Goose Red Blood Cells Research Articles

Does prior vaccination affect the immune response to seasonal influenza vaccination among older adults? Findings from a prospective cohort study in a Northeastern Province of Thailand.

We measured the immunogenicity of seasonal trivalent inactivated influenza vaccines (IIV3) among older Thai adults and the effect of one-year prior vaccination status on immune responses. Adults aged ≥65 years (n = 370) were vaccinated with Southern Hemisphere IIV3 in 2015. Hemagglutination inhibition assays were performed using goose red blood cells on sera collected from the participants at baseline and after 1, 6, and 12 months of vaccination. Prior year vaccination (in 2014) was verified with the national health security office database. We analyzed the associations between prior vaccination and geometric mean titers (GMT) at each time point using generalized linear regression on logged transformed titers, and seroprotection and seroconversion using Log-binomial regression. At baseline, previously vaccinated participants (n = 203) had a significantly higher GMT and seroprotection against all three influenza strains than those previously unvaccinated (n = 167) (all p-values <0.001). Seroprotection rates were similar after one month in both groups for A(H1N1)pdm09 (adjusted risk ratio [aRR] 1.10, 95% CI 0.97-1.25), and A(H3N2) (aRR 1.08, 95% CI 0.87-1.33), but higher in previously vaccinated persons for B (aRR 1.20, 95% CI 1.08-1.32). At 12 months, 50% or more had seroprotection in previously vaccinated group with no difference between previously vaccinated or unvaccinated persons. Seroconversion was lower in the previously vaccinated group for A(H1N1)pdm09 (aRR 0.62, 95% CI 0.43-0.89), but did not differ between the two groups for A(H3N2) (aRR 0.94, 95% CI 0.69-1.28) and B (aRR 0.85, 95% CI 0.60-1.20). Influenza vaccination elicited good humoral response in older Thai adults. While seroconversion seemed attenuated in persons previously vaccinated for influenza A(H1N1)pdm09 (the only vaccine strain not to change), this was not apparent for influenza A(H3N2) and B, and prior vaccination was not associated with any inhibition in seroprotection.

Optimization of Storage Medium for Hematological Reference Samples in External Quality Assessment

Hematological reference samples for external quality assessment (EQA) in hematology are essential for many laboratories in Vietnam, however, the prices are high along with short storing time. This study attempted to establish an optimal formula for the manufacture of hematological reference samples for hematological EQA programs. Human red blood cells (RBCs) were mixed with goose RBCs (pseudo-leucocytes) and goat RBCs (pseudo-platelets) as alternatives for white blood cells (WBCs) and platelets (PLTs), respectively, in different formulas of storage media. The optimal formula was obtained using response surface methodology–central composite design (RSM-CCD). Three main factors affecting the concentrations of RBCs were determined by Plackett–Burmann matrix. The optimal concentrations determined by RSM-CCD were 46% serum, 0.44 g/L neomycin sulfate, and 2.5% glycerol. The model predicted that the maximum number of human RBCs was 4.1 × 1012 cells/L, pseudo-leucocytes was 6.4 × 109 cells/L, and pseudo-platelets was 188 × 109 cells/L. The practical concentrations of RBCs, pseudo-leucocytes, and pseudo-platelets were 4.21 × 1012 cells/L, 6.4 × 109 cells/L, and 187 × 109 cells/L, respectively, which showed 98.93% similarity with theoretical data. This result could be a premise for further study to improve manufacturing of hematological reference samples for hematology EQA programs.

Characteristics of two highly pathogenic avian influenza H5N8 viruses with different pathogenicity in mice

Novel reassortant influenza A (H5N8) viruses are becoming a potential threat not only to the poultry industry but also to public health. Many molecular markers for pathogenicity in mammalian hosts have been identified in other H5 subtype avian influenza viruses (AIVs). However, the pathogenicity of H5N8 AIVs to mammals remains unclear. It is believed that selection of a pair of isolates with a similar genetic background but with different virulence to mammals is a prerequisite for studying the pathogenic mechanism of AIVs. Two avian-origin H5N8 isolates, A/goose/Eastern China/CZ/2013 (CZ13) and A/duck/ Eastern China /JY/2014 (JY14), which shared a similar genetic background (H5 clade 2.3.4.4) and amino acid substitutions that were shown previously to be molecular markers of pathogenicity, were used to determine their biological characteristics and pathogenicity. Hemagglutination assays using α-2,3-sialidase-treated goose red blood cells demonstrated that both viruses exhibited a dual-receptor-binding preference. Viral growth kinetics in vitro indicated that both viruses replicated to high titers in CEF cells (about 108.0 TCID50/mL). In MDCK cells, however, CZ13 replicated efficiently (107.0 TCID50/mL), while JY14 grew to peak titers below 104.0 TCID50/mL. Animal studies indicated that although both viruses were highly virulent in chickens, they exhibited significantly different virulence in mice. CZ13 was highly pathogenic (MLD50 = 101.6EID50), whereas JY14 had low virulence (MLD50>106.5 EID50). Therefore, this pair of viruses can be used to search for unknown molecular markers of virulence and to investigate specific pathogenic mechanisms in mice.

Phylogenetic and biological characterization of three K1203 (H5N8)-like avian influenza A virus reassortants in China in 2014

Three H5N8 avian influenza viruses isolated from domestic geese in China in 2014 were characterized phylogenetically and biologically. Phylogenetic analysis of the complete genomic sequences of the three isolates from this study and those of 61 other H5N8 viruses retrieved from the GISAID platform indicated that, chronologically and geographically, all H5N8 viruses of the Asian H5N1 HA lineage of clade 2.3.4.4 are the direct descendents of the K1203 (H5N8)-like viruses first isolated in China in 2010. The three viruses from this study shared high sequence similarity in all eight gene segments with three other isolates from China in 2013, and two Korean isolates were distinct from the recently circulating reassortants causing outbreaks in Asia, Europe and the United States in 2014 and 2015. In vitro viral growth curves indicated that these H5N8 viruses replicated to high titers in CEF, DEF, MDCK and A549 cells but to significantly lower titers in Vero cells. Pathogenicity studies in vivo indicated that these viruses were all highly virulent to chickens and mallard ducks, while they varied from moderate to high virulence in mice. Additionally, hemagglutination assays using α-2,3-sialidase-treated goose red blood cells and solid-phase direct binding assays with different glycans demonstrated that the three viruses could bind to both avian-type SAα-2,3Gal and human-type SAα-2,6Gal receptors. Our findings confirmed the progenitor nature of the K1203-like viruses in generating recent prevalent clade 2.3.4.4 H5N8 reassortants, which have caused tremendous damage to the poultry industry and are a potential threat to public health.

Receptor specificity and erythrocyte binding preferences of avian influenza viruses isolated from India

IntroductionHemagglutination (HA) and hemagglutination inhibition (HI) assays are conventionally used for detection and identification of influenza viruses. HI assay is also used for detection of antibodies against influenza viruses. Primarily turkey or chicken erythrocytes [red blood cells (RBCs)] are used in these assays, as they are large, nucleated, and sediment fast, which makes it easy to determine the titer. Human influenza viruses agglutinate RBCs from chicken, human, and guinea pig, but not from horse. Human influenza viruses bind preferentially to sialic acid (SA) linked to galactose (Gal) by α 2, 6 linkage (SA α 2, 6-Gal), whereas avian influenza (AI) viruses bind preferentially to SA α 2, 3-Gal linkages. With this background, the present study was undertaken to study erythrocyte binding preferences and receptor specificities of AI viruses isolated from India.Materials and methodsA total of nine AI virus isolates (four subtypes) from India and three reference AI strains (three subtypes) were tested in HA and HI assays against mammalian and avian erythrocytes. The erythrocytes from turkey, chicken, goose, guinea pig and horse were used in the study. The receptor specificity determination assays were performed using goose and turkey RBCs. The amino acids present at 190 helix, 130 and 220 loops of the receptor-binding domain of the hemagglutinin protein were analyzed to correlate amino acid changes with the receptor specificity.ResultsAll tested highly pathogenic avian influenza (HPAI) H5N1 viruses reacted with all five types of RBCs in the HA assay; AI H9N2 and H5N2 viruses did not react with horse RBCs. For H5N1 viruses guinea pig and goose RBCs were best for both HA and HI assays. For H9N2 viruses, guinea pig, fowl and turkey RBCs were suitable. For other tested AI subtypes, avian and guinea pig RBCs were better. Eight isolates of H5N1, one H4N6 and one H7N1 virus showed preference to avian sialic acid receptors. Importantly, two isolates of HPAI H5N1, H9N2 and H11N1 viruses showed receptor specificity preference to both avian and mammalian sialic acid (α-2, 3 and α-2, 6) receptors.ConclusionsUse of different types of RBCs resulted in titer variations in HA and HI assays. This showed that RBCs giving optimum HA and HI titers would increase sensitivity of detection and would be more appropriate for identification and antigenic analysis of AI viruses. Analysis of 16 amino acids in the receptor-binding domain of the hemagglutinin of HPAI H5N1 viruses revealed that the only variation observed was in S221P amino acid position. Two H5N1 viruses showed S221P amino acid change, out of which only one H5N1 virus showed preference to α 2, 6 sialic acid receptor. One H5N1 virus isolate with amino acid S at 221 position, showed preference to α 2,3 as well as α 2,6 sialic acid receptors. This indicated that factor(s) other than S221P mutation in the hemagglutinin are probably involved in determining receptor specificity of H5N1 viruses. This is the first report of receptor specificity and erythrocyte binding preferences of AI viruses from India.

Relative Sensitivities of Japanese Quail to Foreign Red Blood Cell Challenges for Immunotoxicity Testing

Given evidence that the immune system is sensitive to environmental contaminants, evaluating immunocompetence in toxicology studies is increasingly important. By incorporating a test of humoral response into controlled reproductive and developmental studies, more comprehensive results can be gathered to assess the potential for disease. The foreign red blood cells (RBC) challenge is a minimally invasive method for evaluating humoral responses to a foreign antigen. Typically, antibody response is assessed following injections of sheep erythrocytes; however, Japanese quail (Coturnix japonica) are only minimally sensitive to sheep RBC. In the present study, adult Japanese quail were treated with 5% solutions of RBC from goose, goat, cow, donkey, or pig in primary and secondary challenges. After each treatment, plasma samples were taken and antibody responses were measured for total immunoglobulins (Ig), IgG, and IgM. Overall, goose RBC generated the poorest responses in both primary and secondary challenges, while the strongest antibody responses were to pig and donkey RBC. Therefore, pig RBC appear to be a superior antigen for testing humoral response in Japanese quail.

M protein correlates with the receptor-binding specificity of haemagglutinin protein of reassortant influenza A (H1N1) virus.

From the reassortment experiments between A/Aichi/4/92 and A/WSN/33 (WSN) (H1N1) viruses, two different phenotype viruses which contained the haemagglutinin (HA) gene from A/Aichi/4/92 virus and the neuraminidase (NA) gene from WSN virus were obtained. PW13 and PW15 viruses agglutinated chicken red blood cells (CRBC), while PW10 and PW70 viruses did not. However, the expressed HA proteins of these viruses did not adsorb CRBC. The difference in gene constellation between PW13, PW15 and PW10, PW70 viruses was the membrane protein (M) gene. The former two had the M gene from A/Aichi/4/92 virus and the latter two had that from WSN virus. In PW15-infected cells, haemadsorption of CRBC was observed 30 min later than that of goose red blood cells and the M1 protein migrated from the nucleus to the cytoplasm 30 min earlier than adsorption of CRBC was observed. On the other hand, in PW10-infected cells, haemadsorption of CRBC was not observed through the virus replication and the M1 protein stayed in the nucleus after HA and NA activities reached maximum levels. Co-expression of the M and the HA proteins of A/Aichi/4/92 virus did not help the HA protein gain the ability to adsorb CRBC. However, neuraminidase treatment of COS cells expressing the HA protein of A/Aichi/4/92 virus or MDCK cells infected by PW10 virus restored the ability to adsorb CRBC. We discussed the possibility that the M1 protein helped the NA protein in its role to modify the HA protein on the cell surface.

Analysis of the host-specific haemagglutination of influenza A(H1N1) viruses isolated in the 1995/6 season

Two phenotypes of human influenza A(H1N1) virus are currently circulating in Japan. One (group 1) agglutinates both chicken and goose red blood cells (CRBC and GRBC), the other (group 2) agglutinates GRBC but not CRBC. In the 1995/6 season, group 2 viruses accounted for 70% of the H1N1 viruses isolated in MDCK cells. The 1995/6 viruses were located on two branches of the genetic tree. One branch contained both group 1 and group 2 viruses and the other branch contained only group 2 viruses. Group 2 viruses had aspartic acid at residue 225 in the haemagglutinin (HA) protein, the key amino acid residue for group 2 phenotype. The HA protein of group 1 viruses had a change from aspartic acid to asparagine at residue 225 and the expressed HA protein of these viruses adsorbed CRBC. Serial passage of group 2 viruses in MDCK cells or embryonated chicken eggs caused these viruses to gain the ability to agglutinate CRBC. MDCK-adapted viruses had the same amino acid sequences of HA polypeptide as the original ones, but egg-adapted viruses had changed amino acid sequences. The expressed HA protein from one egg-adapted virus that originally belonged to group 2 adsorbed CRBC.

Studies on the molecular basis for loss of the ability of recent influenza A (H1N1) virus strains to agglutinate chicken erythrocytes.

Recent strains of influenza A but not B viruses have lost the ability to agglutinate chicken red blood cells (CRBC). The H1N1 viruses isolated in Japan during the 1991/92 season could be divided into two groups. Group 1 viruses (A/Aichi/4/92 and A/Aichi/7/92) agglutinated goose red blood cells (GRBC) and CRBC, while group 2 viruses (A/Aichi/24/92 and A/Aichi/26/92) did not agglutinate CRBC. There were no amino acid differences between them in the haemagglutinin (HA) polypeptide. Reassortment experiments between a group 1 virus (A/Aichi/4/92) or a group 2 virus (A/Aichi/24/92) and the A/WSN/33 influenza A (H1N1) virus strain suggested that the HA gene products of the viruses of both groups had lost the capacity to agglutinate CRBC. The HA proteins expressed on Cos cells by transfecting the cDNAs of the virus HA gene of A/Aichi/4/92 and A/Aichi/24/92 agglutinated GRBC but not CRBC. These experiments indicated that the HA proteins of H1N1 viruses of both groups isolated in 1992 had lost the ability to agglutinate CRBC even though the group 1 virions showed haemagglutinating capacity with CRBC. By using the cDNAs of the HA gene of seven natural isolates obtained from 1977 to 1992, it was found that the expressed HA proteins of influenza A (H1N1) viruses isolated since 1988 had lost the ability to agglutinate CRBC. Experiments with chimeric and point-mutated HA cDNAs of A/Aichi/24/92 showed that an amino acid change at residue 225, which occurred after 1986, and a cluster of amino acid changes at residues 193, 196 and 197, which occurred before 1986, were responsible for loss of the ability to agglutinate CRBC. Egg-adapted virus derived from A/Aichi/24/92 had one amino acid change at residue 225 compared to the parental virus.

Serodiagnosis of Chagas disease: new reagent for the indirect hemagglutination test (THAI(IAL))

A new reagent was designed to the indirect hemagglutination test (IHATIAL), utilizing goose red blood cells as inert matrix and standardized for the field diagnosis of American trypanosomiasis. The objective was to substitute the lyophilized or frozen reagent of IHAT produced routinely using human erythrocytes in the Adolfo Lutz Institute (São Paulo/Brazil). The standardized reagent presented a long stability in liquid suspension, and was evaluated in 137 serum samples from patient with and without Chagas' disease, by IHATIAL. The diagnostic performance of this test was similar to the IHAT utilizing human erythrocytes and to that of a commercial IHAT kit. The sensitivity was 1.00, specificity 0.98, predictive value of positive 0.96 and of negative 1.00. Different batches of reagent successively produced proved to be reproducible in a quality control method. The new reagent is more economic than the former reagent, it can be produced easily and may be applicable to the seroepidemiologic studies.

Use of goose red blood cells for detection of infection with psittacine beak and feather disease virus by haemagglutination and haemagglutination inhibition

Use of goose red blood cells for detection of infection with psittacine beak and feather disease virus by haemagglutination and haemagglutination inhibition

Inhibition ofBordetella pertussisfilamentous hemagglutinin-mediated cell adherence with monoclonal antibodies

Filamentous hemagglutinin (FHA), a 220-kDa protein located on the surface of Bordetella pertussis, is one of the major cell adhesins of this bacterium. We have produced three hybridoma cell lines that express monoclonal antibodies (mAbs) against FHA: X3C, X3E and X4B. The anti-FHA mAbs X3C and X3E reacted with 220-kDa and 98-kDa FHA protein bands on Western blots. The mAb X4B, which reacted with FHA in ELISA, did not bind to FHA in a Western blot assay. All three mAbs seemed to be directed to the same epitope or to epitopes in close proximity as suggested by competition ELISAs. All three mAbs were able to inhibit the adherence of Chinese hamster ovary cells to purified FHA, and they could also inhibit the FHA-mediated agglutination of goose red blood cells. The attachment of B. pertussis to epithelial cell monolayers was inhibited by the mAb X3C. These antibodies are very useful probes to identify the presence of FHA in bordetellae species and in clinical reagents such as pertussis vaccines, and to characterize the functional domains of this important bacterial adhesin.

Isolation of Dengue Virus with a Human Promonocyte Cell Line

In October-November, 1988 there was an outbreak of dengue fever in the Kaoshiung area of southern Taiwan. We collected 100 serum samples from 96 patients at the onset of their fever for virus cultures and identification. A human promonocyte cell line (HL-CZ) established in our laboratory was used and proved to be susceptible for dengue virus propagation. Type 1 dengue virus in the HL-CZ cell culture was identified by immunofluorescence tests using monoclonal antibodies, and also by hemagglutination tests with goose red blood cells. The density of the virus particles, as measured by sucrose gradient ultracentrifugation, ranged from 1.186 to 1.224 g/ml. The virus yield from this cell culture is comparable with that from the C6/36 mosquito cell line. There was a significant correlation between the antibody responses tested with Western dot blots and hemagglutination inhibition techniques.

Preliminar characterization of a new virus isolated from the spinal fluid of patients with meningitis in São Paulo, Brazil

A total of 138 patients with the age of 4 months to 57 years were attended in different hospitals of São Paulo State with aseptic meningitis. A probable new agent was isolated from the cerebrospinal fluid of 35 of 53 specimens examined. Replication of the agent with similar characteristics was detected by CPE produced in the MDCK cell line. Virus-like particles measuring about 40 nm in diameter were observed by negative staining electron microscopy. No hemagglutinating activity was detected at pH 7.2 by using either human, guinea pig, chicken and at pH ranged 6.0-7.2 with goose red blood cells. The agent was not pathogenic to newborn or adult mice. Virus infectivity as measured by CPE was sensitive to chloroform and not inhibited by BuDR, suggesting that agent is an enveloped virus with RNA genome.

Locus of N-ethylmaleimide action on sugar transport in nucleated erythrocytes.

Goose red blood cells were studied as a model for metabolic regulation of sugar transport. In contrast to their action in human erythrocytes, sulfhydryl-blocking agents such as N-ethylmaleimide (NEM) stimulated 3-O-methylglucose transport markedly in goose red blood cells. The effect of NEM was further enhanced when adenosine 5'-triphosphate (ATP) was first depleted by 2,4-dinitrophenol treatment or anoxia. Only sulfhydryl-blocking agents that enter the cell were effective transport stimulators, and the effect was not altered by substrates of the transporter. In nucleated red blood cell ghosts, NEM inhibited 3-O-methylglucose transport. Results of these studies with intact cells were consistent with the hypothesis that free sulfhydryl groups are essential for regulation of transporter activity rather than for the transport process itself. The locus of NEM action appears to be either on the cytoplasmic side of the membrane or partially located in the cytoplasm. ATP depletion may expose previously masked sulfhydryl groups, producing an enhanced reaction with sulfhydryl-blocking agents and a highly stimulated rate of sugar transport.

Heterophile antibodies. Part I. The specificity of agglutinating and hemolytic sheep red blood cell antibodies in human sera.

Absorption and agglutination inhibition studies carried out on human sera with intact red blood cells, secretory substances and soluble red blood cell membrane extracts show that antibodies against sheep red blood cells possess at least two types of specificities. The agglutinating type has specificities in common with human anti‐A and anti‐B, while the hemolytic antibody does not. Comparative titration studies clearly demonstrate that there is no correlation in the distribution of these two varieties of sheep red blood cell antibodies. Guinea pig and rat erythrocytes share human A‐ and B‐like determinants with sheep red blood cells. Rabbit, horse and goose red blood cells do not possess these blood group factors.

Biological properties of the VSV glycoprotein II. Effects of the host cell and of the glycoprotein carbohydrate composition on hemagglutination

Virions of the Indiana serotype of vesicular stomatitis virus (VSV) grown in HKCC, BHK21-F, and MDBK cells have been found to differ considerably in their ability to agglutinate goose red blood cells and in their pH optimum for hemagglutination. Virions grown in HKCC cells had a specific hemagglutinating activity approximately 20 times greater than those grown in BHK21-F cells, and approximately 40 times greater than those grown in MDBK cells. The differences in activity and pH optima for hemagglutination were also found with the glycoprotein isolated from virions grown in the different cells. Carbohydrate analyses of the glycoproteins isolated from virions grown in the different cells revealed differences in carbohydrate composition. The observed differences in total carbohydrate and sialic acid content of the viral glycoproteins, and their correlation with differences in hemagglutinating activity provide additional evidence for a role of carbohydrate in virus-receptor interactions and suggest a chemical basis for the host-cell modification of the hemagglutinating activity of VSV.

Procedures for the production and separation of H and M antigens in histoplasmin: Chemical and serological properties of the isolated products

Two strains of Histoplasma capsulatum were required to prepare maximum yields of H and of M antigen from histoplasmin. The antigens were separated and partially purified by a series of procedures yielding an overall recovery of 70 to 90% of the individual antigens. Stable products suitable for use as reference products were obtained when the final purification step employed DEAE-cellulose with phosphate buffer elution at increasing molarity and decreasing pH. A final step of purification of each antigen with slab acrylamide gel electrophoresis gave products which were highly reactive and specific in a variety of serological tests with sera from persons with proven cases of histoplasmosis and with natural infections of heterologous deep mycoses. These antigens were maximally active at concentrations of 2 to 16 μg protein in the complement fixation, capillary precipitin, microimmunodiffusion, or immunoelectrophoresis tests; 0.5 μg gave a maximum delayed cutaneous hypersensitivity reaction in homologously infected animals and caused no appreciable reaction in control animals. Although these antigens appeared to be specific when tested with sera from persons with natural infections, the M and H antigens demonstrated the presence of an additional antigen reacting with sera of rabbits immunized with cell membrane and cell particulate fractions of Blastomyces dermatitidis. After purification by electrophoresis, both the H and M antigens of some preparations showed some decomposition and loss of reactivity after storage at 5 C for more than six months. The overall results suggest that the purified H and M antigens of Heiner (12) have multiple serological reactivity and may function in precipitin reactions, complementfixing reactions, hemagglutination of formalin-fixed goose red blood cells, and as antigens for delayed cutaneous tests.

Growth, purification and characterization of Semliki Forest virus in Ehrlich ascites tumor cell suspensions.

The growth of Semliki Forest Virus (SFV) in suspension cultures of Ehrlich Ascites (EA) cells and its purification is described. Large volumes of virus material were concentrated by filtration with DIAFLO XM-300 membrane and precipitation with ammonium sulfate. A combination of protamine sulfate treatment, centrifugation of the virus onto a 50 per cent sucrose cushion, and sedimentation through a 5--30 per cent sucrose density gradient was employed. The purified virus particles were homogeneous as revealed by electron microscopy, by moving boundary electrophoresis, and by polyacrylamide gel electrophoresis. Virus suspensions containing 1 mg/ml of protein had a hemagglutinin titer of 1:12,000 when measured with 0.25 per cent goose red blood cells.

Hemagglutination Inhibition with Arboviruses: Relationship Between Titers and Source of Erythrocytes

Antigens for Grand Arbaud, Hazara, and California arboviruses were able to agglutinate goose and either dog, hamster and guinea pig, or hamster red blood cells (RBC) to the same titer at the same pH; in hemagglutination-inhibition (HI) tests, titers for homologous and related sera were the same with these different types of RBC or occasionally one dilution higher with the mammalian cells. Antigens for St. Louis encephalitis and Eastern equine encephalitis viruses required use of lower antigen dilutions with human, guinea pig, and hamster RBC than with goose RBC. The results of comparative HI testing with these latter antigens and types of RBC indicate that HI titer is not directly related to the antigen dilution used with different types of RBC.