Bin Wang

Oseltamivir resistance in adult oncology and hematology patients infected with pandemic (H1N1) 2009 virus, Australia.

Resistance in virus-infected stem cell transplant recipients illustrates the need for surveillance.

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.

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.

Detection of the rapid emergence of the H275Y mutation associated with oseltamivir resistance in severe pandemic influenza virus A/H1N1 09 infections

In 2009 a new swine-origin influenza virus A/H1N1 (A/H1N1 09) emerged, causing the centurys first pandemic. Most isolates of the new A/H1N1 09 virus are susceptible to neuraminidase inhibitors, but the H275Y mutation in the neuraminidase gene region associated with high-level oseltamivir resistance has been detected. Using rolling circle amplification (RCA) technology, 96 A/H1N1 09-specific RT-PCR positive clinical samples collected from 80 oseltamivir-treated and untreated patients were screened for the presence of the H275Y mutation. Samples positive for 275Y mutation by RCA were cloned and sequenced for confirmation. From 25 patients who had been treated with oseltamivir and remained A/H1N1 09 RT-PCR positive, we identified three (12%) individuals with the H275Y mutation: one immuno-suppressed adult, one immuno-competent adult and one child. Samples collected at multiple time points from the two adults showed a switch from wild-type oseltamivir-sensitive 275H to oseltamivir-resistant 275Y population after 9 days of treatment. The child had the 275Y mutation detected after being persistently A/H1N1 09 RT-PCR positive while receiving oseltamivir treatment. Resistance was not detected in 17 pre-treatment samples and 54 A/H1N1 09 RT-PCR positive outpatients. RCA demonstrates the rapid emergence of the H275Y resistance mutation in individuals with severe A/H1N1 09 infection receiving neuraminidase inhibitors. Rapid detection of oseltamivir resistance in severe infection is essential for patients to receive maximum therapeutic benefit. In the light of emerging resistance, close monitoring and understanding of the nature and dynamics of resistance mutations in newly emerging strains should be a priority.

Tissue-Specific Sequence Alterations in the Human Immunodeficiency Virus Type 1 Envelope Favoring CCR5 Usage Contribute to Persistence of Dual-Tropic Virus in the Brain

ABSTRACT Most human immunodeficiency virus type 1 (HIV-1) strains isolated from the brain use CCR5 for entry into macrophages and microglia. Strains that use both CCR5 and CXCR4 for entry (R5X4 strains) have been identified in the brains of some individuals, but mechanisms underlying the persistence of R5X4 viruses compartmentalized between the brain and other tissue reservoirs are unknown. Here, we characterized changes in the HIV-1 envelope (Env) that enhance the tropism of R5X4 variants for brain or lymphoid tissue. R5X4 Envs derived from the brains of two individuals had enhanced CCR5 usage in fusion assays compared to R5X4 Envs derived from matched spleen or blood, which was associated with reduced dependence on specific residues in the CCR5 N terminus and extracellular loop 1 (ECL1) and ECL3 regions. In contrast, spleen/blood-derived Envs had enhanced CXCR4 usage compared to brain-derived Envs, which was associated with reduced dependence on residues in the CXCR4 N terminus and ECL2 region. Consequently, brain-derived Envs had preferential CCR5 usage for HIV-1 entry into the JC53 cell line, could use either CCR5 or CXCR4 for entry into monocyte-derived macrophages (MDM), and could use CCR5 (albeit inefficiently) for entry into peripheral blood mononuclear cells (PBMC), whereas the entry of spleen-derived Envs was CXCR4 dependent in all three cell types. Mutagenesis studies of Env amino acid variants influencing coreceptor usage showed that S306 in the gp120 V3 region of brain-derived Envs reduces dependence on the CCR5 N terminus and enhances CCR5 usage for HIV-1 entry into PBMC and MDM, whereas R306 in spleen-derived Envs reduces dependence on the CXCR4 N terminus and confers the CXCR4 restricted phenotype. These results identify mechanisms underlying R5X4 HIV-1 persistence in different tissue reservoirs. Tissue-specific changes in the gp120 V3 region that increase the efficiency of CCR5 or CXCR4 usage, and thereby influence coreceptor preference, may enhance the tropism of R5X4 strains for CCR5-expressing macrophage lineage cells in the brain and CXCR4-expressing T cells in lymphoid tissues, respectively.

HIV-1 co-infection superinfection and recombination.

As the human immunodeficiency virus (HIV) pandemic progresses, an increasing number of recombinant viruses have been identified and in many geographical regions they are now the predominating strain. These recombinants are formed when an individual has acquired a co-infection or superinfection with more than one HIV-1 strain or subtype. Thus, dually infected individuals provide opportunities for studying HIV recombinants and viral interactions between infecting strains in vivo. The possible epidemiological, clinical and therapeutic implications of dual infections and recombination are many. Recombination may result in the emergence of more pathogenic and virulent HIV strains with altered fitness, tropism, and resistance to multiple drugs, and may hamper the development of subtype-based vaccines. This review is aimed at providing a more thorough understanding of dual infections (both co-infection and super-infection) and the possible consequences of the emergence of recombinant HIV-1 strains.

Longitudinal Analysis of CCR5 and CXCR4 Usage in a Cohort of Antiretroviral Therapy-Naïve Subjects with Progressive HIV-1 Subtype C Infection

HIV-1 subtype C (C-HIV) is responsible for most HIV-1 cases worldwide. Although the pathogenesis of C-HIV is thought to predominantly involve CCR5-restricted (R5) strains, we do not have a firm understanding of how frequently CXCR4-using (X4 and R5X4) variants emerge in subjects with progressive C-HIV infection. Nor do we completely understand the molecular determinants of coreceptor switching by C-HIV variants. Here, we characterized a panel of HIV-1 envelope glycoproteins (Envs) (nu200a=u200a300) cloned sequentially from plasma of 21 antiretroviral therapy (ART)-naïve subjects who experienced progression from chronic to advanced stages of C-HIV infection, and show that CXCR4-using C-HIV variants emerged in only one individual. Mutagenesis studies and structural models suggest that the evolution of R5 to X4 variants in this subject principally involved acquisition of an “Ile-Gly” insertion in the gp120 V3 loop and replacement of the V3 “Gly-Pro-Gly” crown with a “Gly-Arg-Gly” motif, but that the accumulation of additional gp120 “scaffold” mutations was required for these V3 loop changes to confer functional effects. In this context, either of the V3 loop changes could confer possible transitional R5X4 phenotypes, but when present together they completely abolished CCR5 usage and conferred the X4 phenotype. Our results show that the emergence of CXCR4-using strains is rare in this cohort of untreated individuals with advanced C-HIV infection. In the subject where X4 variants did emerge, alterations in the gp120 V3 loop were necessary but not sufficient to confer CXCR4 usage.

Detection of influenza A H1N1 and H3N2 mutations conferring resistance to oseltamivir using rolling circle amplification

In the event of an influenza pandemic, the use of oseltamivir (OTV) will undoubtedly increase and therefore it is more likely that OTV-resistant influenza strains will also arise. OTV-resistance genotyping using sequence-based testing on viruses isolated in cell culture is time consuming and less likely to detect the low-level presence of drug-resistant virus populations. We have developed a novel rolling circle amplification (RCA) method to achieve the sensitive detection of OTV-resistant viruses from clinical specimens. Using artificially created templates, RCA could detect the presence of OTV-resistant mutations (N2: 119V, 292K, N1: 274Y) even if the population carrying the mutations was <1% of the total. By applying RCA to clinical samples, we identified the emergence of the 274Y mutation in one OTV-treated patient, as well as in seven individuals who were treatment-naïve (confirming community transmission of 274Y-containing resistant influenza A H1N1). These results were further confirmed by neuraminidase region sequencing. In conclusion, RCA technology can provide rapid (<24 h), high-throughput diagnosis of OTV resistance mutations with a high specificity and sensitivity.

Antibody microarray analysis of cell surface antigens on CD4+ and CD8+ T cells from HIV+ individuals correlates with disease stages

BackgroundExpression levels of cell surface antigens such as CD38 and HLA-DR are related to HIV disease stages. To date, the immunophenotyping of cell surface antigens relies on flow cytometry, allowing estimation of 3–6 markers at a time. The recently described DotScan antibody microarray technology enables the simultaneous analysis of a large number of cell surface antigens. This new technology provides new opportunities to identify novel differential markers expressed or co-expressed on CD4+ and CD8+ T cells, which could aid in defining the stage of evolution of HIV infection and the immune status of the patient.ResultsUsing this new technology, we compared cell surface antigen expression on purified CD4+ and CD8+ T cells between 3 HIV disease groups (long-term non-progressors controlling viremia naturally; HIV+ patients on highly active antiretroviral therapy (HAART) with HIV plasma viral loads <50 copies/ml; and HIV+ patients with viremia during HAART) and uninfected controls. Pairwise comparisons identified 17 statistically differential cell surface antigens including 5 novel ones (CD212b1, CD218a, CD183, CD3 epsilon and CD9), not previously reported. Notably, changes in activation marker expression were more pronounced in CD8+ T cells, whereas changes in the expression of cell membrane receptors for cytokines and chemokines were more pronounced in CD4+ T cells.ConclusionOur study not only confirmed cell surface antigens previously reported to be related to HIV disease stages, but also identified 5 novel ones. Of these five, three markers point to major changes in responsiveness to certain cytokines, which are involved in Th1 responses. For the first time our study shows how density of cell surface antigens could be efficiently exploited in an array manner in relation to HIV disease stages. This new platform of identifying disease markers can be further extended to study other diseases.

HIV-1 integrase polymorphisms are associated with prior antiretroviral drug exposure

In a recent summary of integrase sequences, primary integrase inhibitor mutations were rare. In a review of integrase inhibitor-naïve Australian HIV-1 sequences, primary mutations were not identified, although the accessory mutation G140S was detected. A link with previous antiretroviral therapy, intra-subtype B divergence across the integrase gene and transmission of integrase polymorphisms were also noted. Based on these findings, we would recommend ongoing surveillance of integrase mutations, and integrase region sequencing for patients prior to commencement of integrase inhibitors.