Expression of biologically active measles virus hemagglutinin glycoprotein by a recombinant baculovirus.

In this study, one of the measles virus membrane proteins, named hemagglutinin (H) which has a key role in tropism, receptor binding, hemagglutinating activity and also induction of protective immunity against viral infection, was expressed by the baculovirus expression system using specific plasmid (pDONR221) to produce entry clone. Measles Virus (AIK-C strain) genome was extracted from infected Vero cells. H gene was amplified by specific primers during RT-PCR reaction and inserted into the specific plasmid (pDONR221) using BP recombination reaction. Recombinant baculovirus harboring H gene was consequently constructed by LR reaction. Insect cells (Sf9) were infected with recombinant baculovirus. In order to increase viral titer, recombinant baculoviruses were passaged four times in Sf9 cells. Synthesis of H protein was verified by SDS-PAGE, western-blot and indirect immunoflourescene using goat polyclonal antibody against Measles Virus. The results showed that H protein was partially glycosylated, but it appeared to be active in hemagglutination assay.

Morphological study of the role of calcium in the assembly of the rotavirus outer capsid protein VP7.

Maturation of rotavirus occurs in the endoplasmic reticulum (ER), a site of intracellular calcium storage. It was demonstrated previously that calcium plays an important role in the maturation of bovine rotavirus. We used protein A colloidal gold conjugated to an antibody to localize VP7, the outer capsid protein of the simian rotavius SA11, in permeabilized infected cells in the presence and absence of calcium in the culture medium. In medium containing calcium, VP7 was associated with nonenveloped double-shelled particles and membranous structures of the ER. In calcium-free medium, gold particles were not associated with the ER or with virus particles. Gold particles were distributed through the cytoplasm and were mainly associated with granular structures, but did not assemble onto virus particles. Our data suggest that in calcium-free medium, VP7 is synthesized, but does not remain incorporated, in the ER.

Legionella pneumophila replicates within rat alveolar epithelial cells.

Legionella pneumophila replicates in the distal pulmonary airspace, causing legionnaires' pneumonia. Legionella organisms replicate within alveolar macrophages and recruited blood monocytes; however, when these cells are activated, they become potent inhibitors of L. pneumophila proliferation. L. pneumophila may replicate in other cells and thereby avoid the host defenses of macrophages. Experiments demonstrated that L. pneumophila replicate within primary cultures of rat pulmonary alveolar epithelial cells. Double-label immunofluorescent and electron microscopy demonstrated L. pneumophila within epithelial cells. Replication of L. pneumophila required similar numbers of alveolar epithelial cells or alveolar macrophages, required viable epithelial cells, and took place intracellularly. While replication of L. pneumophila occurred in both serum-free and serum-containing media, it was enhanced in the presence of serum. Pulmonary alveolar epithelial cells may represent an alternative site for replication of Legionella species in the terminal airspace and thus clarify some previously unexplained aspects of the pathogenesis of legionnaires' disease.

Pseudomonas aeruginosa exoenzyme S is an adhesion.

Exoenzyme S from Pseudomonas aeruginosa has been studied as an adhesion for glycosphingolipids and buccal cells. Binding of exoenzyme S to gangliotriosylceramide (GalNAc beta 1-4Gal beta 1-4Glc beta 1-1Cer), gangliotetraosylceramide (Gal beta 1-3 GalNAcT beta 1-4 Gal beta 1-4Glc beta 1-1Cer), and lactosylceramide (Gal beta 1-4Glc beta 1-1Cer) separated on thin-layer chromatograms was observed. Binding curves for exoenzyme S with dilutions of gangliotetraosylceramide immobilized on plastic plates were similar to previously reported results for the intact bacteria. Binding of exoenzyme S to sialylated counterparts of these glycosphingolipids was not seen, indicating that the addition of a sialic acid residue interferes with binding. Exoenzyme S and monoclonal antibody to exoenzyme S inhibit the binding of P. aeruginosa to buccal cells. The presence of exoenzyme S on the surface of P. aeruginosa was detected by immunogold labeling of bacteria with antibodies to exoenzyme S. Results of these studies led us to conclude that exoenzyme S is an important adhesion of P. aeruginosa.

Subinhibitory concentrations of tetracycline inhibit surface expression of the Pseudomonas aeruginosa ferripyochelin binding protein in vivo.

The effects of subinhibitory concentrations of tetracycline on surface expression of Pseudomonas aeruginosa ferripyochelin binding protein (FBP) and P. aeruginosa virulence in a pulmonary infection model in rats were examined. Rats were inoculated intratracheally with P. aeruginosa strain DG1 embedded in agar beads. One half of the inoculated animals served as untreated controls while the other half received daily injections of 15 mg/kg tetracycline. FBP was shown to be surface exposed in bacteria isolated from control animals using indirect immunofluorescence and immunoelectron microscopy. No FBP was detectable, however, on the surface of bacteria isolated from the lungs of animals treated with tetracycline. The numbers of bacteria recovered from the lungs of infected animals did not differ between control and tetracycline treated groups, although the degree of pathology observed in tetracycline treated animals was significantly lower than in untreated controls (P = 0.002, one way ANOVA). Sub-inhibitory doses of tetracycline reduced proteolytic activity in vitro, but had no effect on the activities of exotoxin A or exoenzyme S. These studies suggest that exposure to subinhibitory concentrations of tetracycline can repress FBP surface expression as well as proteolytic activity in P. aeruginosa leading to a significant decrease in lung injury during infections due to this organism.

The cell attachment proteins of type 1 and type 3 reovirus are differentially susceptible to trypsin and chymotrypsin.

Purified native sigma 1 proteins from [35S]methionine-labeled reovirions [serotypes 1 (T1) and 3 (T3)] were subjected to limited trypsin and chymotrypsin digestion. It was found that T1 sigma 1 was resistant to both trypsin and chymotrypsin, whereas T3 sigma 1 (49K molecular weight) was cleaved by trypsin to yield a 24K and a 25K fragment, and by chymotrypsin to yield a 42K fragment. The 24K tryptic fragment, but not the 25K tryptic fragment, was shown to possess L-cell binding capacity, and represents the carboxy-terminal half of T3 sigma 1 since it contains the single cysteine residue (amino acid 351) as revealed by tryptic analysis of [35S]cysteine-labeled sigma 1. Neither tryptic fragment was able to bind to glycophorin, the reovirus receptor on human erythrocytes. Thus, the mechanism of reovirus host cell attachment is distinct from that of reovirus hemagglutination. The two tryptic fragments were recognized by different neutralizing monoclonal anti-sigma 1 antibodies, indicating that neutralizing and cell attachment sites are not necessarily equivalent. The 42K chymotryptic fragment of T3 sigma 1 was shown to be generated by a cleavage proximal to the carboxy-terminus. Like intact T3 sigma 1, the 42K protein retained its capacity to bind to both L cells and glycophorin, and was recognized by all the neutralizing monoclonal anti-sigma 1 antibodies tested. Thus, the host cell receptor binding site on T3 sigma 1 is located between the trypsin-sensitive and the chymotrypsin-sensitive sites.

Alteration of pulmonary structure by Pseudomonas aeruginosa exoenzyme S.

Intratracheal administration of purified Pseudomonas aeruginosa exoenzyme S elicited extensive, grossly observable damage in the rat lung within 2 h. Light and electronmicroscopy revealed injury and necrosis of bronchial epithelium, type I pneumocytes and capillary endothelial cells after 1 h; associated haemorrhage, fibrinous exudation and released type II cell lamellar bodies in alveolar lumina after 1-12 h; progressively increasing accumulations of polymorphonuclear leucocytes in the bronchi and alveoli and in alveolar septae (interstitial pneumonia) after 1-12 h; collapse of alveolar septal connective tissue and damage to pulmonary arterioles and venules. Treatment of monolayer cultures of bronchial fibroblasts with purified exoenzyme S elicited vacuolation of the cells with apparent membrane damage as revealed by light and electronmicroscopy. In-vivo production and activity of P. aeruginosa exoenzyme S may be an important pathogenicity determinant in the necrotising lung injury characteristic of P. aeruginosa pneumonia.

Calcium requirement for syncytium formation in HEp-2 cells by respiratory syncytial virus.

Respiratory syncytial virus (RSV) grown in HEp-2 cells in the absence of calcium did not induce cell fusion and syncytium formation. Although the infected cells contained viral antigens, the cytopathic effect (giant cell formation) typical for RSV was not observed in calcium-free cultures. Infectious virus yield was also slightly reduced (less than a one log10 reduction) in the absence of calcium. An analysis of viral proteins synthesized in both the presence and the absence of calcium revealed that the amount of fusion protein (F1) in calcium-free infected cultures was approximately one-third that in calcium-containing infected cultures. These results underscore the necessity of using calcium-containing growth medium for cell culture isolation and diagnosis of RSV.

"Campylobacter cinaedi" bacteremia: case report and laboratory findings.

"Campylobacter cinaedi" was isolated from the blood of a 29-year-old homosexual man with previously diagnosed acquired immune deficiency syndrome. Subculturing of the organism was achieved with the use of 7% lysed horse blood and 10% sheep blood agars at 37 degrees C in a microaerophilic atmosphere. Problems associated with the culturing of this organism are reviewed.

Further analysis of the role of calcium in rotavirus morphogenesis.

Previously we reported that calcium plays an important role in the maturation of bovine rotavirus (M. S. Shahrabadi and P. W. K. Lee, 1986. Virology 152, 298-307). We now demonstrate that the formation of mature double-shelled (L) particles was strictly dependent on the concentration of calcium present in the growth medium. The formation of single-shelled (D) particles did not appear to be a calcium-mediated process. Subsequent labeling studies using 45Ca revealed that calcium was incorporated into the L particles but not the D particles. The previously noted decreased level of the outer capsid protein VP7 (42K) in calcium-deprived cultures was now found to be due to the preferential degradation, and not to the impaired synthesis, of this protein in the absence of calcium. It was further demonstrated that calcium had a stabilizing effect on VP7 and that VP7 synthesized in the presence of calcium was not degraded upon subsequent calcium deprivation. Protein degradation during calcium deprivation was apparently limited to the mature form of VP7 since the unglycosylated precursor (pVP7), formed in the presence of tunicamycin, was found to be stable under this condition. Electron microscopic examination of infected cells revealed that in the presence of calcium, virus maturation took place by the budding of viral cores through the endoplasmic reticulum (ER). No such budding was observed in calcium-deprived cells. In these cells mature virions were absent and membrane fragments could be found associated with viral cores or single-shelled particles.

Purification and characterization of the reovirus cell attachment protein sigma 1.

It has previously been shown that of all the soluble reovirus-specified proteins present in the infected cell lysate, protein sigma 1 alone possesses the capacity to bind to host cells (P.W.K. Lee, E.C. Hayes, and W.K. Joklik, 1981, Virology 108, 156-163). We found that sigma 1 from urea-disrupted reovirus particles was also capable of such specific binding. Reovirions were therefore used as a source of functional sigma 1. Accordingly, a simple procedure has been developed to purify sigma 1 by subjecting urea-disrupted reovirions to DEAE ion-exchange chromatography. Protein sigma 1 thus isolated was electrophoretically homogeneous and the recovery was estimated to be 50 to 60% of the theoretical yield. The purified protein presumably maintained its native conformation since it was recognized by a panel of monoclonal anti-sigma 1 antibodies previously isolated, and was capable of specifically binding to host cell receptors, agglutinating human erythrocytes and inducing neutralization and hemagglutination-inhibition antibodies. Subsequent chemical crosslinking studies revealed the presence of oligomeric (mostly dimeric) sigma 1 forms in the preparation. The amino acid composition of the purified sigma 1 was found to closely match that inferred from the S1 gene sequence. However, attempts to determine its amino-terminal sequence have not been successful. The p/ of the purified protein was determined to be 6.8. Circular dichroic measurements of the purified sigma 1 indicated that 54 and 19% of its residues were arranged in alpha-helical and beta-sheet secondary structures, respectively.

Distribution of porin and lipopolysaccharide antigens on a Pseudomonas aeruginosa permeability mutant.

Pseudomonas aeruginosa common surface antigens were compared in a permeability mutant (PCC118) and its parent (PAO503). The distribution of lipopolysaccharide and porin antigens in the mutant supports the conclusion that beta-lactam permeability was affected by lipopolysaccharide-side chain presentation rather than by a change in porin number.

Bovine rotavirus maturation is a calcium-dependent process.

Bovine rotavirus-infected MA-104 cells maintained in the presence and absence of CaCl2 displayed cytopathic effects (cpe) distinct from each other. Lysates of calcium-free cultures were unable to induce cpe in subsequent passages in MA-104 cells, an observation reflected by the demonstration that virus titers of such lysates were drastically reduced. The minimum concentration of CaCl2 in the growth medium required to maintain maximum virus yield was determined to be approximately 0.17 mM. The period of calcium-dependency for infectious virus formation was between 6 and 12 hr postinfection at 37 degrees, a time corresponding to the entire log phase of virus growth. Viruses produced in the absence of calcium were found to be exclusively incomplete single-shelled particles (D particles), as determined by cesium chloride density gradient analysis, electron microscopy, and SDS-polyacrylamide gel electrophoresis. Subsequent examination of virus-specified proteins in infected cells revealed that there was a reduction in the level of the major outer capsid protein (42K) in the absence of calcium. Thus, total inhibition of mature virus production under this condition could be due to the combined effect of reduced production of the 42K protein and incomplete assembly of the virus.

Rapid diagnosis of rotavirus gastroenteritis by a commercial latex agglutination test.

The Rotalex test, a commercial latex agglutination test for rotavirus, was compared with direct electron microscopy (EM) and the Rotazyme test I, a commercial enzyme immunoassay, for detection of rotavirus in stools of children and neonates. For initial stool specimens from 265 children (less than 3 years old) with diarrhea, the Rotalex test had a sensitivity of 81.7% and specificity of 99.5% compared with EM results. Positive and negative predictive values were 98 and 94.9%, respectively. The Rotalex test was slightly more sensitive and specific than the Rotazyme test. When daily stool specimens from patients with rotavirus gastroenteritis were examined, the sensitivity of the Rotalex test varied depending on the time of stool collection relative to the onset of symptoms. Sensitivity was 100 (20/20), 96 (23/24), and 54% (7/13) during 1 to 4, 5 to 7, and 8 to 18 days, respectively, after the onset of symptoms. The sensitivity of the Rotazyme test varied similarly with days from onset. We also examined 214 EM-negative stool specimens from asymptomatic newborns. False positivity by the Rotalex test was only 3.3% (7/214) compared with 4.2% (9/215) for the Rotazyme test. The Rotalex test was as sensitive and specific as EM for detection of rotavirus during the acute stage of illness and much faster and cheaper than EM or the Rotazyme test. The test appears to be suitable for routine use in small hospitals, emergency wards, or even the physician's office for rapid diagnosis of rotavirus gastroenteritis.

Latex agglutination test for detection of Clostridium difficile toxin in stool samples.

A total of 163 stool specimens were tested for detection of Clostridium difficile and its toxin by cytotoxicity assay with tissue culture, latex agglutination test, and isolation of the organism. From 33 specimens which were positive for toxin by cytotoxicity, 30 were positive by the latex agglutination test; the organism was isolated from 21. The total number of samples which were positive with the latex agglutination test was 44. The predictive value of a positive latex agglutination result relative to the cytotoxicity test was 68%, and the predictive value of a negative result was 97.5%. The specificity and sensitivity of the latex agglutination test relative to the cytotoxicity assay and the low cost and simple facilities required indicate that the latex agglutination test is a useful procedure for screening for C. difficile toxins, provided that positive latex results are confirmed by cytotoxicity assay.

Interaction of Clostridium difficile toxin A with L cells in culture.

Toxin A of Clostridium difficile was purified by column chromatography and acetic acid precipitation. Cells exposed to toxin A showed polarization of nuclei towards one pole of the cells. Toxin A was conjugated to ferritin and applied to L cells to localize binding sites of this toxin to the cell surface. It was found that toxin A conjugate attached to the cell membrane in aggregated form. Antibody specific to toxin A was prepared and used for localization of intracellular toxins in intoxicated cells. Toxin A was found inside the cytoplasm 6 h after cell treatment, mainly in the form of aggregates inside the cytoplasmic vacuoles. At 24 h after exposure, toxin A could be detected within the cytoplasm. Tunicamycin treatment of cells reduced the cell-binding efficiency of toxin A to 50%, but neuraminidase did not effect toxin binding significantly.

Studies on the multiplication of a porcine parvovirus.

A porcine parvovirus has been characterized with regard to its replication in foetal porcine kidney cells and certain biophysical properties. Electron microscopy of infected cells at selected times postinfection revealed that porcine parvovirus replication took place within or near a series of granular intranuclear inclusions which may be contiguous with cellular heterochromatin. Developing virions were observed to aggregate into a nucleolar-like amorphous mass which gradually disrupted as cellular integrity was lost. Purified virions were found to have a buoyant density in CsCl of 1.38 g/ml, while 'empty' particles had a buoyant density of 1.29 g/ml. The particle diameter was calculated to be approximately 22 nm.

Detection and localization of aleutian disease virus and its antigens in vivo by immunoferritin technique.

Tissues from mink infected with aleutian disease virus were examined by the electron microscope for the presence of virus particles. Virus-like particles, measuring 22 nm in diameter, were observed in macrophages of spleen, mesenteric lymph node and in Kupffer cells in liver of mink ten to 13 days after infection. The virus-like particles were usually present in vacuoles inside the cytoplasm of macrophages and Kupffer cells and, occasionally, similar particles were observed inside the nucleus. Cells from uninfected mink did not contain such patricles. To correlate the existence of these virus-like particles with the presence of aleutian disease virus antigen in infected cells, tissues were processed for immunoferritin technique. It was found that aleutian disease virus antigen was present in vacuoles inside the cytoplasm of cells from the infected spleen, lymph node and liver, and that the location was similar to that of the 22 nm virus-like particles. In addition, some viral antigen was also detected as cytoplasmic granular material. The nuclei of some cells also contained aleutian disease virus antigen. The pattern of aleutian disease virus antigen was similar to the distribution of virus-like particles in cells of infected tissue. It is suggested that virus replication occurs inside the nucleus with subsequent accumulation of virus in the vacuoles of the cytoplasm.

Characterization of the protein and nucleic acid of Aleutian disease virus.

Aleutian disease virus (ADV) was extracted and purified from infected mink. Nucleic acid extracted from the virus was examined in an electron microscope. Three different sizes of molecule, with approximate lengths of 1.2, 0.55, and 0.25 micron, were observed. The ratios of the large molecules to the small molecules were similar in all the particles prepared under different conditions. Equilibrium CsCl density gradient centrifugation showed that ADV nucleic acid had a buoyant density of 1.733 g/cm3. In Cs2SO4, ADV had a lower buoyant density than that of double-stranded RNA. These properties and its sensitivity to DNase suggested that ADV contains DNA. Thermal denaturation curves revealed that the DNA of ADV had a single-stranded configuration. Polypeptide analysis of ADV by polyacrylamide gel electrophoresis revealed the presence of four polypepties, with molecular weights of 30,000, 27,000, 20,500, and 14,000. These polypeptides were present in a ratio of 10:3:10:1, respectively. The data suggested that ADV is closely related to the members of the parvovirus groups.