Human immunodeficiency virus mutations during the first month of infection are preferentially found in known cytotoxic T-lymphocyte epitopes.

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.

Wide range of quasispecies diversity during primary hepatitis C virus infection.

Hepatitis C virus (HCV) infections may be initiated by multiple infectious particles, resulting in a genetically heterogeneous viral population, or by a single particle, leading to a clonal population in the initial stage of infection. To determine which of these scenarios is most common, we evaluated the genetic diversity of HCV quasispecies in 12 seronegative subjects with primary infection following community exposures, six acutely infected recipients of HCV-seropositive blood transfusions and six seropositive individuals with infections of undetermined durations. RNA isolated from plasma and a region of the HCV envelope gene including the first hypervariable region (HVR-1) was reverse transcription-PCR amplified and subcloned, and multiple plasmid clones were sequenced. Phylogenetic analysis indicated that all HCV variants clustered by individuals. Genetic distances among HCV variants within recently infected subjects ranged from 1 to 7.8%. On the basis of the estimated mutation rate of HCV in vivo and the Taq polymerase error rate, primary infection viral quasispecies were classified as genetically heterogeneous when the maximum sequence divergence between genetic variants in the same person was >3%. Heterogeneous quasispecies were detected in 4 of 12 preseroconversion subjects, 1 of 6 transfusion recipients, and 4 of 6 seropositive subjects. The high level of viral quasispecies genetic diversity found in at least a third of recently infected individuals is consistent with the transmission of multiple infectious particles. Community-acquired HCV infection, predominantly the result of needle sharing by injection drug users, therefore appears to be frequently initiated by the successful transmission of multiple viral variants.

Primary infection of a male plasma donor with divergent HIV variants from the same source followed by rapid fluctuations in their relative frequency and viral recombination.

The replication of two HIV-1 variants (>4.0% divergent in the env gene) was observed during primary infection of a frequent plasma donor. Phylogenetic analysis indicated that both HIV-1 variants likely originated from the same source. Heteroduplex tracking analysis of the env V3-V5 region indicated that one of these variant emerged in the plasma at the time of seroconversion, 15 days after the initial detection of HIV-1 RNA. Sequencing of the entire protein-coding region of plasma viruses from Days 2, 22, and 31 showed possible regions of recombination in the pol locus occurring within the first month of infection. The very rapid fluctuations of HIV-1 variant frequencies and their recombination during primary infection may reflect changes in their relative fitness in the face of developing immunological responses.

Dynamics of HIV viremia and antibody seroconversion in plasma donors: implications for diagnosis and staging of primary HIV infection.

OBJECTIVES: The characterization of primary HIV infection by the analysis of serial plasma samples from newly infected persons using multiple standard viral assays. DESIGN: A retrospective study involving two sets of archived samples from HIV-infected plasma donors. (A) 435 samples from 51 donors detected by anti-HIV enzyme immunoassays donated during 1984-1994; (B) 145 specimens from 44 donors detected by p24 antigen screening donated during 1996-1998. SETTING: Two US plasma products companies. MAIN OUTCOME MEASURES: The timepoints of appearance of HIV-1 markers and viral load concentrations during primary HIV infection. RESULTS: The pattern of sequential emergence of viral markers in the 'A' panels was highly consistent, allowing the definition and estimation of the duration of six sequential stages. From the 'B' panels, the viral load at p24 antigen seroconversion was estimated by regression analysis at 10 000 copies/ml (95% CI 2000-93 000) and the HIV replication rate at 0.35 log copies/ml/day, corresponding to a doubling time in the preseroconversion phase of 20.5 h (95% CI 18.2-23.4 h). Consequently, an RNA test with 50 copies/ml sensitivity would detect HIV infection approximately 7 days before a p24 antigen test, and 12 days before a sensitive anti-HIV test. CONCLUSION: The sequential emergence of assay reactivity allows the classification of primary HIV-1 infection into distinct laboratory stages, which may facilitate the diagnosis of recent infection and stratification of patients enrolled in clinical trials. Quantitative analysis of preseroconversion replication rates of HIV is useful for projecting the yield and predictive value of assays targeting primary HIV infection.

Comparative sensitivity of HBV NATs and HBsAg assays for detection of acute HBV infection.

BACKGROUND: A study was designed to estimate relative analytic sensitivity and window-period (WP) closure and to project incremental yield of newer HBsAg tests, pooled-sample NAT, and single-sample NAT, compared to currently licensed HBsAg tests. STUDY DESIGN AND METHODS: HBV DNA and HBsAg test results for 23 HBV seroconversion (SC) panels were first analyzed to construct a model of primary HBV viremia. One-hundred representative samples were then selected from 10 panels and coded with 28 analytical controls. All 128 samples were tested by seven HBsAg tests and by four pooled-sample and three single-sample NAT assay formats. Results were analyzed to obtain differential times to HBV detection and combined with HBV incidence rates to project comparative yields. RESULTS: HBV doubling time during the ramp-up phase was estimated at 2.56 days. HBsAg concentrations at cutoff for new tests ranged from 0.07 to 0.12 ng per mL, compared with 0.13 to 0.62 ng per mL for licensed tests. Estimated viral load at cutoff ranged from 102 to 267 IU per mL for new tests and from 363 to 1069 IU per mL for licensed tests. HBsAg tests detected 31 to 63 percent of early ramp-up phase samples in the 100-member seroconversion panel study, while pooled-sample NAT detected 55 to 71 percent and single-sample NAT, 82 to 99 percent. Compared with currently licensed HBsAg assays, newer HBsAg assays would reduce the WP by 2 to 9 days; pooled-sample NAT would reduce the WP by 9 to 11 days; and single-sample NAT would reduce the WP by 25 to 36 days. CONCLUSION: Newer HBsAg tests would be expected to detect an additional 15 to 21 infected units per 107 donations, compared to licensed HBsAg tests. Sensitivity, WP closure, and yield projections for newer HBsAg assays and pooled-sample NAT are comparable. Single-sample NAT would increase yield by 13 to 15 units per 107 donations over pooled-sample NAT and newer HBsAg assays and by 35 to 50 units per 107 donations over currently licensed HBsAg assays.

Homogeneous quasispecies in 16 out of 17 individuals during very early HIV-1 primary infection.

OBJECTIVE: To measure HIV-1 quasispecies diversity in very recently infected male and female plasma donors. METHODS: HIV-1 RNA testing of blood and plasma donations was used to select anti HIV-1 antibody negative, HIV-1 RNA positive plasma samples from 13 males and four females undergoing primary infection. To determine whether these early viral populations were clonal or oligoclonal, heteroduplex mobility assays were performed on multiple independently generated envelope PCR products. Genetically heterogeneous quasispecies where subcloned and their divergent envelope variants sequenced. RESULTS: Because of frequent plasma donations in this population, HIV-1 RNA quasispecies could be studied during very early primary infection. Heteroduplex mobility assays detected the presence of genetically distinct variants in four of the 17 plasma donors. DNA sequence analysis showed that one case was due to a G to A hyper-mutation event and that two cases were caused by the presence of in-frame insertions/deletions resulting in DNA heteroduplex mobility shifts. The early plasma quasispecies of one female contained highly divergent variants differing by up to 6% substitution and multiple insertions/deletions, a level of divergence unlikely to have been generated de novo following transmission. V3 loop sequences analysis indicated the presence of non-syncitium inducing genotypes in 14 out of 17 primary infection cases. CONCLUSION: Plasma viremia is generally genetically homogeneous even during the very early phase of primary infection when viremia is first detected and still rising exponentially. Evidence for the transmission of multiple variants was detected in only one out of four women and none of 13 men undergoing primary infection with subtype B HIV-1.

THE SYNTHESIS OF 5S RIBOSOMAL RNA DURING POLLEN DEVELOPMENT.

Tradescantia paludosa 5S ribosomal RNA (rRNA) has been characterized with respect to its base composition and relative electrophoretic mobility in comparison with that of E. coli. The period of 5S rRNA synthesis during pollen grain development was determined by pulse labeling the RNA synthesized during a 24 hr period of development with 32 P and then chasing in cold medium until pollen maturity. The period of highest 5S rRNA synthesis was found to occur prior to microspore mitosis. During and following mitosis over a period of 4 days there was a sharp decrease in the amount of 5S RNA synthesized and during the last 48 hr of pollen maturation, no 5S rRNA was synthesized.