![]() The first case (C1) concerned a 48-year-old woman, HIV-positive for 10 years, treated by multiple antiretroviral therapy. The 12 tissues represented temporal and frontal lobe, caudate nucleus, thalamus, cerebellum, medulla oblongata, substantia nigra and spinal cord. Twelve postmortem brain tissues from three HIV-positive cases of probable or certain HIV-encephalopathy were provided by the Raymond Escourolle Neuropathology laboratory of the Pitié-Salpêtrière Hospital. In this study, we used UDS to describe HIV diversity in the CNS by sequencing the reverse transcriptase ( RT) gene and the hypervariable V3 loop region of the HIV-1 gp120 envelope protein, to detect minority-resistant variants and to identify HIV-1 tropism in specimens derived from different brain areas in three HIV+ cases of HIV encephalopathy. More specifically, uniquely divergent viral strains were identified in frontal, occipital, parietal, temporal lobes and basal ganglia. Analysis of the envelope gene in either Sanger or single molecule real time (SMRT) sequencing showed viral strains within the CNS evolving independently in different brain areas in patients who died from HIV-encephalopathy. Moreover, phylogenetic studies based on Sanger-sequencing determined brain-specific variants. Ultra-deep sequencing (UDS) detects minority variants that represent up to 1% of the HIV-1 population and that were incriminated for systemic therapeutic failure in treatment-naïve patients. Sanger-sequencing has shown independent evolution of drug resistance mutations to nucleoside and non-nucleoside reverse transcriptase inhibitors and protease inhibitors in different brain areas suggesting that differential drug penetration may occur among them. HIV-persistence in the CNS is principally because of weak penetration of antiretroviral drugs through the blood-brain barrier. The multinucleated giant cell (MGC), formed by cell-to-cell fusion of infected macrophages with microglia is the hallmark of this disease. HIV enters the brain causing HIV-encephalopathy. This study, performed with a powerful sequencing technique, confirmed HIV compartmentalization in the central nervous system already shown by classical sequencing, suggesting that there are several reservoirs within the brain. Furthermore, proportion and distribution of resistance mutations to nucleoside and non-nucleoside reverse transcriptase inhibitors differed among different brain areas of the same case suggesting that penetration of antiretroviral treatment may differ from one compartment to another. Although some cerebral regions shared HIV-variants, most of them harbored a specific HIV-subpopulation reflecting HIV compartmentalization in the central nervous system. Phylogenetic analysis revealed significant inter-regional and intra-regional diversity reflecting persistent HIV-1 viral replication in the different brain areas. Identification of resistant viral variants was performed on Geneious software, according to HIV-1 genotypic drug resistance interpretation's algorithms, 2018 administered by the French Agency for Research on AIDS and Viral Hepatitis. Phylogenetic analysis was performed with Fastree v2.1 using the generalized time-reversible (GTR) model. ![]() Methods:Īfter amplification of the reverse transcriptase and the V3 loop region of the gp120 protein, ultradeep sequencing was performed with Illumina technology. Twelve postmortem brain areas from three cases of possible or certain HIV-encephalopathy were analyzed. To examine viral diversity and resistance mutations in different brain areas in cases of HIV-encephalopathy.
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