Development of an Equine Herpesvirus in Two Cell Culture Systems: Light and Electron Microscopy

Development of equine herpesvirus strain 82A was studied in cells from primary horse kidney (HOK) cultures and an equine dermis (ED) cell strain. HOK and ED cells are equally susceptible to the 82A virus infection and yield about the same amount of infectious virus. Intranuclear inclusions were present in both cell systems, but a ring-shaped syncytial formation was observed only in infected ED cells. Ultrastructural studies revealed the presence of dense granules 30 nm in diameter and characteristic star-like clusters of granules in the infected HOK cells, but these granules were rarely seen in the infected ED cells. Viral nucleocapsids were associated with homogenous nuclear matrices, with moderate electron density in both cell systems. Viral nucleocapsids acquired envelopes by budding into the nuclear vacuoles in both HOK and ED cells. Budding from inner nuclear membranes into perinuclear cisterna or into cytoplasmic vacuoles also was observed frequently in HOK cells but was not seen often in infected ED cells. Multiple, membrane-bound intranuclear inclusions of fibrillar material which may be associated with virus envelopes were seen only in infected ED cells. Enveloped virus particles seen in nuclear vacuoles or perinuclear cisterna were more regular in shape and had a 130-nm diameter, whereas the enveloped virus particles seen in the cytoplasm and extracellular space were more irregular in shape and had a 130- to 160-nm diameter.     

Oncornavirus of guinea pigs. 1. Morphology and distribution in normal and leukemic guinea pig cells.

Two morphologically distinct types of oncornavirus particles were observed in guinea pig cells. In tissues of leukemic guinea pigs, intracisternal A-type particles 90–100-nm diameter, predominated. Extracellular particles with dense core, approximately 90–110 nm in diameter, were seen only occasionally at the intercellular space of the tissues, but were predominant in plasma and sera of the same animals. Placental and fetal tissues obtained from normal guinea pigs showed only intracisternal A-type particles. Cultured guinea pig cells when treated with BrdU revealed many intracytoplasmic A-type particles 90–100-nm diameter. Budding of these A-type particles at the cell membrane to form extracellular enveloped A-type particles was observed. Extracellular virus particles with dense cores, 110–120 nm in diameter, similar to those seen in tissues and plasma of leukemic guinea pigs, were abundant in the BrdU-treated cultures. The morphology and distribution of the intracellular and extracellular virus particles in tissues and tissue cultures derived from leukemic and normal guinea pigs are compared, and the relationships between these virus particles are discussed.     

Oncornavirus of guinea pigs : I. Morphology and distribution in normal and leukemic guinea pig cells

Two morphologically distinct types of oncornavirus particles were observed in guinea pig cells. In tissues of leukemic guinea pigs, intracisternal A-type particles 90–100-nm diameter, predominated. Extracellular particles with dense core, approximately 90–110 nm in diameter, were seen only occasionally at the intercellular space of the tissues, but were predominant in plasma and sera of the same animals. Placental and fetal tissues obtained from normal guinea pigs showed only intracisternal A-type particles. Cultured guinea pig cells when treated with BrdU revealed many intracytoplasmic A-type particles 90–100-nm diameter. Budding of these A-type particles at the cell membrane to form extracellular enveloped A-type particles was observed. Extracellular virus particles with dense cores, 110–120 nm in diameter, similar to those seen in tissues and plasma of leukemic guinea pigs, were abundant in the BrdU-treated cultures. The morphology and distribution of the intracellular and extracellular virus particles in tissues and tissue cultures derived from leukemic and normal guinea pigs are compared, and the relationships between these virus particles are discussed.     

Replication of cytomegalovirus in human epitheloid diploid cell line

Summary Human diploid BAMB cells with epitheloid morphology, which had been derived from amniotic fluid cells, were capable of supporting the replication of human cytomegalovirus (CMV), without prior treatment of the cells with halogenated pyrimidines. The growth of this virus in BAMB cells and in human diploid fibroblastoid (LEP) cells was compared in parallel tests. Virus replication was slower and less efficient in the former than in the latter system. The most characteristic morphological feature of the CMV-infected BAMB cells was the formation of multinucleated giant cells which frequently contained more than a hundred nuclei; such cells were not seen in LEP cultures. The development of ultrastructural changes was slower in BAMB cells than in LEP cells. The additional most marked differences concerned the place of viral envelopment and the production of cytoplasmic dense bodies. While in LEP cells most nucleocapsids were enveloped from the inner leaflet of the nuclear membrane, in the other system a great majority of the particles acquired their envelopes by budding into vacuoles. Cytoplasmic dense bodies were rare in infected LEP cells but very frequent in BAMB cells. Budding of these structures into vacuoles was also observed.With 9 Figures     

An electron and immunoelectron microscopic study of dengue-2 virus infection of cultured mosquito cells: Maturation events

Summary The maturation process of dengue-2 virus in C6/36 mosquito cells was studied by electron microscopy at 12, 16, 24, 48, and 78 hours postinoculation (p.i.) and by immunoelectron microscopy at 48 and 78 hours p.i. Maturing virions appeared within cytoplasmic vacuoles and on the surface of infected cells from 24 hours p.i. onward in close topographical relationship to the dense particles that occurred concurrently in the cytoplasm. The dense particles measured 25 to 35 nm in diameter; the mature virions measured 50 to 55 nm in diameter, with a dense core measuring 30 to 35 nm in diameter covered by a 10 nm-thick membrane envelope. The morphological observations indicated that the dense particles were dengue nucleocapsids assembled in the cytoplasm and that they apparently budded into the vacuolar lumens and the extracellular space at the vacuolar and plasma membranes, acquiring membrane envelopes and becoming mature virions in the process. The virions that budded into the vacuolar lumens were released extracellularly by exocytosis. In the samples tested with dengue-2 polyclonal antibodies, intense immunostaining occurred at the sites of virus budding on the cell surface; host cell membrane and cytoplasm adjacent to the budding virions stained less intensely. In the samples tested with a dengue-2 monoclonal antibody specific for the envelope glycoprotein, budding virions stained rather exclusively, with no staining occurring in adjacent host membrane or cytoplasm.With 10 Figures     

Comparison of guinea pig cytomegalovirus and guinea pig herpes-like virus: growth characteristics and antigentic relationship.

The growth characteristics of guinea pig cytomegalovirus (GPCMV) and guinea pig herpes-like virus (GPHLV) in cell cultures were compared. Guinea pig fibroblast cells were highly susceptible to infection with both viruses, whereas guinea pig kidney cells were sensitive only to GPHLV. No cytopathic effect was observed in the latter cell system after infection with GPCMV,nor was there an increase in virus titer, although the cirus persisted in the kidney cells for 2 to 3 weeks postinfection. Electron microscope studies showed nonvirion tubular structures in GPCMV -infected fibroblast cells, but not in GPHLV- infected cells. Large packages of enveloped nuclear virus particles were commonly seen in GPHLV -infected cells, especially kidney epithelial cells, but none were found in the GPCMV -infected fibroblasts. Complete enveloped extracellular virus particles were present in both virus-cell systems. Both viruses showed narrow host spectra and replicated well only in guinea pig cells although GPHLV multiplied to some degree in rabbit cells. No antigenic relationship could be demonstrated between the two viruses using antisera specific for each virus that was produced in rabbits and guinea pigs. Rabbits produced high neutralizing antibody titers to GPHLV, whereas guinea pigs were the animals of choice for GPCMV antiserum production.     

Ultrastructural development of guinea pig cytomegalovirus in cultured guinea pig embryo cells.

The ultrastructural development of guinea pig cytomegalovirus (GPCMV) in guinea pig embryo cells was studied using electron microscopy. Tubular structures were found in nuclei of virus infected cells, followed by the appearance of intranuclear inclusions containing virus nucleocapsids. While some nucleocapsids were enveloped at the inner nuclear membrane, others were released into the cytoplasm where they were associated with, or within, dense matrix which was subsequently enveloped by cytoplasmic membranes to form enveloped dense virions. Dense bodies without virus capsids were formed in the cytoplasm and enveloped in a similar manner. An involvement of the nuclear pores in the release of unenveloped virus capsids from the nucleus to the cytoplasm was postulated. Evidence that the enveloped dense virions and dense bodies shared common envelope antigen(s) was obtained by immunoelectron microscopy. The similarities and differences in the ultrastructural development of GPCMV and other cytomegaloviruses are discussed.     

Replication of Mayaro virus in Aedes albopictus cells: an electron microscopic study

Summary The replication of Mayaro virus inAedes albopictus cells, was studied by electron microscopy at various times post-infection. In infected cells we observed the presence of cytoplasmic vesicles containing viral nucleocapsids and mature virus particles but at no time did we detect virus budding into such vacuoles. Budding of virus through plasma membrane was rarely observed. Our results are discussed considering the possibility of the release of virus particles to the extracellular space by exocytosis.     

Assembly of enveloped respiratory syncytial virus particles within the cytoplasm of infected vero cells

Summary Electron microscopic examination of syncytia induced by a bovine respiratory syncytial virus strain in Vero cell cultures revealed the presence in the cytoplasm of assembled enveloped virus particles within inclusions of variable sizes. Moreover, budding virus particles were shown occasionally in the intracytoplasmic vesicles. These particles were filamentous in form, about 80–120 nm in diameter, variable in length, containing 12 nm diameter nucleocapsids, and looked like the extracellular particles budding at the plasma membranes. This is the first report on assembly of intracellular virus particles in cells infected by a member of the familyParamyxoviridae. Vero cell-dependent variations appear to be the factor leading to the defect in the late virus replication cycle.     

Ultrastructural development and persistence of guinea pig cytomegalovirus in duct cells of guinea pig submaxillary gland

Summary Salivary glands from Hartley guinea pigs were experimentally infected with guinea pig cytomegalovirus (GPCMV) and examined by light and electron microscopy at different time intervals. Characteristic intranuclear and intracytoplasmic viral inclusions were observed in duct cells of infected animals. Viral inclusion counts and infectivity titers in the salivary gland reached maximum levels by 3 to 4 weeks after infection; infectivity persisted, though at reduced levels, for at least 30 weeks. Electron microscopic examination of viral inclusions revealed several developmental events including nucleocapsid assembly, envelopment of nucleocapsids at the inner nuclear membrane and their enclosure by a thin vacuolar membrane. While contained within cytoplasmic vacuoles, enveloped virions acquired surface spikes. Cytoplasmic vacuoles containing virions subsequently coalesced and discharged mature virions at the cell surface into the lumen of the salivary gland duct. The data indicate that the ultrastructural development of GPCMV in the guinea pig salivary gland shows many similarities to that of human cytomegalovirus in humans. The salivary gland may provide a primary locus for virus shedding and horizontal transmission of cytomegalovirus.With 7 Figures     

Morphology and morphogenesis of a coronavirus infecting intestinal epithelial cells of newborn calves.

The morphology and morphogenesis of virus strain LY-138 recovered from neonatal diarrheic calves were investigated by electron microscopy using negativestaining techniques and ultrathin sectioning. Purified viral particles were spherical in shape and measured 90 nm in average diameter in negatively stained preparations. Pleomorphic forms were also present. The virions had envelopes with petal-shaped projections characteristic of coronaviruses. In ultrathin sections, cores in viral factories were round with a diameter of 50–60 nm. Most of these cores were electron dense but some had an electron-lucent center. In cytoplasmic vacuoles, Golgi vesicles, and on the apical plasmalemma of intestinal epithelial cells, the virions were round or ellipsoidal in shape, measuring 70–80 nm in diameter, and had fine thread-like projections on their surfaces. Uptake of virus occurred through fusion of viral envelopes with the plasmalemma of the microvillous border or by entry into intercellular spaces and interaction with the lateral cell membranes of adjacent intestinal epithelial cells. As a result of this interaction, the lateral cell membranes became altered and ill-defined. During the early stage of infection, the rough andasmooth elements of the endoplasmic reticulum became distended with electron-dense granulofibrillar material. This material accumulated subsequently as well-defined, smooth membrane-bound areas mainly in the apical cytoplasm of infected cells. These structures were considered to be viral factories. The morphogenesis of virus occurred mainly through condensation of the electron-dense, granulo-fibrillar material into viral cores in cytoplasmic viral factories or within the distended cisternes of the rough endoplasmic reticulum. Viral envelopment occurred on membranes of cytoplasmic vacuoles, Golgi vesicles, or in association with membranes of viral factories. Release of virus from infected cells occurred by lysis and fragmentation of the apical plasmalemma and flow of the cytoplasm with its contents into the gut lumen. Release also occurred by digestion and lysis of extruded infected cells or by fusion of virus-containing cytoplasmic vacuoles with the apical plasmalemma and liberation of their contents.     

Morphogenesis of avian infectious bronchitis virus in primary chick kidney cells

Summary Primary chick kidney cells were infected with avian infectious bronchitis virus (IBV) and examined by electron microscopy. Virus particles entered the cells by viropexis and distinction could be made between engulfment by cell processes (phagocytosis) and entry by micropinocytosis in coated transport vesicles.Virus maturation occurred by budding into either the cisternae of the endoplasmic reticulum or cytoplasmic vacuoles, and evidence was obtained to suggest that the viral surface projections could be attached during the budding process. Late in infection large numbers of virus particles were present, mainly in cytoplasmic vacuoles, and the majority were released by cell lysis. Release by fusion of vacuoles with the plasma membrane was also observed, and individual virions could be transported from the endoplasmic reticulum to the surface within coated vesicles.With 10 Figures     

Intra-nuclear localization of two envelope proteins,gB and gD,of herpes simplex virus

Summary The envelopes of herpes simplex virus (HSV) particles are acquired from the inner nuclear membrane (INM) of the infected cell and virus-coded glycoproteins are present in the envelope of mature virions. Our ultrastructural study examined the process of virus envelopment and the targeting of two major viral glycoproteins, gB and gD, to the INM in HSV-infected human embryonic fibroblasts. It was shown that envelopment and transport of virus particles from the nucleus is facilitated by the formation of a dynamic tubulo-reticulum arising from the INM. Capsids were assembled in the nucleus and collected within INM tubules which protruded into the perinuclear space and thence into the cisternae of the endoplasmic reticulum (ER). Envelopment occurred by constriction and fusion of the tubular channel walls, releasing enveloped virions into the ER. Transport to the cell surface took place in membrane-bound compartments and probably followed the normal secretory pathway through the Golgi apparatus. Immunogold probes, tagged with specific monoclonal antibodies, were used to localize gB and gD during the process of virus maturation. Cytoplasmic membranes were not labelled, but probes bound inside the nucleus, mainly at sites of virus assembly. Labelling occurred on the nucleoplasmic side of the INM which surrounded capsids in the process of envelopment, but not on the outside of that membrane, although characteristic gB glycoprotein spikes were labelled on the envelopes of extracellular virus particles and on virions intrans-Golgi transport vesicles just prior to their release from the infected cell. gB was not detected on the surface of enveloped virions in the perinuclear space, or the cisternae of the ER orcis-Golgi, which suggests that the specific epitope was masked during that stage of intracellular processing. gD probes bound to virion envelopes and also to the tegument region of some particles found in both perinuclear and extracellular sites. We postulate that precursor core proteins for both gB and gD are transported first to the nucleus, and then, together with maturing capsids, are targeted to the INM, and later inserted into viral envelopes at the site of budding. Post-translational glycosylation of envelope proteins could occur as virus particles exit the nucleus and travel through the ER and Golgi compartments.     

Extracellular vesicles containing virus-encoded membrane proteins are a byproduct of infection with modified vaccinia virus Ankara

Vaccinia virus is a structurally complex virus that multiplies in the cell cytoplasm. The assembly of Vaccinia virus particles and their egress from infected cells exploit cellular pathways. Most notably, intracellular mature viral particles are enwrapped by Golgi-derived or endosomal vesicles. These enveloped particles, enriched in virus-encoded proteins, migrate to the cell surface where they are released into the extracellular space through fusion of their outer envelope with the cell membrane. We report that baby hamster kidney cells productively infected with the modified vaccinia virus Ankara strain (MVA) also release extracellular vesicles containing virus-encoded envelope proteins but devoid of any virus cargo. Such vesicles were visualized on the cell surface by electron microscopy and immunogold labelling of the B5 envelope protein. A portion of the B5 protein was found to be associated with non-viral material in high speed ultracentrifugation pellets and displayed a buoyant density characteristic of exosomes released by some cell types. An unrelated transmembrane protein (CD40 ligand) encoded by the MVA genome was also incorporated into extracellular vesicles but not into the envelopes that surround extracellular enveloped virus. High speed pellets obtained by centrifugation of culture medium from cells infected with MVA encoding CD40 ligand displayed the ability to induce dendritic cell maturation suggesting that the ligand is on the outer surface of the extracellular vesicles. We propose that the formation of extracellular vesicles after vaccinia virus infection is a byproduct of the pathway leading to the formation of extracellular enveloped virus.     

Unusual findings of viral production (MuMTV) in murine mammary tumors (BALB/cfC3H)

Some unusual findings of the murine mammary tumor virus (MuMTV) are described in cells of BALB/cfC3H mice mammary tumors: (1) a conspicuous endocellular production of B particles by budding of A particles into small and large cytoplasmic vacuoles, into intracytoplasmic lumina, and possibly into vacuoles of secretion; (2) an extracellular production of B particles (in addition to the classic budding via microvilli) by opening at the cell surface of intracytoplasmic lumina containing mature and immature B particles and possibly of small vacuoles containing immature B particles. Morphological data suggest a virus excretion also by the secretory pathway of the neoplastic cell. Golgi complex is often seen in association with the small type of cytoplasmic vacuoles, whereas the large vacuoles are often close to a particular type of dense body, possibly lysosomes. The occurrence of free B particles in the cytoplasm is also reported. The possibility that the observed features are not occasional but are aspects of the normal life of the MuMTV is suggested.     

Antigenicity of mouse hepatitis virus strain 3 subcomponents in C57 strain mice

Summary Morphogenesis of human rotavirus type 2 Wa strain in MA 104 cells was observed. The virus antigen in the cytoplasm was detected by indirect immunofluorescence twelve hours after infection. The cytopathic effect occurred 24 hours after infection when virus particles were detected by EM in the culture fluid as well as in thin sections of the infected cells. Virus particles were observed in the dilated RER, nuclear envelope (perinuclear space), viroplasm, and a lysosome-like body. Three types of virus particles were noted: double-shelled particles 75–85 nm in diameter, single-shelled particles 64–68 nm in diameter and electron-dense nucleoids or cores 32–40 nm in diameter. The outer shell of virus particles was acquired by budding through the membrane of the dilated RER. Tubular structures, similar in diameter to the single-shelled particles, were found in the cytoplasm and nucleus of infected MA 104 cells. Bundles of filaments or the leaflet-like inclusion bodies of membrane-bounded bundles of filaments were found in the cytoplasm and seemed to be associated with virus particles.With 9 Figures     

Histopathologic and ultrastructural studies of disseminated cytomegalovirus infection in strain 2 guinea pigs

Inbred strain 2 guinea pigs developed severe disseminated disease during acute experimental guinea pig cytomegalovirus (GPCMV) infection. A high mortality rate (100%) resulted, with most animals dying between 10 and 14 days after high dose (7.5 X 10(5) TCID50) virus inoculation. Infectious virus was recovered from many tissues, including spleen, lungs, liver, pancreas, heart, adrenals, kidneys, and salivary glands. The rate of GPCMV isolation from these tissues ranged from 50 to 100%. Gross lesions were observed in the spleen, liver, and lungs. On histologic examination, lesions were also seen in many other organs, including heart, pancreas, kidneys, adrenals, brain, intestines, and salivary glands. Intranuclear viral inclusions were present in many cell types of various organs. Under electron microscopic examination, cells with viral inclusions were easily found in the spleen, and liver, but less readily in the lungs, kidneys, salivary glands, and other organs. Most of the intranuclear inclusions consisted of electron-dense fibrils (10 nm diameter), viral nucleocapsids (100 nm), and tubular structures (60 nm diameter). Dense bodies and enveloped dense virions containing single or multiple capsids were present in the cytoplasm of many infected cells. The morphologic developments of GPCMV in these visceral tissues of strain 2 guinea pigs resembled those seen in GPCMV-infected cultured guinea pig cells but differed from those observed in the infected salivary gland duct cells. Strain 2 guinea pigs are a useful animal model for studying disseminated infection in CMV-associated human diseases.     

Ultrastructural study of Mayaro virus replication in BHK-21 cells

Summary The replication of Mayaro virus in BHK-21 cells was studied by electron microscopy. The infected cells show an intense vacuolization and proliferation of membranous structures. At 5 h post-infection, precursor virus particles were seen in the cytoplasm of infected cells. Later, mature virus particles were found outside the cells and budding from the plasma membrane. Enveloped virus particles were also observed inside the vesicles and budding across their membrane. The release of virus particles into the extracellular space by exocytosis was also observed. In a later stage of the infection, inclusion bodies were sometimes present in the cytoplasm of infected cells. We conclude that in BHK-21 cells, budding from the plasma membrane is the main process of Mayaro virus maturation, and in this kind of cell replication differs significantly from that observed inAedes albopictus cells.     

Infection of chick embryo tracheal organ cultures with influenza a2 (Hong Kong) virus

Summary Chick embryo tracheal organ cultures supported the growth of influenza A2 (Hong Kong) virus. Entry of the influenza virus into, and its release from, differentiated cells of the respiratory epithelium was followed with the aid of electron microscopy. Sections prepared from organ cultures revealed the presence of a mucous layer covering the epithelium, and passage of the virus through this mucus was found to be greatly enhanced at higher than 4° C temperatures.Though influenza virus particles readily adsorbed to the cilia, they were not observed to enter them. Entry of the influenza virus was found to take place by disintegration and fusion of the cytoplasmic membrane and the viral envelope. Entry by engulfment of intact particles was not seen.Release of influenza virus particles by budding was first observed 12 hours after the adsorption period. Preparations fixed at 18 and 24 hour intervals following adsorption showed great numbers of particles being released into the mucous layer from the cytoplasmic membranes of the epithelial cells, but not from the cilia.In specimens showing hemadsorption, the guinea pig red blood cells were adsorbed to the cytoplasmic membranes of the epithelial cells or to the immature virions budding from them.Submitted in partial fulfilment of the requirements of the degree of Master of Science, University of Toronto.