Dieter Burger

The complement-requiring neutralization of equine arteritis virus by late antisera

Abstract Neutralization of equine arteritis virus (EAV) by late antibody raised in horses, guinea pigs, rabbits, hamsters, and mice was investigated and found to be complement-dependent. The complement-dependent EAV neutralizing antibody activity was found associated with the IgG fraction of late antisera. Early antisera or their IgM and IgG fractions were ineffective. Analysis of virus-antibody interaction at 37° showed that no appreciable loss of EAV infectivity occurred following incubation with heat-inactivated late antiserum for 20 min. However, upon prolonged incubation, partial reduction in EAV infectivity was detected. The sensitization of EAV (as assessed by anti-IgG) was shown to begin following mixing with antiserum and was essentially completed after 20 min of incubation. Neutralization of sensitized EAV by complement or anti-IgG was instantaneous and not temperature dependent. Analysis of EAV infectivity (in vitro) indicated that specific antibody apparently did not interfere with the early steps of virus-cell interaction. Further studies suggested that following penetration into susceptible cells, sensitized EAV was rendered insensitive to complement inactivation. The present investigation has confirmed the existence of a complement-dependent viral neutralizing antibody in late antisera and, further, has shown that EAV provides a new model for analysis of the complement-dependent neutralization of sensitized virus.

The fate of sensitized equine arteritis virus following neutralization by complement or anti-IgG serum

Abstract The mechanisms involved in the neutralization of infectious equine arteritis virus (EAV)-antibody complexes by complement or anti-IgG serum were investigated. Analysis of label release from 3 H-uridine labeled RNA revealed that after addition of equine or guinea pig complement to sensitized EAV, disruption of the virion with release of RNase-sensitive viral RNA occurred within 30 min at 37 °. In contrast, when sensitized EAV was neutralized with either anti-IgG serum at 37 ° or complement at +2 °, the RNA label remained sedimentable and RNase-resistant. These data and studies with infectivity restored to complement-neutralized EAV-antibody complexes by trypsin treatment seemed to indicate that neutralization of infectious virus-antibody complexes precedes virolysis and takes place by steric hindrance of “critical sites” on the viral envelope. Complement-mediated neutralization is then followed by a temperature-sensitive step, possibly involving late-acting members of the complement series, which leads to virolysis.

The role of sensitizing antibody in the neutralization of equine arteritis virus by complement or anti-IgG serum.

Abstract The dissociability of the infectious equine arteritis virus (EAV)-antibody complexes was investigated before and after neutralization with complement or anti-IgG to determine the possible role of the sensitizing antibody in the multicomponent virus neutralization system. The binding between EAV and sensitizing antibody was irreversible as tested by rapid dilution and sonic vibration. Following digestion of antiserum with trypsin, the antibody retained its capacity to bind to EAV and render it sensitive only to inactivation by anti-IgG. When sensitized EAV was treated with trypsin, about 80% of viral infectivity was no longer sensitive to complement inactivation but remained sensitive to inactivation by anti-IgG. A kinetic study revealed that, when complement-neutralized EAV was treated with trypsin, the maximal recovery of viral infectivity was obtained at 5 min and remained relatively steady for about 150 min. Further analysis indicated that trypsin-mediated reactivation of the complement or anti-IgG-neutralized EAV was accomplished, as expected, by the cleavage of the sensitizing antibody into Fc portion and Fab fragments. The Fab fragments, apparently remained attached to the virion as determined by the sensitivity of the digested neutralized complex to anti-IgG reinactivation and its resistance to complement reinactivation. The present investigation suggests that neutralization of sensitized EAV by complement is dependent on the presence of the complement determinants of the sensitizing antibody, while neutralization by anti-IgG is only dependent on the presence of the antibody determinants. It appears that the role of the sensitizing antibody in the multicomponent EAV-neutralization system is that of providing the specific attachment sites for complement and anti-IgG, the effectors of such viral neutralization.

In vivo and in vitro characteristics of contagious ecthyma virus isolates: Host response mechanism

Three Vero cell culture-adapted contagious ecthyma virus (CEV) isolates were compared by plaque morphology, ability to induce vesicles in skin and in vivo growth curve characteristics by sampling sequentially experimental skin lesions produced in four sheep and one goat. Two of the isolates (CEV-29A and CEV-378) were from outbreaks of ecthyma in sheep and one (CEV-102) from a human case of orf. When replicating in Vero cells, the viruses exhibited similar growth parameters, but were distinguishable from each other on the basis of plaque morphology. In vivo latent periods for these isolates were 48 h (CEV-29A), 96 h (CEV-102), and 120 h (CEV-378). When isolates CEV-102 and CEV-29A were passaged into another sheep, they produced similar patterns of growth. Isolate CEV-102 produced the highest infectivity titer [1.4 X 10(9) plaque forming units (PFU) g-1], followed by CEV-29A (6.8 X 10(7) PFU g-1) and CEV-378 (2.5 X 10(7) PFU g-1). In addition, these viruses varied in their ability to induce vesicle formation. Virus was no longer detectable at the inoculation sites at 288 h post-infection (PI). We conclude that plaque morphology, ability to induce vesicle formation in the skin and growth curves in the skin can be considered as important criteria to differentiate CEV isolates. A comparison of the growth curves of CEV-378 in the skin of sheep and goats suggested differences in virus-host interaction between the two animal species. Since intravenous injection of 1 X 10(9) PFU of CEV failed to produce lesions in the sham-scarified skin of sheep, virus spread via the hematogenous route from one site to another appears unlikely. No virus-neutralizing antibody or interferons were found in serum samples or in skin homogenates collected between 0 and 24 days PI. Virus-neutralizing antibody was present in the circulation as late as 24 days PI. Lymphocytes collected from CEV-exposed sheep as early as 12 days PI responded specifically to stimulation with CEV antigen. As this was about the time when infectious virus disappeared from the sites, we assume that cell-associated immune mechanisms may play a larger role in virus clearance from skin lesions than virus-neutralizing antibody.

Characterization of monoclonal antibodies to bovine herpesvirus type I, Los Angeles strain

We produced monoclonal antibodies (mAbs) against bovine herpesvirus type 1 (BHV-1), Los Angeles strain, and then evaluated them as potential candidates for preparing diagnostic reagents. Of the 318 cell lines expressing antibodies to the virus, 60% (192) secreted IgG and 40% (126) secreted IgM. Twenty-six mAbs were selected based on enzyme-linked immunosorbent assay (ELISA) and virus neutralization (VN) titers and characterized by immunoprecipitation, immunofluorescence and immunoblots. The selected mAbs were assigned to one of four groups based on their immunoprecipitation patterns. Group A (4 mAbs) precipitated a complex of three glycoproteins with molecular weight (MW) 130 kDa, 72 kDa and 55 kDa, which presumably represented gI of BHV-1. Monoclonal antibodies of this group were highly reactive in ELISA but had low VN titers. Group B (4 mAbs) precipitated a glycoprotein with MW of 71 kDa (gIV). This group of mAbs had high VN titers. Group C (16 mAbs) precipitated a 97 kDa glycoprotein (gIII). Monoclonal antibodies of this group had high ELISA but low VN titers. Group D (2 mAbs) precipitated a double band of non-glycosylated proteins with MW of 37/32 kDa; these proteins could not be assigned to any of the antigens of BHV-1 previously described. ELISA and VN titers of this group of mAbs were low. To test the antigenic variability of the antigenic determinants which were recognized by these 4 groups of mAbs, we adapted Madin Darby bovine kidney cell-propagated BHV-1 Los Angeles strain to Crandells feline kidney cell line. After the tenth passage in feline kidney cells, the epitopes on the 37/32 kDa peptide recognized by the mAbs group D were no longer detectable. Additional changes were noted in the electrophoretic mobility of the 130 kDa and 71 kDa glycoproteins (gI) identified by mAb of group A shifted downward. The 71 kDa glycoprotein (gIV) reactive with mAb group B and the 97 kDa (gIII) reactive with mAb group C remained stable. Since clone No. 191 of group B mAb was potent in ELISA, VN, immunoblots and immunofluorescence, and recognized an epitope which did not change under selective pressure, we feel that the mAb produced by this clone are a good candidate for the production of diagnostic reagents.

Analysis of bovine herpesvirus 4 (DN 599) major antigens with monoclonal antibodies and polyclonal immune serum.

SummaryMonoclonal antibodies (MAbs) and polyclonal immune sera were produced and used to identify the major antigens of bovine herpesvirus type 4 (BHV-4). SDS-polyacrylamide gel electrophoresis of immunoprecipitates of radiolabeled lysates from infected cells resolved 24 peptide bands varying from 12kDa to over 300kDa. Six peptides were identified as major viral antigens by immunoprecipitation. Based on the pattern of radioimmunoprecipitation, MAbs were assigned into four groups. Group 1 precipitated a tunicamycinsensitive glycoprotein complex which contained six components (245, 190, 152, 123, and 48/46kDa). Deglycosylation with endoglycosidase F revealed two peptides with Mr of 93 and 38kDa as the basic peptides of the glycoprotein complex. In addition, a 115kDa glycopeptide containing glycan-peptide bonds of mixed type was identified. Group 2 precipitated a non-glycosylated protein complex consisting of three monomers (33/31/30kDa). Groups 3 and 4 reacted with single monomeric non-glycosylated peptides with Mr of 48 and 14kDa, respectively. Although none of the MAbs exhibited significant neutralizing activity, some reacted strongly in immunosorbent and/or immunohistochemical assays, suggesting they may be good candidates for use in diagnostic assays.

Onset, kinetics and duration of in vivo and in vitro delayed hypersensitivity responses of neonatal calves sensitized with mycobacterial components

Abstract Newborn calves developed delayed skin-test responses 10 days after a single intradermal inoculation at birth with 150 μg of purified derivative of Mycobacterium bovis associated with 150 μg of mycobacterial immunopotentiating glycolipid P3 and oil droplets. Development of tuberculin skin-test reactivity was detected simultaneously with increased lymphocyte transformation responses. Longitudinal studies revealed that two of three calves tested at 17 months of age were still skin-test positive. The kinetics of cellular hypersensitivity responses in neonatal calves sensitized with mycobacterial components are compared to the results of acquired cellular resistance and delayed hypersensitivity studies in phylogenetically disparate species. We concluded that bovine neonates have the capacity to rapidly develop cellular immune responses following stimulation with mycobacterial antigens.

Molecular cloning and physical mapping of bovine herpesvirus 4 strain DN 599 and comparison with two American field-isolates

SummaryNinety four percent of the genome of bovine herpesvirus 4 (BHV-4) strain DN 599 was cloned and a physical map was constructed by Southern blot analysis using a library of cloned fragments cleaved with the 3 restriction enzymes (Eco RI, Bam HI, and Hin dIII). The genome length was estimated to be 156.5 kbp±0.7. The genome comprises a region of unique segment (114 kbp) and two flanking segments containing tandem repeats. The size of each repeat was approximately 2.35 kbp and each repeat contained one Eco RI site and two Bam HI sites. We also examined two recent American field-isolates of BHV-4 and compared the Eco RI maps of the two isolates with that of DN 599. We observed the following: (1) insertions or deletions of restriction sites at the periphery of the unique segment; (2) variation in the lengths of junction fragments; (3) variations in the lengths of hypermolar Eco RI fragments containing the repeats; and (4) the Eco RI map of one of the American field-isolates resembles the BHV-4 “Movar type” of Europe.

Heat-stable (STa) enterotoxin of enterotoxigenic Escherichia coli: Binding of the enterotoxin to coagulated milk and casein

Oral or intragastric inoculation of the STa enterotoxin of Escherichia coli has been the standard laboratory test for that toxin. We demonstrated that the severity of the secretory response of 2-4-day-old mice to a single dose of the toxin was influenced by weaning of these mice. After oral application of 50-250 fmol of purified, radioiodinated STa toxin (12-13 Ci mmol-1) approximately 55-65% of the administered toxin bound to the stomach contents. Binding of the toxin did not destroy its biological activity. The binding was pH dependent; stomach contents bound 75% of the toxin at pH 2.3 and only 7.4% at pH 10.1. The principle in the stomach contents which bound the toxin was also demonstrated in murine and bovine milk. Further studies with renin-coagulated milk and commercial casein indicated that milk casein bound the toxin. Based on these findings, we speculate that in cases of food-poisoning involving casein-containing foods STa toxin may be present in the absence of cultivable enterotoxigenic E. coli.