DOI: 10.5061/dryad.dncjsxm44
title: Identification of system-level features in HIV migration within a host

description: Objective: Identify system-level features in HIV migration within a host
 across body tissues. Evaluate heterogeneity in the presence and magnitude
 of these features across hosts.  Method: Using HIV DNA deep
 sequencing data generated across multiple tissues from 8 people with HIV,
 we represent the complex dependencies of HIV migration among tissues as a
 network and model these networks using the family of exponential random
 graph models (ERGMs). ERGMs allow for the statistical assessment of
 whether network features occur more (or less) frequently in viral
 migration than might be expected by chance. The analysis investigates ve
 potential features of the viral migration network: (1) bi-directional ow
 between tissues; (2) preferential migration among tissues in the same
 biological system; (3) heterogeneity in the level of viral migration
 related to HIV reservoir size; (4) hierarchical structure of migration;
 and (5) cyclical migration among several tissues. We calculate the
 Cohran's Q statistic to assess heterogeneity in the magnitude of the
 presence of these features across hosts. The analysis adjusts for missing
 data on body tissues.  Results: We observe strong evidence for
 bi-directional ow between tissues; migration among tissues in the same
 biological system; and hierarchical structure of the viral migration
 network. This analysis shows no evidence for differential level of viral
 migration with respect to the HIV reservoir size of a tissue. There is
 evidence that cyclical migration among three tissues occurs less
 frequently than expected given the amount of viral migration. The analysis
 also provides evidence for heterogeneity in the magnitude that these
 features are present across hosts. Adjusting for missing tissue data
 identifies system-level features within a host as well as heterogeneity in
 the presence of these features across hosts that are not detected when the
 analysis only considers the observed data.  Discussion:
 Identification of common features in viral migration may increase the
 efficiency of HIV cure efforts as it enables targeting specific processes.
description: Blood and tissue samples were collected during a rapid autopsy
 procedure. Genomic DNA was extracted from 5 million
 PBMCs and snap-frozen tissues using QIAamp DNA Mini Kit (Qiagen cat#51306)
 per manufacturer's protocol. After extraction, precipitation was
 performed to concentrate DNA. RNA was extracted from
 blood plasma layering 500-700µl of plasma on top of 200µl of 20% sterile
 filtered sucrose solution. Sample was spun at 23,500xg for 1 hour at 4°C
 to pellet the virus. Supernatant was removed and the pellet resuspended in
 140µl of PBS. RNA was extracted using Qiagen’s QIAamp Viral RNA mini kit
 (cat# 52904) according to the manufacturer’s recommendation. cDNA from HIV
 RNA was generated using Bio-Rad One-Step RT-ddPCR Advanced Kit for Probes
 (cat# 186-4021). <em>Nested PCR</em>. To
 amplify single genome FL env, DNA extracted from antemortem PBMCs and
 post-mortem tissues was diluted using ddPCR quantification data. This
 limited dilution PCR reaction can prevent PCR recombination and ambiguous
 base calls and allow the amplification of viral single genomes.  For HIV
 RNA in blood plasma, cDNA was generated from RNA using SuperScript III
 First Strand Synthesis System (cat# 18080-051). Template cDNA and HIV DNA
 extracted from tissues were diluted until approximately 30% of the
 second-round reactions were positive for the correctly-sized amplification
 product. Primers used for the first round were 5’FENVouter (forward)
 TTAGGCATCTCCTATGGCAGGAA and 3’RENVouter (reverse)
 TCTTAAAGGTACCTGAGGTCTGACTGG. First round PCRs were performed using the
 Advantage 2 PCR Kit from Takara (cat# 639206) following manufacturer’s
 recommendations using the 10X SA Buffer. Cycling conditions were 95C for 1
 min, 35 cycles of 95C 15sec, 57C 30sec, 68C 3min with a final extension at
 68C for 10min.The second round PCR was done using 5’FENVinner:
 GAGCAGAAGACAGTGGCAATGA (forward) and 3’RENVinner: CCACTTGCCACCCATBTTATAGCA
 (reverse). Cycling conditions were 95C for 1 min, 30 cycles of 95C 15sec,
 64C 30sec, 68C 3min with a final extension at 68C for 10min. PCR clean ups
 were done on the second round reaction products using QIAquick PCR
 Purification Kit (cat# 28106). DNA was quantified using Qubit dsDNA HS
 Assay Kit (Invitrogen cat#Q32854). Quality and integrity were measured
 using Genomic DNA Screen Tape in combination with the 2200 TapeStation
 System (Genomic DNA Reagents cat#5067-5366, Genomic DNA Screen Tape Cat
 #5067-5365). <em>Nextera XT Library
 Preparation</em>. Single Genome amplicons were prepared for deep
 sequencing using the Nextera XT DNA Library Preparation Kit (Illumina
 FC-131-1096) with indexing of 96-samples per run (Nextera XT index kit set
 A FC-131-2001) per manufacturer’s protocols.