Screening and partial characterization of lectins from Indonesian seaweeds

Extraction and partial characterization of lectin from Indonesian Padina australis and Padina minor had been carried out. The crude extract of the P. australis and P. minor were examined for hemagglutination activity (HA) using native and trypsin-treated of rabbit and human A, B, O type erythrocytes. Both extracts agglutinated all of the trypsin-treated erythrocytes tested in the HA assay. Strong HA was detected in the crude extract of P. minor with trypsin-treated of human type A and O erythrocytes. However, the sugar-binding specificity study through the quantitative hemagglutination inhibition (HI) assay showed that P. minor extract could not specifically recognize the glycans tested. Apparently, the HA of the P. minor was more due to its co-extracted polyphenols content than its lectin content. On the other hand, the HI assay showed that asialo transferrin human (aTf) and asialo porcine thyroglobulin (aPTG) were the most powerful in inhibiting the HA of P. australis. Those indicated that P. australis protein extract was able to specifically recognized aTf and aPTG. The stability of P. australis and P. minor HA over various temperatures, pH ranges, and divalent cations studies showed that the P. minor HA was stable on a wide range of pH and temperature; not affected by the presence of EDTA, but decreased by Ca2+ and Mg2+ additions showed that P. minor protein extract was not a metallic protein. The HA of P. australis decreased at 60 oC and was inactivated at 90 oC; increased at strong acidic (pH 3 & 4) and strong basic (pH 9 & 10) and dependent by the presence of either EDTA or Ca2+ and Mg2+ divalent cation.

Canine parvovirus (CPV) strains were compared for haemagglutination (HA) activity and antigenicity and were divided into two types by HA activity. Strains Cp49 and 29-F showed temperature-dependent HA, like feline panleukopenia virus (FPLV) and mink enteritis virus (MEV), whereas strains Sp-80 and Y-1 showed temperature-independent HA with erythrocytes from eight species of animals. The results of a cross HA inhibition test using immunized rat sera suggested that of the two types of CPV those showing temperature-dependent HA were antigenically like FPLV and MEV whereas those showing temperature-independent HA were not. This antigenic difference between the two types was confirmed by a HA inhibition test with monoclonal antibodies. A chronological survey revealed that CPV isolates from earlier years have HA activity and antigenicity similar to those of FPLV and MEV, whereas current CPV isolates do not. There are some exceptional isolates from the transitional period which have similar antigenicity to FPLV and MEV but different HA activity. These results suggest that the haemagglutinin of CPV altered from one form resembling that of FPLV to a somewhat different structure during passage in dogs in nature.

A recombinant hemagglutinin-neuraminidase (rHN) protein from Newcastle disease virus (NDV) with hemagglutination (HA) activity was expressed in Spodoptera frugiperda cells using a baculovirus expression system. The rHN protein extracted from infected cells was used as an antigen in a hemagglutination inhibition (HI) test for the detection and titration of NDV-specific antibodies present in chicken sera. The rHN antigen produced high HA titers of 213 per 25 µL, which were similar to those of the NDV antigen produced using chicken eggs, and it remained stable without significant loss of the HA activity for at least 12 weeks at 4℃. The rHN-based HI assay specifically detected NDV antibodies, but not the sera of other avian pathogens, with a specificity and sensitivity of 100% and 98.0%, respectively, in known positive and negative chicken sera (n = 430). Compared with an NDV-based HI assay, the rHN-based HI assay had a relative sensitivity and specificity of 96.1% and 95.5%, respectively, when applied to field chicken sera. The HI titers of the rHN-based HI assay were highly correlated with those in an NDV-based HI assay (r = 0.927). Overall, these results indicate that rHN protein provides a useful alternative to NDV antigen in HI assays.

Determination of Streptococcus gordonii novel adhesins specific to sialidase-treated erythrocytes.

ViroSpot microneutralization assay for antigenic characterization of human influenza viruses

Hemagglutination (HA) activity was recovered from the aqueous phases of commercially and experimentally prepared oil-emulsion (OE) Newcastle disease virus (NDV) vaccines using two methods: aqueous partition and freeze-thaw. Quantitation of the HA activity retrieved by the aqueous partition technique was directly related to the degree of protection conferred to chickens against a strain of velogenic viscerotropic (VV) NDV in nine of the ten vaccines analyzed. One of the ten vaccines yielded high levels of retrieved HA activity but induced low hemagglutination-inhibition (HI) antibody levels and low levels of protection. Therefore, the retrieval and quantitation of HA activity from OE NDV vaccines using the partition technique provides a useful adjunct to vaccine testing methods that doesn't require vaccination and challenge trials. The freeze-thaw method did not yield measurable HA titers for all vaccines with high efficacy, so its use should be restricted to only those vaccines for which it has been demonstrated to be suitable.

Introduction

Effects of proteolytic enzymes on the infectivity, haemagglutinating activity and protein composition of bluetongue virus type 20

Rabbit viral haemorrhagic disease (RVHD) is a highly contagious, highly fatal, peracute and acute viral disease of both wild and domestic rabbits caused by rabbit haemorrhagic disease virus (RHDV). Testing for haemagglutination (HA) activity in processed liver samples is one of the cornerstones for rapid diagnosis of RHDV outbreaks in national rabbitries. However, RHDV isolates exhibiting no HA activity are increasingly being reported. The extent of deviation from classical HA activity patterns for RHDV strains in Egypt has not been investigated. This study compared the HA activity patterns of samples collected from 61 RHDV outbreaks that occurred between 1999 and 2005 to determine whether dependence on HA test (HAT) for diagnosis of RHDV outbreaks needs to be reviewed. All samples were confirmed RHDV positive using SDS-PAGE and western blotting. Using slide HAT, only 36.1% of samples were positive (22 samples). Plate HAT conducted at 4 0 C detected an additional 16 positive samples bringing the total HA-positive samples to 38 (62.3%). Plate HAT conducted at 22 0 C failed to detect additional positive samples. The majority of samples detected after plate HA testing (62.5%) had HA titres comparable to those obtained from slide-HA-positive samples, indicating that the difference in HA activity is dependent on the nature of the HA antigen rather than its presence. Direct detection of HA activity failed in 37.7% of samples despite the presence of classical signs, pathology, and being RT-PCR positive for three different VP60 regions. Experimental infection of seronegative rabbits with 9 HA negative RHDV samples showed that 5 isolates were in-fact HA positive, while only 4 isolates remained HA negative. The increased detection of viruses lacking HA activity and the low HAT sensitivity mandates the use of molecular techniques for rapid confirmation of RHDV diagnosis in the Egyptian environment.

The hemagglutination (HA) activity of porcine epidemic diarrhea virus (PEDV) was investigated. Two cell-adapted strains of PEDV (KPEDV-9 and SM98LVec) were subjected to HA test against erythrocytes of various origin. Both strains showed HA activity with rabbit erythrocytes only after treatment with trypsin or neuraminidase. Optimal conditions for inducing HA activity of PEDV were 2 h incubation at 37°C using phosphate-buffered saline containing 0.1% BSA. These results suggest that the HA activity of PEDV is most likely caused by proteolytic action on it, which could be developed as a new diagnostic method to rapidly detect and differentiate PEDV infections from other enteric diseases.

Seventy-two isolates of Haemophilus paragallinarum were serotyped according to the Page scheme, using a new hemagglutination-inhibition (HI) test. The results were compared with the plate agglutination method conventionally used in the Page scheme. The HI test used washed cells of H. paragallinarum, glutaraldehyde-fixed chicken erythrocytes, and rabbit antisera originally produced for the agglutination method. For 49 of the isolates, there was complete correlation between the results of the HI serotyping test and the previously performed agglutination test--23 were serovar A, two were serovar B, and 24 were serovar C. The other 23 isolates were nontypable by the agglutination test, but 21 of them could be serotyped by the HI method--six as serovar A, two as serovar B, and 13 as serovar C. Nine isolates required treatment of the bacterial cells with hyaluronidase for the expression of hemagglutination (HA) activity. Two isolates did not have HA activity despite hyaluronidase treatment and so could not be serotyped by the HI test.

A lectin was purified from Japanese sea hare Aplysia kurodai by lactosyl-agarose affinity chromatography. The molecular mass of the lectin was determined to be 56 and 32 kDa by SDS-PAGE under non-reducing and reducing conditions, respectively. It was found to agglutinate trypsinized and glutaraldehyde-fixed rabbit and human erythrocytes in the absence of divalent cations. The lectin exhibited stable thermo-tolerance as it retained hemagglutinating activity for 1 h even at 80 degrees C and showed stability at pH 10. By contrast, it was very sensitive at pH less than 5 and in the presence of the sulfhydryl-group preserving reagent, beta-mercaptoethanol. The hemagglutinating activity by the lectin was specifically inhibited by D-galactose, galacturonic acid, methyl-alpha- and methyl-beta-D-galactopyranoside, lactose, melibiose, and asialofetuin. The association rate constant (k(ass)) and dissociation rate constant (k(diss)) were determined for the lectin to be 4.3 x 10(5) M(-1) x sec(-1) and 2.2 x 10(-3) sec(-1), respectively, using a surface plasmon resonance biosensor. The lectin moderately inhibited cell proliferation in the P388 cell line dose dependently. Interestingly, lectin-treated cells did not show a fragmented DNA ladder as is caused by apoptosis, suggesting that the cell proliferation inhibition was caused by another unknown mechanism.

Isolation, purification, characterization and glycan-binding profile of a d-galactoside specific lectin from the marine sponge, Halichondria okadai

Streptococcus gordonii, one of the early colonizers of oral biofilms, is involved in the development of dental caries, periodontal disease, and infective endocarditis. The Hsa adhesin of S. gordonii DL1 has the ability to bind strongly to the terminal sialic acid groups of host glycoproteins via the binding region, nonrepetitive region 2 (NR2), which is important for the pathogenicity of S. gordonii DL1. Low similarity with the NR2 of Hsa homologs among other streptococcal species has been reported. However, the reports have been limited to certain strains. This study attempted to assess frequency of the expression on the bacterial cell surface and to analyze the diversity of Hsa homologs among different wild strains of oral streptococci. We isolated 186 wild-type strains of oral streptococci from healthy volunteers and analyzed their hemagglutinating (HA) activity on human erythrocytes and their Hsa homologs and NR2 homologous regions by dot immunoblotting using anti-Hsa and anti-NR2 antisera, respectively. We found 30 strains reacted with anti-NR2 antiserum (NR2 positive) and determined the sequence of the NR2 regions. Many strains with high HA activity were also NR2 positive, suggesting that the NR2 region may be associated with HA activity. Among the NR2-positive strains, four different amino acid sequence patterns were observed, demonstrating diversity in the NR2 region. Notably, S. gordonii strains frequently possessed Hsa homologs and NR2-like antigens compared with other streptococci. It is speculated that the possessing frequency of Hsa homologs and the amino acid sequence of NR2 region may vary among streptococcal species.

Properties of mouse leukemia viruses: IV. Hemagglutination assay and characterization of hemagglutinating surface components