The cytopathology of herpes simplex virus (HSV) in H.Ep.2 and BHK-21 cells was studied using the techniques of light microscopy, immunofluorescence, electron microscopy, autoradiography and cytogenetics. Both cell types supported rapid growth cycles of HSV resulting in the production of maximum titres after 22 - 24 hours of infection. Cultures treated with 10 µg/ml ara-C or 100 µg/ml IDU at the time of infection showed a 99% decrease in infectious virus production. HSV-infected H.Ep.2 and BHK-21 cells revealed typical virus-induced inclusion bodies and a generalized disorganization of the nucleus and cytoplasm. Syncytia formation was not observed but after 24 hours of infection, nearly 100% of the cells were rounded and often detached from the glass surface. Addition of 10 µg/ml ara-C or 100 µg/ml IDU failed to prevent virus cytopathology but did cause a characteristic cytoplasmic disruption and rounding of uninfected cells. Virus-infected cells also revealed at least four separate immuno-fluorescent elements after exposure to hyperimmune serum prepared in guinea pigs. These elements included small nuclear granules, amorphous nuclear masses, diffuse cytoplasmic antigens, and intense surface fluorescence. The nuclear antigens and cytoplasmic fluorescence appeared after treatment with ara-C or IDU but the surface fluorescence was not produced in the presence of the anti-viral agents. Herpes simplex virus developed in the nucleus of infected H.Ep.2 and BHK-21 cells. The virions were enveloped at the inner lamella of the nuclear membrane and after passing into the cytoplasm, were released from the cells by a process of reverse phagocytosis. Ara-C and IDU allowed the synthesis of certain viral antigens and the development of nuclear cytopathology but completely prevented the formation of infectious HSV particles. Both drugs caused a marked distortion of the mitochondria and endoplasmic reticulum in uninfected cells. DNA synthesis in HSV-infected cells, as measured by ³H-thymidine incorporation, was almost completely inhibited by 4 hours of infection. This early inhibition of cellular DNA synthesis was followed by an immediate increase in ³H-thymidine uptake corresponding to the synthesis of viral DNA. Both cell types showed a brief stimulation of mitosis prior to the complete inhibition observed after 20 hours of infection. Cellular and viral DNA synthesis and mitosis appeared to be inhibited in virus-infected and uninfected cells treated with ara-C or IDU. Infection with HSV resulted in severe chromosomal damage to H.Ep.2 and BHK-21 cells. Chromosomal abnormalities included chromatid gaps and breaks, enhanced secondary constrictions, fragmentation, erosion, and endoreduplication, and were dependent on virus dose and time of infection. The capacity of the virus to induce chromosomal aberrations in cultured cells was UV-inactivated approximately five times less rapidly than the infectious property. Ara-C acted synergistically with the virus to produce a large number of cells with multiple chromosome breaks and also caused a significant number of abnormalities in uninfected cells. In contrast, IDU treatment resulted in few aberrations over and above those produced by HSV and little damage in uninfected cells. It was concluded that HSV was capable of producing severe morphological and genetic alterations in cultured human and hamster cells. The antiviral agents ara-C and IDU were able to completely inhibit virus multiplication but were unable to prevent any of the virus-induced cytopathic effects in vitro. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

In vivo infection with HIV-1 is typically characterized by progressive clinical and immunological deterioration associated with a decrease in CD4 T-cells. The mechanism of CD4 T-cell depletion in vivo and its role in HIV-1 pathogenesis and development of AIDS is not well understood. My research has focused on understanding the mechanism or mechanisms by which HIV-1 induces cell death. To address this aim, a panel of primary HIV-1 isolates were characterized in vitro for replication kinetics, syncytium formation, and cytopathic effects. From this panel two viral isolates, one from patient A and the other patient D, were selected for further evaluation. Patient A was asymptomatic with absolute CD4 cell count of 2302 at the time of virus isolation and remains so nine years later. Patient D was symptomatic with absolute CD4 cell count of 64 and has subsequently died from AIDS. Both of these patients maintained high viral load in vivo. When the viruses were examined in vitro, they also replicated to high titers. However, there were dramatic differences regarding the induction of cell death by HIV-1 isolates. Viruses obtained from patient A did not induce cell death although they replicated to high titers. In contrast, viruses obtained from patient D were extremely cytopathic in PBMCs with comparable viral replication. Therefore, viral replication alone does not predict the single-cell killing capacity of primary HIV-1 isolates. HIV-1 viruses isolated from an individual with normal CD4 T-cell numbers and an individual with CD4 T-cell depletion replicated to equivalent levels in primary CD4 T-cells. However, the virus isolated from the symptomatic individual induced cell death and the virus isolated from the asymptomatic individual was non-cytopathic in vitro. It is known that HIV-1 exists in the host as a group of related viruses known as quasispecies. This diversity allows the virus a broad spectrum of genotypes which result in multiple phenotypic properties. It follows then that a single viral isolate may contain a number of variants which differ in their ability to form syncytia, cell tropism, replication kinetics, as well as cytopathic potential. To address this, biological clones were obtained from each of the patients viral quasispecies and characterized for replication and cytopathicity. These clones, GC6 8-4 (isolated from patient A) and HC4 (isolated from patient D) maintained the same viral phenotype as the parental virus. In other words, HIV-1 biological clone GC6 8-4 was non-syncytium inducing (NSI) and non-cytopathic in vitro. In contrast, HIV-1 biological clone HC4 was syncytium inducing (SI) and cytopathic in vitro. It has been reported that the envelope gene of HIV-1 contains the major determinants of HIV-1 induced CD4 T-cell depletion (17). To understand what may be responsible for the differences in cytopathic behavior between the biological clones, GC6 8-4 and HC4 (42), I analyzed their envelope genes. Chimeric viruses were constructed by switching env regions from V2 through V3 of the biological clones with the corresponding region from the molecular clone NL4-3. These HIV-1 chimeric viruses exhibited similar replication kinetics as well as syncytium inducing abilities. The chimeric virus containing the env region of biological clone, GC6 8-4, was NCP in the single cell killing assay, while the chimeric virus containing the env region of biological clone, HC4, was CP in the single cell killing assay. These studies suggest the presence of a cytopathicity determinant which maps to the envelope region downstream from V2 and through V3 (Stu I at position 6822 to Nhe I at position 7250 based on NL4-3 sequence (101)).