Anterograde transport of HSV-1 and HSV-2 in the visual system.

The anterograde spread of herpesvirus in the visual system subsequent to retinitis has been observed clinically. We compared the ability of two well-studied Herpes simplex virus (HSV) strains to be transported in the anterograde direction in the hamster visual system: strain McIntyre, representing HSV-1, and strain 186, representing HSV-2. Intravitreal injection of HSV-2 labeled more retinorecipient neurons than did HSV-1, suggesting important type differences in the ability of HSV to infect retinorecipient neurons after intravitreal injection. The most likely explanation for our results is that HSV-2 is more efficiently adsorbed than HSV-1 in the retinal ganglion cells. Our results also suggest that HSV may be useful as an anterograde transneuronal tracer for neuroanatomical studies of the visual system.

Somatotopic distribution of corneal afferent neurons in the guinea pig trigeminal ganglion.

The size and somatotopic distribution of corneal afferent neurons in the guinea pig trigeminal ganglion were determined using a retrograde axonal tracing technique. Wheat germ agglutinin-horseradish peroxidase (WGA-HRP) was applied to the central cornea of the guinea pig and the animals were perfusion-fixed 48 h later. In addition, a preliminary study examined corneal afferent neurons in two animals latently infected with the herpes simplex virus by corneal inoculation. The majority of WGA-HRP-labelled neurons were located in the ophthalmic division of the ipsilateral ganglion. A clear dorsoventral somatotopic arrangement of labelled corneal afferent neurons was noted. The size of the neurons averaged 23 microns and the number of cells per ganglion averaged 205. By contrast, the number of labelled neurons in latently infected ganglia averaged less than 50. No size or morphological distinctions could be made between neurons from uninfected or latently infected ganglia. The results of this study have provided for the first time the precise location and somata diameter of primary afferent corneal neurons within the guinea pig trigeminal ganglion.

Thioglycollate elicited macrophages demonstrate enhanced virus replication and depressed bacterial killing.

Thioglycollate elicited peritoneal macrophages of Balb/c mice exhibited minimal antibacterial activity against Listeria monocytogenes but were fully permissive for the replication of ectromelia virus. By comparison, resident and LPS elicited macrophages did not exhibited depressed antibacterial activity nor did they support viral replication. The thioglycollate effects were demonstrated in macrophages cultured in vitro and also in intact Balb/c mice. Mice given thioglycollate intraperitoneally and challenged by the same route suffered overwhelming virus and bacterial infections as a result of early local proliferation within peritoneal macrophages with subsequent spread to the liver. Balb/c mice challenged intravenously with similar doses of the virus of bacterial pathogen after administration of thioglycollate by the i.p. route did not succumb to either infection. Thus the ability of thioglycollate to compromise cellular host defenses against the infectious agents appears to be site specific; i.e. restricted to the peritoneal cavity where exudate macrophages and challenge inocula first come into contact.

The pathogenesis of herpetic ocular disease in the guinea pig.

A detailed study of the pathogenesis of herpetic eye disease in the guinea pig was undertaken to further develop this animal model. Several well-known HSV-1 strains were tested for their ability to produce disease and cause acute and latent infections of the trigeminal ganglion: McKrae, KOS, McIntyre, RE, and Shealey. Two HSV-2 strains failed to cause eye infections. The Shealey strain [HSV-1 (Sh)] produced the most severe eye infections, characterized by epithelial and stromal disease, corneal vascularization and ulcerative blepharitis. Consequently, HSV-1 (Sh) was selected as the prototype strain for this study. The frequency and severity of HSV-1 (Sh) eye disease patterns was determined by a semi-quantitative rating scale, which permitted accurate monitoring of the temporal development of the disease patterns cited above. Virus shedding from infected eyes was also quantified. All of the HSV-1 strains tested established trigeminal ganglionic latency with varying frequency, although HSV-1 (Sh) latency approached 100 percent. The kinetics of acute ganglionic infection by HSV-1 (Sh) was determined, and peak virus titers occurred on the third day after corneal inoculation. This study emphasizes the usefulness of the Guinea pig model for investigations on the pathogenesis, prevention and treatment of herpetic eye infections.

An improved method for detection of ganglionic latency in herpes simplex virus type-1 infected guinea pigs.

A simple method was developed which increased the sensitivity and reliability of detecting latent herpes simplex virus type-1 (HSV-1) in trigeminal ganglia of guinea pigs. Animals were infected with the Shealey strain of HSV-1 immediately following scarification of the cornea and maintained for 30-40 days to ensure that true latency was established. Viral latency was defined as the appearance of infectious virus in ganglia only upon cultivation in vitro. Thus, ganglia from similarly infected animals, homogenized immediately upon removal, did not contain infectious virus. Excised ganglia were incubated intact in high glucose medium and yielded maximal positive results (90-100%) by the twelfth day of incubation. This method was compared with the standard cocultivation technique in which minced fragments of ganglionic tissue were explanted onto Vero cell cultures. Cocultivation yielded a considerably lower latency rate and was more variable (29-57%) than the whole ganglion culture method.

Abortive ectromelia virus infection in peritoneal macrophages activated by Corynebacterium parvum.

We have previously demonstrated that peritoneal macrophages (M phi S) from C3H mice were resistant to in vitro infection by ectromelia virus, following activation by intraperitoneal injection of the immunomodulator Corynebacterium parvum. In contrast, resident and mineral oil-elicited M phi S were fully susceptible to virus infection. This report analyzes the infectious cycle of ectromelia virus in C parvum-activated and mineral oil-elicited M phi S and demonstrates that an abortive infection occurred in the activated M phi S that blocked the infectious cycle prior to the release of DNA from the infecting virions. The kinetics of adsorption of radiolabeled virus were similar in both susceptible and resistant M phi cultures; however, viral-induced incorporation of uridine and thymidine occurred only in the mineral oil-elicited and not the C parvum-activated M phi S. In addition, the late protein hemagglutinin was only detected in infected cultures of susceptible mineral oil-elicited M phi S. An electron micrographic analysis of the infectious cycle indicated that the adsorption of virus to the plasma membrane, uptake into lysosomes, and the primary undercoating and release of viral cores into the M phi cytoplasm were identical in both M phi types. In contrast, secondary uncoating (release of genomic DNA from the viral cores into the cytoplasm) was never detected in infected C parvum M phi S. These data are consistent with our previous findings and with the hypothesis that activation of M phi S by C parvum induces an interferon-mediated resistance to ectromelia virus infection.

Induction of resistance to ectromelia virus infection by corynebacterium parvum in murine peritoneal macrophages.

An in vitro model has been developed to study the replication of ectromelia virus in murine macrophages (M phi). Infection of mineral oil-elicited peritoneal M phi cultures with either the virulent (Moscow) or attenuated (Hampstead) strain of ectromelia virus led to productive infections. The kinetics of virus synthesis was similar to those seen following infection of murine fibroblasts. In contrast, peritoneal M phi s activated by intraperitoneal injection of Corynebacterium parvum vaccine were found to be totally refractory to infection by the attenuated strain and significantly more resistant to the virulent strain of ectromelia virus. Administration of C. parvum doses as small as 7 micrograms were sufficient to induce antiviral activity. M phi resistance became maximal at 5-9 days after C. parvum administration; however, M phi resistance was unstable during in vitro culture. Decay of antiviral activity was detected within the first 24 hr of culture and complete virus susceptibility returned after 5 days in culture. Peritoneal exudate cells (PEC) from C. parvum-immunized mice could induce resistance in susceptible M phi cultures during overnight cocultivation. In addition, cell-free culture supernatants from C. parvum-immune PEC could also induce resistance in susceptible M phi cultures, suggesting that a soluble factor, induced by C. parvum immunization and possessing interferon activity, may account for the intrinsic resistance to ectromelia virus by activated M phi s.

Development of protective immunity against bacterial and viral infections in tumor-bearing mice coincident with suppression of tumor immunity.

This report addresses the question whether Meth A (methylcholanthrene-induced fibrosarcoma) tumor bearing Balb/c mice are able to develop specific antimicrobial immunity. Although specific suppressor T lymphocytes appeared during tumor growth which prevented expression of antitumor immunity, the development of protective immunity to L monocytogenes, S. pneumoniae or ectromelia virus infections was unimpaired. The Meth A tumor produced a soluble immunosuppressive factor which inhibited lymphocyte and macrophage functions in vitro. Tumor growth failed to inhibit the formation of immunoglobulin essential to antipneumococcal immunity, or the development of a specific acquired cellular resistance of primary importance in immunity to listeria and ectromelia virus infections. That tumor growth did not interfere with the development of cell mediated immunity was demonstrated by the effective transfer of antilisteria immunity by immune spleen from tumor-bearing mice.

Impaired resistance to bacterial infection after tumor implant is traced to lactic dehydrogenase virus.

A BALB/c mouse-passaged methylcholanthrene-induced fibrosarcoma tumor caused severe impairment of resistance to systemic listeriosis. Depressed resistance expressed immediately after tumor implantation was traced to inadvertent association of tumor with lactic dehydrogenase virus. Tumor cured of virus was totally inactive.

In situ fixation of cultured mouse peritoneal exudate cells: comparison of fixation methods.

Mouse peritoneal exudate cells grown in vitro on plastic petri dishes were fixed in situ with both glutaraldehyde and osmium tetroxide by a variety of contemporary methods. The goal of the investigation was to determine which method resulted in the best ultrastructural preservation. The parameters being tested included: (a) the method of fixation, i.e. either sequential or simultaneous; (b) the buffer vehicle for fixation, i.e. cocodylate, Mellonig's phosphate, Sorenson's phosphate, or s-collidine; and (c) the temperature of fixation. Results presented indicate that simultaneous fixation is far superior to sequential methods. Samples fixed sequentially at 4 degrees C consistently had better morphological preservation than samples fixed under similar conditions at 23 degrees C. With the exception of s-collidine, which was totally unacceptable for in vitro in situ fixation on plastic, comparable results were noted with different buffer vehicles.

Human lactic dehydrogenase as a marker for human tumor cells grown in athymic mice.

Human tumors implanted s.c. into athymic mice released lactic dehydrogenase (LDH) isoenzymes unique to human tissue. These isoenzymes were readily identified and quantitated in mouse plasma. When injected into mice i.p. or i.v., human LDH isoenzymes were rapidly cleared from the blood circulation, decreasing to within 10% of the initial value in 12 hr. When human tumor cell lines (HEp-2 and T-24) were injected i.v. into heterozygote or athymic mice, they released LDH isoenzymes over a 24-hr period. When these cells were injected by the i.p. route, they released the isoenzymes over the 4-day period studied. Solid tumors of HEp-2, T-24, and SW-733 cells s.c. implanted continuously released amounts of LDH that were directly related to tumor mass. Therefore, the measurement of plasma levels of human LDH isoenzymes in athymic mice is a useful parameter for detecting the presence and growth of human tumors in these animals. Since the bulk of the released LDH is assumed to derive from injured or destroyed human tumor cells, the assay for these isoenzymes should provide a useful marker for determining the effectiveness of experimental antitumor therapy in athymic mice.

Cell surface alterations induced by vaccinia and Newcastle disease viruses (38486).

A comparison was made of HEp-2 cell surface changes induced by NDV or vaccinia virus infection. Three parameters were examined as a function of time after infection: the kinetics of hemadosorption and the appearance of concanavalin (con A) binding sites, and alterations in electrophoretic mobility of single cells. The kinetics of appearance of con A binding sites was strikingly similar for both virus infections, whereas hemadsorption preceded NDA synthesis and followed vaccinia synthesis. These data suggest that in the vaccinia-infected cell the hemadsorption and con A binding sites are different. NDV infection or exposure of sham-infected cells to bacterial neuraminidase significantly reduced their anodal mobilities. This also occurred after enzyme treatment of vaccinia-infected cells. Measurements of the sialic acid content of NDV or sham-infected cells before and after neuraminidase treatment indicated the exposure to the enzyme or NDV materially reduced the sialic acid content of cells. Vaccinia-infected cells contained considerably more sialic acid than did normal cells. For the vaccinia-infected cell a change in surface properties as detected by hemadsorption or increased con A binding was not reflected in a change in electrophoretic mobility.

Origin of the vaccinia virus hemagglutinin.

The relationship between the vaccinia virus hemagglutinin and hemadsorption was examined. Hemagglutinin synthesis was temporally related to the appearance of the hemadsorption reaction. Only chicken erythrocytes, which reacted with hemagglutinin, hemadsorbed to infected cells, and both of these reactions were inhibited by Ca(2+). The distribution of the vaccinia hemagglutinin and 5'-adenosine monophosphatase, a plasma membrane marker enzyme, in sucrose gradients was similar. Plasma membrane ghosts derived from infected cells hemadsorbed erythrocytes and yielded hemagglutinin upon sonic disruption. These data suggest that the majority of vaccinia hemagglutinin is derived from the plasma-membrane of the infected cell.

Poliovirus-induced Cellular Injury.

Protein leakage was used as a quantitative measure of poliovirus-induced cellular injury under suspended cell culture conditions. The requirements for protein leakage were studied in detail and it was established that events early in the infectious cycle which depend upon viral protein synthesis were responsible for cell damage. Extralysosomal beta-glucuronidase appeared in infected cells before the onset of protein leakage and release of newly synthesized virus. Hydrocortisone treatment of infected cells resulted in only a slight delay in the release of beta-glucuronidase from lysosomes and protein and virus from cells. These results suggest that events associated with poliovirus synthesis trigger the release of lysosomal hydrolases which in turn injure the plasma membrane, allowing cytoplasmic proteins and virus to leak out of the cell.