Physico-chemical and serological characterization of five rhabdoviruses infecting fish.

Viruses isolated from fish with viral haemorrhagic septicaemia (VHS), infectious haematopoietic necrosis (IHN), spring viraemia of carp (SVC), swim-bladder inflammation (SBI) and pike fry disease (PFD) have been grown to high titre in fathead minnow cells. While our preparations of the IHN, SVC, SBI and PFD viruses showed typical rhabdovirus morphology with bullet-shaped particles and distinct surface projections, the VHS virus preparations had a less typical rhabdovirus morphology but were pleomorphic with a preponderance of flexuous rods. Using virus labelled with [-3H]-uridine, it was shown that each virus contained RNA which sedimented at 38 to 40 S and was hydrolysed by very low concentrations of ribonuclease. The viruses of SVC, PFD and SBI had a polypeptide composition similar to that of vesicular stomatitis virus, the prototype rhabdovirus, but the IHN and VHS viruses gave a pattern similar to that of rabies virus. In serum neutralization tests the SVC and SBI viruses were indistinguishable. VHS virus showed no serological relationship with the other four viruses but there was a low level of cross-reaction between the PFD, IHN and SVC-SBI viruses.     

The glycoprotein G of rhabdoviruses

Summary Rhabdoviruses show an RNA-containing helically-wound nucleocapsid either enclosed by or enclosing a membrane M protein, surrounded by a lipid bilayer through which dynamic protein trimers made up of noncovalently associated monomers of glycoprotein G (G) project outside. Mature monomeric rhabdoviral G has more than 500 amino acids, 2–6 potential glycosylation sites, 12–16 highly conserved cysteine residues, 2–3 stretches of a–d hydrophobic heptad-repeats, a removed amino terminal hydrophobic signal peptide, a close to the carboxy terminal hydrophobic transmembrane sequence and a carboxy terminal short hydrophylic cytoplasmic domain. Association-dissociation between monomers-trimers and displacement of the trimers along the plane of the lipid membrane, are induced by changes in the external conditions (pH, temperature, detergents, etc.). Throughout conformational changes the G trimers are responsible for the virus attachment to cell receptors, for low-pH membrane fusion and for reacting with host neutralizing monoclonal antibodies (MAbs). Antigenic differences could exist between monomers and trimers, which may have implications for future vaccine developments. The familyRhabdoviridae is made up of theLyssavirus (rabies), theVesiculovirus (vesicular stomatitis virus, VSV) and many rhabdoviruses infecting fish, plants, and arthropod insects. All these reasons make the G of rhabdoviruses an ideal subject to study comparative virology and to investigate new vaccine technologies.     

The morphology and morphogenesis of alcoholic cirrhosis of the liver]

A total of 42 biopsy specimens of the liver (blind and spot) in 32 patients with alcoholic cirrhosis of the liver were investigated. Morphological, portal, postnecrotic, and mixed types of cirrhosis were established. The portal type of cirrhosis is most common. On the basis of repeated analyses of biopsy materials of the liver it may be assumed that the development of cirrhosis of the liver of alcoholic etiology is connected with multiple attacks of acute alcoholic hepatitis. Abstention from alcohol consumption resulted in stifestations of exacerbation of cirrhosis. On the other hand, continuation of alcohol consumption contributed to progressing of cirrhosis, which following several attacks of alcoholic hepatitis, may change its morphological type: portal cirrhosis "transforms" into the postnecrotic or mixed type. The data obtained clarified the role of ethanol in progressing alcoholic cirrhosis of the liver, which according to the initial mecranisms of its development in postnecrotic, since every attack of acute alcoholic hepatitis is accompanied by coagulative (fields of alcoholic hyaline, or Mallory's bodies), or by colliquative (balloon dystrophy) necrosis of hepacytes.     

Phosphoproteins, structural components of rhabdoviruses

Polypeptides derived from purified rabies, vesicular stomatitis, and Kern Canyon viruses, which were labeled with ³H-amino acids and ³²P-phosphate, were subjected to electrophoretic fractionation in order to determine which of the structural proteins is phosphorylated. In the rabies virion, the nucleocapsid protein (N protein) was found to be phosphorylated, whereas the other three virus proteins were not. N protein of free viral nucleocapsids isolated from rabies virus-infected cells was also phosphorylated. The phosphorylation of the N protein of rabies virus is confined to a relatively small terminal segment of the polypeptide chain which can be specifically cleaved off by treatment of the nucleocapsid with trypsin. In vesicular stomatitis virus the minor NS protein component is the only phosphoprotein. In Kern Canyon virus the envelope glycoprotein and the N protein are both phosphorylated, but to a lesser extent than either the N protein of rabies virus or the NS protein of vesicular stomatitis virus. All three rhabdoviruses contain a virion-bound protein kinase. Free nucleocapsids derived from rabies virus-infected cells and purified by equilibrium centrifugation in CsCl solution also contain a protein kinase. Intracellular nucleocapsids of vesicular stomatitis or Kern Canyon viruses do not exhibit protein kinase activity.     

Early stomach cancer: its morphology, histo- and morphogenesis

Macroscopic, histologic and ultrastructural features of an early stomach carcinoma are presented on the basis of literature and the authors' data. Ultrastructural and immunohistochemical data confirm the concept of a common histogenesis of different histologic types of stomach carcinoma, i.e. from reserve cells. Carcinoma, most likely, develops from reserve cells of the foveolate epithelium and metaplastic epithelium of intestinal type (foci of incomplete intestinal metaplasia with sulfomucine secretion). The data are accumulating on the precancerous nature of severe epithelial dysplasia. The development of an early carcinoma from preexisting dysplasia was observed in patients after long follow-up with repeated gastric biopsies.     

Peritoneal endotoxicosis,morphology, and morphogenesis of biosystem involvement in experimental animals

Experimental acute peritonitis was induced by single and repeated injections of 1.5–3% fecal autosuspension in the abdominal cavity of white rats. The blood content of medium-weight molecular proteins, which increases 1.5-2-fold during the course of entry of toxic products into the blood over 1–3 days, was measured. Examination of the vascular system and parenchymatous elements of many organs helped reveal three morphogenetic mechanisms of their injury: hyperfunction of cells, followed by their depletion and death; the direct action of toxic products on membranous structures of cells due to severe impairment of the histohematic barrier at the level of the microcirculation; the destructive effect of hypoxia due to the development of the disseminated intravascular coagulation syndrome. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 118, N ^o 12, pp. 636–639, December, 1994 Presented by D. S. Sarkisov, Member of the Russian Academy of Medical Sciences     

Biochemical, serological, and virulence characterization of clinical and oyster Vibrio parahaemolyticus isolates

In this study, 77 clinical and 67 oyster Vibrio parahaemolyticus isolates from North America were examined for biochemical profiles, serotype, and the presence of potential virulence factors (tdh, trh, and type III secretion system [T3SS] genes). All isolates were positive for oxidase, indole, and glucose fermentation, consistent with previous reports. The isolates represented 35 different serotypes, 9 of which were shared by clinical and oyster isolates. Serotypes associated with pandemic strains (O1:KUT, O1:K25, O3:K6, and O4:K68) were observed for clinical isolates, and 7 (9%) oyster isolates belonged to serotype O1:KUT. Of the clinical isolates, 27% were negative for tdh and trh, while 45% contained both genes. Oyster isolates were preferentially selected for the presence of tdh and/or trh; 34% contained both genes, 42% had trh but not tdh, and 3% had tdh but not trh. All but 1 isolate (143/144) had at least three of the four T3SS1 genes examined. The isolates lacking both tdh and trh contained no T3SS2α or T3SS2β genes. All clinical isolates positive for tdh and negative for trh possessed all T3SS2α genes, and all isolates negative for tdh and positive for trh possessed all T3SS2β genes. The two oyster isolates containing tdh but not trh possessed all but the vopB2 gene of T3SS2α, as reported previously. In contrast to the findings of previous studies, all strains examined that were positive for both tdh and trh also carried T3SS2β genes. This report identifies the serotype as the most distinguishing feature between clinical and oyster isolates. Our findings raise concerns about the reliability of the tdh, trh, and T3SS genes as virulence markers and highlight the need for more-detailed pathogenicity investigations of V. parahaemolyticus.     

Biochemical and serological characterization of Campylobacter cryaerophila.

Sixty-two isolates of Campylobacter cryaerophila were recovered from aborted porcine and bovine fetuses, from porcine, bovine, and equine feces, and from different tissues of a dead piglet. Phenotypic characterization was carried out on all isolates, and the results were compared with those obtained with the reference strains of C. cryaerophila, C. jejuni, C. coli, C. laridis, and C. hyointestinalis. The ability of C. cryaerophila strains to grow under aerobic conditions at 16 degrees C was found to be most useful in differentiating them from strains of other Campylobacter species. Studies were undertaken to develop a serotyping system for C. cryaerophila on the basis of the Lior serotyping system for C. jejuni and C. coli by use of a tube agglutination test with formalinized whole-cell (FWC) and boiled whole-cell (BWC) antigens. Antisera against 18 strains of C. cryaerophila were produced in rabbits. Thirty-five percent of C. cryaerophila strains were typed with the FWC suspension as an antigen, and 61% were typed with the BWC suspension as an antigen. None of the C. cryaerophila strains tested autoagglutinated in saline. BWC antigens of C. jejuni, C. coli, and C. laridis cross-reacted with C. cryaerophila, whereas FWC antigens did not cross-react. Neither FWC nor BWC antigens of C. hyointestinalis reacted with C. cryaerophila antisera.     

Heptad-repeat sequences in the glycoprotein of rhabdoviruses

Two or three regions containing three or more successive newly defined heptads of a–d hydrophobic amino acid repeats have been located in the cDNA-derived amino acid sequences of glycoprotein G of all rhabdoviruses examined (rabies, vesicular stomatitis, fish, and plant rhabdoviruses) by computer search. These new heptad-repeats differ from those previously reported in other viruses because of the presence of all the hydrophobic amino acids in positions a or d, and because they are not predicted to form coiled coils by current methods and thus they have not been detected previously in any rhabdoviruses. The two or three heptad-repeat regions were the only parts of the glycoprotein with at least three successive heptad-repeats in all the rhabdoviral sequences studied and had low sequence variability among the members of each of the rhabdoviral genus but show no sequence similarity among the different genus. All these newly detected heptad repeats were in the vicinity of some of the higher hydrophobic regions in each of the rhabdovirus genera studied and were found mostly, but not always, outside the extra amino acid sequences that occur in the longer insect or plant rhabdovirus glycoprotein G. The correspondence of position and structure of these heptad-repeats among all the rhabdoviruses suggests its participation in common function(s), most probably related to viral fusion with cellular membranes.     

Common antigens of Treponema denticola: chemical, physical, and serological characterization.

A sodium deoxycholate-ethanol extractable antigen (DES-Ag) was obtained from four serotypes of Treponema denticola and characterized by chemical, physical, and serological procedures. The cross-reactivity of these antigens was demonstrated by indirect microhemagglutination, immunodiffusion, and immunoelectrophoresis with hyperimmune rabbit antiserum against each of the T. denticola serotypes. Antiserum against two nonoral treponemes, T. phagedenis biotype Reieter and T. pallidum Nichols strain, did not cross-react with the DES-Ag of T. denticola. Chemical analysis of the DES-Ag indicated that proteins (84%) and hexoses (12%) accounted for 96% of the total dry weight of the antigen. Trace amounts of N-acetylglucosamine (0.6%) were also detected. Fatty acids, including palmitic, oleic, and stearic, were identified by gas-liquid chromatography. Polyacrylamide gel electrophoresis of the DES-Ag of each serotype revealed the presence of two bands. The molecular weights of the bands were estimated to be 58,000 and 31,000 by comparing the electrophoretic mobility of the DES-Ag to that of five protein standards of known molecular weights. Although only a single precipitin line was observed by immunodiffusion when each antigen was reacted against its homologous antiserum, two precipitin lines were evident by immunoelectrophoresis. Antiserum against the DES-Ag of T. denticola was shown to agglutinate whole cells of the homologous serotype. Adsorption of this anti-DES-Ag serum with whole cells of T. denticola resulted in the elimination of precipitating antibodies to the DES-Ag by immunodiffusion. It is concluded that the DES-Ag is a component of the outer sheath of T. denticola.     

Electrophoretic and serological characterization of the lipopolysaccharides of Legionella pneumophila.

Lipopolysaccharide (LPS) is a major constituent of the outer membrane of gram-negative bacteria. We used sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteinase-K-digested cell lysates to provide preliminary data on the LPS chemotypes for 20 strains of Legionella pneumophila (serogroups 1 to 8). The profiles of all strains except Chicago 2 (serogroup 6) were similar in the number, spacing, and size distribution of the bands visualized on silver-stained gels and were indicative of smooth LPS. However, compared with the bands from Salmonella minnesota smooth LPS, their banding pattern was much tighter, with three to four legionella bands for every salmonella band. The proteinase K digest of Chicago 2 was unique in that only two widely separated silver-stained bands were seen. LPS profiles of 10 serogroup 1 strains were identical, and the profile of Knoxville 1 was not altered by extensive in vitro passage. We used immunoblotting to investigate the serological specificities of the LPSs. When a rabbit antiserum prepared against a serogroup 1 strain was used to probe nitrocellulose sheets that bound LPS from strains belonging to eight different serogroups, it recognized only the LPS from the homologous serogroup. Similar results were observed with serogroup 2, 4, and 6 antisera. Our data indicate that L. pneumophila has a smooth-type LPS with an unusual banding pattern and that it is a serogroup-specific antigen.     

Identification, expression, localization and serological characterization of a tryptophan-rich antigen from the human malaria parasite Plasmodium vivax

Plasmodium vivax is most common but non-cultivable human malaria parasite which is poorly characterized at the molecular level. Here, we describe the identification and characterization of a P. vivax Tryptophan-Rich Antigen (PvTRAg) which contains unusually high (8.28%) tryptophan residues and is expressed by all blood stages of the parasite. The pvtrag gene comprises a 978bp open reading frame interrupted by two introns. The first intron is located in the 5'-untranslated region while the second one is positioned 174bp downstream to the ATG codon. The encoded approximately 40kDa protein contains a transmembrane domain near the N-terminus followed by a tryptophan-rich domain with significantly high surface probability and antigenic index. It is localized in the parasite cytoplasm as well as in the cytoplasm of the parasitized erythrocyte. The purified E. coli expressed recombinant PvTRAg protein showed a very high seropositivity rate for the presence of antibodies amongst the P. vivax patients, indicating that the antigen generates significant humoral immune response during the natural course of P. vivax infection. Analysis of various field isolates revealed that the tryptophan-rich domain is highly conserved except for three-point mutations. The PvTRAg could be a potential vaccine candidate since similar tryptophan-rich antigens of P. yoelii have shown protection against malaria in murine model.     

The morphogenesis of human cytomegalovirus. Isolation and polypeptide characterization of cytomegalovirions and dense bodies.

Nucleic acid isolated from subviral particles of Fiji disease virus (FDV) was identified as double-stranded (ds)-RNA by the following properties: (1) Positive orcinol reaction; (2) resistance to ribonuclease (RNase) in 1 × SSC (sodium chloride-sodium citrate buffer) but not in 0.1 × SSC; (3) susceptibility to RNase in 1 × SSC after thermal denaturation; (4) sharp thermal denaturation curve with a melting temperature of 76° in 0.01 × SSC; (5) buoyant density of 1.60 g/cm³ in Cs₂SO₄; and (6) no increase in ultraviolet absorption on treatment with formaldehyde at 37°. On electrophoresis in polyacrylamide gel, FDV-RNA separated into nine RNA segments with a total molecular weight of 15.3 × 10⁶.     

Mouse monoclonal anti-N. I. Production and serological characterization

Two examples of mouse monoclonal anti-N are described. The antibodies were derived from mice immunized with sialoglycoprotein extracts of group O MM ss erythrocyte membranes and the probable stimulus for immunization was glycophorin B associated N-antigen. Both antibodies reacted as direct agglutinins but appeared to recognize different epitopes with one having a greater dependence on sialic acid. The antibodies could prove to be valuable alternatives to those reagents used currently for N-blood grouping.     

New class I in man: serological and molecular characterization

New class I antigens in linkage disequilibrium with HLA-A antigen are demonstrated in PHA T and EBV preferential target cells using human alloantisera. These new antigens are defined as class I antigens by immunoprecipitation of a 41-12 k dimer. The molecule is shown to be distinct from the HLA-A, -B, -C molecule and in particular from the A3 molecule as in sequential immunoprecipitations, the depletion of the HLA-A, -B, -C molecule or A3 molecule (44-12 k) has no effect on the new molecule (41-12 k). Being present on the PHA T cells and lymphoblast lines, these antigens are considered as new epitopes involved in the the cell activation process.