Animal Models of Retroviral Encephalopathies: Feline Model

Michael Podell1, Wayne R. Buck1, Kathleen A. Hayes1, Mikhail A. Gavrilin1, Lawrence E. Mathes1

1 The Ohio State University, Columbus, Ohio
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 9.9
DOI:  10.1002/0471142301.ns0909s17
Online Posting Date:  February, 2002
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Abstract

Human immunodeficiency virus infection in children and adults results in a progressive neurodegenerative disease consistent with a predominant subcortical mediated dementia. Techniques for developing a feline model of the early stages of lentiviralā€associated neurodegeneration are presented. The behavioral, neurophysiologic, immunologic, virologic, and neuropathologic aspects of this model are also described.

     
 
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Table of Contents

  • Basic Protocol 1: Establishment of Peripheral Blood Mononuclear Cell (PBMC) Culture and Propagation of FIV
  • Basic Protocol 2: Determination of Tissue Culture Infectious Dose50 (TCID50)
  • Basic Protocol 3: Quantitative Determination of Proviral (DNA) Load
  • Basic Protocol 4: Quantitative Determination of Viral (RNA) Load
  • Basic Protocol 5: T Lymphocyte Phenotype Evaluation by Flow Cytometry
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Establishment of Peripheral Blood Mononuclear Cell (PBMC) Culture and Propagation of FIV

  Materials
  • Anesthetized cat
  • 1× Dulbecco's calcium/magnesium‐free PBS (CMF‐PBS; 10× stock available from Life Technologies)
  • Percoll (density = 1.056 and 1.077; see recipe)
  • PBMC medium (see recipe)
  • Concanavalin A, tissue culture grade (Sigma; see recipe)
  • 10,000 U/ml recombinant human interleukin‐2 (IL‐2; Boehringer Mannheim/Roche)
  • FIV inoculum: FIV‐MD propagated in PBMC culture, the Maryland isolate of FIV, clade B, which is dual neurotropic and lymphocyto‐ and monocyto‐tropic (Olmsted et al., ). This isolate is currently available only from laboratories working actively in this field. Other infectious molecular FIV clones are available from the AIDS Reagent Program (NIH).
  • 100× polybrene solution (see recipe)
  • Limulus Amebocyte Lysate Assay (LAL) kit (for endotoxin detection) QCL‐1000 (BioWhittaker)
  • 10‐ml Vacutainer blood collection tubes with EDTA
  • 15‐ml and 50‐ml polypropylene conical‐bottom centrifuge tubes
  • 5‐ml sterile glass pipets
  • 37°C, 5% CO 2 humidified incubator
  • 25‐cm2 and 75‐cm2 tissue culture flasks
  • 1.8‐ml freezer vials, sterile
  • Additional reagents and equipment for counting cells using a hemacytometer ( appendix 3B)

Basic Protocol 2: Determination of Tissue Culture Infectious Dose50 (TCID50)

  Materials
  • Virus pool (see protocol 1), 1‐ml aliquots at –70°C
  • PBMC medium (see recipe)
  • 100× polybrene solution (see recipe)
  • Fresh PBMC culture (see protocol 1)
  • FIV Antigen ELISA kit (e.g., PetChek available from IDEXX)
  • 10,000 U/ml recombinant human interleukin‐2 (IL‐2; Boehringer Mannheim/Roche)
  • 37°C water bath
  • 3‐ml sterile polystyrene test tubes with snap caps
  • 1.5‐ml microcentrifuge tubes
  • 24‐well sterile polystyrene tissue culture plates with lids
  • ELISA plate reader with 650‐nm filter
NOTE: It will be necessary to establish a fresh PBMC culture for use in this assay (see protocol 1, steps to ). Maintain sterile conditions and perform assay in a laminar flow or static hood.

Basic Protocol 3: Quantitative Determination of Proviral (DNA) Load

  Materials
  • Homogenization solution with and without 3% (w/v) SDS (see recipe)
  • Frozen brain tissue
  • 20 mg/ml proteinase K (Ambion)
  • 20 mg/ml RNase A (Sigma)
  • Tris‐buffered phenol/CHCl 3/IAA, pH 8.0 (Ambion)
  • Phenol, buffered to pH 8
  • 24:1 (v/v) chloroform/isoamyl alcohol
  • 100% isopropanol
  • 70% and 95% ethanol
  • 1× TE buffer, pH 8.0 ( appendix 2A)
  • 3 mM sodium phosphate, dibasic
  • Competitor plasmid dilution series (must be custom made within the lab according to the procedures outlined by Diehl, )
  • 10× PCR buffer (see recipe)
  • 50 mM MgCl 2
  • 10 mM dNTP (see recipe)
  • 5 U/µl Platinum Taq DNA polymerase (Life Technologies)
  • Primer sequences can be submitted to any number of companies that synthesize custom DNA oligonucletoides:
  •  12.5 µM primer KH3 (5′ GAT CCA AAA ATG GTG TCC 3′; Podell et al., ; Hayes et al., )
  •  12.5 µM ATT primer KH4 (5′ CCT ATT CCC ATA ATC TCT GC 3′; Podell et al., ; Hayes et al., )
  • 60% (w/v) sucrose
  • 50 mM tartrazine
  • Plasmid competitor dilution buffer (see recipe)
  • High resolution agarose (e.g., MetaPhor Agarose, FMC Bioproducts or Agarose SFR, Ambion)
  • 1× TBE ( appendix 2A for 10× TBE)
  • Molecular weight ladder (e.g., Low‐Mass Ladder, Life Technologies)
  • 10 mg/ml ethidium bromide
  • 1.5‐ml microcentrifuge tubes
  • Microcentrifuge tube pestles (e.g., disposable Kontes pellet pestle, VWR; or equivalent)
  • 60°C incubator with shaking or rotating and microcentrifuge rack
  • 1‐ml syringes with 25‐G needles
  • Bunsen burner
  • Pasteur pipet
  • Vacuum aspirator
  • 0.2‐ml PCR tubes in 8‐tube strips with caps
  • Microtiter plate centrifuge (DuPont, Sorvall)
  • Thermal cycler with block for 0.2‐ml thin‐walled PCR tubes and heated lid
  • Horizontal electrophoresis apparatus with automatic buffer recirculation
  • 500‐ml Erlenmeyer flask
  • Orbital shaker
  • AlphaImager 2000 (AlphaInnotech) or equivalent image analysis system
  • Additional reagents and equipment for measurement of DNA concentration by UV spectrophotometry ( appendix 1K), plasmid preparations from bacterial cultures (see Diehl et al., ; and CPMB UNIT ), agarose gel electrophoresis ( appendix 1N)

Basic Protocol 4: Quantitative Determination of Viral (RNA) Load

  Materials
  • Plasma samples from EDTA anticoagulated blood
  • PBS, RNase‐free (see recipe)
  • RNA extraction resuspension buffer (see recipe)
  • 20 mg/ml proteinase K, stored at −70°C
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol, pH 8
  • 24:1 (v/v) chloroform/isoamyl alcohol
  • 10× TE buffer, RNase‐free, pH 7.5 (see recipe)
  • 20 mg/ml glycogen, molecular biology grade (Roche Diagnostics)
  • 10 ng/µl 7.5‐kb synthetic RNA (Life Technologies): diluted in RNase‐free water from stock concentration of 1 µg/µl
  • 70% and 100% ethanol
  • 30 U/µl Prime RNase inhibitor (Eppendorf‐5 Prime)
  • Competitor fragment cloned into pCR3.1 (Invitrogen) or similar vector that contains SP6 and T7 promotors for in vitro RNA synthesis
  • XbaI restriction enzyme with manufacturer‐supplied buffer (Roche Diagnostics)
  • MAXIscript RNA transcription SP6 kit (Ambion) containing:
  •  10× transcription buffer
  •  10 mM ATP
  •  10 mM CTP
  •  10 mM GTP
  •  10 mM UTP
  •  SP6 RNA polymerase
  •  2 U/µl RNase‐free DNase I
  • 5 M ammonium acetate, RNase‐free
  • 3 µg/µl random primers (Life Technologies): dilute to 0.005 pg/µl for use in 20 µg/ml glycogen in DEPC‐treated water
  • 20 U/µl AMV thermostable reverse transcriptase with 5× RT buffer (Roche Diagnostics)
  • 10 mM dNTP (see recipe)
  • 10× PCR buffer (Life Technologies)
  • 25 mM MgCl 2
  • 12.5 µM KH3 primer (see protocol 3)
  • 12.5 µM KH4 primer (see protocol 3)
  • 5 U/µl Platinum Taq DNA polymerase (Life Technologies)
  • 1.5‐ml microcentrifuge tubes
  • 0.2‐ml PCR tubes in 8‐tube strips
  • Thermal cycler with heated lid
  • Positive displacement pipets (Gilson Microman)
NOTE: All solutions should be prepared with sterile deionized water that has been treated with DEPC as described in appendix 2A.CAUTION: DEPC is a suspected carcinogen and should be handled carefully.

Basic Protocol 5: T Lymphocyte Phenotype Evaluation by Flow Cytometry

  Materials
  • Feline whole blood in EDTA
  • 1× CMF‐PBS ( appendix 2A; or available commercially from Life Technologies)
  • Percoll (density = 1.056)
  • PAB: phosphate buffered saline supplemented with 0.2% (w/v) bovine serum albumin and 0.2% (w/v) sodium azide
  • 10× lysing buffer (see recipe)
  • Monoclonal antibody:
  •  Mouse anti‐cat CD5:FITC: working dilution = 1:25 in PAB (Southern  Biotechnology)
  •  Mouse anti‐cat CD4:RPE: working dilution = 1:20 in PAB (Southern  Biotechnology)
  •  Mouse anti‐cat CD8:RPE: working dilution = 1:20 in PAB (Southern  Biotechnology)
  •  Mouse anti‐cat CD21: working dilution = 1:40 in PAB (clone Fe2.8F9; Dr. P.F.  Moore, VM Pathology, Microbiology, Immunology, Univ. of California at  Davis, Davis, CA)
  •  Mouse anti‐cat CD45: working dilution = 1:20 in PAB (clone WC45a; Serotec)
  •  Mouse IgG1 standard FITC (Southern Biotechnology)
  •  Mouse IgG1 standard RPE (Southern Biotechnology)
  •  Goat F(ab′)2 anti‐mouse IgG: working dilution = 1:100 in PAB (Southern  Biotechnology)
  •  (H+L)‐RPE, human adsorbed (Southern Biotechnology)
  • 2% (w/v) paraformaldehyde (see recipe)
  • Pasteur pipets and bulb
  • 15‐ml conical centrifuge tubes
  • 5‐ml and 10‐ml pipets
  • 96‐well U‐bottom plate
  • 12‐multichannel pipet and tips
  • 12 × 75–mm disposable glass culture tubes
  • Parafilm
  • Coulter Epics Elite flow cytometer (Beckman Coulter) equipped with a 15 mW air‐cooled argon ion laser (Cyonics/Uniphase)
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Figures

Videos

Literature Cited

Literature Cited
   Abramo, F., Bo, S., Canese, G., and Poli, A. 1995. Regional distribution of lesions in the central nervous system of cats infected with feline immunodeficiency virus. AIDS Res. Hum. Retroviruses 11:1247‐1253.
   Aylward, E.H., Henderer, J.D., McArthur, J.C., Brettschneider, P.D., Harris, G.J., Barta, P.E., and Pearlson, G.D. 1993. Reduced basal ganglia volume in HIV‐1‐associated dementia: Results from quantitative neuroimaging. Neurology 43:2099‐2104.
   Bell, J.E., Brettle, R.P., Chiswick, A., and Simmonds, P. 1998. HIV encephalitis, proviral load and dementia in drug users and homosexuals with AIDS. Effect of neocortical involvement. Brain 121:2043‐2052.
   Belman, A.L. 1994. HIV‐1‐associated CNS disease in infants and children. Res. Pub. Assoc. Res. Nerv. Mental Dis. 72:289‐310.
   Boche, D., Hurtrel, M., Gray, F., Claessensmaire, M.A., Ganiere, J.P., Montagnier, L., and Hurtrel, B. 1996. Virus load and neuropathology in the FIV model. J. Neurovirol. 2:377‐387.
   Bornstein, R., Nasrallah, H., Para, M., Whitacre, C., Rosenberger, P., and Fass, R. 1993. Neuropsychological performance in symptomatic and asymptomatic HIV infection. AIDS 7:519‐524.
   Buck, W. and Podell, M. 1998. Neuronal loss in FIV‐MD infected cats. J. Neuro‐AIDS 2:69‐77.
   Dal, P.G., McArthur, J.C., Aylward, E., Selnes, O.A., Nance, S.T., Kumar, A.J., Mellits, E.D., and McArthur, J.C. 1992. Patterns of cerebral atrophy in HIV‐1‐infected individuals: Results of a quantitative MRI analysis. Neurology 42:2125‐2130.
   Diehl, L., Mathiason‐DuBard, C., O'Neil, L., Obert, L., and Hoover, E. 1995a. Induction of accelerated feline immunodeficiency virus disease by acute‐phase virus passage. J. Virol. 69:6149‐6157.
   Diehl, L.J., Mathiason‐DuBard, C.K., O'Neil, L.L., and Hoover, E.A. 1995b. Longitudinal assessment of feline immunodeficiency virus kinetics in plasma by use of a quantitative competitive reverse transcriptase PCR. J. Virol. 69:2328‐2332.
   Dow, S., Poss, M., and Hoover, E. 1990. Feline immunodeficiency virus: A neurotropic lentivirus. J. Acquir. Immune Defic. Syndr. 3:658‐668.
   Dow, S.W., Dreitz, M.J., and Hoover, E.A. 1992. Feline immunodeficiency virus neurotropism: Evidence that astrocytes and microglia are the primary target cells. Vet. Immunol. Immunopathol. 35:23‐35.
   Fox, H., Gold, L., Henriksen, S., and Bloom, F. 1997. Simian immunodeficiency virus: A model for neuroAIDS. J. Neurovirol. 4:265‐274.
   Gelman, B. and Guinto, F.C. Jr. 1992. Morphometry, histopathology, and tomography of cerebral atrophy in the acquired immunodeficiency syndrome. Ann. Neurol. 32:31‐40.
   Glass, J.D. and Johnson, R.T. 1996. Human immunodeficiency virus and the brain. Annu. Rev. Neurosci. 19:1‐26.
   Grant, I., Heaton, R., and Atkinson, J. 1995. Neurocognitive disorders in HIV‐1 infection. HNRC Group. HIV Neurobehavioral Research Center. Curr. Topics Microbiol. Immunol. 202:11‐32.
   Greene, W., Meers, J., del Fierro, G., Carnegie, P., and Robinson, W. 1993. Extensive sequence variation of feline immunodeficiency virus env genes in isolates from naturally infected cats. Arch. Virol. 133:51‐62.
   Hall, M., Whaley, R., Robertson, K., Hamby, S., Wilkins, J., and Hall, C. 1996. The correlation between neuropsychological and neuroanatomic changes over time in asymptomatic and symptomatic HIV‐1‐infected individuals. Neurology 46:1697‐1702.
   Hayes, K., Phipps, A., Francke, S., and Mathes, L. 2000. Antiviral therapy reduces viral burden but does not prevent thymic involution in pedicatric cats infected with feline immunodeficiency virus. Antimicrob. Agents Chemother. 44:2399‐2405.
   Hayes, K.A., Lafrado, L.J., Erickson, J.G., Marr, J.M., and Mathes, L.E. 1993. Prophylactic ZDV therapy prevents early viremia and lymphocyte decline but not primary infection in feline immunodeficiency virus‐inoculated cats. J. Acquir. Immune Defic. Syndr. 6:127‐134.
   Hayes, K.A., Wilkinson, J.G., Frick, R., Francke, S., and Mathes, L.E. 1995. Early suppression of viremia by ZDV does not alter the spread of feline immunodeficiency virus infection in cats. J. Acquir. Immune Defic. Syndr. Hum. Retrovirol. 9:114‐122.
   Hurtrel, M., Ganiere, J.P., Guelfi, J.F., Chakrabarti, L., Maire, M.A., Gray, F., Montagnier, L., and Hurtrel, B. 1992. Comparison of early and late feline immunodeficiency virus encephalopathies. AIDS 6:399‐406.
   Ishida, T. and Tomoda, I. 1990. Clinical staging of feline immunodeficiency virus infection. Jpn. J. Vet. Sci. 52:645‐648.
   Jacobson, S., Henriksen, S.J., Prospero‐Garcia, O., Phillips, T.R., Elder, J.H., Young, W.G., Bloom, F.E., and Fox, H.S. 1997. Cortical neuronal cytoskeletal changes associated with FIV infection. J. Neurovirol. 3:283‐289.
   Kibayashi, K., Mastri, A., and Hirsch, C. 1996. Neuropathology of human immunodeficiency virus infection at different disease stages. Hum. Pathol. 27:637‐642.
   Kleinhenz, E.A. and Cohen, S.B. 1991. Accurate determination of pH in organic phenol and phenol: chloroform. Biotechniques 10:740‐741.
   Lipton, S. 1997. Neuropathogenesis of acquired immunodeficiency syndrome dementia. Curr. Opin. Neurol. 10:247‐253.
   Lipton, S.A. 1994. AIDS‐related dementia and calcium homeostasis. Ann. N.Y. Acad. Sci. 747:205‐224.
   Lipton, S.A. 1998. Neuronal injury associated with HIV‐1: Approaches to treatment. Annu. Rev. Pharmacol. Toxicol. 38:159‐177.
   McArthur, J. and Grant, I. 1998. HIV neurocognitive disorders. In The Neurology of AIDS (H.E. Gendelman, S.A. Lipton, L. Epstein, S. Swindells, eds.) pp.499‐523. Chapman and Hall, New York.
   McArthur, J.C., Hoover, D.R., Bacellar, H., Miller, E.N., Cohen, B.A., Becker, J.T., Graham, N.M., McArthur, J.H., Selnes, O.A., and Jacobson, L.P., et al. 1993. Dementia in AIDS patients: Incidence and risk factors. Neurology 43:2245‐2252.
   Meeker, R.B., Theide, B.A., Hall, C., English, R., and Tompkins, M. 1997. Cortical cell loss in asymptomatic cats infected with feline immunodeficiency virus. AIDS Res. Hum. Retrovir. 13:1131‐1140.
   Mitchell, T.W., Buckmaster, P.S., Hoover, E.A., Whalen, L.R., and Dudek, F.E. 1998. Axonal sprouting in hippocampus infected with feline immunodeficiency virus (FIV). J. AIDS Hum. Retrovirol. 17:1‐8.
   Mitchell, T.W., Buckmaster, P.S., Hoover, E.A., Whalen, L.R., and Dudek, F.E. 1999. Neuron loss and axon reorganization in the dentate gyrus of cats infected with feline immunodeficiency virus. J. Comp. Neurol. 411:563‐577.
   Narayan, O. and Cork, L.C. 1987. Lentiviral diseases of sheep and goats: Chronic pneumonia, leukoencephalomyelitis and arthritis. Rev. Infect. Dis. 7:89‐98.
   Nath, A. and Geiger, J. 1998. Neurobiological aspects of human immunodeficiency virus infection: Neurotoxic mechanisms. Prog. Neurobiol. 54:19‐33.
   Navia, B., Cho, E.‐S., Petito, C., and Price, R. 1986. The AIDS dementia complex. II. Neuropathology. Ann. Neurol. 19:525‐535.
   Olmsted, R.A., Langley, R., Roelke, M.E., Goeken, R.M., Adger, J.D., Goff, J.P., Albert, J.P., Packer, C., Laurenson, M.K., and Caro, T.M. 1992. Worldwide prevalence of lentivirus infection in wild feline species: Epidemiologic and phylogenetic aspects. J. Virol. 66:6008‐6018.
   Pedersen, N.C., Ho, E.W., Brown, M.L., and Yamamoto, J.K. 1987. Isolation of a T‐lymphotropic virus from domestic cats with an immunodeficiency‐like syndrome. Science 235:790‐793.
   Phillips, T.R., Prospero‐Garcia, O., Puaoi, D.L., Lerner, D.L., Fox, H.S., Olmsted, R.A., Bloom, F.E., Henriksen, S.J., and Elder, J.H. 1994. Neurologic abnormalities associated with feline immunodeficiency virus infection. J. Gen. Virol. 75:979‐987.
   Podell, M., Oglesbee, M., Mathes, L., Krakowka, S., Olmstead, R., and Lafrado, L. 1993. AIDS‐associated encephalopathy with experimental feline immunodeficiency virus infection. J. Acquir. Immune Defic. Syndr. 6:758‐771.
   Podell, M., Hayes, K., Oglesbee, M., and Mathes, L. 1997. Progressive encephalopathy associated with CD4/CD8 inversion in adult FIV‐infected cats. J. Acquir. Immune Defic. Syndr. Hum. Retrovirol. 15:332‐340.
   Podell, M., Chen, E., and Shelton, D. 1998. Feline immunodeficiency virus associated myopathy in adult cats. Muscle Nerve 21:1680‐1685.
   Podell, M., Maruyama, K., Smith, M., Hayes, K., Buck, W.R., Ruehlmann, D., and Mathes, L. 1999. Frontal lobe injury correlates to altered function in FIV‐infected cats. J. AIDS Hum. Retrovirol. 22:10‐18.
   Podell, M., March, P., Buck, W., and Mathes, L. 2000. The feline model of neuro‐AIDS: Understanding the progression toward AIDS dementia. J. Psychopharmacol . 14:205‐213.
   Poli, A., Abramo, F., Iorio, C., Cantile, C., Carli, M., Pollera, C., Vago, L., Tosoni, A., and Costanzi, C. 1997. Neuropathology in cats experimentally infected with feline immunodeficiency virus: A morphological, immunocytochemical and morphometric study. J. Neurovirol. 3:361‐368.
   Power, C., Moench, T., Peeling, J., Kong, P.‐A., and Langelier, T. 1997. Feline immunodeficiency virus causes increased glutamate levels and neuronal loss in brain. Neuroscience 77:1175‐1185.
   Power, C., Buist, R., Johnston, B., Del Bigio, M.R., Ni, W., Dawood, M.R., and Peeling, J. 1998. Neurovirulence in feline immunodeficiency virus‐infected neonatal cats is viral strain specific and dependent on systemic immune suppresssion. J. Virol. 72:9109‐9115.
   Price, R.W. and Brew, B. 1988a. Infection of the central nervous system by human immunodeficiency virus. Role of the immune system in pathogenesis. Ann. N.Y. Acad. Sci. 540:162‐175.
   Price, R.W. and Brew, B.J. 1988b. The AIDS dementia complex. J. Infect. Dis. 158:1079‐1083.
   Prospero‐Garcia, O., Herold, N., Phillips, T., Elder, J., Bloom, F., and Henriksen, S. 1994. Sleep patterns are disturbed in cats infected with feline immunodeficiency virus. Proc. Natl. Acad. Sci. U.S.A. 91:12947‐12951.
   Ruehlmann, D.R., Chen, E., and Podell, M. 1998. Impaired Physiologic Frontal Cortical Maturation in FIV Infected Cats. American College of Veterinary Internal Medicine, San Diego.
   Saldanha, J., Gannicliffe, A., and Itzhaki, R. 1984. An improved method for preparing DNA from human brain. J. Neurosci. Methods 11:275‐279.
   Salvan, A.M., Vion‐Dury, J., Confort‐Gouny, S., Nicoli, F., Lamaoureux, S., and Cozzone, P.J. 1997. Brain proton magnetic resonance spectroscopy in HIV‐related encephalopathy: Identification of evolving metabolic patterns in relation to dementia and therapy. AIDS Res. Hum. Retrovirol. 13:1055‐1066.
   Sarter, M. and Podell, M. 2000. Preclinical psychopharmacology of AIDS‐associated dementia: Lessons to be learned from the cognitive psychopharmacology of other dementias. J. Psychopharmacol. 14:197‐204.
   Sei, Y., Kustova, Y., Li, Y., Morse, H.C. III., Skolnick, P., and Basile, A. 1998. The encephalopathy associated with murine acquired immunodeficiency syndrome. Ann. N.Y. Acad. Sci. 840:822‐834.
   Silvotti, L., Corradi, A., Brandi, G., Cabassi, A., Bendinelli, M., Magnan, M., and Piedimonte, G. 1997. FIV induced encephalopathy: Early brain lesions in the absence of viral replication in monocyte/macrophages. A pathogenetic model. Vet. Immunol. Immunopathol . 55:263‐271.
   Souaze, F., Ntodou‐Thome, A., Tran, C., Rostene, W., and Forgez, P. 1996. Quantitative RT‐PCR: Limits and accuracy. BioTechniques 21:280‐285.
   Steigerwald, E., Sarter, M., March, P., and Podell, M. 1999. Effects of feline immunodeficiency virus on cognition and behavioral function in cats. J. Acquir. Immune Defic. Syndr. Hum. Retrovirol. 20:411‐419.
   Vinogradov, A.E. 1998. Genome size and GC‐percent in vertebrates as determined by flow cytometry: The triangular relationship. Cytometry 31:100‐109.
   Watson, A., Ranchalis, J., Travis, B., McClure, J., Sutton, W., Johnson, P., Hu, S.‐L., and Haigwood, N. 1997. Plasma viremia in macaques infected with simian immunodeficiency virus: Plasma viral load early in infection predicts survival. J. Virol. 71:284‐290.
  World Health Organization (WHO). 1999. Report on the Global HIV/AIDS Epidemic. UNAIDS/CO.13E.
   Wiley, C.A., Masliah, E., Morey, M., Lemere, C., DeTeresa, R., Grafe, M., Hansen, L., and Terry, R. 1991. Neocortical damage during HIV infection. Ann. Neurol. 29:651‐657.
   Wilfinger, W., Mackey, K., and Chomczynski, P. 1997. Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. BioTechniques 22:474‐476.
   Willett, B.J., Picard, L., Hosie, M.J., Turner, J.D., Adema, K., and Clapham, P.R. 1997. Shared usage of the chemokine receptor CXCR4 by the feline and human immunodeficiency viruses. J. Virol. 71:6407‐6415.
   Yamamoto, J.K., Sparger, E., Ho, E.W., Andersen, P.R., O'Connor, T.P., Mandell, C.P., Lowenstine, L., Munn, R., and Pedersen, N.C. 1988. Pathogenesis of experimentally induced feline immunodeficiency virus infection in cats. Am. J. Vet. Res. 49:1246‐1258.
   Zimmermann, K. and Mannhalter, J. 1996. Technical aspects of quantitative competitive PCR. BioTechniques 21:268‐279.
   Zink, M., Spelman, J., Robinson, R.B., and Clements, J. 1998. SIV infection of macaques: Modeling the progression to AIDS dementia. J. Neurovirol. 4:249‐259.
Internet Resources
   http://www.ncbi.nlm.nih.gov/Entrez/
  The following FIV genome sequences are full length or nearly full length found in GenBank: M25381, E035081, M36968, U11820, U56928, M59418, and X57002.
   http://www.mednav.com/zone/Naids/
  Online journal dedicated to publishing topical reviews in the field of NeuroAIDS with an interactive component to discuss current issues with scientists in the field.
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