Flavien Bernardin

Frequent detection of the parvoviruses, PARV4 and PARV5, in plasma from blood donors and symptomatic individuals

BACKGROUND: Plasma pools used in the manufacture of blood‐ and plasma‐derived medicinal products are frequently contaminated with parvovirus B19. The presence of the novel human parvovirus PARV4 and a related variant PARV5 in manufacturing plasma pools was recently demonstrated. Another recently identified parvovirus, human bocavirus (HBoV), has been identified in respiratory samples from children with lower respiratory tract disease.

Unifying the spatial epidemiology and molecular evolution of emerging epidemics

We introduce a conceptual bridge between the previously unlinked fields of phylogenetics and mathematical spatial ecology, which enables the spatial parameters of an emerging epidemic to be directly estimated from sampled pathogen genome sequences. By using phylogenetic history to correct for spatial autocorrelation, we illustrate how a fundamental spatial variable, the diffusion coefficient, can be estimated using robust nonparametric statistics, and how heterogeneity in dispersal can be readily quantified. We apply this framework to the spread of the West Nile virus across North America, an important recent instance of spatial invasion by an emerging infectious disease. We demonstrate that the dispersal of West Nile virus is greater and far more variable than previously measured, such that its dissemination was critically determined by rare, long-range movements that are unlikely to be discerned during field observations. Our results indicate that, by ignoring this heterogeneity, previous models of the epidemic have substantially overestimated its basic reproductive number. More generally, our approach demonstrates that easily obtainable genetic data can be used to measure the spatial dynamics of natural populations that are otherwise difficult or costly to quantify.

Frequent Detection of Highly Diverse Variants of Cardiovirus, Cosavirus, Bocavirus, and Circovirus in Sewage Samples Collected in the United States

ABSTRACT Untreated sewage samples from 12 cities in the United States were screened for the presence of recently characterized RNA and DNA viruses found at high prevalence in the stool specimens of South Asian children. Genetic variants of human cosaviruses and cardioviruses in the Picornaviridae family and of DNA circoviruses and human bocaviruses were detected, expanding the known genetic diversity and geographic range of these newly identified viruses. All four virus groups were detected in sewage samples of less than a milliliter from multiple U.S. cities. PCR screening of particle-protected viral nucleic acid in sewage samples could therefore rapidly establish the presence and determine the diversity of four newly described enteric viruses in large urban populations. More frequent and deeper sampling of viral nucleic acids in sewage samples could be used to monitor changes in the prevalence and genetic composition of these and other novel enteric viruses.

Clearance of hepatitis C virus RNA from the peripheral blood mononuclear cells of blood donors who spontaneously or therapeutically control their plasma viremia.

We determined whether hepatitis C virus (HCV) RNA could be detected associated with peripheral blood mononuclear cells (PBMC) of seropositive blood donors who had spontaneously or therapeutically cleared their plasma viremia. Blood donor plasma viremia status was first determined with a highly sensitive transcription‐mediated amplification (TMA) test performed in duplicate assays. PBMC from 69 aviremic and 56 viremic blood donors were then analyzed for the presence of HCV RNA with TMA adapted to detect viral RNA in PBMC and with a reverse transcription–nested polymerase chain reaction assay. PBMC‐associated HCV RNA was detected in none of the 69 aviremic donors, including all 6 subjects with a sustained viral response following antiviral therapy. PBMC‐associated HCV RNA was detected in 43 of the 56 viremic donors. The 13 viremic donors with no detectable PBMC‐associated HCV RNA all had very low viral loads (6 positive only in 1 of 2 duplicate plasma TMA assays, 6 with viral loads below 100 HCV RNA copies/mL, and 1 with a viremia of 2700 HCV RNA copies/mL). The absence of detectable PBMC HCV RNA detection in all 69 aviremic donors reported here contrasts with prior studies, possibly as a result of the higher sensitivity of the TMA assay used to test for plasma viremia. Conclusion: Our results indicate that PBMC are unlikely to serve as a long‐lived reservoir of HCV in aviremic subjects. (HEPATOLOGY 2008.)

Transfusion transmission of highly prevalent commensal human viruses

BACKGROUND: Anellovirus species Torque teno virus (TTV), Torque teno mini virus (TTMV), and Torque teno midi virus (TTMDV) and flavivirus GBV‐C are highly prevalent and genetically diverse chronic human viral infections that have not yet been associated with disease.

Human Immunodeficiency Virus Mutations during the First Month of Infection Are Preferentially Found in Known Cytotoxic T-Lymphocyte Epitopes

ABSTRACT The full protein coding region of human immunodeficiency virus (HIV) genomes were sequenced using plasma collected from nine African-Americans prior to seroconversion and 7 to 28 days later. HIV mutations emerged in seven of these subjects at a genomewide rate of 2% per year. The location of nonsynonymous (NS) HIV mutations within these subjects was compared to their potential HLA-A and B types restricted CTL epitopes reported in the Los Alamos National Laboratory HIV immunology database. A statistically significant (P < 0.005) number of the early NS mutations (13.5%) were found within previously reported CTL epitopes. A virus sequencing and reported CTL epitopes database analysis therefore support a model where a significant proportion of very early nonsynonymous HIV mutations are selected by CTL.

Estimate of the total number of CD8+ clonal expansions in healthy adults using a new DNA heteroduplex-tracking assay for CDR3 repertoire analysis.

A T-cell receptor heteroduplex-tracking assay (TCR-HTA) was developed to analyze the sequence diversity of the TCR beta-chain mRNA of each of the 24 T-cell receptor beta-chain variable region (TRBV). TCR-HTA allowed an estimation of the number of expanded CD8 T-cell clones whose distinct CDR3 domain mRNA made up 2% or more of the transcript of each TRBV subfamily. An average of 40 CD8+ clonal expansions (range 34-49) was detected in three healthy adults. Correct sampling of the complex mRNA transcript populations was documented by the reproducible generation of TCR-HTA patterns using independently generated PCR amplicons. The CDR3 sequence of expanded T-cell clones could be rapidly determined by direct sequencing of DNA heteroduplex bands. CD4+ and CD8+ clonal expansions were found predominantly although not exclusively in CD45RO+ CD62L- effector/memory cells and the majority of expanded T-cell clones were stable over a period of at least 6 months. Fewer CD4+ than CD8+ clonal expansions were detected in peripheral blood cells. By providing a high-resolution method for the detection of clonally expanded T-cell clones and by simplifying the pattern generated using traditional DNA heteroduplex analysis, TCR-HTA is shown to be a sensitive method for assessing levels of oligoclonality and changes in TRBV repertoires.

Detection of HIV-1 in alternative specimen types using the APTIMA® HIV-1 RNA Qualitative Assay

Peripheral blood mononuclear cells (PBMCs), saliva, seminal plasma, and dried blood spots were evaluated as specimen types for the APTIMA HIV-1 RNA Qualitative Assay (APTIMA HIV-1 Assay), which employs a target capture step to recover HIV-1-specific sequences from complex specimen types. Analytical sensitivity studies were carried out using samples that were either diluted or eluted with a buffered detergent and spiked with different concentrations of HIV-1 ranging from 1 to 10,000 copies/mL. PBMC samples spiked with HIV-1 had comparable analytical sensitivity to HIV-1 spiked plasma with a 95% limit of detection of 13.1 and 17.2 copies/mL, respectively. Analytical sensitivity in seminal plasma specimens diluted 1:5 and saliva diluted 1:2 was comparable to HIV-1 spiked dilution buffer alone. Whole blood and dried blood spot specimens spiked with HIV-1 had equivalent reactivity at 250 copies/spot (5000 copies/mL). However, the 95% limit of detection values were significantly different (293.7 copies/mL for whole blood and 2384 copies/mL for dried blood spot specimens). No significant effect on analytical sensitivity was observed when one HIV-1 positive dried blood spot punch was pooled with up to 9 HIV-1 negative dried blood spot punches. Together, these studies demonstrate that the APTIMA HIV-1 RNA Qualitative Assay can be used to process a diverse array of specimen types with minimal impact on analytical sensitivity for most specimen types.

Absence of HCV viral recombination following superinfection

Summary.  We sought evidence of viral recombination in five recently hepatitis C virus (HCV) infected young injection drug users who became superinfected with a distinguishable strain of HCV. The entire open reading frame of plasma HCV genomes was reverse transcribed, polymerase chain reaction amplified in two fragments, and directly sequenced. In two cases of same subtype (1a>1a) superinfections the initial and later strains were both sequenced and compared for evidence of recombination. In three cases of superinfection with strains of different genotype/subtype (3a>1a, 1a>3a, 1b>1a), the later time point HCV genomes were sequenced and compared with representative genomes of the initial genotype/subtype. No evidence of intra‐ or inter‐genotype/subtype recombination was detected using six different programs for detecting recombination. We conclude that the generation of viable recombinant HCV genomes able to dominate in the viral quasispecies is a rare event.

Highly Uneven Distribution of Tenofovir-Selected Simian Immunodeficiency Virus in Different Anatomical Sites of Rhesus Macaques

ABSTRACT Antiviral tenofovir monotherapy was used to determine whether drug-selected simian immunodeficiency virus (SIV) variants replaced their wild-type progenitors at the same rate in different tissues of six rhesus macaques. The relative frequencies of drug-resistant and wild-type genotypes were measured longitudinally in blood and in 23 lymphoid and nonlymphoid tissues collected at necropsy. The mutant/wild-type genotype ratio was measured using a heteroduplex tracking assay targeting tenofovir-selected SIV reverse transcriptase codons. After the initiation of tenofovir treatment in animals with high steady-state viremia levels, resistant genotypes emerged in the plasma within 1 to 8 weeks and in five of six cases reached frequencies of nearly 100% within 4 to 25 weeks. The appearance of tenofovir-resistant genotypes in peripheral blood mononuclear cell (PBMC) DNA was generally delayed by 1 to 2 weeks and in one case was completely absent. Necropsies performed 8 to 55 weeks after the initiation of tenofovir treatment showed the frequency of resistant SIV genotypes to be generally higher in tissue RNA than DNA fractions. The frequency of drug-resistant genotypes varied widely between anatomical sites, including different lymph nodes of the same animal. Except for the epidydimis, the tissues with the lowest rates of proviral replacement by tenofovir-resistant genotypes differed between animals. The highly uneven distribution of tenofovir-resistant genotypes in different tissues seen shortly after the initiation of tenofovir monotherapy may reflect differences in local antiviral drug selection pressures and/or the stochastic effect of small effective populations of drug-resistant variants randomly seeding different anatomical sites early in therapy.