Slowly-sedimenting hemagglutinin of the tick-borne encephalitis virus

Ring-shaped particles of 5-10 mm in diameter considered by research workers to be a slowly-sedimenting hemagglutinin (SSHA) of flaviviruses (an antigen immunologically related to virions) were detected in the precipitation band formed in immunoelectrophoresis by the non-virion ("soluble") antigen but not in the precipitation band formed by the virion antigen. The slowly sedimenting (SS) virions of tick-borne encephalitis virus previously found in a SSHA fraction did not differ in the set of structural proteins from virions of the main population (rapidly sedimenting). It is concluded from the foregoing that the hemagglutinating activity of the SS-structures is realized not by a hypothetic SSHA ("natural" ring-shaped fragment of virion envelope, precursor or a by-product of virus morphogenesis, according to other workers) but by SS-virus particles.     

Electrophoretic and sedimentation heterogeneity of the virion antigen of the tick-borne encephalitis virus

Analysis of tick-borne encephalitis virus virion antigen by cross immunoelectrophoresis showed it to be separated into 2 subpopulations of virions differing by their mobility in the electric field. It was demonstrated that in immunoelectrophoresis the formation of precipitation bands typical of virion antigen occurred due to the movement of soluble immune complexes in the electric field. The immunoelectrophoretic characteristics of TBE virus virions are determined by the membrane E protein which was also found to be heterogeneous in its immunoelectrophoretic characteristics. The nucleocapsid of tick-borne encephalitis virus virions formed no precipitation bands in immunoelectrophoresis. The virion antigen not sedimenting in ultracentrifugation was represented by morphologically intact virions the structural protein composition of which differed from that of the structural proteins of virions sedimentable by ultracentrifugation. The possible causes of electrophoretic and sedimentation heterogeneity of tick-borne encephalitis virus virion antigen are discussed.     

Immunochemical characteristics of the antigens of a variant tick-borne encephalitis virus adapted to Hyalomma plumbeum ticks

A comparative study of immunochemical properties of a preparation of the virion antigen of a variant of tick-borne encephalitis (TBE) virus adapted to H. plumbeum ticks (clone 718/574) and the parental TBE strain revealed the following features: (1) immunoelectrodiffusion test revealed no immunoprecipitation rockets directed towards cathode in the clone 718/574 preparation; anodic precipitates differed in shape from those of the parental strain; (2) diffuse precipitation in agar showed a reaction of incomplete identity between the virion antigen of clone 718/574 and the parental strain antigen, the clone 718/574 antigen forming only one precipitation band in contrast to two precipitation bands of the parental strain; (3) immune electron microscopy demonstrated a peculiar nature of the halo-overlay by immunoglobulins of clone 718/574 virions differing from that of the parental strain.     

A comparative study of tick-borne encephalitis virus RNA synthesis in acutely and persistently infected cells

Summary Rate zonal and buoyant density gradient centrifugation did not reveal any difference between tick-borne encephalitis virus virions released from acutely or persistently infected cells. All three RNA species characteristic for flavivirus replication were found both in acutely or persistently infected cells, but increased levels of intracellular 42S RNA polyadenylation was observed in persistently infected cells.With 2 Figures     

Concentration and purification of tick-borne encephalitis virus grown in suspensions of chick embryo cells.

Tick-borne encephalitis (TBE) virus grown in suspensions of chick embryo cells was precipitated by various concentrations of polyethylene glycol (PEG) 6000. Quantitative recovery was obtained at PEG concentrations of 5% and higher. As precipitation of contaminating non-viral protein increases with increasing PEG concentrations, best results with respect to purity and recovery were obtained at 5% PEG 6000. Analysis of virus concentrates by rate zonal centrifugation revealed two peaks--rapidly sedimenting haemagglutinin (RHA) associated with infectivity and slowly sedimenting haemagglutinin (SHA)--characteristic of Flaviviruses. Purification factors after PEG precipitation followed by rate zonal centrifugation in sucrose density gradients ranged from 50 to 200 for HA activity and from 40 to 50 for infectivity.     

The properties of the particles formed in the reproduction of an acute in vitro infection with the tick-borne encephalitis virus adapted to H. plumbeum ticks]

The properties of virions produced in pig embryo kidney (PEK) cells inoculated with tick-borne encephalitis (TBE) virus strain EK-328 which had been passaged in white mice and its variant obtained by passages of TBE in H. plumbeum ticks (clone 718/574 H. pl.17) were found to be different. The clone 718/574 H. pl17 virus particles had no hemagglutinating or precipitating activities, greater sedimentation and density heterogeneity in sucrose density gradient centrifugation, and differences in movements to electrodes in electrophoresis and immune electrophoresis. In mixed infection in PEK cultures with EK-328 strain and clone 718/574 H. pl17, the infective dose-dependent interference was observed which affected the infectious virus titre and the size of cathode antigen precipitate.     

Buoyant density of some togaviruses in sucrose density gradient and capacity of their haemagglutinin fractions to interact with antibody.

The buoyant densities of Western equine encephalomyelitis virus (an Alphavirus) and tick-borne encephalitis (TBE) virus (a Flavivirus) antigens prepared by different methods were studied. Sucrose density centrifugation revealed a heterogeneity in the density of the virions. The sedimentation pattern and height of peaks of the haemagglutinating activities and infectivity, other conditions being equal, depended both on the virus species and properties of its strains and on the mode of preparation and treatment of the virus-containing material. Different haemagglutinating antigen fractions differed in their capacity to interact with specific antibody. It was suggested that the kinetics of a serological reaction and its result depend on the functional activity of the antigen preparation and, in particular, on the proportion in the reaction mixture of virus particles with a dissimilar antigenic structure.     

Evaluation of methods for the determination of Q and K antigens of an 02:K1(L) strain of Escherichia coli.

Tests made on ten colonies from a strain of Escherichia coli O2:K1 demonstrated that bacterial agglutination tests were reliable for identifying the O antigen of serogroup O2 but were unreliable for identifying the K1 antigen. The granular nature of K agglutination was not a reliable characteristic of the L type of K antigen. In contrast, indirect haemagglutination, immunodiffusion and immunoelectrophoresis tests with bacterial extracts gave consistent results with all colonies. The polysaccharide K1 antigen formed a long anodic precipitation line with two peaks, indicating its heterogenous nature, and partial fusion of this line with the O-antigen precipitation line suggested the presence of common serological determinants. In addition, a heat-labile protein antigen, possibly another K antigen, was identified by indirect haemagglutination tests and may have produced a short anodic precipitation line. The results also showed that the K1 antigen was still produced after storage of a culture for 12 years on Dorset-egg medium.     

Demonstration of K88ac and K88ab antigens of Escherichia coli by means of immunoelectrophoresis and immunodiffusion.

Five strains of Escherichia coli were tested for the presence of the K88ac or K88ab antigens by immunoelectrophoresis and immunodiffusion. The K88ac antigen of 0A2 and Sojka Abbotstown gave an anodic line in the immunoelectrophoresis test and a line in immunodiffusion with homologous K88ac antisera. The K88ab antigens of 0G7, 0E68, and Moon 263 also gave anodic lines in immunoelectrophoresis, and were detectable by immunodiffusions. The 0 groups of these strains were also demonstrated by immunoelectrophoresis and immunodiffusion with homologous 0 antisera. Lack of complete inactivation at 100 degrees C of both the K88ac and K88ab antigens was noted in this study.     

Behavior of Escherichia coli K antigens K88ab, K88ac, and K88ad in immunoelectrophoresis, double diffusion, and hemagglutination.

Porcine enteropathogenic Escherichia coli strains were found to possess a variant of the K88 antigen provisionally termed K88ad. We propose to include this antigen into the international E. coli typing scheme. Ultrasonic extracts of field strains of E. coli possessing the K88ab, K88ac, or K88ad antigen and their E. coli K-12 K88+ transconjugants showed a specific K88 precipitation line in immunoelectrophoresis and double diffusion only when grown at 37 degrees C, but not when grown at 18 degrees C. By using agarose gels, K88ab, K88ac, and K88ad antigens showed anodic mobility in immunoelectrophoresis. When using Difco Noble agar gels, K88ad was not mobile or anodic, K88ab was cathodic; K88ac of 17 strains was cathodic and of 24 strains was anodic. The immunoelectrophoretic behavior of a K88 antigen (K88ab, K88ac, or K88ad) did not alter after transfer of the corresponding plasmid to E. coli K-12. Anodic and cathodic K88ac antigens could not be distinguished serologically. The differences between the results obtained in Noble agar gels and agarose gels are due to electro-endosmotic flow. We describe a procedure which increases the detection level of K88+ transconjugants in a mating mixture. It is based on the specific mannose-resistant attachment of K88+ cells to guinea pig erythrocytes.     

Nonstructural protein NV5 of the tick-borne encephalitis virus possesses precipitating activity characteristic for the nonvirion (soluble) antigen

A preparation of tick-borne encephalitis virus proteins obtained by destruction of the infected cells under strong denaturating conditions possesses the precipitating activity in immunodiffusion characteristic of nonvirion ("soluble") antigen. This precipitating activity was shown to be provided by protein P93 (NV5).     

Physical Properties and Antigenic Components of Oriboca Virus

Two complement fixation (CF) components were detected in all of the various preparations (mouse liver, brain, serum, and BHK-21 cell culture) of Oriboca examined by equilibrium density centrifugation. The CF components possessed a buoyant density of 1.21 and 1.18 g/ml, and they were precipitated by treatment with protamine. Analysis of Oriboca virus by rate zonal sedimentation separated three components. The most rapidly sedimenting component was the infectivity which cosedimented with a peak of hemagglutinating (HA) activity designated VHA. A second, more slowly sedimenting, noninfectious HA component (HA-2) was followed by a peak of CF activity which barely moved from the top of the gradient. The single rate zonal CF peak and the two density gradient CF components were broadly cross-reactive serologically with Murutucu antiserum. They could not be identified as Oriboca on the basis of their CF reactions with Murutucu and Oriboca antisera. Oriboca-infective virions possessed an equilibrium density of 1.20 g/ml if derived from BHK-21 cell culture fluid concentrates or mouse serum. Infective virions prepared from mouse brain or liver banded at a buoyant density of 1.17 to 1.18 g/ml. These density differences, along with observed differences in reactivity with protamine and in rate of neutralization, are consistent with the interpretation that these are related to the problem of whether the source preparations of Oriboca represented primarily intracellular or extracellular particles.     

Nonvirion (soluble) antigen of the tick-borne encephalitis virus

Immunoelectrophoretic analysis of the cells destroyed as a result of infection with tick-borne encephalitis virus showed a considerable portion of nonvirion ("soluble") antigen to remain associated with cell membranes and to be released after treatment of the cells with detergents. After treatment with nonionic detergents, the nonvirion antigen showed electrophoretic heterogeneity in immunoelectrophoresis, and after treatment with sodium dodecyl-sulphate behaved as a homogeneous population of protein molecules. Using radioactively labeled carbohydrate precursors, lipids, and RNA, the nonvirion antigen was demonstrated to be a complex structure: a membrane-containing ribonucleoprotein. The immunoprecipitation of proteins by means of antibodies ligated to CNBr-activated sepharose confirmed that protein NV 5 (p93) of tick-borne encephalitis virus was an antigenically active component of the nonvirion antigen. This protein was found to undergo proteolytic cleavage in immunochemical reaction. The possible role of the nonvirion antigen in reproduction of flaviviruses is discussed.     

Characteristics of the precipitation of the antigenic structures of the tick-borne encephalitis virus using polyethylene glycol and ammonium sulfate

When immunoelectrophoretic methods were used for analysis of tick-borne encephalitis (TBE) virus antigens, TBE virus virion antigen (VA) from the virus-containing tissue culture fluid sedimented upon addition of polyethylene glycol (PEG) up to 7% whereas upon addition of ammonium sulphate (AS) the bulk of VA sedimented in the range of 40-70% salt saturation. The non-virion antigens (NA) sedimented at all PEG (up to 22%) and AS (up to 70% saturation) concentrations. In stringent ultracentrifugation procedure (12-13 X 10(6) g X min), the bulk of VA and a small portion of NA are pelleted. Repeated ultracentrifugation results in sedimentation of additional amounts of VA and NA. The sedimentable NA are pelleted by low PEG (up to 5-7%) and AS (up to 20-30%) concentrations and differ in their interaction with antibodies in the course of immunoelectrophoresis from nonprecipitatable ("soluble") NA. Combined sedimentation of antigens with AS (in the range of 40-70% saturation), intensive dialysis and subsequent sedimentation with low and high PEG concentrations produces homogeneous antigenic preparations containing VA or "soluble" NA.     

Immunochemical analysis of streptococcal group A, B, and C carbohydrates, with emphasis on group A.

Streptococcal group A, B, and C carbohydrates were analyzed by counterimmunoelectrophoresis, immunoelectrophoresis, and inhibition of immunoprecipitation. Extracts of streptococci group A or C were shown by counterimmunoelectrophoresis to contain both anodic and cathodic migrating components. In immunoelectrophoresis, group A and C substances formed a continuous precipitation line stretching from the anode to the cathode, suggesting a heterogeneous population of molecules with immunochemical identity. This identity was confirmed by inhibition of immunoprecipitation, in which both anodic and cathodic immunoprecipitates were inhibited by the same constituent sugars: group A-anti-A was inhibited by N-acetylglucosamine, and group C-anti-C was inhibited by N-acetylgalactosamine. Extracts of group B showed only anodic migration in counterimmunoelectrophoresis and a narrow, anodic arc in immunoelectrophoresis. The group B-anti-B reaction was inhibited by rhamnose. Carbohydrates of variant strains of group A streptococci were also analyzed by the same methods. The results suggest that the heterogeneity of group A carbohydrate may have resulted from attachment of various amounts of N-acetylglucosamine to the polyrhamnose backbone.     

Intermolecular association of HANA glycoprotein of Sendai virus in relation to the expression of biological activities

When purified HANA glycoproteins of Sendai virus were centrifuged in a sucrose density gradient in the presence of Triton X-100, neuraminidase activity was distributed into two peaks, a rapidly sedimenting peak H and a slowly sedimenting peak L. The analysis of polypeptides under nonreducing condition showed that peak H contained HANA glycoproteins exclusively in the form of tetramer whereas peak L was in the form of dimer, which suggests that the different sedimentation rate is due primarily to the difference in the mode of intermolecular association of HANA glycoproteins by disulfide linkage. A glycoprotein designated C-HANA with a molecular weight of ～55,000 was also involved in the solubilized HANA preparation and found as tetramer and dimer in peaks H and L, respectively, when analyzed under nonreducing condition. The C-HANA protein was produced only after solubilization of Sendai virions with Triton X-100. The C-HANA was found more in the preparation from egg-grown virions than from LLC-MK₂ cell-grown virions, indicating that production of C-HANA is host cell-dependent. Trypsin treatment of the intact HANA oligomers resulted in the formation of oligomers of the size similar to the C-HANA oligomers. These results suggest that host cell-derived protease(s) present in the purified virus preparation is responsible for the cleavage. In contrast with the intact HANA oligomers, the C-HANA oligomers could neither reconstitute large multivalent structures nor exhibit hemagglutinating activity when centrifuged in sucrose density gradients in the absence of Triton X-100. Though the C-HANA oligomers could adsorb to chicken red blood cells, they were released rapidly from the cells even at a low temperature like monovalent forms of the intact HANA oligomers, suggesting that the formation of multivalent structure is essential for HANA glycoproteins to continue to adsorb to the cells and the C-HANA protein loses the hydrophobic part required for the reconstitution of HANA glycoprotein. In the light of these observations the previous result obtained by several authors who demonstrated an apparent dissociation of hemadsorbing activity from neuraminidase activity of paramyxoviruses can be reasonably explained now.     

Cross-reactions between serum proteins and water soluble liver tissue antigens of the nine-banded armadillo (Dasypus novemcinctus Linn.) and man.

Cross-reactions between serum proteins and water soluble liver antigens of the nine-banded armadillo (Dasypus novemcinctus Linn.) and man were studied by crossed immunoelectrophoresis (CIE). Armadillo serum tested with rabbit antiserum against human serum proteins gave twelve components in CIE. Nine of these cross-reacting proteins were identified and showed partial identity with the corresponding human proteins. The electrophoretic mobility of alpha 2-macroglobulin and Gc-globulin differed in the two species. An ultrasonicate of normal armadillo liver gave twenty-eight anodic and eight cathodic components in CIE. By absorption experiments with armadillo serum, twenty of the former and seven of the latter were shown to be liver tissue components. A combination of CIE and crossed-line immunoelectrophoresis (CLIE) revealed the presence of twelve anodic and six cathodic liver tissue components cross-reacting with man. A cathodic armadillo liver antigen called (CALA-17) showed partial identity with that of man both in tandem and fused rocket immunoelectrophoresis. The implications of the findings are discussed in relation to the use of armadillo-grown M. leprae for skin testing and other purposes in man.     

Electrophoretic separation and differentiation of enzymes from human and from porcine liver

The electrophoretic separations of some human and pig liver enzymes on cellulose acetate and Cellogel were investigated, with reference to their joint occurrence in serum of patients undergoing treatment by extracorporeal pig liver perfusion. In every case it was possible to distinguish between the human and pig enzymes. Pig lactate dehydrogenase isoenzymes occupy a position slightly anodic to the corresponding human bands. The aspartate transaminase band of human is more anodic than that of pig, but their cathodic bands have the same mobility. Alanine transaminase of both human and pig liver extract is shown to exist as two bands each towards the anode. The faster moving human band is more anodic than the corresponding pig band, while the other human band is less anodic. Sorbitol dehydrogenase, alkaline phosphatase, and ornithine carbamoyltransferase all exist as one band each. Human sorbitol dehydrogenase is more cathodic than the pig enzyme, human alkaline phosphatase more anodic than the pig enzyme, while human ornithine carbamoyltransferase is less anodic than the pig enzyme.     

Molecular and ultrastructural analysis of heavy membrane fractions associated with the replication of Kunjin virus RNA

Summary In subcellular extracts of Kunjin virus-infected cells prepared by lysis and differential centrifugation, the viral RNA polymerase, RNA and proteins were associated mainly with cytoplasm. When the cytoplasmic extract (500g supernate) of infected cells labelled for 3 h from 24 h post-infection was further fractionated by rapid centrifugation through a sucrose density gradient, all viral products were located only in dense or heavy membrane fractions, which contained three types of virus-induced morphologically distinct membrane structures. These dense fractions were treated with 0.5% NP 40 and the soluble material was again centrifuged through a sucrose gradient for analyses as before. Viral RNA polymerase activity was retained and was associated with replicative intermediate RNA and some replicative form RNA in the peak enzyme fractions sedimenting at 20 S to 40 S. Enrichment of NS 3 and of the small nonstructural proteins NS 2 A and NS 2 B/NS 4 A was apparent in these fractions which were well separated from the slow sedimenting structural proteins. No detergent-resistant structures in the heavy membrane fractions other than ribosome-like particles were visible. The data show that the RNA polymerase complex cosedimented with virus-induced membrane structures and remained associated with specific nonstructural proteins and replicative intermediate RNA after detergent treatment.     

Fractionation of respiratory syncytial (RS) virus components by centrifugation techniques

Summary After equilibrium centrifugation in CsCl gradients of concentrated RS virus material, infectious particles were recovered in fractions with densities varying between 1.21 to 1.23 g/cc., whereas CF antigen activity mainly accumulated in the density range 1.25 to 1.34 g/cc. Small quantities of CF antigens also appeared at lower densities. Treatment with Tween 80 and ether eliminated all infectivity without a concomitant destruction of CF antigen. The buoyant density distribution of CF activity in such a preparation was identical with that of untreated material except that all low density (1.25 g/cc.) activity had disappeared.Centrifugation of virus material layered on top of a discontinuous CsCl gradient revealed the presence of two populations of particles. One represented rapidly sedimenting particles carrying all infectivity and about 10% of the total CF antigen activity. The other carried all of the remaining CF antigen activity and had the character of a soluble antigen.Supported by grants from the Swedish Medical Research Council (Project No. 16X-748-01).