Nitin K. Saksena

Tracing the origin and history of the HIV-2 epidemic

In this study we date the introduction of HIV-2 into the human population and estimate the epidemic history of HIV-2 subtype A in Guinea-Bissau, the putative geographic origin of HIV-2. The evolutionary history of the simian immunodeficiency virussooty mangabey/HIV-2 lineage was reconstructed by using available database sequences with known sampling dates, and a timescale for this history was calculated by using maximum likelihood methods. The date of the most recent common ancestor of HIV-2 subtype A strains was estimated to be 1940 ± 16 and that of B strains was estimated to be 1945 ± 14. In addition we used coalescent theory to estimate the past population dynamics of HIV-2 subtype A in a rural population of Guinea-Bissau. Parametric and nonparametric estimates of the effective number of infections through time were obtained for an equal sample of gag, pol, and env sequences. Our estimates of the epidemic history of HIV-2 subtype A in Guinea-Bissau show a transition from constant size to rapid exponential growth around 1955–1970. Our analysis provides evidence for a zoonotic transfer of HIV-2 during the first half of the 20th century and an epidemic initiation in Guinea-Bissau that coincides with the independence war (1963–1974), suggesting that war-related changes in sociocultural patterns had a major impact on the HIV-2 epidemic.

HIV-1 nef suppression by virally encoded microRNA

BackgroundMicroRNAs (miRNAs) are 21~25-nucleotides (nt) long and interact with mRNAs to trigger either translational repression or RNA cleavage through RNA interference (RNAi), depending on the degree of complementarity with the target mRNAs. Our recent study has shown that HIV-1 nef dsRNA from AIDS patients who are long-term non-progressors (LTNPs) inhibited the transcription of HIV-1.ResultsHere, we show the possibility that nef-derived miRNAs are produced in HIV-1 persistently infected cells. Furthermore, nef short hairpin RNA (shRNA) that corresponded to a predicted nef miRNA (~25 nt, miR-N367) can block HIV-1 Nef expression in vitro and the suppression by shRNA/miR-N367 would be related with low viremia in an LTNP (15-2-2). In the 15-2-2 model mice, the weight loss, which may be rendered by nef was also inhibited by shRNA/miR-N367 corresponding to suppression of nef expression in vivo.ConclusionsThese data suggest that nef/U3 miRNAs produced in HIV-1-infected cells may suppress both Nef function and HIV-1 virulence through the RNAi pathway.

Independent Evolution of Human Immunodeficiency Virus (HIV) Drug Resistance Mutations in Diverse Areas of the Brain in HIV-Infected Patients, with and without Dementia, on Antiretroviral Treatment

ABSTRACT AIDS dementia complex (ADC) in human immunodeficiency virus (HIV)-infected patients continues to be a problem in the era of highly active antiretroviral therapy (HAART). A better understanding of the drug resistance mutation patterns that emerge in the central nervous system (CNS) during HAART is of paramount importance as these differences in drug resistance mutations may explain underlying reasons for poor penetration of antiretroviral drugs into the CNS and suboptimal concentrations of the drugs that may reside in the brains of HIV-infected individuals during therapy. Thus, we provide a detailed analysis of HIV type 1 (HIV-1) protease and reverse transcriptase (RT) genes derived from different regions of the brains of 20 HIV-1-infected patients (5 without ADC, 2 with probable ADC, and 13 with various stages of ADC) on antiretroviral therapy. We show the compartmentalization and independent evolution of both primary and secondary drug resistance mutations to both RT and protease inhibitors in diverse regions of the CNS of HIV-infected patients, with and without dementia, on antiretroviral therapy. Our results suggest that the independent evolution of drug resistance mutations in diverse areas of the CNS may emerge as a consequence of incomplete suppression of HIV, probably related to suboptimal drug levels in the CNS and drug selection pressure. The emergence of resistant virus in the CNS may have considerable influence on the outcome of neurologic disease and also the reseeding of HIV in the systemic circulation upon failure of therapy.

Rapid and Sensitive Detection of Severe Acute Respiratory Syndrome Coronavirus by Rolling Circle Amplification

ABSTRACT The severe acute respiratory syndrome (SARS) epidemic of 2003 was responsible for 774 deaths and caused significant economic damage worldwide. Since July 2003, a number of SARS cases have occurred in China, raising the possibility of future epidemics. We describe here a rapid, sensitive, and highly efficient assay for the detection of SARS coronavirus (SARS-CoV) in cultured material and a small number (n = 7) of clinical samples. Using rolling circle amplification (RCA), we were able to achieve sensitive detection levels of SARS-CoV RNA in both solid and liquid phases. The main advantage of RCA is that it can be performed under isothermal conditions with minimal reagents and avoids the generation of false-positive results, a problem that is frequently encountered in PCR-based assays. Furthermore, the RCA technology provides a faster, more sensitive, and economical option to currently available PCR-based methods.

Both CD31+ and CD31- Naive CD4+ T Cells Are Persistent HIV Type 1-Infected Reservoirs in Individuals Receiving Antiretroviral Therapy

BACKGROUND Naive T cell recovery is critical for successful immune reconstitution after antiretroviral therapy (ART), but the relative contribution of CD31(+) and CD31⁻ naive T cells to immune reconstitution and viral persistence is unknown. METHODS In a cross-sectional (n = 94) and longitudinal (n = 10) study of human immunodeficiency virus (HIV)-infected patients before and after ART, we examined the ratio of CD31(+) to CD31⁻ naive CD4(+) T cells. In the longitudinal cohort we then quantified the concentration of HIV-1 DNA in each cell subset and performed single-genome amplification of virus from memory and naive T cells. RESULTS Patients receiving ART had a higher proportion of CD31(+) CD4(+) T cells than HIV-1-infected individuals naive to ART and uninfected control subjects (P < .001 and .007, respectively). After 24 months of ART, the proportion of CD31(+) naive CD4(+) T cells did not change, the concentration of HIV-1 DNA in memory CD4(+) T cells significantly decreased over time (P < .001), and there was no change in the concentration of HIV-1 DNA in CD31(+) or CD31⁻ naive CD4(+) T cells (P = .751 and .251, respectively). Single-genome amplification showed no evidence of virus compartmentalization in memory and naive T cell subsets before or after ART. CONCLUSIONS After ART, both CD31(+) and CD31⁻ naive CD4(+) T cells expand, and both subsets represent a stable, persistent reservoir of HIV-1.

Asn 362 in gp120 contributes to enhanced fusogenicity by CCR5-restricted HIV-1 envelope glycoprotein variants from patients with AIDS

BackgroundCCR5-restricted (R5) human immunodeficiency virus type 1 (HIV-1) variants cause CD4+ T-cell loss in the majority of individuals who progress to AIDS, but mechanisms underlying the pathogenicity of R5 strains are poorly understood. To better understand envelope glycoprotein (Env) determinants contributing to pathogenicity of R5 viruses, we characterized 37 full-length R5 Envs from cross-sectional and longitudinal R5 viruses isolated from blood of patients with asymptomatic infection or AIDS, referred to as pre-AIDS (PA) and AIDS (A) R5 Envs, respectively.ResultsCompared to PA-R5 Envs, A-R5 Envs had enhanced fusogenicity in quantitative cell-cell fusion assays, and reduced sensitivity to inhibition by the fusion inhibitor T-20. Sequence analysis identified the presence of Asn 362 (N362), a potential N-linked glycosylation site immediately N-terminal to CD4-binding site (CD4bs) residues in the C3 region of gp120, more frequently in A-R5 Envs than PA-R5 Envs. N362 was associated with enhanced fusogenicity, faster entry kinetics, and increased sensitivity of Env-pseudotyped reporter viruses to neutralization by the CD4bs-directed Env mAb IgG1b12. Mutagenesis studies showed N362 contributes to enhanced fusogenicity of most A-R5 Envs. Molecular models indicate N362 is located adjacent to the CD4 binding loop of gp120, and suggest N362 may enhance fusogenicity by promoting greater exposure of the CD4bs and/or stabilizing the CD4-bound Env structure.ConclusionEnhanced fusogenicity is a phenotype of the A-R5 Envs studied, which was associated with the presence of N362, enhanced HIV-1 entry kinetics and increased CD4bs exposure in gp120. N362 contributes to fusogenicity of R5 Envs in a strain dependent manner. Our studies suggest enhanced fusogenicity of A-R5 Envs may contribute to CD4+ T-cell loss in subjects who progress to AIDS whilst harbouring R5 HIV-1 variants. N362 may contribute to this effect in some individuals.

An examination of signs of disease progression in survivors of the Sydney Blood Bank Cohort (SBBC)

BACKGROUND The Sydney Blood Bank Cohort (SBBC) was infected between 1981 and 1984 with a nef/LTR defective strain of HIV-1. Different responses to HIV-1 infection have emerged between cohort members in the last 5 years. Three recipients (C135, C64 and C49) remain asymptomatic, have normal CD4 T cell counts, below detection (BD) viral loads (VL), remain therapy naive and are termed long-term non-progressors (LTNP). The donor (D36) and the two recipients (C98 and C54) have significantly declining CD4 T cell counts, detectable VL and are now long-term survivors (LTS). In contrast, in the SA cohort, comparison study group for the SBBC, five of 24 remain therapy naïve after 15 years infection with HIV-1 and all have detectable VL. OBJECTIVES This paper examines different outcomes to long-term infection with HIV-1 in the SBBC and provides a brief overview of the therapy naïve in a comparison study group, the SA cohort. STUDY DESIGN Retrospective epidemiological follow-up of the SBBC and the SA cohort has been conducted for >15 years. Analysis of CD4 T cell counts, VL and intermittent monitoring of HIV-specific proliferative responses are reviewed. Viral sequence changes in the SBBC will be considered. RESULTS Prior to therapy D36 had a CD4 T cell count of 160/mm(3) and plasma VL of 9900 copies/ml while C98 had a CD4 T cell count of 387/mm(3) and plasma VL of 11491 copies/ml. After 1 month of therapy, plasma VL was BD (<400 copies/ml) and both showed significant increase in CD4 T cell counts. Molecular changes have occurred in D36 and C98 viral strains, the most recently evolved quasispecies have larger deletions in the nef/LTR region. CONCLUSIONS Infection with nef/LTR deleted HIV-1 has resulted in slower disease progression for the SBBC. The three LTNP have maintained normal low levels of activated CD8 T cells and strong HIV-specific proliferative responses to HIV-1 p24, which are associated with control of viral replication.

First demonstration of a lack of viral sequence evolution in a nonprogressor, defining replication-incompetent HIV-1 infection

It is universally acknowledged that genetic diversity is a hallmark of HIV-1 infection, and it is one of the traits that has considerably hampered the development of an effective vaccine. In a study of full-length HIV-1 genomic sequences (>9 kb), we show unique evidence for complete absence of viral evolution in an individual with truly nonprogressive infection. Gross gene defects were not detected, but the state of replication incompetence was attributed to the presence of stop codons in the structural genes gag p17 and p24 and in pol RT, which emerged as a consequence of G-A hypermutation. These inactivating mutations may have occurred early, soon after infection, during the clonal stage of primary viral replication, since these are the sole archival strains present today. This genetic homogeneity, with <1% variation between strains over an 8-year period, suggests that only limited proviral integration events occurred in this patient. Further study on the antigenic properties of this strain may assist in the development of HIV vaccines and therapeutics.

Double‐Stranded nef RNA Interferes with Human Immunodeficiency Virus Type 1 Replication

RNA interference (RNAi) has been reported to be post‐transcriptional gene silencing (PTGS) by approximately 500 nucleotide‐(nt)‐long double‐stranded (ds) RNA that specifically targets homologous sequences of messenger RNA. In this report, we describe inhibition of HIV‐1 transcription by synthetic dsRNAs constructed with mutated nef genes (nef dsRNAs) derived from long‐term non‐progressors (LTNPs) using cotransfection of the target gene‐expressing plasmid and dsRNA. The effects of nef dsRNAs were examined with luciferase (Luc) reporter which is combined with the HIV‐1 (SF2) LTR in persistently HIV‐1‐infected T cell and macrophage cell lines. At 48 hr, a defective nef dsRNA (556 nt) suppressed Luc activity more potently than did SF2 full‐length nef dsRNA (744 nt), suggesting that approximately 500 nt‐long nef dsRNA could interfere with the HIV‐1 transcription.

Mechanisms of HIV non-progression; robust and sustained CD4+ T-cell proliferative responses to p24 antigen correlate with control of viraemia and lack of disease progression after long-term transfusion-acquired HIV-1 infection

BackgroundElite non-progressors (plasma viral load <50 copies/ml while antiretroviral naive) constitute a tiny fraction of HIV-infected individuals. After 12 years follow-up of a cohort of 13 long-term non-progressors (LTNP) identified from 135 individuals with transfusion-acquired HIV infection, 5 remained LTNP after 23 to 26 years infection, but only 3 retained elite LTNP status. We examined the mechanisms that differentiated delayed progressors from LTNP in this cohort.ResultsA survival advantage was conferred on 12 of 13 subjects, who had at least one host genetic factor (HLA, chemokine receptor or TLR polymorphisms) or viral attenuating factor (defective nef) associated with slow progression. However, antiviral immune responses differentiated the course of disease into and beyond the second decade of infection. A stable p24-specific proliferative response was associated with control of viraemia and retention of non-progressor status, but this p24 response was absent or declined in viraemic subjects. Strong Gag-dominant cytotoxic T lymphocyte (CTL) responses were identified in most LTNP, or Pol dominant-CTL in those with nef-defective HIV infection. CTL were associated with control of viraemia when combined with p24 proliferative responses. However, CTL did not prevent late disease progression. Individuals with sustained viral suppression had CTL recognising numerous Gag epitopes, while strong but restricted responses to one or two immunodominant epitopes was effective for some time, but failed to contain viraemia over the course of this study. Viral escape mutants at a HLA B27-restricted Gag-p24 epitope were detected in only 1 of 3 individuals, whereas declining or negative p24 proliferative responses occurred in all 3 concurrent with an increase in viraemia.ConclusionDetectable viraemia at study entry was predictive of loss of LTNP status and/or disease progression in 6 of 8, and differentiated slow progressors from elite LTNP who retained potent virological control. Sustained immunological suppression of viraemia was independently associated with preserved p24 proliferative responses, regardless of the strength and breadth of the CTL response. A decline in this protective p24 response preceded or correlated with loss of non-progressor status and/or signs of disease progression.