Internalization of human rhinovirus 14 into HeLa and ICAM-1-transfected BHK cells.

Virus adsorption and uptake of human rhinovirus 14 (HRV14) were studied with HeLa cells and baby hamster kidney (BHK) cells which were transfected with the HRV14 receptor intercellular adhesion molecule-1 (ICAM-1). Transmission electron microscopy of HeLa cells revealed that HRV14 was internalized via clathrin-coated pits and -coated vesicles. A minority of virus particles also used uncoated vesicles for entry. The internalization showed the characteristics of receptor-mediated endocytosis. Presence of the carboxylic ionophore monensin inhibited viral uncoating, indicating a pH-dependent entry mechanism. The expression of ICAM-1 on the surface of the ICAM-1 transfected baby hamster kidney cells (BHK-ICAM cells) allowed extensive virus adsorption and internalization through membrane channels. Virus particles were lined up in these channels like pearls on a string, but did not induce a productive infection. Although ICAM-1 was expressed to the same degree on BHK-ICAM and HeLa cells, HRV14 induced neither viral protein and RNA syntheses nor infectious virus progeny in BHK-ICAM cells. ICAM-1 on the transfected BHK cells was a functional active receptor as it rendered these cells permissive to coxsackievirus A21. These results suggest that HRV14 uptake into BHK-ICAM cells is blocked directly in or shortly after its final step of internalization, the uncoating. Our findings underline that the receptor ICAM-1 determines virus uptake into cells, however, is not sufficient to confer susceptibility of BHK cells to HRV14 infection.

Glycoprotein B (gB) of pseudorabies virus interacts specifically with the glycosaminoglycan heparin.

We have previously shown that the pseudorabies virus (PrV) glycoproteins gB and gC (former PrV-gII and PrV-gIII) exhibit heparin-binding properties. While PrV-gC functions as the major adsorption protein, the biological role of the heparin-binding properties of PrV-gB are not understood. We used a gC-deleted PrV-mutant, PrV (dlg92/dltk), to analyse the heparin-binding properties of PrV-gB and the biological role of the PrV-gB-protein in adsorption. PrV-gB was the only glycoprotein of this vaccine strain binding to immobilised heparin in in vitro assays. Presence of the gC-protein was not necessary for the interaction of gB with heparin. Soluble heparin also interfered with adsorption of this mutant virus to a similar extent as it blocked adsorption of wild-type PrV (Ka), but it had only a minor inhibitory effect on infectivity of the mutant strain. These results show that PrV-gB interacts specifically with immobilized heparin and heparin-like structures on the cell surface, but this interaction is not required for a productive infection.

Multicentre study for diagnostic evaluation of an assay for simultaneous detection of antibodies to HIV-1, HIV-2 and HIV-1 subtype 0 (HIV-0).

The aim of the study was to evaluate a new ELISA for detection of HIV-1, HIV-2 and HIV-1 subtype 0 (HIV-0) antibodies. The assay format is based on the antigen sandwich principle. To enable specific detection of HIV-0 antibodies, in addition to HIV-1 and HIV-2 antigens HIV-0 antigen is used for coating the solid phase and for the conjugate. The results show that all 12 HIV-0 samples tested were detected with a high degree of reactivity, as were all the 1,144 anti-HIV-1 and 424 anti-HIV-2 positive samples. The capacity of the test to enable early detection of seroconversions is equivalent to that of other sandwich ELISAs. The specificity of the assay was determined to be 99.89/99.94% (initial/after retest) using 58,366 samples, which is superior to the other ELISAs used for comparison. Even with difficult samples (i.e. samples of African origin, samples known to cause false-positive reactivity in different ELISAs, or samples containing potential interference factors) there were very few false-positive reactions. Therefore, the new assay is well suited for screening blood donations as well as for evaluating samples from patients of different geographic origin.

Cellular receptor structures for pseudorabies virus are blocked by antithrombin III.

Pseudorabies virus (PrV), an alphaherpesvirus of swine, uses cellular heparan sulfate residues as a receptor for attachment. Interaction of the virus with its receptor is mediated by the envelope glycoprotein C (PrV-gC), a protein with heparin-binding properties. We have previously shown that a region of this protein shows structural similarities to the high-affinity heparin-binding site of the serum protease-inhibitor antithrombin III (ATII). In this publication, we describe the effect of ATIII on interaction of PrV with its cellular receptor. ATIII bound specifically to heparan sulfate residues on the surface of herpesvirus-permissive RK13 cells. Binding of ATIII to RK13 cells interfered with adsorption of radioactively labelled PrV to these cells. Enzymatic treatment using heparinase I (E.C. 4.2.2.7) removed the receptor for PrV as well as the receptor for ATIII. Since amino acids 130-137 of the high affinity heparin-binding site of ATIII show structural similarities to amino acids 134-141 of PrV-gC, both sequences were synthesized as synthetic peptides. Although interaction of the peptide derived from ATIII with heparin was significantly stronger, both peptides interacted specifically with heparin in assays in vitro. These results suggest that PrV and ATIII interact with the same structure on the cellular surface.

Low prevalence of hepatitis C virus infection in porphyria cutanea tarda in Germany.

Previous studies from Spain, Italy, and France have demonstrated a high prevalence (71% to 91%) of antibodies against hepatitis C virus in patients with porphyria cutanea tarda (PCT). To determine the role of hepatitis C virus (HCV) in PCT in Germany, we have assessed the prevalence of antibodies against HCV and hepatitis B virus (HBV) in 106 patients (mean age, 60 +/- 14 years) with the disease. Eight of 106 patients (8%) were positive for HCV antibodies and HCV RNA using second-generation enzyme-linked immunosorbent assay (ELISA), recombinant immunoblot assay, and polymerase chain reaction. Antibodies against HBV core antigen were found in 14 patients (13%). Of the patients with antibodies against HCV alanine transaminase (ALT) (aspartate transaminase [AST]) levels above normal occurred in 71% (86%). Because elevated ALT (AST) levels were also found in 51% (64%) of 88 patients without markers of HCV or HBV, we suggest that liver damage in PCT may exist in absence of these viruses. This is supported by the finding that in patients without HCV or HBV markers, higher serum ALT and AST activities were found in patients with overt disease or relapse (ALT, 59 +/- 44 U/L; AST, 37 +/- 21 U/L), whereas patients in remission displayed significantly lower serum enzyme activities (ALT, 16 +/- 8 U/L; AST, 16 +/- 7 U/L), (P < .001). These results indicate that HCV infection does not play a major role in the pathogenesis of PCT in Germany.

A peptide-model for the heparin-binding property of pseudorabies virus glycoprotein III.

The pseudorabies virus glycoprotein III (PrV-gIII) has been identified previously as the major viral component binding to a heparin-like receptor on the surface of target cells. The amino acid sequence of gIII contains three regions corresponding to consensus sequences for heparin binding. A synthetic peptide corresponding to amino acids 134 to 141 of PrV-gIII bound heparin in a dot blot assay. In contrast, a synthetic peptide derived from amino acids 290-299 of PrV-gIII did not bind heparin. We therefore conclude that the region containing amino acid 134-141 is involved in binding to the heparin-like cellular receptor.

The effect of treatment with zidovudine with or without acyclovir on HIV p24 antigenaemia in patients with AIDS or AIDS-related complex.

OBJECTIVE: To evaluate changes in serum HIV p24-antigen levels in a subset of patients who participated in a European/Australian double-blind, placebo-controlled trial evaluating the efficacy of zidovudine (250 mg every 6 h) alone or in combination with acyclovir (800 mg every 6 h) in patients with AIDS, AIDS-related complex (ARC) or Kaposi's sarcoma (KS). DESIGN: Double-blind, placebo-controlled randomized clinical trial of less than or equal to 6 months' therapy. SETTING: Samples were obtained from patients attending teaching hospital outpatient clinics in seven European countries and Australia. SUBJECTS: One hundred and ninety-seven HIV-infected patients (60 with AIDS and 137 with ARC or KS). MAIN OUTCOME MEASURES: Serum HIV p24-antigen levels measured using the Abbott HIV solid-phase enzyme immunoassay. RESULTS: Of 76 ARC/KS patients who were initially HIV p24-antigen-positive, one out of 25 randomized to placebo, eight out of 23 to zidovudine and 11 out of 28 to the zidovudine/acyclovir combination became antigen-negative. The proportion of patients who became antigen-negative was significantly higher in both the zidovudine group (P = 0.016) and the zidovudine/acyclovir group (P = 0.004), compared with the placebo group. There were no statistical differences between the zidovudine and the zidovudine/acyclovir groups. During the trial p24-antigen levels in the zidovudine-treated patients reached their minimum after 4-8 weeks of therapy, and tended to increase gradually thereafter. Disease progression occurred irrespective of whether p24-antigen levels declined during therapy. No association between p24-antigen responses to therapy and baseline disease stage, Karnofsky score or baseline CD4 cell count was detectable. CONCLUSION: Acyclovir does not potentiate the effect of zidovudine on p24-antigen levels. Change in antigen level in response to antiviral therapy needs further investigation before it is used as a surrogate marker for clinical efficacy of antiviral therapy.

Identification of 50- and 23-/25-kDa HeLa cell membrane glycoproteins involved in poliovirus infection: occurrence of poliovirus specific binding sites on susceptible and nonsusceptible cells.

Glycoproteins in the range 50 and 23/25 kDa were identified as poliovirus specific binding sites on HeLa cells with the monoclonal antibody mAb 122. mAb 122 is characterized by its partial inhibiting effect on poliovirus reproduction and adsorption when prebound to HeLa cells. The binding sites are endocytosed in native cells and specific for poliovirus as mAb 122 did not interfere with the adsorption of human rhinovirus type 14 (HRV 14). The poliovirus binding sites are present also on nonprimate so called nonsusceptible cells, e.g., mouse L-cells, as could be shown with sensitive ELISA based binding assays and performance of binding studies with fixed cells at 37 degrees.

A rapid and sensitive micro scale assay for quantitative detection of cell protective effects: application for the isolation of a monoclonal antibody against HeLa cell proteins involved in poliovirus attachment.

A combined assay consisting of a pre-cpe-protection assay and a double-antibody sandwich ELISA for detecting poliovirus was developed on a microtiter scale in order to quantify inhibition of virus replication caused by cell protective antibodies. The system was of high sensitivity and allowed the measurement of the protecting effect caused by a broad range of antibody concentrations before appearance of cytopathic effects. It was applied as a screening test for a large number of hybridomas secreting antibodies specific to the surface of HeLa cells and allowed the identification of four monoclonal antibodies (mAbs) with partial protection activity against poliovirus infection. One of the antibodies, mAb 122, detected SDS-PAGE-separated HeLa cell membrane proteins of 23-25 kDa and 50 kDa by immunoblot, indicating that these proteins are involved in poliovirus adsorption.

Processing of pseudorabies virus glycoprotein gII.

The glycoprotein complex gII of pseudorabies virus was isolated by immunoprecipitation with the monoclonal antibody M5, which was covalently linked to protein A-Sepharose. After sodium dodecyl sulfate-polyarylamide gel electrophoresis under reducing conditions and blotting onto poly(vinylidene difluoride) membrane, its subunits, gIIa, gIIb, and gIIc, were subjected to N-terminal sequencing. gIIa and gIIb start at position 59 and gIIc starts at position 503 according to the amino acid sequence deduced from the gene, indicating that there is one major protein (gIIa) which is cleaved into the two protein fragments gIIb and gIIc. Protein labeling with 14C-amino acids gave no indication that the three proteins (gIIa, gIIb, and gIIc) of the complex are present in equimolar ratios. It seems that gIIa is only a minor component of the complex, whereas gIIb and gIIc are contained in equimolar amounts.

Comparison of heparin-sensitive attachment of pseudorabies virus (PRV) and herpes simplex virus type 1 and identification of heparin-binding PRV glycoproteins.

To determine whether heparan sulphate residues on the cellular surface could serve as an attachment receptor for pseudorabies virus (PRV), the effect of heparin on PRV in plaque reduction and adsorption tests was investigated. PRV was significantly less sensitive to heparin than was herpes simplex virus type 1 (HSV-1). At concentrations of 500 micrograms/ml heparin the number of plaques formed by PRV was reduced to 7% of the untreated control whereas the number of plaques formed by HSV-1 was reduced to below 0.1%. Adsorption of PRV to host cells was also less sensitive to heparin treatment than was adsorption of HSV-1. Experiments concerning the binding sites of PRV showed that heparin binds to the disulphide-linked glycoprotein complex gII (PRV gB), gIII (PRV gC) and probably gV.

Entry of pseudorabies virus into CHO cells is blocked at the level of penetration.

Replication of pseudorabies virus (PrV) in Chinese hamster ovary (CHO) cells, a cell line naturally resistant to infection by herpesviruses, is blocked at the level of penetration. Virions bound to the surface of CHO cells are taken up into cytoplasmic vesicles and degraded.

Recovery of structurally intact and infectious poliovirus type 1 from HeLa cells during receptor-mediated endocytosis.

Poliovirus type 1 enters HeLa cells by receptor-mediated endocytosis as an intact virus. Up to 30 min after adsorption complete virus particles still containing VP4 and sedimenting with 156 S could be recovered from the cells. These virus particles were N-antigenic and infectious. Thirty minutes after adsorption the recovery of intact and infectious virus decreased. This decrease presumably reflects viral uncoating in the acidic endosomes and/or lysosomes because virus particles could be localized in endosomes at this time. The direct involvement of clathrin-coated structures in the endocytosis of poliovirus has been deduced from the enclosure of poliovirus in coated vesicles at 10 min after adsorption. At this time intact and infectious virus could be recovered only after the coated vesicles were disrupted by treatment with 0.5 M Tris at pH 7.0.

Medicion de los anticuerpos contra el virus de la inmunodeficiencia humana: estudio internacional en colaboración para evaluar los sueros de referencia de la OMS

Two preparations of human sera, one reactive against human immunodeficiency virus (HIV) and the other unreactive, were evaluated as potential international reference reagents (IRR) in an international collaborative study. Twenty-one laboratories participated and tested these and five other human sera which were found to range from highly reactive to unreactive. The proposed "positive" IRR was found to react strongly in all immunoassays and gave all the expected bands in immunoblot systems using HTLV-III, LAV-I or similar virus strains as antigens. The "unreactive" serum was judged to be negative by ELISA and immunoblots. The end-points determined by ELISAs varied considerably between laboratories, even between those using the same commercial kit. This variation was reduced somewhat when the reactivities of the samples were expressed relative to the proposed IRR

Measurement of antibodies to human immunodeficiency virus: an international collaborative study to evaluate WHO reference sera.

PIP: 21 laboratories participated in an international collaborative study to evaluate potential international reference reagents for measurement of antibodies to human immunodeficiency virus (HIV). Given inherent differences between the principles of currently used assays (based on enzyme-linked or radioimmunosorbance, immunofluorescence, immunoblotting, or immunoprecipitation) and batch-to-batch variations in the preparations of reagents and kits, there is an urgent need for well-characterized reference materials that can be used to define the reliability and sensitivity of the tests, for quality control of batches, and as common references between laboratories. The 7 human sera tested included 1 reactive against HIV and 1 unreactive. The study was designed to identify the coded preparations that reacted with HIV antibodies and to ascertain the minimum amount of the reactive samples that could be detected in the methods routinely used by the participants. Participants carried out single or duplicate assays for individual manufacturer's ELISAs or by their local methods. The proposed positive international reference reagent (coded A) reacted strongly in all immunoassays and gave all the expected bands in immunoblot systems using HIV-related virus strains as antigens. The unreactive serum (coded E) was negative by ELISA and immunoblots. Although the end-points determined by ELISAs varied considerably between laboratories, even when the same commercial kit was used, this variation was reduced somewhat when the reactivities of the samples were expressed relative to preparation A. It is concluded that the proposed international reference reagent A may have value as a qualitative check on the specificity of assays, in calibrating positive controls included in kits and other assays in arbitrary units, in calibrating detection limits in arbitrary units, and for calibrating immunoblots, especially for defining the optimal amounts of antigen and determining the relative mobilities of the major HIV peptides and glycopeptides.^ieng