In vitro studies

eline neural culture systems have been utilized to study FIV cell tropism and neurovirulence. Dow et al. were the first investigators to isolate virus from cultures of neural cells of cats infected with FIV-Petaluma strain (1). This isolate could infect brain-derived macrophages/microglia and astrocytes but not neurons or brain-derived endothelial cells (BDEC) (1). Effects of FIV on the blood-brain barrier may still be possible, as cellular tropism for cultured feline choroid plexus by the FIV-NCSU₁ strain (2) and BDEC by the FIV-Villefranche strain (3) have been shown. Other viral strains with a variable tropism and infectivity for neural cells include FIV-KYO-1, FIV-TM2, and FIV-TM2 (4). The FIV-MD and pFIV-PPR strains can productively infect feline astrocytes in vitro but only when co-cultured with either FIV-positive lymphocytes or an FIV-susceptible feline T-cell line (5)(6). Others reported only low level infectivity of an isolated feline brain cell line (G355-5) exposed to pFIV-PPR (7).

Determinants of neurovirulence of FIV have been assessed in both enriched and mixed neural cell culture systems. Similar to HIV-1, microglia have historically been implicated as reservoirs of FIV and important contributors to indirect viral effects on brain function. Specifically, the effects of FIV-Petaluma on astroglial and/or macrophage/microglial cells have been the focus of most in vitro investigations. No cytopathic effect of FIV-Petaluma virus on primary cultures of brain-derived macrophage/microglial cells were reported (1), despite a persistent and productive infection of these cell types. A cytopathic effect was observed with FIV-Petaluma (1), compared to a productive, but non-cytopathic effect with the FIV-derived molecular clone (34TF10) (6) in astrocytic cultures.

The increasing evidence of chronic, low level astrocyte infection by several strains of FIV in vitro shifted attention to the astrocyte as another factor in the neurodegenerative process. Astrocyte-mediated glutamate uptake was decreased after FIV-34TF10 in vitro infection (6). Characteristic properties and subcellular functions of astrocytes in culture are also altered after FIV inoculation. A 30% decrease in gap junctional intercellular communication was found in infected versus control astrocytic cultures (5) while mitochondrial membrane potential in FIV-exposed astrocytes was less than 45% of that in normal control cells (8). Conclusions from the combined studies were that reduced glutathione levels may be contributing to free radical damage of cellular membranes and that altered calcium homeostasis could ultimately affect mitochondrial energy production. Although each of these cellular defects indicates astrocyte injury, the precise relationship of these defects to FIV-neuropathogenesis is unclear.

Few studies have examined the direct and indirect effects of FIV or its viral proteins on the functional properties and viability of neurons. Evidence of direct neuronal infection with FIV is lacking, suggesting that neuronal cytotoxic effects are indirectly mediated. Mixed cultures of neurons, astrocytes, and microglia from E40-E57 fetal cat cortex inoculated with FIV-NCSU₁ alone did not exhibit signs of cytotoxicity (9). Addition of 20µM glutamate to the FIV-infected cultures, however, resulted in neuronal swelling in up to 24% of neurons and significantly greater cell death after 24 hours than in non-treated cultures. Excitotoxicity could be prevented with the addition of the NMDA receptor antagonist AP5, indicating mediation by NMDA receptor stimulation. A soluble toxic factor released into the medium by glial cells was believed responsible for the sensitization to glutamate. Others performed a similar study with a primary embryonic fetal neuronal culture model system exposed to either pFIV-PPR virions or envelope proteins (10). Whole virus by itself induced spontaneous neuronal swelling while viral envelope protein induced cell swelling only in the presence of glutamate. Glutamate stimulation of NMDA receptors, as measured by increased intracellular calcium, was enhanced by envelope protein treatment of the cells. This sensitization of neurons to the excitotoxic effects of glutamate has also been reported for HIV-1 envelope proteins. The authors hypothesized that this effect of FIV on neurons in conjunction with the defective glutamate uptake by FIV-infected astrocytes could eventually lead to neuronal death in vivo (10).

Neuropathology

The neuropathology of FIV has been well defined in recent years, and the lesions described are similar in quality to those found in HIV-1 infection (11)(12). Typical neuropathologic changes poorly predict functional alterations in FIV cats (13), comparable to the situation in humans (14)(15). Feline immunodeficiency virus induced brain lesions do not appear to progress rapidly, as studied with classical neuropathological techniques (11). Immune activation of the brain occurs early, as evidenced by widespread astrogliosis at 2 months after intravascular inoculation (11), along with microglial activation (13). A comparative review of FIV neuropathological lesions is discussed below in terms of gray matter and white matter oriented lesions, and lesions characterized by lymphocytic infiltrates.

Gray matter lesions described in FIV-infected cats have consistently included gliosis (11)(12)(16) confirmed with GFAP immunohistochemistry (17)(18), and morphometrical analysis (19). Microglial nodules, one characteristic of HIV-1 encephalitis (20) have been described in a proportion of FIV-infected cats (11)(12)(16)(21)(22). Microglial activation has been confirmed by immunohistochemical methods (17), which, taken with astrogliosis and neuronal loss (13)(23) recapitulates within the cat the changes defined for HIV-1 poliodystrophy (20).

White matter lesions of FIV also parallel those with HIV-1 infection. Astrogliosis is the most consistent finding in FIV cats (18)(19). White matter pallor on myelin stains has been described in cerebellum (11), cerebral white matter (17), and whole brain (12). Vacuolar change of white matter has been described (19), occurring in naturally and experimentally infected cats with consistent myelin splitting and frequent partially demyelinated axons.

A variable incidence of lymphocytic meningeal, choroid plexus, and perivascular infiltrates is present with FIV infection (11)(13)(17)(21)(24). Taken together, these lesions resemble lesions classified by (20) as HIV-1 encephalitis, HIV-1 leukoencephalopathy, vacuolar myelopathy and leukoencephalopathy, lymphocytic meningitis, diffuse poliodystrophy, and cerebral vasculitis. The main exception is the absence of multinucleated giant cells (MNGC), as seen with HIV-1 infection. Although MNGC have been described in a naturally (11) and an experimental infected (22) cat, their presence is rare. The presence of MNGC appears not to be essential for progression to HIV-1 induced dementia (25). Therefore, production of MNGC may not be a determinant of neurovirulence, and therefore, should not be the end-point determinant of neuropathogenicity.

Brain FIV load has been detected as early as 7 days after intravenous inoculation appears to maintain a low level throughout infection times studied to date (12). The cell types infected have been suggested to be of microglial/monocyte origin (17), however, GFAP immunoreactive cells have also been shown to be infected in vivo (26). While some correlation was found between lymph node and brain viral load, the low viral load in the cat brain contrasts with the extent of neurological impairment reported for FIV-infected cats. Overall, severity of neurologic disease may be both strain dependent and duration of infection dependent, where acute phase viremia early in the course of infection can result in a high degree of neurovirulence in selected strains (27).

While neuropathological lesion severity appears to be a poor predictor of neurological function in HIV-1 (14) and FIV (13), morphometric, immunohistochemical, and special histochemical techniques are revealing alterations in brain structure. Neuronal cell loss has been found in FIV-infected brain (17), twice using morphometric techniques (23)(28). This loss appears to correlate to the reduced N-acetyl-aspartate reduction reported, a neuronal marker using proton magnetic resonance spectroscopy (27)(29). Synaptophysin immunoreactivity, which was increased compared to controls during asymptomatic stages, was later found to be decreased below control counts during feline AIDS (23). Neurofilament alterations detected with SMI 32 immunohistochemistry in FIV-infected frontal cortex were similar to alterations found in human neurodegenerative diseases and occurred in the absence of light microscopic lesions (30). Recently, abnormal sprouting of axons of hippocampal dentate granule neurons has been reported in FIV-infected cats (31). These changes may represent a reorganization of axonal architecture that may underlay functional disruption. Further investigation into the mechanisms of neuronal dysfunction may help bridge the gap between clinical signs and the presence of infected cells within the brain.

Conclusions

The studies above demonstrate that several similarities exist between FIV and HIV-1 neuropathogenesis. The mediators of FIV neurotoxicity can be investigated using both in vivo and in vitro systems. More work is clearly needed in this area to understand the interactions of neural cell types during the neurodegenerative process. Experimental infectivity studies allow investigation of the interactions between the immune system and viral kinetics on neurologic function and pathology over a time frame that mimics the natural progression of HIV-1 neuroAIDS in people. Advanced neurobehavioral paradigms can lead to further definition of the precise neuroanatomical, and thus, neurochemical alterations present. Moreover, experimental animal infection is necessary to establish the biologic relevance of in vitro models. Understanding whole virus effects in readily available cell culture systems is a necessary step in developing realistic and rational theories of in vivo events. Investigation of the neuropathology of FIV infection has revealed lesions which are similar to those present in HIV-1 infected brains, particularly during asymptomatic and AIDS-related complex stages. FIV appears to enter the brain early, resulting in persistent glial activation in the presence of a low viral load during the phase analogous to the HIV-1 asymptomatic stage. Importantly, the structural changes in the synaptic organization of the brain now being discovered may explain some of the neurophysiological alterations described in HIV-1 infection of the brain. The FIV model of neuroAIDS is an important model in which to investigate these issues directly.

References

(1) Dow SW, Dreitz MJ, Hoover EA (1992). Feline immunodeficiency virus neurotropism: evidence that astrocytes and microglia are the primary target cells. Vet Immunol Immunopathol 35(1-2):23-35. Medline

(2) Bragg DC, Meeker RB, English RV, Tompkins MB (1998). J Neurovirology 4: 344

(3) Steffan AM, Lafon ME, Gendrault JL, Koehren F, De Monte M, Royer C, Kirn A, Gut JP (1994). Feline immunodeficiency virus can productively infect cultured endothelial cells from cat brain microvessels. J Gen Virol 75 ( Pt 12):3647-53. Medline

(4) Kawaguchi Y, Maeda K, Tohya Y, Furuya T, Miyazawa T, Horimoto T, Norimine J, Kai C, Mikami T (1992). Replicative difference in early-passage feline brain cells among feline immunodeficiency virus isolates. Arch Virol 125(1-4):347-54. Medline

(5) Zenger E, Collisson EW, Barhoumi R, Burghardt RC, Danave IR, Tiffany-Castiglioni E (1995). Laser cytometric analysis of FIV-induced injury in astroglia. Glia 13(2):92-100. Medline

(6) Yu N, Billaud JN, Phillips TR (1998). Effects of feline immunodeficiency virus on astrocyte glutamate uptake: implications for lentivirus-induced central nervous system diseases. Proc Natl Acad Sci U S A 95(5):2624-9. Medline

(7) Phillips TR, Talbott RL, Lamont C, Muir S, Lovelace K, Elder JH (1992). Comparison of two host cell range variants of feline immunodeficiency virus. J Virol 64(10):4605-13. Medline

(8) Danave IR, Tiffany-Castiglioni E, Zenger E, Barhoumi R, Burghardt RC, Collisson EW (1994). J Virol 68(10):6745-50 Feline immunodeficiency virus decreases cell-cell communication and mitochondrial membrane potential. Medline

(9) Meeker RB, English R, Tompkins M (1996). J Neuro-AIDS 1:1-27

(10) Gruol DL, Yu N, Parsons KL, Billaud JN, Elder JH, Phillips TR (1998). Neurotoxic effects of feline immunodeficiency virus, FIV-PPR. J Neurovirol 4(4):415-25. Medline

(11) Hurtrel M, Ganiere JP, Guelfi JF, Chakrabarti L, Maire MA, Gray F, Montagnier L, Hurtrel B (1992). Comparison of early and late feline immunodeficiency virus encephalopathies. AIDS 6(4):399-406. Medline

(12) Boche D, Hurtrel M, Gray F, Claessens-Maire MA, Ganiere JP, Montagnier L, Hurtrel B (1996). Virus load and neuropathology in the FIV model. J Neurovirol 2(6):377-87. Medline

(13) Podell M, Hayes K, Oglesbee M, Mathes L (1997). Progressive encephalopathy associated with CD4/CD8 inversion in adult FIV-infected cats. J Acquir Immune Defic Syndr Hum Retrovirol 15(5):332-40. Medline

(14) Glass JD, Wesselingh SL, Selnes OA, McArthur JC (1993). Clinical-neuropathologic correlation in HIV-associated dementia. Neurology 43(11):2230-7. Medline

(15) Power C, McArthur JC, Johnson RT, Griffin DE, Glass JD, Dewey R, Chesebro B (1995). Distinct HIV-1 env sequences are associated with neurotropism and neurovirulence. Curr Top Microbiol Immunol 202:89-104. Medline

(16) Dow SW, Poss ML, Hoover EA (1990). Feline immunodeficiency virus: a neurotropic lentivirus. J Acquir Immune Defic Syndr 3(7):658-68. Medline

(17) Power C, Moench T, Peeling J, Kong PA, Langelier T (1997). Feline immunodeficiency virus causes increased glutamate levels and neuronal loss in brain. Neuroscience 77(4):1175-85. Medline

(18) Abramo F, Bo S, Canese MG, Poli A (1995). Regional distribution of lesions in the central nervous system of cats infected with feline immunodeficiency virus. AIDS Res Hum Retroviruses 11(10):1247-53. Medline

(19) Poli A, Abramo F, Di Iorio C, Cantile C, Carli MA, Pollera C, Vago L, Tosoni A, Costanzi G (1997). Neuropathology in cats experimentally infected with feline immunodeficiency virus: a morphological, immunocytochemical and morphometric study. J Neurovirol 3(5):361-8. Medline

(20) Budka H, Wiley CA, Kleihues P, Artigas J, Asbury AK, Cho ES, Cornblath DR, Dal Canto MC, DeGirolami U, Dickson D, et al (1991). HIV-associated disease of the nervous system: review of nomenclature and proposal for neuropathology-based terminology. Brain Pathol 1(3):143-52. Medline

(21) Podell M, Oglesbee M, Mathes L, Krakowka S, Olmstead R, Lafrado L (1993). AIDS-associated encephalopathy with experimental feline immunodeficiency virus infection. J Acquir Immune Defic Syndr 6(7):758-71. Medline

(22) Silvotti L, Corradi A, Brandi G, Cabassi A, Bendinelli M, Magnan M, Piedimonte G (1997). FIV induced encephalopathy: early brain lesions in the absence of viral replication in monocyte/macrophages. A pathogenetic model. Vet Immunol Immunopathol 55(4):263-71. Medline

(23) Meeker RB, Thiede BA, Hall C, English R, Tompkins M (1997). Cortical cell loss in asymptomatic cats experimentally infected with feline immunodeficiency virus. AIDS Res Hum Retroviruses 13(13):1131-40. Medline

(24) Phillips TR, Prospero-Garcia O, Puaoi DL, Lerner DL, Fox HS, Olmsted RA, Bloom FE, Henriksen SJ, Elder JH (1994). Neurological abnormalities associated with feline immunodeficiency virus infection. J Gen Virol 75 ( Pt 5):979-87. Medline

(25) McArthur JC, Becker PS, Parisi JE, Trapp B, Selnes OA, Cornblath DR, Balakrishnan J, Griffin JW, Price D (1989). Neuropathological changes in early HIV-1 dementia. Ann Neurol 26(5):681-4. Medline

(26) Gunn-Moore DA, Pearson GR, Harbour DA, Whiting CV (1996). Encephalitis associated with giant cells in a cat with naturally occurring feline immunodeficiency virus infection demonstrated by in situ hybridization. Vet Pathol 33(6):699-703. Medline

(27) Power C, et al. (1998). Paper in press. J Virol 9765456, 9765449

(28) Buck W, Podell M (1998). J Neuro-AIDS 2:69-77

(29) Podell M, Maruyama K, Smith M (1998). J Neurovirol 4:363

(30) Jacobson S, Henriksen SJ, Prospero-Garcia O, Phillips TR, Elder JH, Young WG, Bloom FE, Fox HS (1997). Cortical neuronal cytoskeletal changes associated with FIV infection. J Neurovirol 3(4):283-9. Medline

(31) Mitchell TW, Buckmaster PS, Hoover EA, Whalen LR, Dudek FE (1998). Axonal sprouting in hippocampus of cats infected with feline immunodeficiency virus (FIV). J Acquir Immune Defic Syndr Hum Retrovirol 17(1):1-8. Medline