Severe underquantification of HIV-1 group O isolates by major commercial PCR-based assays.

HIV-1 diversity poses major challenges to viral load assays because genetic polymorphisms can impede nucleic acid detection. In addition to the on-going viral diversification within the HIV-1 group M pandemic, HIV-1 genetic diversity is further increased by non-group M infections, such as HIV-1 groups O (HIV-1-O), N and P. We here conducted a systematic evaluation of commercially available PCR assays to detect HIV-1-O isolates. We collected 25 primary HIV-1-O isolates covering all genetic clusters within HIV-1-O. Subsequently, this panel of isolates was tested on eight commercially available quantitative and five qualitative HIV-1 PCR-based assays in serial dilutions. Sequence analyses were performed for severe cases of underquantification or lack of detection. We observed differences between the assays in quantification that depended on the HIV-1-O isolate's subgroup. All three tested HIV-1-O subgroup IV isolates were underquantified by the Roche CAP/CTM >800-fold compared to the Abbott RealTime assay. In contrast, the latter assay underquantified several subgroup I isolates >200-fold. Notably, the Xpert HIV-1 Viral Load test from Cepheid failed to detect two of the HIV-1-O isolates, whereas the Roche Cobas 8800 assay readily detected all isolates. Comparative sequence analyses identified polymorphisms in the HIV-1-O long-terminal repeat and integrase genes that likely underlie inadequate nucleic acid amplification. Potential viral load underquantification should be considered in therapeutic monitoring of HIV-1-O-infected patients. Pre-clinical assessments of HIV-1 diagnostic assays could be harmonized by establishing improved and internationally standardized panels of HIV-1 isolates that cover the dynamic diversity of circulating HIV-1 strains.

Incidence of CMV co-infection in HIV-positive women and their neonates in a tertiary referral centre: a cohort study.

Co-infection with CMV in HIV-positive pregnant women is associated with perinatal mother-to-child transmission (MTCT) of both viruses. This retrospective study reports on the incidence of maternal and neonatal CMV (presence of anti-CMV IgG and IgM, CMV DNA PCR and/or CMV virus isolation) in high-risk pregnancies due to maternal HIV infection, MTCT of HIV and/or CMV. One hundred and eleven maternal samples and 75 matched neonatal samples were available for HIV and subsequent CMV testing. In this cohort of HIV-positive pregnant women, 96 (86.5 %) serum samples were anti-CMV IgG positive. In nine (9.4 %) of these, anti-CMV IgM was detected, and in none of them a maternal primary CMV infection was suspected. Fifty-seven (51.8 %) maternal serum samples were tested retrospectively by CMV DNA PCR; one sample was positive (0.9 %). All matched neonates were tested for HIV by PCR in the first month of life; HIV transmission was detected in one case. In 74 (67.2 %) of neonates, CMV testing was performed. Sixty-six of these serum samples were tested retrospectively by CMV DNA PCR. Two newborns (2.7 %) showed laboratory markers for CMV infection (one by detection of CMV DNA in plasma, and one by isolation of CMV from a urine sample). In the follow-up, neither of these two showed clinical signs for active CMV disease. We discussed these findings in the light of the national official guidelines. All CMV transmissions occurred due to maternal reinfection or endogenous reactivation. This suggests the success of highly active antiretroviral therapy in preventing MTCT of HIV and CMV disease and highlights the importance of adequate care and follow-up.

Evaluation of two HIV antibody confirmatory assays: Geenius™ HIV1/2 Confirmatory Assay and the recomLine HIV-1 & HIV-2 IgG Line Immunoassay.

The laboratory diagnosis of an HIV infection mainly depends on the detection of HIV-specific antibodies/HIV p24 antigen whereby different algorithms for the confirmation of reactive screening assays exist. The objective of the present study was to compare the performance of two supplemental HIV antibody confirmatory assays: the Geenius™ HIV1/2 Confirmatory Assay and the recomLine HIV-1 & HIV-2 IgG Line Immunoassay. Therefore 279 serum samples previously analyzed for HIV during routine diagnostics at the Institute for Medical Virology, National Reference Center for Retroviruses, University Hospital Frankfurt, were analyzed retrospectively. 96.8% samples had concordant results in both HIV confirmatory assays, whereby the Geenius Assay showed a discrimination rate of 100% while two HIV-1 samples were not typeable with the recomLine Assay. Overall assay sensitivity was 100% in both assays and specificity was 99.0% (recomLine Assay) and 93.4% (Geenius Assay), respectively. The κ-values for both assays indicated high agreement. Overall nine samples had discordant results from which four were from acutely EBV/CMV-infected patients and one from a patient with primary HIV-1 infection during seroconversion. In conclusion, both assays are well suited for the detection, confirmation and discrimination of HIV-1- and -2-specific antibodies.

Detection of herpesvirus EBV DNA in the lower respiratory tract of ICU patients: a marker of infection of the lower respiratory tract?

Epstein-Barr virus (EBV) is a lymphotropic herpesvirus causing clinically self-limiting but lifelong persisting infections. Although several severe diseases (e.g., Hodgkin's disease) are associated with EBV, its role in lower respiratory tract infections is still elusive. The prevalence of EBV, herpes simplex virus (HSV) and cytomegalovirus (CMV) in bronchoalveolar fluid (BAL) samples was evaluated in a retrospective study. BAL samples from 135 patients in the intensive or coronary care unit (ICU/ICC) at University Hospital Frankfurt/Main (Germany) were investigated using an in-house real-time PCR to detect EBV-, HSV- and CMV-specific DNA. Overall, herpesvirus DNA was detected in n = 82/135 BAL samples (60.7 %). Besides mono-infections with either EBV or HSV, concomitant infection with EBV and HSV DNA was most frequent, whereby the relative HSV viral load was typically higher. Patients with HSV-positive BAL required mechanical ventilation on average 5 days longer than patients with HSV-negative BAL (p = 0.006). Additionally, the proinflammatory cytokine IL-6 was significantly elevated in sera of patients positive for EBV in comparison with patients with EBV-negative BAL (p = 0.01). This study demonstrates a high prevalence of herpesviruses in BAL samples of ICU/ICC patients. The detection of one or more herpesvirus in BAL is strongly associated with the duration of ventilation and patient's age. The association between IL-6 levels and EBV detection should be evaluated in further studies.

Evidence for efficient uptake and incorporation of sialic acid by eukaryotic cells.

Sialic acids are the most abundant terminal carbohydrate moiety on cell surface glycoconjugates in eukaryotic cells and are of functional importance for many biological ligand-receptor interactions. It is a widely accepted view that sialic acids cannot be efficiently taken up from the extracellular space by eukaryotic cells. To test this assumption, we cultivated two recently identified human hematopoetic cell lines which are hyposialylated due to a deficiency in de novo sialic acid biosynthesis in the presence of N-acetylneuraminic acid (NeuAc), the most frequently found sialic acid. Surprisingly, NeuAc medium supplementation rapidly and potently compensated for the endogenous hyposialylation in a concentration-dependent manner, resulting in the presentation of cell surface sialoglycans involved in cell adhesion, virus infection and signal transduction. We provide several lines of experimental evidence that all suggest that NeuAc was neither extracellularly incorporated nor degraded to a less complex sugar before uptake. Importantly, NeuAc induced a marked increase in intracellular CMP-NeuAc levels in both human cell lines and in primary cells regardless of the prior sialylation status of the cells. Studies employing 9-[3H]NeuAc revealed an uptake consistent with the observed incorporation of unlabeled NeuAc. We propose the existence of an efficient uptake mechanism for NeuAc in eukaryotic cells.

Efficient biochemical engineering of cellular sialic acids using an unphysiological sialic acid precursor in cells lacking UDP-N-acetylglucosamine 2-epimerase.

Sialic acids comprise a family of terminal sugars essential for a variety of biological recognition systems. N-Propanoylmannosamine, an unphysiological sialic acid precursor, is taken up and metabolized by mammalian cells resulting in oligosaccharide-bound N-propanoylneuraminic acid. N-Propanoylmannosamine, applied to endogenously hyposialylated subclones of the myeloid leukemia HL60 and of the B-cell lymphoma BJA-B, both deficient in UDP-N-acetylglucosamine 2-epimerase, is efficiently metabolized to CMP-N-propanoylneuraminic acid resulting in up to 85% of glycoconjugate-associated sialic acids being unphysiological N-propanoylneuraminic acid. Thus, UDP-N-acetylglucosamine 2-epimerase-deficient cell lines provide an important experimental progress in engineering cells to display an almost homogeneous population of defined, structurally altered sialic acids.

Susceptibility of rat-derived cells to replication by human immunodeficiency virus type 1.

Progress in developing a small animal model of human immunodeficiency virus type 1 (HIV-1) disease would greatly facilitate studies of transmission, pathogenesis, host immune responses, and antiviral strategies. In this study, we have explored the potential of rats as a susceptible host. In a single replication cycle, rat cell lines Rat2 and Nb2 produced infectious virus at levels 10- to 60-fold lower than those produced by human cells. Rat-derived cells supported substantial levels of early HIV-1 gene expression, which was further enhanced by overexpression of human cyclin T1. Rat cells displayed quantitative, qualitative, and cell-type-specific limitations in the late phase of the HIV-1 replication cycle including relative expression levels of HIV-1 Gag proteins, intracellular Gag processing, and viral egress. Nb2 cells were rendered permissive to HIV-1 R5 viruses by coexpression of human CD4 and CCR5, indicating that the major restriction on HIV-1 replication was at the level of cellular entry. We also found that primary rat lymphocytes, macrophages, and microglia expressed considerable levels of early HIV-1 gene products following infection with pseudotyped HIV-1. Importantly, primary rat macrophages and microglia, but not lymphocytes, also expressed substantial levels of HIV-1 p24 CA and produced infectious virions. Collectively, these results identify the rat as a promising candidate for a transgenic small animal model of HIV-1 infection and highlight pertinent cell-type-specific restrictions that are features of this species.

Biosynthesis of N-acetylneuraminic acid in cells lacking UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase.

The first two steps in mammalian biosynthesis of N-acetylneuraminic acid, an important carbohydrate moiety in biological recognition systems, are performed by the bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase. A subclone of the human B lymphoma cell line BJA-B K20, lacking UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase mRNA as well as epimerase activity, displayed hyposialylated, functionally impaired cell surface glycoconjugates. Here we show that this cell line surprisingly still retains N-acetylmannosamine kinase activity. A gel filtration analysis of BJA-B K88 control cells, which express UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, revealed two N-acetylmannosamine kinase activity peaks, one co-eluting with UDP-N-acetylglucosamine 2-epimerase activity and one co-eluting with N-acetylglucosamine kinase. For this enzyme previous studies already showed a ManNAc kinase activity in vitro. In contrast, the hyposialylated BJA-B K20 subclone displayed only the N-acetylmannosamine kinase peak, co-migrating with N-acetylglucosamine kinase. The CMP-N-acetylneuraminic acid content of both K88 and K20 cells and the sialylation of cell surface glycoconjugates of K20 cells could be significantly increased by supplementing the medium with N-acetylmannosamine. This N-acetylmannosamine-induced increase was drastically reduced by co-supplementation with N-acetylglucosamine only in K20 cells. We therefore propose the phosphorylation of N-acetylmannosamine as a hitherto unrecognized role of N-acetylglucosamine kinase in living cells.

Biochemical engineering of the N-acyl side chain of sialic acid: biological implications.

N-Acetylneuraminic acid is the most prominent sialic acid in eukaryotes. The structural diversity of sialic acid is exploited by viruses, bacteria, and toxins and by the sialoglycoproteins and sialoglycolipids involved in cell-cell recognition in their highly specific recognition and binding to cellular receptors. The physiological precursor of all sialic acids is N-acetyl D-mannosamine (ManNAc). By recent findings it could be shown that synthetic N-acyl-modified D-mannosamines can be taken up by cells and efficiently metabolized to the respective N-acyl-modified neuraminic acids in vitro and in vivo. Successfully employed D-mannosamines with modified N-acyl side chains include N-propanoyl- (ManNProp), N-butanoyl- (ManNBut)-, N-pentanoyl- (ManNPent), N-hexanoyl- (ManNHex), N-crotonoyl- (ManNCrot), N-levulinoyl- (ManNLev), N-glycolyl- (ManNGc), and N-azidoacetyl D-mannosamine (ManNAc-azido). All of these compounds are metabolized by the promiscuous sialic acid biosynthetic pathway and are incorporated into cell surface sialoglycoconjugates replacing in a cell type-specific manner 10-85% of normal sialic acids. Application of these compounds to different biological systems has revealed important and unexpected functions of the N-acyl side chain of sialic acids, including its crucial role for the interaction of different viruses with their sialylated host cell receptors. Also, treatment with ManNProp, which contains only one additional methylene group compared to the physiological precursor ManNAc, induced proliferation of astrocytes, microglia, and peripheral T-lymphocytes. Unique, chemically reactive ketone and azido groups can be introduced biosynthetically into cell surface sialoglycans using N-acyl-modified sialic acid precursors, a process offering a variety of applications including the generation of artificial cellular receptors for viral gene delivery. This group of novel sialic acid precursors enabled studies on sialic acid modifications on the surface of living cells and has improved our understanding of carbohydrate receptors in their native environment. The biochemical engineering of the side chain of sialic acid offers new tools to study its biological relevance and to exploit it as a tag for therapeutic and diagnostic applications.

UDP-GlcNAc 2-epimerase: a regulator of cell surface sialylation.

Modification of cell surface molecules with sialic acid is crucial for their function in many biological processes, including cell adhesion and signal transduction. Uridine diphosphate-N-acetylglucosamine 2-epimerase (UDP-GlcNAc 2-epimerase) is an enzyme that catalyzes an early, rate-limiting step in the sialic acid biosynthetic pathway. UDP-GlcNAc 2-epimerase was found to be a major determinant of cell surface sialylation in human hematopoietic cell lines and a critical regulator of the function of specific cell surface adhesion molecules.

Differential sialylation of cell surface glycoconjugates in a human B lymphoma cell line regulates susceptibility for CD95 (APO-1/Fas)-mediated apoptosis and for infection by a lymphotropic virus.

Sialic acid, as a terminal saccharide residue on cell surface glycoconjugates, plays an important role in a variety of biological processes. In this study, we investigated subclones of the human B lymphoma cell line BJA-B for differences in the glycosylation of cell surface glycoconjugates, and studied the functional implications of such differences. With respect to the expression level of most of the tested B cell-associated antigens, as well as the presence of penultimate saccharide moieties on oligosaccharide chains, subclones were phenotypically indistinguishable. Marked differences among subclones, however, were found in the overall level of glycoconjugate sialylation, involving both alpha-2,6 and alpha-2,3-linked sialic acid residues. Accordingly, subclones were classified as highly- (group I) or hyposialylated (group II). The function of two sialic acid-dependent receptor-mediated processes is correlated with the sialylation status of BJA-B subclones. Susceptibility to and binding of the B lymphotropic papovavirus (LPV) was dependent on a high sialylation status of host cells, suggesting that differential sialylation in BJA-B cells can modulate LPV infection via its alpha-2,6-sialylated glycoprotein receptor. CD95-mediated apoptosis, induced by either the human CD95 ligand or a cytotoxic anti-CD95 monoclonal antibody, was drastically enhanced in hyposialylated group II cells. An increase in endogenous sialylation may be one antiapoptotic mechanism that converts tumor cells to a more malignant phenotype. To our knowledge, this is the first report demonstrating that differential sialylation in a clonal cell line may regulate the function of virus and signal-transducing receptors.

Elongation of the N-acyl side chain of sialic acids in MDCK II cells inhibits influenza A virus infection.

The interaction of influenza A virus with sialyated receptor components is one of the best characterized ligand-receptor interactions. We pretreated MDCK II host cells with three different N-acyl-modified sialic acid precursor analogues, N-propanoyl, N-butanoyl or N-pentanoyl D-mannnosamine. Cellular sialic acid biosynthesis yielded 18-35% of new, modified sialic acids on cell surface glycoconjugates, N-propanoyl, N-butanoyl or N-pentanoyl neuraminic acid, respectively. The elongation of the N-acyl group of sialic acids resulted in an inhibition of influenza A virus (strain X31) binding and subsequent infection of up to 80%. In contrast, the sialic acid-independent infection of vesicular stomatitis virus was unaffected in these cells. Molecular modeling studies based on the crystal structure of the influenza A virus hemagglutinin complexed with sialyllactose suggest a steric hindrance of hemagglutinin binding to aliphatically elongated N-acyl groups. We propose that biosynthetic sialic acid modification in conjunction with molecular modeling is a potent tool to further analyze the influenza A virus-receptor interaction.

Biosynthetic modulation of sialic acid-dependent virus-receptor interactions of two primate polyoma viruses.

Sialic acids are essential components of the cell surface receptors of many microorganisms including viruses. A synthetic, N-substituted D-mannosamine derivative has been shown to act as precursor for structurally altered sialic acid incorporated into glycoconjugates in vivo (Kayser, H., Zeitler, R., Kannicht, C., Grunow, D., Nuck, R., and Reutter, W. (1992) J. Biol. Chem. 267, 16934-16938). In this study we have analyzed the potential of three different sialic acid precursor analogues to modulate sialic acid-dependent virus receptor function on different cells. We show that treatment with these D-mannosamine derivatives can result in the structural modification of about 50% of total cellular sialic acid content. Treatment interfered drastically and specifically with sialic acid-dependent infection of two distinct primate polyoma viruses. Both inhibition (over 95%) and enhancement (up to 7-fold) of virus binding and infection were observed depending on the N-acyl substitution at the C-5 position of sialic acid. These effects were attributed to the synthesis of metabolically modified, sialylated virus receptors, carrying elongated N-acyl groups, with altered binding affinities for virus particles. Thus, the principle of biosynthetic modification of sialic acid by application of appropriate sialic acid precursors to tissue culture or in vivo offers new means to specifically influence sialic acid-dependent ligand-receptor interactions and could be a potent tool to further clarify the biological functions of sialic acid, in particular its N-acyl side chain.

Regulation of susceptibility and cell surface receptor for the B-lymphotropic papovavirus by N glycosylation.

The host range of the B-lymphotropic papovavirus (LPV) in cultured human cells is limited to a few B-lymphoma-derived cell lines. The constitutively expressed cell surface receptor for the virus is a major determinant restricting the LPV host range (G. Haun, O. T. Keppler, C. T. Bock, M. Herrmann, H. Zentgraf, and M. Pawlita, J. Virol. 67:7482-7492, 1993). Here we show that human B-lymphoma cells with low-level susceptibility are rendered highly susceptible to LPV infection by pretreatment with the N glycosylation inhibitor tunicamycin but remain nonsusceptible to infection by the related polyomavirus simian virus 40. Among the selective N glycosylation processing inhibitors, deoxymannojirimycin, but not deoxynojirimycin, swainsonine, or castanospermine, could mimic the effect of tunicamycin. Tunicamycin treatment also induced a drastic enhancement of the cells' LPV-binding capacity, indicating that the induction of LPV susceptibility might be mediated by an increase in the number of functional cell surface receptors and/or by increased receptor affinity. Sialidase sensitivity of the tunicamycin-induced LPV receptor showed that oligosaccharides carrying terminal sialic acids are necessary for binding and are likely to be O linked. The constitutive LPV receptor is also sialic acid dependent, which points to a possible identity with the sialic acid-dependent tunicamycin-induced LPV receptor. We conclude that removal or modification of certain N-linked oligosaccharides in human B-lymphoma cells can enhance expression or functional activity of the sialylated LPV receptor.

The cell surface receptor is a major determinant restricting the host range of the B-lymphotropic papovavirus.

The B-lymphotropic papovavirus (LPV) productively infects only a subset of human B-lymphoma-derived cell lines while transfection of the viral genome yields infectious viral particles in a much wider variety of human hematopoietic cell lines. We have analyzed the contribution of a putative LPV receptor on the cell surface of B-cell lines in restricting the virus host range. In order to establish a quantitative virus binding assay for LPV, infectious virus particles were highly purified by metrizamide equilibrium density centrifugation and used as immunogens to raise seven mouse monoclonal antibodies specific for LPV VP1. Virus particle binding was quantitated in an indirect, nonradioactive assay with an LPV VP1-specific enzyme-linked immunosorbent assay. Binding of LPV particles to permissive human B-lymphoma cell line BJA-B occurred within minutes. Kinetics and capacity of binding were similar at 4 and 37 degrees C. A BJA-B cell was estimated to bind approximately 600 virus particles at conditions under which 50% of the administered virus was bound. The sialidase and trypsin sensitivities of the cellular virus binding moiety show that sialylated and proteinaceous components are necessary components of the LPV receptor on BJA-B cells. Despite a high binding capacity of BJA-B cells for simian virus 40, LPV binding was not significantly affected by a 20-fold excess of simian virus 40 particles, indicating that these related polyomaviruses do not bind to the same receptor on BJA-B cells. Reduction of LPV binding to sialidase-pretreated BJA-B cells was accompanied by a similar reduction of infection, indicating that virus binding may be a limiting factor in the LPV replicative cycle. The two highly LPV-permissive human B-lymphoma cell lines BJA-B and Namalwa displayed high virus binding whereas low and nonpermissive hematopoietic cell lines showed reduced or undetectable virus binding. We conclude that the inability of LPV particles to productively infect the nonpermissive human hematopoietic cell lines analyzed is probably due to the absence or insufficient expression of a functional cell surface receptor.

Human Golgi beta-galactoside alpha-2,6-sialyltransferase generates a group of sialylated B lymphocyte differentiation antigens.

The role of the human beta-galactoside alpha-2,6-sialyltransferase (hu alpha-2,6-ST) in the generation of B cell surface antigens was investigated by selecting subclones of COS cells (monkey kidney epithelial cells) constitutively expressing a transfected cDNA which encodes the hu alpha-2,6-ST (COS alpha-2,6-ST cells). Expression of hu alpha-2,6-ST in COS cells was sufficient to generate sialylated cell surface epitopes on different glycosylated antigens recognized by monoclonal antibodies to CDw75, CD76, and the unclustered monoclonal antibodies HB-4 and EBU-65. These epitopes were sensitive to sialidase treatment and are likely to contain terminal alpha-2,6-linked sialic acid residues. A novel antiserum raised against bacterially expressed hu alpha-2,6-ST fusion protein was used to localize the sialyltransferase in two cell lines with high expression of either endogenous (B cell line JOK-1) or recombinant (COS alpha-2,6-ST cells) hu alpha-2,6-ST. In both cell lines, the enzyme was detected only intracellularly in the juxtanuclear region and not on the cell surface. In contrast, CDw75, formerly proposed to be identical with an alpha-2,6-ectosialyltransferase, was strongly expressed on the cell surface. The different expression patterns show that neither the CDw75 antigen nor any of the other sialylated antigens analyzed is identical with the hu alpha-2,6-ST. Furthermore, the presence of a surface-expressed alpha-2,6-ST appears unlikely in these cell lines. We propose that CDw75, CD76, HB-4, and EBU-65 represent a unique group of B cell differentiation antigens the production of which requires the enzymatic activity of alpha-2,6-ST.