Structural homology between hemagglutinin (HA) of measles virus and the active site of long neurotoxins

The amino acid sequence of a carboxy terminal domain corresponding to the end of the outer envelope projection of the hemagglutinin glycoprotein (HA) of measles and subacute sclerosing panencephalitis viruses has a high degree of homology with the active domain of long neurotoxins, which specifically binds to the nicotinic acetylcholine receptor. The homology in amino acid sequence of HA to this domain of neurotoxin, as well as native alpha-bungarotoxin (BTx), was confirmed by the following evidence: a) rabbit anti-HA monospecific sear reacted with BTx in ELISA, b) HA dose-dependently blocked the binding of radio-labeled BTx in competitive radioimmunoassay, and c) antibody to a synthetic peptide of the active domain of BTx precipitated HA in radioimmunoprecipitation. In addition, SSPE patients had significantly high titers of antibody to BTx than healthy children who had been previously infected with measles. This epitope of HA may play an important role in the transsynaptic spreading of the virus in the brain.     

Cloning and characterization of the cDNA encoding the HA protein of a hemagglutination-defective measles virus strain

cDNA clones corresponding to the mRNA for the hemagglutinin of the hemagglutination-defective strain AK-1 of measles virus were isolated and characterized. Compared with the prototype Edmonstron strain, 60 nucleotide substitutions that resulted in 18 amino acid changes were detected. An additional potential N-linked glycosylation site was added by point mutation, which was supported by the observation that the hemagglutinin of the AK-1 strain was stained more heavily after NaDodSO₄PAGE and periodic acid-Schiff (PAS) staining than the Edmonston strain. Computer-assisted analysis revealed that three reverse turns in the secondary structure had disappeared in the hemagglutinin of the AK-1 strain. Moreover, one of these structural changes occurred in the closely glycosylated region at amino acid residues 168–240, which appeared to be a biologically important functional domain. The isoelectric point calculated from the predicted amino acid sequence became about 1 pH unit more basic in the AK-1 strain than the Edmonston strain. This present study is the first sequence analysis of the hemagglutinin gene in a hemagglutination-defective strain of the measles virus.     

Genetic characterization of wild type measles virus isolated in Croatia during the 2003-2004 outbreak

Viral epidemiology is determined by the movement of infected people within and between geographical areas. The genetic characterization of wild-type isolates combined with standard epidemiological methods may enable the identification of the source and transmission pathways and permit differentiation between indigenous and imported viruses. We investigated the genetic characteristics of the wild-type measles virus isolated in Croatia during a 2003-2004 outbreak. The results of this study indicate the presence of the D4 measles virus genotype in Europe. The isolated virus is closely related to virus isolates from the India-like subgroup of the D4 measles virus genotype. The virus responsible for this outbreak differs in the hemagglutinin gene sequence from other virus strains belonging to the D4 genotype. The hemagglutinin gene sequence also differs when compared to viruses from other genotypes that are known to circulate in Europe and from vaccine strains.     

Molecular analysis of structural protein genes of the Yamagata-1 strain of defective subacute sclerosing panencephalitis virus. III. Nucleotide sequence of the hemagglutinin gene

The full-length cDNA corresponding to the mRNA for the hemagglutinin (H) protein of the Yamagata-1 strain of the subacute sclerosing panencephalitis (SSPE) virus was cloned and the nucleotide sequence was determined. The mRNA corresponding to the H protein was composed of 1952 nucleotides and contained a single large open reading frame, which encoded 620 amino acids with a predicted molecular weight of 69,723. This cDNA clone expressed the H protein in Cos 7 cells, and the transfected cells showed hemadsorption. The nucleotide and amino-acid sequence homology with the Edmonston strain of MV were 98.0% and 96.6%, respectively. The deduced amino acid sequence had a single hydrophobic domain near the N-terminus that was long enough to serve as an anchor in the membrane. Five potential glycosylation sites were found on the H protein at identical positions as in the H protein of MV. Cysteine and proline were located at almost identical positions as those of the H protein of MV. In addition, monoclonal antibody study revealed that three epitopes, including the domains that were involved in the biological activities of the H protein of MV, were conserved on the Yamagata-1 strain. These results suggested that the H protein of the Yamagata-1 strain of defective SSPE virus is structurally and functionally similar to that of the Edmonston strain of MV.     

Detection of IgG antibodies to measles virus using monoclonal antibodies for antigen-capture in enzyme immunoassay

An enzyme immunoassay (EIA) for the detection of IgG antibodies to measles viral hemagglutinin (H) has been developed. Monoclonal antibodies were employed as antigen-capture reagents on a polystyrene ball solid phase. The antigen-capture EIA was significantly more sensitive than hemagglutination inhibition (HAI) and somewhat more sensitive than indirect immunofluorescence and indirect EIA for the detection of antibodies to measles virus. The importance of selecting a monoclonal antibody with a high binding affinity, and controlling for non-specific adherence of antigen or antibodies to the solid phase were demonstrated. This method offers not only greater sensitivity than HAI, but also the practicality of reagents capable of standardization and long term storage.     

Sequence analysis of the hemagglutinin gene of measles virus isolates in Denmark 1997-1998: no evidence of persistent circulation of measles virus in Denmark

The hemagglutinin-coding region of 17 virus samples from 12 measles cases in Denmark during 1997-1998 was analysed by partial nucleotide sequencing. The cases appeared as three sporadic cases and two epidemics, both with a limited time course and geographical distribution. The measles strains identified from the three sporadic cases and two epidemics could be allocated to five different previously well-defined sequence groups consistent with the assumption that cases of measles in Denmark are due to repeated introduction from abroad rather than persistent circulation of strains in the population.     

Effect of carboxylic ionophores on measles virus hemagglutinin protein

Summary We have studied the effect of two carboxylic ionophores, monensin and laidlomycin, on the replication of measles virus in KB cells. The yield of infectious virus was markedly depressed at the concentrations of the ionophores which had no effect on overall viral protein synthesis. The ionophores selectively blocked the migration of hemagglutinin (H) glycoprotein from Golgi apparatus to the cell surface. As a result, H glycoprotein is prevented from being converted from incompletely glycosylated form to the mature form.The inhibitory effect on the transport and glycosylation of H was reversed, although gradually, upon the removal of the ionophores.With 6 Figures     

Radioimmunoassay for the detection of virus-specific IgA antibodies in saliva

The use of a sensitive and versatile radioimmunoassay (RIA) for detection of mumps-specific IgA and measles-specific IgA in unconcentrated saliva samples is described. The samples were obtained either by expectoration or by swabbing of the oral cavity, with or without stimulation of secretion, and were inactivated and clarified before testing. Mumps-specific IgA antibodies were detected as early as one day after onset of illness and peaked at 1-2 weeks after onset. Measles-specific salivary IgA antibodies were detected in 15-month old children 2-3 weeks after immunization. These results suggest that the RIA technique may be useful for early diagnosis of viral infections and for confirmation of response to immunization without the need for a blood sample, as well as for the study of the secretory immune response in very young and older subjects.     

Bovine coronavirus hemagglutinin protein

Treatment of purified bovine coronavirus (Mebus strain) with pronase destroyed the integrity of virion surface glycoproteins gp140, gp120, gp100, reduced the amount of gp26 and destroyed the hemagglutinating activity of the virus. Bromelain, on the other hand, destroyed the integrity of gp120, gp100 and gp26 but failed to remove gp140 and failed to destroy viral hemagglutinating activity. These experiments suggest that gp140 is the virion hemagglutinin. Immunoblotting studies using monospecific antiserum demonstrate that gp140 is a disulfide-linked dimeric structure reducible to monomers of 65 kDa.     

Intracellular processing and antigenic maturation of measles virus hemagglutinin protein

Summary The intracellular processing and antigenic maturation of the measles virus (MV) hemagglutinin (H) protein in virus infected cells were probed with murine monoclonal antibodies (Mabs) that reacted with continuous and discontinuous epitopes. The antibodies distinguished between the immature, cotranslational monomeric form of the protein and the mature, dimeric hemagglutinin structure. This was evidenced by testing of immunoreactivity of the Mabs with synthetic peptides, by in vitro synthesized H protein analysis, and by pulse-chase analysis of gel separated monomeric and dimeric forms of the H protein. Time kinetics analysis showed that the protein was synthesized as monomers and most of them were converted into dimers witht _1/2 about 30 min. The H protein remained endoglycosidase H (Endo H) sensitive up to 30 min and started to acquire partial resistance to Endo H between 30 and 60 min (t _1/2 about 60 min) after synthesis. Oligomerization of the H protein was unaffected in virus infected cells treated with a compound (carbonylcyanide m-chlorophenylhydrazone, CCCP) that blocks transport from the endoplasmic reticulum (ER) to the Golgi complex. These results suggest that the H protein dimerization takes place in the ER before its transport to the medial Golgi complex. The Mabs specific for discontinuous epitopes reacted with the H protein in cells treated with CCCP. Thus conformational antigenic epitope formation appears to take place in the ER.     

Genetic and antigenic characterization of measles viruses that circulated in Korea during the 2000-2001 epidemic

Despite the marked reduction in the incidence of measles in Korea by the introduction of measles vaccine, a large measles epidemic occurred during 2000-2001. During the epidemic, more than 55,000 measles cases were reported and at least 7 children were dead. In this study, we analyzed the genetic and antigenic properties of 15 measles viruses that isolated during the epidemic. Sequence analyses of entire hemagglutinin (H) and nucleoprotein (N) genes of the viruses indicated that all Korean isolates had a high degree of homology (>99.8%) when compared with each other. They differed from other wild-type viruses by as much as 6.8% in the H gene and 6.5% in the N gene at the nucleotide level. The deduced amino acid variability was up to 6.4% for the H protein and up to 6.5% for the N protein. Phylogenetic analysis of nucleotide sequences and deduced amino acid sequences of the H and N genes revealed that all Korean viruses were grouped into the genotype H1. This strongly demonstrated that single genotype of measles virus has been circulated in Korea during the 2000-2001 epidemic. Plaque reduction neutralizing antibody titers against vaccine strains, Edmonston and Schwarz, and recently isolated Korean strains were measured using sera from vaccinees and recently infected children. Although sera of recently infected children demonstrated higher neutralizing antibody titers against wild-type strains than against vaccine strains, both sera neutralized both strains and the reciprocal geometric mean titers (GMTs) were not significantly different against both strains.     

Maturation of measles virus hemagglutinin glycoprotein

Summary The processing of measles virus hemagglutinin glycoprotein (H) in infected cells was studied by pulse-chase method and two-dimensional isoelectric focusing and SDS-polyacrylamide slab gel electrophoresis. H glycoprotein was synthesized initially as polypeptides smaller than H glycoprotein present in the virions. They were then processed into a cohort of polypeptides of larger molecular size and with reduced charge. The change was associated with the expression of H glycoprotein on the cell surface. The removal of sialic acid from carbohydrate chain of H glycoprotein resulted in the shift of isoelectric point to a more basic range. The entire process of maturation of H glycoprotein required approximately 5 hours.Carbohydrate content in H was determined to be approximately 12 per cent by weight. Mannose, galactose, fucose, N-acetylglucosamine, and N-acetylneuraminic acid were the constituent monosaccharides.With 4 Figures     

麻疹病毒血凝素和融合蛋白基因的克隆与表达及其重组蛋白的免疫学评价

采用逆转录－多聚酶链反应（ＲＴ－ＰＣＲ）的方法从麻疹病毒Ｅｄｍｏｎｓｔｏｎ株基因组中扩增出血凝素Ｈ基因和融合蛋白Ｆ基因，并利用转移质粒将这两个基因分别重组到杆状病毒多角体蛋白启动子（ＰＨ）控制之下，获得重组病毒ｖＢＭＶＨ和ｖＢＭＶＦ。重组杆状病毒感染Ｓｆ９昆虫细胞，表达的重组蛋白分别具有血凝、血溶活性。红细胞吸附抑制试验、免疫印迹、酶联免疫试验结果显示重组蛋白在生物学、生化学特性与天然蛋白类似，并能被天然麻疹免疫血清（人、鼠、兔）所识别。动物实验表明，重组蛋白具有诱生中和抗体的能力。重组血凝素免疫血清还具有血凝抑制抗体活性。这些结果说明杆状病毒－昆虫细胞体系表达的麻疹病毒血凝素和融合蛋白具有较好的生物学活性及免疫原性和抗原性。     

Examination of the immunological relationship between measles virus and canine distemper virus using monospecific measles antisera

Indirect immunofluorescence titrations were performed with measles virus, the Rockborn strain of canine distemper virus (CDV), and a large plaque variant of the Onderstepoort strain of CDV (Ond-LP) using monospecific antisera prepared against either the haemagglutinin (anti-HA), the haemolysin (anti-HL), or the ribonucleoprotein (anti-RNP) of measles virus. Tests with anti-HA showed that the Rockborn strain of CDV was more closely related to measles virus than Ond-LP. The ribonucleoprotein antigens of the CDV strains were closely related to each other but were both related to and distinct from measles virus RNP. The use of measles anti-HL serum demonstrated that CDV possesses an antigenically related acetone-sensitive component equivalent to the haemolysin of measles virus. Absorption of human convalescent serum with excess quantities of acetone-fixed CDV antigens had no effect on measles-specific anti-HA, HL, or RNP activity in the serum. Absorption with measles antigens on the other hand, totally removed all measles and CDV-specific HA and RNP activity. CDV was not neutralised by any of the monospecific antisera when tested either as individual antisera or as mixtures. Our results demonstrate the occurrence of antigenic variation between different strains of CDV, they also reveal unique antigenic determinants in both measles virus and CDV.     

Radioimmunoassay of measles virus antibodies in SSPE

A sensitive radioimmunological assay (RIA) was introduced for detection of measles virus IgG and IgM antibodies. The hyperimmune response to measles virus could be demonstrated more precisely by RIA than by haemagglutination inhibition (HI). The ratio between RIA and HI antibody titres was decidedly higher in sera and cerebrospinal fluids (CSF) of patients with subacute sclerosing panencephalitis (SSPE) than in those of other groups tested.     

Solid-phase radioimmunoassay determination of virus-specific IgM antibody levels in a follow-up of patients with naturally acquired measles infections

The question of the exact disappearance time or possible persistence of measles-specific IgM antibodies after naturally acquired measles virus infections was studied with a sensitive solid-phase radioimmunoassay (RIA) method. A total of 30 patients were analyzed with follow-up times varying from 4.5 to 8 months; all were measles IgM positive in the first serum specimen obtained after the onset of rash. In 29 of 30 patients, the measles IgM declined to undetectable levels by approximately 90 days. The remaining patient developed postmeasles encephalitis, however, and was found to have a prolonged measles IgM antibody response. For comparison, the measles-specific IgG response was also studied and was found to develop only slightly later than the IgM response, with levels then remaining high and stable up to 8 months later. Although apparent measles IgM antibodies were found in 1 of 64 nonmatched adult controls, they were due to the presence of high levels of IgM-class rheumatoid factor. The data presented indicate that measles IgM antibodies begin to decline soon after the onset of rash and reach negative levels 1 to 3 months later; in complicated infections, however, measles IgM antibody synthesis may not terminate normally.     

Characterization of epitopes on the measles virus hemagglutinin

Measles virus hemagglutinin epitopes were analyzed using nine monoclonal antibodies (MAbs) in competitive binding assays (CBA) and by in vitro selection of antigenic variants. In CBA the nine MAbs formed four different but partially overlapping binding groups. In vitro selected variants were analyzed by radioimmune precipitation assay, hemagglutination inhibition (HI), and neutralization assays. Seven operationally distinct MAb antigenic sites could be delineated: two sites were defined by two MAbs to each and five sites by single MAbs. Variants which had lost reactivity to different combinations of MAbs were generated by a branched sequential selection procedure. The most epitopically modified variants had lost reactivity in all assays to eight MAbs. None of these variants showed any major changes in hemagglutination nor neurotropism per se. Only minor changes were detected by HI in reactivity of these variants with human measles antiserum indicating that the epitopic changes were not of major epidemiological significance at least in terms of HI.     

Synthesis of measles virus-specific IgM antibodies and IgM-class rheumatoid factor in relation to clinical onset of subacute sclerosing panencephalitis

Thirty-seven serum and 37 cerebrospinal fluid (CSF) specimens from 27 patients with subacute sclerosing panencephalitis (SSPE) were tested for measles virus (MV) IgM antibodies and IgM-class rheumatoid factor (RF) to determine if a temporal relationship exists between SSPE onset and these immunoglobulins. Sensitive solid-phase radioimmunoassays were used for immunoglobulin detection. Five of six MV IgM-positive CSF, both MV IgM-positive sera and six out of eight sera with elevated RF levels were collected within 1 yr of SSPE onset. Also collected during this time were six other sera having high binding in the MV IgM assay, which was not due to MV-specific IgM antibodies. Two of these sera had as high or higher binding of IgM to a Vero cell control antigen, suggesting involvement of an IgM-class autoantibody. The other four sera had false-positive MV IgM assay results due to RF interference. RF interference was dependent on both the titer and avidity of the MV IgG antibodies involved. Three conclusions can be drawn from these results. First, MV IgM antibodies and elevated RF levels are not markers for acute SSPE, despite the tendency for their synthesis at this stage of the disease. Second, immunoassay analysis of viral IgM antibodies must employ an appropriate control antigen to account for background IgM binding. Finally, even if RF levels are normal or near normal, a false-positive IgM immunoassay result can still occur if antigen-specific IgG antibodies in the same sample have the right combination of titer and avidity.     

Radioimmunoassay and characterization of woodchuck hepatitis virus core antigen and antibody

Solid-phase radioimmunoassays for woodchuck hepatitis virus core antigen (WHcAg) and antibody (anti-WHc) were developed. WHcAg in woodchuck liver homogenates was characterized by ultracentrifugation in CsCl gradients; both heavy (1.35 g/cm3) and light (1.31 g/cm3) cores were obtained from the liver of an animal during acute WHV infection, which is consistent with observations in hepatitis B virus infection in man. Endpoint titers of anti-WHc were higher in chronic WHV carriers than in animals recovered from acute infections. Both IgM and IgG anti-WHc antibodies were produced by infected woodchucks. A survey of colony woodchucks demonstrated that 88/89 animals having one or more markers of past or ongoing WHV infection were positive for anti-WHc. Thus, serum anti-WHc appears to be a sensitive marker of WHV infection.