Analysis of Soluble Bovine Viral Diarrhoea Virus Antigens and Serological Relationship to Virus Structural Glycoproteins

Abstract

IntroductionPapers demonstrating bovine viral diarrhoea virus (BVDV)-specific antibodies (10) in sera from recovered cattle by using "soluble" antigen preparations, or describing the production of vaccines against BVDV (7, 17) and studies of serological relationship (1-3, 8, 13, 18) between BVDV and hog cholera virus (HCV) obviously deal with soluble antigens which differ in their chemical and biological properties. Differences have been found with regard to composition (3, lo), stability (3, lo), and buoyant density (3, 7,9).The following studies investigate whether BVDV structural glycopolypeptides (1 5 ) display serological identity with the soluble antigen possessing nucleoside tri-and -diphosphase (XTPase) activity (2) and which is serologically related to HCV (SA) (1,3,13). Material and MethodsThe cytopathogenic BVDV strains Oregon C 24 V and NADL, propagated in primary calf testis cell cultures and labelled with 3%-methionine in some of these experiments, were purified as described (15). Crude soluble antigen preparations were obtained by pelleting cell debris and virus (SW 27, 26 000 rpm, 4 h, + 4 "C). High-molecular proteins were removed by 40 O/o (w/v) ammonium sulphate precipitation. Purified SA was isolated from crude preparations according to V A N AERT et al. (3).Abbreviations used: BVDV = bovine viral diarrhoea virus; H C V = hog holera virus; SA = soluble enzymatically active and HCV-related BVDV antigen; IDT = immunodiffusion test; IE = immunoelectrophoresis; MCIE = micro countercurrent immunoelectrophoresis; XTPasc = enzymatically active for nucleoside tri-and diphosphate digestion; mol. wt. = molecular weight; BAS-BVDV = bovine anti-BVDV serum; PAS-HCV = porcine anti-HCV serum. U. S.Subsequently, each preparation of the soluble antigens was mixed with Triton X-190(1 'Yo, w/v), sonicated in an ice-bath for 45 sec, incubated overnight at + 4 OC and sonicated once more. After exhaustive dialysis (3 days a t + 4 "C) against PBS with 0.1 O/o Triton X-100 (w/v) and low-speed centrifugation (4,000 xg, 30 min, + 4 "C) a portion of each sample was assayed for precipitating activities and mol. wt. determination in the SDS-PAGE. The rest was taken for affinity chromatography on protein A-Sepharose.For this purpose, bovine anti-BVDV serum (BAS-BVDV) and porcine anti-HCV serum (PAS-HCV) of naturally or experimentally infected animals (3,11,13) were dialyzed against PBS, adsorbed onto protein A-Sepharose CL-4 B (Deutsche Pharmacia GmbH, D-7800 Freiburg) after thorough washing and resuspension in PBS. Excessive and nonreacting proteins were removed and the beads then mixed with soluble antigen preparations and gently shaken for 30 min at room temperature. After an incubation period overnight at + 4 OC, the beads were extensively washed on a suction filter with PBS and finally once with double-distilled water, and then transferred into small plastic tubes. SDS and 2-mercaptoethanol-containing T E N buffer (pH 7.5) were added and the mixtures heated in a boiling water-bath and subjected to SDS-PAGE (15).Infecti...

Differences in Reaction Behaviour of Structural Polypeptides of Bovine Viral Diarrhoea Virus (BVDV) with Antisera against BVDV and Hog Cholera Virus (HCV)

“…A., Brussels, Belgium). All the sera used showed high precipitating activities and cross-reactivities in the I D T (14) and good immunofluorescence as well as high titres of virus neutralization ( 9 , but lacked any reaction in the IDT with non-infected cell homogenates as controls (14). IgG was isolated from these sera by salt precipitation, gel filtration and subsequent ion-exchange (13).…”

“…For degradation and test procedures (immunoprecipitation in vitro, IDT, micro countercurrent immunoelectrophoresis -MCIE -, affinity chromatography on protein A-Sepharose), the viral preparations were mixed with Triton X-100, sonicated, and dialyzed as described before (14). Details of the methods and materials used for IDT, MCIE and the isolation of the immune-complexes have been described in previous papers (14,16,20). For affinity chromatography, 15 mg. protein A-Sepharose CL-4 B (Deutsche Pharmacia GmbH, Freiburg) was swollen, washed and resuspended in 1OOpl.…”

“…The properties of the SA (1,2,8,15) as well as of the precipitating HCV antigen (1, 10-12) have been reported previously. These two antigens could not be identified as viral structural proteins (12,14,17,18). A relationship between BVDV and HCV particles was demonstrated by DINTER (5) using bovine and porcine hyperimmune sera in cross-neutralization tests.…”