Jerome Schwartz

Latent herpes simplex virus infection of mice. Infectious virus in homogenates of latently infected dorsal root ganglia.

C-57 albino weanling mice were latently infected with herpes simplex virus (Mp strain, type 1) by inoculation of 104 plaque forming units in the right hind footpad. The virus was demonstrable in explant cultures of the sacral dorsal root ganglia of these mice for as long as 18 months following inoculation. In addition, the virus was detectable when homogenates of these latently infected ganglia were placed on to differentiated organotypic cultures of fetal mouse dorsal root ganglia for as long as 8 months following inoculation of the mice. Virus was not demonstrable in these homogenates when they were placed on to Hela cells. The results suggest that during herpes simplex virus latent infection in mice there is continuous synthesis of infectious virus, probably in a highly localized area, which is detectable if a sensitive indicator substrate, such as these organotypic cultures, is used.

Comparative effects of herpes simplex virus types 1 and 2 in organotypic cultures of mouse dorsal root ganglion.

Mature organized cultures of mouse dorsal root ganglion (MDRG) were infected with herpes simplex virus, type 1 (HSV 1) and type 2 (HSV 2). Onset of infectious virus production occurred faster and reached higher levels in HSV 2-infected cultures. Neurons, supporting cells and myelin were affected in both types of infection, but morphological changes occurred significantly earlier and more dramatically with the type 2 infection. The pattern of myelin changes was distinctly different in the two types of infection. Within 20 hours post infection nerve cells infected with HSV 2 developed several types of intranuclear inclusions consisting of membranes and filaments; no such neuronal inclusions were seen with HSV 1 infection. HSV 2 infection showed frequent, large, membranous inclusions in supporting cell nuclei whereas, only rare, small inclusions of this type were seen in supporting cells infected with HSV 1. The observations demonstrate that the two virus types produce different virus replication pattern and different morphologic changes in long term cultures of MDRG. There appears to be a differential response of neurons and non-neuronal elements to the virus in the tissue substrate. Viral latency was not induced in this system by direct inoculation of the virus under the conditions described.

Growth of Herpes Simplex Virus in Transformed Glial and Neuronal Cells in Tissue Culture: Ultrastructural Studies

The production of herpes simplex virus by transformed neuronal and glial cells in continuous tissue culture was studied by plaque formation techniques and electron microscopy. It was found that i) transformed neuronal cells routinely produced 100–1000 fold more infectious virus particles than do similarly infected transformed glial cells, and ii) in the electron microscope, envelopment of nucleocapsids at the glial cell nuclear membrane is inefficient with few enveloped particles produced. In the glial cells, infection causes a loss of integrity of the nuclear membrane. The differential response of these neuronal and glial cells in continuous tissue culture to HSV infection may reflect different responses occurring in these cell types in vivo.

Herpes simplex virus types 1 and 2 in organotypic cultures of mouse central and peripheral nervous system. 2. Electron microscopic observations of myelin degeneration.

Organotypic cultures of mouse spinal cord with attached dorsal root ganglia, which contain both central and peripheral myelin in the one unit of tissue, were infected with HSV 1 or HSV 2 and studied using electron microscopy. Intranuclear viral nucleocapsids and intrcytoplasmic enveloped particles were found in the Schwann cells associated with peripheral myelin and in oligodendroglia associated with central myelin. Degeneration of peripheral myelin most commonly involved an asymmetrical swelling of the myelin lamellae, whereas degeneration of central myelin was characterized by a more generalized swelling resulting in separation of the myelin lamellae. Degeneration of both central and peripheral myelin was found in the presence of intact axons which were indistinguishable from those in controls.

Herpes Simplex Virus Types 1 and 2 in Organotypic Cultures of Mouse Central and Peripheral Nervous System

Mature mouse spinal cord-ganglion cultures, which contain both peripheral and central nervous system as one unit, were infected with herpes simplex virus type 1 (HSV 1) or type 2 (HSV 2) and observed by bright field microscopy for up to 72 hours. There was degeneration of both central and peripheral myelin in cultures infected with either virus, but the pattern of peripheral myelin degeneration associated with HSV 1-infected cultures was different from that in HSV 2-infected cultures. Type 1 was characterized by focal dilatations; type 2 by ‘sausage-shaped’ swellings, and the cytopathic effect of HSV 2 both began (6 hours p.i.) and was completed (36 hours p.i.) earlier than in cultures infected with HSV 1 (12 hours and 48 hours p.i. respectively). In central nervous tissue, the appearance of degenerating myelin after infection with HSV 1 was indistinguishable from that in HSV 2-infected cultures, but the rate of myelin loss was greater in cultures infected with the type 2 virus. Evidence is presented which suggests that, at least in the peripheral nervous system, myelin degeneration did not appear to be dependent on neuronal or axonal dysfunction or death, but was a direct result of virus infection.

Further Characterization of Brain Actin by Electron Microscopy

The physical state of actin in nerve ending preparations and its relationship to the membranes was studied at the ultrastructural level by negative staining with uranyl acetate before and after treatment with muscle heaving meromyosin (HMM). Actin prepared from synaptosomal or synaptic membrane preparations did not polymerize to fiber formation as readily as striated muscle actin under the same conditions. Treatment of these brain actin preparations with HMM, however, resulted in formation of fibers characteristically decorated with arrowheads which were quite similar to those formed with muscle actin. Treatment of the synaptosomal or synaptic membrane fractions themselves with HMM caused the formation of numerous decorated fibers although fibers were not evident before HMM treatment. This did not occur with the presynaptic vesicle fraction. The studies suggest that at least part of the actin is associated with synaptic membranes and is in a partially polymerized or non-polymerized state; polymerization can be induced by HMM.

Further Characterization of Suckling Mouse Cataract Agent (SMCA): A Slow, Persistent Infection of the Nervous System

Summary Experiments were designed to further characterize the nature of suckling mouse cataract agent (SMCA). The agent is sensitive to heat, trypsin and ultraviolet inactivation, but is stable over the pH range 5.8 to 10. Penicillin, kanamycin, streptomycin and rifampin have no effect on the replication of the agent while BUdR, actinomycin D and hydroxyurea are inhibitory. By filtration the size of the infectious particle was determined to be between 200 and 500 nm. Ultra-structural studies upon infected allantoic fluids reveal unique particles not present in fluids infected with a series of RNA and DNA viruses. The response of the agent to drugs indicates that it is not a bacterial agent and is probably viral. Filtration experiments and ultrastructural studies favor a viral nature. The authors acknowledge the expert technical assistance of Eva Nagy as electron microscopy technician and James E. Pounds who grew and titered the agent.

Herpes Simplex Virus Encephalitis in Suckling Mice: Ultrastructural Studies of Virus Replication

Ultrastructural studies concerning the replication and release of a neuro-adapted strain of herpes simplex virus were carried out on acutely encephalitic mice. It was found that envelopment of nucleocapsid particles at the nuclear membrane of brain cells was inefficient, resulting in accumulation of un-enveloped nucleocapsids in the cell cytoplasm. These nucleocapsids were associated with arrays of microtubules within cytoplasmic processes. Enveloped particles were rarely present in tissue spaces. Those that were seen appeared to be in a state of disintegration. This replication process of herpes simplex virus in the murine encephalitis model differs from that observed in tissue culture cells. The implications of these differences are discussed.

Schilder's disease Virology studies concerning dense core particles

Virologic studies were done of biopsy and autopsy materials from a patient with Schilders disease. Electron microscopy showed that previously reported membrane-bound, dense core particles were present in affected cells. Although these particles strongly resemble viruses, no virus could be demonstrated in these materials as studied by appropriate animal inoculation and tissue culture techniques.

Complement-mediated changes in morphology of hepatitis B antigen-antibody complexes.

Abstract. The size distribution of particles of purified hepatitis B antigen (HB Ag) was altered by the action of complement. Prior to exposure to complement 94% of round particles of HB Ag were in a size distribution of 10–18 nm with a modal diameter of 15 nm. The modal diameter of particles treated with complement increased to 19 nm and particles larger than 30 nm were prominent. This effect on the morphology of HB Ag may be due to an enzymatic action of complement.