Hemagglutination by Rabies Virus

Goose erythrocytes were agglutinated by five strains of rabies virus grown in monolayer cell cultures at pH 6.4 and at 0 to 4 C. Hemagglutination was not affected by the cell type in which the virus was grown. Prerequisites for occurrence of hemagglutination are absence of hemagglutination inhibitors (such as those contained in bovine serum) and a relatively high virus concentration (> 10(6) plaque-forming units of virus per ml). "Soluble" hemagglutinin was not present in crude preparations of extracellular virus. Treatment of purified preparations of extracellular virus with Tween 80 and ether did not result in release of a "soluble" hemagglutinin. The hemagglutinating property of extracellular virus seemed to be conditioned by the integrity of its coat. Preparations of infectious intracellular virus exhibited about 15 times lower hemagglutinating activity than extracellular virus. This decreased hemagglutinating activity did not seem to be caused by binding of hemagglutination inhibitors to the virus particles. Rabies virus can be quantitatively adsorbed onto and eluted from erythrocytes. Erythrocytes pretreated with rabies virus retained their ability to be agglutinated by the same virus strain. The reaction with rabies virus of erythrocytes treated with the receptor-destroying enzyme or KIO(4) was the same as that of nontreated erythrocytes. The hemagglutinating component of rabies virus, therefore, does not exhibit neuraminidase activity. Treatment of extracellular virus by various agents indicated that the hemagglutinating component consists of protein or lipoprotein. Sulfhydryl groups present in the viral hemagglutinin are essential for hemagglutination.     

Distribution of Cell Receptors and Simple Sugar Inhibitors During Encephalomyocarditis Virus-Cell Union

Encephalomyocarditis (EMC) virus was studied for ability to agglutinate erythrocytes of various species. Human, rat, and guinea pig erythrocytes, as well as those from young rabbits, were readily agglutinated. Cells from older rabbits absorbed virus poorly, and showed little agglutination. Uptake of virus by rabbit brain also diminished with age. Various mouse tissues absorbed virus about equally well. Hemagglutination-inhibition studies demonstrated that a number of simple sugars, particularly glucose and galactosamine, interfered with uptake of virus by cells. Dextran sulfates were highly active inhibitors of EMC hemagglutination. Attempts to localize the site of action of the sugars on virus or cell are described. Treatment of virus with periodate or p-chloromercurobenzoate, and acetylation of virus, inhibited hemagglutination, but acetylation of semipurified receptor did not. Clarification of the nature of the virus-cell union will require studies to identify possible specific sugars in the virus capsid and the cell receptor.     

Screening of a high growth influenza B virus strain in Vero cells

Due to the insufficient supply of embryonated chicken eggs, the preparation of large quantities of inactivated influenza vaccines will require an alternative virus culture system after the emergence or reemergence of a pandemic influenza virus. The Vero cell is one of the ideal options since it was used for producing many kinds of human vaccines. However, most of the influenza viruses can not grow well in Vero cells. To develop a new influenza vaccine with Vero cells as a substrate, the virus needs to adapt to this cell substrate to maintain high growth characteristics. By serial passages in Vero cells, the B/Yunnan/2/2005va (B) strain was successfully adapted to Vero cells, with the hemagglutination titer (HAT) of the virus reaching 1:512. The high growth characteristic of this strain is stable up to 21 passages. The strain was identified by hemagglutination inhibition (HAI) test and sequencing respectively; the HA₁ gene sequence of the virus was cloned and analyzed. The screening and establishment of high growth B virus provides an important tool for influenza vaccine production in Vero cells.     

Characteristics of Sendai virus receptors in a model membrane

The adsorption of Sendai virus to liposomes of different compositions was studied. Liposomes prepared with only phosphatidylcholine and cholesterol, and liposomes prepared with phosphatidylcholine and cholesterol plus phosphatidic acid or phosphatidyl serine did not adsorb virus. Phosphatidyleholine-cholesterol liposomes containing also stearyl amine or ganglioside did, however, adsorb virus. The ability of the adsorbing liposomes to compete with erythrocytes for virus was measured by hemagglutination inhibition. Liposomes containing ganglioside, but not those containing stearyl amine, inhibited hemagglutination. When the molar ratio of ganglioside N-acetyl neuraminic acid to phosphatidylcholine was less than 0.02, ganglioside liposomes did not inhibit hemagglutination. As the ratio increased from 0.02 to 0.05, the liposomes caused increasing amounts of hemagglutination inhibition, but with further increases in the ratio the hemagglutination inhibition remained constant. It is concluded that gangliosides can serve as Sendai receptors and that a multiplicity of receptors is needed for virus binding.     

Hemagglutination-Inhibition Method and Immunofluorescence Staining with Venezuelan Equine Encephalomyelitis Virus

Hemagglutination and fluorescent antibody (FA) are compared for the direct detection of virus devoid of host cells. A determination was made of the minimal number of tissue plaque-forming units of Venezuelan equine encephalomyelitis virus that could be detected by the hemagglutination technique. Similar concentrations of the virus in bovine albumin borate saline, Brain Heart Infusion broth (Difco), and demineralized water were tested by the FA technique. Somewhat higher concentrations of the virus in bovine albumin borate saline were used in the hemagglutination-inhibition test. The quantitative hemagglutination procedure employed for these studies was carried out at 37 C for 75 min with variations in concentration of goose red cells. As a result of lowering the red cell concentration, smaller concentrations of virus were detected. The direct FA staining procedure applied to slide preparations containing known numbers of tissue culture plaque-forming units of virus was negative. Adsorbed viral antigen on agglutinated goose erythrocytes was visualized by direct and indirect FA techniques.     

Sendai virus stimulates chemiluminescence in mouse spleen cells.

Sendai virus stimulates chemiluminescence within a few seconds after it is added to a suspension of mouse spleen cells. Virus rendered non infectious by irradiation with ultraviolet light induces a similar burst of chemiluminescence. Heating or pronase treatment of the virus abrogate this reaction, as does sonication of the cells before the addition of the virus. The ability of the virus to stimulate chemiluminescence is correlated with its hemagglutination, neuraminidase, cell fusion and hemolytic properties. It is suggested that Sendai virus-induced chemiluminescence is initiated by the interaction of the virus envelope spike glycoproteins with the cell membrane.     

Capsid region involved in hepatitis A virus binding to glycophorin A of the erythrocyte membrane

Hepatitis A virus (HAV) has previously been reported to agglutinate human red blood cells at acidic pHs. Treatment of erythrocytes with different enzymes and chemical reagents indicated that HAV attachment is mediated through an interaction with sialylglycoproteins. HAV hemagglutination could be blocked by incubating the virus with glycophorin A, indicating that this sialylglycoprotein is the erythrocyte receptor. The number of receptors used was estimated to be around 500 per cell. At the same time, HAV-induced hemagglutination could also be blocked by either monoclonal antibody H7C27 or an anti-VP3(102-121) ascitic fluid, indicating that lysine 221 of VP1 and the surrounding VP3 residues lining the capsid pit are involved in HAV binding to erythrocytes.     

Isolation and identification of dengue viruses by combined use of C6/36 cells and the immune adherence hemagglutination test

Dengue viruses were isolated in a mosquito cell clone, C6/36, and their serotypes were identified by the immune adherence hemagglutination (IAHA) test. Even when viruses were not cytopathogenic, the IAHA test detected virus growth in the cells.     

Lipid vesicle-cell interactions. I. Hemagglutination and hemolysis

The interaction of lipid vesicles (liposomes) of several different compositions with erythrocytes has been investigated. Lecithin liposomes, rendered positively charged with stearylamine, exhibit potent hemagglutination activity in media containing low concentrations of electrolytes. The hemagglutination titer is found to be a linear function of the zeta potential of the lipid vesicles. Hemagglutination is reduced when the surface potential of the cells is made more positive by pH adjustment or enzyme treatment. Similarly, hemagglutination is reduced by increasing concentrations of electrolytes. Hemagglutination is examined theoretically and is shown to be consistent with vesicle-cell interactions that are due to only electrostatic forces. Vesicles containing lysolecithin in addition to lecithin and stearylamine cause lysis of erythrocytes, provided the lipids of the vesicles are above the crystal-liquid crystal phase transition temperature. In addition, hemolysis requires close juxtaposition of the vesicle to the cell membrane; vesicles precoated with antibodies exhibit severely diminished hemolytic activities, only a small fraction of which can be attributed to a reduction in hemagglutination titer. Evidence is presented indicating that a single vesicle is sufficient to lyse one cell. With regard to hemagglutination and hemolysis, lipid vesicles of simple composition mimic paramyxoviruses such as Sendai virus.     

Genetic reassortants for identification of the genome segment coding for the bluetongue virus hemagglutinin.

Two bluetongue virus (BTV) serotypes isolated in Australia and two selected reassortants derived from cells coinfected with these viruses have been used to identify the gene coding for the virus hemagglutinin. The parent viruses had characteristic hemagglutination patterns: BTV type 20 agglutinated sheep erythrocytes only; and BTV type 21 agglutinated sheep, bovine, human, and goose erythrocytes. Analysis of the two virus clones that had reassorted in genes coding for the outer capsid polypeptides demonstrated that hemagglutination and hemagglutination inhibition are functions associated with the outer capsid protein (VP2), which is encoded by genome segment 2.     

筛选抗甲3型（H3N2）流感病毒单克隆抗体ELISA间接方法的建立

用血凝抑制实验方法,虽可直接筛选到抗甲_3型流感病毒血凝素(H)单克隆抗体,但检测出的杂交瘤培养物上清中有小牛血清,做血凝抑制时还需用受体破坏酶处理去除非特异性抑制物。为减少麻烦我们建立了便于大批检测和筛选抗甲_3型(H_3N_2)流感病毒单克隆抗体的ELISA间接方法。     

Effect of the DnaK chaperone on the conformational quality of JCV VP1 virus‐like particles produced in Escherichia coli

Protein nanoparticles such as virus‐like particles (VLPs) can be obtained by recombinant protein production of viral capsid proteins and spontaneous self‐assembling in cell factories. Contrarily to infective viral particles, VLPs lack infective viral genome while retaining important viral properties like cellular tropism and intracellular delivery of internalized molecules. These properties make VLPs promising and fully biocompatible nanovehicles for drug delivery. VLPs of human JC virus (hJCV) VP1 capsid protein produced in Escherichia coli elicit variable hemagglutination properties when incubated at different NaCl concentrations and pH conditions, being optimal at 200 mM NaCl and at pH range between 5.8 and 7.5. In addition, the presence or absence of chaperone DnaK in E. coli cells influence the solubility of recombinant VP1 and the conformational quality of this protein in the VLPs. The hemagglutination ability of hJCV VP1 VLPs contained in E. coli cell extracts can be modulated by buffer composition in the hemagglutination assay. It has been also determined that the production of recombinant hJCV VP1 in E. coli is favored by the absence of chaperone DnaK as observed by Western Blot analysis in different E. coli genetic backgrounds, indicating a proteolysis targeting role for DnaK. However, solubility is highly compromised in a DnaK^? E. coli strain suggesting an important role of this chaperone in reduction of protein aggregates. Finally, hemagglutination efficiency of recombinant VP1 is directly related to the presence of DnaK in the producing cells. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:744–748, 2014     

重组细胞高产型H3N2猪流感病毒株的拯救

为拯救出一株能够在动物传代细胞中高水平复制的H3N2亚型猪流感疫苗株,利用反向遗传操作技术,将A/Goose/Dalian/3/01(H9N2)毒株的PB1、PA、NP、M、NS基因和A/PR/8/34毒株的PB2基因作为内部基因与猪流感病毒A/Swine/Henan/S4/01(H3N2)毒株的HA、NA基因进行重组,成功拯救出了具有高度细胞适应性毒株rH3N2株,该毒株接毒MDCK细胞60h后,血凝价可以达到1∶512,表明该毒株具有高度适应细胞繁殖特性,为H3N2亚型猪流感病毒细胞培养型疫苗的研制奠定了基础。     

A single-amino-acid substitution in polyomavirus VP1 correlates with plaque size and hemagglutination behavior.

The plaque size and hemagglutination characteristics of five cloned wild-type strains of polyomavirus were determined. The strains fell into two groups, those with large or small plaques, each with distinctive hemagglutination behavior at different temperatures and pHs. The nucleotide sequence of VP1, the major capsid protein of the virus, was determined for each of the viral strains. The PTA (large-plaque) and RA (small-plaque) strains differed only at residue 92 of VP1, where there is a glutamic acid or glycine, respectively (R. Freund, A. Calderone, C. J. Dawe, and T. L. Benjamin, J. Virol. 65:335-341, 1991). The same amino acid difference in VP1 correlated with plaque size and hemagglutination properties of the other sequenced viruses. Mutagenesis converting amino acid 92 from glutamic acid to glycine converted the plaque size and hemagglutination behavior of the large-plaque PTA strain to that of a small-plaque strain. Furthermore, PTA and RA VP1 proteins produced in Escherichia coli behaved as their parental viruses did in hemagglutination assays. These results demonstrate that amino acid residue 92 of VP1 is involved in determining the plaque size and hemagglutination behavior of polyomavirus and strongly suggest that this region of the VP1 polypeptide interacts directly with cell receptors.     

Herpes simplex virus type 1-induced hemagglutination: glycoprotein C mediates virus binding to erythrocyte surface heparan sulfate.

We recently reported that herpes simplex virus type 1 (HSV-1) can cause agglutination of murine erythrocytes (E. Trybala, Z. Larski, and J. Wisniewski, Arch. Virol. 113:89-94, 1990). We now demonstrate that the mechanism of this hemagglutination is glycoprotein C-mediated binding of virus to heparan sulfate moieties at the surface of erythrocytes. Hemagglutination was found to be a common property of all gC-expressing laboratory strains and clinical isolates of HSV-1 tested. Mutants of HSV-1 deficient in glycoprotein C caused no specific hemagglutination, whereas their derivatives transfected with a functional gC-1 gene, thus reconstituting gC expression, regained full hemagglutinating activity. Hemagglutination activity was inhibited by antibodies against gC-1 but not by antibodies with specificity for glycoproteins gB, gD, or gE or by murine antiserum raised against the MP strain of HSV-1, which is gC deficient. Finally, purified gC-1 protein, like whole HSV-1 virions, showed high hemagglutinating activity which was inhibited by heparan sulfate and/or heparin and was completely prevented by pretreatment of erythrocytes with heparitinase, providing evidence that gC-1 mediates hemagglutination by binding to heparan sulfate at the cell surface. Thus, HSV-1-induced hemagglutination is gC-1 dependent and resembles the recently proposed mechanism by which HSV-1 attaches to surface heparans on susceptible cells, providing a simple model for initial events in the virus-cell interaction.     

Mutant of Polyoma Virus with Impaired Adsorption to BHK Cells

A mutant of polyoma virus PY235 has an impaired adsorption to guinea pig red blood cells and BHK-21 hamster cells. Adsorption to 3T3 mouse cells is much less inhibited. These altered adsorption properties are responsible for the apparent inability of PY235 to cause cell transformation or hemagglutination.     

Effect of β-Propiolactone on Sendai Virus

The biological properties (infectivity, hemagglutination, hemolysis, cell fusion, neuraminidase) of Sendai virus were dissociated on the basis of sensitivity to beta-propiolactone, by freeze-thawing, by heating at different temperatures, and by adsorption-elution with formalinized chicken erythrocytes. Possible mechanisms whereby beta-propiolactone selectively destroys viral infectivity are discussed.     

H1N1亚型猪流感病毒的拯救

利用8质粒拯救系统成功拯救出了猪流感病毒毒株A/Swine/TianJin/01/2004(H1N1)(A/S/TJ/04)。将猪流感病毒8个基因节段经RT-PCR合成cDNA后, 分别克隆到RNA聚合酶I/II双向表达载体PHW2000中, 构建成8个重组质粒。用8个重组质粒共转染COS-1细胞, 30 h后加入TPCK-胰酶至终浓度0.5 mg/mL。共转染48小时后收获COS-1细胞及其上清, 经尿囊腔接种9日龄SPF鸡胚。收获死亡鸡胚尿囊液并继续用SPF鸡胚传3代, 得到有感染性的病毒。经血凝、血凝抑制验、测序分析、电镜观察等均证实了A/S/TJ/04猪流感病毒的成功拯救。这是目前国内首次报道拯救出H1N1亚型猪流感病毒, 为进一步研究猪流感病毒基因组结构与功能的关系、流感跨种传播的机制以及构建新型猪流感疫苗株奠定了基础。     

树类-细小病毒的初步研究

从树肾细胞培养物中分离出3株病毒,嗜在对数分裂期的TL细胞上复制,产生细胞病变和血凝素抗原,能凝集豚鼠和小鼠红细胞。交互血凝抑制实验表明3株病毒同属一个血清型。免疫酶染色显示抗原首先出现于细胞核。电镜观察负染标本病毒形态近似圆形和六角形,无胞膜,直径约30nm。血清学检查大多数树血清中含有该病毒抗体,证明是树的一种潜在病毒。初步鉴定为类细小病毒。     

H1N1亚型猪流感病毒的拯救

利用8质粒拯救系统成功拯救出了猪流感病毒毒株A/Swine/TianJin/01/2004(H1N1)(A/S/TJ/04)。将猪流感病毒8个基因节段经RT-PCR合成cDNA后, 分别克隆到RNA聚合酶I/II双向表达载体PHW2000中, 构建成8个重组质粒。用8个重组质粒共转染COS-1细胞, 30 h后加入TPCK-胰酶至终浓度0.5 mg/mL。共转染48小时后收获COS-1细胞及其上清, 经尿囊腔接种9日龄SPF鸡胚。收获死亡鸡胚尿囊液并继续用SPF鸡胚传3代, 得到有感染性的病毒。经血凝、血凝抑制验、测序分析、电镜观察等均证实了A/S/TJ/04猪流感病毒的成功拯救。这是目前国内首次报道拯救出H1N1亚型猪流感病毒, 为进一步研究猪流感病毒基因组结构与功能的关系、流感跨种传播的机制以及构建新型猪流感疫苗株奠定了基础。