scholarly journals Single genome amplification and direct amplicon sequencing of Plasmodium spp. DNA from ape fecal specimens

2010 ◽  
Author(s):  
Weimin Liu
Keyword(s):  
Amplicon Sequencing ◽  
Single Genome Amplification ◽  
Genome Amplification ◽  
Single Genome

scholarly journals The efficiency of single genome amplification and sequencing is improved by quantitation and use of a bioinformatics tool

2009 ◽  
Vol 162 (1-2) ◽  
pp. 280-283 ◽  
Author(s):  
David M. Butler ◽  
Mary E. Pacold ◽  
Parris S. Jordan ◽  
Douglas D. Richman ◽  
Davey M. Smith
Keyword(s):  
Bioinformatics Tool ◽  
Single Genome Amplification ◽  
Genome Amplification ◽  
Single Genome

scholarly journals Drug Resistance Mutation Frequency of Single-Genome Amplification-Derived HIV-1 Polymerase Genomes in the Cerebrospinal Fluid and Plasma of HIV-1-Infected Individuals under Nonsuppressive Therapy

2020 ◽  
Vol 94 (20) ◽  
Author(s):  
Leslie St. Bernard ◽  
Jeremy Abolade ◽  
Hiroshi Mohri ◽  
Martin Markowitz ◽  
Teresa H. Evering
Keyword(s):  
Drug Resistance ◽  
Cerebrospinal Fluid ◽  
Antiretroviral Therapy ◽  
Longitudinal Analyses ◽  
Resistance Mutations ◽  
Single Genome Amplification ◽  
Genome Amplification ◽  
Drug Resistance Mutations ◽  
Single Genome ◽  
Hiv 1

ABSTRACT HIV-1 evolution in the cerebrospinal fluid (CSF) and plasma may result in discordant drug resistance mutations (DRMs) in the compartments. Single-genome amplification (SGA) was used to generate partial HIV-1 polymerase genomes in paired CSF and plasma samples from 12 HIV-1-positive participants in the CNS HIV Antiretroviral Therapy Effects Research (CHARTER) study who were classified as neurocognitively unimpaired or with various degrees of HIV-associated neurocognitive disorders (HAND). Subjects were viremic on combination antiretroviral therapy (cART). HIV-1 DRMs and phylogenetic characteristics were determined using the Stanford HIVdb program and phylogenetic analyses. Individual DRMs were identified more frequently in plasma than in paired CSF (P = 0.0078). Significant differences in the ratios of DRMs in CSF and plasma were found in 3 individuals with HAND (3/7 = 43%). Two HAND subjects (2/7 = 29%) demonstrated one DRM in CSF not identified in paired plasma. Longitudinal analyses (n = 4) revealed significant temporal differences in the ratios of DRMs in the compartments. Statistically significant differences in the frequency of DRMs in the CSF and plasma are readily found in those on nonsuppressive cART. While compartment-based DRM discordance was largely consistent with increased drug-selective pressures in the plasma, overrepresentation of DRMs in the central nervous system (CNS) can occur. Underlying mechanisms of HAND are complex and multifactorial. The clinical impact of DRM discordance on viral persistence and HAND pathogenesis remains unclear and warrants further investigation in larger, longitudinal cohorts. IMPORTANCE Several antiretroviral agents do not efficiently enter the CNS, and independent evolution of HIV-1 viral variants in the CNS and plasma can occur. We used single-genome amplification (SGA) in cross-sectional and longitudinal analyses to uniquely define both the identity and relative proportions of drug resistance mutations (DRMs) on individual HIV-1 polymerase genomes in the cerebrospinal fluid (CSF) and plasma in individuals with incomplete viral suppression and known neurocognitive status. Statistically significant differences in the ratio of DRMs in the CSF and plasma were readily found in those on nonsuppressive cART, and overrepresentation of DRMs in the CNS can occur. Although questions about the clinical significance of DRM discordance remain, in the quest for viral eradication, it is important to recognize that a significant, dynamic, compartment-based DRM ratio imbalance can exist, as it has the potential to go unnoticed in the setting of standard clinical drug resistance testing.

scholarly journals Tracking the culprit: HIV-1 evolution and immune selection revealed by single-genome amplification

2009 ◽  
Vol 206 (6) ◽  
pp. 1215-1218 ◽  
Author(s):  
Zabrina L. Brumme ◽  
Bruce D. Walker
Keyword(s):  
Immune Response ◽  
Hiv Infection ◽  
Cell Response ◽  
Cd8 T Cell ◽  
T Cell Response ◽  
Immune Selection ◽  
Single Genome Amplification ◽  
Genome Amplification ◽  
Single Genome ◽  
Hiv 1

Early control of HIV-1 infection is determined by a balance between the host immune response and the ability of the virus to escape this response. Studies using single-genome amplification now reveal new details about the kinetics and specificity of the CD8+ T cell response and the evolution of the virus during early HIV infection.

scholarly journals Deciphering Human Immunodeficiency Virus Type 1 Transmission and Early Envelope Diversification by Single-Genome Amplification and Sequencing

2008 ◽  
Vol 82 (8) ◽  
pp. 3952-3970 ◽  
Author(s):  
Jesus F. Salazar-Gonzalez ◽  
Elizabeth Bailes ◽  
Kimmy T. Pham ◽  
Maria G. Salazar ◽  
M. Brad Guffey ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Immune Escape ◽  
Single Genome Amplification ◽  
Genome Amplification ◽  
Single Genome ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Accurate identification of the transmitted virus and sequences evolving from it could be instrumental in elucidating the transmission of human immunodeficiency virus type 1 (HIV-1) and in developing vaccines, drugs, or microbicides to prevent infection. Here we describe an experimental approach to analyze HIV-1 env genes as intact genetic units amplified from plasma virion RNA by single-genome amplification (SGA), followed by direct sequencing of uncloned DNA amplicons. We show that this strategy precludes in vitro artifacts caused by Taq-induced nucleotide substitutions and template switching, provides an accurate representation of the env quasispecies in vivo, and has an overall error rate (including nucleotide misincorporation, insertion, and deletion) of less than 8 × 10−5. Applying this method to the analysis of virus in plasma from 12 Zambian subjects from whom samples were obtained within 3 months of seroconversion, we show that transmitted or early founder viruses can be identified and that molecular pathways and rates of early env diversification can be defined. Specifically, we show that 8 of the 12 subjects were each infected by a single virus, while 4 others acquired more than one virus; that the rate of virus evolution in one subject during an 80-day period spanning seroconversion was 1.7 × 10−5 substitutions per site per day; and that evidence of strong immunologic selection can be seen in Env and overlapping Rev sequences based on nonrandom accumulation of nonsynonymous mutations. We also compared the results of the SGA approach with those of more-conventional bulk PCR amplification methods performed on the same patient samples and found that the latter is associated with excessive rates of Taq-induced recombination, nucleotide misincorporation, template resampling, and cloning bias. These findings indicate that HIV-1 env genes, other viral genes, and even full-length viral genomes responsible for productive clinical infection can be identified by SGA analysis of plasma virus sampled at intervals typical in large-scale vaccine trials and that pathways of viral diversification and immune escape can be determined accurately.

scholarly journals Infant Transmitted/Founder HIV-1 Viruses from Peripartum Transmission are Neutralization Resistant to Paired Maternal Plasma

2017 ◽  
Author(s):  
Amit Kumar ◽  
Claire E. P. Smith ◽  
Elena E. Giorgi ◽  
Joshua Eudailey ◽  
David R. Martinez ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Maternal Plasma ◽  
Pediatric Hiv ◽  
Viral Population ◽  
Single Genome Amplification ◽  
Genome Amplification ◽  
Implementation Challenges ◽  
Amplification Method ◽  
Single Genome ◽  
Hiv 1

AbstractDespite extensive genetic diversity of HIV-1 in chronic infection, infant HIV-1 infection involves selective transmission of a single or few maternal virus variants. These transmitted/founder (T/F) variants are of particular interest, as a maternal or infant HIV vaccine should raise envelope (Env)-specific IgG responses capable of blocking this group of viruses. However, the maternal or infant factors that contribute to selection of infant T/F viruses are not well understood. In this study, we isolated HIV-1envgenes by single genome amplification from 16 mother-infant transmitting pairs from the U.S. pre-antiretroviral era Women Infant Transmission Study (WITS). Infant T/F and representative maternal non-transmitted Env variants from plasma were identified and used to generate pseudoviruses for paired maternal plasma neutralization sensitivity analysis. Eighteen out of 21 (85%) infant T/F Env pseudoviruses were neutralization resistant to paired maternal plasma. Yet, all infant T/F viruses were neutralization sensitive to a panel of HIV-1 broadly neutralizing antibodies and variably sensitive to heterologous plasma neutralizing antibodies. Moreover, infant T/F pseudoviruses were overall more neutralization resistant compared to maternal non-transmitted plasma variants (p=0.012). Altogether, our findings suggest that autologous neutralization of circulating viruses by maternal plasma antibodies select for neutralization-resistant viruses that initiate peripartum transmission, raising the spector that enhancement of this response at the end of pregnancy could further reduce infant HIV infection risk.Author SummaryMother to child transmission (MTCT) of HIV-1 can occur during pregnancy (in utero), at the time of delivery (peripartum) or by breastfeeding (postpartum). With the availability of anti-retroviral therapy (ART), rate of MTCT of HIV-1 have been significantly lowered. However, significant implementation challenges remains in resource-poor areas, making it difficult to eliminate pediatric HIV. An improved understanding of the viral population (escape variants from autologous neutralizing antibodies) that lead to infection of infants at time of transmission will help in designing immune interventions to reduce vertical HIV-1 transmission. Here, we selected 16 HIV-1-infected mother-infant pairs from WITS cohort (from pre anti-retroviral era), where infants became infected peripartum. HIV-1envgene sequences were obtained by the single genome amplification method. The sensitivity of these infant Env pseudoviruses against paired maternal plasma and a panel of broadly neutralizing monoclonal antibodies (bNAbs) was analyzed. We demonstrated that the infant T/F viruses were more resistant against maternal plasma than non-transmitted maternal variants, but sensitive to most (bNAbs). Signature sequence analysis of infant T/F and non-transmitted maternal variants revealed the potential importance of V3 and MPER region for resistance against to paired maternal plasma. These findings provide insights for the design of maternal immunization strategies to enhance neutralizing antibodies that target V3 region of autologous virus populations, which could work synergistically with maternal ARVs to further reduce the rate of peripartum HIV-1 transmission.

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