Laser Capture Microdissection Assessment of Virus Compartmentalization in the Central Nervous Systems of Macaques Infected with Neurovirulent Simian Immunodeficiency Virus

Abstract

bNonhuman primate-simian immunodeficiency virus (SIV) models are powerful tools for studying the pathogenesis of human immunodeficiency virus type 1 (HIV-1) in the brain. Our laboratory recently isolated a neuropathogenic viral swarm, SIVsmH804E, a derivative of SIVsmE543-3, which was the result of sequential intravenous passages of viruses isolated from the brains of rhesus macaques with SIV encephalitis. Animals infected with SIVsmH804E or its precursor (SIVsmH783Br) developed SIV meningitis and/or encephalitis at high frequencies. Since we observed macaques with a combination of meningitis and encephalitis, as well as animals in which meningitis or encephalitis was the dominant component, we hypothesized that distinct mechanisms could be driving the two pathological states. Therefore, we assessed viral populations in the meninges and the brain parenchyma by laser capture microdissection. Viral RNAs were isolated from representative areas of the meninges, brain parenchyma, terminal plasma, and cerebrospinal fluid (CSF) and from the inoculum, and the SIV envelope fragment was amplified by PCR. Phylogenetic analysis of envelope sequences from the conventional progressors revealed compartmentalization of viral populations between the meninges and the parenchyma. In one of these animals, viral populations in meninges were closely related to those from CSF and shared signature truncations in the cytoplasmic domain of gp41, consistent with a common origin. Apart from magnetic resonance imaging (MRI) and positron-emission tomography (PET) imaging, CSF is the most accessible assess to the central nervous system for HIV-1-infected patients. However, our results suggest that the virus in the CSF may not always be representative of viral populations in the brain and that caution should be applied in extrapolating between the properties of viruses in these two compartments.

Characterization of Simian Immunodeficiency Virus (SIV) That Induces SIV Encephalitis in Rhesus Macaques with High Frequency: Role of TRIM5 and Major Histocompatibility Complex Genotypes and Early Entry to the Brain

“…PBMCs from SIV-naive, healthy rhesus macaques were separated from whole blood, stimulated with 5 g of phytohemagglutinin (PHA) per ml and 10% interleukin-2 (IL-2) (Advanced Biotechnologies, Columbia, MD) for 3 days, and maintained in RPMI 1640 medium containing 10% fetal calf serum (FCS) and 10% IL-2. Rhesus macaque MDMs were obtained from rhesus macaque PBMCs as previously described (11). Briefly, fresh PBMCs were incubated with anti-nonhuman-primate CD14 magnetic beads (Miltenyi Biotec, Auburn, CA) and positively selected with magnetically activated cell sorting (MACS) separation columns (Miltenyi Biotec).…”

“…CSF and postmortem brain samples are the most accessible samples from HIV-1-infected individuals. However, as we have previously reported, the viral population in the CSF in simian immunodeficiency virus (SIV)-infected macaques does not always reflect the viral populations in the brain parenchyma, presumably the site of direct neuropathology (11,12). In addition, analysis of the human brain is restricted to postmortem samples and thus only gives cross-sectional information at the terminal stages of infection, which is not informative in addressing events in the acute or chronic phase of infection (13).…”

Enhanced antagonism of BST-2 by a neurovirulent SIV envelope

“…Env sequences of SIVsmH445, SIVsmH631Br, and SIVsmH783Br viral stocks were characterized in the previous study by the single-genome amplification method (15,48). The Env sequence of the SIVsm804E viral stock was characterized as previously described (14). Briefly, total RNA was extracted from the viral stock, and reverse transcription was performed by ThermoScript RT-PCR system (Thermo Fisher Scientific) using the primer 9341-R (See Supplemental Table 1 for primer sequences).…”

“…Virus replication of the isolates was evaluated in rhesus macaque PBMCs and MDMs as described previously (14,15). PBMCs from SIV-naive, healthy rhesus macaques were separated from whole blood, stimulated with 5 μg of phytohemagglutinin (PHA) per ml and 10% IL-2 (Advanced Biotechnologies) for 3 days, and maintained in RPMI 1640 medium containing 10% FCS and 10% IL-2.…”

“…Although it may not directly reflect the disease progression in HIV-1-infected individuals, nonhuman primate models enable access to CNS samples at all stages of disease progression, something that cannot be done with human patients for ethical reasons. Our group has recently reported on the isolation of a neurovirulent strain of simian immunodeficiency virus (SIV) African primate lentivirus (sm), SIVsm804E, that induced neuropathology in 83% of infected rhesus macaques when animals with restrictive MHC or TRIM alleles were eliminated from the cohort (14,15). This virus isolate was generated by sequential passage of the nonneurovirulent parental virus SIVsmE543-3 through rhesus macaques.…”