Takeshi Kurata

A Genetic Determinant Required for Continuous Reinfection of Adjacent Cells on Large Plasmid in S. flexneri 2a

We have identified a region (virG) on the 230 kb virulence plasmid of S. flexneri that is required for cell-to-cell spread of the bacterium. Tn5 insertions into this region result in avirulent mutants that can initially invade and multiply in epithelial cells, but tend to lose active movement and tend to localize within the cytoplasm, where they are gradually extinguished without infecting adjacent cells. The virG region was localized to within 4 kb and may contain a single cistron. Sequences hybridizing to this region were found in all intact virulence plasmids of Shigellae and enteroinvasive E. coli.

Pathology of the human spinal ganglia in varicella-zoster virus infection.

SummarySpinal ganglia from a patient who died on the 6th day of varicella infection were examined by immunofluorescence and electron microscopy, and were compared with spinal ganglia from a patient dying on the 17th day of herpes zoster infection. In herpes zoster, typical intranuclear inclusion bodies were found in neurons, satellite cells and fibroblast-like cells of the ganglia, which contained numerous naked virus particles. In varicella, few changes were found by light microscopy but viral antigen was detected in a few neurons and satellite cells by immunofluorescence. Electron microscopy revealed scattered virus particles near the nuclear membrane of a neuron, satellite cells and capsular cells and enveloped particles in the cytoplasma of satellite cells. The particles in the nuclei were mostly naked virions with specific crescent-like inner-nuclear structure; those in the cytoplasm had complete and incomplete envelopes and showed pleomorphism. A “virus-like” intranuclear filament found in mononuclear cells in herpes zoster and a “plexiform vermicellar array” found in the nuclei of neurons in varicella are at present considered to be non-specific nuclear changes caused probably by viral infections.

Detection of Viral Antigens in Formalin‐fixed Specimens by Enzyme Treatment

Enzyme treatment (protease or trypsin) was applied to formalin-fixed paraffin-embedded materials and virus-infected cultured cells to detect viral antigens by immunofluorescence. The viral antigens were demonstrated in several organs of autopsy or biopsy cases of which diagnoses had been established by immunofluorescence or virus isolation using frozen materials, or suspected on the basis of serology and/or histopathological findings. These included herpes simplex, varicella-zoster, cytomegalo, subacute sclerosing panencephalitis, progressive multifocal leukoencephalopathy, Japanese B encephalitis, measles, acute hemorrhagic conjunctivitis, Lassa and Korean hemorrhagic fever. Antigen could be recovered also in virus-infected cells (herpes simplex, measles, Lassa, Ebola, Marburg, Rift Valley, Congo and Korean Hemorrhagic fever) by enzyme treatment after periods of formalin fixation of four weeks and storage of three months. In herpes simplex virus-infected mouse brain, antigen was detected after fixation for three months in formalin.

Experimental Infection in Newborn Mice and Rats by Hemorrhagic Fever with Renal Syndrome (HFRS) Virus

Newborn mice and rats were inoculated intracerebrally (ic) or intraperitoneally (ip) with Hantaan virus (76–118 strain) or HFRS‐related virus (B‐1 strain). The mortality and the influence on the increase of body weight in newborn mice were higher in the groups infected with the 76–118 strain than in the groups infected with the B‐1 strain, while the B‐1 strain was more virulent in rats than the 76–118 strain. Virus isolation from rats inoculated with either strain was attempted 7 and 11 weeks after inoculation. Virus could be isolated from various organs of rats infected with the B‐1 strain, while it was recovered from only the brain and lungs of rats infected with the 76–118 strain. Viral antigen was readily detected in various organs of rats infected with the B‐1 strain, but the amount and distribution of antigens were less in rats infected with the 76–118 strain. Our results suggest that the virulence of HFRS‐related virus is variable, depending on the species of infected animals as well as on the. virus strains. The virus also persists in the injected animals with high titers of antibodies for at least 11 weeks.

Distribution of gentamicin by immunofluorescence in the guinea pig inner ear

SummaryWe studied the distribution of gentamicin in the inner ear, brain and kidney of the guinea pig following intraperitoneal administration or perfusion of gentamicin through the perilymphatic space. The resulting histopathologcial changes were examined by immunofluorescence using antigentamicin antiserum. After perfusion of gentamicin through the perilymphatic space, specific fluorescence was found in the cochlea, and was especially prominent in the outer hair cells, basilar membrane and basilar crest. Although no fluorescence was observed in the cochlea following intraperitoneal administration of high doses of gentamicin, type I hair cells in the vestibule were seen to be selectively stained with the antibody. Furthermore, some of the vestibular ganglion cells, Purkinje cells and unidentified nuclei in the brain stem were also stained. In particular, fine granules showing relatively intense fluorescence were recognized in the cytoplasm of the stained cells. In the cortex of kidney, only proximal tubular cells were stained with intense fluorescence. Our results suggest that the aminoglycoside antibiotics have two sites of action: one is the cell membrane of the sensory hair cells and the other is the cytoplasm.

Loss of membrane excitability after herpes simplex virus infection in tissue-cultured nerve cells from adult mammals

Dorsal root ganglion cells of adult guinea-pigs in vitro were inoculated with herpes simplex virus (HSV). Intracellular recording with a glass microelectrode revealed that HSV-infection caused a marked decrease in membrane excitability of the nerve cells within 24 h, which could be explained as a decrease in Na+ channel activity in the plasma membrane; while changes in other physiological properties (resting membrane potential, membrane resistance and capacitance) remain small. Viral antigens were detected in only 10-15% of the HSV-infected nerve cells. These indicate that the loss of the membrane excitability occurs much earlier than any other change in the HSV-infected nerve cells.

Analysis of viral antigens in giant cells of measles pneumonia by immunoperoxidase method.

The localization of measles virus proteins was analyzed by immunoperoxidase method using both monospecific and monoclonal antibodies. In Vero cells infected with the Edmonston or EB-L strain, the former being a laboratory strain and the latter a fresh isolate from a measles patient, nucleocapsid protein was located in the nuclei, and matrix protein, phosphoprotein, haemagglutinin and fusion protein were located in the cytoplasm. In the lung tissues of eight cases with measles giant cell pneumonia, the similar findings were obtained. The presence of haemagglutinin on the surface of giant cells at the luminal side was also noticed. Histopathologically, measles giant cells had nuclear and cytoplasmic eosinophilic inclusion bodies with some differences in appearance. The significance of localization of viral proteins is discussed in comparison with histopathological findings in measles giant cells.

Specific reduction in Na currents after infection with herpes simplex virus in cultured mammalian nerve cells

Tissue-cultured nerve cells originating from dorsal root ganglia of adult guinea-pigs were infected with type 2 herpes simplex virus (HSV) in vitro, and changes in membrane properties were examined (48 h later) by conventional electrophysiological technique using a glass microelectrode. The nerve cells infected with HSV (HSV-NC) failed to generate full-sized Na spikes, which was associated with a marked reduction in the maximum rate of rise of the Na spike, i.e. Na currents. Some SV-NC failed to generate any Na spike. By contrast, the HSV-NC generated unchanged, full-sized Ca spikes when bathed in a Na-free solution containing tetraethylammonium+. Changes in resting membrane properties, such as resting potential, input resistance and capacitance, remained small after the infection. We thus consider that HSV reduces Na currents in a specific fashion in this early period of the infection.

Morphological and physiological studies on cultured nerve cells from guinea pigs infected with herpes simplex virus in vivo

Adult guinea pigs were inoculated with type 2 herpes simplex virus (HSV) on the whole back skin, and nerve cells from the dorsal root ganglia (DRG) 6 days after infection were grown in tissue culture. Morphological and physiological properties of the cultured nerve cells from HSV-infected animals (HSV-NC) were compared to those of nerve cells of DRG from the control, non-virus infected animals (CON-NC). During the early period of the culture (0-4 days) growth of nerve cells and non-neuronal cells from the HSV infected animals was essentially the same as that from the control animals. HSV-antigen was present in only a small percentage of the HSV-NC by immunofluorescence (IF). Electrophysiological examination revealed that most of the HSV-NC exhibited a reduced capability of generating spikes, which was quantitatively described as a reduction in the Vmax of the Na spikes. This was interpreted as a reduction in the number of Na channel molecules in the plasma membrane of the HSV-NC, while the resting membrane properties of the same cells, such as the resting membrane potential, input resistance and capacitance, were essentially the same as those of the CON-NC. On 4-5 days in culture, some HSV-NC regained a full capability to generate Na spikes. We considered these nerve cells to have overcome the HSV infection and were now entering a latent period of HSV infection. About 1/3 of the HSV-NC still remained incapable of generating Na spikes. Viral antigen was detected in only 10% of the nerve cells. In the late stage of the culture, HSV infection in vitro was first observed as lysis of non-neuronal cells growing close to some HSV-NC. Nerve cells then started to lose their neurites and became spherical. Finally on 8-9 days most of the cells, including the nerve cells, were lost from the dishes as a result of a generalized infection of supporting cells, i.e. fibroblasts and Schwann cells. This study confirms our previous finding that the electrophysiological technique is much more sensitive than the IF method for the detection of HSV-infection in nerve cells. The results indicate that some nerve cells infected with HSV overcome the infection in vitro. This is interpreted as the entering of these nerve cells into a latent period of HSV infection in vitro.

Experiments on herpes simplex viral infections of the facial nerve in the tympanic cavity.

SummaryAn experimental protocol was developed to study viral infection of the facial nerve. Facial palsy was induced in a guinea pig model by inoculating type 1 herpes simplex virus (HSV-1) directly onto the facial nerve in the temporal bone. Partial removal of the bony wall of the fallopian canal was effected, and virus was placed on the nerve after incising its sheath on the right side and without incision of the sheath on the left side. All animals exhibited subsequent bilateral facial palsies, but with severe changes occurring on the right side. The animals were then sacrificed sequentially following infection. Immunofluorescence and histopathological studies revealed the presence of HSV antigen, infiltration of inflammatory cells, hemorrhage, and degeneration of the right facial nerve. In the left nerves, a slight hemorrhage was recognized without cellular infiltration or HSV antigens. These results suggest that the intact facial nerve is rather resistant to HSV infection, but this defense mechanism is easily destroyed by damage to the facial nerve sheath.