Successful application of hyperbranched multidisplacement genomic amplification to detect HIV-1 sequences in single neurons removed from autopsy brain sections by laser capture microdissection.
To confirm studies suggesting that HIV-1 infects neurons and to determine whether CD8(+) T lymphocytes traffic to HIV-1-infected neurons, we used laser capture microdissection to remove hippocampal neurons with and without perineuronal CD8(+) T cells from AIDS patients with HIV-1 encephalitis (HIVE) or without HIVE and from normal controls. We used hyperbranched multidisplacement amplification for whole gene amplification (MDA-WGA) plus two rounds of PCR to amplify housekeeping sequences (HK(+)) and, in HK(+) samples, to amplify HIV-1 gag, nef, and pol sequences. Sample size and, in single neurons, MDA-WGA correlated with housekeeping gene amplification (P < 0.05), whereas patient group and postmortem interval did not (P > 0.05). Neuronal viral sequences correlated with HIVE (43% vs. 13% and 0 in non-HIVE and controls, respectively) and, in HIVE cases, with perineuronal CD8(+) T lymphocytes (70% in CD8(+) samples vs. 37% of CD8(-) samples). Our results suggest that MDA-WGA is a useful technique when analyzing DNA from single cells from autopsy brains, supporting prior studies that show that neurons may contain HIV-1 neuronal sequences in vivo. The association between neuronal infection and perineuronal CD8(+) T cells supports our hypothesis that these cells specifically traffic to infected neurons but raises the possibility that CD8(+) T cells, if infected, could transmit virus to neurons.
Department of Pathology (R5), Miller School of Medicine, Jackson Memorial Hospital, 1611 NW 12 Avenue, Miami, FL 33136, USA.
Jorge E
JE
Torres-Muñoz
Mariana
M
Núñez
Carol K
CK
Petito
D000163:Acquired Immunodeficiency Syndrome / Q000473:pathology / Q000821:virology; D000328:Adult; D001344:Autopsy; D001921:Brain / Q000378:metabolism / Q000473:pathology / Q000821:virology*; D016827:CD8 Antigens / Q000032:analysis; D018414:CD8-Positive T-Lymphocytes / Q000473:pathology / Q000821:virology; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D015497:HIV-1 / Q000235:genetics*; D006624:Hippocampus / Q000473:pathology / Q000821:virology; D006801:Humans; D007150:Immunohistochemistry; D007834:Lasers; D008297:Male; D042282:Microdissection / Q000295:instrumentation / Q000379:methods; D008875:Middle Aged; D009474:Neurons / Q000378:metabolism / Q000473:pathology / Q000821:virology*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*
D016827:CD8 Antigens; D004279:DNA, Viral