Publications by authors named "Susanna L Lamers"

52 Publications

Emergence of an early SARS-CoV-2 epidemic in the United States.

medRxiv 2021 Feb 8. Epub 2021 Feb 8.

The emergence of the early COVID-19 epidemic in the United States (U.S.) went largely undetected, due to a lack of adequate testing and mitigation efforts. The city of New Orleans, Louisiana experienced one of the earliest and fastest accelerating outbreaks, coinciding with the annual Mardi Gras festival, which went ahead without precautions. To gain insight into the emergence of SARS-CoV-2 in the U.S. and how large, crowded events may have accelerated early transmission, we sequenced SARS-CoV-2 genomes during the first wave of the COVID-19 epidemic in Louisiana. We show that SARS-CoV-2 in Louisiana initially had limited sequence diversity compared to other U.S. states, and that one successful introduction of SARS-CoV-2 led to almost all of the early SARS-CoV-2 transmission in Louisiana. By analyzing mobility and genomic data, we show that SARS-CoV-2 was already present in New Orleans before Mardi Gras and that the festival dramatically accelerated transmission, eventually leading to secondary localized COVID-19 epidemics throughout the Southern U.S.. Our study provides an understanding of how superspreading during large-scale events played a key role during the early outbreak in the U.S. and can greatly accelerate COVID-19 epidemics on a local and regional scale.
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http://dx.doi.org/10.1101/2021.02.05.21251235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7872376PMC
February 2021

Persistence of HIV transmission clusters among people who inject drugs.

AIDS 2020 11;34(14):2037-2044

Department of Medicine, Johns Hopkins University.

Objective: We investigated the duration of HIV transmission clusters.

Design: Fifty-four individuals newly infected at enrollment in the ALIVE cohort were included, all of whom had sequences at an intake visit (T1) and from a second (T2) and/or a third (T3) follow-up visit, median 2.9 and 5.4 years later, respectively.

Methods: Sequences were generated using the 454 DNA sequencing platform for portions of HIV pol and env (HXB2 positions 2717-3230; 7941-8264). Genetic distances were calculated using tn93 and sequences were clustered over a range of thresholds (1--5%) using HIV-TRACE. Analyses were performed separately for individuals with pol sequences for T1 + T2 (n = 40, 'Set 1') and T1 + T3 (n = 25; 'Set 2'), and env sequences for T1 + T2 (n = 47, 'Set 1'), and T1 + T3 (n = 30; 'Set 2').

Results: For pol, with one exception, a single cluster contained more than 75% of samples at all thresholds, and cluster composition was at least 90% concordant between time points/thresholds. For env, two major clusters (A and B) were observed at T1 and T2/T3, although cluster composition concordance between time points/thresholds was low (<60%) at lower thresholds for both sets 1 and 2. In addition, several individuals were included in clusters at T2/T3, although not at T1.

Conclusion: Caution should be used in applying a single threshold in population studies where seroconversion dates are unknown. However, the retention of some clusters even after 5 + years is evidence for the robustness of the clustering approach in general.
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http://dx.doi.org/10.1097/QAD.0000000000002662DOI Listing
November 2020

Molecular surveillance of methicillin-resistant Staphylococcus aureus genomes in hospital unexpectedly reveals discordance between temporal and genetic clustering.

Am J Infect Control 2021 01 18;49(1):59-64. Epub 2020 Jun 18.

BioInfoExperts, LLC, Thibodaux, LA.

Background: The objective of this study was to identify sources and linkages among methicillin-resistant Staphylococcus aureus infections using whole-genome sequencing (WGS).

Methods: A total of 56 samples were obtained from all patients with a confirmed MRSA infection over 6 months at University of Florida-Health Jacksonville. Samples were cultured and sequenced; data was analyzed on an automated cloud-based platform. Genetic Clusters were defined as <40 single nucleotide polymorphisms. Temporal Clusters were defined as ≥5 MRSA cases over 3 days.

Results: We found 7 Genetic Clusters comprising 15 samples. Four Genetic Clusters contained patients with non-overlapping stays (3-10 weeks apart), 3 of which contained patients who shared the same Unit. We also found 5 Temporal Clusters comprising 23 samples, although none of the samples were genetically related.

Discussion: Results showed that temporal clustering may be a poor indicator of genetic linkage. Shared epidemiological characteristics between patients in Genetic Clusters may point toward previously unidentified hospital sources. Repeated observation of related strains is also consistent with ongoing MRSA transmission within the surrounding high-risk community.

Conclusions: WGS is a valuable tool for hospital infection prevention and control.
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http://dx.doi.org/10.1016/j.ajic.2020.06.180DOI Listing
January 2021

HIV-1 Subtype Distribution and Diversity Over 18 Years in Rakai, Uganda.

AIDS Res Hum Retroviruses 2020 06;36(6):522-526

Laboratory of Immunoregulation, Division of Intramural Research, NIAID, NIH, Baltimore, Maryland, USA.

The Rakai Community Cohort Study in south central Uganda has surveyed people aged 15-49 since 1994. Antiretroviral therapy (ART) was introduced in 2004. HIV p24 and gp41 subtype distribution and viral diversity were studied from blood samples collected at three surveys in 1994-1995, 2002-2003, and 2008-2009, which were compared with a new survey round from 2011 to 2012. These included 1364 HIV+ individuals. For both p24 and gp41 domains, the genetic diversity within subtypes A and D was significantly increasing in the pre-ART era and decreased between the last two survey rounds in the ART era ( < .01). This study suggests that despite ongoing mixing of viral subtypes, an association with the introduction of ART to a reduction of intra-subtype viral genomic diversity may be occurring, which can be explored in ongoing studies.
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http://dx.doi.org/10.1089/AID.2020.0062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7476391PMC
June 2020

Recombination Analysis of Near Full-Length HIV-1 Sequences and the Identification of a Potential New Circulating Recombinant Form from Rakai, Uganda.

AIDS Res Hum Retroviruses 2020 06 2;36(6):467-474. Epub 2020 Mar 2.

The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

The Phylogenetics And Networks for Generalized HIV Epidemics in Africa (PANGEA-HIV) consortium has been vital in the generation and examination of near full-length HIV-1 sequences generated from Sub-Saharan Africa. In this study, we examined a subset ( = 275) of sequences from Rakai, Uganda, collected between August 2011 and January 2015. Sequences were initially screened with COMET for subtyping and then evaluated using bootscanning and phylogenetic inference. Among 275 sequences, 38.6% were subtype D, 19.3% were subtype A, 2.9% were subtype C, and 39.3% were recombinant. The recombinants were structurally diverse in the number of breakpoints observed, the location of recombinant segments, and represented subtypes, with AD recombinants accounting for the majority of all recombinants (29.8%). Within the AD subpopulation, we identified a potential new circulating recombinant form in five individuals where the polymerase gene was subtype D and most of was subtype A (D-A junctures at HXB2 6760 and 8709). While the breakpoints were identical for the viruses from these individuals, the viral fragments did not cluster together. These results suggest selection for a viral strain where properties of the subtype A and subtype D portions of the virus confer a survival advantage. The continued study of recombinants will increase our breadth of knowledge for the genetic diversity and evolution of HIV-1, which can further contribute to our understanding toward a universal HIV-1 vaccine.
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http://dx.doi.org/10.1089/AID.2019.0150DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7262644PMC
June 2020

Longitudinal Antibody Responses in People Who Inject Drugs Infected With Similar Human Immunodeficiency Virus Strains.

J Infect Dis 2020 02;221(5):756-765

Laboratory of Immunoregulation, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.

Background: Multiple factors influence the human immunodeficiency virus (HIV) antibody response produced during natural infection, leading to responses that can vary in specificity, strength, and breadth.

Methods: People who inject drugs identified as recently infected with HIV (n = 23) were analyzed for clustering of their viral sequences (genetic distance, <2%). Longitudinal antibody responses were identified for neutralizing antibody (Nab) potential, and differences in antibody subclass, specificity, and Fc receptor ligation using pseudovirus entry and multiplexed Fc array assays, respectively. Responses were analyzed for differences between subject groups, defined by similarity in the sequence of the infecting virus.

Results: Viral sequences from infected individuals were grouped into 3 distinct clusters with 7 unclustered individuals. Subjects in cluster 1 generally had lower antibody response magnitudes, except for antibodies targeting the V1/V2 region. Subjects in clusters 2 and 3 typically had higher antibody response magnitudes, with the Fv specificity of cluster 2 favoring gp140 recognition. NAb responses differed significantly between clusters for 3 of 18 pseudoviruses examined (P < .05), but there were no differences in overall NAb breadth (P = .62).

Discussion: These data demonstrate that individuals infected with similar viral strains can generate partially similar antibody responses, but these do not drastically differ from those in individuals infected with relatively unrelated strains.
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http://dx.doi.org/10.1093/infdis/jiz503DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026888PMC
February 2020

Inconsistent temporal patterns of genetic variation of HCV among high-risk subjects may impact inference of transmission networks.

Infect Genet Evol 2019 07 22;71:1-6. Epub 2019 Feb 22.

Department of Medicine, Johns Hopkins University, Baltimore, MD, United States; NIAID, NIH, Baltimore, MD, United States.

Hepatitis-C Virus (HCV) sequences are often used to establish networks of people who inject drugs (PWID). However, the degree to which within-host evolutionary dynamics affect those inferences has not been carefully studied. Here, we analyzed 702 longitudinally-sampled HCV E1 sequences from 88 HCV+ people who inject drugs (PWID) in the Baltimore Before and After Acute Study of Hepatitis (BBAASH) cohort. Individuals were tested for HCV RNA over multiple visits to the clinic, and the HCV E1 gene was sequenced for HCV+ samples. Genetic clustering was performed on the full set of sequences using a 3% genetic distance threshold to define epidemiological linkage. Maximum-likelihood (ML) phylogenies were inferred to assess evolutionary relationships. We found 22 clusters containing sequences sampled over five or more years (long-term clusters, LTC), of which 17 had >1 subject. In six of the multi-subject LTC, one subject had a sequence sampled >3 years earlier or later than the next-closest subject in the cluster (time-gap LTC). ML trees showed that, in three of the time-gap LTC, two subjects had identical sequences despite 7-10 years separating the sampling times. In four of the time-gap LTC for whom additional data were available, the subject with the later detected shared variant had both different variants and visits with no detectable HCV RNA (RNA-) prior to the appearance of the shared variant. In the subject with the earlier detection of the shared variant, different variants and RNA- visits were also detected in multiple cases subsequent to appearance of the shared variant. Complex patterns of shared viral variation among PWID reflect on-going re-infection, multiple transmission partners, and/or inconsistent detection of viral variants. Our results suggest that transmission events are currently underestimated by analysis of sequences at a single point in time.
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http://dx.doi.org/10.1016/j.meegid.2019.02.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6620775PMC
July 2019

Emerging Patterns in HIV-1 gp120 Variable Domains in Anatomical Tissues in the Absence of a Plasma Viral Load.

AIDS Res Hum Retroviruses 2019 06 10;35(6):588-596. Epub 2019 Apr 10.

6 Departments of Laboratory Medicine, Pathology and Medicine, The University of California at San Francisco, San Francisco, California.

The HIV envelope protein contains five hypervariable domains (V1-V5) that are fundamental for cell entry. We contrasted modifications in the variable domains derived from a panel of 24 tissues from 7 subjects with no measurable plasma viral load (NPVL) to variable domains from 76 tissues from 15 subjects who had a detectable plasma viral load (PVL) at death. NPVL subject's V1 and V2 domains were usually highly length variable, whereas length variation in PVL sequences was more conserved. Longer V1s contained more charged residues, whereas longer V2s were more glycosylated. Structural analysis demonstrated V1/V2 charge, and N-site additions/subtractions were localized to the CD4 binding pocket. Diversified envelopes in tissues during therapy may represent a mechanism for HIV persistence in tissues, as binding pocket complexity is associated with HIV that may escape neutralization, whereas shorter envelopes are associated with increased infectivity. Further analysis of tissue-derived envelope sequences may enable better understanding of potential immunological approaches targeting the persistent HIV reservoir.
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http://dx.doi.org/10.1089/AID.2018.0267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6588100PMC
June 2019

Quantitation of the latent HIV-1 reservoir from the sequence diversity in viral outgrowth assays.

Retrovirology 2018 07 5;15(1):47. Epub 2018 Jul 5.

Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Baltimore, MD, USA.

Background: The ability of HIV-1 to integrate into the genomes of quiescent host immune cells, establishing a long-lived latent viral reservoir (LVR), is the primary obstacle to curing these infections. Quantitative viral outgrowth assays (QVOAs) are the gold standard for estimating the size of the replication-competent HIV-1 LVR, measured by the number of infectious units per million (IUPM) cells. QVOAs are time-consuming because they rely on culturing replicate wells to amplify the production of virus antigen or nucleic acid to reproducibly detectable levels. Sequence analysis can reduce the required number of culture wells because the virus genetic diversity within the LVR provides an internal replication and dilution series. Here we develop a Bayesian method to jointly estimate the IUPM and variant frequencies (a measure of clonality) from the sequence diversity of QVOAs.

Results: Using simulation experiments, we find our Bayesian approach confers significantly greater accuracy over current methods to estimate the IUPM, particularly for reduced numbers of QVOA replicates and/or increasing actual IUPM. Furthermore, we determine that the improvement in accuracy is greater with increasing genetic diversity in the sample population. We contrast results of these different methods applied to new HIV-1 sequence data derived from QVOAs from two individuals with suppressed viral loads from the Rakai Health Sciences Program in Uganda.

Conclusions: Utilizing sequence variation has the additional benefit of providing information on the contribution of clonality of the LVR, where high clonality (the predominance of a single genetic variant) suggests a role for cell division in the long-term persistence of the reservoir. In addition, our Bayesian approach can be adapted to other limiting dilution assays where positive outcomes can be partitioned by their genetic heterogeneity, such as immune cell populations and other viruses.
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http://dx.doi.org/10.1186/s12977-018-0426-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6034329PMC
July 2018

Ultradeep single-molecule real-time sequencing of HIV envelope reveals complete compartmentalization of highly macrophage-tropic R5 proviral variants in brain and CXCR4-using variants in immune and peripheral tissues.

J Neurovirol 2018 08 23;24(4):439-453. Epub 2018 Apr 23.

Bioinfoexperts, LLC, 718 Bayou Ln, Thibodaux, LA, 70301, USA.

Despite combined antiretroviral therapy (cART), HIV+ patients still develop neurological disorders, which may be due to persistent HIV infection and selective evolution in brain tissues. Single-molecule real-time (SMRT) sequencing technology offers an improved opportunity to study the relationship among HIV isolates in the brain and lymphoid tissues because it is capable of generating thousands of long sequence reads in a single run. Here, we used SMRT sequencing to generate ~ 50,000 high-quality full-length HIV envelope sequences (> 2200 bp) from seven autopsy tissues from an HIV+/cART+ subject, including three brain and four non-brain sites. Sanger sequencing was used for comparison with SMRT data and to clone functional pseudoviruses for in vitro tropism assays. Phylogenetic analysis demonstrated that brain-derived HIV was compartmentalized from HIV outside the brain and that the variants from each of the three brain tissues grouped independently. Variants from all peripheral tissues were intermixed on the tree but independent of the brain clades. Due to the large number of sequences, a clustering analysis at three similarity thresholds (99, 99.5, and 99.9%) was also performed. All brain sequences clustered exclusive of any non-brain sequences at all thresholds; however, frontal lobe sequences clustered independently of occipital and parietal lobes. Translated sequences revealed potentially functional differences between brain and non-brain sequences in the location of putative N-linked glycosylation sites (N-sites), V1 length, V3 charge, and the number of V4 N-sites. All brain sequences were predicted to use the CCR5 co-receptor, while most non-brain sequences were predicted to use CXCR4 co-receptor. Tropism results were confirmed by in vitro infection assays. The study is the first to use a SMRT sequencing approach to study HIV compartmentalization in tissues and supports other reports of limited trafficking between brain and non-brain sequences during cART. Due to the long sequence length, we could observe changes along the entire envelope gene, likely caused by differential selective pressure in the brain that may contribute to neurological disease.
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http://dx.doi.org/10.1007/s13365-018-0633-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7281851PMC
August 2018

Urogenital Chlamydia trachomatis multilocus sequence types and genovar distribution in chlamydia infected patients in a multi-ethnic region of Saratov, Russia.

PLoS One 2018 11;13(4):e0195386. Epub 2018 Apr 11.

Department of Pathology, Department of Microbiology & Immunology, University of Texas Medical Branch (UTMB), Galveston, Texas, United States of America.

Background: This is the first report to characterize the prevalence and genovar distribution of genital chlamydial infections among random heterosexual patients in the multi-ethnic Saratov Region, located in Southeast Russia.

Methods: Sixty-one clinical samples (cervical or urethral swabs) collected from a random cohort of 856 patients (7.1%) were C. trachomatis (CT) positive in commercial nucleic acid amplification tests (NAATs) and duplex TaqMan PCRs.

Results: Sequence analysis of the VDII region of the ompA gene revealed seven genovars of C. trachomatis in PCR-positive patients. The overall genovars were distributed as E (41.9%), G (21.6%), F (13.5%), K (9.5%), D (6.8%), J (4.1%), and H (2.7%). CT-positive samples were from males (n = 12, 19.7%), females (n = 42, 68.8%), and anonymous (n = 7, 11.5%) patients, with an age range of 19 to 45 years (average 26.4), including 12 different ethnic groups representative of this region. Most patients were infected with a single genovar (82%), while 18% were co-infected with either two or three genovars. The 1156 bp-fragment of the ompA gene was sequenced in 46 samples to determine single nucleotide polymorphisms (SNP) among isolates. SNP-based subtyping and phylogenetic reconstruction revealed the presence of 13 variants of the ompA gene, such as E (E1, E2, E6), G (G1, G2, G3, G5), F1, K, D (D1, Da2), J1, and H2. Differing genovar distribution was identified among urban (E>G>F) and rural (E>K) populations, and in Slavic (E>G>D) and non-Slavic (E>G>K) ethnic groups. Multilocus sequence typing (MLST) determined five sequences types (STs), such as ST4 (56%, 95% confidence interval, CI, 70.0 to 41.3), ST6 (10%, 95% CI 21.8 to 3.3), ST9 (22%, 95% CI 35.9 to 11.5), ST10 (2%, 95% CI 10.7 to 0.05) and ST38 (10%, 95% CI 21.8 to 3.3). Thus, the most common STs were ST4 and ST9.

Conclusion: C. trachomatis is a significant cause of morbidity among random heterosexual patients with genital chlamydial infections in the Saratov Region. Further studies should extend this investigation by describing trends in a larger population, both inside and outside of the Saratov Region to clarify some aspects for the actual application of C. trachomatis genotype analysis for disease control.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0195386PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5895025PMC
July 2018

Complex patterns of Hepatitis-C virus longitudinal clustering in a high-risk population.

Infect Genet Evol 2018 03 16;58:77-82. Epub 2017 Dec 16.

Department of Medicine, Johns Hopkins University, Baltimore, MD, United States; NIAID, NIH, Baltimore, MD, United States. Electronic address:

We investigated longitudinal viral clustering among and within subjects in a highly networked cohort of people who inject drugs (PWID). All subjects had estimated dates of infection and two or more E1 sequences (bp 943-1288 relative to H77) with 1 to 14years of follow up. Two methods (HIV-TRACE and PhyloPart) were used to determine clusters. Genetic distance thresholds were determined by comparing intra-and inter-host distances. Additional phylogenetic analysis was performed on subjects with complicated viral histories. At the optimal threshold of 3.9%, HIV-TRACE found 77 clusters and PhyloPart found 63 clusters, of which 27 and 32 contained multiple subjects, respectively. Furthermore, 1/3 of the subjects had sequences in different clusters over the course of the study, including some cases in which a later-sampled sequence matched a cluster detected much earlier in the infection, despite being separated by RNA-negative lab visit and detection of sequences in different clusters. A detailed phylogenetic analysis of four subjects with such patterns showed that in all four cases, the earlier and later variants grouped closely on the tree, and did not group with concurrent sequences from any other subject. These observations suggest that subjects are either experiencing rapid and recurring infection-clearance-reinfection cycles from the same source, or a single transmission event produces a chronic infection that may go undetected and/or co-circulate with different viruses from separate transmission events. Furthermore, our results show the utility of using longitudinal sampling to obtain a more comprehensive view of the viral linkages in high-risk populations.
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http://dx.doi.org/10.1016/j.meegid.2017.12.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5807204PMC
March 2018

Early Epstein-Barr Virus Genomic Diversity and Convergence toward the B95.8 Genome in Primary Infection.

J Virol 2018 01 2;92(2). Epub 2018 Jan 2.

Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA

Over 90% of the world's population is persistently infected with Epstein-Barr virus. While EBV does not cause disease in most individuals, it is the common cause of acute infectious mononucleosis (AIM) and has been associated with several cancers and autoimmune diseases, highlighting a need for a preventive vaccine. At present, very few primary, circulating EBV genomes have been sequenced directly from infected individuals. While low levels of diversity and low viral evolution rates have been predicted for double-stranded DNA (dsDNA) viruses, recent studies have demonstrated appreciable diversity in common dsDNA pathogens (e.g., cytomegalovirus). Here, we report 40 full-length EBV genome sequences obtained from matched oral wash and B cell fractions from a cohort of 10 AIM patients. Both intra- and interpatient diversity were observed across the length of the entire viral genome. Diversity was most pronounced in viral genes required for establishing latent infection and persistence, with appreciable levels of diversity also detected in structural genes, including envelope glycoproteins. Interestingly, intrapatient diversity declined significantly over time ( < 0.01), and this was particularly evident on comparison of viral genomes sequenced from B cell fractions in early primary infection and convalescence ( < 0.001). B cell-associated viral genomes were observed to converge, becoming nearly identical to the B95.8 reference genome over time (Spearman rank-order correlation test; = -0.5589, = 0.0264). The reduction in diversity was most marked in the EBV latency genes. In summary, our data suggest independent convergence of diverse viral genome sequences toward a reference-like strain within a relatively short period following primary EBV infection. Identification of viral proteins with low variability and high immunogenicity is important for the development of a protective vaccine. Knowledge of genome diversity within circulating viral populations is a key step in this process, as is the expansion of intrahost genomic variation during infection. We report full-length EBV genomes sequenced from the blood and oral wash of 10 individuals early in primary infection and during convalescence. Our data demonstrate considerable diversity within the pool of circulating EBV strains, as well as within individual patients. Overall viral diversity decreased from early to persistent infection, particularly in latently infected B cells, which serve as the viral reservoir. Reduction in B cell-associated viral genome diversity coincided with a convergence toward a reference-like EBV genotype. Greater convergence positively correlated with time after infection, suggesting that the reference-like genome is the result of selection.
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http://dx.doi.org/10.1128/JVI.01466-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5752928PMC
January 2018

The Spleen Is an HIV-1 Sanctuary During Combined Antiretroviral Therapy.

AIDS Res Hum Retroviruses 2018 01 30;34(1):123-125. Epub 2017 Nov 30.

5 The AIDS and Cancer Specimen Resource, University of California San Francisco , San Francisco, California.

Combined antiretroviral therapy (cART) does not eradicate HIV, which persists for years and can re-establish replication if treatment is stopped. The current challenge is identifying those tissues harboring virus through cART. Here, we used HIV env-nef single genome sequencing and HIV gag droplet digital PCR (ddPCR) to survey 50 tissues from five subjects on cART with no detectable plasma viral load at death. The spleen most consistently contained multiple proviral and expressed sequences (4/5 participants). Spleen-derived HIV demonstrated two distinct phylogenetic patterns: multiple identical sequences, often from different tissues, as well as diverse viral sequences on long terminal branches. Our results suggested that ddPCR may overestimate the size of the tissue-based viral reservoir. The spleen, a lymphatic organ at the intersection of the immune and circulatory systems, may play a key role in viral persistence.
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http://dx.doi.org/10.1089/AID.2017.0254DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5771542PMC
January 2018

Brain-specific HIV Nef identified in multiple patients with neurological disease.

J Neurovirol 2018 02 23;24(1):1-15. Epub 2017 Oct 23.

The University of California, San Francisco, CA, USA.

HIV-1 Nef is a flexible, multifunctional protein with several cellular targets that is required for pathogenicity of the virus. This protein maintains a high degree of genetic variation among intra- and inter-host isolates. HIV Nef is relevant to HIV-associated neurological diseases (HAND) in patients treated with combined antiretroviral therapy because of the protein's role in promoting survival and migration of infected brain macrophages. In this study, we analyzed 2020 HIV Nef sequences derived from 22 different tissues and 31 subjects using a novel computational approach. This approach combines statistical regression and evolved neural networks (ENNs) to classify brain sequences based on the physical and chemical characteristics of functional Nef domains. Based on training, testing, and validation data, the method successfully classified brain Nef sequences at 84.5% and provided informative features for further examination. These included physicochemical features associated with the Src-homology-3 binding domain, the Nef loop (including the AP-2 Binding region), and a cytokine-binding domain. Non-brain sequences from patients with HIV-associated neurological disease were frequently classified as brain, suggesting that the approach could indicate neurological risk using blood-derived virus or for the development of biomarkers for use in assay systems aimed at drug efficacy studies for the treatment of HIV-associated neurological diseases.
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http://dx.doi.org/10.1007/s13365-017-0586-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5792318PMC
February 2018

Eradication of HIV from Tissue Reservoirs: Challenges for the Cure.

AIDS Res Hum Retroviruses 2018 01 7;34(1):3-8. Epub 2017 Aug 7.

5 The AIDS and Cancer Specimen Resource , San Francisco, California.

The persistence of HIV infection, even after lengthy and successful combined antiretroviral therapy (cART), has precluded an effective cure. The anatomical locations and biological mechanisms through which the viral population is maintained remain unknown. Much research has focused nearly exclusively on circulating resting T cells as the predominant source of persistent HIV, a strategy with limited success in developing an effective cure strategy. In this study, we review research supporting the importance of anatomical tissues and other immune cells for HIV maintenance and expansion, including the central nervous system, lymph nodes, and macrophages. We present accumulated research that clearly demonstrates the limitations of using blood-derived cells as a proxy for tissue reservoirs and sanctuaries throughout the body. We cite recent studies that have successfully used deep-sequencing strategies to uncover the complexity of HIV infection and the ability of the virus to evolve despite undetectable plasma viral loads. Finally, we suggest new strategies and highlight the importance of tissue banks for future research.
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http://dx.doi.org/10.1089/AID.2017.0072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5771544PMC
January 2018

Paucity of Intact Non-Induced Provirus with Early, Long-Term Antiretroviral Therapy of Perinatal HIV Infection.

PLoS One 2017 8;12(2):e0170548. Epub 2017 Feb 8.

Department of Pediatrics-Infectious Diseases, Johns Hopkins University School of Medicine, Baltimore, MD, United States of America.

The latent reservoir is a major barrier to HIV eradication. Reservoir size is emerging as an important biomarker to assess the likelihood of HIV remission in the absence of antiretroviral therapy (ART) and may be reduced by earlier initiation of ART that restricts HIV spread into CD4+ T cells. Reservoir size is traditionally measured with a quantitative viral outgrowth assay (QVOA) that induces replication-competent HIV production through in vitro stimulation of resting CD4+ T cells. However, the recent identification of replication-intact, non-induced proviral genomes (NIPG) suggests the QVOA significantly underestimates (by 62-fold) latent reservoir size in chronically-infected adults. Whether formation and persistence of Intact, NIPG is thwarted by early ART initiation and long-term virologic suppression in perinatal infection is unclear. Here, we show that the latent reservoir in 11 early treated, long-term suppressed perinatally infected children and adolescents was not inducible by QVOA and dominated by defective, NIPG. Single genome analysis of 164 NIPG from 232 million cultured resting CD4+ T cells revealed no replication-intact, near-full length sequences. Forty-three (26%) NIPG contained APOBEC3G-mediated hypermutation, 115 (70%) NIPG contained large internal deletions, one NIPG contained nonsense mutations and indels, and 5 (3%) NIPG were assigned as "Not Evaluable" due to multiple failed sequencing attempts that precluded further classification. The lack of replication competent inducible provirus and intact NIPG in this cohort indicate early, long-term ART of perinatal infection leads to marked diminution of replication-competent HIV-1 reservoirs, creating a favorable state towards interventions aimed at virologic remission.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0170548PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5298215PMC
August 2017

Identifying Transmission Clusters with Cluster Picker and HIV-TRACE.

AIDS Res Hum Retroviruses 2017 03 13;33(3):211-218. Epub 2016 Dec 13.

5 Laboratory of Immunoregulation, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health , Bethesda, Maryland.

We compared the behavior of two approaches (Cluster Picker and HIV-TRACE) at varying genetic distances to identify transmission clusters. We used three HIV gp41 sequence datasets originating from the Rakai Community Cohort Study: (1) next-generation sequence (NGS) data from nine linked couples; (2) NGS data from longitudinal sampling of 14 individuals; and (3) Sanger consensus sequences from a cross-sectional dataset (n = 1,022) containing 91 epidemiologically linked heterosexual couples. We calculated the optimal genetic distance threshold to separate linked versus unlinked NGS datasets using a receiver operating curve analysis. We evaluated the number, size, and composition of clusters detected by Cluster Picker and HIV-TRACE at six genetic distance thresholds (1%-5.3%) on all three datasets. We further tested the effect of using all NGS, versus only a single variant for each patient/time point, for datasets (1) and (2). The optimal gp41 genetic distance threshold to distinguish linked and unlinked couples and individuals was 5.3% and 4%, respectively. HIV-TRACE tended to detect larger and fewer clusters, whereas Cluster Picker detected more clusters containing only two sequences. For NGS datasets (1) and (2), HIV-TRACE and Cluster Picker detected all linked pairs at 3% and 4% genetic distances, respectively. However, at 5.3% genetic distance, 20% of couples in dataset (3) did not cluster using either program, and for >1/3 of couples cluster assignment were discordant. We suggest caution in choosing thresholds for clustering analyses in a generalized epidemic.
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http://dx.doi.org/10.1089/AID.2016.0205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5333565PMC
March 2017

Low Rates of Transmitted Drug Resistance Among Newly Identified HIV-1 Seroconverters in Rural Rakai, Uganda.

AIDS Res Hum Retroviruses 2017 05 28;33(5):448-451. Epub 2016 Nov 28.

1 Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health , Bethesda, Maryland.

We investigated the rate of transmitted drug resistance (TDR) among HIV-1 seroconverters identified from the Rakai Community Cohort Study (RCCS) survey, a population-based cohort in Rakai District, Uganda. Participants aged 15-49 are interviewed at study visits approximately every 12-18 months and provided a serological sample. Antiretroviral therapy (ART) has been provided free of charge since 2004. RCCS participants with documented negative HIV-1 serology between January 2011 and August 2012 and confirmed seroconversion between November 2012 and October 2013 were included in this analysis. Serum was genotyped for HIV drug resistance mutations in reverse transcriptase and protease genes. Mutations were classified according to the 2009 World Health Organization surveillance of transmitted HIV-1 drug resistance update. Seventy-five (75) seroconverters were identified and genotyped. The mean age was 28 years (range 18-49) and the majority were male, n = 44 (58%). The HIV-1 subtype frequencies were A = 19 (25%), D = 44 (59%), C = 4 (5%), A/D recombinant = 5 (7%), and C/D recombinant = 3 (4%). The majority (72/75, 96%) of individuals were infected with wild-type virus with no evidence of TDR. Two individuals had a single non-nucleoside reverse transcriptase inhibitor mutation each, K101E and K103N, and one had a single protease inhibitor mutation, M46I. No mutations were identified involving nucleoside reverse transcriptase inhibitors. In conclusion, almost 10 years after the introduction of ART in rural Uganda, rates of TDR remain low. Ongoing surveillance for TDR remains an important public health priority and should be conducted among known seroconverters to estimate TDR.
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http://dx.doi.org/10.1089/AID.2015.0370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5439436PMC
May 2017

HIV-1 Evolutionary Patterns Associated with Metastatic Kaposi's Sarcoma during AIDS.

Sarcoma 2016 29;2016:4510483. Epub 2016 Aug 29.

The AIDS and Cancer Specimen Resource, University of California at San Francisco and the Department of Laboratory Medicine, Pathology, and Medicine, University of California at San Francisco, 1001 Poterero Ave, Bldg 3, Rm 207, UCSF Box 1317, San Francisco, CA 94110, USA.

Kaposi's sarcoma (KS) in HIV-infected individuals can have a wide range of clinical outcomes, from indolent skin tumors to a life-threatening visceral cancer. KS tumors contain endothelial-related cells and inflammatory cells that may be HIV-infected. In this study we tested if HIV evolutionary patterns distinguish KS tumor relatedness and progression. Multisite autopsies from participants who died from HIV-AIDS with KS prior to the availability of antiretroviral therapy were identified at the AIDS and Cancer Specimen Resource (ACSR). Two patients (KS1 and KS2) died predominantly from non-KS-associated disease and KS3 died due to aggressive and metastatic KS within one month of diagnosis. Skin and visceral tumor and nontumor autopsy tissues were obtained (n = 12). Single genome sequencing was used to amplify HIV RNA and DNA, which was present in all tumors. Independent HIV tumor clades in phylogenies differentiated KS1 and KS2 from KS3, whose sequences were interrelated by both phylogeny and selection. HIV compartmentalization was confirmed in KS1 and KS2 tumors; however, in KS3, no compartmentalization was observed among sampled tissues. While the sample size is small, the HIV evolutionary patterns observed in all patients suggest an interplay between tumor cells and HIV-infected cells which provides a selective advantage and could promote KS progression.
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http://dx.doi.org/10.1155/2016/4510483DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5019946PMC
September 2016

HIV DNA Is Frequently Present within Pathologic Tissues Evaluated at Autopsy from Combined Antiretroviral Therapy-Treated Patients with Undetectable Viral Loads.

J Virol 2016 10 29;90(20):8968-83. Epub 2016 Sep 29.

National Neurological AIDS Bank, Department of Neurology, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, California, USA David Geffen School of Medicine and Olive View-UCLA Medical Center, Department of Neurology, Los Angeles, California, USA.

Unlabelled: HIV infection treatment strategies have historically defined effectiveness through measuring patient plasma HIV RNA. While combined antiretroviral therapy (cART) can reduce plasma viral load (pVL) to undetectable levels, the degree that HIV is eliminated from other anatomical sites remains unclear. We investigated the HIV DNA levels in 229 varied autopsy tissues from 20 HIV-positive (HIV(+)) cART-treated study participants with low or undetectable plasma VL and cerebrospinal fluid (CSF) VL prior to death who were enrolled in the National Neurological AIDS Bank (NNAB) longitudinal study and autopsy cohort. Extensive medical histories were obtained for each participant. Autopsy specimens, including at least six brain and nonbrain tissues per participant, were reviewed by study pathologists. HIV DNA, measured in tissues by quantitative and droplet digital PCR, was identified in 48/87 brain tissues and 82/142 nonbrain tissues at levels >200 HIV copies/million cell equivalents. No participant was found to be completely free of tissue HIV. Parallel sequencing studies from some tissues recovered intact HIV DNA and RNA. Abnormal histological findings were identified in all participants, especially in brain, spleen, lung, lymph node, liver, aorta, and kidney. All brain tissues demonstrated some degree of pathology. Ninety-five percent of participants had some degree of atherosclerosis, and 75% of participants died with cancer. This study assists in characterizing the anatomical locations of HIV, in particular, macrophage-rich tissues, such as the central nervous system (CNS) and testis. Additional studies are needed to determine if the HIV recovered from tissues promotes the pathogenesis of inflammatory diseases, such as HIV-associated neurocognitive disorders, cancer, and atherosclerosis.

Importance: It is well-known that combined antiretroviral therapy (cART) can reduce plasma HIV to undetectable levels; however, cART cannot completely clear HIV infection. An ongoing question is, "Where is HIV hiding?" A well-studied HIV reservoir is "resting" T cells, which can be isolated from blood products and succumb to cART once activated. Less-studied reservoirs are anatomical tissue samples, which have unknown cART penetration, contain a comparably diverse spectrum of potentially HIV-infected immune cells, and are important since <2% of body lymphocytes actually reside in blood. We examined 229 varied autopsy specimens from 20 HIV(+) participants who died while on cART and identified that >50% of tissues were HIV infected. Additionally, we identified considerable pathology in participants' tissues, especially in brain, spleen, lung, lymph node, liver, aorta, and kidney. This study substantiates that tissue-associated HIV is present despite cART and can inform future studies into HIV persistence.
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http://dx.doi.org/10.1128/JVI.00674-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5044815PMC
October 2016

HIV Maintains an Evolving and Dispersed Population in Multiple Tissues during Suppressive Combined Antiretroviral Therapy in Individuals with Cancer.

J Virol 2016 10 29;90(20):8984-93. Epub 2016 Sep 29.

The AIDS and Cancer Specimen Resource, University of California at San Francisco, San Francisco, California, USA Departments of Laboratory Medicine, Pathology, and Medicine, University of California at San Francisco, San Francisco, California, USA

Unlabelled: While combined antiretroviral therapy (cART) can result in undetectable plasma viral loads, it does not eradicate HIV infection. Furthermore, HIV-infected individuals while on cART remain at an increased risk of developing serious comorbidities, such as cancer, neurological disease, and atherosclerosis, suggesting that during cART, tissue-based HIV may contribute to such pathologies. We obtained DNA and RNA env, nef, and pol sequences using single-genome sequencing from postmortem tissues of three HIV(+) cART-treated (cART(+)) individuals with undetectable viral load and metastatic cancer at death and performed time-scaled Bayesian evolutionary analyses. We used a sensitive in situ hybridization technique to visualize HIV gag-pol mRNA transcripts in cerebellum and lymph node tissues from one patient. Tissue-associated virus evolved at similar rates in cART(+) and cART-naive (cART(-)) patients. Phylogenetic trees were characterized by two distinct features: (i) branching patterns consistent with constant viral evolution and dispersal among tissues and (ii) very recently derived clades containing both DNA and RNA sequences from multiple tissues. Rapid expansion of virus near death corresponded to wide-spread metastasis. HIV RNA(+) cells clustered in cerebellum tissue but were dispersed in lymph node tissue, mirroring the evolutionary patterns observed for that patient. Activated, infiltrating macrophages were associated with HIV RNA. Our data provide evidence that tissues serve as a sanctuary for wild-type HIV during cART and suggest the importance of macrophages as an alternative reservoir and mechanism of virus spread.

Importance: Combined antiretroviral therapy (cART) reduces plasma HIV to undetectable levels; however, removal of cART results in plasma HIV rebound, thus highlighting its inability to entirely rid the body of infection. Additionally, HIV-infected individuals on cART remain at high risk of serious diseases, which suggests a contribution from residual HIV. In this study, we isolated and sequenced HIV from postmortem tissues from three HIV(+) cART(+) individuals who died with metastatic cancer and had no detectable plasma viral load. Using high-resolution evolutionary analyses, we found that tissue-based HIV continues to replicate, evolve, and migrate among tissues during cART. Furthermore, cancer onset and metastasis coincided with increased HIV expansion, suggesting a linked mechanism. HIV-expressing cells were associated with tissue macrophages, a target of HIV infection. Our results suggest the importance of tissues, and macrophages in particular, as a target for novel anti-HIV therapies.
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http://dx.doi.org/10.1128/JVI.00684-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5044847PMC
October 2016

HIV-1 Sequence Data Coverage in Central East Africa from 1959 to 2013.

AIDS Res Hum Retroviruses 2016 09 2;32(9):904-8. Epub 2016 Aug 2.

5 Department of Epidemiology, Bloomberg School of Public Health, Johns Hopkins University , Baltimore, Maryland.

Central and Eastern African HIV sequence data have been most critical in understanding the establishment and evolution of the global HIV pandemic. Here we report on the extent of publicly available HIV genetic sequence data in the Los Alamos National Laboratory Sequence Database sampled from 1959 to 2013 from six African countries: Uganda, Kenya, Tanzania, Burundi, the Democratic Republic of Congo, and Rwanda. We have summarized these data, including HIV subtypes, the years sampled, and the genomic regions sequenced. We also provide curated alignments for this important geographic area in five HIV genomic regions with substantial coverage.
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http://dx.doi.org/10.1089/AID.2016.0079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5028903PMC
September 2016

Evolution of Neuroadaptation in the Periphery and Purifying Selection in the Brain Contribute to Compartmentalization of Simian Immunodeficiency Virus (SIV) in the Brains of Rhesus Macaques with SIV-Associated Encephalitis.

J Virol 2016 07 10;90(13):6112-6126. Epub 2016 Jun 10.

Department of Pathology, Immunology, and Laboratory Medicine, University of Florida, Gainesville, Florida, USA

Unlabelled: The emergence of a distinct subpopulation of human or simian immunodeficiency virus (HIV/SIV) sequences within the brain (compartmentalization) during infection is hypothesized to be linked to AIDS-related central nervous system (CNS) neuropathology. However, the exact evolutionary mechanism responsible for HIV/SIV brain compartmentalization has not been thoroughly investigated. Using extensive viral sampling from several different peripheral tissues and cell types and from three distinct regions within the brain from two well-characterized rhesus macaque models of the neurological complications of HIV infection (neuroAIDS), we have been able to perform in-depth evolutionary analyses that have been unattainable in HIV-infected subjects. The results indicate that, despite multiple introductions of virus into the brain over the course of infection, brain sequence compartmentalization in macaques with SIV-associated CNS neuropathology likely results from late viral entry of virus that has acquired through evolution in the periphery sufficient adaptation for the distinct microenvironment of the CNS.

Importance: HIV-associated neurocognitive disorders remain prevalent among HIV type 1-infected individuals, whereas our understanding of the critical components of disease pathogenesis, such as virus evolution and adaptation, remains limited. Building upon earlier findings of specific viral subpopulations in the brain, we present novel yet fundamental results concerning the evolutionary patterns driving this phenomenon in two well-characterized animal models of neuroAIDS and provide insight into the timing of entry of virus into the brain and selective pressure associated with viral adaptation to this particular microenvironment. Such knowledge is invaluable for therapeutic strategies designed to slow or even prevent neurocognitive impairment associated with AIDS.
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http://dx.doi.org/10.1128/JVI.00137-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4907235PMC
July 2016

On the Physicochemical and Structural Modifications Associated with HIV-1 Subtype B Tropism Transition.

AIDS Res Hum Retroviruses 2016 08 1;32(8):829-40. Epub 2016 Jun 1.

4 Department of Laboratory Medicine, Pathology, and Medicine, and the AIDS and Cancer Specimen Resource, University of California , San Francisco, California.

HIV-1 enters immune cells via binding the viral envelope to a host cell CD4 receptor, and then a secondary co-receptor, usually CCR5 (R5) or CXCR4 (X4), and some HIV can utilize both co-receptors (R5X4). Although a small set of amino-acid properties such as charge and sequence length applied to HIV-1 envelope V3 loop sequence data can be used to predict co-receptor usage, we sought to expand the fundamental understanding of the physiochemical basis of tropism by analyzing many, perhaps less obvious, amino-acid properties over a diverse array of HIV sequences. We examined 74 amino-acid physicochemical scales over 1,559 V3 loop sequences with biologically tested tropisms downloaded from the Los Alamos HIV sequence database. Linear regressions were then calculated for each feature relative to three tropism transitions (R5→X4; R5→R5X4; R5X4→X4). Independent correlations were rank ordered to determine informative features. A structural analysis of the V3 loop was performed to better interpret these findings relative to HIV tropism states. Similar structural changes are required for R5 and R5X4 to transition to X4, thus suggesting that R5 and R5X4 types are more similar than either phenotype is to X4. Overall, the analysis suggests a continuum of viral tropism that is only partially related to charge; in fact, the analysis suggests that charge modification may be primarily attributed to decreased R5 usage, and further structural changes, particularly those associated with β-sheet structure, are likely required for full X4 usage.
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http://dx.doi.org/10.1089/AID.2015.0373DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4971420PMC
August 2016

The meningeal lymphatic system: a route for HIV brain migration?

J Neurovirol 2016 06 16;22(3):275-81. Epub 2015 Nov 16.

University of California and the AIDS and Cancer Specimen Resource, San Francisco, CA, USA.

Two innovative studies recently identified functional lymphatic structures in the meninges that may influence the development of HIV-associated neurological disorders (HAND). Until now, blood vessels were assumed to be the sole transport system by which HIV-infected monocytes entered the brain by bypassing a potentially hostile blood-brain barrier through inflammatory-mediated semi-permeability. A cascade of specific chemokine signals promote monocyte migration from blood vessels to surrounding brain tissues via a well-supported endothelium, where the cells differentiate into tissue macrophages capable of productive HIV infection. Lymphatic vessels on the other hand are more loosely organized than blood vessels. They absorb interstitial fluid from bodily tissues where HIV may persist and exchange a variety of immune cells (CD4(+) T cells, monocytes, macrophages, and dendritic cells) with surrounding tissues through discontinuous endothelial junctions. We propose that the newly discovered meningeal lymphatics are key to HIV migration among viral reservoirs and brain tissue during periods of undetectable plasma viral loads due to suppressive combinational antiretroviral therapy, thus redefining the migration process in terms of a blood-lymphatic transport system.
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http://dx.doi.org/10.1007/s13365-015-0399-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4868798PMC
June 2016

Identification of dual-tropic HIV-1 using evolved neural networks.

Biosystems 2015 Nov 28;137:12-9. Epub 2015 Sep 28.

University of California at San Francisco, Department of Laboratory Medicine and The AIDS and Cancer Specimen Resource, San Francisco, CA 94143, United States.

Blocking the binding of the envelope HIV-1 protein to immune cells is a popular concept for development of anti-HIV therapeutics. R5 HIV-1 binds CCR5, X4 HIV-1 binds CXCR4, and dual-tropic HIV-1 can bind either coreceptor for cellular entry. R5 viruses are associated with early infection and over time can evolve to X4 viruses that are associated with immune failure. Dual-tropic HIV-1 is less studied; however, it represents functional antigenic intermediates during the transition of R5 to X4 viruses. Viral tropism is linked partly to the HIV-1 envelope V3 domain, where the amino acid sequence helps dictate the receptor a particular virus will target; however, using V3 sequence information to identify dual-tropic HIV-1 isolates has remained difficult. Our goal in this study was to elucidate features of dual-tropic HIV-1 isolates that assist in the biological understanding of dual-tropism and develop an approach for their detection. Over 1559 HIV-1 subtype B sequences with known tropisms were analyzed. Each sequence was represented by 73 structural, biochemical and regional features. These features were provided to an evolved neural network classifier and evaluated using balanced and unbalanced data sets. The study resolved R5X4 viruses from R5 with an accuracy of 81.8% and from X4 with an accuracy of 78.8%. The approach also identified a set of V3 features (hydrophobicity, structural and polarity) that are associated with tropism transitions. The ability to distinguish R5X4 isolates will improve computational tropism decisions for R5 vs. X4 and assist in HIV-1 research and drug development efforts.
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http://dx.doi.org/10.1016/j.biosystems.2015.09.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4921197PMC
November 2015

Tracking the Emergence of Host-Specific Simian Immunodeficiency Virus env and nef Populations Reveals nef Early Adaptation and Convergent Evolution in Brain of Naturally Progressing Rhesus Macaques.

J Virol 2015 Aug 3;89(16):8484-96. Epub 2015 Jun 3.

Department of Pathology, Immunology, and Laboratory Medicine, University of Florida, Gainesville, Florida, USA Emerging Pathogens Institute, University of Florida, Gainesville, Florida, USA

Unlabelled: While a clear understanding of the events leading to successful establishment of host-specific viral populations and productive infection in the central nervous system (CNS) has not yet been reached, the simian immunodeficiency virus (SIV)-infected rhesus macaque provides a powerful model for the study of human immunodeficiency virus (HIV) intrahost evolution and neuropathogenesis. The evolution of the gp120 and nef genes, which encode two key proteins required for the establishment and maintenance of infection, was assessed in macaques that were intravenously inoculated with the same viral swarm and allowed to naturally progress to simian AIDS and potential SIV-associated encephalitis (SIVE). Longitudinal plasma samples and immune markers were monitored until terminal illness. Single-genome sequencing was employed to amplify full-length env through nef transcripts from plasma over time and from brain tissues at necropsy. nef sequences diverged from the founder virus faster than gp120 diverged. Host-specific sequence populations were detected in nef (~92 days) before they were detected in gp120 (~182 days). At necropsy, similar brain nef sequences were found in different macaques, indicating convergent evolution, while gp120 brain sequences remained largely host specific. Molecular clock and selection analyses showed weaker clock-like behavior and stronger selection pressure in nef than in gp120, with the strongest nef selection in the macaque with SIVE. Rapid nef diversification, occurring prior to gp120 diversification, indicates that early adaptation of nef in the new host is essential for successful infection. Moreover, the convergent evolution of nef sequences in the CNS suggests a significant role for nef in establishing neurotropic strains.

Importance: The SIV-infected rhesus macaque model closely resembles HIV-1 immunopathogenesis, neuropathogenesis, and disease progression in humans. Macaques were intravenously infected with identical viral swarms to investigate evolutionary patterns in the gp120 and nef genes leading to the emergence of host-specific viral populations and potentially linked to disease progression. Although each macaque exhibited unique immune profiles, macaque-specific nef sequences evolving under selection were consistently detected in plasma samples at 3 months postinfection, significantly earlier than in gp120 macaque-specific sequences. On the other hand, nef sequences in brain tissues, collected at necropsy of two animals with detectable infection in the central nervous system (CNS), revealed convergent evolution. The results not only indicate that early adaptation of nef in the new host may be essential for successful infection, but also suggest that specific nef variants may be required for SIV to efficiently invade CNS macrophages and/or enhance macrophage migration, resulting in HIV neuropathology.
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http://dx.doi.org/10.1128/JVI.01010-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4524235PMC
August 2015

Genome Sequencing and Analysis of Geographically Diverse Clinical Isolates of Herpes Simplex Virus 2.

J Virol 2015 Aug 27;89(16):8219-32. Epub 2015 May 27.

Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA.

Unlabelled: Herpes simplex virus 2 (HSV-2), the principal causative agent of recurrent genital herpes, is a highly prevalent viral infection worldwide. Limited information is available on the amount of genomic DNA variation between HSV-2 strains because only two genomes have been determined, the HG52 laboratory strain and the newly sequenced SD90e low-passage-number clinical isolate strain, each from a different geographical area. In this study, we report the nearly complete genome sequences of 34 HSV-2 low-passage-number and laboratory strains, 14 of which were collected in Uganda, 1 in South Africa, 11 in the United States, and 8 in Japan. Our analyses of these genomes demonstrated remarkable sequence conservation, regardless of geographic origin, with the maximum nucleotide divergence between strains being 0.4% across the genome. In contrast, prior studies indicated that HSV-1 genomes exhibit more sequence diversity, as well as geographical clustering. Additionally, unlike HSV-1, little viral recombination between HSV-2 strains could be substantiated. These results are interpreted in light of HSV-2 evolution, epidemiology, and pathogenesis. Finally, the newly generated sequences more closely resemble the low-passage-number SD90e than HG52, supporting the use of the former as the new reference genome of HSV-2.

Importance: Herpes simplex virus 2 (HSV-2) is a causative agent of genital and neonatal herpes. Therefore, knowledge of its DNA genome and genetic variability is central to preventing and treating genital herpes. However, only two full-length HSV-2 genomes have been reported. In this study, we sequenced 34 additional HSV-2 low-passage-number and laboratory viral genomes and initiated analysis of the genetic diversity of HSV-2 strains from around the world. The analysis of these genomes will facilitate research aimed at vaccine development, diagnosis, and the evaluation of clinical manifestations and transmission of HSV-2. This information will also contribute to our understanding of HSV evolution.
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http://dx.doi.org/10.1128/JVI.01303-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4524243PMC
August 2015