Rhabdovirus-like and closterovirus-like particles in ultrathin sections of Ribes species with symptoms of blackcurrant reversion and gooseberry veinbanding diseases

In attempts to determine the causal agents of blackcurrant reversion (BCRD) and gooseberry veinbanding (GVBD) diseases of Ribes species, details of the ultrastructure of different kinds of tissue from plants affected with these different diseases were studied. In three of 12 blackcurrant plants affected with BCRD, leaves and flowers of plants showing symptoms typical of the severe (R) form of the disease, contained rhabdovirus-like particles c. 65–80 nm × 215–485 nm. They were seen most often in the nucleus of cells as single particles but were also found in clusters or rafts. In leaves, these virus-like particles (VLPs) were present only in cells associated with the xylem parenchyma where they occurred as membrane-bound clusters within the nucleus. In flowers, they were also found in phloem parenchyma cells in the peripheral cytoplasm and very occasionally in the cytoplasm of epidermal cells. All non-nuclear VLPs were membrane-bound, either singly or in groups and the membrane seemed to be part of the endoplasmic reticulum. The proportion of vascular cells containing these VLPs was very low (< 1%). In a few cells, smaller bacilliform particles, c. 40–50 nm × 200–250 nm, were found in the nucleus together with the larger particles. Double-membrane bodies, detected in fig leaves affected with fig mosaic (the agent of which is also mite-transmitted), were not detected in any BCRD-affected plants. In leaf tissue of one of three gooseberry and one of two blackcurrant plants affected with GVBD, two kinds of VLPs were found. Rhabdovirus-like particles, similar to those in BCRD-affected material, were present in the nuclei, perinuclear space and cytoplasm of xylem parenchyma cells. They were c. 60–72 nm × 155–230 nm but there was no evidence of the smaller rhabdovirus-like particles detected in a few cells of BCRD-affected tissues. The second kind of VLP was found in noncrystalline masses, with a mean centre-centre spacing of c. 10 nm, in the cytoplasm of phloem cells. These particles, together with other ultrastructural changes, were typical of those reported for aphid-transmitted closteroviruses. No badnavirus-like particles, reported previously from GVBD-affected plants, were observed in any of the plants studied. The significance of these findings in relation to these two important diseases of commercial Ribes species is discussed.     

Light and electron microscopical study of Nosema eurytremae

A nuclear-polyhedrosis virus (NPV) of the silkworm, Bombyx mori, which forms an icosahedral inclusion body, was transmitted to larvae of the rice stem borer, Chilo suppressalis. Serial passages of Bombyx NPV in the alternate host by injecting the supernatant of diseased hemolymph produced inclusion bodies with cuboidal and other shapes that differed from the original shape formed in Bombyx. These different shapes increased with times of passages, and after the twelfth passage, only cuboidal inclusion bodies were formed. The icosahedral inclusion bodies in B. mori and the cuboidal inclusion bodies in C. suppressalis occluded singly enveloped virions of the same size (350 × 75 nm), but the cuboidal inclusion bodies contained only a few virions and a large number of membraneous spherical structures. The formation process of the cuboidal inclusion body differed from that of the icosahedral. At first, irregularly branched inclusion bodies containing “vacant” spaces appeared in the infected nuclei. The bodies grew larger with the deposition of protein in the spaces between the branches, and this was accompanied with the occlusion of a large number of membraneous structures formed in the vicinity of the inclusion bodies, which became cuboidal in shape.     

Ultrastructural investigation on a strain of a cytoplasmic-polyhedrosis virus with nuclear inclusions

A strain of a cytoplasmic-polyhedrosis virus causes the formation of crystalline inclusions almost entirely in the nucleus, and only rarely in the cytoplasm, of the midgut epithelial cells of the silkworm Bombyx mori. It also differs from the typical strain in causing the hypertrophy of the nucleoli and the formation of dense reticulum and spherical bodies in the nucleus. The virus particles and the virogenic stromata of the new strain resemble those of the typical strain. The cytoplasmic inclusions contain virus particles, while the nuclear inclusions do not. When the infected larvae are kept at 30°C for 15 hr or at 35°C for 3–15 hr, the nuclear inclusions break up into particles of 70–250 nm in diameter. The particles are dispersed in the cells but not present in the spaces previously occupied by the decomposed inclusions.     

A baculovirus of the European skipper, Thymelicus lineola (Lepidoptera: Hesperiidae)

The nuclear polyhedrosis virus (NPV) of the European skipper, Thymelicus lineola, observed for the first time in Quebec in 1974, is highly pathogenic for its host. The infected larvae fill with pure polyhedra and die within 4 to 10 days. All tissues were infected except nerve cells and silk glands, but all nuclei of infected cells were filled with polyhedra. Biochemical analyses revealed that important metabolic disturbances occurred in infected larvae, such as serious modifications in the activity of certain enzymes. Polyhedra measured from 350 to 1330 nm in diameter and contained up to about 80 single-enveloped virions which measured to 270 × 58 nm. Abnormally short and abnormally long particles were also observed. Safety tests on mammals, fish, and beneficial insects revealed that this virus had no effect on these organisms, thus, it was recommended for the control of T. lineola outbreaks.     

Ultrastructure of the midgut of the worker honey bee Apis mellifera heavily infected with Nosema apis

Midgut epithelial cells from healthy bees possessed numerous mitochondria, strands of endoplasmic reticulum, evenly distributed ribosomes, zymogen granules, and two kinds of lipid inclusions. In heavily infected midguts of honey bees, Apis mellifera, all epithelial cells were observed to be infected with Nosema apis. Cells of the entire midgut were packed with mature spores and, in some cases, mixed with immature stages. Spores were not found among cells of the brush border and basal infolding. Muscle cells and tracheal end cells of the midgut were not infected. The cytoplasm of the infected cell contained a large number of vacuoles, numerous large inclusion bodies, and aggregated ribosomes. Signs of extensive lysis were observed within the heavily infected cells, although the cell membranes were intact.     

A nonoccluded virus of the army cutworm

A nonoccluded virus was isolated from larvae of the army cutworm, Euxoa auxiliaris. Infected larvae became lethargic and shrunken, and death usually occurred 12–20 days after infection. The primary site of viral infection and replication appeared to be the nuclei of midgut epithelial cells; however, virus replication also occurred in cells of the tracheal matrix and in muscle. Nuclei in early stages of the infection contained large granular areas with the chromatin scattered near the nuclear membrane. These areas differentiated into viral particles that measured 24 nm and formed crystalline arrays, occasionally 10 μm long. Disruption of the nuclear membrane liberated these arrays of particles into the cytoplasm. Fluorescence microscopy studies indicated that the viral particles contained DNA. The crystalline arrays were Feulgen positive. The virus also infected larvae of the armyworm, Pseudaletia unipuncta, and corn carworm, Heliothis zea, in laboratory tests.     

Pleistophora oncoperae sp.n. (Protozoa: Microsporida) from Oncopera alboguttata (Lepidoptera: Hepialidae) in Australia

Pleistophora oncoperae sp.n. is described from adults and larvae of Oncopera alboguttata and O. rufobrunnea. The main site of infection was muscle, though fat body and connective tissue were also infected. Fresh pansporoblasts measured about 25 μm in diameter and contained 16 to 32 or more spores with a mean size of 5.9 × 3.1 μm. Macrospores measuring 7.7 × 4.4 μm were also seen. The mean polar filament length was 158 μm; ultrastructural studies showed that the filament is normally arranged in 14 coils (range, 13 to 20) at an angle of 53.5° to the axis of the spore. The species was found to be distinct from all previously described Pleistophora reported from Lepidoptera.     

Establishment of a persistent baculovirus infection in a lepidopteran cell line

The paper describes the first overt attempt to establish an insect cell line (Spodoptera frugiperda), persistently infected with its homologous baculovirus. The persistently infected cells were morphologically different and grew to a higher density than the noninfected parent line. The parent line, however, had a shorter doubling time. Persistently infected cells were passaged 40 times over 10 months; they still continued to produce infectious virus and polyhedral inclusion bodies. However, the infectious viral titer was ca. 100 times lower in the persistently infected line than in the parent line; also, the number of inclusion bodies was reduced ca. 98%. Interference with both homologous and heterologous baculoviruses was demonstrated in the persistently infected cell line. Sevently percent of the persistently infected cells contained antigens for S. frugiperda nuclear polyhedrosis virus, ca. 1% of the cells showed infectious viral centers, and ca. 3% of the cells contained inclusion bodies. Although the inclusion bodies from the persistently infected cells were infectious for S. frugiperda larvae, they were about 3 times less infectious than the inclusion bodies produced in the parent line.     

Intranuclear disease of uncertain etiology in larvae of the blackfly,Simulium vittatum

An intranuclear disease of uncertain etiology has been found in larvae of the blackfly, Simulium vittatum (Diptera: Simuliidae). The disease, which causes little mortality, is characterized by a lack of pigmentation in the cuticle and the occurrence of melanotic deposits in the cytoplasm of fat body and midgut epithelial cells. A single, dense lacunate inclusion develops within the nuclei of these cells, ranging from 20 to 30 μm in midgut epithelial cells, and averaging 18 μm in fat body cells. The disease has been reproduced in the laboratory by feeding first-instar blackfly larvae on homogenates of diseased larvae. However, attempts to isolate a causative agent have not been successful. The cytopathology of the disease resembles the cytopathology caused by densonucleosis viruses, but particles, either free or in aggregations, typical of the virions of this virus type have not been identified in infected nuclei.     

Production of porcine parvovirus virus-like particles using silkworm larvae

Porcine parvovirus (PPV) is a significant causative agent of porcine reproductive failure, causing serious economic losses in the swine industry. PPV is a nonenveloped virus, and its capsid is assembled from three viral proteins (VP1, VP2, and VP3). The major capsid protein, VP2, is the main target for PPV neutralizing antibodies and vaccine development. In this study, PPV-VP2 protein was expressed in silkworm larvae, and its antigenicity and production were compared with those in B. mori cells (Bm5). The recombinant VP2 protein was expressed successfully in silkworm larvae and Bm5 cells with a size of approximately 64 kDa. The formation of virus-like particles (VLPs) by recombinant PPV-VP2 was confirmed through transmission electron microscopy. The recombinant PPV-VP2 protein assembled into spherical particles with diameters ranging from 20 to 22 nm. The antigenicity of PPV-VLPs was comparatively analyzed between Bm5 cells and silkworm larvae by ELISA, hemagglutination and hemagglutination inhibition assays. Consequently, it was confirmed that the PPV-VLPs produced in the silkworm larvae were more antigenic than VLPs produced in Bm5 cells. Therefore, it is expected that economical and effective vaccine development will be possible by mass production of PPV-VLPs in silkworm larvae.     

Norovirus Binding to Intestinal Epithelial Cells Is Independent of Histo-Blood Group Antigens

Human noroviruses (NoVs) are a major cause of non-bacterial gastroenteritis. Although histo-blood group antigens (HBGAs) have been implicated in the initial binding of NoV, the mechanism of that binding before internalization is not clear. To determine the involvement of NoVs and HBGAs in cell binding, we examined the localization of NoV virus-like particles (VLPs) and HBGAs in a human intestinal cell line and the human ileum biopsy specimens by immunofluorescence microscopy. The localizations of Ueno 7k VLPs (genogroup II.6) and each HBGA (type H1-, H2- and Le^b-HBGAs) on the human intestinal cell line, Caco-2, were examined by confocal laser-scanning microscopy. To explore any interactions of NoVs and HBGAs in vivo, fresh biopsy specimens from human ileum were directly incubated with NoV VLPs and examined by immunofluorescence microscopy. We found that VLP binding depended on the state of cell differentiation, but not on the presence of HBGAs. In differentiated Caco-2 cells, we detected no type H1 HBGAs, but VLPs bound to the cells anyway. We incubated fresh biopsies of human ileum directly with VLPs, a model that better replicates the in vivo environment. VLPs mainly bound epithelial cells and goblet cells. Although the incubations were performed at 4°C to hinder internalization, VLPs were still detected inside cells. Our results suggest that VLPs utilize molecule(s) other than HBGAs during binding and internalization into cells.     

STUDIES ON MORPHOLOGY AND DEVELOPMENT OF OEDMEUS ASIATICUS ENTOMOPOXVIRUS PROPAGATED IN OEDALEUS INFERNALIS*

Abstract The morphological characteristics and development of Oedaleus asiaticus entomopoxvirus propagated in Oeddeus infernalis are reported. This virus mainly infected host's fat bodies and hemocytes. Three kinds of spheroids were observed during propagation: big spheroid (30. 41 μm × 25. 40 μm), ellipsoid (6. 58 μm × 4. 78 μm) and small spheroid (3. 35 μm × 2. 60 μm). The virions embeded in them were oval, measuring 230 nm × 176 nm. The typical characteristic of poxviruses as spherical units with the mulberry-like surface could be seen under transmission electron microscope. The lateral body was cylinder-shaped. The rope-like substances in the core folded back only once; therefore two spots in transverse sections were seen. The development of the virions included four stages: the appearance of viro-plasm, the formation of spherical particles, the differentiation of core and capsid. The grasshoppers only in the same genus could be infected by this virus.     

A picornavirus-like pathogen of Cotylogaster occidentalis (Trematoda: Aspidogastrea), an intestinal parasite of freshwater mollusks

Nonoccluded, icosahedral picornavirus-like (PVL) particles, 23 nm in diameter, forming paracrystalline arrays were seen in the cytoplasm of various cells in Cotylogaster occidentalis. Viral inclusions were visible in live specimens and in sections prepared for light and electron microscopy. All worms examined over a 2-year period were found to be infected. Infections were naturally acquired and susceptibility was not associated with any particular developmental stages. Development of viral inclusions involved an increase in the inclusion volume, progressive accumulation and condensation of materials into the interior of the inclusions, and formation of multilamellar membrane networks. Virus particles were observed in the stroma of the inclusions in association with multilamellar spherical bodies. Mature PVL particles aggregated into polygonally shaped paracrystalline arrays. When such arrays occurred in the surface tegument, local disruption of the tegumentary membrane may liberate these particles into the environment. PVL particle production did not exhaust glycogen content of infected cells and did not appear to affect short-term survival of the parasite outside the molluscan host.     

VIRUS-LIKE PARTICLES IN HYDRURUS (CHRYSOPHYCEAE)1,2

Virus-like particles (VLPs) have been found in cells of the colonial, chrysophycean alga Hydrurus foetidus (Vill.) Trev. The infected cells, probably zoospores, were discovered in material fixed directly in the field. The VLPs appear either hexagonal or pentagonal when sectioned and are therefore presumed to be icosahedra, They are 50–60 nm diam. and demonstrate distinct trilaminar coals. The VLPs were observed in the cytoplasm, but not in the chloroplast or nucleus.     

Bioassay of nucleopolyhedrosis virus against larval instars of Malacosoma neustria

The relative susceptibility of third- and fourth-instar Malacosoma neustria larvae to a nucleopolyhedrosis virus was studied by bioassay in the laboratory. Larval mortality increased with increased dosage, whereas the dosage-incubation relationship was reversed. Larval age inversely affected mortality and incubation. The computed LD₅₀'s for third- and fourth-instar larvae were 1,405 and 12,320 polyhedral inclusion bodies (PIB's)/larva. The median lethal doses calculated as number of PIB's/mg body weight showed that third-instar larvae were only twice more susceptible to virus than fourths. The LT₅₀ values for 3 × 10⁵, 3 × 10⁴, and 3 × 10³ PIB's/larva were 5.9, 6.58, and 8.15 days, respectively, in third-instar assay; the corresponding figures for the two highest concentrations were 9.3 and 10.7 days in the older larvae. Lethally infected individuals died after one or, exceptionally, two molts. No correlation was found between pupal weight or adult emergence of survivors and the virus dose administered to the larvae.     

Effect of Acanthocephala infection on the reproductive potential of crustacean intermediate hosts

The effect of a naturally acquired infection by three acanthocephalan parasites Dentitruncus truttae, Echinorhynchus truttae, and Polymorphus minutus on the reproductive potential of their intermediate host, Echinogammarus tibaldii (Amphipoda) from Lake Piediluco (Centre of Italy) was assessed. During May 2007, 1135 amphipods were collected from two different samplings and examined for larval helminths. Forty-five amphipods were infected and of those, 16 were infected with D. truttae (intensity = 1-3 larvae), 15 with E. truttae (intensity = 1-2 larvae), and 14 with P. minutus (intensity = 1 larva). The sex ratio was nearly 1:1 in all examined amphipods. One female infected with D. truttae contained six eggs in the brood pouch and another female infected with E. truttae contained five eggs. However, none of the eight female amphipods harbouring P. minutus larva contained eggs in their brood pouch. Uninfected females of the same size and body length as that of the infected females contained between 20 and 32 eggs. No acanthocephalan species were found to co-occur.     

The endogenous development of two new species of Isospora Schneider, 1881 (Apicomplexa: Eimeriidae) from Thai geckoes

Two new species of Isospora are described from Thai geckoes. I. platyurusi n. sp. in Cosymbotus platyurus from Chiang Mai and Khon Kaen and I. uptoni n. sp. in Hemidactylus frenatus from Chiang Mai. Oöcysts of I. platyurusi are spherical to subspherical, 19–22.5 × 16.5–21.5 μm and have two ovoid sporocysts of 11.5–14 × 7.5–9 μm. Oöcysts of I. uptoni are spherical to subspherical, 24–31.5 × 20–27.5 μm and have two ovoid sporocysts of 12.5–15 × 9–11.5 μm. Both species have sporocysts with Stieda bodies and undergo endogenous development in the nucleus of the host gut epithelial cells. On the completion of merogony and gamogony, the host nucleus is reduced to a thin envelope.     

Pathology of Spiroplasma floricola in Galleria mellonella larvae

Spiroplasma floricola strain 23-6, originally isolated from tulip tree flowers, was injected into larvae of the greater wax moth. Histopathology and cytopathology of disease larvae were studied by histochemical, fluorescent antibody, and electron microscopical methods. The gut was empty, polysaccharides in fat and muscle tissue were reduced, the fat body was broken down, and phospholipids were depleted in larvae 4 days after injection. Fluorescein conjugated S. floricola antibody was adsorbed onto hemocytes, sarcolemma, gut epithelial membrane, and the cortex of the ventral ganglia. By electron microscopy, spiroplasmas were found in hemocoel, hemocytes, pericardial cells, connective tissues, basement membranes, epidermal cells of the cuticle, the neural lamella, and the peripheral glial cells of the ventral nerve cord, and on midgut and epidermal membranes. It is postulated that the cytopathological effects induced in the body of the insect released nutritional elements that allow extensive reproduction of S. floricola.     

Isolation of an iridovirus from two terrestrial isopods,the pill bug, Armadillidium vulgare, and the sow bug, Porcellio dilatatus

An iridovirus was isolated from two terrestrial isopods (class Crustacea, order Isopoda), the pill bug, Armadillidium vulgare, and the sow bug, Porcellio dilatatus, collected in southern California. The isolates have been designated Type 31 (from A. vulgare) and Type 32 (from P. dilatatus). Diseased isopods were recognized by a characteristic blue discoloration of the normally gray cuticle. Based on the relative number of virions observed in diseased cells, viral replication was most extensive in epidermal, muscle, and adipose tissue. Additionally, small clusters of midgut epithelial cells were heavily infected in many specimens, although replication throughout this tissue was never observed. Nerve and reproductive tissues were lightly infected. Infection was not observed in hemocytes or the hepatopancreatic caeca. Virions of both isolates measured ca. 125 nm in diameter in ultrathin sections and 141 nm in negatively stained preparations, and formed paracrystalline arrays in heavily infected cells. The isolation of a typical iridovirus from isopods further demonstrates that the natural host range of this virus group extends beyond the class Insecta.     

A new virus disease in the housefly, Musca domestica (Diptera)

Reovirus particles were isolated from adults in laboratory colonies of the housefly, Musca domestica. These particles were spherical in outline, 57–76 nm in diameter, and were found only in hemocyte cytoplasm, where virions have been disclosed by a new technique. Virions were present in large numbers, and viral inclusion bodies were identified. The virus particles had pentagonal and hexagonal shapes resembling a simple icosahedral structure. The virus was shown to be infectious and pathogenic to adult flies through injection or by feeding them suspensions from flies that had died of the virus. Electron micrographs of midgut sections from infected flies showed that the midgut cells were packed with dark undulating threads which were not present in uninfected flies. However, no virus particles or inclusion bodies could be seen in these cells. On the basis of their association with infected flies, and the similarity to results from other studies on reoviruses and insect viruses, it is suggested that these threads are an alternative replicative form of the reovirus. When the virus suspensions from heavily infected flies were dialyzed against weak alkaline solutions, the threads showed an inner component of coiled material, 12 nm in diameter, inside an envelope with a diameter of 50–83 nm, mean 60.3 ± 7.5, composed of subunits 7–8 nm long and 7–8 nm across.