Hemagglutinating Activity Research Articles

Hemagglutinins from the green algae, Chlorophyta

The extracts from 30 species of the green algae were examined for hemagglutination activity with a variety of different animal and human erythrocytes which were untreated and treated with enzymes. Almost all extracts showed the activity at least toward one type of erythrocytes tested. Strong activity was detected in the extracts from 5 Chlorophyta species (Anadyomene plicata, Avrainvillea erecta, Boodlea struveoides, Halimeda velasquezii and Halimeda discoidea) with enzyme-treated rabbit and sheep erythrocytes. The hemagglutinins of some active species were examined for carbohydrate binding specificity, pH and temperature stability, and effect of divalent cation on their hemagglutination activities. All of the hemagglutinins had no affinity for monosaccharides, except hemagglutinin of Codium arabicum whose activities were inhibited by N-acetyl-D-galactosamine and N-acetyl-D-glucosamine. On the other hand, hemagglutination activities of the hemagglutinins were inhibited by some glycoproteins. The inhibition profiles with glycoproteins were different, depending on hemagglutininproducing species, and suggest the presence of lectins specific for high mannose N-glycans, complex N-glycans, or O-glycans. The hemagglutination activities of the green algal hemagglutinins were stable in a wide range of pH and temperature, and independent on divalent cations. Therefore, Vietnamese green algae may promise as a valuable source of new lectins for application.

The industrial process of solvent extraction of castor oil reduces the toxicity of the meal

Castor (Ricinus communis L.) oil is an important raw material for the chemical industry. The industrial process of castor oil extraction exposes the material to chemical, thermal, and mechanical treatments that influence the properties of the proteins in the meal. This study tested the hypothesis that the regular industrial process of castor oil extraction with solvent changes properties of the meal like the profile of proteins, the nematicide efficiency, and its toxicity. The changes made by the process of oil extraction create opportunities for adding value to this by-product. Samples of castor cake and meal were collected along the process of oil extraction under the regular operation of an industry. The samples were analyzed for chemical composition, hemagglutination activity, nematicide efficiency, and toxicity for feeding broilers and sheep. It was found that the step of solvent evaporation is responsible for the most relevant modifications in the meal, with significant changes in the protein profile, loss of hemagglutination capacity, and reduction of toxicity and nematicide efficiency. The castor meal produced just after the solvent evaporation has the toxicity reduced to a level that was tolerable to be used in the ration of small ruminants without detectable loss of performance or clinical signs of intoxication. The castor meal was toxic for broilers. The nematicide capacity of castor meal against larvae of Haemonchus contortus is reduced after the step of solvent extraction, but it is still significant and detected in doses as low as 5 mg/g. When castor meal was separated in fractions by sieving, the fine fraction (particles smaller than 0.5 mm) had high protein content (60.2%), low fiber content (6.0%), and high water holding capacity (164.2% of its dry weight).

Effects of histamine on the neuroendocrine-immune regulatory network in the Pacific white shrimp, Litopenaeus vannamei

In shrimp culture, feed made from stale fishmeal and decomposition of dead shrimp easily produces large amounts of histamine in the water environment, which will cause disease outbreaks. Simultaneously, in the internal environment of crustaceans, histamine is a short-acting biogenic amine that can act as a neurotransmitter and regulate immunity. To investigate the effects of histamine on the neuroendocrine-immune network of crustaceans, a histamine injection experiment was carried out on Litopenaeus vannamei in this study. The results showed that the neuroendocrine factors, including hormones (corticotrophin-releasing hormone, adrenocorticotropic hormone) and biogenic amines (dopamine, noradrenaline), in the histamine injection groups increased significantly at 6 h, while the 5-hydroxytryptamine concentration was significantly decreased at 12 h. The intracellular signaling pathways, including the PKC pathway (phospholipase C, diacylglycerol and protein kinase C), the RAP1 pathway (RAS-associated protein and integrin αIIbβ3), the PI3K/PKB pathway (phosphoinositide 3-kinase, phosphatidylinositol (3,4,5)-trisphosphate and protein kinase B) and the MAPK pathway (rat sarcoma protein and extracellular signal-regulated kinase) in the histamine injection groups all decreased significantly after 6 h and shared a similar trend. The immune parameters, including total hemocyte count, cell viability, phagocytic activity, phenoloxidase activity of hemocyte, antibacterial activity, bacteriolytic activity and hemagglutinating activity, all decreased significantly in the histamine injection group after 6 h, while phenoloxidase activity of plasma increased significantly. In addition, the antioxidant parameters (superoxide dismutase, catalase and glutathione peroxidase) in the histamine injection group were significantly increased. These results indicated that histamine might regulate the prophenoloxidase system via the PKC pathway, further influence the antibacterial and bacteriolytic activities, regulate hemagglutinating activity via the PKC-RAP1 pathway, regulate the total hemocyte count and cell viability via the PKC -PI3K/PKB pathway, and regulate phagocytic activity via the PKC -PI3K/PKB -MAPK pathway. Meanwhile, histamine may also affect the activities of antioxidant enzymes via the RAP1 pathway and the PKC-PI3K/PKB pathway. Therefore, it may be concluded that the neuroendocrine-immunoregulatory network plays a principal role in histamine regulation and causes immune and antioxidant defense in L. vannamei.

Partial Purification and Characterization of the Lectins of Two Varieties of Phaseolus coccineus (Ayocote Bean)

In this study, a partial purification and characterization of the lectins from two varieties of Phaseolus coccineus (black and purple ayocote bean) was carried out. A partial purification of the lectins was performed by affinity chromatography on fetuin-agarose. The lectins’ characterization showed that the hemagglutination activity was only inhibited by the fetuin and that the saccharides (mono-, di- and tri-saccharides) do not influence their hemagglutination capacity. A difference in the relative molecular weight between the two Phaseolus varieties was observed, 28.5 kDa for the black variety and 30.6 kDa for the purple variety. In the lectins of both Phaseolus varieties, ions were detected in their structure and influenced their hemagglutination activity, principally to the ion Ca+2; on the other hand, only the lectins of the purple variety of the Phaseolus, had a detectable ion, Cu+2. The lectins showed thermostability in the temperature range of 25 and 70 °C, on the other hand, in the pH range between 3 and 10, the lectins showed hemagglutination activity. According to the obtained results, the structural differences between the lectins obtained from both Phaseolus varieties influence their hemagglutination activity.

Hemagglutinating properties of a Streptococcus gordonii strain expressing sialic acid-binding adhesin homolog with low binding site similarity to that of strain DL1

ObjectivesThe Hsa adhesin of Streptococcus gordonii strain DL1 was previously identified as a hemagglutinin that binds specifically to sialoglycoconjugates. We recently found that among oral streptococcal species, S. gordonii strains most frequently express Hsa homologs on the bacterial cell surface. However, the effect of amino acid sequence diversity of nonrepetitive region 2 (NR2), a putative binding site of Hsa, on antigenicity and hemagglutinating (HA) properties is unclear due to difficulties in DNA sequencing the NR2 coding region. The aim of this study was to elucidate the similarity of the low NR2 antigenicity Hsa homolog of strain NDU1118 to that of strain DL1 and the association of the homolog with HA properties of the strain. MethodsThe hsa homolog of NDU1118 was sequenced using a long-read next-generation sequencer, and the Hsa homolog was assessed by alignment analysis of the deduced amino acid sequences. The hsa mutant of NDU1118 was generated by insertion of the erythromycin resistance gene. The HA properties of the wild type and the hsa mutant were assessed with human erythrocytes. ResultsThe NR2 amino acid sequence of the NDU1118 Hsa homolog was almost identical to that of the S. gordonii M99 Hsa homolog, also known as GspB, and less similar to that of DL1 Hsa. The hsa mutation of NDU1118 induced reduction of HA activity in untreated erythrocytes, but surprisingly increased lactose-inhibitable HA activity in neuraminidase-treated erythrocytes. ConclusionsThe results suggest the existence of an adhesin other than the Hsa homolog on the cell surface of NDU1118.

Effect of Graded Levels of Dietary Emamectin Benzoate on Immunity, Enzyme Activity, and Withdrawal Period in Labeo rohita Juveniles (Hamilton, 1822)

This study was conducted to evaluate the effect of dietary emamectin benzoate (EB) levels on immune responses, serum enzyme activities, and retention of EB in muscle tissue to establish the withdrawal period in rohu, Labeo rohita juveniles (avg. wt. 18 ± 0.11 g). To ascertain this, 450 healthy L. rohita juveniles were fed with EB in graded doses viz., at 50 (1x), 125 (2.5x), 250 (5x), and 375 (7.5x) μg kg-1 of fish biomass day-1, respectively, in triplicate for 21 days through a basal feed. Upon completion of 21 days, the same experimental fish were fed with basal feed (without EB) for another 14 days to measure the retention of EB in the muscle tissue. Each 7-day interval, five fish were randomly sampled. The results showed that respiratory burst activity, myeloperoxidase activity, bacterial haemagglutination, and haemolysis activities were improved significantly ( p < 0.05 ) in 1x and 2.5x dose fed group from 7th day onwards until 14th day. Few selected enzyme activities viz., LDH and ALP were found to be significantly ( p < 0.05 ) high in the fish fed with EB at 5x and 7.5x dose compared to 1x. The LC-MS/MS study of the experimental fish discloses that EB was retained in muscle tissue at a dose-dependent manner and significantly ( p < 0.05 ) lowest level ( 0.341 ± 0.03 ppb) was retained in the fish fed with 1x dose of EB. Hence, 1x dose of EB may be adhered to treat L. rohita juveniles through feed with 14 days of withdrawal period.

Knockdown of <i>FLT4</i>, <i>Nup98</i>, and <i>Nup205</i> cellular genes as a suppressor for the viral activity of Influenza A/WSN/33 (H1N1) in A549 cell culture

Objectives. To evaluate the effect of cellular genes FLT4, Nup98, and Nup205 on the reproduction of the influenza A virus in A549 human lung cancer cell line.Methods. The work was carried out using the equipment of the center for collective use of the I.I. Mechnikov Research Institute of Vaccines and Sera (Russia). The virus-containing fluid was collected within three days from the moment of transfection and infection and the intensity of viral reproduction was assessed by viral titration and hemagglutination reaction. The viral RNA concentration was determined by real-time reverse-transcription polymerase chain reaction (RT-PCR). To calculate statistically significant differences between groups, the nonparametric Mann–Whitney test was used.Results. In cells treated with small interfering RNAs (siRNAs) targeted at FLT4, Nup98, and Nup205 genes, a significant decrease in their expression and indicators of viral reproduction (virus titer, hemagglutinating activity, viral RNA concentration) was observed at a multiplicity of infection (MOI) = 0.1. Additionally, it was found that a decrease in the expression of target genes using siRNA does not lead to a significant decrease in cell survival. The viral titer in cells treated with siRNA FLT4.2, Nup98.1, and Nup205 on the first day was lower by an average of 1.0 lg, and on the second and third days, by 2.2–2.3 lg, compared to cells treated with nonspecific siRNA. During real-time RT-PCR, a significant decrease in the concentration of viral RNA was observed with siRNA Nup98.1 (up to 190 times) and Nup205 (up to 30 times) on the first day, 26 and 29 times on the second day, and 6 and 30 times on the third day, respectively. For FLT4.2 siRNA, the number of viral RNA copies decreased by 23, 18, and 16 times on the first, second, and third days. Similar results were obtained when determining the hemagglutinating activity of the virus. The hemagglutinating activity on the third day most strongly decreased in cells treated with siRNA Nup205 and FLT4.2 (16 times). In cells treated with siRNA FLT4.1, Nup98.1, and Nup98.2, hemagglutinating activity decreased by 8 times.Conclusions. In the present study, three cellular genes (FLT4, Nup98, and Nup205) were identified—the decrease in the expression of which effectively suppresses viral reproduction— and the original siRNA sequences were obtained. The results obtained are important for creating therapeutic and prophylactic medication, whose action is based on the RNA interference mechanism.

Comparative study on immunological responses in Scylla serrata and Litopenaeus vannamei upon WSSV infection

The present study was directed towards elucidation of immunological markers in WSSV-infected mud crabs through a comparative study with whiteleg shrimp. Parameters analysed including serum protein levels, bacteriolytic activity, haemagglutination activity, phenoloxidase activity and total haemocyte count. Analyses of above said parameters revealed that during the course of WSSV infection, serum haemagglutinating and phenoloxidase activity in mud crabs did not show any significant change in contrast with the whiteleg shrimps. Possibly, such distinct immune response against WSSV may play an important role in pathogenicity caused by WSSV in the mud crab S. serrata and may contribute to the resistivity against WSSV by these crabs.

Nematicidal effect of a lectin preparation from Artocarpus heterophyllus (Moraceae) on larvae and adults of Haemonchus contortus

Haemonchus contortus is a hematophagous parasite that causes damage to ruminant production worldwide. This study reported the in vitro nematicidal effect of a lectin preparation (LP) isolated from Artocarpus heterophyllus seeds on larvae and adults of H. contortus. The protein extraction was in phosphate-buffered saline followed by protein precipitation with ammonium sulfate 70% and dialysis. The dialyzed protein fraction was chromatographed to obtain isolated LP. The LP was characterized by hemagglutinating activity (HA) assays, protein dosage and polyacrylamide gel electrophoresis. The motility index of H. contortus in the third larval stage (unsheathed L3 larvae) and adult stage was evaluated. The HA inhibition of LP by mannose and galactose as well as the electrophoretic profile indicated the presence of the lectins ArtinM and Jacalin. The motility index of H. contortus was significantly reduced (p < 0.001) during the first 8 h of LP exposure, both in larvae (lowest index 8.3% with LP at 1 mg mL−1) and female adults (lowest index 20% with LP at 500 μg mL−1; index 40% with LP at 1 mg mL−1). This research revealed that the LP has potential for being utilized in the development of natural nematicides.

Nutritional and Phytochemical Properties of Aqueous Leaf Extract of Mistletoe (Tapinanthus Bangwensis) Grown on Orange Tree

Background: African mistletoe (Tapinanthus bagwensis) is a parasitic plant commonly used in folk medicine. The bioconstituents and thus its nutritional and ethnomedicinal uses may be greatly affected by ecological interactions between the plant and host. Objectives: This study investigated the proximate and phytochemical compositions of aqueous leaf extract of Tapinanthus bangwensis grown on orange tree. Methods: Standard methods of the Association of Official Analytical Chemists (AOAC) was used to evaluate the proximate and mineral elements compositions of Tapinanthus bangwensis aqueous leaf extract. Vitamins content and presence of lectin was quantified using high performance liquid chromatography and via hemagglutinating activity respectively. Phytochemicals were determined using spot test and spectrophotometrically. Results: The proximate analysis revealed that T. bangwensis aqueous leaf extract contained total carbohydrate (12.72 ± 0.1 %), crude fat (9.65±0.3%), protein (6.8 ± 0.4%), ash (3.5 ± 0.1%), moisture (0.13 ± 0.2%) and crude fibre (67.2 ± 0.1%). The minerals (mg/ 100 g dry weight) present were K (147.3 ± 0.01) &gt; Ca (90.70 ± 0.025) &gt; P (62.9 ± 0.025) &gt; Na (54.6 ± 0.03) &gt; Mg (43.05 ± 0.02) &gt; Mn (34.00 ± 0.01) &gt; Zn (24.90 ± 0.015) &gt; Iodine (2.05 ± 0.01). Vitamins A, D, B2, E, K, B12 and Folic acid were relatively abundant compared to other vitamins. The extract possessed lectin depicted by hemagglutination activity on different animal species’ blood samples. The phytochemicals (mg/100 g) concentration was tannin (108.65 ± 0.04), phenol (66.7 ± 0.01), flavonoid (44.58 ± 0.03), cyanogenic glycoside (24.53 ± 0.02) and saponin (2.40 ± 0.02). Conclusion: Given these nutritional, phytochemical constituents and their concentrations, T. bangwensis aqueous leaf extract could be a good nutrient source for consumers and might be beneficial in maintaining good health.

Purification, characterization, and immunomodulatory activity of a lectin from the seeds of horse chestnut (Aesculus hippocastanum L.)

This work aimed to purify, characterize, and evaluate the immunomodulatory activity of a lectin from Aesculus hippocastanum seeds (AhSL). The lectin was isolated from saline extract through chromatography on a DEAE-Sephadex column. Protein homogeneity and subunit composition were evaluated with polyacrylamide gel electrophoresis (PAGE) under native and denaturing (SDS-PAGE) conditions, respectively. AhSL was evaluated for trypsin inhibitor activity, carbohydrate-binding specificity, and the effects of cations (Ca2+, Mn2+, and Mg2+), temperature, and pH on its hemagglutinating activity. Lectin stability under heating and pH variation was evaluated through fluorometric analysis. The activity of this lectin (12.5 µg/mL) in modulating cytokine release and nitric oxide production by mouse splenocytes was assessed. Isolated AhSL (purification fold: 68.8) showed a single polypeptide band in PAGE for acidic proteins and two polypeptide bands of approximately 68 and 124 kDa in SDS-PAGE. Its hemagglutinating activity was inhibited by monosaccharides, disaccharides, and glycoproteins, but was not affected by cations, and remained stable after heating (30–100 °C) and under pH 4.0–8.0. Fluorometric analysis revealed that the overall lectin conformation was not altered after heating to 100 °C and remained stable under various pH values. AhSL also showed trypsin inhibitor activity (specific activity: 1,280.31 U/mg) and induced the release of interleukins 10 and 6, tumor necrosis factor α, and nitric oxide by splenocytes. In conclusion, AhSL is a bioactive protein with promising biotechnological potential, which may find applications as immunological targeting or antitumor agents.

Antimicrobial Analysis of Biosynthesized Lectin-Conjugated Gold Nanoparticles

To enhance the bioactivity of molecules through nanoparticles is being tested which has potential use in sustained-release drug delivery systems and to enhance the therapeutic effectiveness of drugs. Our current investigation s is to conjugate lectin to that of a gold nanoparticle (GNP) surface without disturbing the bioactive properties and enhances the antibacterial activity of lectin. Au-lectin nanoparticles were checked for their hemagglutination activity, characterized by transmission electron microscopy (TEM) and UV-visible spectrophotometer. The antibacterial effect of nanoparticle lectin, Au salt nanoparticle, and conjugated Au-lectin was estimated by Kirby-Bauer disc method; MICs were determined by microbroth dilution and compared with ciprofloxacin. These tests were done using known species of bacterial strain of multidrug resistant. The hemagglutination activity of lectin was improved to fourfold after purification. Lectin and Au nanoparticles combined had a significant effect on the inhibition of bacterial growth. No significant differences were observed in the inhibition zone diameters from killed bacteria and its supernatant towards any of the tested organisms. Lectin-conjugated gold particles showed good efficacy as antimicrobial agents and the nanoparticle-killed bacteria to work against the viable population of the same bacterium and/or other bacterial species too.

Effect of feeding solid state fermented mahua oil cake (Bassia latifolia) on growth performance, enzymatic and immunological parameters in rohu (Labeo rohita)

Solid-state fermentation of mahua oil cake (Bassia latifolia) was carried out by yeast Saccharomyces cerevisiae and then it was incorporated as feed ingredient for production of fish feed for rohu (Labeo rohita). To evaluate the solid-state fermented mahua oil cake as feed ingredient in rohu, two types of fish feeds were produced i.e. a control feed using raw mahua oil cake and treatment feed using solid-state fermented mahua oil cake. A feeding trial of 60 days’ duration was conducted to know the effect of feeding fish with fermented mahua oil cake during which growth performance, digestibility of nutrients, intestinal enzyme activity and non-specific immune parameter of fish fed on fermented mahua oil cake. Solid-state fermentation of mahua oil cake by yeast improved the quality of mahua oil cake by increase in crude protein (CP), decrease in crude fibre (CF) and anti-nutritional factors like saponin and tannin. Use of fermented mahua oil cake as feed ingredient in rohu resulted in significant improvement (P<0.05) of growth performance and digestibility of nutrients (dry matter, crude protein, crude fibre and ether extract) compared to control fed with raw mahua oil cake. Similarly, significantly higher (P<0.05) intestinal enzyme activity like amylase and protease and non-specific immune parameters like lysozyme, myeloperoxidase, bacterial agglutination, hem-agglutination and respiratory bust activity (P<0.05) were recorded in fish fed on fermented mahua oil cake compared to fish fed on raw mahua oil cake. This showed that, feeding of solid state fermented mahua oil cake increased the growth performance, enzyme activity and immunological parameters in rohu compared to mahua oil cake without solid state fermentation.

Screening of Haemagglutination Activity of Some Common Vegetables

Screening of Haemagglutination Activity of Some Common Vegetables

Preliminary report on the hemagglutinating activity of the Scorpaena plumieri fish venom

The scorpionfish Scorpaena plumieri is one of the most venomous fish species in the Brazilian coast. Amongst many biological activities, the S. plumieri fish venom (SpV) promotes hemagglutination. Although this activity appears to be associated to the presence of C-type lectins in the venom, it has not yet been chemically or functionally characterized. In the present work we sought to advance the characterization of the hemagglutinating activity associated to this venom. By fractionating SpV through saline precipitation followed by size exclusion chromatography we obtained two purified fractions - HF1 and HF3 - with Ca2+-dependent agglutinating activity against rabbit erythrocytes, which remained stable upon storage at 4 and -80oC. HF1 and HF3 were bacteriostatic against Gram-positive bacteria (Staphylococcus aureus), displaying minimum inhibitory concentration (MIC) of 50 and 200 μg/mL, respectively. In addition, a resazurin-based viability assay revealed that both fractions, at doses up to 370 μg/mL, were cytotoxic against tumor and non-tumor cell lines. Finally, a tendency towards edema formation could be detected when the fractions - particularly HF1 - were injected into mice footpads. We believe our data contribute to a better understanding of the biological properties of the so often neglected fish venoms.

Способность экстрактов некоторых съедобных грибов подавлять репродукцию вируса гриппа

In this work, extracts from the mycelium of some xylophytic fungi: Pleurotus eryngii, Auricularia auriculajudae, Tremella fuciformis were obtained, the toxicity of the obtained extracts was evaluated, and the antiviral activity was researched. The influenza virus was grown in the allantois cavity of chicken embryos. The infectious titer of the influenza virus was determined by titration on chicken embryos. The presence of the virus was judged by the reaction of hemagglutinating activity. The virus infectivity titer was calculated using the Reed-Muench method. The hemagglutinating activity of viruses was determined by a standard method using 0.75 % suspension of chicken red blood cells. The main criterion for studying the specific antiviral effect of compounds was the CTI (chemical-therapeutic index), that is determined by the ratio of the average toxic concentration of the substance (TC50) to the average effective viral inhibitory concentration (EC50). The antiviral activity of the obtained extracts was studied on a model of human, animal and bird influenza viruses, in the dose range from 0.025 mcg/ml to 1.25 mcg/ml using the methodological recommendations of the “Guidelines for conducting Preclinical studies of medicines”. While studying the acute toxicity of 3 obtained fungi extracts, it was found that all studied fungi compounds with a single intragastric injection to white mongrel mice, did not show a toxic effect in the studied dose range. During the study of the ability to suppress the reproduction of various strains of the influenza virus, it was found that extracts of the fungi Auricularia auricula-judae and Tremella fuciformis have possessed antiviral properties, and extracts exceed in terms of CTI to commercial anti-influenza drugs.

Sensory Characteristics and Nutritional Quality of Food Products Made with a Biofortified and Lectin Free Common Bean (Phaseolus vulgaris L.) Flour.

Common beans (Phaseolus vulgaris L.) are an important source of nutrients with beneficial effects on human health. However, they contain lectins, that limit the direct use of flour in food preparations without thermal treatment, and phytic acid, that reduces mineral cation bioavailability. The objectives of this research were: to obtain biofortified snacks and a cream using an untreated common bean flour devoid of active lectins (lec−) and with reduced content of phytic acid (lpa) and to evaluate the sensorial appreciation for these products. The main results of the present work were: the products with the lpa lec− flour did not retain residual hemagglutinating activity due to lectins; they showed higher residual α-amylase inhibitor activity (from 2.2 to 135 times), reduced in vitro predicted glycemic index (about 5 units reduction) and increased iron bioavailability compared to the products with wild type flour; products with common bean flour were less appreciated than the reference ones without this flour, but the presence of an intense umami taste can be a positive attribute. Results confirmed that the use of the lpa lec− flour has important advantages in the preparation of safe and nutritionally improved products, and provide useful information to identify target consumers, such as children and elderly people.

Expression and diversity of the sialic acid-binding adhesin and its homologs associated with oral streptococcal infection.

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.

Amphotericin b effect on the sensitivity to influenza infection of WI-38 VA-13 cells with IFITM3 gene knockout

BACKGROUND: The application of CRISPR/Cas9 is one of the most rapidly developing areas in biotechnology. This method was used to obtain clones of а human origin cell line with knockout of one or more genes of the IFITM family, representing host restriction factors for influenza infection. Amphotericin B has previously been shown to promote influenza infection by blocking IFITM3 function.&#x0D; AIM: The aim of this study was to evaluate the effect of amphotericin B on the sensitivity of IFITM knockout cells to influenza A virus infection.&#x0D; MATERIALS AND METHODS: WI-38 VA-13 cells and mutant clones with IFITM3 knockout (F3 clone) or IFITM1, IFITM3 knockout (clone E12) were infected with influenza virus A/PR/8/34 (H1N1) in the presence or absence of amphotericin B. Forty-four hours after infection, the culture medium was taken to determine the infectious activity of the virus by titration in the MDCK cell culture, as well as the hemagglutinating activity of the virus. The infected cells were stained with fluorescently labeled antibodies against the viral NP protein, and the number of NP-positive cells was determined by flow cytometry.&#x0D; RESULTS: The addition of amphotericin B increased the hemagglutinating and infectious activity of the virus in WI-38 VA-13cells, while the difference was insignificant for clones with IFITM gene knockout. A similar dependency was obtained for the percent of infected cells.&#x0D; CONCLUSIONS: Mutant cells with a knockout of one or several genes of the IFITM family were equally susceptible to influenza infection regardless of the addition of amphotericin B, which confirms the crucial importance of a defect in the IFITM3 protein in increasing the permissiveness of cells to influenza A virus.

Assessment of the hemagglutinating activity of the Porcine orthorubulavirus

Blue eye disease (BED) in pigs is caused by Porcine orthorubulavirus (PRV) of the Paramyxoviridae family. It is an endemic disease in swine production in the central region of Mexico and causes nervous signs and high mortality in suckling pigs, pneumonia in growing pigs, orchitis in boars and mummification during gestation. PRV hemagglutinates most red blood cells (RBCs) of domestic species. For serological diagnosis, the hemagglutination inhibition test is used, and in this test, guinea pig, bovine and chicken RBCs have been commonly used. In this investigation, hemagglutination with PRV was evaluated using the RBCs of seven domestic species (chicken, bovine, horse, pig, dog, guinea pig and rabbit). In the hemagglutination test, the following parameters were evaluated: temperature (25 °C and 37 °C), bottoms of the wells (V and U), erythrocyte concentration (0.5%, 0.75%, and 1%), and reading time (15, 30, 45, 60 and 90 min). Significant differences (P < 0.001) were found in most of the evaluated treatments. The best hemagglutination results were obtained with chicken, bovine and horse RBCs. The hemagglutination titer is higher (2 dilutions) when using chicken RBCs than when using bovine or horse RBCs. If chicken RBCs are used in the inhibition of hemagglutination, the test will be more sensitive, while it is more specific when bovine or horse RBCs are used. The hemagglutination readings are imprecise when using RBCs from dogs, pigs, guinea pigs and rabbits. RBCs from these species should not be used for the diagnosis or investigation of PRV.