Comparative Studies on the Soluble Components of Adenovirus Types 9 and 15 and the Intermediate Strain 9-15

Five different soluble components of adenovirus types 9, 9-15, and 15 have been identified. These are: (i) a slowly sedimenting, trypsin-resistant, incomplete hemagglutinin (HA). (This component was demonstrable by hemagglutination-enhancement (HE) tests in the presence of heterotypic antisera against members of Rosen's subgroups II and III, but not of subgroup I); (ii) a slowly sedimenting, trypsin-resistant, complete HA, causing only a partial agglutination of cells; (iii) a rapidly sedimenting, incomplete HA, demonstrable by HE tests in the presence of heterotypic antisera against members of all Rosen's subgroups. (Trypsin treatment of this component caused a conversion into slowly sedimenting incomplete HA); (iv) a group-specific complement-fixing (CF) antigen devoid of HA activity; and (v) a rapidly sedimenting, trypsin-sensitive, complete HA, which in the electron microscope was found to represent a dodecahedral aggregate of 12 pentons (a dodecon). On the basis of their biological and physicochemical characteristics, the first four components were interpreted to represent (i) fibers, (ii) a polymer of a few, probably two, fibers, (iii) pentons, and (iv) hexons, respectively. The length of fibers extending from dodecons and virions was estimated to be 11 to 14 nm. A similar value was suggested from exclusion chromatography experiments. Adenovirus types 9 and 15 fibers were recovered in a position intermediate to that of fibers of types 3 and 4, the lengths of which are 10 and 17 nm, respectively. The sequence of elution of different components of types 9 and 9-15 from an anion exchanger was fibers, fiber-aggregate, pentons, hexons, and dodecons. Type 15 components appeared in the same order except for the fact that dodecons eluted before hexons. The molarities of NaCl required to elute the different types 9 and 9-15 components, excluding hexons, were identical. They were distinctly different from those of the corresponding type 15 components. However, hexons of all three serotypes eluted in proximity to each other and there was a slight tendency for type 9-15 hexons to take a position intermediate to those of types 9 and 15.     

Physical and Biological Properties of Dengue-2 Virus and Associated Antigens

Dengue virus suspensions from mouse brain and cell culture were fractionated into three components by rate zonal centrifugation in sucrose gradients. Infectious virus sedimented in a single zone and possessed hemagglutinating (HA) and complement fixing (CF) activity. Electron micrographs showed the virion to be a spherical particle 48 to 50 nm in diameter with 7-nm spherical structures on its surface. Buoyant density in CsCl of virions from mouse brain was estimated at 1.22 g/cm(3) and from cell culture at 1.24 g/cm(3). During centrifugation of virions in CsCl, an additional HA component appeared with a buoyant density of 1.18 g/cm(3). It was shown in electron micrographs to consist of virion fragments. A noninfectious component with HA and CF activity sedimented in sucrose more slowly than intact virus, had a buoyant density of 1.23 g/cm(3) in CsCl, and appeared as "doughnut" forms measuring 13.8 to 14 nm in diameter. A third component, with CF activity and no HA activity, sedimented very little in sucrose gradients. Particles of the same size and shape as the spherical subunits on the surface of the virion were observed in electron micrographs.     

A monoclonal antibody to an oocyte-specific poly(A) RNA-binding protein

Xenopus oocyte-specific poly(A) RNA-binding proteins were isolated and used to prepare monoclonal antibodies. One antibody was used to characterize one particular antigen by immunoblot analysis. The antigen had a molecular weight of 56,000 was oocyte-specific, and decreased in amount during oogenesis. The antigen was localized in the cytoplasm throughout oogenesis and sedimented mainly at 40-60 S. The antigen also was shown to bind poly(A) RNA following chromatography of ribonucleoprotein particles on oligo(dT)-cellulose. The antibody was used to immunoadsorb nontranslating ribonucleoprotein particles. Fifty-five per cent of the poly(A) RNA sedimenting between 40-60 S was shown to be bound by the antigen. The further use of this antibody in attempting to examine other components of the ribonucleoprotein particle is discussed.     

Different forms of simian virus 40 large tumor antigen varying in their affinities for DNA

In various permissive monkey cell lines infected with simian virus 40 there are two major forms of large T antigen which differ in their rate of sedimentation through sucrose gradients. The lighter (5 to 7S) form sedimented slightly more rapidly than the 4S tRNA marker, whereas the heavier (16S) form sedimented slightly more slowly than the 18S rRNA marker. The small t antigen did not form complexes which sedimented as rapidly as those formed by the large T antigen. The 16S T antigen form was converted to the slowly sedimenting 5 to 7S form in the presence of 1.0 M NaCl. The majority of large T antigen synthesized in cell-free protein-synthesizing systems primed by mRNA isolated from infected cells sedimented as the 5 to 7S form even when premixed with excess quantities of cellular T antigen. The formation of the 16S form in infected cells did not require ongoing viral or cellular DNA replication because considerable quantities of this T antigen class were produced in the presence of DNA synthesis inhibitors, such as cytosine arabinoside. Both 5 to 7S and 16S forms could be isolated separately and, therefore, each could be analyzed as to its individual properties. The 5 to 7S T antigen form bound more efficiently and tightly to DNA and had specific affinity for sequences at the viral origin of replication, whereas the 16S form bound less efficiently to DNA and exhibited very little specificity for origin-containing DNA sequences. It is therefore likely that the active DNA-binding species of T antigen isolated from infected cells is the 5 to 7S form.     

Specific subtyping of influenza A virus using a recombinant hemagglutinin protein expressed in baculovirus

Influenza A viruses are subtyped according to antigen characterization of hemagglutinin (HA) and neuraminidase surface glycoproteins. The hemagglutination inhibition (HI) assay using reference antiserum is currently applied to serologic screening of subtype-specific antibodies in sera. The reference antiserum is made by injecting chickens with live or inactivated whole virus preparations. Nonspecific inhibitors of antisera prepared by the conventional method may affect the specificity of HI assay. In this study, highly pure recombinant proteins generated using baculovirus expression vector system based on full-length of HA (HAF) and antigenic region of HA₁ genes of H9 subtype, and also inactivated whole virus were used to immunization of chickens. Measurable antibody titers were present for treated birds after 3 weeks and generally increased after each boost. The performance of the prepared antisera was evaluated by testing a panel of known standard strains of influenza virus representing five HA subtypes. Relative to the conventional method using whole virus immunization and recombinant HAF protein, the antiserum prepared by recombinant HA₁ had a specificity of 100% for all tested subtypes. The antiserum prepared by expression of HA1 protein in baculovirus has the potential for rapid and specific HA subtyping of influenza viruses without producing antibodies specific to other viral proteins.     

Biophysical Comparison of Two Canine Adenoviruses

The two canine adenoviruses, infectious canine hepatitis (ICH) virus and infectious canine laryngotracheitis (ICL) virus (also designated as Toronto A26/61 virus), were studied with respect to their morphology and the biological properties of their soluble components. The two viruses were found to be composed of soluble components similar to, and carrying biological activities corresponding to, those of the human adenoviruses after fractionation by rate zonal centrifugation and anion-exchange chromatography. The following soluble components were identified: hexons (carrying a common group-specific complement-fixing antigen), penton oligomers (complete soluble hemagglutinin), and penton and fiber monomers (incomplete soluble hemagglutinins). The latter were indicated by hemagglutination enhancement with selected antisera directed against human adenovirus soluble components. Elution characteristics of corresponding components of the two viruses in anion-exchange chromatography experiments were distinctly different. Electron microscopic examination of purified virions and soluble components revealed the fiber component of ICL virus to be 35 to 37 nm in length, and that of ICH virus to be 25 to 27 nm long.     

Soluble Components of Adenovirus Type 8

Soluble components of type 8 adenovirus have been studied. Four different components were isolated by anion-exchange chromatography and purified by further chromatographic procedures, by zonal centrifugation, and by erythrocyte absorption and elution. The four components exhibited the following characteristics. (i) Fiber antigen was trypsin-resistant and functioned as incomplete hemagglutinin (agglutinated rat and human erythrocytes only in the presence of certain types of adenovirus antisera). (ii) The penton was trypsin-sensitive, exerted a cytotoxic effect, and also showed incomplete hemagglutination, being active in the presence of a majority of heterotypic adenovirus antisera studied. (iii) The group-specific hexon antigen reacted in complement fixation reaction and gel precipitation with sera prepared against other types of adenoviruses, besides showing characteristics indicating the presence of a type-specific antigen component. (iv) The soluble complete hemagglutinin was trypsin-sensitive, displayed cytotoxic effect, adsorbed easily to human and rat erythrocytes, and could be eluted from them by means of receptor-destroying enzyme. The three hemagglutinins of adenovirus type 8 proved to be highly unstable, and their demonstration was only successful by using a large quantity of freshly prepared concentrated virus material. Considering these conditions, a method was developed for their concentration and purification.     

Self-assembly of simian virus 40 large T antigen oligomers by divalent cations.

In simian virus 40-transformed cells, simian virus 40 large T antigen can be detected in different forms separable by sucrose density gradient centrifugation. In our experiments, light forms sedimented around 5 to 7S, oligomers such as tetramers were detected around 16S, and higher aggregates sedimented in a broad distribution reaching above 23S. The oligomers sedimenting at and above 16S could be disassembled into the slowly sedimenting 5 to 7S forms by chelating agents [EDTA or ethylene bis(oxonitrilo)tetraacetate]. After the addition of divalent cations (CaCl2 or MgCl2) in excess of chelating agents, oligomeric forms reassembled and appeared in a sedimentation pattern resembling that observed before treatment with chelating agents. Time course studies permitted the identification of the 5 to 7S forms as precursors upon pulse-labeling (15 min); the 16S and higher oligomers were identified as the successors after a 14-h chase. Treatment of extracts of pulse-chase-labeled cells with chelating agents again disassembled the oligomers, whereas pulse-labeled precursors did not change their 5 to 7S sedimentation pattern. Adding an excess of divalent cations reassembled the pulse-chase-labeled T antigen to oligomers but did not influence the sedimentation behavior of pulse-labeled 5 to 7S precursors. It is therefore reasonable to assume that a posttranslational modulation induces divalent cation binding, leading finally to the oligomerization of T antigen. Thus, some of the multifunctional activities of T antigen can be dictated by divalent cation binding properties.     

A membrane antigen of rabbit thymus cells

Rabbit cells, bearing a thymus-specific antigen, which we call rabbit thymus lymphocyte antigen (RTLA), could be detected with a suitably absorbed heterologous antiserum (goat). In the presence of complement, the RTLA antiserum lysed more than 95% of thymus cells, 70 ± 6% of lymph node cells, 46 ± 10% of spleen cells and 12 ± 7% of bone marrow cells. The number of direct or indirect hemolytic spleen plaques was not reduced by treatment with RTLA antiserum and complement, but was greatly diminished by an unabsorbed thymus antiserum which killed more than 90% of bone marrow cells. RTLA-bearing subpopulations of spleen cells were characterized by velocity sedimentation analysis and were distinguished from Ig receptor bearing subpopulations. The antiserum concentration could be so adjusted that the cytotoxicity against bone marrow was not manifested, while the cytotoxicity against other cell populations remained unchanged. The latter were identified by thymidine incorporation induced by treatment with antibody directed against rabbit light chain allotype. A small subpopulation of thymus cells did not have RTLA antigen and sedimented with a velocity distinct from that of the peak of RTLA-bearing cells.     

Multiple Group-specific Antigen Components of Avian Tumor Viruses Detected with Chicken and Hamster Sera

Multiple group-specific (gs) components of the avian leukosis-sarcoma viruses were detected by immunodiffusion (Ouchterlony) tests with sera from hamsters bearing tumors induced by sarcoma viruses and with sera from adult chickens immunized with avian sarcoma or leukosis viruses. Immune hamster sera detected up to four components, whereas chicken sera detected at least one. The hamster and chicken sera identified a similar antigen, as indicated by reactions of identity. Relatively few chicken sera containing neutralizing antibody to avian sarcoma or leukosis viruses reacted in immunodiffusion with the gs antigen. The gs components were released from the virion by various means of disruption, including freezing and thawing. Tests with tissues from normal chickens and from chickens with Marek's disease failed to demonstrate any reactions with hamster or chicken gs antiserum.     

猪流感病毒抗原表位基因表达载体构建与免疫原性分析

根据猪流感病毒血凝素蛋白基因(Heamuglutinine, HA)的核苷酸序列, 设计、筛选HA蛋白氨基酸序列的主要表位多肽4个, 将4个片段以柔性连接串联成模拟蛋白, 核苷酸约为300 bp, 体外扩增该模拟蛋白基因, 插入到原核表达载体pET30a(+)中, 转染宿主菌诱导表达, 结果获得分子量为20 kD的表达蛋白, 该蛋白可与抗His-tag抗体、抗猪流感病毒H1N1、H3N2亚型高免血清发生免疫学反应。纯化后免疫小鼠, ELISA及血凝抑制(Heamuglutinine inhibitor, HI)试验检测, 小鼠产生针对多肽抗原的血清抗体, 同时还可检测到H1N1、H3N2亚型SIV血凝抗体。流氏细胞仪检测免疫组外周血淋巴细胞高于对照组, 说明该模拟蛋白具有与H1N1、H3N2亚型猪流感病毒相似的免疫原性及反应原性, 为H1N1、H3N2血清亚型猪流感病毒疫苗研制提供了新手段。     

猪流感病毒抗原表位基因表达载体构建与免疫原性分析

根据猪流感病毒血凝素蛋白基因(Heamuglutinine, HA)的核苷酸序列, 设计、筛选HA蛋白氨基酸序列的主要表位多肽4个, 将4个片段以柔性连接串联成模拟蛋白, 核苷酸约为300 bp, 体外扩增该模拟蛋白基因, 插入到原核表达载体pET30a(+)中, 转染宿主菌诱导表达, 结果获得分子量为20 kD的表达蛋白, 该蛋白可与抗His-tag抗体、抗猪流感病毒H1N1、H3N2亚型高免血清发生免疫学反应。纯化后免疫小鼠, ELISA及血凝抑制(Heamuglutinine inhibitor, HI)试验检测, 小鼠产生针对多肽抗原的血清抗体, 同时还可检测到H1N1、H3N2亚型SIV血凝抗体。流氏细胞仪检测免疫组外周血淋巴细胞高于对照组, 说明该模拟蛋白具有与H1N1、H3N2亚型猪流感病毒相似的免疫原性及反应原性, 为H1N1、H3N2血清亚型猪流感病毒疫苗研制提供了新手段。     

Role of carbohydrate in biological functions of Friend murine leukemia virus gp71.

Purified gp71 of Friend murine leukemia virus (FLV) can interfere with virus infection, absorb neutralizing antibody, and in the presence of group-specific anti-gp71 antibody, hemagglutinate sheep erythrocytes. Interference by FLV gp71 with several murine leukemia viruses (MuLV) was tested in the XC and S + L- assay systems. Treatment of gp71 with trypsin or Pronase eliminated its interfering capacity. However, treatment with neuraminidase or a mixture of glycosidase enzymes, which left the major serological properties of gp71 intact, did not reduce the interference potential of gp71 for FLV or AKR MuLV. The capacity of gp71 to absorb type- or group-specific virus-neutralizing antibodies was similarly affected by the various enzyme treatments. In contrast, indirect hemagglutination by gp71 was abolished not only by proteases but also by treatment with glycosidase enzymes, although neuraminidase had no effect. Preliminary data indicate that infectivity of FLV or xenotropic MuLV was not affected by short treatment with glycosidase enzymes.     

Hemagglutinin (HA) proteins from H1 and H3 serotypes of influenza A viruses require different antigen designs for the induction of optimal protective antibody responses as studied by codon-optimized HA DNA vaccines

Effective antibody responses provide crucial immunity against influenza virus infection. The hemagglutinin (HA) protein is the major target of protective antibody responses induced by viral infection and by vaccination with both inactivated and live-attenuated flu vaccines, but knowledge about the optimal designs of protective HA antigens from different flu serotypes is still limited. In this study, we have significantly improved the immunogenicity of HA-expressing DNA vaccines by using codon-optimized HA sequences for either an H1 serotype (A/NewCal/20/99) or an H3 serotype (A/Panama/2007/99) human influenza A virus and then used these constructs as model antigens to identify the optimal HA antigen designs to elicit high-level protective antibody responses. Two forms of HA antigen, a wild-type, full-length HA and a secreted form with transmembrane (TM) domain-truncated HA, were produced. Both forms of HA DNA vaccines, from either H1 or H3 serotypes, were able to elicit high levels of HA-specific immunoglobulin G responses in immunized rabbits as measured by enzyme-linked immunosorbent assay. Interestingly, the abilities of H1 HA and H3 HA antigens to elicit hemagglutination inhibition (HI) and neutralizing antibody (NAb) responses differ. For the H1 HA antigens, the full-length HA induced significantly higher HI and NAb responses than did the TM-truncated HA. For the H3 HA antigen, both the full-length HA and TM-truncated HA induced high levels of HI and NAb responses. These data indicate that H1 and H3 antigens have different expression requirements for the induction of an optimal protective antibody response and that the structure integrity of HA antigens is critical for eliciting type-specific protective antibody responses. Our findings will have an important impact on future subunit-based flu vaccine development.     

In Vitro Product of a Ribonucleic Acid Polymerase Induced by Influenza Virus

The ribonucleic acid (RNA)-dependent RNA polymerase induced in the microsomal fraction of cells infected with influenza virus synthesized a mixture of single-and double-stranded RNA in vitro. The single-stranded RNA sedimented mainly in the 8S region on sucrose density gradients, with a smaller proportion of the RNA sedimenting at 18S. This sedimentation pattern corresponds closely to that of incomplete influenza virus RNA. The double-stranded RNA formed in vitro sedimented at 11S, but molecules which may be replicative intermediate, sedimenting at 14 to 20S, were also detected in the in vitro reaction product. Similar species of RNA were detected in vivo by pulse-labeling infected cells at the time of polymerase harvest, but the proportion of each RNA species was different, most of the RNA being single-stranded and sedimenting in the 18S region. An 11S double-stranded RNA was also synthesized in vivo. Pulse chase analysis of the double-stranded RNA synthesized in vitro showed that most is stable, and only a small proportion turns over during the reaction. A proportion of the RNA formed in vitro could be annealed to RNA formed in infected cells and to RNA extracted from purified virus.     

Immunological and Other Biological Characteristics of Pentons of Human Adenoviruses

Comparative hemagglutination-enhancement (HE) tests demonstrated diversified patterns of antigenic specificities both in the fiber and vertex capsomer part of pentons of human adenovirus types 3, 11 (subgroup I), 9, 15 (II), 1, 2, 4, 5, 6 (III), and 12. All fibers contained a type-specific antigen. Subgroup II and III fibers, in addition, contained specificities both unique for each subgroup and also common to the two subgroups. Fibers of serotypes 4 and 12 displayed a somewhat deviating behavior. All vertex capsomers tested shared a group-specific part. This was the only antigenic specificity demonstrable for serotype 12. Maximal penton HE titers of all sera were reached in tests with incomplete hemagglutinin of type 11. In addition, maximal HE activity of sera against individual serotypes also was recorded against pentons of other members of the same subgroup. Antigen characteristics of vertex capsomers of type 4 indicated a closer relationship to subgroup I than to subgroup III. The toxin activity of pentons was more sensitive to trypsin treatment than their capacity to function as incomplete hemagglutinin. Homotypic antipenton sera, unabsorbed or absorbed with homotypic fibers to remove antibodies against this component, and, to a varying extent, also heterotypic antipenton sera could neutralize toxin activity. Antifiber sera could neutralize toxin activity of pentons carrying short fibers (10 nm, type 3) but not of those carrying long fibers (28 to 31 nm, type 2). It is concluded that toxin activity is carried by a specific part of vertex capsomers and that cell detachment can be brought about via a direct contact between this component and cell membranes. Fiber-mediated attachment does not seem to be necessary for this biological activity to become expressed.     

Evidence for an Association of the 15-kDa Proteolipid of Mediatophore with a 14-kDa Polypeptide

The present report shows that mediatophore, a nerve terminal membrane protein that translocates acetylcholine on calcium action, forms a complex with a 14-kDa polypeptide. The complex was identified based on the following results. (a) A polyclonal antimediatophore antiserum that immunoprecipitates activity precipitates both the 15- and 14-kDa polypeptides. (b) After HPLC purification of mediatophore, both antigens were found in the same peak. (c) After 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate solubilization of presynaptic membranes or of the purified mediatophore, an immunoaffinity column made with the anti-14-kDa antigen monoclonal antibody retained both the 14-kDa and the 15-kDa polypeptide. Similarly, immunoprecipitation experiments using protein A-coated beads sedimented an immunocomplex in which both antigens were found. (d) The 14-kDa antigen could be localized in the synaptosomal membrane where mediatophore and its 15-kDa component are found.     

Velocity sedimentation profiles of normal and regenerating primitive erythroid burst-forming units: relation to phase of cell cycle and growth in vitro

Abstract. The primitive burst-forming unit-erythroid (BFU-e) derived from normal and regenerating murine bone marrow was examined by velocity sedimentation at unit gravity. An increase in the modal sedimentation velocity and the percentage of rapidly sedimenting BFU-e was found in regenerating marrow as compared to normal marrow. Neither hypertransfusion-induced plethora nor administration of erythropoietin (Ep) during regeneration altered the changes from normal in the velocity sedimentation profile observed during regeneration. Separated marrow cells were pooled as rapidly sedimenting and slowly sedimenting and then examined for percentage of BFU-e in DNA synthesis and growth response in vitro to increasing concentrations of a partially purified Ep preparation. The percentage of BFU-e in DNA synthesis as determined by tritiated thymidine killing does not correspond to the BFU-e growth response to Ep in vitro . No difference in growth was noted between BFU-e from rapidly and slowly sedimenting normal marrow cells despite an increased percentage in DNA synthesis of normal BFU-e which sedimented rapidly. No significant difference in the percentage of BFU-e in DNA synthesis was found between the rapidly and slowly sedimenting subpopulations of regenerating BFU-e, but the latter had a reduced growth response to low concentrations of Ep.     

Simian virus 40 large T antigen oligomers: analysis of electrophoresis in the absence of detergent.

Large T antigen of simian virus 40 is found as monomeric and oligomeric species in transformed cells. These can be demonstrated in cell extracts by velocity centrifugation in sucrose gradients. We analyzed them further in a transformed human line cell (SV80) and a transformed mouse line cell (SVT2). Individual fractions from sucrose gradients were subjected to polyacrylamide gel electrophoresis in the absence of detergent. T-antigen species were then detected by protein blotting and antibody overlay with polyclonal anti-D2 T antibody or monoclonal Pab419, Pab101, or Pb1700 antibody. The rapidly sedimenting species (14S and larger) of large T antigen from both cell lines reproducibly showed two major bands with estimated molecular weights of 670,000 and 850,000. A third band of 1,200,000 was more prominent in SVT2 cells than in SV80 cells. In SV80 cells the slowly sedimenting species of large T antigen (5S to 11S) contained two reproducible bands. A band with a molecular weight of 95,000 was the predominant one in all fractions between 5S and 11S. A relatively minor band with a molecular weight of 230,000 was found in fractions between 9S and 11S. The low-molecular-weight forms were seen in SVT2 cells only when a prominent peak at 5S to 7S was present, that is, when extracts were stored before analysis. In fresh extracts, the low-molecular-weight bands and slowly sedimenting forms were absent.     

Production of antibody to individual polypeptides derived from purified hepatitis B surface antigen.

Purified preparations of hepatitis B surface antigen (HBsAg) were solubilized with sodium dodecyl sulfate and urea under reducing conditions and subsequently fractionated by preparative sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis (PAGE). Pools of the individual fractions eluted from the preparative PAGE were concentrated and purified further by analytical PAGE. Five purified polypeptides were isolated from HBsAg, types adw and ayw, with molecular weights of 19,000, 24,000, 27,000, 35,000, and 40,000. Each preparations was emulsified in Freund complete adjuvant and injected into guinea pigs. Antibody to each HBsAg type was measured by radioimmunoassay. The 19,000 molecular weight polypeptide derived from ayw particles and the 27,000 molecular weight subunit obtained from both types failed to elicit an antibody response. The other three polypeptides derived from the ayw particles elicited group-specific antibody responses. Similar group-specific reactivities were observed in the testing of anti-adw 35,000 and anti-adw 40,000 molecular weight polypeptide sera. However, guinea pigs immunized with the 19,000 and the 24,000 molecular weight polypeptides of the adw type produced antibody that reacted preferentially with adw particles. This indicates that either these subunits carry predominately d determinants or that, because of the low levels of material used for inoculation, no immune response or an undetectable one was elicited to the a or w components.