Catherine A. Macken

PERSISTENCE OF HIV-1 TRANSCRIPTION IN PERIPHERAL-BLOOD MONONUCLEAR CELLS IN PATIENTS RECEIVING POTENT ANTIRETROVIRAL THERAPY

BACKGROUND AND METHODS Although potent antiretroviral therapy can control infection with human immunodeficiency virus type 1 (HIV-1), a long-lived reservoir of infectious virus persists in CD4+ T cells. We investigated this viral reservoir by measuring the levels of cell-associated viral DNA and messenger RNA (mRNA) that are essential for HIV-1 replication. Approximately every 6 months, we obtained samples of peripheral-blood mononuclear cells from five men with long-standing HIV-1 infection who had had undetectable levels of plasma HIV-1 RNA for 20 months or more during treatment with potent antiretroviral drugs. RESULTS Before treatment, plasma levels of HIV-1 RNA correlated with the levels of cell-associated unintegrated HIV-1 DNA and unspliced viral mRNA. After treatment, plasma levels of HIV-1 RNA fell by more than 2.7 log to undetectable levels. The decrease in cell-associated integrated and unintegrated HIV-1 DNA and mRNA occurred in two phases. The first phase occurred during the initial 500 days of treatment and was characterized by substantial decreases in the levels of DNA and mRNA, but not to undetectable levels. The concentrations of cell-associated unintegrated viral DNA, integrated proviral DNA, and unspliced viral mRNA decreased by 1.25 to 1.46 log. The second phase occurred during the subsequent 300 days or more of treatment and was characterized by a plateau in the levels of HIV-1 DNA and unspliced mRNA. After an initial rapid decline, the ratio of unspliced to multiply spliced viral mRNA (a measure of active viral transcription) stabilized and remained greater than zero at each measurement. CONCLUSIONS Despite treatment with potent antiretroviral drugs and the suppression of plasma HIV-1 RNA to undetectable levels for 20 months or more, HIV-1 transcription persists in peripheral-blood mononuclear cells. Unless the quasi-steady state levels of HIV DNA and mRNA eventually disappear with longer periods of therapy, these findings suggest that HIV-1 infection cannot be eradicated with current treatments.

The decay of the latent reservoir of replication-competent HIV-1 is inversely correlated with the extent of residual viral replication during prolonged anti-retroviral therapy.

Replication-competent HIV-1 can be isolated from infected patients despite prolonged plasma virus suppression by anti-retroviral treatment. Recent studies have identified resting, memory CD4+ T lymphocytes as a long-lived latent reservoir of HIV-1 (refs. 4,5). Cross-sectional analyses indicate that the reservoir is rather small, between 103 and 107 cells per patient. In individuals whose plasma viremia levels are well suppressed by anti-retroviral therapy, peripheral blood mononuclear cells containing replication-competent HIV-1 were found to decay with a mean half-life of approximately 6 months, close to the decay characteristics of memory lymphocytes in humans and monkeys. In contrast, little decay was found in a less-selective patient population. We undertook this study to address this apparent discrepancy. Using a quantitative micro-culture assay, we demonstrate here that the latent reservoir decays with a mean half-life of 6.3 months in patients who consistently maintain plasma HIV-1 RNA levels of fewer than 50 copies/ml. Slower decay rates occur in individuals who experience intermittent episodes of plasma viremia. Our findings indicate that the persistence of the latent reservoir of HIV-1 despite prolonged treatment is due not only to its slow intrinsic decay characteristics but also to the inability of current drug regimens to completely block HIV-1 replication.

Kinetics of Influenza A Virus Infection in Humans

ABSTRACT Currently, little is known about the viral kinetics of influenza A during infection within an individual. We utilize a series of mathematical models of increasing complexity, which incorporate target cell limitation and the innate interferon response, to examine influenza A virus kinetics in the upper respiratory tracts of experimentally infected adults. The models were fit to data from an experimental H1N1 influenza A/Hong Kong/123/77 infection and suggest that it is important to include the eclipse phase of the viral life cycle in viral dynamic models. Doing so, we estimate that after a delay of ∼6 h, infected cells begin producing influenza virus and continue to do so for ∼5 h. The average lifetime of infected cells is ∼11 h, and the half-life of free infectious virus is ∼3 h. We calculated the basic reproductive number, R0, which indicated that a single infected cell could produce ∼22 new productive infections. This suggests that antiviral treatments have a large hurdle to overcome in moderating symptoms and limiting infectiousness and that treatment has to be initiated as early as possible. For about 50% of patients, the curve of viral titer versus time has two peaks. This bimodal behavior can be explained by incorporating the antiviral effects of interferon into the model. Our model also compared well to an additional data set on viral titer after experimental infection and treatment with the neuraminidase inhibitor zanamivir, which suggests that such models may prove useful in estimating the efficacies of different antiviral therapies for influenza A infection.

Detection of Influenza Viruses Resistant to Neuraminidase Inhibitors in Global Surveillance during the First 3 Years of Their Use

ABSTRACT Emergence of influenza viruses with reduced susceptibility to neuraminidase inhibitors (NAIs) develops at a low level following drug treatment, and person-to-person transmission of resistant virus has not been recognized to date. The Neuraminidase Inhibitor Susceptibility Network (NISN) was established to follow susceptibility of isolates and occurrence of NAI resistance at a population level in various parts of the world. Isolates from the WHO influenza collaborating centers were screened for susceptibilities to oseltamivir and zanamivir by a chemiluminescent enzyme inhibition assay, and those considered potentially resistant were analyzed by sequence analysis of the neuraminidase genes. During the first 3 years of NAI use (1999 to 2002), 2,287 isolates were tested. Among them, eight (0.33%) viruses had a >10-fold decrease in susceptibility to oseltamivir, one (0.22%) in 1999 to 2000, three (0.36%) in 2000 to 2001, and four (0.41%) in 2001 to 2002. Six had unique changes in the neuraminidase gene compared to neuraminidases of the same subtype in the influenza sequence database. Although only one of the mutations had previously been recognized in persons receiving NAIs, none were from patients who were known to have received the drugs. During the 3 years preceding NAI use, no resistant variants were detected among 1,054 viruses. Drug use was relatively stable during the period, except for an approximate 10-fold increase in oseltamivir use in Japan during the third year. The frequency of variants with decreased sensitivity to the NAIs did not increase significantly during this period, but continued surveillance is required, especially in regions with higher NAI use.

The value of a database in surveillance and vaccine selection

Abstract The Influenza Sequence Database (ISD) is a curated, specialized database of influenza genomic and protein sequences, and influenza-specific sequence analysis tools that have a web interface available to the public http://www.flu.lanl.gov ). Contents are primarily those influenza sequences that have been deposited in GenBank. A growing number of sequences are deposited directly into the ISD by WHO surveillance sites around the world. The core function of the interface is a database search. Analysis tools are a combination of developments by ISD staff and adaptation of existing, freely available software. Future developments of the database and its web site will be driven by user needs and by output from our in-house research. Projects underway include a layered, secure interface with the database to allow variable access privileges to sensitive data, and clickable mapping of a homology model if Influenza B hemagglutinin.

Influenza Research Database: an integrated bioinformatics resource for influenza research and surveillance

Please cite this paper as: Squires et al. (2012) Influenza research database: an integrated bioinformatics resource for influenza research and surveillance. Influenza and Other Respiratory Viruses 6(6), 404–416.

The Ariel Project: A Prospective Cohort Study of Maternal-Child Transmission of Human Immunodeficiency Virus Type 1 in the Era of Maternal Antiretroviral Therapy

In a prospective cohort study, clinical and biologic factors that contribute to maternal-child transmission of human immunodeficiency virus type 1 (HIV-1) were studied. HIV-infected pregnant women and their infants were evaluated prospectively according to a standardized protocol. Of 204 evaluable women, 81% received zidovudine during their pregnancy. The infection rate among the 209 evaluable infants was 9.1%. By univariate analysis, histologic chorioamnionitis, prolonged rupture of membranes, and a history of genital warts were significantly associated with transmission. Additional factors associated with transmission that approached significance included a higher maternal virus load at delivery and the presence of cocaine in the urine. In a logistic regression model, histologic chorioamnionitis was the only independent predictor of transmission. Despite a significantly higher transmission rate at one site, no unique viral genotype was found at any site. Thus, chorioamnionitis was found to be the major risk factor for transmission among women receiving zidovudine.

Multiple measures of HIV burden in blood and tissue are correlated with each other but not with clinical parameters in aviremic subjects.

Objectives: To determine the levels of residual HIV DNA and RNA in blood and gut reservoirs in aviremic patients, assess correlations among compartmental measurements of HIV burden, and evaluate association with clinical parameters. Design: Cross-sectional analysis of baseline data only, on 40 patients enrolled in phase II study evaluating efficacy of autologous gene-modified CD4+ and CD8+ T cells. All patients were on stable antiretroviral regimen with undetectable plasma HIV RNA (< 50 copies/ml). Methods: Measurements repeatedly performed over 8–12 weeks pre-intervention: blood HIV DNA, analysis of rectal mucosa-associated lymphoid tissue for both HIV RNA and HIV DNA, and quantitative co-culture of HIV from CD8-depleted peripheral blood mononuclear cells (PBMC). Results: Quantifiable levels of HIV detected in compartments despite undetectable levels of plasma HIV RNA: HIV co-culture of PBMC (88%), blood HIV DNA (95%), rectal biopsy HIV DNA (95%), rectal biopsy HIV RNA (65%). A significant correlation existed among various measures of HIV burden (HIV co-culture, blood HIV DNA, rectal biopsy HIV RNA and DNA) but not between assays and clinical parameters [duration of highly active antiretroviral therapy (HAART), type of HAART]. All assays had comparable or less variability than in plasma viral load assays; HIV co-culture had the highest coefficient of variability whereas the blood HIV DNA assay had the lowest and was considered the most reliable assay. Conclusions: The data support safety, feasibility and high compliance of quantifying reservoirs of residual HIV in treated subjects with undetectable plasma HIV RNA. Lack of correlation between levels of HIV in residual reservoirs and duration of HAART suggests treatment-mediated viral suppression alone does not lead to reproducible decay in HIV reservoirs.

BioHealthBase: informatics support in the elucidation of influenza virus host–pathogen interactions and virulence

The BioHealthBase Bioinformatics Resource Center (BRC) (http://www.biohealthbase.org) is a public bioinformatics database and analysis resource for the study of specific biodefense and public health pathogens—Influenza virus, Francisella tularensis, Mycobacterium tuberculosis, Microsporidia species and ricin toxin. The BioHealthBase serves as an extensive integrated repository of data imported from public databases, data derived from various computational algorithms and information curated from the scientific literature. The goal of the BioHealthBase is to facilitate the development of therapeutics, diagnostics and vaccines by integrating all available data in the context of host–pathogen interactions, thus allowing researchers to understand the root causes of virulence and pathogenicity. Genome and protein annotations can be viewed either as formatted text or graphically through a genome browser. 3D visualization capabilities allow researchers to view proteins with key structural and functional features highlighted. Influenza virus host–pathogen interactions at the molecular/cellular and systemic levels are represented. Host immune response to influenza infection is conveyed through the display of experimentally determined antibody and T-cell epitopes curated from the scientific literature or as derived from computational predictions. At the molecular/cellular level, the BioHealthBase BRC has developed biological pathway representations relevant to influenza virus host–pathogen interaction in collaboration with the Reactome database (http://www.reactome.org).

Surveillance for neuraminidase-inhibitor-resistant influenza viruses in Japan, 1996-2007

BACKGROUND High usage of the neuraminidase inhibitor (NAI) oseltamivir in Japan since 2003 led the Neuraminidase Inhibitor Susceptibility Network to assess the susceptibility of community isolates of influenza viruses to oseltamivir and zanamivir. METHODS Isolates were tested by the enzyme inhibition assay and by neuraminidase (NA) sequence analysis. RESULTS Among 1,141 A(H3N2) viruses and 171 type B viruses collected in Japan during the 2003-2004 season, 3 (0.3%) A(H3N2) isolates showed high 50% inhibitory concentrations (IC(50)) to oseltamivir. Each possessed a known resistance NA mutation at R292K or E119V. During the 2004-2005 season, no resistance was found among 567 influenza A(H3N2) or 60 A(H1N1) isolates, but 1 of 58 influenza B isolates had an NAI resistance mutation (D197N). Sequence analysis found that 4 (3%) of 132 A(H1N1) viruses from 2005-2006 had known NA resistance mutations (all H274Y), but no additional resistant isolates were detected from that or the subsequent 2006-2007 season. Concurrent testing of a selection of 500 influenza B viruses from 2000 to 2006 showed significant variations between seasons in both oseltamivir and zanamivir IC(50) values, but no persistent increases over this period. CONCLUSIONS Our findings suggested possible low-level transmission of resistant variants from oseltamivir-treated patients in several seasons in Japan but no sustained reductions in NAI susceptibility or consistently increased frequency of detecting resistant variants for any strain or subtype, despite high levels of drug use. In particular, although oseltamivir-resistant A(H1N1) viruses with the H274Y mutation spread globally in 2007-2008, we found little evidence for increasing levels of resistant A(H1N1) variants in Japan in preceding years.