Biosynthesis and morphogenesis of group C rotavirus in swine testicular cells

Summary Polypeptide synthesis and morphogenesis of a group C rotavirus (AmC-1) adapted to a continuous swine testicular cell line was examined. SDS-PAGE analysis of³⁵S methionine labeled infected cell lysates revealed 9 viral polypeptides (122, 98, 79, 78, 43, 41, 35, 24, and 20 kD). Viral polypeptide synthesis appeared to be maximal at 7–10 h post infection. Purified group C virus grown in the presence of trypsin was found to contain seven structural polypeptides (122, 98, 79, 53, 43, 41, and 30 kD) by protein blotting and five polypeptides (98, 79, 78, 43, and 41 kD) by immunoprecipitation with a hyperimmune rabbit antisera. Tunicamycin treatment, Concanavalin A binding, protein blotting, endo-H treatment and^2,6H-mannose labeling suggested that group C rotavirus contains one structural glycoprotein (41 kD) with a corresponding precursor mol. wt. of 37 kD and one not previously identified non-structural glycoprotein (24 kD) with a corresponding precursor mol. wt. of 20 kD. Electron microscopy of infected swine testicular cells revealed an assembly process for group C rotavirus similar to group A, with single-shelled particles budding through the rough endoplasmic reticulum with concomitant acquisition of a transient membrane.     

Human immune response to cytomegalovirus structural polypeptides studied by immunoblotting

To define better the human immune response to individual structural proteins of human cytomegalovirus (HCMV), 55 human sera with different IgG and IgM titres were studied for their reactivity with HCMV structural polypeptides separated by SDS-PAGE and electrotransferred to nitrocellulose paper. The results obtained showed that antibody titres detected by immunoassay correlate with the intensity and the number of polypeptides reacting by immunoblotting (IB). The IB profiles of HCMV polypeptides reacting with different sera having the same antibody titres show considerable variation. Sera with high levels of IgG antibody and that are IgM-positive frequently react with 155, 149, 82.5, 74.5, 67, 57, 55, 38.5, and 28 kD polypeptides; all these sera react with 155, 67, 57, 55, 38.5 kD polypeptides. Sera with high levels of IgG antibody but that are IgM negative frequently react with all these polypeptides, with the exception of 149 and 74.5. Only 155 and 28 kD polypeptides were recognized by all sera of this group. The sera with moderate levels of IgG antibody preferentially recognize 155, 110, 82.5, 62, 55, 38.5 and 28 kD polypeptides. The sera with low levels of antibody reacted especially with 155 and 62 kD polypeptides. IgM antibody seems to recognize preferentially 155, 110, 67, 57, 55, 38.5 kD polypeptides.     

Differential isotype recognition of two centromere associated polypeptides by immunoblotting in connective tissue disease.

On investigating the immunoblotting profile of 65 systemic sclerosis patients, a 140 kD polypeptide was recognised by sera from 16, when immunoblotted against a nuclear-enriched K562 cell sonicate. All 16 sera contained anticentromere antibodies (ACA) detected by immunofluorescence (IF) and 15 of 16 also recognized a 19 kD polypeptide on immunoblotting. Two ACA positive sera failed to recognize the 140 kD polypeptide but one of these recognized the 19 kD polypeptide. The 140 kD polypeptide identified a group with more limited skin involvement (P less than 0.05) and all 16 had Raynaud's phenomenon. The sera from three of 100 systemic lupus erythematosus (SLE) patients also recognized both polypeptides. On investigating the isotype specificity, the 140 kD polypeptide was strongly detected by an IgM autoantibody and the 19 kD polypeptide by an IgG autoantibody.     

High prevalence of anti-mitochondrial antibodies among patients with some well-defined connective tissue diseases.

A sensitive enzyme linked immunosorbent assay for determination of low levels of anti-mitochondrial antibodies (AMA) has been developed. With this method, sera from patients with primary biliary cirrhosis (PBC) and patients with different connective tissue diseases were investigated. Ninety percent of PBC sera were found to harbour high levels of AMA and a high proportion of patients with systemic lupus erythematosus (SLE), but also other patients with connective tissue diseases were found to have low affinity or low concentrations of AMA in their sera. AMA positive sera were further investigated with sodium dodecyl sulphate polyacrylamide gel electrophoresis and immunoblotting technique. PBC showed reactivity to 70, 50 and 45 kD mitochondrial polypeptides. SLE sera showed reactivity to 70 and 45 kD polypeptides and furthermore to a 65 kD polypeptide. Many of the AMA positive sera from patients with connective tissue diseases reacted to a 65 kD polypeptide.     

A comparative analysis of the polypeptides of the the Sindbis and Karelian fever viruses

In pulse-chase experiments with Karelian fever virus-infected cells, proteins were found with molecular weights of 130, 98, 78, and 62 kD of which the first, second and fourth were classified as polypeptide precursors of the structural proteins of virion. The molecular weights of proteins E1, E2 and C of 52, 47 and 34 kD, respectively, as well as isoelectric points of isolated glycoproteins (pI E1 = 6.3, pI E2 = 8.4) were similar in KFV (strain Leiv-9298) and Sindbis virus (strain AR339). The antigenic similarity of the strains under study in neutralization test with hyperimmune sera, the identity of physicochemical characteristics of the structural proteins of KFV and prototype Sindbis virus strain suggest a close relationship of the Leiv-9298 strain to the Afro-European variants of Sindbis virus.     

Polypeptides and structure of African swine fever virus

Extracellular and intracellular African swine fever virus (ASFV) was purified using a two-phase aqueous polymer system. Both the structure of the virus and the polypeptides present during the purification procedure were studied. After PEG/dextran phase separation and centrifugation through 20% (w/v) Ficoll, 79% of input infectivity was recovered as semi-purified virus. The density of the virus after equilibrium centrifugation in sucrose was 1.19 g/ml. The envelope of the virion consisting of a unit membrane was removed from the virion after centrifugation in sucrose. Removal of envelope was associated with the loss of a 230 kilodalton (kd) glycoprotein from the virion. Disruption of the viral surface structure resulted in a loss of infectivity. Eighteen of the most prominent of the 33 polypeptides of extracellular or cell free (CF) virus were those with molecular weights of 230, 195, 165, 155, 150, 125, 116, 97, 92, 73, 62, 58, 50, 45, 35, 33, 25 and 11 kd, while the fourteen most prominent polypeptides in intracellular or cell associated (CA) virus were 103, 97, 92, 84, 73, 62, 58, 54, 47, 45, 35, 33, 25 and 17 kd. The 45 kd polypeptide may be actin which copurifies with the virus. No major differences were found in the number or size of proteins among three isolates of ASFV. Electron micrographs of thin sections of ASFV show the capsid to consist of a distinct double layer of closely packed capsomeres enclosed on both sides with a semi-transparent layer. Cell associated virus measured from side-to-side 188 nm and vertex-to-vertex 212 nm. The capsid encloses an inner core composed of a dense nucleoid surrounded by a 40-48 nm layer of core protein.     

小鼠早期胚胎细胞的中间丝蛋白质成分的性质研究

用选择性抽提和ＤＧＤ包埋去包埋剂电镜技术、间接免疫荧光和免疫印迹技术研究了小鼠早期胚胎细胞的中间丝分布及其蛋白质成分的性质。本研究结果表明，小鼠早期胚胎细胞中存在由角蛋白组成的中间丝网络结构和角蛋白库，３种角蛋白的分子量分别为５０ｋＤ、５３ｋＤ和５７ｋＤ。     

Identification and mapping of the early gene products of adenovirus type 12

Cytoplasmic RNA was prepared from KB cells early after lytic infection with adenovirus type 12 and virus-specific RNAs were selected by hybridization to endo R·HindIII fragments of Ad12 DNA. The selected RNAs were translated in cell-free systems derived from wheat germ or rabbit reticulocytes and the protein products were analyzed by gel electrophoresis. It was found that the leftmost early region (region EI) of the genome (0–11%) coded for polypeptides with apparent molecular weights (kilodaltons) of 60, 41, a series of proteins between 39 and 23, 19, 17, 16, and 15 kD. The 41-kD protein maps on the left side of region EI, and the 19- and 60-kD proteins probably on the right side of EI. Early region II (62–68%) coded for polypeptides of 60, 52, and 46 kD which probably represent the single-stranded DNA-binding protein and two proteolytic breakdown products. We could not map unambiguously a protein in early region III, but we suspect that a 23-kD protein (and possibly a 16-kD protein) are encoded by this region. Early region IV (91–100%) coded for polypeptides of 14.5 and 13.5 kD. The virus-specific RNA from an Adl2-transformed hamster cell line directed the synthesis of 60-, 41-, 19-, 16-, 15-, 14.5-, and 13.5-kD polypeptides, suggesting that, in addition to early region 1, early region IV is also expressed. A remarkable similarity was observed between proteins synthesized in vitro by Adl2- and Ad5-specific RNA.     

Marek's disease virus gene clones encoding virus-specific phosphorylated polypeptides and serological characterization of fusion proteins

Marek's disease virus (MDV) gene clones, RA2 and GA8, constructed inE. coli bacteriophage lambda-gt11 (gt11) were identified by a monoclonal antibody (MAb), H19.47, against a putative transformation-related viral antigen consisting of a complex of three phosphorylated polypeptides, pp41, pp38, and pp24. Both recombinants have a MDV-DNA insert of about 0.5 kb and are mapped to the region ofBamHI-H orEcoRI-X fragments of the MDV genome by Southern blot hybridization. Immunoblot and immunoprecipitation with H19.47 identified a recombinant beta-galactosidase-MDV 140-kD fusion protein for RA2 and a 127-kD fusion protein for GA8.Immunoprecipitation of³⁵S-methionine-labeled, MDV-infected chicken embryo fibroblasts (CEF) with antisera against RA2 and GA8 fusion proteins recognized five polypeptides, of which three (p41, p38, and p24) are specified by H19.47 and the remaining two, p135 and p20, have not been previously identified. Immunoprecipitation of³²P-phosphate-labeled or³H-glucosamine-labeled, GA-MDV-infected CEF with the antiserum against RA2 fusion protein identified a phosphorylated polypeptide of 38 kD and two glycoproteins of 60 and 49 kD, respectively. The antisera against recombinant fusion proteins thus revealed the existence of epitopes common to the phosphorylated polypeptides and other MDV-specific polypeptides.Sera from chickens or mice hyperimmunized with the purified fusion proteins reacted with serotype 1, MDV-infected CEF in the fluorescent antibody (FA) test to significant titers. These immune sera did not react with either serotype II or III, indicating the serotype specificity of the phosphorylated polypeptides.Requests for reprints should be addressed to Lucy F. Lee, USDA, Agricultural Research Service, Regional Research Laboratory, 3606 East Mount Hope Road, East Lansing, MI 48823, USA.     

Class specific immunoglobulin response to individual polypeptides of Chlamydia trachomatis, elementary bodies, and reticulate bodies in patients with chlamydial infection.

Sera from 10 women with Chlamydia trachomatis culture positive cervicitis and sera from six men with C trachomatis positive non-gonococcal urethritis were studied for the presence of IgG, IgM, and IgA antibodies to polypeptides of C trachomatis elementary bodies and reticulate bodies using immunoblotting techniques. All the sera with IgG, IgM, or IgA immunoglobulins specific to C trachomatis recognised the major outer membrane protein (MOMP) of elementary bodies. IgG antibodies also detected several other proteins, whereas IgM immunoglobulins recognised only MOMP and proteins of 60 kD, 62 kD, and 66 kD. The IgA reacted with MOMP and the 60 kD and 62 kD proteins in elementary bodies. Class specific antibody response against the proteins of reticulate bodies was similar to that observed for elementary body antigens--with one substantial difference: no reaction was observed in the 60 kD and 62 kD positions. This suggests that 60 kD and 62 kD proteins are deficient in reticulate bodies.     

Comparative cytogenetic study of a continuous human cell line and its subline susceptible and resistant, respectively, to coxsackie B3 virus

Cytogenetic characteristics of the J-96 human cell line and its J-41 subline, highly susceptible and resistant, respectively, to coxsackie B3 virus, were compared. The J-41 subline, as compared to the original J-96 line, had fewer chromosomes in the modal class cells (54-57 and 58-62, respectively) mostly at the expense of normal chromosomes. In most J-41 cells chromosome 21 was eliminated and the number of homologues of chromosomes 2, 9, 11, and 12 was reduced. The percentage of marker chromosomes in the J-41 subline (31.3) was slightly higher than in the J-96 line (24.3). The relationship between differences in the karyotypes and properties of the cultures such as resistance to coxsackie B3 virus, capacity to produce virus-induced interferon and to acquire an antiviral state after treatment with interferon were discussed.     

Immunoblotting analysis of class-specific antibody response in patients with primary HSV infection

Class-specific immune response in acute herpes simplex virus (HSV) infections to individual virus specified polypeptide antigens was analysed by immunoblotting. The HSV specified glycoproteins B (gB), C (gC), and D (gD) were detected. IgG-antibody response was shown to develop to various other virus specific polypeptides as well. The IgM- and IgA-antibody responses remained restricted to only a few HSV specific proteins, namely VP 13 (80 kD), VP 20 (52 kD), and VP 23 (40 kD) and to the low molecular weight polypeptides.     

Modulation in response to temperature of Mayaro virus proteosynthesis in Aedes Albopictus cells

Incubation of Aedes albopictus cells infected with Mayaro virus at 37 degrees C causes inhibition of virus replication. During the first hour post infection (p.i.) incubation at 37 degrees C inhibited cellular and virus proteosynthesis. A preferential translation of heat shock proteins 82 kD and 70 kD was observed. After incubations longer than 1 hr at 37 degrees C, a switch to normal pattern of cell protein synthesis occurred without recovery of virus proteosynthesis. In addition, preferential synthesis of three major virus proteins of 62 kD, 50 kD and 34 kD was observed, when infected cells incubated at 37 degrees C were shifted down to 28 degrees C.     

Antibodies for autonomous parvoviruses of lower animals detected in human serum

Summary Isolates of ADV replicate to rather high quantities in lungs from neonatally infected mink kits. The virus was analysed for polypeptide composition, and for the first time high molecular weight polypeptides have been observed inin vivo produced ADVs. These polypeptides are analogous to those ofin vitro produced ADVs. The molecular weights of the structural polypeptides of the low virulence Pullman ADV and the highly virulent DK and Utah I isolates of ADV were found to be 88kD and 78kD andin vivo produced ADV-G polypeptides were found to be 85kD and 75kD, the same molecular weights as those described forin vitro produced ADV-G. Presence of the ADV coded, non-structural polypeptide with the molecular weight of 71kD (p71) was also demonstrated in the lung tissue from mink kits.With 4 Figures     

Monoclonal antibodies against the nucleoprotein of mumps virus: their binding characteristics and cross-reactivity with other paramyxoviruses

Twelve monoclonal antibodies (MoAbs) directed against the nucleoprotein (NP) of mumps virus were analysed for their binding characteristics. Competitive binding in enzyme-linked immunosorbent assay divided them into eight groups. Two of the MoAbs recognized exclusively 66 kD polypeptide of NP, two recognized 66 kD and 60 kD, and one recognized 66 kD (and 60 kD to a lesser extent) in Western blot assays under either denaturing or partially denaturing conditions. Under partially denaturing condition, another five MoAbs reacted faintly but the remaining two did not react at all. Under denaturing condition, on the other hand, these seven MoAbs showed little reactivity with any polypeptide. Furthermore, denaturation resulted in formation of other polypeptides 55 kD, 50 kD, and 43 kD which all were detected by MoAbs reacting with 66 kD and/or 60 kD. Previously demonstrated antigenic cross-reactivity among the NPs of mumps virus and those of human parainfluenza viruses type 2 and type 4 in radioimmunoprecipitation assay using polyclonal antisera was confirmed by an anti-NP MoAb which showed little reactivity in denaturing Western blot assay.     

Clinical and biochemical significance of toxin production by Aeromonas hydrophila.

Production of cytotoxin and enterotoxin by Aeromonas strains obtained from stools of 50 children in Mexico and Texas and from blood of 9 children with sepsis was determined. Results were correlated with clinical features of infected children as well as with biochemical traits of Aeromonas strains. Cytotoxin was produced by 40 of 42 Aeromonas strains (95%) isolated from stools of children with diarrhea, by all 8 isolates from stools of well children, and by all 9 isolates from children with sepsis. There was no difference in the quantities (amount of cytotoxin per milligram of protein required to kill 50% of the cells) of cytotoxin produced and in clinical manifestations among the groups. None of the isolates produced a toxin that could be neutralized by antiserum raised against Shiga toxin produced by Shigella dysenteriae 1 60R. Heat-labile-like enterotoxin (LT) was produced by 26 of 42 stool isolates (62%), while only 1 of the 42 isolates (2%) produced enterotoxinlike activity in suckling mice; 65% of the cytotoxin-producing strains also produced an LT-like material. All strains from blood produced LT-like material, and 2 of 6 (33%) produced activity in suckling mice. All strains produced hemolysin; 37 of 57 (65%) were Voges-Proskauer positive; 27 of 57 (47%) were lysine decarboxylase positive by API 20E strips, none were positive for lysine decarboxylose production by lysin-iron agar slants at 24 h, but 17 of 54 (31%) were positive at 48 h. There was no correlation between biochemical reactions and enterotoxin or cytotoxin production. There appears to be no correlation between toxin production by Aeromonas spp. and gastroenteritis.     

Analysis of polypeptide composition and antigenic components of Rickettsia tsutsugamushi by polyacrylamide gel electrophoresis and immunoblotting.

Polyacrylamide gel electrophoresis of lysates of purified Rickettsia tsutsugamushi revealed as many as 30 polypeptide bands, including major bands corresponding to molecular sizes of 70, 60, 54 to 56, and 46 to 47 kilodaltons. Compared with the polypeptide composition of the rickettsiae of Gilliam, Karp, and Kato strains and a newly isolated Shimokoshi strain, the major polypeptide in the Kato strain (54-56K) and in the Karp strain (46-47K) migrated a little faster and slower, respectively, than the corresponding polypeptides in the other strains. The largest major polypeptide (54-56K) was digestible by the treatment of intact rickettsiae with trypsin and variable in content in separate preparations, suggesting that the polypeptide exists on the rickettsial surface and is easily degraded during the handling of these microorganisms. Several surface polypeptides of rickettsiae, including the 54-56K and 46-47K polypeptides, were detected by radioiodination of intact rickettsiae followed by polyacrylamide gel electrophoresis of the lysate; however, the 70K and 60K polypeptides were not labeled. Immunoblotting experiments with hyperimmune sera prepared in guinea pigs against each strain demonstrated that the 70K, 54-56K, and 46-47K polypeptides showed antigenic activities. The 54-56K polypeptide appeared to be strain specific, whereas the 70K and 46-47K polypeptides cross-reacted with the heterologous antisera.     

Polypeptide composition of the delta-antigen

Studies by polyacrylamide gel electrophoresis and immunoblotting using 125I-labeled antibody to delta-antigen showed the particles of hepatitis delta virus particles to include 2 polypeptides with molecular weights of 26 and 22 KD carrying epitopes of delta-antigen. In hepatocytes, the delta-antigen is presented by a complex of polypeptides of which the main ones are also polypeptides with molecular weights of 26 and 22 kD.