Short communication: HIV-1 gag genetic variation in a single acutely infected participant defined by high-resolution deep sequencing.

Acute HIV-1 infection is characterized by the rapid generation of highly diverse genetic variants to adapt to the new host environment. Understanding the dynamics of viral genetic variation at this stage of infection is critical for vaccine design efforts and early drug treatment. Here, using a high-resolution deep sequencing approach targeting the HIV-1 gag region, we reveal very early immune pressure with dramatic subpopulation shifts in a single acutely infected participant providing further insight into the genetic dynamics of acute HIV-1 infection.

HIV-1 quasispecies delineation by tag linkage deep sequencing.

Trade-offs between throughput, read length, and error rates in high-throughput sequencing limit certain applications such as monitoring viral quasispecies. Here, we describe a molecular-based tag linkage method that allows assemblage of short sequence reads into long DNA fragments. It enables haplotype phasing with high accuracy and sensitivity to interrogate individual viral sequences in a quasispecies.

Ineffectual targeting of HIV-1 Nef by cytotoxic T lymphocytes in acute infection results in no functional impairment or viremia reduction.

The human immunodeficiency virus type 1 (HIV-1) accessory protein Nef is heavily targeted by CD8(+) T lymphocytes (CTLs) during acute infection and therefore is included in many candidate vaccines. We investigated whether CTL targeting of Nef during acute infection contributes to immune control by disrupting the function of Nef. The sequence and function of Nef in parallel with CTL responses were assessed longitudinally from peak viremia until the viremia set point in a cohort of six subjects with acute infection.

HIV-1 Nef sequence and functional compartmentalization in the gut is not due to differential cytotoxic T lymphocyte selective pressure.

The gut is the largest lymphoid organ in the body and a site of active HIV-1 replication and immune surveillance. The gut is a reservoir of persistent infection in some individuals with fully suppressed plasma viremia on combination antiretroviral therapy (cART) although the cause of this persistence is unknown. The HIV-1 accessory protein Nef contributes to persistence through multiple functions including immune evasion and increasing infectivity.

A new system to measure and compare hepatitis C virus replication capacity using full-length, replication competent viruses.

Measuring the in vitro replication capacity of viruses is an important tool for assessing the effects of selective pressure of immune responses and drug therapy. Measuring hepatitis C virus (HCV) replication capacity utilizing primarily sub-genomic reporter constructs is limited. To overcome some of these limitations a quantitative reverse transcriptase PCR (RT-qPCR) was designed to measure simultaneously the growth rate of 2 whole genome HCV variants under identical culture conditions.

Combination of allele-specific detection techniques to quantify minority resistance variants in hepatitis B infection: a novel approach.

Detection of minor variant viral quasispecies of the rtV173L+rtL180M+rtM204V combination mutation in the hepatitis B virus (HBV) polymerase mediating both lamivudine resistance and vaccine escape is potentially important for tracking the development and evolution of resistance within both individuals and populations. A highly sensitive and specific assay to quantitate HBV genomes was developed with this mutation combination directly from viral DNA in serum using allele-specific quantitative PCR with locked nucleic acid primers and a minor groove binder probe. This combination of primers and probe yields a linear detection range down to 150 copies.

Partial escape of HIV-1 from cytotoxic T lymphocytes during chronic infection.

Viral mutational escape from CD8(+) cytotoxic T lymphocytes (CTLs) is typically considered to be a dichotomous process and uncommon during chronic HIV-1 infection. Ex vivo passaging of HIV-1 from persons with chronic infection, however, revealed the evolution of many fixed substitutions within and around CTL-targeted regions, with an associated increase in replicative capacity. This indicates an evolution of mutations during chronic HIV-1 infection that trade replicative fitness for incomplete evasion of CTLs, or "partial escape.

Immune selection in vitro reveals human immunodeficiency virus type 1 Nef sequence motifs important for its immune evasion function in vivo.

Human immunodeficiency virus type 1 (HIV-1) Nef downregulates major histocompatibility complex class I (MHC-I), impairing the clearance of infected cells by CD8(+) cytotoxic T lymphocytes (CTLs). While sequence motifs mediating this function have been determined by in vitro mutagenesis studies of laboratory-adapted HIV-1 molecular clones, it is unclear whether the highly variable Nef sequences of primary isolates in vivo rely on the same sequence motifs. To address this issue, nef quasispecies from nine chronically HIV-1-infected persons were examined for sequence evolution and altered MHC-I downregulatory function under Gag-specific CTL immune pressure in vitro.

Human leukocyte antigen class I haplotypes of human immunodeficiency virus-1-infected persons on Likoma Island, Malawi.

Human leukocyte antigen (HLA) allele frequencies vary between different human populations, with implications both for the evolutionary pressures shaping those populations as well as for the outcome of new infectious epidemics. We defined HLA class I types in a well-described cohort of persons on Likoma Island in Malawi, a population for which there are lacking data on allelic frequencies. The profile of HLA frequencies was similar but phylogenetically distinct from those of other sub-Saharan African populations in neighboring regions.

Risk factors for infection and colonization with community-associated methicillin-resistant Staphylococcus aureus in the Los Angeles County jail: a case-control study.

Background: Community-associated methicillin-resistant Staphylococcus aureus (MRSA) infections and outbreaks occur in correctional facilities, such as jails and prisons. Spread of these infections can be extremely difficult to control. Development of effective prevention protocols requires an understanding of MRSA risk factors in incarcerated persons.

Simultaneous assessment of CD4 and MHC-I downregulation by Nef primary isolates in the context of infection.

The HIV-1 Nef protein plays a key role in pathogenesis, as demonstrated by strong selective pressure to maintain its open reading frame, and disease attenuation when it is deleted. Among myriad cellular effects attributed to Nef, downregulation of cell surface CD4 and major histocompatibility complex class I (MHC-I) proteins are the best documented. However, few data regarding primary isolate Nef functions are available, and most studies have been performed using transient transfections to express Nef driven by a non-physiologic promoter.

Functional adaptation of Nef to the immune milieu of HIV-1 infection in vivo.

Nef-mediated down-regulation of MHC class I (MHC-I) molecules on HIV-1-infected cells has been proposed to enhance viral persistence through evasion of host CTLs. This conclusion is based largely on demonstrations that Nef from laboratory HIV-1 strains reduces the susceptibility of infected cells to CTL killing in vitro. However, the function and role of Nef-mediated MHC-I down-regulation in vivo have not been well described.

Primary human immunodeficiency virus type 1 (HIV-1) infection during HIV-1 Gag vaccination.

Vaccination for human immunodeficiency virus type 1 (HIV-1) remains an elusive goal. Whether an unsuccessful vaccine might not only fail to provoke detectable immune responses but also could actually interfere with subsequent natural immunity upon HIV-1 infection is unknown. We performed detailed assessment of an HIV-1 gag DNA vaccine recipient (subject 00015) who was previously uninfected but sustained HIV-1 infection before completing a vaccination trial and another contemporaneously acutely infected individual (subject 00016) with the same strain of HIV-1.

Thymus-derived leukemia-lymphoma in mice transgenic for the Tax gene of human T-lymphotropic virus type I.

Adult T-cell leukemia-lymphoma (ATLL) is a group of T-cell malignancies caused by infection with human T-lymphotropic virus type I (HTLV-I). Although the pathogenesis of ATLL remains incompletely understood, the viral regulatory protein Tax is centrally involved in cellular transformation. Here we describe the generation of HTLV-I Tax transgenic mice using the Lck proximal promoter to restrict transgene expression to developing thymocytes.

Comparison of CREB- and NF-kappaB-mediated transactivation by human T lymphotropic virus type II (HTLV-II) and type I (HTLV-I) tax proteins.

The function of the transactivator protein Tax from HTLV-II subtype A, subtype B, Brazilian subtype C, and African subtype D isolates was compared to that of Tax from an HTLV-I isolate. HTLV-II subtypes A, B, and C were less active in the transactivation of a NF-kappaB reporter compared to HTLV-I Tax in 293T but not Jurkat T cells. In both cell types there were no significant differences between the functions of HTLV-II B, C, and D and HTLV-I Tax proteins on either a full-length HTLV-I LTR or a 21-bp repeat reporter, suggesting that there is equivalent CREB-mediated transactivation between the viruses and these subtypes.