First observation of herpes-like virus particles in northern pike, Esox lucius L., associated with bluespot-like disease in Ireland.

Circular whitish granular lesions, 5-12 mm in diameter, were observed on the skin and fins of a wild northern pike, Esox lucius, caught in a lake in the Republic of Ireland. Histological examination of the lesions revealed hypertrophied cells in the epidermis with deeply basophilic enlarged nuclei and dark-staining granular material in the cytoplasm. Transmission electron microscopy of these cells revealed naked hexagonal herpes-like virus nucleocapsids (97 +/- 7 nm) in their nuclei while the cytoplasm contained multiple aggregates of enveloped viral particles. This is the first report of herpes-like virus particles in northern pike originating outside North America, where esocid herpesvirus-1 (EsHV-1) has previously been reported. Shared clinical, histological, morphological and epidemiological findings suggest that the observed particles in this report may also be EsHV-1.

Comparison of the structures of three circoviruses: chicken anemia virus, porcine circovirus type 2, and beak and feather disease virus.

Circoviruses are small, nonenveloped icosahedral animal viruses characterized by circular single-stranded DNA genomes. Their genomes are the smallest possessed by animal viruses. Infections with circoviruses, which can lead to economically important diseases, frequently result in virus-induced damage to lymphoid tissue and immunosuppression. Within the family Circoviridae, different genera are distinguished by differences in genomic organization. Thus, Chicken anemia virus is in the genus Gyrovirus, while porcine circoviruses and Beak and feather disease virus belong to the genus CIRCOVIRUS: Little is known about the structures of circoviruses. Accordingly, we investigated the structures of these three viruses with a view to determining whether they are related. Three-dimensional maps computed from electron micrographs showed that all three viruses have a T=1 organization with capsids formed from 60 subunits. Porcine circovirus type 2 and beak and feather disease virus show similar capsid structures with flat pentameric morphological units, whereas chicken anemia virus has stikingly different protruding pentagonal trumpet-shaped units. It thus appears that the structures of viruses in the same genus are related but that those of viruses in different genera are unrelated.

Isolation and characterisation of rhabdovirus from wild common bream Abramis brama, roach Rutilus rutilus, farmed brown trout Salmo trutta and rainbow trout Oncorhynchus mykiss in Northern Ireland.

Rhabdovirus was isolated from wild common bream Abramis brama during a disease outbreak with high mortality in Northern Ireland during May 1998. Rhabdovirus was also isolated at the same time from healthy farmed rainbow Oncorhynchus mykiss and brown trout Salmo trutta on the same stretch of river and 11 mo later from healthy wild bream and roach Rutilus rutilus in the same river system. Experimental intra-peritoneal infection of bream and mirror carp Cyprinus carpio var specularis with 2 of these isolates produced low mortality rates of < or = 12%. Serological testing of these isolates by virus neutralisation indicated that they were antigenically closely related to pike fry rhabdovirus (PFRV) but not to spring viraemia of carp virus (SVCV), while testing by enzyme-linked immunosorbent assay indicated them to be antigenically different from both. Comparison of nucleotide sequence data of a 550 base pair segment of the viral glycoprotein generated by reverse transcription-polymerase chain reaction indicated a high (> or = 96.6%) degree of similarity between these isolates and a previous Northern Ireland isolate made in 1984, a 1997 isolate from bream in the Republic of Ireland and an earlier Dutch isolate from roach. In contrast, similarity between these isolates and PFRV was < 82.4%, indicating that these viruses belong to 2 distinct genogroups, while similarity to SVCV was even lower (< 67.4%).

Bovine adenovirus type 10: properties of viruses isolated from cases of bovine haemorrhagic enterocolitis.

The isolation, serological classification and growth properties of adenoviruses isolated from fatal cases of haemorrhagic enterocolitis in calves are described. Four viruses, from different submissions, were isolated in cultures of calf testis cells and were identified as adenoviruses by electron microscopy. The four isolates were serologically identical and were classified as bovine adenovirus type 10 in cross-neutralisation tests with other bovine, ovine and porcine adenovirus species. Their growth in the nucleus of infected cells was accompanied by the production of typical adenovirus-associated inclusions. A serological survey to determine the incidence of infection with the virus in cattle in Northern Ireland demonstrated the presence of low levels of neutralising antibodies in 55 per cent of cattle over two years old, although only 8 per cent were positive at a 1/500 dilution of serum.

Identification of a 24 kDa protein expressed by chicken anaemia virus.

Antisera raised against oriented peptide conjugates were used to identify and partially characterize a 24 kDa protein product expressed by chicken anaemia virus (CAV). The peptides derived from the N and C termini of the protein were shown to react against the native protein, expressed within virus-infected cells, by immunofluorescence, immunoperoxidase and immunogold thin section electron microscopy techniques. The protein product was located by immunogold single labelling in intranuclear inclusions similar to those described previously for the 13 kDa CAV protein, which causes apoptosis. The 24 kDa protein was co-localized to the nuclear inclusions with the CAV 13 kDa protein by simultaneous dual labelling immunogold electron microscopy. Following isolation of the CAV proteins by nuclei isolation and SDS-PAGE, the antisera were used to probe for the protein by immunoblotting. The antisera recognized an expressed protein product of apparent molecular mass 30 kDa. An immunofluorescence time course study of CAV protein expression was carried out and the peptide antisera reacted against the protein at 12 h post-infection. Antisera against the 13 kDa protein reacted at similar times post-infection. This was in contrast to antisera raised against the 52 kDa capsid protein which is detectable by immunofluorescence only after 24 h. The 13 kDa and 24 kDa proteins thus appear to be early antigens produced by CAV during infection.

Studies on a new enterovirus-like virus isolated from chickens.

During investigations into an outbreak of respiratory distress in broilers chicks, a small round virus was isolated following inoculation into chicken embryos. The isolate, designated 612, was identified as an enterovirus-like virus on the basis of its size and morphology, resistance to chloroform and to treatment at pH 3.0, and intracytoplasmic replication in cell culture. The virus produced a partial cytopathic effect following inoculation into chick embryo kidney cell cultures and viral antigens could be detected by immunostaining. The preferred culture method for 612 virus was by inoculation onto the CAM of chick embryos. Cross-immunofluorescence indicated that the virus is not antigenically related to five previously identified chicken enterovirus/enterovirus-like virus serogroups. Following experimental inoculation of 1-day-old male broilers a number of which had maternal antibody to 612, growth retardation ranging from 9.6 to 20.4% was detected. Serological studies demonstrated antibody to 612 virus was widespread in commercial chicken flocks in N. Ireland.

A single chicken anemia virus protein induces apoptosis.

Chicken anemia virus (CAV) causes cytopathogenic effects in chicken thymocytes and cultured transformed mononuclear cells via apoptosis. Early after infection of chicken mononuclear cells, the CAV-encoded protein VP3 exhibits a finely granular distribution within the nucleus. At a later stage after infection, VP3 forms aggregates. At this point, the cell becomes apoptotic and the cellular DNA is fragmented and condensed. By immunogold electron microscopy VP3 was shown to be associated with apoptotic structures. In vitro, expression of VP3 induced apoptosis in chicken lymphoblastoid T cells and myeloid cells, which are susceptible to CAV infection, but not in chicken embryo fibroblasts, which are not susceptible to CAV. Expression of a C-terminally truncated VP3 induced much less pronounced apoptosis in the chicken lymphoblastoid T cells.

Immuno-gold labelling of turkey rhinotracheitis virus.

The morphology and morphogenesis of five viruses isolated in Great Britain, France and South Africa from turkeys with rhinotracheitis were examined. The five isolates were antigenically related by immunofluorescence and were indistinguishable by negative contrast and thin section electron microscopy. Negative contrast electron microscopy of infected Vero cell cultures revealed ortho- or paramyxovirus-like particles and helical nucleocapsids 14 nm in diameter with a pitch of 6 nm. The viral nature of these structures was confirmed by immuno-gold labelling, using a hyperimmune rabbit antiserum to TRT virus and 15 nm gold-labelled goat anti-rabbit IgG. Ultrastructural changes characteristics of paramyxovirus infection were observed in Vero cell cultures infected with each of the five TRT virus isolates. These alterations, which included areas of localised thickening of plasma membrane, associated cytoplasmic inclusions of nucleocapsids and budding virus particles also labelled specifically following immunogold staining. These observations are in accord with the suggestion that TRT virus is an avian pneumovirus.

Chicken anemia agent: an electron microscopic study.

Particles of chicken anemia agent (CAA) negatively stained with uranyl acetate were found to be 26.5 nm in diameter. The surface detail evident on the particles indicated that the virus capsid was composed of 32 structural subunits arranged as in a class P = 3 icosahedron with a triangulation number of 3. Using mouse monoclonal antibodies to CAA and a gold-labeled goat anti-mouse IgG, CAA-specific structures were observed by thin-section electron microscopy in infected MDCC-MSB1 cells and in thymic lymphocytes from experimentally infected chicks. These consisted of electron-dense, granular, non-membrane-bound nuclear inclusions, which were often ring-shaped, and cytoplasmic accumulations of microtubules. Aggregates of virus-like particles were sometimes observed in the nuclei of infected MDCC-MSB1 cells. The nucleolar involvement that is characteristic of the morphogenesis of parvoviruses was not observed with CAA.

Electron microscopic visualisation of the in vitro phagocytosis of group B streptococci by bovine polymorphonuclear leucocytes.

The phagocytosis of group B streptococci by bovine blood polymorphonuclear leucocytes, after 30 minutes at 37 degrees C, was visualised using scanning and transmission electron microscopy. Several polymorphonuclear leucocytes were seen to have phagocytosed more than one bacterium, despite the initial ratio of bacteria to cells being unity. All bacteria within the cells appeared to be viable and some were undergoing multiplication.

Detection of viruses in avian faeces by direct electron microscopy.

A total of 151 specimens of turkey and chicken faeces and intestinal contents were examined for the presence of viruses by electron microscopy. Viruses were detected in 48 of these specimens (32%). The most frequently observed viruses were rotaviruses and enterovirus-like particles. Rotavirus infection was associated with outbreaks of diarrhoea in turkeys, but symptomless rotavirus infection was seen in broiler chickens. Adeno-viruses and infectious bursal disease virus were also observed in turkey faeces. The best method for preparing faecal material for examination employed initial purification by extraction with a fluorocarbon, followed by concentration in the ultracentrifuge. Examination of the pooled contents of the caeca and large intestine gave better results than examination of small intestinal contents. It is concluded that direct electron microscopic examination of faeces has considerable potential as a diagnostic technique in avian virology.

Ultrastructural studies of the replication of fowl adenoviruses in primary cell cultures.

The replication of four fowl adenovirus strains (FAV-1, strain Phelps; FAV-5, strains 340 and TR-22; and FAV-7, strain YR-36), in primary chick kidney cell cultures, is described. Differences were found in the distribution of virus particles and virus associated inclusions between viruses belonging to different cytopathology subgroups. Thus in cells infected with FAV-1 (Phelps) and FAV-5 (340) (i.e. subgroup 1) virus particles, as they increased in number, tended to become distributed peripherally, close to the nuclear membranes, with the virus associated inclusions in the centre of the nucleus. With FAV-5 (TR-22) and FAV-7 (YR-36) (i.e. subgroup 2) virus particles and associated inclusions became concentrated initially in the central nuclear area later increasing to fill the whole nucleus, with virus particles and inclusions completely intermixed. The virus-associated inclusions were found to be identical to those described in human adenovirus infected cells and the same nomenclature was adopted. Other inclusions found in infected nuclei, included tubular structures and inclusions composed of granular particles.

Isolation of an adeno associated virus from sheep. Brief report.

An adenovirus and a spherical virus 20--24 nm diameter were isolated from ovine faeces. The small virus replicated in the nucleus, and was associated especially with the nucleolus. It haemagglutinated guinea pig and human erythrocytes, was thermostable and required an adenovirus for replication. It is concluded that this represents the first recorded isolate of an ovine AAV.

Diagnosis of avian viral diseass by electron microscopy.

Clinical material from avian species was examined directly by electron microscopy for the presence of viruses. Mycoplasma-like and coronavirus-like particles were found in chicken feces. These particles did not appear to be associated with disease and were not propagated in the laboratory. Infectious bursal disease virus was readily detected in impression smears of bursas from experimentally infected birds. Poxviruses were demonstrated in smears made from canarypox lesions. Difficulty in distinguishing intact particles of Newcastle disease virus from mycoplasmas and orthomyxoviruses was resolved by treating viral preparations with deoxycholate. After treatment, Newcastle disease virus was lysed, rendering the nucleocapsid visible, whereas influenza virus was mainly unaffected. Viral particles that were recognized only with difficulty by direct elecron microscopic examination were more easily identified using immunoelectron microscopy.

Synthesis of coreless, probably defective virus particles in cell cultures infected with rotaviruses.

PK-15 cells infected with pig and lamb rotavirus strains which were not adapted to serial growth in cell cultures were examined by electron microscopy. A major difference between virus morphogenesis in the initial passage in PK-15 cells and in intestinal epithelial cells was the generation of large numbers of coreless virus particles in PK-15 cells. The numbers of coreless particles increased with increasing multiplicity of infection. Infectious virus was synthesized in PK-15 cells, but a variable decrease in infectivity titre occurred between 12 and 24 hours after infection. It is suggested that synthesis of defective interfering particles or an inhibitory substance such as interferon might account for this decrease.

Reovirus-like agent (rotavirus) from lambs.

Rotavirus particles were demonstrated by electron microscopy in the feces of lambs with diarrhea. Rotavirus antigen was synthesized in cell cultures infected with filtrates of the diarrheic feces, but the virus was not adapted to grow serially in cell cultures. An antigenic relationship between rotaviruses from lambs, pigs, and calves was demonstrated by immunofluorescence. Colostrum-deprived lambs were infected with the lamb rotavirus, and the virus was passaged in lambs. Viral replication occurred in the villous epithelial cells of the small intestine, and the virus was excreted in the feces up to 78 h postinfection. Diarrhea was not observed in the experimentally infected lambs.

The morphogenesis of a cytopathic bovine rotavirus in Madin-Darby bovine kidney cells.

The morphogenesis of a cytopathic bovine rotavirus isolate was examined in MDBK cells. Distension of cisternae of the rough endoplasmic reticulum was seen 14 h post-infection (p.i.), but few virus particles were present. Many virus particles were observed within distended cisternae of the endoplasmic reticulum 22 h p.i., and matrices of viroplasm were found close to developing virus particles. The virus particles measured 68 to 72 nm in diam. and consisted of an electron-dense nucleoid surrounded by a single less electron-dense shell. In contrast, many of the virus particles observed 54 h p.i. possessed an additional, outer, electron-dense shell. These double-shelled particles were 76 to 82 nm in diam. and acquired the outer shell by a budding process. Virus was released from the cell by discharge through breaks in the plasma membranes of damaged cells.