Genetic shift of env V3 loop viral sequences in patients with HIV-associated neurocognitive disorder during antiretroviral therapy.

The development of human immunodeficiency virus type 1 (HIV)-associated neurocognitive disorder (HAND) involves the adaptation of viral sequences coding for the V3 loop of the env protein. The plasma and cerebrospinal fluid (CSF) may contain viral populations from various cellular sources and with differing pathogenicity. Combination antiretroviral therapy (cART) may alter the relative abundance of these viral populations, leading to a genetic shift. We characterized plasma and CNS viral populations prior to and during cART and relate the findings to viral elimination kinetics and the clinical phenotype. Longitudinal plasma and CSF samples of five chronically infected HIV patients, four of whom had HAND, and one seroconverter were analyzed for V3 sequences by RT-PCR and sequence analysis. In the chronically infected patients, pre-cART plasma and CSF viral sequences were different irrespective of viral elimination kinetics and clinical phenotype. cART induced replacement of plasma viral populations in all subjects. CSF viral populations underwent a clear genetic shift in some patients but remained stable in others. This was not dependent on the presence of HAND. The genetic shift of CSF V3 sequences was absent in the two subjects whose CSF viral load initially increased during cART. In one patient, pre- and post-treatment CSF sequences were closely related to the post-treatment plasma sequences, suggesting a common cellular source. We found heterogeneous patterns of genetic compartmentalization and genetic shift over time. Although these did not closely match viral elimination kinetics and clinical phenotype, the results imply different patterns of the dynamics and relative contribution of compartment-specific virus populations in chronic HIV infection.

Real-time PCR assay for the detection of tanapox virus and yaba-like disease virus.

The yatapoxvirus genus contains three members: tanapox virus (TPV), yaba-like disease virus (YLDV) and yaba monkey tumor virus (YMTV), two of which (TPV and YLDV) may infect humans. However, only a very small number of patients have been diagnosed with TPV outside Africa. Given the increased international travel and the similarity of clinical signs during the early stages of a TPV/YLDV infection as compared to diseases caused by agents of potential biological warfare, such as smallpox, monkeypox, tularemia and anthrax, the rapid and reliable recognition of a TPV/YLDV infection is crucial. A real-time PCR assay using TaqManchemistry was developed in order to identify unambiguously TPV/YLDV. Primers and probe targeting a 101bp region of the PstI L fragment of TPV, initial optimisations steps were carried out with YLDV DNA as template. Using probit regression analysis, the lower limit of detection was calculated to be ca. 8 copies per assay. A total of five TPV strains, one YDLV strain and scab-derived DNA from a patient with a TPV infection yielded specific amplification, whereas the DNA of YMTV was not amplified. Various viral and bacterial pathogens (n=29) associated with rash-causing illnesses were not detected using this assay.

Infection of cells expressing CXCR4 mutants lacking N-glycosylation at the N-terminal extracellular domain is enhanced for R5X4-dualtropic human immunodeficiency virus type-1.

BACKGROUND: Infection with human immunodeficiency virus type-1 (HIV-1) requires binding of the viral envelope gp120 to CD4 and to the CXCR4 coreceptor. Both, gp120 and CXCR4 are subject to N-glycosylation. Here we investigated the influence of the N-linked glycans g1 and g2 present on CXCR4 for HIV-1 infection. METHODS: The two CXCR4 N-glycosylation sites g1 (NYT) and g2 (NVS) were mutated by changing the first or third amino acids N or T/S to Q and A respectively (g1; N11Q or T13A; g2, N176Q or S178A). Human osteosarcoma cells (GHOST) expressing human CD4 and the various CXCR4 glycosylation mutants were tested for infection using NL4-3-based viruses with X4, R5 or R5X4-tropism differing only in the V3 loop region. RESULTS: All constructed cell lines expressing the various CXCR4 glycomutants showed similar permissiveness for the X4-monotropic virus and no change in the coreceptor specificity that allows infection of a CCR5-dependent R5-monotropic virus. Interestingly, the removal of glycan g1 significantly enhanced the permissiveness of GHOST cells for the R5X4 dualtropic virus. GHOST cells expressing the CXCR4-g1 or CXCR4-g1/2 mutants were infected at higher rates by the R5X4-dualtropic virus compared to cells expressing CXCR4-wt or CXCR4-g2 coreceptors. CONCLUSION: Our present observations underscore a role for glycans present on the CXCR4 coreceptor in the entry process of HIV-1. The data will help to better understand the multifaceted mechanism of HIV infection and the selective forces which drive HIV-1 evolution from mono- to dual-tropism.