scholarly journals Population Genetic Analysis of the Protease Locus of Human Immunodeficiency Virus Type 1 Quasispecies Undergoing Drug Selection, Using a Denaturing Gradient-Heteroduplex Tracking Assay

2001 ◽  
Vol 75 (14) ◽  
pp. 6729-6736 ◽  
Author(s):  
Laurence Doukhan ◽  
Eric Delwart
Keyword(s):  
Genetic Diversity ◽  
Genetic Variants ◽  
Population Genetic ◽  
Protease Gene ◽  
Plasma Viremia ◽  
Viral Quasispecies ◽  
Immunodeficiency Virus ◽  
Pcr Products ◽  
Hiv 1

ABSTRACT Monitoring the evolution of human immunodeficiency virus type 1 (HIV-1) drug resistance requires measuring the frequency of closely related genetic variants making up the complex viral quasispecies found in vivo. In order to resolve both major and minor (≥2%) protease gene variants differing by one or more nucleotide substitutions, we analyzed PCR products derived from plasma viral quasispecies by using a combination of denaturing gradient gel electrophoresis and DNA heteroduplex tracking assays. Correct population sampling of the high level of genetic diversity present within viral quasispecies could be documented by parallel analysis of duplicate, independently generated PCR products. The composition of genetically complex protease gene quasispecies remained constant over short periods of time in the absence of treatment and while plasma viremia fell >100-fold following the initiation of protease inhibitor ritonavir monotherapy. Within a month of initiating therapy, a strong reduction in the genetic diversity of plasma viral populations at the selected protease locus was associated with rising plasma viremia and the emergence of drug resistance. The high levels of protease genetic diversity seen before treatment reemerged only months later. In one patient, reduction in genetic diversity at the protease gene was observed concomitantly with an increase in diversity at the envelope gene (E. L. Delwart, P. Heng, A. Neumann, and M. Markowitz, J. Virol. 72:2416-2421, 1998), indicating that opposite population genetic changes can take place in different HIV-1 loci. The rapid emergence of drug-resistant HIV-1 was therefore associated with a strong, although only transient, reduction in genetic diversity at the selected locus. The denaturing gradient-heteroduplex tracking assay is a simple method for the separation and quantitation of very closely related, low-frequency, genetic variants within complex viral populations.

scholarly journals Induction of a Striking Systemic Cytokine Cascade prior to Peak Viremia in Acute Human Immunodeficiency Virus Type 1 Infection, in Contrast to More Modest and Delayed Responses in Acute Hepatitis B and C Virus Infections

2009 ◽  
Vol 83 (8) ◽  
pp. 3719-3733 ◽  
Author(s):  
Andrea R. Stacey ◽  
Philip J. Norris ◽  
Li Qin ◽  
Elizabeth A. Haygreen ◽  
Elizabeth Taylor ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Hepatitis B ◽  
Human Immunodeficiency Virus Type ◽  
Plasma Viremia ◽  
Cytokines And Chemokines ◽  
Immunodeficiency Virus ◽  
Acute Hiv ◽  
Cytokine Cascade ◽  
Hiv 1

ABSTRACT Characterization of the immune responses induced in the initial stages of human immunodeficiency virus type 1 (HIV-1) infection is of critical importance for an understanding of early viral pathogenesis and prophylactic vaccine design. Here, we used sequential plasma samples collected during the eclipse and exponential viral expansion phases from subjects acquiring HIV-1 (or, for comparison, hepatitis B virus [HBV]or hepatitis C virus [HCV]) to determine the nature and kinetics of the earliest systemic elevations in cytokine and chemokine levels in each infection. Plasma viremia was quantitated over time, and levels of 30 cytokines and chemokines were measured using Luminex-based multiplex assays and enzyme-linked immunosorbent assays. The increase in plasma viremia in acute HIV-1 infection was found to be associated with elevations in plasma levels of multiple cytokines and chemokines, including rapid and transient elevations in alpha interferon (IFN-α) and interleukin-15 (IL-15) levels; a large increase in inducible protein 10 (IP-10) levels; rapid and more-sustained increases in tumor necrosis factor alpha and monocyte chemotactic protein 1 levels; more slowly initiated elevations in levels of additional proinflammatory factors including IL-6, IL-8, IL-18, and IFN-γ; and a late-peaking increase in levels of the immunoregulatory cytokine IL-10. Notably, there was comparatively little perturbation in plasma cytokine levels during the same phase of HBV infection and a delayed response of more intermediate magnitude in acute HCV infection, indicating that the rapid activation of a striking systemic cytokine cascade is not a prerequisite for viral clearance (which occurs in a majority of HBV-infected individuals). The intense early cytokine storm in acute HIV-1 infection may have immunopathological consequences, promoting immune activation, viral replication, and CD4+ T-cell loss.

scholarly journals Changes in Human Immunodeficiency Virus Type 1 Fitness and Genetic Diversity during Disease Progression

2005 ◽  
Vol 79 (14) ◽  
pp. 9006-9018 ◽  
Author(s):  
Ryan M. Troyer ◽  
Kalonji R. Collins ◽  
Awet Abraha ◽  
Erika Fraundorf ◽  
Dawn M. Moore ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Human Immunodeficiency Virus ◽  
Disease Progression ◽  
Human Immunodeficiency Virus Type ◽  
Ex Vivo ◽  
Relative Fitness ◽  
Viral Diversity ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT This study examined the relationship between ex vivo human immunodeficiency virus type 1 (HIV-1) fitness and viral genetic diversity during the course of HIV-1 disease. Primary HIV-1 isolates from 10 patients at different time points were competed against control HIV-1 strains in peripheral blood mononuclear cell (PBMC) cultures to determine relative fitness values. Patient HIV-1 isolates sequentially gained fitness during disease at a significant rate that directly correlated with viral load and HIV-1 env C2V3 diversity. A loss in both fitness and viral diversity was observed upon the initiation of antiretroviral therapy. A possible relationship between genotype and phenotype (virus replication efficiency) is supported by the parallel increases in ex vivo fitness and viral diversity during disease, of which the correlation is largely based on specific V3 sequences. Syncytium-inducing, CXCR4-tropic HIV-1 isolates did have higher relative fitness values than non-syncytium-inducing, CCR5-tropic HIV-1 isolates, as determined by dual virus competitions in PBMC, but increases in fitness during disease were not solely powered by a gradual switch in coreceptor usage. These data provide in vivo evidence that increasing HIV-1 replication efficiency may be related to a concomitant increase in HIV-1 diversity, which in turn may be a determining factor in disease progression.

scholarly journals Human Immunodeficiency Virus Type 1 Monoclonal Antibodies Suppress Acute Simian-Human Immunodeficiency Virus Viremia and Limit Seeding of Cell-Associated Viral Reservoirs

2015 ◽  
Vol 90 (3) ◽  
pp. 1321-1332 ◽  
Author(s):  
Diane L. Bolton ◽  
Amarendra Pegu ◽  
Keyun Wang ◽  
Kathleen McGinnis ◽  
Martha Nason ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Clinical Trials ◽  
Monoclonal Antibodies ◽  
Antiretroviral Therapy ◽  
Envelope Glycoprotein ◽  
Plasma Viremia ◽  
Immunodeficiency Virus ◽  
Acute Hiv ◽  
Hiv 1

ABSTRACTCombination antiretroviral therapy (cART) administered shortly after human immunodeficiency virus type 1 (HIV-1) infection can suppress viremia and limit seeding of the viral reservoir, but lifelong treatment is required for the majority of patients. Highly potent broadly neutralizing HIV-1 monoclonal antibodies (MAbs) can reduce plasma viremia when administered during chronic HIV-1 infection, but the therapeutic potential of these antibodies during acute infection is unknown. We tested the ability of HIV-1 envelope glycoprotein-specific broadly neutralizing MAbs to suppress acute simian-human immunodeficiency virus (SHIV) replication in rhesus macaques. Four groups of macaques were infected with SHIV-SF162P3 and received (i) the CD4-binding-site MAb VRC01; (ii) a combination of a more potent clonal relative of VRC01 (VRC07-523) and a V3 glycan-dependent MAb (PGT121); (iii) daily cART, all on day 10, just prior to expected peak plasma viremia; or (iv) no treatment. Daily cART was initiated 11 days after MAb administration and was continued for 13 weeks in all treated animals. Over a period of 11 days after a single administration, MAb treatment significantly reduced peak viremia, accelerated the decay slope, and reduced total viral replication compared to untreated controls. Proviral DNA in lymph node CD4 T cells was also diminished after treatment with the dual MAb. These data demonstrate the virological effect of potent MAbs and support future clinical trials that investigate HIV-1-neutralizing MAbs as adjunctive therapy with cART during acute HIV-1 infection.IMPORTANCETreatment of chronic HIV-1 infection with potent broadly neutralizing HIV-1 MAbs has been shown to significantly reduce plasma viremia. However, the antiviral effect of MAb treatment during acute HIV-1 infection is unknown. Here, we demonstrate that MAbs targeting the HIV-1 envelope glycoprotein both suppress acute SHIV plasma viremia and limit CD4 T cell-associated viral DNA. These findings provide support for clinical trials of MAbs as adjunctive therapy with antiretroviral therapy during acute HIV-1 infection.

scholarly journals Characterization of V3 Sequence Heterogeneity in Subtype C Human Immunodeficiency Virus Type 1 Isolates from Malawi: Underrepresentation of X4 Variants

1999 ◽  
Vol 73 (8) ◽  
pp. 6271-6281 ◽  
Author(s):  
Li-Hua Ping ◽  
Julie A. E. Nelson ◽  
Irving F. Hoffman ◽  
Jody Schock ◽  
Suzanna L. Lamers ◽  
...  
Keyword(s):  
Basic Amino Acid ◽  
Coreceptor Usage ◽  
Sequence Variability ◽  
Subtype C ◽  
Subtype B ◽  
Immunodeficiency Virus ◽  
Pcr Products ◽  
V3 Region ◽  
Hiv 1

ABSTRACT We have examined the nature of V3 sequence variability among subtype C human immunodeficiency virus type 1 (HIV-1) sequences from plasma-derived viral RNA present in infected men from Malawi. Sequence variability was assessed by direct sequence analysis of the V3 reverse transcription-PCR products, examination of virus populations by a subtype C V3-specific heteroduplex tracking assay (V3-HTA), and selected sequence analysis of molecular clones derived from the PCR products. Sequence variability in V3 among the subtype C viruses was not associated with the presence of basic amino acid substitutions. This observation is in contrast to that for subtype B HIV-1, where sequence variability is associated with such substitutions, and these substitutions are determinants of altered coreceptor usage. Evolutionary variants in subtype C V3 sequences, as defined by the V3-HTA, were not correlated with the CD4 level in the infected person, while such a correlation was found with subtype B V3 sequences. Viruses were isolated from a subset of the subjects; all isolates used CCR5 and not CXCR4 as a coreceptor, and none was able to grow in MT-2 cells, a hallmark of the syncytium-inducing phenotype that is correlated with CXCR4 usage. The overall sequence variability of the subtype C V3 region was no greater than that of the conserved regions of gp120. This limited sequence variability was also a feature of subtype B V3 sequences that do not carry the basic amino acid substitutions associated with altered coreceptor usage. Our results indicate that altered coreceptor usage is rare in subtype C HIV-1 isolates in sub-Saharan Africa and that sequence variability is not a feature of the V3 region of env in the absence of altered coreceptor usage.

scholarly journals Simultaneous Detection of Multiplex-Amplified Human Immunodeficiency Virus Type 1 RNA, Hepatitis C Virus RNA, and Hepatitis B Virus DNA Using a Flow Cytometer Microsphere-Based Hybridization Assay

2000 ◽  
Vol 38 (3) ◽  
pp. 1066-1071 ◽  
Author(s):  
J.-P. Defoort ◽  
M. Martin ◽  
B. Casano ◽  
S. Prato ◽  
C. Camilla ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Flow Cytometer ◽  
Rt Pcr ◽  
B Virus ◽  
Immunodeficiency Virus ◽  
Pcr Products ◽  
Hiv 1

The feasibility of performing a multiplex assay for the detection of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) RNAs and hepatitis B virus (HBV) DNA is demonstrated. This assay is based (i) on the coamplification of a 142-bp fragment from thegag region of the HIV-1 genome and a 142-bp HIV-1 quantitation standard fragment, a 244-bp fragment from the 5′ noncoding region of the HCV genome, and a 104-bp fragment from the pre-C and C gene regions of the HBV genome, using three sets of specific primers; (ii) on the capacity of these four biotinylated PCR products to hybridize to their specific oligonucleotide probe-coated microspheres; and (iii) on the ability of the flow cytometer to discriminate between distinct fluorescent-microsphere categories. Absence of cross-hybridization between the unrelated oligonucleotide probes and PCR products generated by the multiplex reverse transcription-PCR (RT-PCR) and the highly sensitive detection method allowed us to assess unambiguously the HIV-1 viral load and the infectious status of 35 serologically well-established clinical samples and 20 seronegative blood donor plasma samples tested. The results indicate that multiplex RT-PCR and flow cytometer microsphere-based hybridization assays, when combined, provide a rapid, sensitive, and specific method for the quantitation and detection of the major viral agents of infectious diseases in a single plasma sample.

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