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The isoelectric point of the K99 antigen in partially purified preparations isolated from Escherichia coli B41 was 4.2. Electrofocused K99 antigen hemagglutinated guinea pig and sheep erythrocytes and gave a single precipitin line on diffusion against antisera to E. coli B41 and absorbed factor K99 antisera.
Toxigenic strains of Escherichia coli isolated from humans were studied for adherence to human buccal mucosal epithelial cells. The E. coli strains were labeled with 3H-amino acids or fluorescein isothiocyanate. Toxigenic E. coli strains varied in their ability to adhere in the presence of mannose. Of 32 toxigenic strains examined, 52% bound to the buccal cells, whereas none of 8 control strains did so (Mann-Whitney U test, P =0.007). The control strains were nontoxigenic E. coli isolates from humans, enterotoxigenic E. coli isolates from animals, and E. coli K-12 containing the K88 or K99 plasmid; these strains exhibited only background-level adherence in this assay. Among the toxigenic E. coli strains that bound to human buccal mucosal cells, there was no correlation with mannose-resistant hemagglutination (MR-HA) of guinea pig and human erythrocytes. Screening 32 strains, we found the following phenotypes: (i) MR-HA+, buccal adherent; (ii) MR-HA+, buccal nonadherent; (iii) MR-HA-, buccal adherent. Presumably the third group represents strains with another type(s) of surface attachment components not involved in the MR-HA reaction. Our findings indicate that a number of bacterial surface structures can function in MR-HA and buccal adherence.
Rabbit antisera were raised against plaque-purified influenza virus strains of A/Victoria/75 and A/Texas/77 isolated from Seattle influenza patients. The antigenic specificity of hemagglutinins was compared by hemagglutination inhibition (HI) and kinetic neutralization tests. Anti-A/Victoria/75 had equally high HI titers and neutralization rate constants (kappa values) for A/Victoria/75 and A/Texas/77. In contrast, anti-A/Texas/77 had a high HI titer and kappa value to A/Texas/77 and a low HI titer and kappa value to A/Victoria/75. Similar results were obtained with antisera to recombinants with hemagglutinin specific for A/Victoria/3/75 or A/Texas/1/77 and with irrelevant neuraminidase. Seven wild-type isolates, three each of A/Texas and A/Victoria, and one strain characterized as a bridging strain were tested by HI and kinetic neutralization. Characterization as A/Texas or A/Victoria was confirmed by the results. No significant difference in neuraminidase specific for A/Victoria/75 or A/Texas/77 was hown when recombinants with an irrelevant hemagglutinin were compared by the neuraminidase inhibition test. These results suggest that A/Victoria/75 strains are "senior" to A/Texas/77 strains. The epidemiological implications of this observation are discussed.
The uropathogenic Escherichia coli KS52 strain expresses a mannose-resistant hemagglutinin involving an erythrocyte recognition site distinct from the alpha-digalactoside glycosphingolipid receptor identified for the uropathogenic E. coli strains specifying a P adhesin. The KS52 strain showed three major properties. (i) It agglutinated human erythrocytes of all tested blood groups. (ii) Hemagglutinin activity was found both in the supernatant fluid L-broth cultures and in cells grown on L-agar plates. (iii) No fimbriae in organisms grown on L-agar plates were detected by electron microscopy. Whole-cell DNA from the KS52 strain was size fractionated and cloned into the pHC79 cosmid vector. Three recombinant cosmids expressing a mannose-resistant hemagglutination (MRHA) phenotype were characterized and used to subclone the smallest DNA fragment able to confer the same MRHA properties as the parent strain. A 6.7-kilobase chromosomal DNA fragment cloned in pBR322 (pIL14) was shown to be necessary for host-cell MRHA expression and uroepithelial cell adherence. The insert encoded the production of a 16,000-dalton hemagglutinin. This polypeptide could be detected in culture supernatant fluids, in E. coli minicells harboring the pIL14 plasmid, and, by immunoblotting, in the KS52 strain and E. coli whole cells harboring the pIL14 plasmid. No homology was detected by Southern hybridization between the cloned insert and the DNA of the operon responsible for MRHA in the P-specifying, fimbriate strains (pap operon).
Forty-five strains of Campylobacter pylori isolated from gastric biopsy specimens showed distinct hemagglutination activity. The activity was partially decreased by treatment with heat, trypsin, or an alkylating agent and was inhibited by porcine gastric mucin but not by various compounds, including D-mannose.
The mechanisms by which Porphyromonas gingivalis, a gram-negative anaerobic bacterium, is pathogenic for the periodontium remain largely hypothetical. Invasion of host tissues by P. gingivalis is believed to require adhesion of the bacterium to host cells. The aim of this study was to use monoclonal antibodies (MAbs) to characterize the bacterial cell surface component(s) acting as a ligand binding to a receptor on epithelial cells. Surface antigens of P. gingivalis ATCC 33277 were obtained as a glass bead-EDTA extract (GBE), and antiserum against the GBE was produced in rabbits. Epithelial cell membrane proteins (ECMP) were prepared from a homogenate of the SK-MES-1 cell line with Triton X-100. The antigen/ligand profile of GBE was resolved by crossed immunoaffinity electrophoresis by using ECMP in the first-dimension gel. The migration of one immunoprecipitate (IP) was retarded, indicating a ligand-receptor interaction between a surface antigen of P. gingivalis and a complementary binding site on the epithelial cell membrane. The corresponding IP in the GBE/anti-GBE immunoelectrophoresis profile was excised from replicate gels to immunize mice for production of MAbs specific for the bacterial ligand. Five MAbs were obtained and tested for reactivity with GBE in immunoblots and for inhibition of the interaction between GBE and ECMP. Immunoblots revealed polypeptides at 28, 42, 43, and 49 kDa. Inhibition tests were positive for all five MAbs. These results are conclusive evidence that the MAbs recognize functional epitopes involved in the adherence of P. gingivalis to epithelial cells and that the adhesins are likely associated with fimbriae and the hemagglutinating adhesin HA-Ag2.
We have cloned the chromosomal hemolysin determinants from Escherichia coli strains belonging to the four O-serotypes O4, O6, O18, and O75. The hemolysin-producing clones were isolated from gene banks of these strains which were constructed by inserting partial Sau3A fragments of chromosomal DNA into the cosmid pJC74. The hemolytic cosmid clones were relatively stable. The inserts were further subcloned either as SalI fragments in pACYC184 or as BamHI-SalI fragments in a recombinant plasmid (pANN202) containing cistron C (hlyC) of the plasmid-encoded hemolysin determinant. Detailed restriction maps of each of these determinants were constructed, and it was found that, despite sharing overall homology, the determinants exhibited minor specific differences in their structure. These appeared to be restricted to cistron A (hlyA), which is the structural gene for hemolysin. In the gene banks of two of these hemolytic strains, we could also identify clones which carried the genetic determinants for the mannose-resistant hemagglutination antigens Vb and VIc. Both of these fimbrial antigens were expressed in the E. coli K-12 clones to an extent similar to that observed in the wild-type strains. These recombinant cosmids were rather unstable, and, in the absence of selection, segregated at a high frequency.
Escherichia coli isolated from cases of bacteremia and from a variety of urinary tract infections were characterized according to serotype (O:H antigenicity), K type (possession of K1, K2, K3, K12, or K13), hemagglutination (HA) type, and production of beta-hemolysin. Results obtained with the bacteremia and urinary tract infection isolates were similar except for more hemolytic isolated from urine than from blood (42 versus 29%) and more K1+ isolates from blood than from urine (50 versus 29%). A close correlation was found between Ha type VI (production of fimbriae which mediate mannose-resistant HA of human and African green monkey erythrocytes) and the production of hemolysin or K1 capsular antigen or both. Most (95 of 98, or 95%) of the HA type VI+ blood isolates and most (146 of 164, or 89%) of the HA type VI+ urine isolates produced hemolysin or K1 or both, in contrast to 22 and 26%, respectively, of those belonging to HA types other than HA type VI. Also, 76% of all hemolytic and 70% of all K1+ isolates belonged to HA type VI. Remarkably few of the HA type VI+ isolates (13%) and even fewer of the HA type VI- isolates (3%) produced both K1 and hemolysin; these belonged mainly to serotypes O16:H6, O18:H7 and O2:H4. Other major serogroups were usually K1+/hemolysin- (O1, O7) or K1-/hemolysin+ (O2, O4, O6). At least 74% (262 of 351) and possibly as many as 83% (293 of 351) of those isolates which produced mannose-resistant HA of human erythrocytes were classified as HA type VI+; 31 isolates produced mannose-resistant HA with all erythrocytes tested. Taking serogroup and serotype into consideration, we conclude that the E. coli fimbrial hemagglutinin(s) responsible for the HA type VI phenotype will prove to be the same as the virulence-associated mannose-resistant adhesins of uropathogenic E. coli which other investigators have characterized as unique fimbrial antigens detectable by mannose-resistant HA of human erythrocytes.
Hemagglutination titers of La Crosse virus antigens prepared in BHK-21 suspension cell cultures were substantially increased by precipitating the antigens with polyethylene glycol.
To evaluate the usefulness of the indirect hemagglutination (IHA) test in the epidemiological investigation of shigellosis, single serum specimens were tested from 50 patients with Shigella dysenteriae 1 (Shiga bacillus) infections, 103 asymptomatic contacts of these cases, 267 adult and 100 student control, and serum specimens collected during two outbreaks caused by S. sonnei and one outbreak due to S. flexneri 6. In patients with S. dysenteriae 1, 74% demonstrated titers of greater than or equal to 1:40, with 50% showing titers of greater than or equal to 1:160, whereas in the controls 10.4% had titers of greater than or equal to 1:40 and only 0.3% had titers of greater than or equal to 1:160. IHA titers in serum specimens collected from patients with S. sonnei and S. flexneri 6 were too low to be considered diagnostic for individual patients, but were useful in analysis of group results. Groups of ill individuals yielded titers significantly higher than non-ill groups; however, titers from ill groups were usually less than 1:40. The IHA test for S. dysenteriae 1 antibodies serves as a valuable adjunct to the diagnosis of Shiga bacillus dysentery. In our laboratory, an IHA titer of 1:40 or 1:80 is a "borderline positive." Shiga bacillus dysentery is strongly indicated when IHA titers are greater than or equal to 1:60.
The immune adherence hemagglutination (IAHA) test has been used successfully to detect antibody to herpes simplex (HS) virus and varicella-zoster (V-Z) virus. Comparative studies between the complement-fixation (CF) test and the IAHA test revealed that, in most cases, the IAHA test was more sensitive than the CF test. Furthermore, diagnosis on the basis of a fourfold change in antibody titer was made more rapidly by the IAHA test. The IAHA test was found to be a very simple and practical technique requiring only a few hours for completion compared with the conventional CF test which required up to 24 h. In addition, both sera and cerebrospinal fluids could be tested in very low dilutions in the IAHA test, so that very low levels of antibody could be detected. Also, the IAHA test detected antibody to V-Z virus more frequently than did the CF test in adults with a history of varicella occurring 9 to 30 years prior to sampling. The level of cross-reaction between HS and V-Z viruses was examined by both the CF test and the IAHA test, and no major differences between the two techniques were found.
Previously described techniques for detection of hepatitis A antigen (HA Ag) and antibody (anti-HA) have required purified HA Ag and expensive equipment. Herein is described an enzyme-linked immunosorbent assay (ELISA) for specific detection of HA Ag in human stool filtrates and of anti-HA in sera by using selected HA Ag-containing human stool filtrates as the antigen source. Because human stools often react nonspecifically in serological tests for HA Ag, blocking with preexposure and hyperimmune anti-HA sera from a chimpanzee inoculated with hepatitis A virus was used to confirm specific detection of HA Ag. The sensitivity of ELISA was found to be comparable to that of solid-phase radioimmunoassay (SPRIA) and immune electron microscopy (IEM). Of 37 acute-phase stools collected from nine patients, 16 were positive for HA Ag by ELISA. In 13 of these, HA Ag particles were found by IEM, and an additional 3 stools negative by ELISA contained HA Ag particles by IEM. Eight control stools were negative by both ELISA and IEM. Anti-HA was measured in sera by demonstrating its ability to block binding of the enzyme conjugate to HA Ag in a stool without detectable nonspecificity. This test (blocking ELISA) was as sensitive and specific as blocking SPIRA, IEM, and immune adherence hemagglutination and, like SPRIA and IEM, detected early-developing antibody. The ELISA is simple to perform and requires only a minimum of equipment. It is useful for screening stools for HA Ag and for monitoring HA Ag during purification, as well as for detecting early and late anti-HA in sera.
An enzyme immunossay (EIA) was adapted for detection of antibody to varicella-zoster virus, and its sensitivity and specificity were compared with those of neutralization, immune adherence hemagglutination (IAHA), and complement fixation tests. Test sera showed little nonspecific reactivity in the EIA system, and valid results could usually be obtained at serum dilutions as low as 1:8. Demonstration of the presence or absence of varicella-zoster viral antibody by EIA showed 94% correlation with results obtained in neutralization tests, but EIA titers were 2- to 16-fold higher than neutralizing antibody titers. Results by IAHA showed 87% correlation with those obtained by neutralization. No false positive IAHA results were seen, but a number of false negative IAHA results were seen at the 1:8 serum dilution, particularly in older individuals. With increasing age (>40 years), and presumably increased time from varicella infection, neutralizing antibody levels generally declined to 1:8 or 1:16, EIA levels fell to 1:128 or 1:256, and IAHA and complement fixation antibody titers were usually <1:8 or 1:8. EIA and IAHA were as reliable as the neutralization and complement fixation tests for serodiagnosis of varicella and zoster infections. All tests demonstrated heterotypic varicella-zoster antibody titer rises in selected patients with initial herpes simplex virus infections, but fewer heterotypic responses were seen by EIA than by the other methods. EIA offers a rapid, sensitive, and specific method for varicella-zoster antibody assay that is applicable to use in a clinical setting.
The close genetic and antigenic relatedness among the group 2 coronaviruses human coronavirus OC43 (HCoV-OC43), bovine coronavirus (BCoV), and porcine hemagglutinating encephalomyelitis virus (PHEV) suggests that these three viruses with different host specificities diverged fairly recently. In this study, we determined the complete genomic sequence of PHEV (strain PHEV-VW572), revealing the presence of a truncated group 2-specific ns2 gene in PHEV in comparison to other group 2 coronaviruses. Using a relaxed molecular clock approach, we reconstructed the evolutionary relationships between PHEV, BCoV, and HCoV-OC43 in real-time units, which indicated relatively recent common ancestors for these species-specific coronaviruses.
Newcastle disease virus (Herts strain), grown in embryonated eggs or in a line of bovine kidney cells, was purified and then separated by sucrose density gradient centrifugation into infectious (IH) and noninfectious hemagglutinating (NIH) particles. These particles were morphologically similar, although the average size of IH was twice that of NIH particles. The activity of hemagglutinin per milligram of virus protein was two- to threefold higher in NIH particles than in IH particles, whereas the specific activity of neuraminidase did not differ in the two particle types. This was consistent with the observed particle size difference. The distribution of the major proteins in IH and NIH particles from egg-grown virus, determined by polyacrylamide gel electrophoresis (PAGE), was significantly different. In IH particles the molar ratio of protein 1 (74,000 daltons) to proteins 2 and 3 (56,000 daltons): protein 6 (41,000 daltons) was 1.0:2.5:2.5; in NIH particles the ratio was 1.0:0.6:1.0. When Newcastle disease virus was grown in bovine kidney cells, the molar ratio of proteins in IH particles resembled that of of egg-grown virus. However, in NIH particles from bovine kidney cells, only protein bands corresponding to protein 1 and proteins 2-3 were present and their molar ratio was 1.0:0.6. Protein 6 was marginally detectable in these particles. Analysis of the proteins in [3H]isoleucine- and [14C]glucosamine-labeled virus showed proteins 1 and 2 (glycoproteins) present in the ratio of 1.0:0.5; protein 3, the nucleoprotein, was not detected. These results are compatible with previous findings by others that NIH particles are deficient in RNA and nucleoprotein antigen, and suggest that formation of discrete particles of Newcastle disease virus by budding requires at most minimal amounts of proteins 3 or 6. The fatty acid composition of egg-grown IH and NIH particles was not significantly different and resembled that of normal allantoic fluid.
Transfer of phospholipid from the envelope of hemagglutinating virus of Japan (HVJ) to erythrocyte (RBC) membrane and the virus-induced transfer of phospholipid between RBC membranes were studied using spin-labeled phosphatidylcholine (PC). The transfer of PC from membranes labeled densely with PC to unlabeled membranes was followed by the peak height increase in the electron spin resonance spectrum. The two kinds of transfer reactions took place very rapidly as reported previously. To obtain further details, the transfer reactions were studied with HVJ, HVJ inactivated by trypsin, HVJ harvested early, HVJ grown in fibroblast cells, the fibroblast HVJ activated by trypsin, influenza virus, and glutaraldehyde-treated RBCs. The results demonstrated that the viral F glycoprotein played a crucial role in the transmembrane phospholipid movements as well as in the fusion and hemolysis of RBCs. The transfer from HVJ to RBC's occurred partially through an exchange mechanism not accompanying the envelope fusion. This was shown by a decrease in the exchange broadening of the electron spin resonance spectrum of released spin-labeled HVJ (HVJ) and also by an increase in the ratio of PC to viral proteins incorporated into RBC membranes. HVJ modified RBC membrane so as to be able to exchange its phospholipids with those of inactive membranes such as fibroblast HVJ, influenza virus, glutaraldehyde-treated RBC'S, and phosphatidylcholine vesicles. HVJ affected the fluidity of RBC membranes markedly, the environments around PC being much fluidized. The virus-induced fusion was discussed based on close apposition of the membranes by HANA proteins and on the destabilization and fluidization of RBC membranes by F glycoproteins.
The array of Bacteroides gingivalis W83 antigens revealed by crossed immunoelectrophoresis includes one antigen that is associated with an erythrocyte-binding capacity, termed the hemagglutinating adhesin HA-Ag2. This antigen was excised from crossed-immunoelectrophoresis plates to produce two polyclonal antisera, VL 011 and WL 303, whose restricted specificity for HA-Ag2 was assessed using crossed immunoelectrophoresis, crossed immunoelectrophoresis with an intermediate gel, and crossed immunoaffinoelectrophoresis. Both antisera, when used to probe blots of an EDTA cell surface extract of B. gingivalis W83, reacted with two bands, at 33 and 38 kDa, which were also detected by a monoclonal antibody (Naito et al. 1985. Infect. Immun. 50: 231-235), specific for a hemagglutinin of B. gingivalis. Antiserum WL 303 was used to examined by immunoblotting the distribution of HA-Ag2 among a variety of human and animal strains of B. gingivalis. All human strains tested showed two major bands at 33 and 38 kDa in the EDTA cell surface extract, and at 43 and 49 kDa in outer membrane preparations. Only one band, at 29 kDa, was detected in EDTA cell surface extracts from the animal strains, while the outer membrane preparation of a single strain showed a positive reaction. We concluded that HA-Ag2 is an antigen common to human and animal strains of B. gingivalis and that its subunits may show heterogeneity in apparent molecular mass.
While investigating myoblast fusion using enveloped viruses, we unexpectedly found that the production of hemagglutinating virus of Japan (HVJ; Sendai virus) was suppressed temperature dependently in quail myoblasts transformed with a temperature-sensitive Rous sarcoma virus, which proliferate at 35.5 degrees but differentiate at 41 degrees; viral production was normal at 35.5 degrees but suppressed at 41 degrees irrespective of the species of host cells. The production of some viruses, i.e. measles virus, influenza virus, herpes simplex virus type 1 and poliovirus, was also markedly suppressed at 41 degrees, suggesting that a temperature of 41 degrees affects viral infection generally. To clarify the mechanism of the suppression, the infectious pattern of HVJ was examined both at 37 degrees and at 41 degrees in LLC-MK2 cells. The synthesis of HVJ-specific proteins was inhibited at the transcriptional level at 41 degrees, although viral penetration by envelope fusion was not affected. The transcriptional inhibition was also seen in quail fibroblasts, which do not express a 70-kD heat shock protein (HSP70), suggesting that HSP70 is dispensable for the inhibition of viral gene transcription at 41 degrees. Further, when the infected cells were incubated at 41 degrees after the viral proteins had been synthesized at 37 degrees, viral production was also inhibited. Immunofluorescent staining of the cells exposed to 41 degrees showed that HVJ envelope proteins formed large aggregates on the cell surface, into which both M and NP proteins were assembled. Under the electron microscope, HVJ virions appeared normal even at 41 degrees, but were detected in clusters on the cell surface, unlike at 37 degrees. These observations suggested that the release of HVJ virions from the cell surface was inhibited for some reason at 41 degrees. Consequently, it was indicated that two steps, viral gene transcription and the release of virions, were inhibited at 41 degrees.