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Substiuted sialic acid prosthetic groups as determinants of viral hemagglutination.

Inhibitors of hemagglutination by type A2 influenza virus and a recently isolated strain of type B influenza virus were separated by sucrose density gradient centrifugation and agarose gel filtration from horse serum. Using selected reagents, it was demonstrated that the active substituent on the horse serum inhibitor of A2 influenza virus was 4-O-acetyl-N-acetylneuraminic acid; however, the active substituent on the inhibitor of the influenza B virus was shown to be N-acetylneuraminic acid (NANA). Sodium metaperiodate treatment of a component of horse serum resulted in a 10 to 15-fold enhancement of inhibitory activity against the type B virus, whereas the A2 inhibitor was completely destroyed. Since this enhancement did not occur with influenza B viruses isolated prior to 1965, it was considered that this sensitivity to an oxidized NANA glycoside may have been a reflection of an antigenic change which occurred at that time. The use of different virus strains and selected chemical reagents to define the important sialic acid prosthetic groups active in inhibition was described.

Animals↗

Hemagglutination activity of Lactobacillus acidophilus group lactic acid bacteria.

The cells of 28 strains of the Lactobacillus acidophilus group were evaluated for hemagglutination (HA) activity. The activity was found in the surface layer (SL) protein fraction extracted by 2 M guanidine hydrochloride. The most SL proteins from the A group strains (L. acidophilus (A1), L. crispatus (A2), L. amylovorus (A3), and L. gallinarum (A4)) showed HA activity, but the proteins from the B group strains (L. gasseri (B1) and L. johnsonii (B2)) showed no activity. The SL proteins from the A group strains were composed in common of a main component having molecular mass of about 40-45 kDa on SDS-PAGE. The SL proteins from JCM 1034 strain that showed the highest HA activity was fractionated by CM-Toyopearl ion-exchange chromatography. The highest HA activity was detected in the major protein of 41 kDa. This protein was purified and shown to be composed of about 50% of hydrophobic amino acids. The HA activity of the protein (1034 lectin) was specifically inhibited by fetuin and bovine lactoferrin at the concentrations of 80 and 160 micrograms/ml, respectively. The removal of N-acetylneuraminic acid from fetuin significantly decreased the inhibitory activity.

Amino Acids↗

Hemagglutination with porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome (PRRS) virus grown on MARC-145 cell cultures was tested for hemagglutination (HA) with erythrocytes from a variety of species at 4 degrees C, room temperature and 37 degrees C. HA was observed at all temperatures with mouse erythrocytes but not with cattle, sheep, goat, horse, swine, guinea pig, mongolian gerbil, goose and chicken erythrocytes. The HA activity was enhanced by treatment of virus materials with Tween 80 followed by treatment with ether. The HA titer and HA pattern of virus materials treated with Tween 80 and ether (TE) were 4- to 8-fold higher and more clear than those of the virus materials without TE treatment. The optimum conditions consisted in pretreatment of virus material with Tween 80 at a final concentration of 0.06-0.125% (v/ v) for 15-60 min followed by treatment with ether at a concentration of 50% (v/v) for 5-15 min in ice bath with continuous shaking. The curve of active virus production in intra- and extracellular virus samples resembled that of HA production although it rose somewhat earlier in intracellular virus samples. The HA reaction was inhibited by specific antiserum. HI antibody titers of individual pig sera showed a significant positive correlation with their neutralizing antibody titers.

Animals↗

Effect of heparin on hemagglutination by equine arteritis virus.

Heparin inhibited hemagglutination (HA) by equine arteritis virus (EAV) as well as did HA by Aujeszky's disease virus (ADV), but failed to inhibit HA by parainfluenza virus type 3 (PIV-3). The minimal concentration of heparin required to inhibit 8 HA U of EAV was 0.1 U/ml. In addition, most EAV hemagglutinin was retained by heparin acrylic beads, as was ADV hemagglutinin, but was not PIV-3 hemagglutinin. Mouse erythrocytes failed to combine with the HA inhibitory factor of heparin. However, mouse erythrocytes treated with heparinase had greatly reduced agglutinability by EAV. All these findings suggest that a heparin-like molecule on the surface of mouse erythrocytes serves as the virus-cell receptor.

Animals↗

Human serum non-antibody inhibitors towards SA-11 rotavirus hemagglutination.

In the present work the non-antibody inhibitory activity of human serum towards SA-11 rotavirus hemagglutination (HA) has been studied. Among human serum protein components, inhibitory activity was recovered in fractions III (beta-globulin), IV (alpha-globulin) and IV (glycoprotein). Additional experiments performed with purified lipoprotein sub-classes and their lipid and apolipoprotein moieties demonstrated the ability of HDL and, HDL1 to prevent SA-11 HA.

Animals↗

Detection of Epstein-Barr virus early antigen-D and its antibodies by passive hemagglutination.

These data describe the use of a passive hemagglutination test (PHAT) for the detection of antibodies to the Epstein-Barr virus (EBV) associated antigen, early antigen-D (EA-D). EA-D was obtained from EBV-superinfected RPMI 64-10 cell preparations which showed by immunofluorescent staining (IF) that 25-35% of the cells contained EA-D. Solubilization of EA-D was achieved by sonic oscillation, treatment of the pellet-associated material with triton X 100, ammonium sulfate precipitation, and chromatography on diethylaminoethyl celluiose. Successful use of the PHAT for detection of antibodies to EA-D was dependent on the degree of purity of the partially purified antigen. Throughout these studies preparations from uninfected 64-10 cells were used as control reagents. More than 100 human antisera from patients with EBV-associated diseases, which had been previously tested by standard IF procedures were assayed by PHAT. A good correlation was found between IF and PHAT titers for EA-D and, in addition, the PHAT was 50-100 times more sensitive. Most human sera gave no agglutination of red cells coated with control preparations. An inhibition (blocking) of PHAT was developed which was useful in the purification and characterization of EA-D. These data indicate that the PHAT may provide a rapid, reliable, objective and sensitive method for studying EA-D and possibly other virus-associated antigens.

Antibodies, Viral↗

Evaluation of an indirect hemagglutination inhibition technique for identifying antibody in animals experimentally and naturally exposed to fungi.

Rabbits were exposed to aerosols containing spores of Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, or of a Penicillium sp. Sera from these rabbits were tested by indirect hemagglutination (IHA) and by IHA inhibition. The serologic reactions with the rabbit sera were compared to reactions with sera from cattle naturally exposed to airborne microorganisms. By three months of age, most cattle had positive IHA reactions to A. fumigatus and Penicillium antigens. The IHA inhibition tests indicated that antibody production in 12 of the 20 cattle probably resulted from exposure to A. flavus. One calf reacted as if sensitized by A. niger. Two were totally nonreactive. Five of the cattle had reactions that were not identifiable relative to the reactions in rabbits.

Aerosols↗

Hemagglutination with pseudorabies virus. Brief report.

Pseudorabies virus grown in CPK cell cultures was tested for hemagglutination (HA) with erythrocytes of a variety of species at 4 degrees C, 25 degrees C and 37 degrees C. HA was observed at all temperatures with mouse erythrocytes but not with cattle, sheep, goat, swine, cat, rabbit, guinea pig, rat, mongolian gerbil, chicken, and goose erythrocytes. Mice showed a strain variation in agglutinability of their erythrocytes, requiring selection of mice to obtain erythrocytes for HA. The HA reaction was inhibited by specific antiserum. Some factors involved in the HA and HA-inhibition (HI) were investigated and standard HA and HI tests were established. HI antibody titers of individual pig sera showed a significant positive correlation with their neutralizing antibody titers.

Animals↗

Hemagglutination with ovine rotavirus. Brief report.

The virus was grown in MA 104 cells, a stable cell line derived from embryonic rhesus monkey kidney, and tested for hemagglutination (HA) with erythrocytes of a variety of species at 4 degrees C, room temperature and 37 degrees C. HA was observed at all temperatures with chicken, sheep, rabbit, guinea pig and human erythrocytes but not with horse, cattle, goat, swine and goose erythrocytes. HA reaction was inhibited by specific antiserum. Some factors involved in the HA and HA-inhibition (HI) were investigated and standard HA and HI tests were worked out. The HI test as well as neutralization test clearly distinguished ovine rotavirus from bovine, human, simian, equine, porcine and lapine rotaviruses.

Animals↗

Detection of bovine coronavirus in feces by reversed passive hemagglutination.

A reversed passive hemagglutination (RPHA) method was developed for the detection of bovine coronavirus in fecal specimens. Sheep erythrocytes fixed with glutaraldehyde, and then treated with tannic acid were coated with anti-bovine coronavirus rabbit antibodies purified by affinity chromatography using bovine coronavirus linked to Sepharose 4B. The RPHA test was carried out by a microtiter method. Erythrocytes coated with purified specific antibodies were agglutinated by bovine coronavirus, but not by bovine rotavirus or enterovirus. The reaction was inhibited by antiserum to bovine coronavirus, confirming the specificity of the reaction. The RPHA test detected bovine coronavirus in 13 of 22 fecal specimens (59 per cent), from natural cases of diarrhea, while the positive rates were only 14 per cent (3/22) and 22 per cent (5/22) for immunofluorescent staining of primary cultures of calf kidney cells infected with the specimens, and immune electron microscopy respectively. The advantages of the RPHA method are its simplicity, high sensitivity and rapidity.

Animals↗

Relation of virus particle counts to the hemagglutinating activity of influenza virus suspensions measured by the HA pattern test and by use of the photometric HCU method.

The ratios of the number of virus (n.v.p.) to the hemagglutinating activity were determined for a series of influenza A and B strains using both the photometric HCU method and the HA pattern test. The logarithms of the ratios of n.v.p./hcu ranged from 7.06 to 7.54 and the logarithms of the ratios of n.v.p./HA from 6.87 to 7.56. While the n.v.p./HA ratios of different strains showed significant differences in a number of cases, such differences were not found for the n.v.p./HCU ratios with the only exception of the strain B/Hong Kong/8/73 differing uniformly from the other strains tested. Since the standard deviation of HCU values ranged from 0.006 to 0.039 log units and that of HA titers from 0.076 to 0.272 log units, it was concluded that the differences of n.v.p./HA ratios found were due to the range of variance of HA tests and did not reflect strain specific differences. The degree of purity of virus suspensions was found not to influence measurably the log n.v.p./HCU values.

Hemagglutination Tests↗

Appearance of slow-reacting, complement-requiring hemagglutination-inhibiting antibody in swine infected with pseudorabies virus.

Slow-reacting, complement-requiring hemagglutination-inhibiting (HI) antibody was detected in sera from pigs infected with pseudorabies virus; approximately 16 hemolytic units of complement were necessary for the detection of such antibody. Higher HI antibody titers were obtained when antigen and serum were allowed to incubate before addition of complement than when all three components were incubated at the same time. A HI test was developed in which antigen-serum mixtures were incubated at 4 degrees C for 48 h and then with complement at 37 degrees C for 1 h; this gave an improved sensitivity over the previous incubation without complement.

Animals↗

Passive hemagglutination procedure for group A streptococcal polysaccharide.

A passive hemagglutination procedure for detection of antibody to group A beta-hemolytic streptococcus polysaccharide is described. The polysaccharide was conjugated to human gamma globulin and the complex adsorbed onto stabilized human erythrocytes. Antigen coated cells were used to measure antibody levels in normal human sera and in rabbit antisera.

Animals↗

Simple serological diagnosis of arboviruses: a simplified diluent system for Japanese encephalitis virus hemagglutination-inhibition tests using formalinized chick erythrocytes.

Formalinized chick erythrocytes agglutinated with Japanese encephalitis virus antigens over a wider range of pH (6.0-7.2) than fresh erythrocytes (pH 6.4-6.8). The virus antigens were unstable below pH 7.0 and therefore the use of the formalinized erythrocytes made it possible for a one-diluent system of the hemagglutination-inhibition tests to be used. In these tests phosphate-buffered saline at pH 7.0 was used throughout as a diluent for serum, antigen and erythrocytes, although the conventional system, using fresh erythrocytes, requires two buffers of different pHs, one an alkaline diluent for serum and antigen and the other an acidic diluent for erythrocytes.

Animals↗

Rat alpha 1 macroglobulin inhibits hemagglutination by influenza C virus.

Purified alpha 1-macroglobulin (RMG) isolated from rat plasma was found to be a potent inhibitor of hemagglutination by influenza C virus. Neuraminidase treatment of purified RMG reduced its inhibitory activity by more than 80% indicating that sialic acid is required for maximal HI-activity. The inhibitory activity of RMG was shown to be sensitive to the receptor-destroying activity (RDA) of influenza C virus. Methylation analysis of the glycopeptides of RMG indicated the presence of only one major type of oligosaccharide which is a complex N-linked oligosaccharide with a biantennary structure. Comparison of the glycopeptides before and after neuraminidase treatment revealed that the oligosaccharides are terminated by sialic acid residues attached to galactose residues at position C-6. Methylation analysis was also performed on RMG which had lost its inhibitory activity upon incubation with RDA of influenza C virus. No difference between the glycopeptides of native and inactive RMG could be detected. Galactose was found to be substituted at position C-6 in both samples, indicating that also the oligosaccharides of inactive RMG are terminated by sialic acid. The implications of these results are discussed.

Animals↗

Use of the hemagglutinating virus of Japan (HVJ)-liposome method for analysis of infiltrating lymphocytes induced by hepatitis B virus gene expression in liver tissue.

We previously developed a method for introducing foreign genes into liver tissue using liposomes with incorporated hemagglutinating virus of Japan (HVJ, Sendai virus), and found that liver cells transfected with the E. coli beta-galactosidase gene or the gene for hepatitis B virus (HBV) surface protein (HBsAg) expressed these proteins in vivo. Here, we analyzed cellular reactions leading to hepatitis in the liver by expressing the genes of HBV in vivo. Lymphocytes were eluted directly from liver transfected with the HBsAg genes and shown to be cytotoxic only to cells expressing HBsAg in vitro. These lymphocytes were identified as cytotoxic T lymphocytes with the CD4- CD8+ phenotype. Transfer of these lymphocytes to transgenic mice with the whole HBV genome led to elevation of the serum glutamic-pyruvic transaminase (SGPT) level, indicating the induction of hepatitis due to the cytotoxic T lymphocytes in vivo. Similarly, direct transfer of the gene for the HBV secretory core protein (HBeAg) induced expression of HBeAg in hepatocytes and the appearance of antibody against HBeAg in the serum. However, using this system, we found that the lymphocytes infiltrating the transfected liver showed no cytotoxicity specific for HBeAg. These results indicate that expression of HBsAg, one of the components of virions, in animal liver induced hepatitis efficiently through generation of specific cytotoxic T lymphocytes (CTL) without any expression of the other viral components. This in vivo experimental system should be useful for evaluating how expression of a given gene induces cellular reactions and intrinsic functions in the living body.

Animals↗

Hemagglutinating activity of Curcuma plants.

Crude proteins obtained by Mg/NP-40 extraction from Thai medicinal plants of the Curcuma species exhibited agglutination activity against rabbit erythrocytes. A crude extract from Salingalinthong, a Thai Curcuma specie, exhibited the strongest hemagglutinating activity, 2 x 10(-5) mg/ml.

Animals↗

Isolation of a novel legumin-like lectin with potent hemagglutinating activity from seeds of the Chinese chestnut Castanea mollisima.

A novel mannose- and glucose-specific lectin with high hemagglutinating activity was isolated from seeds of the Chinese chestnut Castanea mollisima. The lectin possessed a molecular mass of 140 kDa and was made up of two subunits, one with a molecular mass of 31 kDa and another with a molecular mass of 32 kDa. They exhibited substantial homology in N-terminal sequence to the storage protein legumin. The lectin was unstable in the presence of acid and alkali and at temperatures above 50 degrees C, but it was unaffected by various salts. The lectin was purified with a procedure involving ion exchange chromatography on CM-Sepharose, Q-Sepharose and Resource Q and gel filtration on Superose 12.

Animals↗