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     

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 studies of the envelopment and release of guinea pig herpes-like virus in cultured cells

The process of envelopment and release of guinea pig herpes-like virus was examined in both infected guinea pig kidney and thymus tissue culture cells by electron microscopy. The majority of the nucleocapsids were enveloped by budding into nuclear vacuoles; some were enveloped by budding from the inner nuclear membrane. Budding into cytoplasmic vacuoles was also seen. Many enveloped virus particles inside the nuclear vacuoles were pear shaped with a tail-like structure. Approximately 23% of pear-shaped virus particles were seen in the infected thymus fibroblastic cells, but only 6% were found in the infected epithelial cells. The envelopes of all nuclear enveloped virus particles appeared as smooth membranes, while the majority of particles exhibiting fuzzy and thick dense envelopes were seen in the cytoplasm or extracellular space. The average diameter of the cytoplasmic or extracellular enveloped virus particles was approximately 167 nm, and the average diameter of the nuclear enveloped virus particles was about 146 nm.Data also showed that mature nuclear virus particles were first released into perinuclear cisterna and then traveled through cytoplasmic channels to the extracellular space.     

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 localization of viral antigen in nuclear inclusions of cytomegalovirus infected guinea pig cells

Summary Intranuclear localization of viral antigens in guinea pig cytomegalovirus (GPCMV) infected guinea pig embryo (GPE) cells was investigated by cross-reactive indirect immunoperoxidase and immunoferritin techniques utilizing guinea pig antisera to GPCMV. Following primary fixation with 4 percent paraformal-dehyde, a brief treatment of infected cells with 0.25 percent trypsin was found to enhance penetration of antibodies and the conjugates. Ferritin or horseradish peroxidase conjugated goat anti-rabbit IgG was used as a secondary antibody that cross reacted with guinea pig immunoglobulins in order to reduce non-specific immunochemical reactions. Using light microscopy following immunoperoxidase staining, GPCMV antigens in an intranuclear location were not discernable when the infected cells were stained without pretreatment with trypsin, however intranuclear GPCMV antigens could be visualized after the fixed cells were treated with trypsin for 2–4 minutes prior to addition of the antiserum. Electron microscopic examination following indirect immunoferritin staining revealed viral antigens localized on viral capsids and on scattered electrondense amorphous matrices but not on the surrounding tubular structures or fibrils. The possibility that tubular structures may be a host cell product produced in response to GPCMV infection is discussed.With 3 Figures     

Electron microscopy of rabbit embryo fibroblasts infected with herpesvirus isolates from Clethrionomys glareolus and Apodemus flavicollis

Electron microscopy of rabbit embryo fibroblasts infected with five herpesvirus isolates from murine rodents (Clethrionomys glareolus and Apodemus flavicollis) revealed morphological changes characterized by the formation of intranuclear vacuoles, in which envelopment of nucleocapsids took place. The herpesvirus nucleocapsids were formed in the vicinity of intranuclear granular inclusions. Margination of chromatin, disaggregation of nucleoli and, in some isolates, the occurrence of tubular structures with a diameter of 33--35 nm characterized the ultrastructural changes in the nuclei. In the cytoplasm, membraneous vacuoles, containing numerous naked nucleocapsids and, in some isolates, large electron-dense bodies, containing non-enveloped nucleocapsids, were formed. Extracellular spaces contained either enveloped virions which, besides nucleocapsids, contained in their envelope a part of the dense material from intracytoplasmic dense bodies, or viral envelope-like structures without nucleocapsids partially filled with dense material from intracytoplasmic dense bodies. Ultrastructural development of these isolates in REF resembled that of freshly isolated herpes simplex type 1 viruses but some morphological changes resembled those characteristic of cytomegaloviruses.     

Comparison of guinea pig cytomegalovirus and guinea pig herpes-like virus: pathogenesis and persistence in experimentally infected animals.

The pathogenesis of guinea pig cytomegalovirus (GPCMV) and guinea pig herpes-like virus (GPHLV) in guinea pigs was compared. Animals were inoculated with the two viruses by different routes and sacrificed after varying periods of time. GPCMV was consistently isolated from salivary gland 2 weeks postinoculation and thereafter following intraperitoneal or subcutaneous incoulaton. Virus was less frequently found in other tissues including blood, spleen, and kidney. Intranuclear inclusions were seen in tissue sections of salivary gland after inoculation with GPCMV- infected tissue suspension, but were only rarely found after inoculation with tissue culture virus. In GPHLV-infected guinea pigs, consistent latent infection of leukocytes and other tissues was detected by cocultivation techniques. Intranuclear inclusions were not found in the spleen, salivary gland, or other infected tissues after GPHLV infection with either tissue culture virus or infected tissue suspension. Guinea pigs inoculated with GPCMV produced high titers of specific neutralizing antibody to the homologous virus; those inoculated with GPHLV developed long-term viremia accompanied by minimal neutralizing antibody levels to the virus.     

Detection of guinea pig cytomegalovirus nucleic acids in cultured cells with biotin-labelled hybridization probes

Biotin labelled hybridization probes prepared from recombinant plasmids containing segments of the guinea pig cytomegalovirus (GPCMV) genome were used to detect GPCMV nucleic acids in guinea pig cells by in situ hybridization. The time course of GPCMV infection was assessed in two cultured cell types, guinea pig embryo (GPE) cells and 104C1 cells, a transformed and cloned guinea pig cell line. Detection of GPCMV nucleic acids was accomplished in both cell types with individual GPCMV DNA fragments and with mixtures of GPCMV DNA fragments. When compared to other established methods of GPCMV detection, the method of in situ hybridization enabled the detection of a higher percentage of positive cells early during the course of the infection. In addition, differences in the replication cycle of GPCMV in the two cultured cell lines could be demonstrated. These findings will facilitate future studies of GPCMV tissue tropism in vivo.     

Electron microscopy of porcine cytomegalovirus

Summary Porcine cytomegalovirus was examined by electron microscopy in the nasal mucosa of piglets from a natural epizootic of the disease with the object to determine the localization of nucleocapsids arranged in crystalline arrays in the cell and to investigate the structure of most common viral forms. The study revealed the following. 1. The morphogenesis of porcine cytomegalovirus is marked by intranuclear formation of nucleocapsids arranged in crystalline arrays. Large quantities of crystals found in the cytoplasm were apparently extruded from the nucleus at late stages of infection; 2. nucleocapsids in the cytoplasm were coated with an electron dense amorphous material. This coat was also seen at the surface of capsids in cytoplasmic and extracellular enveloped particles, but was absent from the surface of capsids located in the nucleus; 3. the majority of enveloped virus particles showed an electron translucent zone separating the capsid from the envelope, and 4. the outer envelope of cytoplasmic and extracellular particles had projections and showed a clear unit membrane structure, whereas the outer envelope of intranuclear particles and those located in the perinuclear cisterna had the appearance of an electron dense single membrane.     

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.     

Identification of essential and non-essential genes of the guinea pig cytomegalovirus (GPCMV) genome via transposome mutagenesis of an infectious BAC clone

We report application of a transposition methodology that allows the easy characterization and mutation of genes encoded on an infectious bacterial artificial chromosome (BAC) clone. We characterized mutants generated by transposome (Tn) mutagenesis of a BAC clone of guinea pig cytomegalovirus (GPCMV). A pool of Tn mutant GPCMV BACs were screened initially by restriction profile analysis to verify they were full-length, and subsequently GPCMV BAC DNA from individual mutants was transfected onto guinea pig lung fibroblast cells in order to generate virus. Tn GPCMV BAC mutants were classed as either essential or non-essential gene insertions, depending upon their ability to regenerate viable, replication-competent virus. Representative mutants were more fully characterized. Analysis by sequencing the Tn insertion site on the mutated BACs, and by regeneration of virus using transfection of guinea pig fibroblasts (GPL), demonstrated that a recombinant with a Tn insertion in the UL35 homolog gene (GP35) was a non-essential gene for viral replication in tissue culture. A mutant with an insertion in the UL46 homolog (GP46) was nonviable, a phenotype which could be rescued by homologous recombination of BAC DNA with wild-type UL46 sequences, suggesting an essential role of this putative capsid gene in virus replication.     

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.     

Herpes simplex virus infection of the human sensory neuron

Summary Herpes simplex virus (HSV) type 1 was used to infect cultures of human embryonic dorsal root ganglion cells. Infected cultured were studied by electron microscopy. Viral nucleocapsids were observed to be internalized into neuronal cells bodies and neuritic extensions by fusion of the viral envelope and the plasma membrane. No signs of internalization by endocytosis were noted. Nucleocapsids were transported in neurites and were within 2 hrs post-infection found located near the microtubules and close to the nuclear pores in the perikaryon.A primary envelopment of nucleocapsids occurred at the inner lamina of the nuclear membrane and virions appeared between the two laminae. Presence of non-enveloped nucleocapsids outside the nuclear membrane and in close contact with the endoplasmic reticulum suggested that nucleocapsids could pass to the cytoplasmic side probably by de-envelopment at the outer nuclear membrane. A secondary envelopment occurred at the endoplasmic reticulum where the virions also became enclosed in transport vesicles. Enveloped virus appearing in the cytoplasm of neurons and neuritic extensions was always found only inside these transport vesicles.During their passage through the cytoplasm the virion-transport vesicle complexes were surrounded by smaller lysosome-like vesicles possibly derived from the Golgi apparatus. Fusion reactions between vesicles with virions and the smaller vesicles seemed to occur. We discuss if in this way the virion-transport vesicle complexes might be provided with glycosyl transferases and substrates necessary for maturation and completion of glycosylation of the viral envelope glycoproteins. The transport vesicles seemed essential for egress of virions from the infected cell by releasing virus when fusing with the plasma membrane.     

The fine structure of the guinea pig visceral yolk sac placenta

The structure of the guinea pig visceral yolk sac from 26 days of gestation to term was studied by transmission and scanning electron microscopy. Particular emphasis was placed on the columnar endoderm cells of the villous portion of the yolk sac. The apical cytoplasm of the endoderm cells contained numerous membrane invaginations, endocytic vesicles, dense tubules and large vacuoles which appeared to form an interrelated absorptive system. The saccular invaginations of the apical cell membrane were specialized by the development of both an amorphous extracellular coat and an internal coat. Both the endocytic vesicles and dense tubules were thought to be derived from the saccular invaginations following detachment of the latter from the cell surface — the endocytic vesicles forming by fusion of saccules creating progressively larger structures, and the dense apical tubules forming by a process involving fluid loss from the saccules. Large vacuoles were present deeper in the apical cytoplasm; these probably were formed by fusion of smaller vesicles. The supranuclear cytoplasm contained numerous dense droplets and a Golgi zone. The possible relationships of the droplets to the vacuoles was discussed.     

Completely assembled virus particles detected by transmission electron microscopy in proximal and mid-axons of neurons infected with herpes simplex virus type 1, herpes simplex virus type 2 and pseudorabies virus

The morphology of alphaherpesviruses during anterograde axonal transport from the neuron cell body towards the axon terminus is controversial. Reports suggest that transport of herpes simplex virus type 1 (HSV-1) nucleocapsids and envelope proteins occurs in separate compartments and that complete virions form at varicosities or axon termini (subassembly transport model), while transport of a related alphaherpesvirus, pseudorabies virus (PRV) occurs as enveloped capsids in vesicles (assembled transport model). Transmission electron microscopy of proximal and mid-axons of primary superior cervical ganglion (SCG) neurons was used to compare anterograde axonal transport of HSV-1, HSV-2 and PRV. SCG cell bodies were infected with HSV-1 NS and 17, HSV-2 2.12 and PRV Becker. Fully assembled virus particles were detected intracellularly within vesicles in proximal and mid-axons adjacent to microtubules after infection with each virus, indicating that assembled virions are transported anterograde within axons for all three alphaherpesviruses.     

Protease degradation of Autographa californica nuclear polyhedrosis virus proteins

The Autographa californica nuclear polyhedrosis virus (Ac-NPV) occlusion bodies produced in vivo contained a protease which degraded occlusion body matrix protein and polypeptides of enveloped nucleocapsids during alkaline dissolution. The protease activity was required for complete dissociation of occlusion body matrix protein from the viral membrane during alkali liberation of the virus from occlusion bodies. In addition protease activity was required for the subsequent removal of the viral membrane from the nucleocapsids by Nonidet P40 treatments. Protease activity was not detected in occlusion bodies isolated from tissue culture cells or heat-treated (80 degrees , 45 min) occlusion bodies isolated from insect larvae. When occluded Ac-NPV virions were purified in the absence of protease activity, 26- polypeptides with molecular weights ranging from 115,000 to 15,000 were identified. The occlusion body matrix protein had an apparent molecular weight of 33,000.     

Differences in the morphology of herpes simplex virus infected cells: I. Comparative scanning and transmission electron microscopic studies on HSV-1 infected HEp-2 and chick embryo fibroblast cells.

Infection with herpes simplex virus type 1 (HSV-1) induces different morphological changes in different cell lines. This is demonstrated by comparative scanning (SEM and transmission (TEM) electron microscopic investigations of cell cultures prepared under identical conditions. SEM of HSV-1 infected HEp-2 cells reveals a slightly altered cell surface: only the number of the microvilli is reduced. Large amounts of released virions are detectable adhering to the outer plasma membrane. Ultra-thin sections show typical virus maturation steps in the nuclei (formation of nucleocapsids and virus budding from the inner lamella of the nuclear membrane) and in the cytoplasm (egress of enveloped nucleocapsids through membranous structures). HSV-infected primary chick embryo fibroblast (CEF) cells are characterized by crumpled and rough surfaces without virus particles adhering to the membrane. Ultra-thin sections exhibit atypical virus maturation with many unenveloped nucleocapsids within the cytoplasm. The distribution of HSV-induced antigen(s) on the surface of the infected cells is identical in the two cell systems as determined by the peroxidase labelling technique. The c.p.e. (as seen by phase contrast light microscopy) is similar in both HEp-2 and CEF cells: both fusion and rounding up is induced in the infected cells.     

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     

The process of baculovirus deproteinization

The localization and function of alkaline protease inside the supervirion capsids (granules, inclusion bodies) of granulosis virus and nuclear polyhedrosis virus have been determined with the help of electron microscopy. It has been shown that deproteinization of baculoviruses proceeds in several steps. The first two stages (the release of virus from supervirion capsids and the release of nucleocapsids from the internal and external coats, were accomplished in the intestine of the insects, while the third stage (the release of nucleic molecules from the nucleocapsids) took place in the nucleoplasm of intestinal epithelial cells.