are spherical, 50-70 nm in diameter, including an envelope with surface projections that incorporate two or three polypeptides, usually glycosylated; (ii) the nucleocapsid comprises a core protein and a single strand of positive-sense RNA, molecular weight about 4 X 10(6); where characterized, the RNA has an m7G 'cap' at the 5' end and is polyadenylated at the 3' end; (iii) maturation occurs by budding of spherical nucleocapsids 30-35 nm in diameter, with proven or presumed icosahedral symmetry, through cytoplasmic membranes. Where characterized, translation of structural proteins occurs on subgenomic messenger RNA(s); these appear to represent the 3' end of the genome. Nearly all alphavirus species are transmitted by mosquitoes. Transmission also occurs transovarially (Alphavirus) or transplacentally (Rubivirus and Pestivirus). Members of a genus are serologically related, but are not related to members of other genera.

Electron microscopic observation of a newly isolated flavivirus-like virus from field-caught mosquitoes.

Of many unidentified virus strains which were isolated from field-caught mosquitoes by using C6/36 cells (a virus-sensitive clone of Aedes albopictus cells), three strains which formed small size plaques (SP virus) in C6/36 cells were investigated by electron microscopy. Although the SP virus strains did not react with antisera against known arboviruses in serological tests, they closely resembled flaviviruses in morphology. However, when they were compared to Japanese encephalitis (JE) virus, several differences in morphogenesis were observed. Proliferating membranous structures and electron-dense amorphous areas involving precursors of the virus were observed only in cells infected with the SP virus strains. Enlarged areas of endoplasmic reticulum containing mature virions were often observed adjacent to these structures. Since the SP virus strains were isolated from wild mosquitoes and multiplied only in mosquito cells, it seems appropriate to classify them as insect viruses which resemble togaviruses morphologically.

Simian haemorrhagic fever (SHF): new virus isolate from a chronically infected patas monkey.

A new strain of simian haemorrhagic fever (SHF) virus was isolated from chronically infected patas monkey no. 248 (P-248) in USU-104 cells. The P-248 isolate had the same size, morphology and cytoplasmic site of replication as the prototype LVR strain. However, the P-248 isolate caused a persistent infection without noticeable cytopathology in USU-104 cells rather than the strongly lytic infection produced by prototype LVR virus. The capacity of P-248 virus to produce a persistent, non-lytic infection of USU-104 cells was a very stable characteristic of the isolate. Extensive serial passage of this isolate through USU-104 cells (over 50 passages) and rhesus monkeys (six passages) failed to unmask virus with lytic properties for USU-104 cells. Culture medium from persistently infected cultures assayed in rhesus monkey peritoneal mononuclear phagocytes, where measurable cytopathology occurs, was found to contain about 10(5) to 10(6) TCID50/ml of cell-free P-248 virus. Immunolabelling techniques showed only a low percentage of infected cells in persistently infected cultures. The mechanism of persistence of the P-248 isolate in USU-104 cells has not been determined but evidence suggests it does not involve interferon or defective interfering particles.