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Inborn errors of TLR3-dependent type I IFN immunity in cortical neurons underlie forebrain herpes simplex virus-1 (HSV-1) encephalitis (HSE) due to uncontrolled viral growth and subsequent cell death. We report an otherwise healthy patient with HSE who was compound heterozygous for nonsense (R422*) and frameshift (P493fs9*) RIPK3 variants. Receptor-interacting protein kinase 3 (RIPK3) is a ubiquitous cytoplasmic kinase regulating cell death outcomes, including apoptosis and necroptosis. In vitro, the R422* and P493fs9* RIPK3 proteins impaired cellular apoptosis and necroptosis upon TLR3, TLR4, or TNFR1 stimulation and ZBP1/DAI-mediated necroptotic cell death after HSV-1 infection. The patient’s fibroblasts displayed no detectable RIPK3 expression. After TNFR1 or TLR3 stimulation, the patient’s cells did not undergo apoptosis or necroptosis. After HSV-1 infection, the cells supported excessive viral growth despite normal induction of antiviral IFN-β and IFN-stimulated genes (ISGs). This phenotype was, nevertheless, rescued by application of exogenous type I IFN. The patient’s human pluripotent stem cell (hPSC)–derived cortical neurons displayed impaired cell death and enhanced viral growth after HSV-1 infection, as did isogenic RIPK3-knockout hPSC-derived cortical neurons. Inherited RIPK3 deficiency therefore confers a predisposition to HSE by impairing the cell death–dependent control of HSV-1 in cortical neurons but not their production of or response to type I IFNs.

Invasion of the brain by herpes simplex virus 1 (HSV1) can lead to the development of herpes simplex encephalitis (HSE) that is often associated with significant morbidity and mortality regardless of therapeutic intervention. Both virus and host immune factors dictate HSE onset and progression. Because programmed cell death pathways including necroptosis are important antiviral defense mechanisms in HSV1-associated peripheral diseases, they might also play critical roles in HSV1 neuropathogenesis. HSV1-encoded ICP6 prevents receptor-interacting protein kinase 3 (RIPK3)-mediated necroptosis during infection of human cells, but it also acts as a species-dependent inducer of necroptosis in murine cells and thereby restricts virus replication. We therefore used an established mouse model of HSE to investigate RIPK3-mediated necroptosis impact on HSV1 neuropathogenesis. Following corneal HSV1 inoculation, RIPK3 knockout mice showed increased susceptibility to HSE when compared with wildtype mice indicating RIPK3 helps to limit HSE progression. RIPK3-mediated defense against HSE was found to be independent of the kinase domain necessary to drive necroptosis implicating that a death independent function of RIPK3 protects against HSE. Conversely the pro-necroptotic kinase function RIPK3 served to limit viral replication in corneal tissue implicating a tissue-specific RIPK3 function in limiting HSV1. Further evaluation of the kinase-independent mechanism to restrict HSE revealed that the RIPK3 signaling partner, caspase 8, contributes to limiting HSE neuropathogenesis. Increased HSE susceptibility from loss of caspase 8 and RIPK3 correlated with decreased levels of chemokines, cytokines, and antiviral lymphocytes recruitment to the brain. We conclude that RIPK3 contributes toward host control of HSV1 replication in a tissue-specific fashion. Whereas RIPK3-mediated necroptosis restricts virus replication within the cornea, kinase-independent induction of inflammation by RIPK3 in collaboration with caspase 8 restricts virus replication within the brain during HSE neuropathogenesis.

Human cytomegalovirus (HCMV) generates a significant clinical burden worldwide, particularly among the immune compromised. In approximately 30% of untreated HIV/AIDS patients without access or sufficient response to antiretroviral therapies, for example, HCMV causes a sight-threatening retinitis. To study the mechanisms of AIDS-related HCMV retinitis, our lab has for many years used a mouse model in which a mixture of mouse retroviruses induces murine AIDS after approximately 10 weeks, rendering otherwise resistant mice susceptible to opportunistic pathogens. This immunodeficiency combined with subretinal inoculation of murine cytomegalovirus yields a reproducible model of the human disease, facilitating the discovery of many clinically relevant virologic and immunologic mechanisms of retinal destruction which we summarize in this review.

Interleukin-1α (IL-1α) is an alarmin involved in the recruitment of macrophages and neutrophils during tissue inflammation. IL-1α can undergo cleavage by proteases, such as calpain-1, that enhances IL-1α binding to its receptor, although proteolytic cleavage is not necessary for biological activity. Macrophages and neutrophils are involved in the retinal inflammation associated with development of AIDS-related human cytomegalovirus (HCMV) retinitis. We therefore performed studies to test the hypothesis that IL-1α gene expression is stimulated intraocularly during retinitis development using two mouse models of murine cytomegalovirus (MCMV) retinitis that differ in method of immunosuppression, one by retrovirus-induced immunosuppression (MAIDS) and the other by corticosteroid-induced immunosuppression. MCMV-infected eyes of groups of retinitis-susceptible mice with MAIDS of 10 weeks duration (MAIDS-10 mice) and retinitis-susceptible corticosteroid-treated mice showed significant stimulation of IL-1α mRNA. Western blot analysis confirmed IL-1α protein production within the MCMV-infected eyes of MAIDS-10 mice. Whereas significant intraocular calpain-1 mRNA and protein production were also observed within MCMV-infected eyes of MAIDS-10 mice, the MCMV-infected eyes of retinitis-susceptible corticosteroid-treated mice showed a pattern of mRNA synthesis equivalent to that found within the MCMV-infected eyes of healthy mice that fail to develop retinitis. Our findings suggest a role for the alarmin IL-1α in the pathogenesis of MCMV retinitis in immunosuppressed mice. These findings may extend to the pathogenesis of HCMV retinitis in patients with AIDS or other forms of immunosuppression.

AIDS-related human cytomegalovirus retinitis remains a leading cause of blindness worldwide. We compared two C57BL/6 mouse models of experimental murine cytomegalovirus (MCMV) retinitis for intraocular expression of suppressors of cytokine signaling (SOCS)1 and SOCS3, host proteins that are inducible negative feedback regulators of cytokine signaling. These mouse models differed in method of immune suppression, one by retrovirus-induced immune suppression (MAIDS) and the other by corticosteroid-induced immune suppression. Following subretinal injection of MCMV to induce retinitis, intraocular SOCS1 and SOCS3 were only mildly stimulated, and often without significance, within MCMV-infected eyes during the progression of MCMV retinitis in corticosteroid-immunosuppressed mice, contrary to MCMV-infected eyes of mice with MAIDS that showed significant high stimulation of SOCS1 and SOCS3 expression in agreement with previous findings. Frequency and severity of retinitis as well as amounts of intraocular infectious MCMV in corticosteroid-immunosuppressed mice were also unexpectedly lower than values previously reported for MAIDS animals during MCMV retinitis. These data reveal a major difference between two mouse models of experimental MCMV retinitis and suggest a possible link between the amplitude of SOCS1 and SOCS3 stimulation and severity of disease in these models.

The mechanisms that contribute to retinal tissue destruction during the onset and progression of AIDS-related human cytomegalovirus (HCMV) retinitis remain unclear. Evidence for the stimulation of multiple cell death pathways including apoptosis, necroptosis, and pyroptosis during the pathogenesis of experimental murine cytomegalovirus (MCMV) retinitis in mice with retrovirus-induced immunosuppression (MAIDS) has been reported. Parthanatos is a caspase-independent cell death pathway mediated by rapid overactivation of poly (ADP-ribose) polymerase-1 (PARP-1) and distinct from other cell death pathways. Using the MAIDS model of MCMV retinitis, studies were performed to test the hypothesis that intraocular MCMV infection of mice with MAIDS stimulates parthanatos-associated messenger RNAs (mRNAs) and proteins within the eye during the development of retinal necrosis that takes place by 10 days after MCMV infection. MCMV-infected eyes of MAIDS mice exhibited significant stimulation of PARP-1 mRNA and proteins at 3 days after infection but declined thereafter at 6 and 10 days after infection. Additional studies showed the intraocular stimulation of mRNAs or proteins before MCMV retinitis development for two additional participants in parthanatos, polymer of ADP-ribose and poly (ADP-ribose) glycohydrolase. These results provide new evidence for a role for parthanatos during MAIDS-related MCMV retinitis that may also extend to AIDS-related HCMV retinitis.

Pyroptosis is a caspase-dependent programmed cell death pathway that initiates and sustains inflammation through release of pro-inflammatory cytokines interleukin (IL)-1β and IL-18 following formation of gasdermin D (GSDMD)-mediated membrane pores. To determine the possible pathogenic contributions of pyroptosis toward development of full-thickness retinal necrosis during AIDS-related human cytomegalovirus retinitis, we performed a series of studies using an established model of experimental murine cytomegalovirus (MCMV) retinitis in mice with retrovirus-induced immunosuppression (MAIDS). Initial investigations demonstrated significant transcription and translation of key pyroptosis-associated genes within the ocular compartments of MCMV-infected eyes of mice with MAIDS. Subsequent investigations compared MCMV-infected eyes of groups of wildtype MAIDS mice with MCMV-infected eyes of groups of caspase-1(−/−) MAIDS mice, GSDMD(−/−) MAIDS mice, or IL-18(−/−) MAIDS mice to explore a possible contribution of pyroptosis towards the pathogenesis of MAIDS-related MCMV retinitis. Histopathologic analysis revealed typical full-thickness retinal necrosis in 100% of MCMV-infected eyes of wildtype MAIDS mice. In sharp contrast, none (0%) of MCMV-infected eyes of MAIDS mice that were deficient in either caspase-1, GSDMD, or IL-18 developed full-thickness retinal necrosis but instead exhibited an atypical pattern of retinal disease characterized by thickening and proliferation of the retinal pigmented epithelium layer with relative sparing of the neurosensory retina. Surprisingly, MCMV-infected eyes of all groups of deficient MAIDS mice harbored equivalent intraocular amounts of infectious virus as seen in MCMV-infected eyes of groups of wildtype MAIDS mice despite failure to develop full-thickness retinal necrosis. We conclude that pyroptosis plays a significant role in the development of full-thickness retinal necrosis during the pathogenesis of MAIDS-related MCMV retinitis. This observation may extend to the pathogenesis of AIDS-related HCMV retinitis and other AIDS-related opportunistic virus infections.

Herpes simplex virus 1 (HSV-1) is a widespread global pathogen, of which the strain KOS is one of the most extensively studied. Previous sequence studies revealed that KOS does not cluster with other strains of North American geographic origin, but instead clustered with Asian strains. We sequenced a historical isolate of the original KOS strain, called KOS63, along with a separately isolated strain attributed to the same source individual, termed KOS79. Genomic analyses revealed that KOS63 closely resembled other recently sequenced isolates of KOS and was of Asian origin, but that KOS79 was a genetically unrelated strain that clustered in genetic distance analyses with HSV-1 strains of North American/European origin. These data suggest that the human source of KOS63 and KOS79 could have been infected with two genetically unrelated strains of disparate geographic origins. A PCR RFLP test was developed for rapid identification of these strains.

Human cytomegalovirus (HCMV) is an opportunistic human herpesvirus that causes a sight-threatening retinitis in immunosuppressed patients, especially those with AIDS. Using an established model of experimental murine cytomegalovirus (MCMV) retinitis in mice with retrovirus-induced immunodeficiency (MAIDS), we have been attempting to define with greater clarity the immunologic mechanisms that contribute to the progression of AIDS-related HCMV retinitis in the unique immunosuppressive setting of HIV infection. Toward this end, we provide herein a comprehensive assessment of immune response gene expression during the onset and development of MAIDS-related MCMV retinitis employing NanoString nCounter. In so doing, we analyzed and compared the intraocular expressions of 561 immune response genes within MCMV-infected eyes of groups of healthy mice, MCMV-infected mice with MAIDS of 4 weeks’ (MAIDS-4) duration, and MCMV-infected eyes of mice with MAIDS of 10 weeks’ (MAIDS-10) duration. These animal groups show a progression of retinal disease from absolute resistance to retinitis development in healthy mice to the development of classic full-thickness retinal necrosis in MAIDS-10 mice but through an intermediate stage of retinal disease development in MAIDS-4 mice. Our findings showed that increased susceptibility to MCMV retinitis during the progression of MAIDS is associated with robust upregulation or downregulation of a surprisingly large number of immune response genes that operate within several immune response pathways often unique to each animal group. Analysis of 14 additional immune response genes associated with programmed cell death pathways suggested involvement of necroptosis and pyroptosis during MAIDS-related MCMV retinitis pathogenesis. Use of the NanoString nCounter technology provided new and unexpected information on the immunopathogenesis of retinitis within MCMV-infected eyes of mice with retrovirus-induced immunosuppression. Our findings may provide new insights into the immunologic events that operate during the pathogenesis of AIDS-related HCMV retinitis., Author summary The immunodeficiency caused by HIV during the progression of AIDS creates a unique systemic immunosuppressive environment that often leads to the development of opportunistic virus infections. Among these is a sight-threatening retinitis caused by human cytomegalovirus (HCMV). While the clinical and histopathologic features of AIDS-related HCMV retinitis have been well documented, the immunologic mechanisms that continue to operate during HIV-induced immunosuppression and contribute to the onset and development of retinal tissue destruction caused by ocular HCMV infection in patients with AIDS remain unexplored. Herein, we report the first comprehensive analysis of a total of 571 immune response genes that are expressed during the pathogenesis of experimental murine cytomegalovirus (MCMV) retinitis in mice at different stages of retrovirus-induced immunosuppression (MAIDS) who exhibit different susceptibilities to retinal disease development. Our finding that different patterns of immune response gene expression are observed at different stages of retrovirus immunosuppression during increased susceptibility to retinal necrosis development adds new knowledge to our understanding of the contributions of unique innate and adaptive immune pathways towards the pathogenesis of AIDS-related HCMV retinitis and may extend to other AIDS-related opportunistic virus infections.

Human cytomegalovirus (HCMV) is a species-specific β-herpesvirus that infects for life up to 80% of the world's population and causes severe morbidity in at-risk immunocompromised populations. Suppressors of cytokine signaling (SOCS)1 and SOCS3 are host proteins that act as inducible negative feedback regulators of cytokine signaling and have been implicated in several ocular diseases and viral infections. We recently found in our mouse model of experimental cytomegalovirus retinitis that subretinally-injected murine cytomegalovirus (MCMV) stimulates ocular SOCS1 and SOCS3 during retrovirus-induced immune suppression of murine AIDS (MAIDS), and that infiltrating macrophages are prominent cellular sources of retinal SOCS1 and SOCS3 expression. Herein we investigate possible virologic mechanisms whereby MCMV infection may stimulate SOCS1 and/or SOCS3 expression in cell culture. We report that infection of IC-21 mouse macrophages with MCMV propagated through the salivary glands of BALB/c mice, but not from tissue culture in C57BL/6 fibroblasts, transiently stimulates SOCS1 and SOCS3 mRNA transcripts, but not SOCS5 mRNA. Viral tegument proteins are insufficient for this stimulation, as replication-deficient UV-inactivated MCMV fails to stimulate SOCS1 or SOCS3 in IC-21 macrophages. By contrast, infection of murine embryonic fibroblasts (MEFs) with either productive MCMV or UV-inactivated MCMV significantly stimulates SOCS1 and SOCS3 mRNA expression early after infection. Treatment of MCMV-infected IC-21 mouse macrophages with the antiviral drug ganciclovir significantly decreases MCMV-stimulated SOCS3 expression at 3 days post-infection. These data suggest cell type-specific, different roles for viral immediate early or early gene expression and/or viral tegument proteins in the early stimulation of SOCS1 and SOCS3 during MCMV infection. Furthermore, putative biphasic stimulation of SOCS3 during late MCMV infection of IC-21 mouse macrophages may occur by divergent virologic mechanisms.

AIDS-related human cytomegalovirus (HCMV) retinitis remains a major ophthalmologic problem worldwide. Although this sight-threatening disease is well characterized clinically, many pathogenic issues remain unresolved, among them a basic understanding of the relative roles of cell death pathways during development of retinal tissue destruction. Using an established model of experimental murine cytomegalovirus (MCMV) retinitis in mice with retrovirus-induced immunosuppression (MAIDS), we initially investigated MCMV-infected eyes for evidence of apoptosis-associated molecules in mice with MAIDS of 4 weeks' (MAIDS-4) and 10 weeks' (MAIDS-10) duration, which were resistant and susceptible to retinal disease, respectively, but which harbored equivalent amounts of infectious MCMV. Whereas MCMV-infected eyes of MAIDS-4 mice showed little evidence of apoptosis-associated molecules, MCMV-infected eyes of MAIDS-10 mice showed significant amounts of tumor necrosis factor alpha (TNF-α), TNF receptors 1 and 2, active caspase 8, active caspase 3, TNF-related apoptosis-inducing ligand (TRAIL), TRAIL-R(DR5), Fas, and Fas ligand mRNAs and/or proteins, all detected at peak amounts prior to development of most severe retinal disease. Immunohistochemical staining showed macrophages, granulocytes (neutrophils), Müller cells, and microglial cells as TNF-α sources. Remarkably, quantification of apoptosis by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay suggested that apoptosis contributed minimally to retinal disease in MCMV-infected eyes of MAIDS-10 mice. Subsequent studies demonstrated that MCMV-infected eyes of MAIDS-10 mice, but not MAIDS-4 mice, showed evidence of significant increases in molecules associated with two additional cell death pathways, necroptosis (receptor-interacting protein 1 [RIP1] and RIP3 mRNAs) and pyroptosis (caspase 1, interleukin 1β [IL-1β], and IL-18 mRNAs). We conclude that apoptosis, necroptosis, and pyroptosis participate simultaneously during MAIDS-related MCMV retinitis, and all may play a role during AIDS-related HCMV retinitis.

Although cell-mediated cytotoxicity effectively kills target virus-infected cells, no careful consideration has been given to the fate of infectious progeny virus contained within target cells following the cytolytic event. To address this issue with respect to murine cytomegalovirus (MCMV) pathogenesis, we developed a rapid semiquantitative assay for infectious MCMV based on expression of beta-galactosidase using a LacZ-expressing recombinant MCMV. Simultaneous use of this assay in combination with a modified cell-mediated cytotoxicity assay revealed that cytotoxicity of MCMV-infected target cells mediated by MCMV-immune splenic cells does not lead to inactivation of intracellular infectious virus.

Purpose To determine if prior exposure to pathogens associated with vascular disease, cytomegalovirus, Chlamydia pneumoniae , and Helicobacter pylori correlates with neovascular age-related macular degeneration (AMD). Design An experimental study. Methods Setting Institutional. Bascom Palmer Eye Institute, October 2001 to December 2002. Patient population 150 patients (47 neovascular amd, 36 dry amd, and 67 non-amd controls) were included in the study. exclusion criteria included hiv infection, malignancy, recent acute illness requiring hospitalization within 6 months, or immunosuppressive illness. Procedure Serum samples were obtained for analysis of cytomegalovirus, chlamydia pneumoniae , and helicobacter pylori igg antibody titers by elisa. Main outcome measure Comparison of the distribution of igg titers between patients with wet amd, dry amd, and controls. Results The average cytomegalovirus IgG titer was higher in patients with wet AMD versus controls (p = 0.02, Student t-test, two-tailed) and patients with dry AMD (p = 0.06). Twenty-six (55%) of 47 subjects with wet AMD had high cytomegalovirus IgG titers compared with 14 (39%) of 36 patients with dry AMD (odds ratio [OR] = 2.23, 95% confidence interval [CI] = 0.77 to 6.44) and 23 (34%) of 67 control patients (OR = 2.49, 95% CI=0.98 to 6.33). There was no major difference in the distribution of titers for Chlamydia pneumoniae IgG and Helicobacter pylori IgG in wet and dry AMD patients. Five of 47 patients with wet AMD (11%) had high antibody titers to all three pathogens, compared with only 1 of 36 patients with dry AMD (3%) (OR = 4.17, 95% CI=0.46 to 37.36). Conclusions There was a significant association of high cytomegalovirus IgG titer with neovascular AMD compared with dry AMD and control patients. Chronic infection with cytomegalovirus may be a novel risk factor for the progression from dry to neovascular AMD.

(2004). AIDS-related cytomegalovirus retinitis: Lessons from the laboratory. Current Eye Research: Vol. 29, No. 2-3, pp. 91-101.

Experiments were performed to explore the ability of murine cytomegalovirus (MCMV) to disseminate to the eye following intravenous inoculation and to cause infection of ocular tissues and necrotizing retinitis in C57BL/6 mice with retrovirus-induced immunodeficiency syndrome (MAIDS). Although infectious virus could be detected in whole eye homogenates of mice with MAIDS at 10 days after intravenous MCMV inoculation, a recombinant MCMV (RM461) that carries an MCMV IE1 promoter-LacZ insert was used as a tracer virus to confirm direct infection of ocular tissues. Evidence for MCMV replication (determined by RM461-induced expression of β-galactosidase) was consistently observed in the nonpigmented epithelium of the ciliary body of the eyes of MAIDS animals at 14 days after infection. In sharp contrast, however, the neurosensory retina was spared and necrotizing retinitis failed to develop. These findings demonstrate that systemic MCMV infection of mice with MAIDS results in ocular MCMV infection without development of ocular MCMV disease. Conversion of occult subclinical MCMV infection of ocular tissues to overt clinical MCMV retinitis may require as yet unidentified cofactor(s). Identification of these cofactors could lead to more innovative therapeutic approaches for prevention and/or treatment of CMV retinitis in patients with AIDS.

Interleukin-17 (IL-17), a proinflammatory cytokine produced by CD4+ Th17 cells, has been associated with the pathogenesis of several autoimmune diseases including uveitis. The fate of IL-17 during HIV/AIDS, however, remains unclear, and a possible role for IL-17 in the pathogenesis of AIDS-related diseases has not been investigated. Toward these ends, we performed studies using a well-established animal model of experimental murine cytomegalovirus (MCMV) retinitis that develops in C57/BL6 mice with retrovirus-induced immunosuppression (MAIDS). After establishing baseline levels for IL-17 production in whole splenic cells of healthy mice, we observed a significant increase in IL-17 mRNA levels in whole splenic cells of mice with MAIDS of 4-weeks (MAIDS-4), 8-weeks (MAIDS-8), and 10-weeks (MAIDS-10) duration. In contrast, enriched populations of splenic CD4+ T cells, splenic macrophages, and splenic neutrophils exhibited a reproducible decrease in levels of IL-17 mRNA during MAIDS progression. To explore a possible role for IL-17 during the pathogenesis of MAIDS-related MCMV retinitis, we first demonstrated constitutive IL-17 expression in retinal photoreceptor cells of uninfected eyes of healthy mice. Subsequent studies, however, revealed a significant decrease in intraocular levels of IL-17 mRNA and protein in MCMV-infected eyes of MAIDS-10 mice during retinitis development. That MCMV infection might cause a remarkable downregulation of IL-17 production was supported further by the finding that systemic MCMV infection of healthy, MAIDS-4, or MAIDS-10 mice also significantly decreased IL-17 mRNA production by whole splenic CD4+ T cells. Based on additional studies using IL-10 −/− mice infected systemically with MCMV and IL-10 −/− mice with MAIDS infected intraocularly with MCMV, we propose that MCMV infection downregulates IL-17 production via stimulation of suppressor of cytokine signaling (SOCS)-3 and interleukin-10.

Interleukin-4 (IL-4) and interleukin-10 (IL-10) are key cytokines whose increased production during systemic HIV infection has been associated with decreased cellular immunity during AIDS. We examined whether HIV-induced stimulation of IL-4 or IL-10 production leads to increased susceptibility to AIDS-related human cytomegalovirus retinitis. It was confirmed that there were increased amounts of IL-4 and IL-10 mRNA levels in mice with MAIDS of 10 weeks duration when most susceptible to MCMV retinitis. Surprisingly, however, MCMV-infected eyes of IL-4 –/– and IL-10 –/– mice with MAIDS of 8 weeks duration exhibited retinitis and infectious virus equivalent to that observed in MCMV-infected eyes of wild-type mice with MAIDS. We conclude that neither IL-4 nor IL-10 alone play a role in increased susceptibility to MAIDS-related MCMV retinitis, but may work collectively with other retrovirus-induced immunosuppressive factors to allow for retinal disease.

OBJECTIVE To investigate the diagnostic value of cerebrospinal fluid (CSF) culture for opportunistic viruses from HIV-1-infected individuals. METHODS A 4-year prospective study was conducted using a participant population consisting of 186 HIV-1-infected individuals without neurologic disease, 73 HIV-1-infected individuals with encephalopathy, myelopathy, and/or peripheral neuropathy, and 10 controls. CSF samples recovered at 1-year intervals were subjected to virus culture using technique commonly used in the clinical laboratory setting. RESULTS CSF samples obtained from only 15 of the 269 (5.6%) participants yielded an opportunistic virus upon culture. Cytomegalovirus, herpes simplex virus types 1 and 2, adenovirus, and presumptive enteroviruses were identified. No consistent correlation was observed between the detection of an opportunistic virus within a CSF sample and the presence or future development of neurologic disease. However, a significant correlation was observed between culture of virus from CSF and the future development of abnormal CD4+ (chi 2, P = 0.0286) and CD8+ (chi 2, P = 0.0018) lymphocyte counts in HIV-1-infected participants without neurologic disease. CONCLUSION These results show that culture of CSF to screen for opportunistic viruses is neither diagnostic nor predictive of neurologic disease in HIV-1-infected individuals. Nevertheless, the presence of virus within CSF may be an indicator of HIV-1-mediated immune dysfunction and a predictor for future development of abnormal CD4+ and/or CD8+ lymphocyte counts.

We examined the axonal transport of two strains of herpes simplex virus 1 (HSV-1) within the central nervous system of cebus monkeys. Each strain was injected into the "arm area" of the primary motor cortex. One strain, HSV-1(McIntyre-B), was transported transneuronally in the retrograde direction. It infected neurons at sites known to project to the arm area of the primary motor cortex (e.g., ventrolateral thalamus). In addition, "second-order" neurons were labeled in the deep cerebellar nuclei (dentate and interpositus) and in the globus pallidus (internal segment). This result supports the concept that the arm area of the primary motor cortex is a target of both cerebellar and basal ganglia output. In contrast, the other strain, HSV-1(H129), was transported transneuronally in the anterograde direction. It infected neurons at sites known to receive input from the arm area of the primary motor cortex (e.g., putamen, pontine nuclei). In addition, "third-order" neurons were labeled in the cerebellar cortex (granule and Golgi cells) and in the globus pallidus (largely the external segment). Our observations suggest that strain differences have an important impact on the direction of transneuronal transport of HSV-1. Furthermore, it should be possible to examine the organization of cerebellar and basal ganglia loops with cerebral cortex by exploiting transneuronal transport of HSV-1 and virus strain differences.

Four monoclonal antibodies directed against glycoprotein B of herpes simplex virus were evaluated for their ability to immunize mice passively against acute virus-induced neurological illness and death when administered intraperitoneally 2 hours prior to footpad challenge with type 1 or type 2 virus. Two monoclonal antibodies, H120 and H157, failed to reduce the severity of neurological disease in infected animals. In contrast, H233 and H368 antibodies provided significant protection in type-common and type-specific fashions, respectively. A direct correlation was observed between in vitro neutralization and in vivo protection. These results provide the first in vivo evidence that glycoprotein gB of herpes simplex virus expresses both type-common and type-specific determinants during the evolution of acute virus-induced neurological disease.

Monolayer cultures of primary rat astrocytes grown with or without dibutyryl cyclic AMP (dBcAMP) for two weeks or longer were infected with round plaque-forming (Rd) or syncytia-forming (Syn) variants of herpes simplex virus type 1 (HSV-1). Infection with HSV-1 did not stimulate synthesis of glial fibrillary acidic protein (GFAP) or alter the general organization of the intermediate (glial) filaments in astrocyte cultures. However, the dBcAMP-treated astrocytes produced 10- to 100-fold lower titers of cell-free progeny HSV-1 than the untreated astrocyte cultures. Radiolabeled amino acid or glucosamine incorporated into acid precipitable cellular or viral glycoproteins was decreased by 10-25% in dBcAMP-treated astrocytes. Distinctive cell-rounding or syncytial cytopathology was produced by HSV-1 strains infecting untreated astrocytes, but the infected dBcAMP-treated astrocytes displayed only cell-rounding cytopathology. The dBcAMP-related effects on HSV-1 infection were specific to primary astrocyte cultures; they were not observed in HSV-1-infected human fibroblast cultures treated with dBcAMP. Comparison of HSV-1 infection of untreated versus dBcAMP-treated astrocytes suggests that the dBcAMP-induced "reactive" or differentiated state of the astrocyte can affect expression of virus-induced cytopathology and virus-specific polypeptide synthesis. The dBcAMP-treated primary astrocyte culture may afford a non-neoplastic, differentiated in vitro system for studying HSV-neural cell interactions.

Purpose: To test if nicotine counteracts the dampening effect of human cytomegalovirus (HCMV) infection of NF-κB in retinal pigment epithelial (RPE) cells, thereby increasing the permissiveness of RPE cells for HCMV replication. Methods: Human ARPE-19 cells were transfected with NF-κB luciferase DNA, inoculated with HCMV at 24 hr post-transfection, and maintained in the absence or presence of a physiologic dose of nicotine at 1 hr prior to HCMV inoculation. Results: Whereas HCMV-infected ARPE-19 cells without nicotine treatment showed a dramatic decrease in NF-κB levels, nicotine treatment reduced this decrease but did not abolish it completely. Nicotine treatment of uninfected ARPE-19 cells had no effect on baseline NF-κB levels. Conclusions: Treatment of HCMV-infected ARPE-19 cells with nicotine at a physiologic dose dampened the downregulation of NF-κB observed in HCMV-infected ARPE-19 cells without nicotine treatment. We conclude that nicotine can serve as a cofactor to stimulate productive, lytic rep...

Cytokines are small proteins produced by T lymphocytes that mediate immune responses. Those produced by the CD4+ Th1 subset induce cell-mediated immunity, whereas those produced by the CD4+ Th2 subset are more efficient at stimulating immunoglobulin production. The goal of cytokine immunotherapy is prevention or reduction of disease progression through stimulation of cell-mediated immunity (i.e., immune reconstitution) by administration of an exogenous Th1 cytokine such as interleukin-2 (IL-2). Cytokine immunotherapy has its origins in cancer immunobiology where IL-2 has been used successfully to manage several human cancers (metastatic melanoma, acute myelogenous leukemia, and metastatic renal cell carcinoma). More recent work has demonstrated cytokine immunotherapy to be effective at improving immune responses in patients with HIV-1 disease. To explore cytokine immunotherapy for sight-threatening AIDS-related human cytomegalovirus (HCMV) retinitis, we developed a mouse model of experimental murine cytomegalovirus (MCMV) retinitis that employs mice with MAIDS, a retrovirus-induced immunodeficiency syndrome. Systemic cytokine immunotherapy with IL-2, but not with interleukin-12 (IL-12), provides absolute protection against MAIDS-related MCMV retinitis by stimulation of the perforin-mediated pathway of cytotoxicity used by natural killer cells and cytotoxic CD8+ T cells to kill virus-infected cells. Our findings warrant additional studies on the use of cytokine immunotherapy for management of HCMV retinitis (and possibly other opportunistic infections) during HIV-1-induced immunodeficiency. We envision systemic cytokine immunotherapy as an altemative or adjunct to traditional antiviral chemotherapy for optimal management of AIDS-related HCMV retinitis.

To correlate tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) synthesis with histopathologic disease and virus replication within murine cytomegalovirus (MCMV)-infected eyes during progression of murine acquired immunodeficiency syndrome (MAIDS).Groups of normal mice and mice with MAIDS of 2-weeks (MAIDS-2), 4-weeks (MAIDS-4), and 12-weeks (MAIDS-12) duration were infected uniocularly with MCMV by subretinal MCMV injection. MCMV-inoculated eyes from all mice were subjected to histopathologic analysis, quantitative plaque assay, or cytometric bead array analysis for quantification of TNF-alpha and IFN-gamma.Whereas MCMV-inoculated eyes of normal, MAIDS-2, and MAIDS-4 mice were resistant to MCMV retinitis, all MCMV-inoculated eyes of MAIDS-12 mice developed retinitis. Surprisingly, MCMV-inoculated eyes of MAIDS-4 mice without retinitis harbored high amounts of infectious virus at a level equivalent to that of MCMV-inoculated eyes of MAIDS-12 mice that developed retinitis. Intraocular TNF-alpha levels were consistently approximately 50% greater in MCMV-inoculated eyes of MAIDS-12 mice when compared with TNF-alpha levels of normal, MAIDS-2, and MAIDS-4 mice. In contrast, intraocular INF-gamma levels within MCMV-inoculated eyes progressively declined as animals became susceptible to retinitis.An inverse relationship exists between TNF-alpha and INF-gamma production within MCMV-inoculated eyes during MAIDS evolution that is characterized by an increase in intraocular TNF-alpha levels and a concomitant decrease in intraocular INF-gamma levels. Susceptibility of MCMV-inoculated eyes to virus replication and development of necrotizing retinitis are independent events with susceptibility to MCMV replication preceding susceptibility to MCMV retinitis by several weeks. Time of Th1/Th2 shift in cytokine profile appears to be a crucial event in the pathogenesis of MAIDS-related MCMV retinitis.

Adoptive transfer studies were performed to test the hypothesis that the perforin cytotoxic pathway is more important than the Fas/FasL cytotoxic pathway in protection against experimental murine cytomegalovirus (MCMV) retinitis. Splenic immune cells from donor MCMV-immunized normal mice or gld mice deficient in Fas/FasL-mediated cytotoxicity significantly reduced the frequency and severity of MCMV retinitis following subretinal MCMV challenge when transferred into recipient PKO mice deficient in perforin-mediated cytotoxicity. In sharp contrast, splenic cells from donor MCMV-immunized PKO mice failed to provide protection against MCMV retinitis when transferred into recipient PKO mice. Protection was not achieved, however, in recipient mice with retrovirus-induced immunodeficiency (MAIDS), even when splenic cells originated from MCMV-immunized normal mice.

To determine if murine cytomegalovirus (MCMV)-infected eyes of mice with murine acquired immunodeficiency syndrome (MAIDS) that are destined to develop MCMV retinitis display elevated intraocular levels of interleukin-4 (IL-4) mRNA when compared with uninfected eyes of mice with MAIDS and unmanipulated, uninfected, eyes of normal healthy mice.Groups of C57BL/6 mice with MAIDS and normal C57BL/6 mice were infected uniocularly with MCMV by subretinal MCMV injection. IL-4 levels in individual spleens collected five days later from groups of MAIDS mice and normal mice were assessed by quantitative ELISA. MCMV-infected eyes and uninfected contralateral eyes from another group of mice with MAIDS were also collected at five days postinfection and individually subjected to competitive RT-PCR assay and real-time RT-PCR assay for detection and quantification of IL-4 mRNA. Unmanipulated eyes from normal C57BL/6 mice served as controls.IL-4 mRNA was detected at a level of 9.7 +/- 3.4 pg mRNA per 1000 ng total RNA in 100% of MCMV-infected eyes of mice with MAIDS by competitive RT-PCR assay, but could not be detected in any of the uninfected eyes of MAIDS mice. In comparison, the more sensitive technique of real-time RT-PCR assay detected copies of IL-4 cDNA in both MCMV-infected eyes and uninfected eyes of MAIDS mice. MCMV-infected eyes showed a 16-fold increase in the number of IL-4 cDNA copies when compared with uninfected eyes. Neither technique detected IL-4 mRNA in unmanipulated eyes of normal mice. As expected, spleen cells from mice with MAIDS expressed significantly greater levels of IL-4 when compared with spleen cells from normal mice.MCMV-infected mice with MAIDS exhibited an expected preferential activation of Th2 cells as determined by increased levels of IL-4 in spleen cells when compared with spleen cells of normal mice. MCMV-infected eyes destined to develop retinitis during MAIDS also showed increased levels of detectable IL-4 mRNA when compared with uninfected eyes of mice with MAIDS. It is therefore possible that IL-4 synthesis by Th2 CD4+ T cells during retrovirus-induced immunosuppression serves to inhibit the perforin cytotoxic pathway that subsequently allows susceptibility to MCMV retinitis during MAIDS.

AIDS-related human cytomegalovirus (HCMV) retinitis continues to be a chronic ophthalmologic problem among human immunodeficiency virus type 1 (HIV-1)-infected patients who do not respond to highly active antiretroviral therapy. Although HCMV retinitis occurs during HIV-1-induced immunosuppression, the precise effector mechanism(s) that fails during the immunopathogenesis of AIDS to allow onset and progression of HCMV retinal disease remains unclear. We therefore performed a series of experiments to explore the relative roles of distinct pathways of lymphocyte-mediated cytotoxicity in either resistance or susceptibility to experimental murine cytomegalovirus (MCMV) retinitis in mice. Whereas mutant C57BL/6 mice deficient in the Fas/FasL cytotoxic pathway (gldmice) were identical to normal C57BL/6 mice and exhibited absolute resistance to retinal necrosis following subretinal MCMV inoculation, knockout C57BL/6 mice deficient in the perforin cytotoxic pathway (PKO mice) were susceptible to MCMV retinitis. Susceptibility of PKO mice to MCMV retinitis correlated with increased ocular MCMV titers when compared with ocular MCMV titers ofgldand normal mice. Since mice with retrovirus-induced immunodeficiency syndrome (MAIDS) exhibited a frequency and severity of MCMV retinitis that were equivalent to those observed in PKO mice, we hypothesized that susceptibility to MCMV retinitis during MAIDS correlates with a decrease in the perforin cytotoxic pathway. To test this hypothesis, we developed a quantitative competitive reverse transcription-PCR assay to measure mouse perforin mRNA levels in the splenic T lymphocytes and MCMV-inoculated eyes of normal mice or mice with MAIDS. Perforin mRNA levels in splenic T lymphocytes were significantly decreased during MAIDS, by ∼100-fold, from perforin mRNA levels in normal mice. Moreover, MCMV-inoculated eyes destined to develop retinitis during MAIDS also showed a significant decrease in perforin mRNA levels from the perforin mRNA levels of MCMV-inoculated eyes of normal mice destined to be resistant to retinitis. As expected, perforin mRNA could not be detected in unmanipulated and uninfected eyes of normal mice. These results provide the first evidence that the perforin cytotoxic pathway is more important than the Fas/FasL cytotoxic pathway in providing resistance to experimental MCMV retinitis and that loss of the perforin cytotoxic pathway predisposes to MCMV retinitis.

AIDS-related human cytomegalovirus retinitis continues to be an important sight-threatening disease in AIDS patients who do not respond to highly active antiretroviral therapy. We have shown previously that systemic cytokine immunotherapy with interleukin-2 (IL-2) will protect against experimental murine cytomegalovirus (MCMV) in mice with a murine retrovirus-induced immunodeficiency syndrome (MAIDS). Since IL-2 serves as a Th1 immunoregulatory cytokine, we hypothesized that IL-2-induced protection against MCMV retinitis during MAIDS would correlate with a measurable increase in the number of natural killer (NK) cells and/or CD8+ T cells that infiltrate the eye in response to MCMV infection of the retina. We therefore performed a study to quantify and compare the number of NK cells and CD8+ T cells that infiltrate MCMV-infected eyes in untreated and IL-2-treated mice with MAIDS at 3 days and 5 days after subretinal MCMV inoculation. Double-label flow cytometric analysis revealed the detection of measurable numbers of both NK cells and CD8+ T cells in MCMV-infected eyes of untreated MAIDS mice destined to develop retinitis. In contrast, IL-2 immunotherapy during MAIDS correlated with a 10-fold increase by day 5 after inoculation in the number of CD8+ T cells in MCMV-infected eyes destined to be resistant to retinitis. However, IL-2 immunotherapy during MAIDS had no appreciable effect on the number of NK cells that infiltrated MCMV-infected eyes. Taken together, our findings suggest that function of cytotoxic lymphocytes that infiltrate the eye may be more important than absolute numbers of cytotoxic lymphocytes that infiltrate the eye when assessing the protective effects of IL-2 immunotherapy on MCMV retinitis during MAIDS.

Passive-transfer studies were performed to assess the ability of antibody alone to reduce the frequency and/or severity of necrotizing retinitis caused by murine cytomegalovirus (MCMV) in C57BL/6 mice with retrovirus-induced immunodeficiency syndrome (MAIDS). Initial experiments showed a gradual decline in the ability of mice to initiate humoral immunity during the evolution of MAIDS so that neither MCMV-specific IgM nor IgG could be detected during late-stage MAIDS. Passively administered hyperimmune MCMV immunoglobulin, however, could be detected within the serum of mice with MAIDS for at least 9 days after intraperitoneal injection and protected these animals in preliminary experiments from systemic MCMV disease and death when administered 24 h prior to intraperitoneal challenge with a lethal dose of virus. Nonetheless, passive transfer of hyperimmune MCMV serum to mice with MAIDS failed to reduce intraocular MCMV titers, frequency of retinitis, or severity of retinitis when administered 24 h prior to subretinal MCMV inoculation. Whereas whole eyes of MAIDS animals that received normal mouse serum and were injected subretinally with MCMV had an ocular MCMV titer of 4.3 log10 and a frequency of retinitis of 89% (severity score = 55%), whole eyes of antibody-treated mice with MAIDS had an ocular MCMV titer of 4.3 log10 and a frequency of retinitis of 87% (severity score = 57 %). Passive transfer of a neutralizing MCMV-specific monoclonal antibody also failed to reduce the frequency or severity of MCMV retinitis when administered to mice with MAIDS prior to subretinal MCMV inoculation. Our findings suggest that antibody immunotherapy alone will not be effective therapeutically for cytomegalovirus retinitis in patients with AIDS.

The neovascular (wet) form of age-related macular degeneration (AMD) leads to vision loss due to choroidal neovascularization (CNV). Since macrophages are important in CNV development, and cytomegalovirus (CMV)-specific IgG serum titers in patients with wet AMD are elevated, we hypothesized that chronic CMV infection contributes to wet AMD, possibly by pro-angiogenic macrophage activation. This hypothesis was tested using an established mouse model of experimental CNV. At 6 days, 6 weeks, or 12 weeks after infection with murine CMV (MCMV), laser-induced CNV was performed, and CNV severity was determined 4 weeks later by analysis of choroidal flatmounts. Although all MCMV-infected mice exhibited more severe CNV when compared with control mice, the most severe CNV developed in mice with chronic infection, a time when MCMV-specific gene sequences could not be detected within choroidal tissues. Splenic macrophages collected from mice with chronic MCMV infection, however, expressed significantly greater levels of TNF-α, COX-2, MMP-9, and, most significantly, VEGF transcripts by quantitative RT-PCR assay when compared to splenic macrophages from control mice. Direct MCMV infection of monolayers of IC-21 mouse macrophages confirmed significant stimulation of VEGF mRNA and VEGF protein as determined by quantitative RT-PCR assay, ELISA, and immunostaining. Stimulation of VEGF production in vivo and in vitro was sensitive to the antiviral ganciclovir. These studies suggest that chronic CMV infection may serve as a heretofore unrecognized risk factor in the pathogenesis of wet AMD. One mechanism by which chronic CMV infection might promote increased CNV severity is via stimulation of macrophages to make pro-angiogenic factors (VEGF), an outcome that requires active virus replication., Author Summary Neovascular age-related macular degeneration (AMD) is the leading cause of vision loss in the elderly. Onset of AMD is due to local production of vascular endothelial growth factor (VEGF) that promotes formation of new blood vessels in the retina, thereby leading to retinal tissue destruction and blindness. Since a clinical study by us showed that AMD patients have high amounts of antibodies to human cytomegalovirus (HCMV), we postulated that infection with HCMV might be a risk factor for AMD. To investigate this possibility, mice were infected with murine cytomegalovirus (MCMV), and at various times after infection, subjected to laser treatment of the eye to induce choroidal neovascularization, an experimental model of AMD. Most severe CNV developed in mice with chronic MCMV infection, a time when MCMV gene sequences could not be detected within eye tissues. However, splenic macrophages collected from mice with chronic MCMV infection produced high levels of gene transcripts to several pro-angiogenic factors including VEGF. MCMV infection of mouse macrophages in culture also produced high amounts of VEGF. Stimulation of VEGF production in vivo and in vitro was sensitive to antiviral treatment. Chronic HCMV infection may therefore promote AMD by stimulation of VEGF production by activated macrophages.

Although various methods of immunosuppression have been used to enhance susceptibility of mice to murine cytomegalovirus (MCMV) retinitis, none reproduce the unique complexity of immune deficiency experienced by patients during the progression of AIDS. C57BL/6 mice are susceptible to a retrovirus-induced murine acquired immune deficiency syndrome (MAIDS), characterized by progressive immune dysfunction which shares many features with AIDS. We therefore evaluated the frequency and severity of MCMV retinitis in C57BL/6 mice with MAIDS. Following subretinal inoculation of MCMV, nearly 90% of mice with MAIDS developed a necrotizing retinitis 8 to 10 days postinfection, whereas retinitis was observed in only 8% of age-matched immunocompetent control mice. Histopathologic analysis of the retinitis that developed in mice with MAIDS revealed features similar to those found in AIDS-related CMV retinitis. Eyes from MAIDS animals also contained on average higher amounts of infectious virus when compared with eyes from control animals. We conclude that retrovirus-induced immunosuppression increases susceptibility of C57BL/6 mice to MCMV retinitis.

Intra-visual cortex inoculation of 10(2) plaque-forming units of herpes simplex virus type 1 (KOS-63) induced physiologic and morphologic retinal changes in 62.3% (33/53) of infected animals; of these, 91% were bilateral. In contrast, inoculation of the same viral titers into the frontal lobe induced retinal alterations in only 13.3% (2/15). Initially, there was a decrease of the b-wave amplitude and retinal sensitivity and necrotic changes of the ganglion cells and nuclei in the inner nuclear layer. Immunoperoxidase staining for virus-specific antigens showed positive staining of the same cell type. Over time, there was a progressive decrease in the electroretinogram until it was extinguished and the retina was replaced by gliotic tissue. Parallel viral recovery studies demonstrated detectable infectious virus in one of eight eyes on day 2 after inoculation and in three of eight eyes on day 4. Thereafter, there was an increase in the percentage of eyes with infectious virus and a concomitant increase in viral titers. Immunoperoxidase staining of brain sections obtained on days 6 through 8 demonstrated virus-specific antigens on cells in the lateral geniculate nuclei and the suprachiasmatic nuclei bilaterally.

We explored a possible role for the basic fibroblast growth factor (FGF) receptor during herpes simplex virus type 1 (HSV-1) infection of primary mouse astrocytes, glial cells of the central nervous system known to express FGF receptors. Plaque reduction experiments showed that treatment of astrocyte monolayers with human recombinant basic FGF failed to inhibit HSV-1 infectivity, although treatment with either heparin or poly-l-lysine reduced it by approximately 100%. Identical results were obtained when monolayers of human embryonic lung fibroblasts or African green monkey kidney cells were treated with FGF, heparin or poly-l-lysine prior to HSV-1 infection. We conclude that the basic FGF receptor is not involved in the uptake of HSV-1 during productive infection of astrocytes. Our findings suggest that this molecule is not the predominant cellular receptor for HSV-1 among vertebrate cells and plays no role in defining HSV-1 neurotropism in vivo.

Human neural cell aggregate cultures were prepared from dissociated fetal brain tissue and maintained in rotation culture. After 35 days in culture, aggregates had the histologic appearance of dense, immature, neural cells in a tightly packed neuropil. Electron microscopy revealed ultrastructural features suggestive of immature neurons and neuroglia. In addition, neuron-specific enolase and glial fibrillary acidic protein associated with radial glial cells were detected within the aggregates by immunoperoxidase staining. When infected with a laboratory-adapted strain of cytomegalovirus (CMV), [AD169], cells containing large, bizarre, nuclei and CMV-induced intranuclear inclusion bodies were dispersed throughout the aggregates at 16 days postinfection. In situ hybridization using a CMV-specific DNA probe and electron microscopy confirmed the presence of virus sequences as well as virus particles at histologic sites of cytopathology. In sharp contrast, aggregate cultures infected with a CMV strain recovered from the retina of an acquired immune deficiency syndrome (AIDS) patient with CMV retinitis and encephalitis displayed distinct foci of cytopathology at 23 days postinfection, a pattern not observed in CMV [AD169]-infected aggregates. Our findings suggest that human neural cell aggregates represent a a promising multicellular non-neoplastic culture system in which to study the replication of human neurotropic viruses within neural tissue.

Herpes simplex encephalitis (HSE) is characterized by focal lesions of hemorrhage and necrosis, primarily in the inferior temporal lobe. Since immunosuppressed patients with HSE lack the focal inflammatory changes and temporal lobe localization, it has been suggested that the immune system participates in the pathogenesis of HSE. Evaluation of this hypothesis has been impeded by the lack of an immunologically defined animal model that resembles the human disease. Toward this end, 10 strains of inbred mice were infected intranasally with a neurovirulent clinical isolate of herpes simplex virus type 1. Most mice died without localizing signs of disease in the central nervous system. However, a significant number of SJL mice had a pattern of encephalitis highly reminiscent of that described in humans. To our knowledge, this is the first murine model that faithfully mimics this human disease, and thus it affords the opportunity to study the immunopathogenesis of HSE.

✓ The present studies were undertaken to determine if viral particles can be delivered across the rat blood-brain barrier (BBB). Osmotic BBB modification with intracarotid mannitol (25%) was immediately followed by bolus intracarotid administration of 0.5 ml purified, ultraviolet-inactivated, herpes simplex virus type 1 endogenously labeled with 35S-labeled methionine (2.0 × 106 cpm, approximately 5 × 108 plaque-forming units/ml). After 60 minutes, intravascular virus was cleared by saline perfusion and the animals were sacrificed. A marked increase (fourfold, p ≤ 0.02) in radioactivity was observed in the ipsilateral brain hemisphere when compared to control animals without barrier modification. Administration of intravenous virus immediately after BBB modification displayed no difference in delivery when compared to intracarotid saline-infused controls (without BBB modification) suggesting the importance of a first-pass phenomenon. There were no significant differences in serum concentrations among intracarotid or intravenous groups. These preliminary studies suggest the possibility of delivering viral particles across the BBB with osmotic disruption, which may permit delivery of genetic material in replication-defective viral vectors in the feline model of GM2-gangliosidosis.

Alterations in host defense mechanisms that predispose to infection can be broadly classified into three major categories: abnormalities of cell-mediated immunity (T-lymphocytes and macrophages), abnormalities of humoral immunity (immunoglobulins), and abnormal neutrophils. Currently, the most common cause of abnormalities in the cell-mediated arm of immunity is the acquired immunodeficiency syndrome (AIDS), but among other causes, abnormalities in this system are also associated with lymphoma, particularly Hodgkin’s disease, corticosteroid and antineoplastic therapy, organ transplantation, and malnutrition.

Within a 5-month period, 2 homosexual men with persistent lymphadenopathy developed clinical findings consistent with herpes simplex virus (HSV) encephalitis. These signs included abrupt change in mental status, seizures, cerebrospinal fluid pleocytosis, and localized electroencephalographic abnormalities showing temporal lobe involvement. Initial computed tomographic scans were unremarkable. Treatment with adenine arabinoside was instituted and temporal lobe biopsies were performed. Although virus-specific antigens were detectable in only 1 patient, cultures of biopsy tissue from both patients yielded HSV type 2 organisms. Spiking fevers persisted and the patients failed to improve, prompting administration of acyclovir. Both patients recovered gradually after their second course of antiviral therapy and survived with severe neurological deficits. These patients should raise concerns about an increased incidence of type 2 encephalitic illness among homosexual men with persistent lymphadenopathy or acquired immune deficiency syndrome. In addition, the importance of using HSV type 2 antibody in the immunofluorescence test of brain biopsy tissue for rapid diagnosis of the disease is emphasized.

Over a one-year period the cerebrospinal fluid (CSF) obtained from a series of homosexual men immunocompromised with either Hodgkin's disease or acquired immune deficiency syndrome (AIDS) was cultured to assess the frequency with which infectious viruses could be recovered. Of 58 patients examined, 4 (6.9%) had CSF cultures that showed a cytopathology consistent with a virus infection. All isolates proved to be herpesviruses. Cytomegalovirus (CMV) and varicella-zoster virus were isolated from CSF obtained from 2 patients with neurological features consistent with a subacute encephalitis common among AIDS patients. CMV was also recovered from the CSF of an AIDS patient who developed an ascending myelitis of herpesvirus origin. Finally, a CSF sample obtained from an immunodeficient homosexual man who showed no detectable neurological abnormalities consistently yielded herpes simplex virus type 1 in culture. These results suggest that seeding of the CSF with infectious virus is an uncommon event in this patient population. However, our experience should not dissuade attempts to culture viruses from CSF in similar cases. Successful isolations may prove beneficial in the diagnosis of an accompanying neurological illness and facilitate treatment with antiviral therapy when indicated.

We performed several experiments to define possible factors that influence the viability of herpes simplex virus type 1 on the applanation tonometer. Infectious virus could be detected on experimentally inoculated tonometer heads for up to two hours during natural drying. If tonometers were kept moist the virus could be detected more than eight hours later. Several ophthalmic solutions, including topical anesthetics, dilating agents, and a fluorescein solution, showed only minimal antiviral activities. Wiping of virus-infected tonometer heads with a dry tissue was ineffective and allowed residual infectious virus to remain. However, no infectious virus could be detected on infected tonometer heads that had been swabbed with 70% isopropyl alcohol. Our results raised concerns regarding effective disinfection of tonometers after eye examinations. We concluded that the applanation tonometer should be swabbed routinely with alcohol after each patient examination to ensure complete virus inactivation and to prevent accidental transmission of virus in the clinical setting.

Monoclonal antibodies HCl and HD1, directed against herpes simplex virus type 1 (HSV-1) glycoproteins gC and gD, respectively, were evaluated for their ability to passively immunize mice against acute virus-induced neurological disease after footpad inoculation with HSV-1 or herpes simplex virus type 2 (HSV-2). Control virus-infected mice receiving a single intraperitoneal injection of normal serum died within 7 to 10 days after the spread of virus from footpad to spinal cord and brain. However, a single intraperitoneal injection of either HCl or HD1 antibody protected mice from neurological illness and death when administered to HSV-1 (strain HTZ)-infected mice at either 2 h before virus challenge or at 24 h after virus inoculation. To determine the in vivo specificity of the antibodies, passive transfer studies were performed with mice infected with the MP strain of HSV-1, a mutant of HSV-1 (mP) which is defective in the production of glycoprotein gC. Whereas HD1 antibody decreased the incidence of neurological illness in MP- and mP-infected mice, HCl antibody, which protected mP-infected animals, failed to protect mice infected with the MP strain. When HD1 antibody was administered to HSV-2 (strain G)-infected mice at either 2 h before virus challenge or at 6 h (but not 24 h) after virus inoculation, 100% of the infected animals receiving HD1 antibody survived. In contrast, 100% of HSV-2 (strain G)-infected animals passively immunized with HCl antibody developed neurological illness and died. These results provide in vivo evidence that the HSV-induced glycoprotein gC expresses type-specific antigenic determinants, whereas glycoprotein gD expresses type-common determinants.

The authors have isolated herpes simplex virus type 1 (HSV-1) from the vitreous of two patients with acute retinal necrosis. Clinical and laboratory data suggest that one case represented a primary HSV-1 infection, whereas the other case appeared to be a recurrent HSV-1 infection. In the primary case, changes on magnetic resonance imaging (MRI) suggest spread of the virus posteriorly to both optic tracts and the lateral geniculate ganglia. This case shares many features with the "von Szily" experimental model for HSV retinitis in the mouse.