Publications by authors named "Scott Sherrill-Mix"

38 Publications

Predictors of Nonseroconversion after SARS-CoV-2 Infection.

Emerg Infect Dis 2021 Jun 30;27(9). Epub 2021 Jun 30.

Not all persons recovering from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection develop SARS-CoV-2-specific antibodies. We show that nonseroconversion is associated with younger age and higher reverse transcription PCR cycle threshold values and identify SARS-CoV-2 viral loads in the nasopharynx as a major correlate of the systemic antibody response.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3201/eid2709.211042DOI Listing
June 2021

Detection of SARS-CoV-2 RNA using RT-LAMP and molecular beacons.

Genome Biol 2021 06 3;22(1):169. Epub 2021 Jun 3.

Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.

Background: Rapid spread of SARS-CoV-2 has led to a global pandemic, resulting in the need for rapid assays to allow diagnosis and prevention of transmission. Reverse transcription-polymerase chain reaction (RT-PCR) provides a gold standard assay for SARS-CoV-2 RNA, but instrument costs are high and supply chains are potentially fragile, motivating interest in additional assay methods. Reverse transcription and loop-mediated isothermal amplification (RT-LAMP) provides an alternative that uses orthogonal and often less expensive reagents without the need for thermocyclers. The presence of SARS-CoV-2 RNA is typically detected using dyes to report bulk amplification of DNA; however, a common artifact is nonspecific DNA amplification, which complicates detection.

Results: Here we describe the design and testing of molecular beacons, which allow sequence-specific detection of SARS-CoV-2 genomes with improved discrimination in simple reaction mixtures. To optimize beacons for RT-LAMP, multiple locked nucleic acid monomers were incorporated to elevate melting temperatures. We also show how beacons with different fluorescent labels can allow convenient multiplex detection of several amplicons in "single pot" reactions, including incorporation of a human RNA LAMP-BEAC assay to confirm sample integrity. Comparison of LAMP-BEAC and RT-qPCR on clinical saliva samples showed good concordance between assays. To facilitate implementation, we developed custom polymerases for LAMP-BEAC and inexpensive purification procedures, which also facilitates increasing sensitivity by increasing reaction volumes.

Conclusions: LAMP-BEAC thus provides an affordable and simple SARS-CoV-2 RNA assay suitable for population screening; implementation of the assay has allowed robust screening of thousands of saliva samples per week.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13059-021-02387-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8173101PMC
June 2021

CD4 receptor diversity represents an ancient protection mechanism against primate lentiviruses.

Proc Natl Acad Sci U S A 2021 Mar;118(13)

Lukuru Wildlife Research Foundation, Tshuapa-Lomami-Lualaba Project, BP 2012, Kinshasa, Democratic Republic of the Congo.

Infection with human and simian immunodeficiency viruses (HIV/SIV) requires binding of the viral envelope glycoprotein (Env) to the host protein CD4 on the surface of immune cells. Although invariant in humans, the Env binding domain of the chimpanzee CD4 is highly polymorphic, with nine coding variants circulating in wild populations. Here, we show that within-species CD4 diversity is not unique to chimpanzees but found in many African primate species. Characterizing the outermost (D1) domain of the CD4 protein in over 500 monkeys and apes, we found polymorphic residues in 24 of 29 primate species, with as many as 11 different coding variants identified within a single species. D1 domain amino acid replacements affected SIV Env-mediated cell entry in a single-round infection assay, restricting infection in a strain- and allele-specific fashion. Several identical CD4 polymorphisms, including the addition of -linked glycosylation sites, were found in primate species from different genera, providing striking examples of parallel evolution. Moreover, seven different guenons ( spp.) shared multiple distinct D1 domain variants, pointing to long-term trans-specific polymorphism. These data indicate that the HIV/SIV Env binding region of the primate CD4 protein is highly variable, both within and between species, and suggest that this diversity has been maintained by balancing selection for millions of years, at least in part to confer protection against primate lentiviruses. Although long-term SIV-infected species have evolved specific mechanisms to avoid disease progression, primate lentiviruses are intrinsically pathogenic and have left their mark on the host genome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.2025914118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8020793PMC
March 2021

SARS-CoV-2 Genomic Variation in Space and Time in Hospitalized Patients in Philadelphia.

mBio 2021 01 19;12(1). Epub 2021 Jan 19.

Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA

The severe acute respiratory coronavirus 2 (SARS-CoV-2) is the cause of the global outbreak of COVID-19. The epidemic accelerated in Philadelphia, PA, in the spring of 2020, with the city experiencing a first peak of infections on 15 April, followed by a decline through midsummer. Here, we investigate spread of the epidemic in the first wave in Philadelphia using full-genome sequencing of 52 SARS-CoV-2 samples obtained from 27 hospitalized patients collected between 30 March and 17 July 2020. Sequences most commonly resembled lineages circulating at earlier times in New York, suggesting transmission primarily from this location, though a minority of Philadelphia genomes matched sequences from other sites, suggesting additional introductions. Multiple genomes showed even closer matches to other Philadelphia isolates, suggestive of ongoing transmission within Philadelphia. We found that all of our isolates contained the D614G substitution in the viral spike and belong to lineages variously designated B.1, Nextstrain clade 20A or 20C, and GISAID clade G or GH. There were no viral sequence polymorphisms detectably associated with disease outcome. For some patients, genome sequences were determined longitudinally or concurrently from multiple body sites. In both cases, some comparisons showed reproducible polymorphisms, suggesting initial seeding with multiple variants and/or accumulation of polymorphisms after infection. These results thus provide data on the sources of SARS-CoV-2 infection in Philadelphia and begin to explore the dynamics within hospitalized patients. Understanding how SARS-CoV-2 spreads globally and within infected individuals is critical to the development of mitigation strategies. We found that most lineages in Philadelphia had resembled sequences from New York, suggesting infection primarily but not exclusively from this location. Many genomes had even nearer neighbors within Philadelphia, indicating local spread. Multiple genome sequences were available for some subjects and in a subset of cases could be shown to differ between time points and body sites within an individual, indicating heterogeneous viral populations within individuals and raising questions on the mechanisms responsible. There was no evidence that different lineages were associated with different outcomes in patients, emphasizing the importance of individual-specific vulnerability.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/mBio.03456-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7829343PMC
January 2021

Heightened resistance to host type 1 interferons characterizes HIV-1 at transmission and after antiretroviral therapy interruption.

Sci Transl Med 2021 01;13(576)

Laboratory of Molecular Immunology, Rockefeller University, New York, NY 10065, USA.

Type 1 interferons (IFN-I) are potent innate antiviral effectors that constrain HIV-1 transmission. However, harnessing these cytokines for HIV-1 cure strategies has been hampered by an incomplete understanding of their antiviral activities at later stages of infection. Here, we characterized the IFN-I sensitivity of 500 clonally derived HIV-1 isolates from the plasma and CD4 T cells of 26 individuals sampled longitudinally after transmission or after antiretroviral therapy (ART) and analytical treatment interruption. We determined the concentration of IFNα2 and IFNβ that reduced viral replication in vitro by 50% (IC) and found consistent changes in the sensitivity of HIV-1 to IFN-I inhibition both across individuals and over time. Resistance of HIV-1 isolates to IFN-I was uniformly high during acute infection, decreased in all individuals in the first year after infection, was reacquired concomitant with CD4 T cell loss, and remained elevated in individuals with accelerated disease. HIV-1 isolates obtained by viral outgrowth during suppressive ART were relatively IFN-I sensitive, resembling viruses circulating just before ART initiation. However, viruses that rebounded after treatment interruption displayed the highest degree of IFNα2 and IFNβ resistance observed at any time during the infection course. These findings indicate a dynamic interplay between host innate responses and the evolving HIV-1 quasispecies, with the relative contribution of IFN-I to HIV-1 control affected by both ART and analytical treatment interruption. Although elevated at transmission, host innate pressures are the highest during viral rebound, limiting the viruses that successfully become reactivated from latency to those that are IFN-I resistant.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/scitranslmed.abd8179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7923595PMC
January 2021

A cone-beam computed tomographic evaluation of alveolar bone dimensional changes and the periodontal limits of mandibular incisor advancement in skeletal Class II patients.

Angle Orthod 2020 05;90(3):330-338

Objectives: To evaluate the presence of dehiscences and changes in alveolar bone height and width in the area of the mandibular central incisors pre- and post-orthodontic treatment.

Materials And Methods: In 60 skeletal Class II patients, cone-beam computed tomographic (CBCT) images were obtained and the patients were divided into four groups based on the presence of dehiscences at pre- and post-orthodontic treatment. The alveolar bone height and width were measured on CBCT in cross section along the long axis of the teeth. Lateral cephalograms were analyzed.

Results: The changes in L1-NB and IMPA appeared to be correlated with vertical bone loss and dehiscence. Alveolar bone height appeared to follow a segmented relationship with these two variables, with changes below a threshold (L1-NB = 0.71 mm, IMPA = 3.02°) having relatively minimal or no effect on bone loss but with changes beyond the threshold correlated with extensive bone loss. Similarly, increases in L1-NB or IMPA correlated with decreases in alveolar bone width (L1-NB: -0.25 mm/mm, IMPA: -0.07 mm/°) and increased the probability of developing dehiscences, with an estimated 50% probability of vertical bone loss at a L1-NB change of 2.00 mm or, equivalently, an IMPA change of 8.02° was estimated.

Conclusions: When treating skeletal Class II patients, the limits of incisor proclination/protraction are less than previously thought. To prevent undesired periodontal outcomes, careful three-dimensional diagnosis is advisable. Furthermore, when excessive protrusion and/or proclination is planned, additional treatment modalities, including orthognathic surgery, tooth extraction, and corticotomy with bone graft, should be considered.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2319/080219-510.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8032294PMC
May 2020

A Summary of the Fifth Annual Virology Education HIV Microbiome Workshop.

AIDS Res Hum Retroviruses 2020 11 7;36(11):886-895. Epub 2020 Sep 7.

Department of Epidemiology, The George Washington University, Washington, District of Columbia, USA.

In October of 2019, researchers and community members from around the world met at the NIH for the fifth annual International Workshop on Microbiome in HIV. New research was presented on the role of the microbiome on chronic inflammation and vaccine design, interactions of genetics, environment, sexual practice and HIV infection with the microbiome and the development and clinical trials of microbiome-based therapeutic approaches intended to decrease the probability of HIV acquisition/transmission or ameliorate sequelae of HIV. The keynote address by Dr. Jacques Ravel focused on his work on the vaginal microbiome and efforts to improve the analysis and resolution of microbiome data.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/AID.2020.0121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7869876PMC
November 2020

The stepwise assembly of the neonatal virome is modulated by breastfeeding.

Nature 2020 05 15;581(7809):470-474. Epub 2020 Apr 15.

Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

The gut of healthy human neonates is usually devoid of viruses at birth, but quickly becomes colonized, which-in some cases-leads to gastrointestinal disorders. Here we show that the assembly of the viral community in neonates takes place in distinct steps. Fluorescent staining of virus-like particles purified from infant meconium or early stool samples shows few or no particles, but by one month of life particle numbers increase to 10 per gram, and these numbers seem to persist throughout life. We investigated the origin of these viral populations using shotgun metagenomic sequencing of virus-enriched preparations and whole microbial communities, followed by targeted microbiological analyses. Results indicate that, early after birth, pioneer bacteria colonize the infant gut and by one month prophages induced from these bacteria provide the predominant population of virus-like particles. By four months of life, identifiable viruses that replicate in human cells become more prominent. Multiple human viruses were more abundant in stool samples from babies who were exclusively fed on formula milk compared with those fed partially or fully on breast milk, paralleling reports that breast milk can be protective against viral infections. Bacteriophage populations also differed depending on whether or not the infant was breastfed. We show that the colonization of the infant gut is stepwise, first mainly by temperate bacteriophages induced from pioneer bacteria, and later by viruses that replicate in human cells; this second phase is modulated by breastfeeding.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-020-2192-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7263352PMC
May 2020

Single-cell transcriptional landscapes reveal HIV-1-driven aberrant host gene transcription as a potential therapeutic target.

Sci Transl Med 2020 05;12(543)

Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT 06519, USA.

Understanding HIV-1-host interactions can identify the cellular environment supporting HIV-1 reactivation and mechanisms of clonal expansion. We developed HIV-1 SortSeq to isolate rare HIV-1-infected cells from virally suppressed, HIV-1-infected individuals upon early latency reversal. Single-cell transcriptome analysis of HIV-1 SortSeq cells revealed enrichment of nonsense-mediated RNA decay and viral transcription pathways. HIV-1 SortSeq cells up-regulated cellular factors that can support HIV-1 transcription ( and ) or promote cellular survival ( and ). HIV-1-host RNA landscape analysis at the integration site revealed that HIV-1 drives high aberrant host gene transcription downstream, but not upstream, of the integration site through HIV-1-to-host aberrant splicing, in which HIV-1 RNA splices into the host RNA and aberrantly drives host RNA transcription. HIV-1-induced aberrant transcription was driven by the HIV-1 promoter as shown by CRISPR-dCas9-mediated HIV-1-specific activation and could be suppressed by CRISPR-dCas9-mediated inhibition of HIV-1 5' long terminal repeat. Overall, we identified cellular factors supporting HIV-1 reactivation and HIV-1-driven aberrant host gene transcription as potential therapeutic targets to disrupt HIV-1 persistence.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/scitranslmed.aaz0802DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7453882PMC
May 2020

CpG Frequency in the 5' Third of the Gene Determines Sensitivity of Primary HIV-1 Strains to the Zinc-Finger Antiviral Protein.

mBio 2020 01 14;11(1). Epub 2020 Jan 14.

Institute of Molecular Virology, Ulm University Medical Center, Ulm, Germany

CpG dinucleotide suppression has been reported to allow HIV-1 to evade inhibition by the zinc-finger antiviral protein (ZAP). Here, we show that primate lentiviruses display marked differences in CpG frequencies across their genome, ranging from 0.44% in simian immunodeficiency virus SIVwrc from Western red colobus to 2.3% in SIVmon infecting mona monkeys. Moreover, functional analyses of a large panel of human and simian immunodeficiency viruses revealed that the magnitude of CpG suppression does not correlate with their susceptibility to ZAP. However, we found that the number of CpG dinucleotides within a region of ∼700 bases at the 5' end of the gene determines ZAP sensitivity of primary HIV-1 strains but not of HIV-2. Increased numbers of CpGs in this region were associated with reduced mRNA expression and viral protein production. ZAP sensitivity profiles of chimeric simian-human immunodeficiency viruses (SHIVs) expressing different HIV-1 genes were highly similar to those of the corresponding HIV-1 strains. The frequency of CpGs in the identified region correlated with differences in clinical progression rates. Thus, the CpG frequency in a specific part of , rather than the overall genomic CpG content, governs the susceptibility of HIV-1 to ZAP and might affect viral pathogenicity Evasion of the zinc-finger antiviral protein (ZAP) may drive CpG dinucleotide suppression in HIV-1 and many other viral pathogens but the viral determinants of ZAP sensitivity are poorly defined. Here, we examined CpG suppression and ZAP sensitivity in a large number of primate lentiviruses and demonstrate that their genomic frequency of CpGs varies substantially and does not correlate with ZAP sensitivity. We further show that the number of CpG residues in a defined region at the 5' end of the gene together with structural features plays a key role in HIV-1 susceptibility to ZAP and correlates with differences in clinical progression rates in HIV-1-infected individuals. Our identification of a specific part of as a major determinant of HIV-1 susceptibility to ZAP restriction provides a basis for future studies of the underlying inhibitory mechanisms and their potential relevance in the pathogenesis of AIDS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/mBio.02903-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6960287PMC
January 2020

CD19-targeting CAR T cell immunotherapy outcomes correlate with genomic modification by vector integration.

J Clin Invest 2020 02;130(2):673-685

Department of Microbiology.

Chimeric antigen receptor-engineered T cells targeting CD19 (CART19) provide an effective treatment for pediatric acute lymphoblastic leukemia but are less effective for chronic lymphocytic leukemia (CLL), focusing attention on improving efficacy. CART19 harbor an engineered receptor, which is delivered through lentiviral vector integration, thereby marking cell lineages and modifying the cellular genome by insertional mutagenesis. We recently reported that vector integration within the host TET2 gene was associated with CLL remission. Here, we investigated clonal population structure and therapeutic outcomes in another 39 patients by high-throughput sequencing of vector-integration sites. Genes at integration sites enriched in responders were commonly found in cell-signaling and chromatin modification pathways, suggesting that insertional mutagenesis in these genes promoted therapeutic T cell proliferation. We also developed a multivariate model based on integration-site distributions and found that data from preinfusion products forecasted response in CLL successfully in discovery and validation cohorts and, in day 28 samples, reported responders to CLL therapy with high accuracy. These data clarify how insertional mutagenesis can modulate cell proliferation in CART19 therapy and how data on integration-site distributions can be linked to treatment outcomes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI130144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994131PMC
February 2020

Virus structures constrain transmission modes.

Nat Microbiol 2019 11 29;4(11):1778-1780. Epub 2019 Jul 29.

Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA.

Here we investigate links between the structures of viruses and routes of transmission. Viruses show a wide range of different structures, and the transmission of viruses between vertebrate hosts can take place through many different routes. We compiled a database of 243 virus-host combinations and report a statistical analysis that documents the associations between structures and routes of transmission-for example, viruses that are transmitted by the faecal-oral mode of infection are rarely enclosed in a lipid envelope.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41564-019-0523-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6814542PMC
November 2019

CD4 receptor diversity in chimpanzees protects against SIV infection.

Proc Natl Acad Sci U S A 2019 02 4;116(8):3229-3238. Epub 2019 Feb 4.

Sanaga-Yong Chimpanzee Rescue Center, In Defense of Animals-Africa, Portland, OR 97204.

Human and simian immunodeficiency viruses (HIV/SIVs) use CD4 as the primary receptor to enter target cells. Here, we show that the chimpanzee CD4 is highly polymorphic, with nine coding variants present in wild populations, and that this diversity interferes with SIV envelope (Env)-CD4 interactions. Testing the replication fitness of SIVcpz strains in CD4 T cells from captive chimpanzees, we found that certain viruses were unable to infect cells from certain hosts. These differences were recapitulated in CD4 transfection assays, which revealed a strong association between CD4 genotypes and SIVcpz infection phenotypes. The most striking differences were observed for three substitutions (Q25R, Q40R, and P68T), with P68T generating a second N-linked glycosylation site (N66) in addition to an invariant N32 encoded by all chimpanzee CD4 alleles. In silico modeling and site-directed mutagenesis identified charged residues at the CD4-Env interface and clashes between CD4- and Env-encoded glycans as mechanisms of inhibition. CD4 polymorphisms also reduced Env-mediated cell entry of monkey SIVs, which was dependent on at least one D1 domain glycan. CD4 allele frequencies varied among wild chimpanzees, with high diversity in all but the western subspecies, which appeared to have undergone a selective sweep. One allele was associated with lower SIVcpz prevalence rates in the wild. These results indicate that substitutions in the D1 domain of the chimpanzee CD4 can prevent SIV cell entry. Although some SIVcpz strains have adapted to utilize these variants, CD4 diversity is maintained, protecting chimpanzees against infection with SIVcpz and other SIVs to which they are exposed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1821197116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6386711PMC
February 2019

Lack of detection of a human placenta microbiome in samples from preterm and term deliveries.

Microbiome 2018 10 30;6(1):196. Epub 2018 Oct 30.

Department of Microbiology, University of Pennsylvania School of Medicine, 3610 Hamilton Walk, Philadelphia, PA, 19104-6076, USA.

Background: Historically, the human womb has been thought to be sterile in healthy pregnancies, but this idea has been challenged by recent studies using DNA sequence-based methods, which have suggested that the womb is colonized with bacteria. For example, analysis of DNA from placenta samples yielded small proportions of microbial sequences which were proposed to represent normal bacterial colonization. However, an analysis by our group showed no distinction between background negative controls and placenta samples. Also supporting the idea that the womb is sterile is the observation that germ-free mammals can be generated by sterile delivery of neonates into a sterile isolator, after which neonates remain germ-free, which would seem to provide strong data in support of sterility of the womb.

Results: To probe this further and to investigate possible placental colonization associated with spontaneous preterm birth, we carried out another study comparing microbiota in placenta samples from 20 term and 20 spontaneous preterm deliveries. Both 16S rRNA marker gene sequencing and shotgun metagenomic sequencing were used to characterize placenta and control samples. We first quantified absolute amounts of bacterial 16S rRNA gene sequences using 16S rRNA gene quantitative PCR (qPCR). As in our previous study, levels were found to be low in the placenta samples and indistinguishable from negative controls. Analysis by DNA sequencing did not yield a placenta microbiome distinct from negative controls, either using marker gene sequencing as in our previous work, or with shotgun metagenomic sequencing. Several types of artifacts, including erroneous read classifications and barcode misattribution, needed to be identified and removed from the data to clarify this point.

Conclusions: Our findings do not support the existence of a consistent placental microbiome, in either placenta from term deliveries or spontaneous preterm births.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s40168-018-0575-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6208038PMC
October 2018

HIV-1 latent reservoir size and diversity are stable following brief treatment interruption.

J Clin Invest 2018 07 18;128(7):3102-3115. Epub 2018 Jun 18.

University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Background: The effect of a brief analytical treatment interruption (ATI) on the HIV-1 latent reservoir of individuals who initiate antiretroviral therapy (ART) during chronic infection is unknown.

Methods: We evaluated the impact of transient viremia on the latent reservoir in participants who underwent an ATI and at least 6 months of subsequent viral suppression in a clinical trial testing the effect of passive infusion of the broadly neutralizing Ab VRC01 during ATI.

Results: Measures of total HIV-1 DNA, cell-associated RNA, and infectious units per million cells (IUPM) (measured by quantitative viral outgrowth assay [QVOA]) were not statistically different before or after ATI. Phylogenetic analyses of HIV-1 env sequences from QVOA and proviral DNA demonstrated little change in the composition of the virus populations comprising the pre- and post-ATI reservoir. Expanded clones were common in both QVOA and proviral DNA sequences. The frequency of clonal populations differed significantly between QVOA viruses, proviral DNA sequences, and the viruses that reactivated in vivo.

Conclusions: The results indicate that transient viremia from ATI does not substantially alter measures of the latent reservoir, that clonal expansion is prevalent within the latent reservoir, and that characterization of latent viruses that can reactivate in vivo remains challenging.

Trial Registration: ClinicalTrials.gov NCT02463227FUNDING. Funding was provided by the NIH.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI120194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6026010PMC
July 2018

Allometry and Ecology of the Bilaterian Gut Microbiome.

mBio 2018 03 27;9(2). Epub 2018 Mar 27.

Division of Gastroenterology, Hepatology, and Nutrition, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.

Classical ecology provides principles for construction and function of biological communities, but to what extent these apply to the animal-associated microbiota is just beginning to be assessed. Here, we investigated the influence of several well-known ecological principles on animal-associated microbiota by characterizing gut microbial specimens from bilaterally symmetrical animals () ranging from flies to whales. A rigorously vetted sample set containing 265 specimens from 64 species was assembled. Bacterial lineages were characterized by 16S rRNA gene sequencing. Previously published samples were also compared, allowing analysis of over 1,098 samples in total. A restricted number of bacterial phyla was found to account for the great majority of gut colonists. Gut microbial composition was associated with host phylogeny and diet. We identified numerous gut bacterial 16S rRNA gene sequences that diverged deeply from previously studied taxa, identifying opportunities to discover new bacterial types. The number of bacterial lineages per gut sample was positively associated with animal mass, paralleling known species-area relationships from island biogeography and implicating body size as a determinant of community stability and niche complexity. Samples from larger animals harbored greater numbers of anaerobic communities, specifying a mechanism for generating more-complex microbial environments. Predictions for species/abundance relationships from models of neutral colonization did not match the data set, pointing to alternative mechanisms such as selection of specific colonists by environmental niche. Taken together, the data suggest that niche complexity increases with gut size and that niche selection forces dominate gut community construction. The intestinal microbiome of animals is essential for health, contributing to digestion of foods, proper immune development, inhibition of pathogen colonization, and catabolism of xenobiotic compounds. How these communities assemble and persist is just beginning to be investigated. Here we interrogated a set of gut samples from a wide range of animals to investigate the roles of selection and random processes in microbial community construction. We show that the numbers of bacterial species increased with the weight of host organisms, paralleling findings from studies of island biogeography. Communities in larger organisms tended to be more anaerobic, suggesting one mechanism for niche diversification. Nonselective processes enable specific predictions for community structure, but our samples did not match the predictions of the neutral model. Thus, these findings highlight the importance of niche selection in community construction and suggest mechanisms of niche diversification.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/mBio.00319-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5874926PMC
March 2018

Wild bonobos host geographically restricted malaria parasites including a putative new Laverania species.

Nat Commun 2017 11 21;8(1):1635. Epub 2017 Nov 21.

Department of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.

Malaria parasites, though widespread among wild chimpanzees and gorillas, have not been detected in bonobos. Here, we show that wild-living bonobos are endemically Plasmodium infected in the eastern-most part of their range. Testing 1556 faecal samples from 11 field sites, we identify high prevalence Laverania infections in the Tshuapa-Lomami-Lualaba (TL2) area, but not at other locations across the Congo. TL2 bonobos harbour P. gaboni, formerly only found in chimpanzees, as well as a potential new species, Plasmodium lomamiensis sp. nov. Rare co-infections with non-Laverania parasites were also observed. Phylogenetic relationships among Laverania species are consistent with co-divergence with their gorilla, chimpanzee and bonobo hosts, suggesting a timescale for their evolution. The absence of Plasmodium from most field sites could not be explained by parasite seasonality, nor by bonobo population structure, diet or gut microbiota. Thus, the geographic restriction of bonobo Plasmodium reflects still unidentified factors that likely influence parasite transmission.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-017-01798-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5696340PMC
November 2017

Guidelines for Genome-Scale Analysis of Biological Rhythms.

J Biol Rhythms 2017 Oct 3;32(5):380-393. Epub 2017 Nov 3.

5 Center for Integrative Genomics, Génopode, University of Lausanne, Lausanne, Switzerland.

Genome biology approaches have made enormous contributions to our understanding of biological rhythms, particularly in identifying outputs of the clock, including RNAs, proteins, and metabolites, whose abundance oscillates throughout the day. These methods hold significant promise for future discovery, particularly when combined with computational modeling. However, genome-scale experiments are costly and laborious, yielding "big data" that are conceptually and statistically difficult to analyze. There is no obvious consensus regarding design or analysis. Here we discuss the relevant technical considerations to generate reproducible, statistically sound, and broadly useful genome-scale data. Rather than suggest a set of rigid rules, we aim to codify principles by which investigators, reviewers, and readers of the primary literature can evaluate the suitability of different experimental designs for measuring different aspects of biological rhythms. We introduce CircaInSilico, a web-based application for generating synthetic genome biology data to benchmark statistical methods for studying biological rhythms. Finally, we discuss several unmet analytical needs, including applications to clinical medicine, and suggest productive avenues to address them.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1177/0748730417728663DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5692188PMC
October 2017

Optimizing methods and dodging pitfalls in microbiome research.

Microbiome 2017 05 5;5(1):52. Epub 2017 May 5.

Division of Gastroenterology, Hepatology, and Nutrition, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, 19104, USA.

Research on the human microbiome has yielded numerous insights into health and disease, but also has resulted in a wealth of experimental artifacts. Here, we present suggestions for optimizing experimental design and avoiding known pitfalls, organized in the typical order in which studies are carried out. We first review best practices in experimental design and introduce common confounders such as age, diet, antibiotic use, pet ownership, longitudinal instability, and microbial sharing during cohousing in animal studies. Typically, samples will need to be stored, so we provide data on best practices for several sample types. We then discuss design and analysis of positive and negative controls, which should always be run with experimental samples. We introduce a convenient set of non-biological DNA sequences that can be useful as positive controls for high-volume analysis. Careful analysis of negative and positive controls is particularly important in studies of samples with low microbial biomass, where contamination can comprise most or all of a sample. Lastly, we summarize approaches to enhancing experimental robustness by careful control of multiple comparisons and to comparing discovery and validation cohorts. We hope the experimental tactics summarized here will help researchers in this exciting field advance their studies efficiently while avoiding errors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s40168-017-0267-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5420141PMC
May 2017

Associations of the vaginal microbiota with HIV infection, bacterial vaginosis, and demographic factors.

AIDS 2017 04;31(7):895-904

aDepartment of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania bDepartment of Cellular and Molecular Biology, Feinberg School of Medicine, Northwestern University cCORE Center, Stroger Hospital of Cook County, Chicago, Illinois, USA.

Objective: We sought to investigate the effects of HIV infection on the vaginal microbiota and associations with treatment and demographic factors. We thus compared vaginal microbiome samples from HIV-infected (HIV+) and HIV-uninfected (HIV-) women collected at two Chicago area hospitals.

Design: We studied vaginal microbiome samples from 178 women analyzed longitudinally (n = 324 samples) and collected extensive data on clinical status and demographic factors.

Methods: We used 16S rRNA gene sequencing to characterize the bacterial lineages present, then UniFrac, Shannon diversity, and other measures to compare community structure with sample metadata.

Results: Differences in microbiota measures were modest in the comparison of HIV+ and HIV- samples, in contrast to several previous studies, consistent with effective antiretroviral therapy. Proportions of healthy Lactobacillus species were not higher in HIV- patients overall, but were significantly higher when analyzed within each hospital in isolation. Rates of bacterial vaginosis were higher among African-American women and HIV+ women. Bacterial vaginosis was associated with higher frequency of HIV+. Unexpectedly, African-American women were more likely to switch bacterial vaginosis status between sampling times; switching was not associated with HIV+ status.

Conclusion: The influence of HIV infection on the vaginal microbiome was modest for this cohort of well suppressed urban American women, consistent with effective antiretroviral therapy. HIV+ was found to be associated with bacterial vaginosis. Although bacterial vaginosis has previously been associated with HIV transmission, most of the women studied here became HIV+ many years before our test for bacterial vaginosis, thus implicating additional mechanisms linking HIV infection and bacterial vaginosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/QAD.0000000000001421DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5370567PMC
April 2017

Resistance to type 1 interferons is a major determinant of HIV-1 transmission fitness.

Proc Natl Acad Sci U S A 2017 01 9;114(4):E590-E599. Epub 2017 Jan 9.

Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104;

Sexual transmission of HIV-1 is an inefficient process, with only one or few variants of the donor quasispecies establishing the new infection. A critical, and as yet unresolved, question is whether the mucosal bottleneck selects for viruses with increased transmission fitness. Here, we characterized 300 limiting dilution-derived virus isolates from the plasma, and in some instances genital secretions, of eight HIV-1 donor and recipient pairs. Although there were no differences in the amount of virion-associated envelope glycoprotein, recipient isolates were on average threefold more infectious (P = 0.0001), replicated to 1.4-fold higher titers (P = 0.004), were released from infected cells 4.2-fold more efficiently (P < 0.00001), and were significantly more resistant to type I IFNs than the corresponding donor isolates. Remarkably, transmitted viruses exhibited 7.8-fold higher IFNα2 (P < 0.00001) and 39-fold higher IFNβ (P < 0.00001) half-maximal inhibitory concentrations (IC) than did donor isolates, and their odds of replicating in CD4 T cells at the highest IFNα2 and IFNβ doses were 35-fold (P < 0.00001) and 250-fold (P < 0.00001) greater, respectively. Interestingly, pretreatment of CD4 T cells with IFNβ, but not IFNα2, selected donor plasma isolates that exhibited a transmitted virus-like phenotype, and such viruses were also detected in the donor genital tract. These data indicate that transmitted viruses are phenotypically distinct, and that increased IFN resistance represents their most distinguishing property. Thus, the mucosal bottleneck selects for viruses that are able to replicate and spread efficiently in the face of a potent innate immune response.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1620144114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5278458PMC
January 2017

Structural Basis for Inhibitor-Induced Aggregation of HIV Integrase.

PLoS Biol 2016 Dec 9;14(12):e1002584. Epub 2016 Dec 9.

Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

The allosteric inhibitors of integrase (termed ALLINIs) interfere with HIV replication by binding to the viral-encoded integrase (IN) protein. Surprisingly, ALLINIs interfere not with DNA integration but with viral particle assembly late during HIV replication. To investigate the ALLINI inhibitory mechanism, we crystallized full-length HIV-1 IN bound to the ALLINI GSK1264 and determined the structure of the complex at 4.4 Å resolution. The structure shows GSK1264 buried between the IN C-terminal domain (CTD) and the catalytic core domain. In the crystal lattice, the interacting domains are contributed by two different dimers so that IN forms an open polymer mediated by inhibitor-bridged contacts; the N-terminal domains do not participate and are structurally disordered. Engineered amino acid substitutions at the inhibitor interface blocked ALLINI-induced multimerization. HIV escape mutants with reduced sensitivity to ALLINIs commonly altered amino acids at or near the inhibitor-bound interface, and these substitutions also diminished IN multimerization. We propose that ALLINIs inhibit particle assembly by stimulating inappropriate polymerization of IN via interactions between the catalytic core domain and the CTD and that understanding the interface involved offers new routes to inhibitor optimization.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pbio.1002584DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5147827PMC
December 2016

Comparison of placenta samples with contamination controls does not provide evidence for a distinct placenta microbiota.

Microbiome 2016 Jun 23;4(1):29. Epub 2016 Jun 23.

Department of Microbiology, Perelman School of Medicine at the University of Pennsylvania, 3610 Hamilton Walk, Philadelphia, PA, 19104-6076, USA.

Background: Recent studies have suggested that bacteria associated with the placenta-a "placental microbiome"-may be important in reproductive health and disease. However, a challenge in working with specimens with low bacterial biomass, such as placental samples, is that some or all of the bacterial DNA may derive from contamination in dust or commercial reagents. To investigate this, we compared placental samples from healthy deliveries to a matched set of contamination controls, as well as to oral and vaginal samples from the same women.

Results: We quantified total 16S rRNA gene copies using quantitative PCR and found that placental samples and negative controls contained low and indistinguishable copy numbers. Oral and vaginal swab samples, in contrast, showed higher copy numbers. We carried out 16S rRNA gene sequencing and community analysis and found no separation between communities from placental samples and contamination controls, though oral and vaginal samples showed characteristic, distinctive composition. Two different DNA purification methods were compared with similar conclusions, though the composition of the contamination background differed. Authentically present microbiota should yield mostly similar results regardless of the purification method used-this was seen for oral samples, but no placental bacterial lineages were (1) shared between extraction methods, (2) present at >1 % of the total, and (3) present at greater abundance in placental samples than contamination controls.

Conclusions: We conclude that for this sample set, using the methods described, we could not distinguish between placental samples and contamination introduced during DNA purification.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s40168-016-0172-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4917942PMC
June 2016

Gene activity in primary T cells infected with HIV89.6: intron retention and induction of genomic repeats.

Retrovirology 2015 Sep 17;12:79. Epub 2015 Sep 17.

Department of Microbiology, Perelman School of Medicine at the University of Pennsylvania, 425 Johnson Pavilion, 3610 Hamilton Walk, Philadelphia, PA, 19104, USA.

Background: HIV infection has been reported to alter cellular gene activity, but published studies have commonly assayed transformed cell lines and lab-adapted HIV strains, yielding inconsistent results. Here we carried out a deep RNA-Seq analysis of primary human T cells infected with the low passage HIV isolate HIV89.6.

Results: Seventeen percent of cellular genes showed altered activity 48 h after infection. In a meta-analysis including four other studies, our data differed from studies of HIV infection in cell lines but showed more parallels with infections of primary cells. We found a global trend toward retention of introns after infection, suggestive of a novel cellular response to infection. HIV89.6 infection was also associated with activation of several human endogenous retroviruses (HERVs) and retrotransposons, of interest as possible novel antigens that could serve as vaccine targets. The most highly activated group of HERVs was a subset of the ERV-9. Analysis showed that activation was associated with a particular variant of ERV-9 long terminal repeats that contains an indel near the U3-R border. These data also allowed quantification of >70 splice forms of the HIV89.6 RNA and specified the main types of chimeric HIV89.6-host RNAs. Comparison to over 100,000 integration site sequences from the same infected cell populations allowed quantification of authentic versus artifactual chimeric reads, showing that 5' read-in, splicing out of HIV89.6 from the D4 donor and 3' read-through were the most common HIV89.6-host cell chimeric RNA forms.

Conclusions: Analysis of RNA abundance after infection of primary T cells with the low passage HIV89.6 isolate disclosed multiple novel features of HIV-host interactions, notably intron retention and induction of transcription of retrotransposons and endogenous retroviruses.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12977-015-0205-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4574318PMC
September 2015

Covalent Modification of Bacteriophage T4 DNA Inhibits CRISPR-Cas9.

mBio 2015 Jun 16;6(3):e00648. Epub 2015 Jun 16.

Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA

Unlabelled: The genomic DNAs of tailed bacteriophages are commonly modified by the attachment of chemical groups. Some forms of DNA modification are known to protect phage DNA from cleavage by restriction enzymes, but others are of unknown function. Recently, the CRISPR-Cas nuclease complexes were shown to mediate bacterial adaptive immunity by RNA-guided target recognition, raising the question of whether phage DNA modifications may also block attack by CRISPR-Cas9. We investigated phage T4 as a model system, where cytosine is replaced with glucosyl-hydroxymethylcytosine (glc-HMC). We first quantified the extent and distribution of covalent modifications in T4 DNA by single-molecule DNA sequencing and enzymatic probing. We then designed CRISPR spacer sequences targeting T4 and found that wild-type T4 containing glc-HMC was insensitive to attack by CRISPR-Cas9 but mutants with unmodified cytosine were sensitive. Phage with HMC showed only intermediate sensitivity. While this work was in progress, another group reported examples of heavily engineered CRISRP-Cas9 complexes that could, in fact, overcome the effects of T4 DNA modification, indicating that modifications can inhibit but do not always fully block attack.

Importance: Bacteria were recently found to have a form of adaptive immunity, the CRISPR-Cas systems, which use nucleic acid pairing to recognize and cleave genomic DNA of invaders such as bacteriophage. Historic work with tailed phages has shown that phage DNA is often modified by covalent attachment of large chemical groups. Here we demonstrate that DNA modification in phage T4 inhibits attack by the CRISPR-Cas9 system. This finding provides insight into mechanisms of host-virus competition and also a new set of tools that may be useful in modulating the activity of CRISPR-Cas9 in genome engineering applications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/mBio.00648-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4471564PMC
June 2015

Power and sample-size estimation for microbiome studies using pairwise distances and PERMANOVA.

Bioinformatics 2015 Aug 29;31(15):2461-8. Epub 2015 Mar 29.

Department of Biostatistics and Epidemiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.

Motivation: The variation in community composition between microbiome samples, termed beta diversity, can be measured by pairwise distance based on either presence-absence or quantitative species abundance data. PERMANOVA, a permutation-based extension of multivariate analysis of variance to a matrix of pairwise distances, partitions within-group and between-group distances to permit assessment of the effect of an exposure or intervention (grouping factor) upon the sampled microbiome. Within-group distance and exposure/intervention effect size must be accurately modeled to estimate statistical power for a microbiome study that will be analyzed with pairwise distances and PERMANOVA.

Results: We present a framework for PERMANOVA power estimation tailored to marker-gene microbiome studies that will be analyzed by pairwise distances, which includes: (i) a novel method for distance matrix simulation that permits modeling of within-group pairwise distances according to pre-specified population parameters; (ii) a method to incorporate effects of different sizes within the simulated distance matrix; (iii) a simulation-based method for estimating PERMANOVA power from simulated distance matrices; and (iv) an R statistical software package that implements the above. Matrices of pairwise distances can be efficiently simulated to satisfy the triangle inequality and incorporate group-level effects, which are quantified by the adjusted coefficient of determination, omega-squared (ω2). From simulated distance matrices, available PERMANOVA power or necessary sample size can be estimated for a planned microbiome study.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/bioinformatics/btv183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4514928PMC
August 2015

A reverse transcription loop-mediated isothermal amplification assay optimized to detect multiple HIV subtypes.

PLoS One 2015 12;10(2):e0117852. Epub 2015 Feb 12.

Department of Microbiology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

Diagnostic methods for detecting and quantifying HIV RNA have been improving, but efficient methods for point-of-care analysis are still needed, particularly for applications in resource-limited settings. Detection based on reverse-transcription loop-mediated isothermal amplification (RT-LAMP) is particularly useful for this, because when combined with fluorescence-based DNA detection, RT-LAMP can be implemented with minimal equipment and expense. Assays have been developed to detect HIV RNA with RT-LAMP, but existing methods detect only a limited subset of HIV subtypes. Here we report a bioinformatic study to develop optimized primers, followed by empirical testing of 44 new primer designs. One primer set (ACeIN-26), targeting the HIV integrase coding region, consistently detected subtypes A, B, C, D, and G. The assay was sensitive to at least 5000 copies per reaction for subtypes A, B, C, D, and G, with Z-factors of above 0.69 (detection of the minor subtype F was found to be unreliable). There are already rapid and efficient assays available for detecting HIV infection in a binary yes/no format, but the rapid RT-LAMP assay described here has additional uses, including 1) tracking response to medication by comparing longitudinal values for a subject, 2) detecting of infection in neonates unimpeded by the presence of maternal antibody, and 3) detecting infection prior to seroconversion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0117852PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4326360PMC
November 2015

Phylogeography and molecular epidemiology of an epidemic strain of dengue virus type 1 in Sri Lanka.

Am J Trop Med Hyg 2014 Aug 5;91(2):225-34. Epub 2014 May 5.

University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania; Genetech Research Institute, Colombo, Sri Lanka; North Colombo Teaching Hospital, Ragama, Sri Lanka; Department of Zoology, University of Colombo, Colombo, Sri Lanka; Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, California

In 2009, a severe epidemic of dengue disease occurred in Sri Lanka, with higher mortality and morbidity than any previously recorded epidemic in the country. It corresponded to a shift to dengue virus 1 as the major disease-causing serotype in Sri Lanka. Dengue disease reached epidemic levels in the next 3 years. We report phylogenetic evidence that the 2009 epidemic DENV-1 strain continued to circulate within the population and caused severe disease in the epidemic of 2012. Bayesian phylogeographic analyses suggest that the 2009 Sri Lankan epidemic DENV-1 strain may have traveled directly or indirectly from Thailand through China to Sri Lanka, and after spreading within the Sri Lankan population, it traveled to Pakistan and Singapore. Our findings delineate the dissemination route of a virulent DENV-1 strain in Asia. Understanding such routes will be of particular importance to global control efforts.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4269/ajtmh.13-0523DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4125241PMC
August 2014

HIV latency and integration site placement in five cell-based models.

Retrovirology 2013 Aug 16;10:90. Epub 2013 Aug 16.

Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA.

Background: HIV infection can be treated effectively with antiretroviral agents, but the persistence of a latent reservoir of integrated proviruses prevents eradication of HIV from infected individuals. The chromosomal environment of integrated proviruses has been proposed to influence HIV latency, but the determinants of transcriptional repression have not been fully clarified, and it is unclear whether the same molecular mechanisms drive latency in different cell culture models.

Results: Here we compare data from five different in vitro models of latency based on primary human T cells or a T cell line. Cells were infected in vitro and separated into fractions containing proviruses that were either expressed or silent/inducible, and integration site populations sequenced from each. We compared the locations of 6,252 expressed proviruses to those of 6,184 silent/inducible proviruses with respect to 140 forms of genomic annotation, many analyzed over chromosomal intervals of multiple lengths. A regularized logistic regression model linking proviral expression status to genomic features revealed no predictors of latency that performed better than chance, though several genomic features were significantly associated with proviral expression in individual models. Proviruses in the same chromosomal region did tend to share the same expressed or silent/inducible status if they were from the same cell culture model, but not if they were from different models.

Conclusions: The silent/inducible phenotype appears to be associated with chromosomal position, but the molecular basis is not fully clarified and may differ among in vitro models of latency.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1742-4690-10-90DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3765678PMC
August 2013

Plasmodium falciparum-like parasites infecting wild apes in southern Cameroon do not represent a recurrent source of human malaria.

Proc Natl Acad Sci U S A 2013 Apr 8;110(17):7020-5. Epub 2013 Apr 8.

Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

Wild-living chimpanzees and gorillas harbor a multitude of Plasmodium species, including six of the subgenus Laverania, one of which served as the progenitor of Plasmodium falciparum. Despite the magnitude of this reservoir, it is unknown whether apes represent a source of human infections. Here, we used Plasmodium species-specific PCR, single-genome amplification, and 454 sequencing to screen humans from remote areas of southern Cameroon for ape Laverania infections. Among 1,402 blood samples, we found 1,000 to be Plasmodium mitochondrial DNA (mtDNA) positive, all of which contained human parasites as determined by sequencing and/or restriction enzyme digestion. To exclude low-abundance infections, we subjected 514 of these samples to 454 sequencing, targeting a region of the mtDNA genome that distinguishes ape from human Laverania species. Using algorithms specifically developed to differentiate rare Plasmodium variants from 454-sequencing error, we identified single and mixed-species infections with P. falciparum, Plasmodium malariae, and/or Plasmodium ovale. However, none of the human samples contained ape Laverania parasites, including the gorilla precursor of P. falciparum. To characterize further the diversity of P. falciparum in Cameroon, we used single-genome amplification to amplify 3.4-kb mtDNA fragments from 229 infected humans. Phylogenetic analysis identified 62 new variants, all of which clustered with extant P. falciparum, providing further evidence that P. falciparum emerged following a single gorilla-to-human transmission. Thus, unlike Plasmodium knowlesi-infected macaques in southeast Asia, African apes harboring Laverania parasites do not seem to serve as a recurrent source of human malaria, a finding of import to ongoing control and eradication measures.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1305201110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3637760PMC
April 2013
-->