Cytopathology of satellite tobacco mosaic virus and its helper virus in tobacco

Ultrastructural responses of tobacco cells infected with a newly discovered satellite virus (STMV) that has an isometric morphology and is associated with rigid rodshaped tobacco mosaic virus (TMV) were studied in situ. In cells infected with TMV alone,TMV particles occurred as crystalline arrays in the cytoplasm and were usually associated with TMV-characteristic X bodies. In cells infected with both TMV and STMV, particles of STMV occurred only in cells that contained TMV particles, which suggests a correlation between the satellite and helper virus presence. However, the replication and/or accumulation sites of STMV appear to be independent from its helper virus. Unlike TMV particles, STMV particles were associated with several cytopathic structures such as granular inclusions, membranous vesicles of 50–80 nm, and myelin-like bodies which were all bounded by a single common membrane, No X bodies occurred in cells containing STMV particles, and the mitochondria possessed abnormal tubular structures containing flocculent material.     

A CORRELATED HISTOCHEMICAL AND ELECTRON MICROSCOPIC STUDY OF THE INTRANUCLEAR CRYSTALLINE AGGREGATES OF ADENOVIRUS (RI-APC VIRUS) IN HELA CELLS

HeLa cells in tissue cultures infected with types 3, 4, or 7 of adenovirus (RI-APC virus) were studied in order to correlate certain histochemical and electron microscopic findings. Adjacent thin (ca. 0.05 µ) and thick (2–4 µ) sections of osmium-fixed, methacrylate-embedded cells were cut; by mapping the sections the same cells could be identified with both the electron and the light microscope. Intranuclear crystalline aggregates seen with the electron microscope to be composed of ordered arrays of viral particles were found by means of the Feulgen reaction to contain DNA. DNA is therefore assumed to be a constituent of the viral particle. The virus appeared to develop from an osmiophilic Feulgen-negative matrix. Displacement of nuclear chromatin occurred during this process. A Feulgen-azure staining method was found to permit clear distinction between viral and nuclear (host) DNA in thick sections.     

Electron microscope study on the replication of Autographa nuclear polyhedrosis virus and Spodoptera nuclear polyhedrosis virus in Spodoptera exigua

A comparative study of Spodoptera nuclear polyhedrosis virus (NPV) and Autographa NPV replication in Spodoptera exigua revealed some cytopathologic differences. Infection with Spodoptera NPV was accompanied by electron-dense intranuclear granules. Autographa infection within the midgut led to secretion within the lumens of the goblet cells of paracrystalline arrays of small, round particles, 9.5 nm in diameter. Autographa virus was also observed in various stages of possible replication within the cytoplasm.     

Nuclear membrane changes in herpes simplex virus-infected BHK-21 cells as seen by freeze-fracture.

The freeze-fracture technique, which produced high-resolution replicas of large internal faces of membranes, was used for an ultrastructural study of the nuclei of herpes simplex virus-infected BHK-21 cells and mock-infected controls. Crystalline arrays of viral nucleocapsids were found in the nucleoplasm of infected cells, and numerous nuclear membrane "blebs" and protrusions were observed. The numerous areas of membrane distortions were not found to contain nuclear pores. In addition, specific areas of normal protein intramembranous particles are deleted from certain areas of the nuclear membrane as a result of herpes simplex virus, type 2, infection.     

THE MACROMOLECULAR PARACRYSTALLINE LATTICE OF INSECT VIRAL POLYHEDRAL BODIES DEMONSTRATED IN ULTRATHIN SECTIONS EXAMINED IN THE ELECTRON MICROSCOPE

Thin sections of polyhedra obtained from gipsy moth larvae infected with P. dispar virus and from silkworm larvae infected with B. mori virus revealed viral particles contained within a pseudohexagonal, macromolecular, paracrystalline lattice. The gipsy moth virus occurs in bundles of one to eight rods enclosed by a limiting membrane. The particles of the silkworm virus, although generally occurring singly, also possess a limiting membrane. The macromolecules appear to be dense, discrete particles when cross-sectioned and to form dense bands by superimposition when longitudinally or obliquely sectioned at certain angles. Calculations of macromolecular size have been made.     

Fine structure of epithelial cells of Lieberkühn's crypts in feline panleukopenia.

Electron microscopic observation was carried out on epithelial cells of Lieberkühn's crypts of cats naturally affected with feline panleukopenia. The most important change was the replication of feline panleukopenia. The most important change was the replication of feline panleukopenia virus in the nucleus with associated alterations in the lining epithelial cells of the crypts. In these cells in the early stage of infection, virus particles 20 nm in average diameter were found either singly or in small regularly arrayed clusters everywhere in the markedly swollen nucleus. In the course of infection, the nucleus of infected cells became rather atrophic with a marked margination of chromatin granules. Its major portion was occupied with masses of fine fibrillar substance. It was a "viral matrix area" in which appeared a large compact aggregate of virus particles showing a crystalline array. At the same time, the outer membrane of the nuclear envelope partially extended and disrupted. Membranous elements related to it in the cytoplasm were regularly distributed almost always with particles indistinguishable from the virus particles in the nucleus. From these results it was suggested that the major portion of the infected nucleus, or the site of viral replication, might correspond to the amphophilic intranuclear inclusion body revealed by light microscopy.     

Transgenic protein production in silkworm silk glands requires cathepsin and chitinase of Autographa californica multicapsid nucleopolyhedrovirus

The silkworm Bombyx mori represents an established in vivo system for the production of recombinant proteins. Baculoviruses have been extensively investigated and optimised for the expression of high protein levels inside the haemolymph of larvae and pupae of this lepidopteran insect. Current technology includes deletion of genes responsible for the activity of virus-borne proteases, which in wild-type viruses, cause liquefaction of the host insect and enhance horizontal transmission of newly synthesised virus particles. Besides the haemolymph, the silk gland of B. mori provides an additional expression system for recombinant proteins. In this paper, we investigated how silk gland can be efficiently infected by a Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV). We demonstrated that the viral chitinase and the cysteine protease cathepsin are necessary to permit viral entry into the silk gland cells of intrahaemocoelically infected B. mori larvae. Moreover, for the first time, we showed AcMNPV crossing the basal lamina of silk glands in B. mori larvae, and we assessed a new path of infection of silk gland cells that can be exploited for protein production.     

An iridescent virus fromPopillia japonica (Col.: Scarabaeidae)

A very low incidence (<0.01%) of a blue iridovirus (IV) was found in larvae of the Japanese beetle,Popillia japonica Newman, that were sampled over a two year period on Terceira Island (Azores, Portugal). In the most heavily infected larvae, a deep blue iridescence was observed, particularly in the fat body. Transmission electron microscopy revealed the characteristic crystalline arrays of the hexagonal virus particles in the cytoplasm of fat body cells, tracheal matrix, muscle, hypodermis and blood cells. Crystals of the virus particles were also observed freely circulating in the hemolymph. The average diameter of negatively stained purified virus particles was 157 nm. Similarities and differences with other IVs found in the Scarabaeidae are discussed. Considering the broad host range of some of the iridescent viruses, the relatively recent invasion of Terceira byP. japonica, and the rarity of the virus in the beetle, it is probable that the infection was the result of transmission from another species of soil-inhabiting arthropod. Its value as a potential biological control agent ofP. japonica is negligible.     

Aspects of the Encapsidation of Simian Virus 40 Deoxyribonucleic Acid

Growing subcloned CV1-cells were infected with simian virus 40, and the time course of virus formation was determined. When infected cells were fractionated into cytoplasmic and nuclear fractions, most of the progeny virus particles were recovered in the cytoplasmic extract and not in the nuclei. This result was independent of the technique used for the preparation of nuclei and of the time after infection at which the extracts were prepared. Leakage of the virions from the nucleus occurred during the course of cell fractionation, suggesting that the nuclear membrane of the infected cells is damaged. Virions were found to accumulate in a nonlinear fashion, at the time when the number of viral deoxyribonucleic acid (DNA) molecules increases linearly with time after infection. This suggests that the size of the intracellular pool of capsid proteins increases constantly during the late phase of virus replication. Progeny viral DNA to become encapsidated is withdrawn at random from the pool of replicated DNA molecules.     

Viral release and membrane acquisition by budding through the nuclear envelope of hemocytes of the gypsy moth, Porthetria dispar

Viral particles of the nuclear polyhedrosis virus (Baculovirus) of the gypsy moth, Porthetria dispar, appear to be released from hemocyte nuclei by budding through both inner and outer lamellae of the nuclear envelope. As a result of budding, the virus particle acquires its envelope from the inner lamella of the nuclear envelope. The outer lamella, which forms a membrane-limited vesicle around the enveloped particles, may fuse with the plasma membrane during viral release from host cells by exocytosis. These observations differ from two other reported cases of nuclear budding in NPV-infected cells in that the process occurred in the absence of nuclear inclusion bodies.     

Electron Microscopy of Measles Virus Replication

Replication of measles virus in HeLa cells was examined by electron microscopy with ultrathin sectioning and phosphotungstic acid negative staining methods. The cytoplasmic inclusion bodies consisted of masses of helical nucleocapsid which was similar in structure to the nucleocapsid found in measles virions. The cytoplasmic helical nucleocapsid appeared to align near the HeLa cell membrane, and the membrane differentiated into the internal membrane of the viral envelope and the outer layer of the short projections. The viral particles were released by a budding process involving incorporation into the viral envelope of membrane which was contiguous to but morphologically altered from the membrane of the HeLa cells. The intranuclear inclusion bodies were composed of tubular structures similar to those found in the cytoplasmic inclusion bodies. These structures aggregated to crystalline arrangement. The relationship between nuclear inclusion body and replication of measles virus was not clear.     

Cytopathology of the soybean looper,Pseudoplusia includens,infected with the Pseudoplusia includens icosahedral virus

Ultrastructural responses of soybean looper cells of various tissues infected with Pseudoplusia includens icosahedral virus (PIIV), a newly characterized RNA virus [Y. C. Chao, H. A. Scott, and S. Y. Young (1983)J. Gen. Virol.64, 1835–1838], were studied in situ. Most cells of fat body and epidermis consistently contained virus particles and associated cytopathic structures. Virus particles, corresponding to those of purified PIIV in morphology and size, always occurred in the cytoplasm either in membrane-bound virogenic stroma and/or freely in the ground cytoplasm. Virogenic stroma, which appeared to be distinct from those induced by other insect viruses, consisted of electron-dense matrix material, in which virus particles were embedded, and membranous vesicles, 70 or 80 nm in diameter, containing nucleic acid-like fibrils. Virus particles in virogenic stroma occurred as hexagonally arranged crystalline arrays made up primarily of homogeneously dense particles, while those in the ground cytoplasm were dispersed randomly and had an electron-lucent central core. Extremely large numbers of virus particles were also located in noncellular cuticle layers of the integument.     

Replication of Epstein-Barr virus: ultrastructural and immunofluorescent studies of P3HR1-superinfected Raji cells.

We have studied by means of electron microscopy and immunofluorescence the different steps of the replication of the P3HR1 strain of Epstein-Barr virus in Raji cells. The virus entered the cell by fusion of the viral envelope with the plasma membrane, followed by the disintegration of the capsid. In some cases, the migration of nucleocapsids toward the nuclear membrane was observed. The synthesis of new virions began as early as 7 h after infection (in the case of a high multiplicity of infection [MOI]-800 particles per cell) and took place in low-electron-density areas of the nucleus. A viral envelope was acquired by budding either through the nuclear membrane or more often through membranes of the Golgi apparatus or cytoplasmic vacuoles. Comparing immunofluorescence and electron microscopic data a good correlation was found between the presence of early antigen and ultrastructurally altered cells, as well as between the presence of viral capsid antigen and virus-producing cells. With different MOIs, different types of viral cycles were observed: at a low MOI (less than or equal to 50 particles per cell), a nonproducer cycle was induced, with early antigen synthesis only; at a higher MOI (100 particles per cell), a transient production of a small amount of virions was observed, and at a high MOI (greater than or equal to 300 particles per cell), a productive cycle was the rule.     

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 Three-Dimensional Comparison of Tick-Borne Flavivirus Infection in Mammalian and Tick Cell Lines

Tick-borne flaviviruses (TBFV) are sustained in nature through cycling between mammalian and tick hosts. In this study, we used African green monkey kidney cells (Vero) and Ixodes scapularis tick cells (ISE6) to compare virus-induced changes in mammalian and arthropod cells. Using confocal microscopy, transmission electron microscopy (TEM), and electron tomography (ET), we examined viral protein distribution and the ultrastructural changes that occur during TBFV infection. Within host cells, flaviviruses cause complex rearrangement of cellular membranes for the purpose of virus replication. Virus infection was accompanied by a marked expansion in endoplasmic reticulum (ER) staining and markers for TBFV replication were localized mainly to the ER in both cell lines. TEM of Vero cells showed membrane-bound vesicles enclosed in a network of dilated, anastomosing ER cisternae. Virions were seen within the ER and were sometimes in paracrystalline arrays. Tubular structures or elongated vesicles were occasionally noted. In acutely and persistently infected ISE6 cells, membrane proliferation and vesicles were also noted; however, the extent of membrane expansion and the abundance of vesicles were lower and no viral particles were observed. Tubular profiles were far more prevalent in persistently infected ISE6 cells than in acutely infected cells. By ET, tubular profiles, in persistently infected tick cells, had a cross-sectional diameter of 60–100 nm, reached up to 800 nm in length, were closed at the ends, and were often arranged in fascicle-like bundles, shrouded with ER membrane. Our experiments provide analysis of viral protein localization within the context of both mammalian and arthropod cell lines as well as both acute and persistent arthropod cell infection. Additionally, we show for the first time 3D flavivirus infection in a vector cell line and the first ET of persistent flavivirus infection.     

Pathological changes in Fenneropenaeus indicus experimentally infected with white spot virus and virus morphogenesis

To demonstrate pathological changes due to white spot virus infection in Fenneropenaeus indicus, a batch of hatchery bred quarantined animals was experimentally infected with the virus. Organs such as gills, foregut, mid-gut, hindgut, nerve, eye, heart, ovary and integument were examined by light and electron microscopy. Histopathological analyses revealed changes hitherto not reported in F. indicus such as lesions to the internal folding of gut resulted in syncytial mass sloughed off into lumen, thickening of hepatopancreatic connective tissue with vacuolization of tubules and necrosis of rectal pads in hindgut. Virus replication was seen in the crystalline tract region of the compound eye and eosinophilic granules infiltrated from its base. In the gill arch, dilation and disintegration of median blood vessel was observed. In the nervous tissues, encapsulation and subsequent atrophy of hypertrophied nuclei of the neurosecretory cells were found. Transmission electron microscopy showed viral replication and morphogenesis in cells of infected tissue. De novo formed vesicles covered the capsid forming a bilayered envelop opened at one end inside the virogenic stroma. Circular vesicles containing nuclear material was found fused with the envelop. Subsequent thickening of the envelop resulted in the fully formed virus. In this study, a correlation was observed between the stages of viral multiplication and the corresponding pathological changes in the cells during the WSV infection. Accordingly, gill and foregut tissues were found highly infected during the onset of clinical signs itself, and are proposed to be used as the tissues for routine disease diagnosis.     

Development of Coliphage T5: Ultrastructural and Biochemical Studies

Electron microscopic studies of Escherichia coli infected with bacteriophage T5(+) have revealed that host nuclear material disappeared before 9 min after infection. This disappearance seemed to correspond to the breakdown of host deoxyribonucleic acid (DNA) into acid-soluble fragments. Little or no host DNA thymidine was reincorporated into phage DNA, except in the presence of 5-fluorodeoxyuridine (FUdR). Progeny virus particles were observed in the cytoplasm 20 min postinfection. Most of these particles were in the form of hexagonal-shaped heads or capsids, which were filled with electron-dense material (presumably T5 DNA). A small percentage (3 to 4%) of the phage heads appeared empty. On rare occasions, crystalline arrays of empty heads were observed. Nalidixic acid, hydroxyurea, and FUdR substantially inhibited replication of T5 DNA. However, these agents did not prevent virus-induced degradation of E. coli DNA. Most of the phage-specified structures seen in T5(+)-infected cells treated with FUdR or with nalidixic were in the form of empty capsids. Infected cells treated with hydroxyurea did not contain empty capsids. When E. coli F was infected with the DO mutant T5 amH18a (restrictive conditions), there was a small amount of DNA synthesis. Such cells contained only empty capsids, but their numbers were few in comparison to those in cells infected under permissive conditions or infected with T5(+). The cells also failed to lyse. These results confirm other reports which suggest that DNA replication is not required for the synthesis of late proteins. The data also indicate that DNA replication influences the quantity of viral structures being produced.     

Nuclear alterations in a baculovirus-like infection of midgut epithelial cells in the stick insect, Bacillus rossius

In the epithelial cell nuclei of the posterior part of the midgut of the stick insect, Bacillus rossius, a symptomless virus infection is most commonly found. The virus particles consist of rod-shaped DNP nucleocapsids singly enveloped by a membrane and often packed in a pseudocristalline pattern along their major axis; occlusion bodies of any kind are not formed. On the basis of size, structural characteristics, and localization, the virus is thought to be a baculovirus-like virus. The viral infection, which is asynchronous in different nuclei, causes the formation of electron-light virogenic areas, the swelling of the nucleus, the nuclear chromatin lysis, the production of highly variable numbers of virus particles and, eventually, the extrusion of the whole infected nucleus into the gut lumen. Cellular DNA synthesis does not seem to be stimulated by the virus, RNA synthesis is apparently maintained as long as some cellular chromatin is present in the infected nucleus. Nucleolar segregation invariably occurs at late stages of infection, while major cytoplamic alterations have not been noticed until nuclear elimination and cell death occur.     

Locally Produced IL-10 Limits Cutaneous Vaccinia Virus Spread

Skin infection with the poxvirus vaccinia (VV) elicits a powerful, inflammatory cellular response that clears virus infection in a coordinated, spatially organized manner. Given the high concentration of pro-inflammatory effectors at areas of viral infection, it is unclear how tissue pathology is limited while virus-infected cells are being eliminated. To better understand the spatial dynamics of the anti-inflammatory response to a cutaneous viral infection, we first screened cytokine mRNA expression levels after epicutaneous (ec.) VV infection and found a large increase the anti-inflammatory cytokine IL-10. Ex vivo analyses revealed that T cells in the skin were the primary IL-10-producing cells. To understand the distribution of IL-10-producing T cells in vivo, we performed multiphoton intravital microscopy (MPM) of VV-infected mice, assessing the location and dynamic behavior of IL-10 producing cells. Although virus-specific T cells were distributed throughout areas of the inflamed skin lacking overt virus-infection, IL-10⁺ cells closely associated with large keratinocytic foci of virus replication where they exhibited similar motility patterns to bulk antigen-specific CD8⁺ T cells. Paradoxically, neutralizing secreted IL-10 in vivo with an anti-IL-10 antibody increased viral lesion size and viral replication. Additional analyses demonstrated that IL-10 antibody administration decreased recruitment of CCR2⁺ inflammatory monocytes, which were important for reducing viral burden in the infected skin. Based upon these findings, we conclude that spatially concentrated IL-10 production limits cutaneous viral replication and dissemination, likely through modulation of the innate immune repertoire at the site of viral growth.     

Characterization of cricket paralysis virus-induced polypeptides in Drosophila cells

Cricket paralysis virus purified from Galleria mellonella larvae was shown to be similar to virus purified from Drosophila melanogaster cells. Cricket paralysis virus contained three major structural polypeptides of similar molecular weight (around 30,000), had a buoyant density of 1.344 g/ml, and had a capsid diameter of 27 nm. Twenty virus-induced polypeptides could be detected in CrPV-infected Drosophila cells. Two major polypeptides found in the infected cells corresponded to two structural viral polypeptides (VP1 and VP3), whereas the third major intracellular polypeptide was the apparent precursor of the third viral structural polypeptide (VP2). Three of the primary virus-induced polypeptides had molecular weights of 144,000, 124,000, and 115,000. These and other polypeptides were chased into lower-molecular-weight proteins when excess cold methionine was added after a short [³⁵S]methionine pulse. Although cricket paralysis virus has a number of characteristics in common with the mammalian enteroviruses, the extremely fast processing of high-molecular-weight polypeptides into viral proteins seems atypical. Also, no VP4 (8,000 to 10,000 molecular weight) has been found in the virus particles.